Chemistry: molecular biology and microbiology – Apparatus – Mutation or genetic engineering apparatus
Reexamination Certificate
2000-11-08
2003-09-09
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Mutation or genetic engineering apparatus
C435S173600, C204S290120, C204S290130
Reexamination Certificate
active
06617154
ABSTRACT:
TECHNICAL FIELD
The present invention relates to improved electrodes for use in generating an electrical field in a saline solution. In particular, the invention relates to erosion resistant electrodes for use in a variety of applications, such as in an electroporation device for the encapsulation of biologically-active substances in various cell populations. More particularly, the present invention relates to improved electrodes for use in 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
The present invention provides that an electrode surface may be protected from wear, such as erosion and pitting, due to internally generated electrical signals occurring in a saline solution. In particular, a pulsed electrical signal such as generated by the electroporation device described herein, normally causes accelerated erosion and inoperability of the electrodes, and furthermore contaminates the solution and cells with metal ions. The present invention provides electrodes that can be subjected to frequent pulses of electrical charge in a saline solution, as in an electroporation apparatus, and have substantially increased useful terms over conventional electrodes, without contamination of the products of interest.
Previous powdered porous metal nitride coatings, such as titanium nitride (TiN), on electrodes used in gaseous environments have not addressed the unique problems associated with electrodes used to generate electric fields in an aqueous saline solution. In particular, the advantages previously taught for using an electrode having a powdered porous nitride coating are disadvantageous when used in aqueous biological saline environments. The porosity of such prior art electrode coatings does not serve to protect the exposed portions of the electrode surface from surface ion erosion and pitting which are normally accelerated during electric signal emission in an aqueous saline solution.
The use of a metal nitride coating has previously been taught to protect surgical implants or instruments used in a biological system from corrosion and wearing due to externally generated forces, such as salts or friction. However, when using electrodes in an aqueous biological saline environment, internally delivered forces of charged particles (electrons and protons) emanating from the surface of the electrode cause accelerated pitting and erosion of the metal surface of the electrode. Nitriding electrode surfaces has been proposed for improving signal detection in biological systems, such as in pacemaker detection of intracardiac signals, however, not for electric signal generation or stimulation in biological systems, which presents the unique pitting and erosion problems described above. More specifically, the unique demands on a pair of electrodes sending rapid and reversing pulses of high voltage electrical signals in an electroporation chamber, as described herein, pose a problem heretofore unsolved in the art.
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.
The major function of red blood cells is to transport oxygen from the lungs to the tissues of the body, and transport carbon dioxide from the tissues to the lungs for removal. Very little oxygen is transported by the blood plasma because oxygen is only sparingly soluble in aqueous solutions. Most of the oxygen carried by the blood is transported by the hemoglobin of the erythrocytes. Erythrocytes in mammals do not contain nuclei, mitochondria or any other intracellular organelles, and they do not use oxygen in their own metabolism. Red blood cells contain about 35 percent by weight hemoglobin, which is responsible for binding and transporting oxygen.
Hemoglobin is a protein having a molecular weight of approximately 64,500 Daltons. It contains four polypeptide chains and four heme prosthetic groups in which iron atoms are bound in the ferrous state. Normal globin, the protein portion of the hemoglobin molecule, consists of two &agr; chains and two &bgr; chains. Each of the four chains has a characteristic tertiary structure in which the chain is folded. The four polypeptide chains fit together in an approximately tetrahedral arrangement, to constitute the characteristic quaternary structure of hemoglobin. There is one heme group bound to each polypeptide chain which can reversibly bind one molecule of molecular oxygen. When hemoglobin combines with oxygen, oxyhemoglobin is formed. When oxygen is released, the oxyhemoglobin is reduced to deoxyhemoglobin.
Delivery of oxygen to tissues depends upon a number of factors including, but not limited to, the volume of blood flow, the number of red blood cells, the concentration of hemoglobin in the red blood cells, the oxygen affinity of the hemoglobin and, in certain species, on the molar ratio of intraerythrocytic hemoglobins with high and low oxygen affinity. The oxygen affinity of hemoglobin depends on four factors as well, namely: (1) the partial pressure of oxygen; (2) the pH; (3) the concentration of the allosteric effective 2,3-diphosphoglycerate (DPG) in the hemoglobin; and (4) the concentration of carbon dioxide. In the lungs, at an oxygen partial pressure of 100 mm Hg, approximately 98% of circulating hemoglobin is saturated with oxygen. This represents the total oxygen transport capacity of the blood. When fully oxygenated, 100 ml of whole mammalian blood can carry about 21 ml of gaseous oxygen.
The effect of the partial pressure of oxygen and the pH on the ability of hemoglobin to bind oxygen is best illustrated by examination of the oxygen saturation curve of hemoglobin. An oxygen saturation curve plots the percentage of total oxygen-binding sites of a hemoglobin molecule that are occupied by oxygen molecules when solutions of the hemoglobin molecule are in equilibrium with different partial pressures of oxygen in the gas phase.
The oxygen saturation curve for hemoglobin is sigmoid. Thus, binding the first molecule of oxygen increases the affinity of the remaining hemoglobin for binding additional oxygen molecules. As the partial pressure of oxygen is increased, a plateau is approached at which each of the hemoglobin molecules is saturated and contains the upper limit of four molecules of oxygen.
The reversible binding of oxygen by hemoglobin is accompanied by the release of protons, according to the equation:
HHb
+
+O
2
⇄HbO
2
+H
+
Thus, an increase in the pH will pull the equilibrium to the right and cause hemoglobin to bind more oxygen at a given partial pressure. A decrease in the pH will decrease the amount of oxygen bound.
In the lungs, the partial pressure of oxygen in the air spaces is approximately 90 to 100 mm Hg and the pH is also high relative to normal blood pH (up to 7.6). Therefore, hemoglobin will tend to become almost maximally saturated with oxygen in the lungs. At that pressure and pH, hemoglobin is approximately 98 percent saturated with oxygen. On the other hand, in the capillaries in the interior of the peripheral tissues, the partial pressure of oxygen is only about 25 to 40 mm Hg and the pH is also relatively low (about 7.2 to 7.3). Because muscle cells use oxygen at a high rate thereby lowering the local concentration of oxygen, the release of some of the bound oxygen to the tissue is favored. As the blood passes through the capillaries in the muscles, oxygen will be released from the nearly saturated hemoglobin in the red blood cells into the blood plas
Beisner William H.
Fulbright & Jaworski LLP
MaxCyte, Inc.
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