Plasma Activation of Biological Materials for Surface Modification

Abstract

Disclosed in certain embodiments is a composition comprising a biological material activated by oxygen plasma and a ligand bound to a surface of the biological material.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No. 61/479,627 filed Apr. 27, 2011, the disclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The presently disclosed subject matter includes compositions comprising biological material with pharmacologically active agent(s) bound to a surface thereof, either directly or through intermediary layers, and methods for producing said compositions.

BACKGROUND

It is desirable to alter the surface chemistry of tissue and other biological materials in order to control the body's interaction with it after implantation or grafting. One reason is to prevent infection. Another is to reduce inflammatory response. Yet another is to prevent the rejection of implanted biological material.

In some cases, alteration of surface chemistry can be achieved by soaking the material in a compatible solution of a pharmacological agent. A number of issues arise from this approach. Once implanted, the pharmacological solution will diffuse from the material into the patient. Generally speaking, a higher concentration that is necessary for local efficacy must be used due to this diffusion effect. The surgeon must therefore balance the total dosage of pharmacological agent with the necessary amount required to have the desired local effect. In some cases, concentration level required may cause undesirable side effects in the patient.

In addition, once implanted, it is impossible to control the rapid elution rate of the bioactive agent form the implanted tissue into the implant site and from there, into the patient. It is generally desirable for the bioactive agent to remain within the implanted or grafted tissue for a certain amount of time. Further, depending on the pharmacological agent, the amount required in solution for local efficacy may make the implantation or grafting procedure prohibitively expensive.

Accordingly, there remains a need in the art for biological materials suitable for implantation or grafting into living mammals, and methods of creating the same.

SUMMARY OF THE INVENTION

It is an object of certain embodiments of the present invention to provide biological materials with surface modifications to allow for conjugation with a ligand.

It is an object of certain embodiments of the present invention to provide biological materials conjugated with an antimicrobial agent.

It is an object of certain embodiments of the present invention to provide biological materials that have reduced incidence of infection upon implantation or grafting into a patient.

It is an object of certain embodiments of the present invention to provide biological materials that have reduced incidence of rejection upon implantation or grafting into a patient.

It is an object of certain embodiments of the present invention to provide biological materials that have improved stability and integrity upon implantation or grafting into a patient.

It is an object of certain embodiments of the present invention to provide methods of manufacturing modified biological materials as disclosed herein.

It is an object of certain embodiments of the present invention to provide methods of dehydrating biological materials.

The above objects of the invention, and others, may be achieved by the present invention which in certain embodiments is directed to a composition comprising a biological material and a ligand bound to a surface of the biological material. The biological material can be, e.g., collagen, tissue, or bone. In certain embodiments, the tissue is acellular dermal tissue.

In certain embodiments, the present invention is directed to a composition comprising a biological material activated by oxygen plasma. Optionally, a ligand is bound to the activated biological material.

In certain embodiments, the present invention is directed to a composition comprising acellular tissue; a coupling agent bound to the acellular tissue; and a pharmacological agent bound to the coupling agent.

In certain embodiments, the present invention is directed to a method of treating biological material comprising: contacting biological material with oxygen plasma to form activated biological material. In other embodiments, a ligand can be bound to the activated biological material.

Where bonding is referenced herein, such bonding can be achieved through any type of chemical bond, including, without limitation, covalent bonding, polar covalent bonding, ionic bonding, hydrogen bonding, van der Waals forces and a combination thereof.

In certain embodiments, the present invention is directed to a process for dehydrating a biological material (e.g., an acellular biological material).

In certain embodiments, the present invention is directed to the implantation or grafting of a modified biological material as disclosed herein in a patient in need thereof.

In certain embodiments, the present invention is directed to a method of performing reconstructive surgery in a patient in need thereof comprising implanting or grafting a modified biological material as disclosed herein.

In certain embodiments, the present invention is directed to a method of administering a drug to a patient in need thereof comprising implanting or grafting a biological material conjugated with pharmacological agent or bioactive agent as disclosed herein.

