Title:  Biologically-active polymers

United States Patent:  6,184,030

Appl. No.:  023257

This invention relates to biologically-active polymers that are useful for analyte detection and isolation and delivery of substances. The biologically-active polymers are capable of specifically and reversibly binding to analytes, including molecules and cells. The biologically-active polymers are also capable of releasing substances upon electrical stimulation. The present invention provides compositions comprising biologically-active polymer membranes and methods for making these biologically-active polymer membranes that may be specifically designed to selectively bind cells and specific cell types, to affect cell growth characteristics, and to modulate cellular differentiation. These biologically-active polymer membranes may be controlled electrically to induce controlled cellular differentiation and modulate the cell growth cycle. These biologically-active polymers have many applications in biological and chemical fields.

SUMMARY OF THE INVENTION

In its broadest respects, the present invention is a biologically-active polymer composition and method for detecting, isolating and/or purifying an analyte. The analyte can be found in a liquid in soluble or insoluble form or it can be a particle in a liquid. The analyte may also be in a gas phase. Analytes that can be detected, purified or isolated according to the present invention include, but are not limited to, atoms, ions, molecules, cells, cell organelles, and other particulates. In one preferred embodiment of the present invention, the composition and method are used to isolate stem cells from a suspension of cells collected from blood or bone marrow.

In another aspect, the biologically-active polymer of the present invention is a composition and method for making polymer membranes that have characteristic and specific properties for cell adhesion, growth and differentiation. These polymer membranes may be specifically designed and controlled to possess characteristic properties for cell adhesion, growth and differentiation.

Thus, in one embodiment, the biologically-active polymer of the present invention provides a method of isolating and purifying immature bone marrow cells. The present invention provides a composition and method for preparing a cell population useful for stem cell transplantation that is substantially pure immature marrow cells and substantially free of mature myeloid and lymphoid cells. The present invention also provides a method of collecting donations useful for stem cell transplantation that avoids the disadvantages of conventional marrow harvesting techniques. The present invention provides a therapeutic method of transplanting stem cells that can extend the use of stem cell transplantation to the treatment of non-fatal diseases.

These and other objects of the present invention are achieved by one or more of the following embodiments. One embodiment of the present invention provides a suspension of human cells comprising pluripotent lympho-hematopoietic stem cells substantially free of mature lymphoid and myeloid, cells, as well as therapeutic methods employing such a cell suspension.

In another embodiment, the present invention may be used to isolate stem cells unrelated to the lympho-hematopoietic system. These stem cells may be derived from ectoderm, mesoderm, and endoderm. These stem cells may include germ cells such as, but not limited to, oogonia and spermatogonia, myoblasts, fibroblasts, osteoblasts and neuroblasts.

The biologically-active polymer of the present invention provides a polymer film that is capable of specifically binding an analyte, such as a stem cell. Although not essential in the practice of the present invention, when a voltage is applied to the polymer film binding of the analyte can be increased. The bound stem cell can then be released by reversing the voltage across the film. The present invention also includes an electrolyte that can be used to capture the analyte on the polymer film. The present invention also encompasses a method of making the polymer film that is capable of specifically capturing an analyte.

In one embodiment, the polymer film of the present invention has antibodies incorporated therein so that, when a voltage is applied across the polymer film, the antibodies will bind the analyte to which the antibody is directed. Some antibodies or other analytes require a certain background current to bind antigens or analyte recognition molecules, respectively. Next, the voltage is partially or completely reversed, the antibodies are no longer capable of binding the analyte, and any analyte that was bound to the antibodies bound to the polymer film is partially or completely released depending on the degree of voltage reversal. These analytes may be in a gaseous phase, in a liquid phase, in solution, insoluble, and possibly not bound to another entity. Alternatively, these analytes may be bound to other molecules, organelles and cells in a manner that permits recognition by the antibody bound to the polymer film.

In another embodiment, a polymer film can be prepared that does not have any antibody incorporated therein, but can still specifically bind to an analyte normally recognized by the antibody. In preparing the polymer film, an antibody (or other molecule) that specifically recognizes the desired analyte is used to impart to the polymer film the ability to recognize and bind the analyte. The antibody is then released from the polymer film, but the film itself can still recognize the analyte.

It is to be understood that non-antibody analytes can be used to impart recognition capability to the polymer film for any other analyte that is recognizable by the analyte used to impart recognition capability. Accordingly, in another embodiment of the present invention, an analyte such as a biological molecule is used to impart to the polymer film the ability to recognize and bind any antibody, receptor, organelle or cell surface molecule that is normally recognized by the biological molecule used to impart recognition capability to the polymer film.

It is also understood that the scope of the present invention is not limited to biological molecules or biological analytes. Any molecule may be employed to impart recognition capability to the polymer film. Accordingly the present invention encompasses the ability to use these films to bind all atoms, ions, molecules, inorganic molecules, organic molecules and complexes of inorganic and organic molecules.

In another embodiment of the present invention, several polymer films, each specific for an individual analyte are arranged in series or matrix to provide the ability to perform multiple analyte determinations in a single sample. These polymer films may be located in a patient or employed in vitro. The data from these membranes may be displayed, printed, stored in a data storage means, input into a computer, sent to a remote data storage means or a computer, or input into a trained neural network. This embodiment is also present in the form of an easy to use home test kit which is configured to provide data to or about the patient, and may also be configured to transmit the data to a remote location such as the office of a health care provider, a health maintenance organization, a hospital, a centralized data analysis facility, or elsewhere.

