Richard O. Cliff

Controlled release of transforming growth factor-β 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.

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.

Circulation persistence and biodistribution of lyophilized liposome-encapsulated hemoglobin: an oxygen-carrying resuscitative fluid.

Objective: To evaluate the circulation persistence and organ biodistribution of a freezedried, oxygen carrying resuscitative fluid: liposome‐encapsulated hemoglobin. Design: Randomized, animal studies. Setting: Accredited animal research facilities. Subjects: Normal female Balb/c mice and male New Zealand rabbits. Interventions: Two groups of normal female Balb/c mice were injected in the tail vein with either lyophilized liposome‐encapsulated hemoglobin (n = 9) that was reconstituted just before administration, or with unlyoph ilized liposomeencapsulated hemoglobin (n = 9) as a comparison. Two groups of male New Zealand rabbits were injected in the ear vein with either lyophilized 99mTc‐liposome‐encapsulated hemoglobin (n = 6) or unly oph ilized 99mTc‐liposome‐encapsulated hemoglobin as a comparison (n = 6). After injection, mice were anesthetized by brief inhalation of halothane followed by blood sampling through the retro‐orbital sinus. Rabbits were anesthetized 30 mins before liposome‐encapsulated hemoglobin administration with an intramuscular injection of a 5:1 mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg). Rabbits were then dynamically imaged for 90 mins, housed, and at 20 hrs, imaging again followed by autopsy and tissue sampling to validate imaged organ biodistributions. Measurements: Circulation persistence in the mouse was measured by removing a blood sample at various time points up to 24 hrs after injection. The blood sample was centrifuged in a hematocrit capillary tube and the disappearance of the sedimented liposome‐encapsulated hemoglobin fraction was measured. The change in the sedimented fraction of the liposomes with time was used to generate circulation persistence profiles in mice. The circulation persistence and organ biodistribution of 99mTc‐liposome‐encapsulated hemoglobin was measured by circling regions of interest on computer‐generated gamma camera images. These image intensities were then calculated as a function of total injected dose which was measured from a known volume and activity of 99mTc‐liposomeencapsulated hemoglobin. Actual tissue uptake was estimated from images by subtracting blood pool contribution which was measured by injecting 99mTc‐labeled rabbit red cells. Imaged organ biodistribution was validated at 20 hrs by measuring activity in weighed portions of tissue after autopsy. Main Results: The mean circulation half‐life of liposome‐encapsulated hemoglobin in mice injected at a dose of 1.0 g phospholipid/kg mouse and 1.95 g hemoglobin/kg was approximately 10.4 ± 0.5 (sd) hrs. The circulation half‐life of lyophilized liposome‐encapsulated hemoglobin was 10.7 ± 0.7 hrs. The circulation profiles demonstrate a rapid removal phase over the first 4 hrs after injection, followed by a secondary slow removal measured up to 24 hrs. The rapid removal phase of liposome‐encapsulated hemoglobin and lyophilized liposome‐encapsulated hemoglobin in the rabbit (injected at the same dose) indicated that lyophilized liposome‐encapsulated hemoglobin persists longer than the unlyophilized form in the first 4 hrs after injection. The organ biodistributions of unlyophilized 99mTc‐liposome‐encapsulated hemoglobin and lyophilized 99mTc‐liposome‐encapsulated hemoglobin in the rabbit demonstrate that the reticuloendothelial system is the primary site of removal, with significant uptake of lyophilized 99mTc‐liposome‐encapsulated hemoglobin by the liver (15.6 ± 1.0%), bone marrow (12.6 ± 1.6%), and spleen (9.7 ± 1.1%). The kidneys showed little accumulation of unlyophilized 99mTc‐liposome‐encapsulated hemoglobin or lyophilized 99mTc‐liposome‐encapsulated hemoglobin (1.6 ± 0.2% and 1.8 ± 0.1%, respectively), an important result for the efficacy and safety of this hemoglobin‐based blood substitute. Conclusion: The present results suggest that liposome‐encapsulated hemoglobin (and lyophilized liposome‐encapsulated hemoglobin) have pharmacokinetics that enable oxygen delivery during early treatment for hemorrhagic shock. The organ biodistribution demonstrates that the monocyte phagocytic system is principally involved with the slow removal of liposomeencapsulated hemoglobin (and lyophilized liposome‐encapsulated hemoglobin) over the course of 24 hrs. The lyophilized liposome‐encapsulated hemoglobin has similar pharmacokinetics to freshly prepared liposome‐encapsulated hemoglobin and could be an important storage strategy for the utilization of liposome‐encapsulated hemoglobin in areas where stored blood is unavailable. (Crit Care Med 1994; 22:142‐150)

Physiological responses, organ distribution, and circulation kinetics in anesthetized rats after hypovolemic exchange transfusion with technetium-99m-labeled liposome-encapsulated hemoglobin.