In describing the present invention, the following terms are to be used as indicated below. As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a pharmacological agent” includes a single pharmacological agent as well as a mixture of two or more different pharmacological agents.

As used herein, “biological material” means any material derived in whole or in part from an organism, including, without limitation, soft tissue sources such connective and non-connective tissue. Examples of connective tissue includes, without limitation, tendons, ligaments, fascia, dermal tissue, fat, dura, pericardia, fibrous tissues and synovial membranes. Examples of non-connective tissue includes, without limitation, muscles, blood vessels and nerves. “Biological material” also includes hard tissue sources such as bone and cartilage. In certain embodiments, such materials may have been harvested from a living organism and then submitted to further processing and/or chemical treatment. The living organism could be comprised of eukaryotic or prokaryotic cells. Recombinant proteins, which can be derived from bacteria such as E. coli and are produced from recombinant DNA, can also be modified with the present invention.

“Acellular biological material” refers to biological material from which all, or substantially all, viable cells and detectable subcellular components and/or debris from cell death have been removed.

In some embodiments, the acellular biological material utilized in the present invention has a concentration of viable cells that is less that about 5%, less than about 3%, less than about 1% or less than about 0.5% of the concentration in the original biological material from which the acellular biological material was derived. In other embodiments, the acellular biological material has an amount of viable cells that is less that about 3%, less than about 1%, less than about 0.5% or less than about 0.2% of the total weight of the acellular material.

In some embodiments, the acellular biological materials utilized in the present invention comprise less than about 40%, less than about 25%, less than about 10%, or less than about 5% of nucleic acid that was present in the original cellularized biological material from which the acellular biological material was derived. In other embodiments, the acellular biological material has an amount of nucleic acid that is less than about 25%, less than about 10%, less than about 5%, or less than about 2% of the total weight of the acellular material

“Activated biological material” refers to biological material that has been contacted with an activating agent (e.g., oxygen plasma) to provide reactive functional groups on the surface of the material.

“Pharmacological agent” or “bioactive agent” means any agent that can be bound to activated biological material. Examples of bioactive or pharmacological agents include, without limitation, (i) those of an anti-infective nature, such as antimicrobials, antibiotics, antifungals, antiseptics, disinfectants, and preservatives; (ii) immunosuppressant drugs such as glucocorticoids, antibodies, ciclosporin, tacrolimus, calcineurin inhibitors, and sirolimus; and (iii) agents to mediate and induce cellular/tissue growth such as bone morphogenic proteins (BMP), epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I and II), TGF-D, and vascular endothelial growth factor (VEGF).

“Anti-infective” refers to anything that is capable of destroying or inhibiting the microorganism growth, including, without limitation, antimicrobial agents such as antibacterial and antifungal agents.

“Coupling agent” means an agent capable of forming a bond between the surface of an activated biological material and a ligand (such as a pharmacological or bioactive agent). This bond may be achieved by first forming a bond between the surface of the activated biological material and the coupling agent, and then forming a bond between the coupling agent and the ligand. In other embodiments, the ligand can be bonded to the coupling agent, and the resultant conjugate is bound to the biological material. Alternatively, the coupling agent may facilitate bonding directly between the surface of an activated biological material and the ligand.

The term “oxygen plasma” means an oxygen source having a portion of the molecules ionized.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic diagram of one embodiment of the present invention.

FIG. 2 depicts retention of gentamicin under conditions of soaking in an infinite sink in PBS for 28 days versus untreated control. The Y-axis is nanograms per sample core. The X-axis is time.

FIG. 3 depicts the results of elution studies of (i) samples treated according to the method of the present invention, (ii) untreated samples, (iii) samples not treated with plasma but soaked in gentamicin, and (iv) samples dehydrated and exposed to plasma and then soaked in gentamicin. FIG. 3 demonstrates that soaked samples eluted practically all of their gentamicin by the end of the first day and thereafter were equivalent to untreated samples. Samples treated with plasma according to the present invention showed a burst elution of unbound material followed by a steady state of gentamicin over the 14 days of the experiment. Drug retention was measured by soaking the test samples in buffer of sufficient volume to be considered an infinite sink.