In another embodiment of the invention, the analyte-detection polymer films may be arranged in a configuration so that electrically activated flow gates are located at the outflow side of the sample transmission tube. Since each polymer film can detect the analyte in a very short time and transmit this information to a computer, the computer can be programmed to open or close the flow gate depending on the next analyte that should be measured. For example, in the differential diagnosis of a particular condition involving several variables, detection of analyte A at a certain concentration X might indicate that the measurement of analyte C should be performed and not analyte B. If analyte C were present at a threshold concentration then analyte D should be measured and not analyte E. A computer programmed in this fashion would receive the results from polymer film A as indicating a concentration greater than or equal to X and then open gate A to direct the sample flow to polymer film C. If the measurement at polymer film C were below threshold, then the computer would open gate C to direct sample flow to polymer film E.

It is also within the scope of the present invention to employ trained neural networks housed in a computer to analyze patterns in the data obtained from the detection and measurement of analytes. These trained neural networks assist the health care provider in the analysis of the analyte data output of the analyte detection polymer films.

Still a further embodiment of the present invention involves a polymer film, or series of polymer films to which specific substances are reversibly bound. Such polymer films may be implanted into a patient or used in vitro. These polymer films release substances upon activation through changes in voltage. Furthermore, the amount and duration of substance released may be adjusted by altering the signal to the polymer film.

Yet another embodiment of the present invention is a system for detection and measurement of analytes and subsequent release of substances which may provide a therapeutic or other desired effect.

Accordingly, it is an object of the present invention to provide a polymer film that is capable of binding a specific analyte and, when desired, will release the analyte from the polymer film.

It is a further object of the present invention to provide a polymer film that can quantitatively or qualitatively detect the presence of an analyte.

Another object of the present invention is to provide a device comprising an assembly of several polymer films, each specifically designed to bind an analyte, so that several analytes may be measured in a single sample.

Another object of the present invention is to provide a device comprising an assembly of several polymer films, each specifically designed to bind an analyte, so that several analytes may be measured in a single sample, wherein the degree of binding is measured as a change in voltage which is output to a data storage device which may optionally analyze the data and optionally transmit the results to a nearby or remote data receiving station for inspection by a health care provider.

It is another object of the present invention to provide a plurality of analyte specific polymer films in a sample flow through system equipped with computer activated gates positioned on the outflow side of the polymer film such that a computer may direct the flow of sample to specific polymer films.

Yet another object of the present invention is to provide a device for home use comprising several polymer films, each specifically designed to bind an analyte, so that several analytes may be measured in a single sample.

Still another object of the present invention is to provide for the analysis of data produced from a plurality of analyte specific polymer films.

It is another object of the present invention to provide a polymer film that is capable of binding and collecting analytes, thereby providing a means to isolate and purify analytes.

Another object of the present invention is to provide a polymer film that is capable of releasing substances upon electrical stimulation of the polymer film.

It is another object of the present invention to provide a polymer film that can be implanted within a patient and is capable of releasing substances upon electrical stimulation of the polymer film.

It is another object of the present invention to provide a polymer film that is capable of binding cells and releasing them when the polymer film is activated electrically.

It is another object of the present invention to provide a polymer film that is capable of binding and collecting stem cells.

It is another object of the present invention to provide a polymer film that is capable of binding and collecting lymphohematopoietic stem cells.

It is another object of the present invention to provide a polymer film that is capable of binding and collecting bacteria.

Another object of the present invention is to provide polymer membranes that bind cells and a method for making these polymer membranes.

It is further an object of the present invention to provide polymer membranes that bind cells and promote cell growth and a method for making these polymer membranes.

It is further an object of the present invention to provide polymer membranes that bind cells and promote cell growth and differentiation and a method for making these polymer membranes.

It is an object of the present invention to provide polymer membranes that have characteristic and preselected properties for cell adhesion, growth and differentiation and a method for making these polymer membranes.

It is further an object of the present invention to provide polymer membranes and a method for making polymer membranes that favor the growth and differentiation of a homogeneous population of cells when initially exposed to a stem cell population.

It is another object of the present invention to provide polymer membranes and a method for making polymer membranes that favor the selection of a specific type of cell when initially exposed to a heterogeneous population of cells.

It is another object of the present invention to provide polymer membranes and a method for making polymer membranes that favor the selection and growth of a specific type of cell when initially exposed to a heterogeneous population of cells.

Still another object of the present invention to provide polymer membranes and a method for making polymer membranes that favor the selection, growth and differentiation of a specific type of cell when initially exposed to a heterogeneous population of cells.

It is yet another object of the present invention to provide polymer membranes and a method for making polymer membranes that favor a specific type of growth and differentiation of cells.

It is another object of the present invention to provide a method by which the polymer membranes can be controlled electrically to induce controlled cellular differentiation and modulate the cell growth cycle.

Claim 1 of 23 Claims

What is claimed is:

1. A polymer composition comprising a polymer and an antibody, wherein the polymer is selected from the group consisting of polymers of pyrrole, thiophene, aniline, and combinations thereof, said polymer composition being adapted to bind specifically to an analyte and to release the analyte, and said polymer composition is prepared by a process comprising:

mixing an aqueous solution of acetonitrile and an electrolyte to make a mixture wherein the electrolyte is p-toluene sulfonic acid, naphthalene sulfonic acid, tetraacetyl ammonium toluene sulfonate, or a combination of p-toluene sulfonic acid and naphthalene sulfonic acid;

increasing pH of the mixture through addition of base to the mixture;

inserting an electrode into the mixture;

adding one or more monomers to the mixture, wherein the one or more monomers is pyrrole, thiophene, analine, or a combination thereof;

adding an antibody to the mixture, wherein the antibody is capable of binding to the analyte;

applying a voltage to the electrode, wherein the voltage produces a direct current; and,

altering the voltage.

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