Patent Application
20210324327
Incorporation of electroactive elements, including conducting polymers, graphene, and carbon nanotubes in a biomaterial has a significant effect on cellular adhesion, proliferation, and differentiation, as well as allowing for both the possibility of electrical stimulation and sensing to both augment and better understand the environment of regenerating tissue. However, achieving a complex three-dimensional (3D) structure, biologically relevant mechanical properties, and cell binding domains along with the electroactive element is an ongoing challenge. Complex composites of synthetic and/or natural polymers with conducting elements, electrospun fibers coated with conducting elements, or 3D printing and bioprinting have been proposed. However, at best, these approaches achieve a “compromise” material that is generally lacking in one of the three aforementioned attributes.
Biocompatible electronically conductive polymers and biological substrates funcationalized with the electronically conductive polymers are provided. Also provided are methods of functionalizing the biological substrates and methods of using the functionalized biological substrates in tissued engineering applications, including cell cultures.
One embodiment of a biocompatible electronically conductive polymer has: a poly(ethylenedioxythiophene) backbone; a set of first functional groups pendant from the poly(ethylenedioxythiophene) backbone, wherein the first functional groups comprise free succinimide groups, free maleimide groups, or a combination thereof; and a set of second functional groups pendant from the poly(ethylenedioxythiophene) backbone, wherein the second functional groups increase the solubility of the polymer in water and act as electrical conductivity-enhancing dopants.
The methods functionalize a biological substrate having free amine groups with a polymer that includes: a poly(ethylenedioxythiophene) backbone; a set of first functional groups pendant from the poly(ethylenedioxythiophene) backbone, wherein the first functional groups comprise free succinimide groups, free maleimide groups, or a combination thereof; and a set of second functional groups pendant from the poly(ethylenedioxythiophene) backbone, wherein the second functional groups increase the solubility of the polymer in water. One embodiment of such a method includes the steps of: reacting the free succinimide groups, the free maleimide groups, or both, of the polymer with the free amine groups of the biological substrate, thereby covalently bonding the polymer to the biological substrate.
One embodiment of a functionalized biological substrate includes: a biological substrate; a polymer having a poly(ethylenedioxythiophene) backbone; a set of first functional groups pendant from the poly(ethylenedioxythiophene) backbone, wherein the first functional groups comprise free succinimide groups, free maleimide groups, or a combination thereof; and a set of second functional groups pendant from the poly(ethylenedioxythiophene) backbone, wherein the second functional groups increase the solubility of the polymer in water. The polymer is covalently bonded to the biological substrate by covalent bonds formed by reactions between the succinimide groups of the polymer and amine groups of the biological substrate.
One embodiment of a method of growing biological tissue includes the steps of seeding a functionalized biological substrate and culturing the seeded functionalized biological substrate in a cell culture medium. The functionalized biological substrate includes: a biological substrate; a polymer having a poly(ethylenedioxythiophene) backbone; a set of first functional groups pendant from the poly(ethylenedioxythiophene) backbone, wherein the first functional groups comprise free succinimide groups, free maleimide groups, or a combination thereof; and a set of second functional groups pendant from the poly(ethylenedioxythiophene) backbone, wherein the second functional groups increase the solubility of the polymer in water. The polymer is covalently bonded to the biological substrate by covalent bonds formed by reactions between the succinimide groups of the polymer and amine groups of the biological substrate.
Illustrative embodiments of the invention will hereafter be described with reference to the accompanying drawings.
Conjugated, electrically conducting polymers (CPs) with the ability to covalently graft onto collagen are provided. Also provided are methods of functionalizing biological tissues and other biological substrates with the CPs, and methods of using the functionalized biological substrates as cell and tissue growth scaffolds that harness the passive therapeutic benefits of CPs and use the enhanced conductivity provided by the scaffolds to stimulate cell growth and proliferation through the bulk of the biological substrate.