FIG. 4 depicts the long-term efficacy of gentamicin in samples treated according to the method of the present invention. FIG. 4 demonstrates that explanted tissue retains antimicrobial activity even after almost 7 days of implantation time

FIG. 5 depicts a graph that demonstrates gentamicin load is significantly improved after introducing short vacuum during the CDI activation step of the method of the present invention.

FIG. 6 depicts the antimicrobial efficacy of samples in which a short vacuum has been introduced during the CDI activation step of the method of the present invention versus samples prepared without vacuum.

FIG. 7 depicts bacterial outgrowth results. N=3 per group, per timepoint. Error bars represent the standard deviation.

FIG. 8 depicts results from a fibroblast assay. N=4 per group. Error bars represent the standard deviation.

FIG. 9 depicts cell viability results using an MTS assay. Relative cell number was extrapolated from a standard curve produced from dermal fibroblasts grown on TCPS. N=4 for all groups. Error bars represent the standard deviation.

FIG. 10 depicts the results of a gentamicin quantification assay using an ELISA kit.

FIG. 11 depicts the results of killing assays for samples treated according to a method of the present invention using 4 different bacteria. For all groups, n=3 and error bars represent the standard deviation.

DETAILED DESCRIPTION

Harvested biological material is often utilized for implantation or grafting in a host organism for a variety of reasons such as reconstructive surgery (e.g., hernia repair or external burn treatment). A common source of biological material is dermal tissue. The source of the dermal tissue can be from another area of the patient's body, called an autograft, obtained from another person (e.g., donor skin from cadavers called an allograft, or from an animal (e.g., porcine or bovine source), called a xenograft.

Implanted or grafted biological material is susceptible to complications (e.g., infection) which can lead to failure of the procedure and issues to the patient. Accordingly, it is desirable to have the biological material functionally modified (e.g., conjugated or bound to an anti-infective agent) prior to implantation or grafting.

In certain embodiments, the biological material is animal tissue. Animal tissue is mainly made of Type I collagen, which contains a very limited number of functional groups for bioconjugation (approximately 5%). In instances where it is desirable for these tissues to be processed, altered or derivitized for implantation or grafting into an organism, this lack of functional groups can present challenges. For example, it may be desirable to treat implanted tissues with antibiotic agents prior to implantation to prevent infection at the surgery site. However, the lack of functional groups to which these agents can bind makes it difficult for the tissue to retain the agents long enough for the agents to impart the desired effect—i.e., preventing infection.

The present invention provides a solution to this problem by creating functional groups on the surface of biological material after exposure to an activating agent. Preferably, the activating agent is oxygen plasma. By virtue of the present invention, biological material (e.g., tissue) with surface functional groups such as hydroxyl and carboxyl are created after contact with oxygen plasma. This creation of functional groups creates a surface on the biological material for the binding of ligands such as antimicrobial and antibiotic agents. The plasma-generated surface reactive hydroxyl and carboxyl groups may be formed by breaking hydrocarbon bonds on the surface of the biological material through bombardment of oxygen plasma. Preferably, the treatment does not disrupt the integrity of the bulk tissue underneath, presenting minimal challenges to post-implant integration.

One method of the present invention involves contacting a biological material with oxygen plasma to, for example, remove the water shell that is tightly associated with the biological material and activate the exposed surface to generate more functional groups for chemical modification. Ligands may then be bound to these functional groups. Ligands may include coupling agents and pharmacological or bioactive agents. In some embodiments, a coupling agent is used to facilitate bonding (e.g., covalent bonding) between the activated biological material and a pharmacological or bioactive agent (such as an antibiotic). For example, plasma-generated surface reactive groups are bound to a coupling agent. Ligands are then directly conjugated onto the coupling agent biological material conjugate. In a certain embodiment, a coupling agent such as 1,1′-carbonyldiimidazole can be used to create a bond between the activated surface groups and free amine groups and the antibiotic gentamicin. Another coupling agent is a mixture of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and N-Hydroxysuccinimide (NHS). In certain embodiments, the biological material is dehydrated prior to activation and/or ligand binding.