ABSTRACT Physiological responses and circulation properties of liposome-encapsulated hemoglobin (LEH) labeled with technetium-99m (99mTc) were measured in rats after a 10% (170 mg/kg hemoglobin, 430 mg/kg phospholipid) or a 50% (450 mg/kg hemoglobin, 2.3 g/kg phospholipid) hypovolemic exchange transfusion (n = 5 per exchange group). Mean arterial pressure returned to baseline values (105 ± 8 mmHg) by 90 min post-infusion for both groups. By 20 h, mean arterial pressure remained at baseline values for the 10% group, but dropped to 30 ± 14 mmHg for the 50% group. For both groups, bradycardia was seen after the exchange period, but heart rate recovered by 30 min for the 10% group and by 90 min for the 50% group. The 99mTc-LEH remained in circulation longer for the 50% group (18.2 h half-life) than for the 10% group (2.4 h half-life). Removal of 99mTc-LEH from the bloodstream was via the liver and spleen. At 20 h, 99mTc-LEH accumulation in these organs was greater for the 10% group (liver, 36.2 ± 1.7%; spleen, 37.5 ± 2.5%) than for the 50% group (liver, 17.0 ± 1.4%; spleen, 17.1 ± 1.4%). The data show that there is less clearance of 99mTc-LEH from the bloodstream by the reticuloendothelial system after a 50% hypovolemic exchange transfusion, thus supporting the possible use of LEH as an oxygen-carrying resuscitative fluid in situations of severe blood loss.

A Comparative Study of the Accurate Measurement of Endotoxin in Liposome-Encapsulated Hemoglobin

We have examined three different methods of endotoxin determination utilizing the Limulus Amebocyte Lysate (LAL) assay to accurately determine endotoxin levels in Liposome Encapsulated Hemoglobin (LEH), 1) the gel-clot method, 2) chromogenic spectroscopic-based LAL, and 3) the turbidimetric method which determines endotoxin levels in solutions based on the time needed to reach a specific degree of turbidity. Both the chromogenic and turbidimetric methods require significant dilution of the LEH preparation before accurate measurement can be made. We have tested the levels of endotoxin in LEH solutions using these methods and measured LEH, liposome, and hemoglobin samples spiked with known amounts of endotoxin. A comparison of the three methods shows that the absolute value of endotoxin measured in LEH by the three methods can vary significantly. However, within any one assay the spiked amount of endotoxin in the sample can be accurately measured. The accuracy of these methods may also be complicated by the binding of endotoxin to LEH. This was evident by mixing free endotoxin with LEH followed by centrifugation to separate the LEH. Biological activity of endotoxin bound to LEH was measured by exposure to RAW264.7 followed by the expression of tumor necrosis factor.

Complement Activation and Thromboxane Secretion by Liposome-Encapsulated Hemoglobin in Rats in Vivo: Inhibition by Soluble Complement Receptor Type 1

Intravenous administration of liposome-encapsulated hemoglobin (LEH) in rats led to an early (within 15 min) decline of hemolytic complement (C) activity in the plasma along with a significant, parallel rise in thromboxane B2 (TXB2) levels. The TXB2 response was inhibited by co-administration of soluble C receptor type 1 (sCR1) with LEH, as well as by C depletion with cobra venom factor. These observations provide evidence for a causal relationship between LEH-induced C activation and TXB2 release, and suggest that sCR1 could be useful in attenuating the acute respiratory, hematological and hemodynamic side effects of LEH described earlier in the rat.

