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The present invention is a method for releasing an active agent into a use environment by disposing this active agent within the lumen of a population of tubules. and disposing the tubules into a use environment, either directly or in some matrix such as a paint in contact with the use environment. The tubules have a preselected release profile to provide a preselected release rate curve. The preselected release profile may be achieved by controlling the length or length distribution of the tubules or by placing degradable endcaps over some or all of the tubules in the population, or by combinations of these methods. The invention is also a population of tubules having a preselected release profile to provide a preselected release rate curve., Supersedes PAT-APPL-523 725-95.

A process and medium are disclosed for the lyophilization of platelets. During lyophilization, carbohydrate-load platelets are supercooled while suspended in a buffer solution including a biocompatible polymer that serves to preserve the structure of the platelets. The supercooled platelets are then frozen at a temperature below the glass transition temperature of the suspension. A vacuum is placed on the frozen suspension to remove most of the water therefrom. Then, the temperature of the platelets is increased to the supercooled temperature while the vacuum is maintained. After being sealed under vacuum, the lyophilized platelets may be reconstituted to form viable, transfusable platelets. The reconstituted platelets have a high aggregation index, retain normal agglutination and degranulation capability, and are able to participate in clot formation., Supersedes PAT-APPL-546 464-95.

A process for forming spatially oriented neo-vascular capillaries comprising: (a) providing a combination ultra-thin film (UTF) pattern of at least one cell adhesion promoter and at least one cell adhesion inhibitor wherein the cell adhesion promoters nave a linewidth of between about 50-490 microns; (b) seeding the combination UTF pattern with endothelial cells at an initial seeding cell density; (c) adding a first medium for incubating the seeded endothelial cells until the endothelial cells are grown to confluency; (d) replacing the first medium with a second medium at confluency; and (e) allowing the endothelial cells to differentiate into spatially oriented neo-vascular capillaries., Supersedes PAT-APPL-234 034, AD DO16 321

One aspect of the present invention is a method for storing tissues and cells (typically erythrocytes) having the step of: (1) stabilizing the cells with a reversible stabilizing agent. This method typically will have the additional steps of; (2) loading the cells with a cryoprotectant, and typically; (3) storing the cells in liquid, frozen, or dry state. This method will also typically have the additional step of; and (4) prior to use, reversing the stabilization reaction. Preferably, the erythrocytes are pre-treated with Co to complex the hemoglobin with Co. It is anticipated that a practical method according to the invention will include reoxygenation of the enthrocytes, and also washing out reagents prior to in vivo use. Another aspect of the present invention is an erythrocyte that has had its shape stabilized by the reversible crosslinking of proteins in the erythrocyte, such as the structural proteins of the cytoskeleton. Another aspect of the invention is a population of such reversibly crosslinked erythrocytes. Another aspect of the invention is the in vivo use of such erythrocytes, after the reversal of the crosslinking reaction. The use of more gentle, reversible crosslinking as described below is desirable to result in the recovery of erythrocyte deformability and extended post-transfusion survival.

Aprocess and medium are disclosed for the lyophilization of cells, specifically red blood cells and platelets, and cell-like matter, which comprises the use of solution including a carbohydrate, and biocompatible polymers to permit reconstitution of transfusably useful cells which are viable by the measure of ATP and 2,3 DPG., Supersedes PAT-APPL-497 708-95, AD D017 689.

Incubation of liposome-encapsulated Hb (LEH) with rat serum at 37C led to accelcelerated decay of serum complement (C) hemolytic activity (CH50). Empty liposomes (L) caused less expressed decrease of CH50, whereas free Hb had no effect on C hemolytic activity. The LEH- and L-induced increase in C consumption was unlikely due to endotoxin (LPS) contamination, as spiking of rat serum with LPS caused reduction in CH50 only at levels significantly higher than detectable in LEH or L. The LPS- induced C consumption was not potentiated by free hemoglobin.