Intermediary layers (e.g., coupling agents) that may be used to create a series of bonds between the plasma-activated tissue surface and the further ligand preferably have one or more of the following qualities: (i) in its final processed state it is biocompatible; (ii) it is reactive with the activated tissue substrate upon application; (iii) after application, the intermediary layer is reactive with the bioactive agent or subsequent intermediary layer; and (iv) in its final processed state, it is substantially as flexible as its underlying substrate, which may be achieved, for example, through the thinness of the layer.

Certain embodiments of the present invention include a composition comprising a biological material and a ligand bound (e.g., via covalent bonding) to a surface of the biological material. The biological material may be, for example, soft tissue, collagen, bone, dermal tissue, or any other suitable tissue. In certain embodiments, the biological material is acellular. The ligand can, for example, be a pharmacological agent, a biologically active molecule, or a coupling agent.

In certain embodiments, the present invention includes a composition comprising acellular tissue, a coupling agent bound to the acellular tissue, and pharmacological agent bound to the coupling agent.

The biological material can be derived from any suitable biological source, including, without limitation, mammalian, avian, reptilian, amphibian and bacteria. In certain embodiments, the mammalian source is selected from humans, primates (e.g., monkeys, chimpanzees, gorillas, gibbons and orangutan), livestock (e.g., pigs, cows, horses, goats, sheep), dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. In certain embodiments, the mammalian source is a human cadaver. In other embodiments, the mammalian source is a living human. Examples of avian sources include chicken, turkey, duck and goose.

The biological material is preferably acellular as to avoid the rejection of the biological material from the host. One source of acellular tissue that can be modified according to the present invention is FlexHD® commercially available from Ethicon. Further examples of acellular biological materials are described in U.S. Patent Application Publication No. 2006/0275377; International Application No. PCT/US08/52882; International Application No. PCT/US08/52884, and International Application No. PCT/US08/52885, which are incorporated herein by reference in their entireties.

In certain embodiments, the present invention is directed to dehydrated biological materials and methods of preparing the same. In certain embodiments, the dehydrated biological material can optionally be activated and/or ligand bound as disclosed herein. The dehydration process can include, for example, lyophilization or a solvent exchange process. In some embodiments, the biological material has been dehydrated via a solvent exchange process comprising soaking the biological material in a solvent. In further embodiments, the biological material has been dehydrated via a solvent exchange process comprising soaking the biological material in a hydrophilic solvent followed by soaking the biological material in an organic solvent. In some embodiments, the biological material has been dehydrated via a solvent exchange process comprising soaking the biological material in a hydrophilic solvent followed by soaking the biological material in an organic solvent under vacuum.

In a particular embodiment, the solvent exchange process comprises soaking the biological material in a solvent miscible with or capable of forming an azeotrope with water. The biological material can then be optionally placed under a vacuum with or without heat.

In another embodiment, the solvent exchange process comprises soaking the biological material in a solvent miscible with or capable of forming an azeotrope with water followed by soaking the biological material in a volatile organic solvent. Preferably, the boiling point of the organic solvent at atmospheric pressure is less than or equal to about 80° C., less than or equal to about 70° C., or less than or equal to about 80° C. The biological material can then be optionally placed under a vacuum with or without heat.

An exemplary solvent exchange process comprises soaking the biological material in an organic hydrophilic solvent miscible with water, such as ethanol, iso-propanol, or any other azeotrope-forming solvent to extract the water from the biological material. This can be performed for one or more cycles (e.g., 2 or 3 cycles). The tissue can then be soaked in organic solvents with lower boiling points, such as dichloromethane or tetrahydrofuran, for one or more cycles (e.g., 2 or 3 cycles) to replace the previous solvent. After draining out the solvent, the biological material may be dried. In one embodiment, the sample is placed under vacuum with or without gentle heating, to remove any residual solvent that remains.

