Alan S. Rudolph

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.

Fabrication and selective surface modification of 3‐dimensionally textured biomedical polymers from etched silicon substrates

A new method is described for producing biomedically relevant polymers with precisely defined micron scale surface texture in the x, y, and z planes. Patterned Si templates were fabricated using photolithography to create a relief pattern in photoresist with lateral dimensions as small as 1 micron. Electroless Ni was selectively deposited in the trenches of the patterned substrate. The Ni served as a resilient mask for transferring the patterns onto the Si substrate to depths of up to 8.5 microns by anisotropic reactive ion etching with a fluorine-based plasma. The 3-dimensional (3-D) textured silicon substrates were used as robust, reusable molds for pattern transfer onto poly (dimethyl siloxane), low density poly (ethylene), poly (L-lactide), and poly (glycolide) by either casting or injection molding. The fidelity of the pattern transfer from the silicon substrates to the polymers was 90 to 95% in all three planes for all polymers for more than 60 transfers from a single wafer, as determined by scanning electron microscopy and atomic force microscopy. Further, the 3-D textured polymers were selectively modified to coat proteins either in the trenches or on the mesas by capillary modification or selective coating techniques. These selectively patterned 3-D polymer substrates may be useful for a variety of biomaterial applications.

Encapsulation of hemoglobin in a bicontinuous cubic phase lipid

The effects of encapsulating bovine hemoglobin (BHb) in the bicontinuous cubic phase formed by monooleoylglycerol and water was investigated with Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction. Cubic phase was formed in the presence of 1-10 wt% BHb. Studies using X-ray diffraction reveal that at 0.5-2.5 wt% BHb, the cubic phase structure is characterized by the double diamond lattice (Pn3m). At 2.5-5 wt% BHb, coexistence of two cubic phase structures, Pn3m and the gyroid lattice (Ia3d), was observed while at BHb, concentrations higher than 5 wt% the gyroid structure persists. FTIR shows there is an increase in intensity of the free nu C = O (1745 cm-1) and a corresponding decrease in the intensity of the hydrogen bonded nu C = O (1720 cm-1) as the BHb concentration is increased. The nu C-O-CO peak shifts from 1183 cm-1 to 1181 cm-1 as the concentration of BHb raised from 2.5 to 10 wt% indicating BHb may induce subtle changes in the interfacial region of cubic phase monoolein. The bandwidth of the nu asCH2 stretch (2926 cm-1) increased in the presence of 5 wt% BHb compared to samples with 2.5 or 10 wt% BHb. The increase in frequency of the nu sCH2 stretch (2854 cm-1) induced by increasing temperature 20 to 60 degrees C was dampened when BHb was present compared to samples heated in isotonic buffer. Analysis of the amide I band at 1650 cm-1 showed that the secondary structure of BHb is not affected by encapsulation in monoolein. In vitro release studies showed that 45% of the entrapped BHb was released after 144 h at 37 degrees C. The porous nature of bulk cubic phase was further demonstrated by diffusion of K2Fe(CN)6 and conversion of 73% of the oxyhemoglobin to methemoglobin after 1 h. These results suggest that the cubic phase may be useful for encapsulation of Hb as a red cell substitute and for the encapsulation and delivery of other bioactive agents.

Lyophilized liposome encapsulated hemoglobin: evaluation of hemodynamic, biochemical, and hematologic responses.

ObjectiveTo characterize the hemodynamic, biochemical, and hematologic responses to the administration of the oxygen-carrying fluid lyophilized liposome-encapsulated hemoglobin in the conscious, normovolemic rat. DesignProspective, randomized trial. SettingAnimal laboratory, Jefferson Medical College. SubjectsEighty-four male Sprague-Dawley rats. InterventionsCatheters were introduced into the right atrium (through the jugular vein) and both femoral arteries of test animals, and a thermistor was placed in the ascending aorta through the left common carotid artery for infusion of lyophilized liposome-encapsulated hemoglobin, blood collection, and blood pressure (BP) and cardiac output determinations. Measurements and Main ResultsLyophilized liposome-encapsulated hemoglobin (n = 8) infusion (1 mL/min iv) at 1 or 6 mL/kg (10% of estimated blood volume) had no detectable effect on BP, cardiac output, total peripheral resistance, and heart rate during the 5-hr observation period. The infusion also had no effect on hematocrit, leukocyte count, and serum tumor necrosis factor-α concentrations. Survival at 7 days was 100% (n = 20). Lyophilized liposomeencapsulated hemoglobin caused transient (2-hr) thrombocytopenia (-24 ± 9% vs. a Ringers lactate control group, p < .01), and marginally increased serum thromboxane B2 concentrations (14.6 ± 6 pg/100 μL, p < .01). ConclusionsThese data suggest that lyophilized liposome-encapsulated hemoglobin can be safely administered to conscious rats, supporting the development of this substance as a potential blood substitute. (Crit Care Med 1994; 22:480–485)