The described CPs are biocompatible water soluble, conjugated polymers that allow for covalent attachment to a biological substrate via reactions with reactive functional groups on the substrate under mild conditions. For use in tissue growth and/or cell culture applications, the CPs are also cytocompatible. Use of a water-soluble conjugated polymer is particularly advantageous because it allows for renal clearance of the conducting element in vivo. As used herein, the term biocompatible refers to a material that does not have a significant negative impact on tissue growth and viability and/or a material that, if implanted in a living biological entity (e.g., a mammal, such as a human), does not cause an adverse reaction in that biological entity. As used herein, the term cytocompatible refers to a material that is biocompatible and, more specifically, that does not have an adverse effect on the growth and viability of biological cells.
The CPs are characterized by: a poly(ethylenedioxythiophene) backbone; a set of first functional groups pendant from the poly(ethylenedioxythiophene) backbone, wherein the first functional groups comprise chemical groups that react with a biological substrate to covalently graft the CPs to a the biological substrate; and a set of second functional groups pendant from the poly(ethylenedioxythiophene) backbone, wherein the second functional groups increase the water solubility of the CP. For the purposes of this disclosure a functional group is considered to increase the water solubility of the CP if a CP having the pendant functional groups is more soluble in water than a CP that lacks the pendant functional groups, but, otherwise, has the same structure. In some embodiments, the first functional groups comprise free succinimide groups, free maleamide groups, free thiol groups, or a combination of two or more thereof. In some embodiments, the second functional groups comprise sulfate groups. The poly(ethylenedioxythiophene) backbone may consist of only ethylenedioxythiophene units. As used herein, the term “free” indicates that the functional group is reactive and can form a covalent bond. By way of illustration, one example of a CP comprises free succinimide groups to impart the CP with covalent grafting ability and sulfate groups to impart water solubility to the CP.
The first and second functional groups can be attached to the polymer backbone by organic linkers comprising carbon and hydrogen and, optionally, nitrogen, oxygen, and/or sulfur atoms. The organic linkers and pendant groups can be incorporated into a polymer using reactive organic linker molecules. The organic linker molecules include a reactive group that reacts with the monomers that make up the CP backbone or that reacts with the CP backbond itself and a second reactive group that reacts with the first or second functional groups in order to covalent attached those functional groups to the CP. The first and second reactive groups may be separated by a linker chain. The linker chain may be a substituted or unsubstituted alkyl chain, a substituted or unsubstituted alkenyl chain, or a substituted or unsubstituted alkynyl chain. By way of illustration, substituted or unsubstituted C1-C6 alkyl, alkenyl, or alkynyl chains may be used as the linker chain. Substituted or unsubstituted heteroalkyl, heteroalkenyl, or heteroalkynyl chains can also be used as linker chains, where heteroalkyl, heteroalkenyl, or heteroalkynyl chains refer to alkyl, alkenyl, and alkynyl chains (including C1-C6 alkyl, alkenyl, or alkynyl chains), respectively, in which the carbon chain is interrupted by one or more heteroatoms, such as nitrogen, oxygen, and/or sulfur atoms. Alkoxy chains are illustrative examples of heteroalkyl chains. In some examples of the conductive polymers, the functional groups pendant from the electrically conductive polymers consist of only the first and second functional groups. This includes examples in which the side-chains extending from the polymer backbone consist of only the linker chains and first and second functional groups listed herein.
One embodiment of a CP has the structure:
where A represents:
and B represents:
and n and m represent the number of repeat units for the respective units in the polymer backbone. As illustrated by the CP above, the polymers may be oxidized in order to dope them with charge-balancing counterions, which enhance the electrical conductivity of the CP. In the doped CPs the water solubility enhancing group has a negative change and acts as a counterion dopants that stabilize the positive charges on the polymer backbone. By enhanced electrical conductivity, it is meant that the doped polymers are more electrically conductive than a polymer that lacks the water solubility enhancing functional groups, but is otherwise the same. Methods for making the CP shown above are described in the Example and illustrated in
The synthesis of the CPs can be carried out using a Pd catalysis to make a co-polymer with more controlled stoichiometry, relative to that provided by oxidative polymerizations. This can be carried out without the use of extremely oxidizing or acidic conditions, and, therefore, is suitable for functionalizing allograft tissue. The ratio of the monomers bearing the water solubility increasing groups (for example, sulfate groups) to the monomers bearing surface grafting groups (for example, succinimide groups) will vary depending on the desired level of water solubility and the desired strength of surface bonding. Generally, however, the CPs will contain a greater number of the monomers bearing the water solubility-increasing groups. By way of illustration, in various embodiments of the CPs, the mole ratio of first functional groups to second functional groups is in the range from about 1:1 to about 1:100. This includes embodiments in which the mole ratio of first functional groups to second functional groups is in the range from about 1:2 to 1:20 and further includes embodiments in which the mole ratio of first functional groups to second functional groups is in the range from about 1:5 to 1:15.