The solvent for a solvent exchange process used to prepare the biological material of the present invention can be, for example, one or more of ethanol, n-propanol, iso-propanol, n-butanol, sec-butanol, iso-butanol, tert-butanol, allyl alcohol, benzyl alcohol, furfuryl alcohol, cyclohexanol, benzyl alcohol, tetrahydrofuran, chloroform, methyl ethyl ketone, benzene, ethyl acetate, cyclohexane, benzene, carbon tetrachloride, ethylene chloride, acetonitrile, toluene, n-hexane, n-heptane, carbon disulfide, diethyl ketone, n-propyl acetate, methanol, acetone, aqueous mixtures thereof, and combinations thereof. In certain embodiments, the solvent is an organic solvent and can be, for example, one or more of dichloromethane, tetrahydrofuran ethyl ether, methyl t-butyl ether, pentane, hexane, aqueous mixtures thereof, and combinations thereof.

In embodiments that subject the biological material to a solvent exchange process, the biological material may be placed under vacuum at room temperature or with heat.

In certain embodiments of the present invention, the ligand that is bound to the biological material is a coupling agent. The coupling agent can, for example, be 1,1′-carbonyldiimidazole, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, N,N′-Disuccinimidyl carbonate, N-hydroxysuccimimidyl chloroformate, isocyanate, Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate, (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, O-(Benzotriazol-1-yl)-N,N,N,′N′-tetramethyluronium hexafluorophosphate, O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, (7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 1-Hydroxybenzotriazole, N,N′-Dicyclohexylcarbodiimide, N,N′-Diisopropylcarbodiimide, Diethyl azodicarboxylate or N,N′-Di-tert-butylcarbodiimide, salts thereof, derivatives thereof and combinations thereof.

In some embodiments, a second ligand is bound to the composition of the present invention. The second ligand can, for example, be a pharmacological agent that is bound (e.g., covalently bound) to the biological material via the first ligand. The pharmacological agent can be, for example, an antimicrobial agent such as an antibiotic. In certain embodiments, the antibiotic has an available nucleophilic group.

In certain embodiments, the antimicrobial agent is selected from one or more of amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, geldanamycin, herbimycin, loracarbef, ertapenem, doripenem, imipenem/cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, ticarcillin, amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanate, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfonamidochrysoidine, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, linezolid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, rifaximin, thiamphenicol, tigecycline, tinidazole, salts thereof, derivatives thereof, and combinations thereof.

In certain embodiments, the antimicrobial agent of the present invention is selected from one or more of chlorhexidine, biguanides, quaternary ammonium compounds, salts thereof, derivatives thereof, and combinations thereof.

The attachment of the ligand can be performed, e.g., by contact with an aqueous, non-aqueous or partial aqueous solution of the ligand. The solution can contain the ligand, e.g., in an amount from about 0.0001% to about 99% w/w. In particular embodiments, the ligand solution (e.g., gentamycin or 1,1′-carbonyldiimidazole) comprises from about 0.0001% to about 50%, from about 0.001 to about 25%, from about 0.01 to about 10% or from about 0.1 to about 5% ligand.

In certain embodiments, the source of the oxygen plasma can be O₂, air, or a combination thereof. In other embodiments, the source of oxygen plasma is any gaseous mixture that has a minimum percent of oxygen to provide a suitable surface of functional groups on the biological material after contact to allow for further chemical modification

In certain embodiments, a ligand such as an antimicrobial or antibiotic agent is directly bound to the surface functional groups of the biological material without the use of intermediary layers such as coupling agents.

In certain embodiments, the compositions, biological materials, or activated biological materials are sterilized, i.e., they are substantially free of living microorganisms such as bacteria and viruses.

In certain embodiments of the inventive composition comprising a pharmacological agent, the composition maintains between about 5% and 95% of the pharmacological agent after soaking in an infinite sink in phosphate buffer saline for 24 hours, for 4 days, for 7 days, or for 14 days.

In further embodiments of the inventive composition comprising a pharmacological agent, the composition maintains between about 50% and 95% of the pharmacological agent after soaking in an infinite sink in phosphate buffer saline for 24 hours, for 4 days, for 7 days, or for 14 days.