A new salutary resuscitative fluid: liposome encapsulated hemoglobin/hypertonic saline solution.

Low-volume resuscitation with hypertonic (7.5%) saline (HTS) is an evolving therapeutic modality for patients with hemorrhagic shock. This solution has been shown to exert protective hemodynamic effects in models of controlled hemorrhagic shock and in several clinical trials. However, HTS has no oxygen-carrying capacity and therefore does not improve oxygen delivery directly. One of the leading strategies in developing an oxygen-carrying resuscitative fluid is the encapsulation of hemoglobin within phospholipid vesicles (LEH). This preparation has the advantage of being blood type and antigen free, easily adaptable to scale-up production, and remarkably stable with a long shelf life. We therefore tested the hypothesis that lyophilized LEH reconstituted with HTS will improve tissue oxygenation and survival in rats exposed to a lethal controlled hemorrhagic shock. Shock was induced by withdrawal of 70% of blood volume and therapy (n = 10-16) with HTS (5 mL/kg), LEH (5 mL/kg), lactated Ringers solution (vol:vol = 1:3), LEH-HTS (5 mL/kg), or oxygen (100%) was initiated 15 minutes later. The LEH-HTS improved skeletal muscle oxygen tension directly measured using a thin-film chamber oxygen sensor (PO2 87 +/- 13 mm Hg vs. 40-50 mm Hg in other groups, p < 0.05). This was associated with improved blood pressure, reduced acidosis, and increased survival at 24 hours (75% vs. 6%-25% in other groups, p < 0.05). In conclusion, the study demonstrates a remarkably salutary effect of LEH reconstituted with HTS as a blood substitute in the treatment of hemorrhagic shock.

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.

Biologically engineered microstructures : controlled release applications

The area of self-assembled ultrafine particulate-based composites (nano composites) has been a major thrust in advanced material development. In this paper we report on the application of biologically derived, self-assembled cylindrical microstructures to form advanced composite materials for controlled release applications. These microstructures (we call them tubules) have many applications in the material sciences. This paper will focus on the potential for rationally controlling the fabrication of submicron microstructures for controlled release applications.

Complement activation in vitro by the red cell substitute, liposome- encapsulated hemoglobin: mechanism of activation and inhibition by soluble complement receptor type 1

BACKGROUND: Liposome‐encapsulated hemoglobin (LEH) has been developed as an emergency blood substitute, yet its effect on human complement has never been explored. Considering that complement activation is a major pathogenic factor in the respiratory distress syndrome that often develops in trauma and shock, LEH‐induced complement activation may be a critical safety issue. STUDY DESIGN AND METHODS: Various LEH and corresponding empty liposomes were incubated with normal human sera, and various markers of complement activation (serum levels of C4d, Bb, SC5b‐9, and CH50; C5a‐induced granulocyte aggregation; membrane deposition of C3b) were measured. Incubations were also performed in the presence of (ethylene‐bis[oxyethylenenitrilo]tetraacetic acid) (EGTA) and Mg++ (EGTA/Mg++) and soluble complement receptor type 1. RESULTS: LEH and liposomes activated human complement, as indicated by significant changes in one or more markers. The effect was primarily due to the presence of the phospholipid vehicle; small, unilamellar, highly homodispersed vesicles induced the greatest degree of complement activation. Complement activation was partially inhibited by EGTA/Mg++. The latter finding, together with the parallel increases in serum C4d and Bb, suggests activation of both the classical and alternative pathways. Soluble complement receptor type 1 (0.05‐20 micrograms/mL) efficiently inhibited all vesicle‐induced complement activation. CONCLUSION: Because of complement activation, the use of LEH for transfusion may require careful evaluation of safety. Soluble complement receptor type 1 may be useful as a prophylactic agent for complement activation‐related complications of liposome infusions.

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 lipids 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.

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.