Transient changes in the mononuclear phagocyte system following administration of the blood substitute liposome-encapsulated haemoglobin

We have examined the effects of administration of the blood substitute, liposome-encapsulated haemoglobin (LEH), in the normovolaemic rat. Test groups included LEH, lyophilized EH, the liposome vehicle, unencapsulated haemoglobin and normal saline, which were injected into the tail vein (n = 6; n = 3 for sham and saline groups). Administration of LEH (2.5 g phospholipid, 1.25 g haemoglobin/kg rat) was followed by blood sampling at 2 h, 24 h, 1 wk and 2 wk. Blood samples were analysed for alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, total and indirect bilirubin, serum creatinine, albumin, total protein, lipase, cholesterol, blood urea nitrogen, haematocrit, haemoglobin and differential white blood cell counts. Observed effects following injection were mild and transient, with baseline values recovered at 1 wk. Alanine aminotransferase increased moderately in the LEH group at 24 h to 601 +/- 143 IU/dl (P < 0.0001), with a return to baseline at 1 wk. Aspartate aminotransferase showed a smaller increase from 46 +/- 5 to 162 +/- 40 at 24 h and also returned to baseline at the 1 wk measurement (P < 0.001). The transient increase in serum transaminases was not observed for the lyophilized LEH group. Tissue sections showed accumulation of liposome groups in resident macrophages of the liver and spleen. Incubation of an adherent population of human peripheral blood monocytes with LEH in culture did not elicit the production of the inflammatory cytokine, tumour necrosis factor. Pre-incubation of monocytes with LEH prior to exposure to endotoxin did, however, result in a reduced expression of this inflammatory cytokine.

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.

Technological Development of Lipid-Based Microcylinders: Biocompatibility and Controlled Release

We have developed a lipid-based microcylinder for the controlled release of biological response modifiers. Lipid microcylinders are composed of 1,2-ditricosa-10,12-diynoyl-sn-glycerol-3-phosphocholine which form hollow cylinders 50–250 μm in length with a diameter approximately 0.5–1.0 μm. Amphiphilic molecules such as ganglioside (5–6 mol%) can be incorporated into the bilayers of the microcylinder, modulating surface characteristics of the structure. In this study, we have examined the biocompatibility of lipid microcylinders and lipid microcylinders containing 6 mol% ganglioside. The interaction of microcylinders with peripheral blood monocytes and three cell lines: U937, a histocytic monocyte; K562, an erythroblast, and; a Jurkat derivative, a lymphoblast was assessed. Toxicity, as measured by proliferative status of the cell lines, was not evident at lipid concentrations up to 100 μg/ml lipid. Peripheral blood monocytes were observed to closely associate, but not engulf lipid microcylinders. However, when ganglioside was present in the lipid microcylinders this interaction was markedly decreased. We have begun to measure the release rates of growth factors such as transforming growth factor-beta (TGF-β) from lipid microcylinders. Release of TGF-β from the microcylinders follows first-order kinetics. The rate of release can be modulated by increasing the temperature which results in a thermotrophic phase transition of the lipid at 43 °C.

Use of oxygen-15-labeled molecular oxygen for oxygen delivery studies of blood and blood substitutes

Blood substitutes are being developed as a temporary replacement fluid and oxygen carrier for patients with severe blood loss due to trauma, shock or combat casualty (1,2). These substitutes are not meant to replace blood for they do not have properties bestowed by white blood cells, platelets and other plasma factors. Within the field of blood substitute research, there is a great need for developing techniques to determine the effectiveness of various blood substitutes in relationship to their ability to pick up oxygen as it passes through the lungs or deliver oxygen when it reaches the tissues. These comparisons must be performed to monitor the effectiveness of the various substitutes between each other or as a group against the native oxygen carrier, red blood cells (RBC). For widespread use, these methods to determine effectiveness in relationship to oxygen transport properties must be relatively non-invasive and physiologic, as well as applicable to human patients by being easy to perform in a clinical setting and use non-toxic agents. Moreover, these techniques must be able to distinguish the tissue oxygenation by the blood substitute in the presence of other oxygen carriers, namely RBC. Many techniques including frozen myocardial spectroscopy, microelectrodes, phosphorescence quenching, magnetic resonance spectroscopy, near-infrared spectroscopy, NADH fluorescence and electron spin resonance have been summarized for their usefulness in assessing oxygenation either directly or indirectly by Wagner and Scheid (3).