In other embodiments of the inventive composition comprising a pharmacological agent, the composition maintains at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the pharmacological agent after soaking in an infinite sink in phosphate buffer saline for 24 hours, for 4 days, for 7 days, or for 14 days.

In further embodiments of the inventive composition comprising a pharmacological agent, the amount of pharmacological agent is maintained after soaking in an infinite sink in phosphate buffer saline for 4 days, for 7 days, or for 14 days is within about 10%, within about 15%, within about 20%, or within about 25% of the amount of pharmacological agent present in the composition at 24 hours.

The modified biological materials disclosed herein can be used in reconstructive surgery including but not limited to hernia repair, breast reconstruction, abdominal wall repair, chest wall repair, urological repair, bone and cartilage implantation, gynecological repair, plastic surgery, tendon repair, burn and wound treatment and vein/artery repair.

The modified biological materials disclosed herein are optionally packaged in a sterile container for transport and storage.

The following examples are set forth to assist in understanding the invention and should not be construed as specifically limiting the invention described and claimed herein. Such variations of the invention, including the substitution of all equivalents now known or later developed, which would be within the purview of those skilled in the art, and changes in formulation or minor changes in experimental design, are to be considered to fall within the scope of the invention incorporated herein.

EXAMPLES

Example 1

Solvent Exchange

FlexHD® samples were immersed in absolute ethanol for 15 minutes with gentle shaking. This step was repeated until a total of 3 cycles had been completed. The samples were then immersed in dichloromethane for 15 minutes with gentle shaking. This step was repeated once more so that a total of 2 cycles were completed. The samples were then placed in a vacuum oven at 30° C. for 1 hour to remove residual solvent.

Example 2

Plasma Activation

Lyophilized acellular biological material, or a sample dehydrated according to Example 1, was placed inside a plasma chamber using air or oxygen as the process gas. When the pressure inside the chamber was below 2 Torr, the plasma process was started. The samples were treated by plasma for 0.5 to 5 minutes depending on sample size, with the radio frequency level set to “HIGH” (30W).

Example 3

Attachment of Coupler to Activated Sample

Immediately after processing according to Example 2, samples were immersed in 50 mL of tetrahydrofuran (THF) containing 175 mg of 1,1′-carbonyldiimidazole (CDI) for 30 minutes in a 50 mL Falcon tube at room temperature to yield samples to which CDI was chemically bound to the samples. For lyophilized samples, this example was carried out in a desiccator connected to a vacuum pump, vacuumed to a pressure of about 30-60 Torr for about 5-15 minutes to remove air bubbles inside the sample.

Example 4

Attachment of Antibiotic

The CDI-bound samples of Example 3 were rinsed with dry THF and immersed in 50 mL of an aqueous gentamicin solution (10 mg/mL) at room temperature for 2 hours to bind the gentamicin to the sample. Following the gentamicin conjugation, the tissue samples were washed with deionized water and then soaked in 1× PBS for at least 16 hours to remove any unbound gentamicin.

Example 5

Gentamicin Retention vs. Untreated Control

Samples prepared according to Example 4 were soaked in an infinite sink in PBS for 28 days. Untreated controls were also soaked under the same conditions. Gentamicin content (in nanograms per sample) was measured in vitro at 0, 24, 48, 168, 336, and 672 hours (Day 0, Day 1, Day 2, Day 7, Day 14, and Day 28, respectively). FIG. 2 depicts the retention of gentamicin in samples taken at these points in time. As shown, the gentamicin content per sample dropped drastically between Day 0 and Day 1, which reflects the initial elution of unbound gentamicin from the sample. The amount of gentamicin per sample remained steadily between about 1500 ng and 500 ng from Day 1 to Day 28.

