Flow electroporation chamber and method

Abstract

The present invention relates to a method and apparatus for the encapsulation of substances and drugs into cells and platelets. The present invention is also related to the incorporation of thrombus dissolving drugs, such as tissue plasminogen activator and streptokinase into platelets using the apparatus described herein. The treated platelets can then be used to treat patients suffering from a thrombus blocking a blood vessel. The present invention is also related to a preparation of red blood cells that has a stable right shift of the oxygen dissociation curve.

Description

TECHNICAL FIELD The present invention relates to methods and apparatus for the encapsulation of biologically-active substances in various cell populations. More particularly, the present invention relates to a method and apparatus for the encapsulation of allosteric effectors of hemoglobin in erythrocytes by electroporation to achieve therapeutically desirable changes in the physical characteristics of the intracellular hemoglobin. BACKGROUND OF THE INVENTION In the vascular system of an adult human being, blood has a volume of about 5 to 6 liters. Approximately one half of this volume is occupied by cells, including red blood cells (erythrocytes), white blood cells (leukocytes), and blood platelets. Red blood cells comprise the majority of the cellular components of blood. Plasma, the liquid portion of blood, is approximately 90 percent water and 10 percent various solutes. These solutes include plasma proteins, organic metabolites and waste products, and inorganic compounds. Th...

AI Technical Summary

Benefits of technology

The present invention relates to a method and apparatus for the encapsulation of biologically-active substances in various cell populations. More specifically, the present invention provides an electroporation chamber that may form part of an automated, self-contained, flow apparatus for encapsulating compounds or compositions, such as inositol hexaphosphate, in red blood cells, thereby reducing the affinity of the hemoglobin for oxygen and enhancing the delivery of oxygen by red blood cells to tissues. Encapsulation is preferably achieved by electroporation; however, it is contemplated that other methods of encapsulation may be used in practicing the present invention. The method and apparatus, including the electroporation chamber, of the present invention, is equally suited to the encapsulation of a variety of biologically-active substances in various cell populations.

It is another object of the present invention to provide a method and apparatus that allows continuous encapsulation of biologically-active substances in a population of cells or vesicles.

Problems solved by technology

Current methods of electroporation make the procedure commercially impractical on a scale suitable for commercial use.

The drawbacks associated with the liposomal technique include poor reproducibility of the IHP concentrations incorporated in the red blood cells and significant hemolysis of the red blood cells following treatment.

Additionally, commercialization is not practical because the procedure is tedious and complicated.

Treatment of the red blood cells according to the process disclosed results in a cell with unaffected activity.

The osmotic pulse technique has several shortcomings including the fact that the technique is tedious, complicated and unsuited to automation.

In addition, the results are typically unpredictable and unreliable.

For these reasons, the osmotic pulse technique has had little commercial success.

However, the results obtained by Mouneimne and his colleagues indicate that approximately 20% of the retransfused cells were lost within the first 24 hours of transfusion.

The electroporation methods disclosed in the prior art are not suitable for processing large volumes of sample, nor use of a high or repetitive electric charge.

In addition, the stability of the P50 right shift as well as the stability of the red blood cells has not proved adequate for clinical use.

Furthermore, the methods are not suitable for use in a continuous or “flow” electroporation chamber.

Continuous use of a “static” chamber results in over heating of the chamber and increased cell lysis.

Furthermore, the existing technology is unable to incorporate a sufficient quantity of IHP in a sufficient percentage of the cells being processed to dramatically change the oxygen carrying capacity of the blood.

In addition, the prior art methods require elaborate equipment and are not suited for loading red blood cells of a patient at the point of care.

Thus, the procedure is time consuming and not suitable for use on a commercial scale.

The acute symptoms and pathology of many cardiovascular diseases, including congestive heart failure, ischemia, myocardial infarction, stroke, intermittent claudication, and sickle cell anemia, result from an insufficient supply of oxygen in fluids that bathe the tissues.

Likewise, the acute loss of blood following hemorrhage, traumatic injury, or surgery results in decreased oxygen supply to vital organs.

Without oxygen, tissues at sites distal to the heart, and even the heart itself, cannot produce enough energy to sustain their normal functions.

The result of oxygen deprivation is tissue death and organ failure.

Although the attention of the American public has long been focused on the preventive measures required to alleviate heart disease, such as exercise, appropriate dietary habits, and moderation in alcohol consumption, deaths continue to occur at an alarming rate.

This leads to inadequate oxygenation of their tissues and subsequent complications.

Often the amount of blood which can be drawn and stored prior to surgery limits the use of autologous blood.

Typically, a surgical patient does not have enough time to donate a sufficient quantity of blood prior to surgery.

As each unit requires a period of several weeks between donations and can not be done less than two weeks prior to surgery, it is often impossible to sequester an adequate supply of blood.

Although it is evident that methods of enhancing oxygen delivery to tissues have potential medical applications, currently there are no methods clinically available for increasing tissue delivery of oxygen bound to hemoglobin.

The natural regulation of DPG synthesis in vivo and its relatively short biological half-life, however, limit the DPG concentration and the duration of increased tissue PO2, and thus limit its therapeutic usefulness.

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