The substrate grafting groups on the CPs can react with various reactive functionalities on a biological substrate to form covalent bonds to the substrate. Suitable reactive functionalities include, for example, nucleophilic groups, such as free amine groups and free hydroxy groups. By way of illustration, succinimide groups and maleimide groups can react with free amine groups on a biological substrate having free amine groups to form covalent bonds to the substrate. This can be accomplished by infusing the biological substrate with the polymer in the presence of phosphate buffer saline (PBS), as illustrated in the Example and shown in the upper panel of
The biological substrates to which the CPs can be grafted are substrates that are composed of biomolecules (e.g., biological tissues) or that incorporate biomolecules into their structures. For example, the biomolecules may be proteins, such as collagen, which has free amines and is a primary component of extracellular matrix (ECM). This is significant because decellularized ECM (dECM) harvested from humans and other animals preserves the macro- and micro-structure of the tissues from which it is harvested and, thus, has found a wide range of both clinical and pre-clinical applications.
The biohybrid composites that result from grafting the CPs onto a biological substrate have augmented electroactivity, which facilitates in vitro cell proliferation and enables bulk electrical stimulation to enhance tissue regeneration. The tissue growth or regeneration may be passive—that is, without the application of an external electrical bias, or active—that is, carried out under the influence of an applied electrical bias. Electrical recording devices and/or electrical stimulation devices can be integrated directly into the biological sample to monitor and/or promote tissue growth.
Moreover, when the CP material binds to native tissues, such as collagen-containing human allograft, the native tissue architecture and presence of native bioactive molecules, such as growth factors, remain undisturbed, which renders the functionalized tissue well-suited for use as allograft implants.
The CP-grafted biological substrates can be used as biological cell and tissue growth scaffolds by seeding the functionalized biological substrates with biological cells, such as human mesenchymal cells (hMSCs), and culturing the seeded biological substrates in a cell culture medium, including a differentiation medium or a proliferation medium. Alternatively, the CP-grafted substrates can be implanted into an animal (e.g., human) to enable in vivo cell growth, differentiation, and proliferation. Tissues that can be grown using the tissue growth scaffolds include dermal, neural, osteo, chondral, cardiac, and osteochondral tissue.
A conducting conjugated polymer was synthesized according the reaction scheme shown in
The conducting conjugated polymer was grafted to a collagen-containing substrate by simply soaking the collagen-containing substrate in a PBS solution of the polymer, as shown in
L929 fibroblast cells were cultured on the acellular collagen sponges grafted with the CP of
Rat BMSC cells were also cultures on the acellular collagen sponges grafted with the CP of
The up-regulation of some osteogenic-relevant genes on the CP-functionalized ACS (here the CP is referred to as PEDOT for simplicity) was measured in both osteogenic and standard media after one week (
The word “illustrative” is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “illustrative” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Further, for the purposes of this disclosure and unless otherwise specified, “a” or “an” can mean “only one” or can mean “one or more.”
The foregoing description of illustrative embodiments of the invention has been presented for purposes of illustration and of description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention. The embodiments were chosen and described in order to explain the principles of the invention and as practical applications of the invention to enable one skilled in the art to utilize the invention in various embodiments and with various modifications as suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
The present application claims priority to U.S. provisional patent application No. 63/010,181 that was filed Apr. 15, 2020, and U.S. provisional patent application No. 63/010,156 that was filed Apr. 15, 2020, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63010181 | Apr 2020 | US | |
| 63010156 | Apr 2020 | US |