Liposome encapsulated hemoglobin is being developed as hem an artificial resuscitative fluid for in vivo oxygen delivery. in the present report, we examine the effect of accumulation of liposome encapsulated hemoglobin on the structure of reticuloendothelial organs following administration of liposome encapsulated bovine hemoglobin in the normovolemic rat. We have also examined the administration of the liposome vehicle, tetrameric bovine hemoglobin, and liposome encapsulated bovine hemoglobin that had been lyophilized with 300 mM trehalose and rehydrated just before injection. Following injection into the tail vein, rats were sacrificed and liver, spleen, kidney, and lung harvested at 2 h, 24 h, 1 week, and 2 weeks for analysis. Gross pathologic findings of animals injected with liposome en may capsulated hemoglobin showed statistically significant splenomegaly with a waxy parenchymal pallor at early time points. Microscopic findings indicate that the liver and spleen are principally involved with liposome encapsulated hemoglobin removal over the course of 24 h with transient cytoplasmic vacuolization in tissue resident phagocytes as evidenced by both light and electron microscopic examination. Presence of liposome encapsulated hemoglobin in these vacuoles was confirmed by oil red 0 and prussian blue stains. Splenic weight was observed to decline after 24 h but still remained significant above sham-treated controls at 2 weeks and could be correlated with increased hematopoietic activity.

Incubation of liposome-encapsulated Hb (LEH) with rat serum at 37 deg C led to accelerated decay of serum complement (C) hemolytic activity (CH50). Empty liposomes (L) caused less expressed decrease of CH50, whereas free Hb had no effect on C hemolytic activity. The LEH- and L-induced increase in C consumption was unlikely due to endotoxin (LPS) contamination, as spiking of rat serum with LPS caused reduction in CH50 only at levels significantly higher than detectable in LEH or L. The LPS-induced C consumption was not potentiated by free 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., Prepared in cooperation with Geo-Centers, Inc.,Fort Washington, Md.

The invention is directed to a composition comprising a permeabilizing agent, a preserving agent, and a buffered solvent. This composition is used to prepare the cells for lyophilization cells and to rehydrate the cells to recover them from lyophilization. The process of this invention comprises adding the permeabilizing agent and the preserving agent in a buffered solution to red blood cells, agitating the combination for a period of time sufficient to allow permeation of the preserving agent into the cell, shell freezing the mixture, and lyophilizing the mixture. The dry lyophilized material can then be stored. The cells can be rehydrated using the same composition of permeability agent, preserving agent and buffered solvent., Supersedes PAT-APPL-659 765-91, AD-D015 031.

The invention relates to a 99m-Tc-labeled liposome encapsulated protein and to a highly efficient method of radiolabeling liposome encapsulated protein. In particular, a 99mTc carrier is used to label preformed liposome encapsulated hemoglobin. The liposome-encapsulated 99MTc labeled hemoglobin is highly stable in vitro and in vivo and is suitable for a variety of clinical uses, including biodistribution imaging studies. The invention also relates to a method of using technetium-99mTc-labeled liposome encapsulated hemoglobin to label neutrophils. A kit method which could be used for the convenient preparation of 99mTc-labeled liposome encapsulated hemoglobin for clinical use is also disclosed.

A hemoglobin-containing blood substitute preparation is protected from oxidation to methemoglobin. The protected preparation comprises between 10mM and 60 mM of a compound selected from the group consisting of glutathione(GSH), nicotinamide adenine dinucleotide (NADH), nicotine adenine dinucleotide phosphate (NADPH) and mixtures of these in the aqueous phase of a hemoglobin-containing fluid. Oxidized hemoglobin in a preparation can be restored by adding between 10 mM and 60 mM of a compound selected from a group consisting of glutathione (GSH), nicotinamide adenine dinucleotide (NADH), nicotine adenine dinucleotide phosphate(NADPH) and mixtures of these to a hemoglobin-containing fluid in which the hemoglobin has converted in whole or part to methemoglobin, and storing the fluid at storage temperatures for blood products above the freezing point of the fluid for a time sufficient to convert methemoglobin to hemoglobin., Supersedes PAT-APPL-233 303-88, AD-D013 894.