Example 6

Gentamicin Retention vs. Soaked Control

Samples prepared according to Example 4 were compared with: (i) samples that were soaked in gentamicin but not treated with oxygen plasma or CDI, (ii) samples that were dehydrated and exposed to air plasma (but not treated with CDI) and then soaked in gentamicin, and (iii) samples that were not treated at all. All samples were soaked in an infinite sink in PBS for 14 days. Gentamicin content (in nanograms per sample) was measured in vitro at 0, 24, 96, 168, and 336 hours (Day 0, Day 1, Day 4, Day 7, and Day 14, respectively). FIG. 3 depicts the retention of gentamicin in samples taken at these points in time. As shown, the gentamicin content of the treated sample dropped drastically between Day 0 and Day 1, which reflects the initial elution of unbound gentamicin from the sample. The amount of gentamicin in the treated sample remained steadily between about 70 ng and 85 ng from Day 1 to Day 14. After Day 0, the control samples drastically dropped to no more than about 10 ng of gentamicin from Day 1 to Day 14. This data demonstrates that preparing a sample according to the direct plasma process described in Examples 1-4 enables it to retain significantly more gentamicin than just soaking the sample in gentamicin in the absence of CDI and oxygen plasma.

Example 7

Bacterial Outgrowth with E. Coli

Samples prepared according to Example 4, as well as untreated controls, were implanted into non-inoculated subcutaneous pockets of rats for a set amount of time and then explanted. The treated and untreated samples were then evaluated in an outgrowth assay in which bacteria were seeded onto the samples and then placed in bacterial growth media. FIG. 4 depicts bacterial growth in each of the treated and untreated samples at Day 0, Day 4, Day 7, and Day 14. As shown, the explanted treated tissue retains antimicrobial activity after almost 7 days of implantation time.

It is noted that although the in vitro data from Examples 5 and 6 demonstrate that gentamicin was retained in the sample for at least 28 days, the in vivo data of this Example 7 shows cessation of efficacy after 7 days. This can be explained by the natural remodeling process going on in the organism's body. As soon as the sample is implanted, it becomes coated with serum proteins as the body begins to incorporate and remodel it. Over time, the foreign material will become coated with new connective tissue and nascent vasculature. In the case of the explanted tissue, by Day 7, the sample has already been coated with connected tissue and is well on its way to being remodeled. In other words, although the gentamicin in the matrix of the sample is likely still bound, it has been sequestered by the newly formed connective tissue and is not exposed to the bacterial colonies in the outgrowth assay.

Example 8

Attachment of Antibiotic Using a Vacuum

Plasma-activated samples were placed in solution containing CDI. For lyophilized samples, the solution was placed in a desiccator connected to a vacuum pump, and the desiccator was vacuumed to a pressure of about 30 to 60 Torr for 5 to 15 minutes to remove air bubbles inside the sample to achieve better solution penetration. After 30 minutes to 2 hours of activation under agitation, samples were immersed in another solution containing a target bioactive molecule for 2 to 8 hours under agitation in order to attach the molecule to the surface of the sample. The samples were then washed with deionized water 5 to 10 times to remove any unattached molecules.

FIG. 5 shows that gentamicin retention is significantly improved after introducing a short vacuum during the CDI activation step. The antimicrobial efficacy is also enhanced dramatically in the bacteria outgrowth assay, as shown in FIG. 6.

Example 9

Bacterial Outgrowth Assay

An overnight culture of E. coli was diluted down to OD₆₀₀ nm=0.03 (approximately 10⁶ CFU/mL) in LB (Lennox) broth (Fisher) and 20 μL of this bacterial dilution was pipetted onto 6 mm biopsy punches of untreated human dermis as a control or plasma gentamicin-treated dermis. The samples incubated for 10 minutes at room temperature before each one was placed into a sterile 50 mL Erlenmeyer flask containing 10 mL LB media. The flasks were shaken at 37° C. at 225 RPM in an orbital shaker (Lab-Line), and optical density measurements were taken every hour to monitor bacterial growth until turbidity was achieved or no growth was observed.

As depicted in FIG. 7, at 2, 3, and 4 hours of outgrowth assay, there were elevated numbers of E. coli in the untreated tissue compared with the treated samples. The control samples showed an exponential growth pattern while the treated samples showed no growth, indicative of complete bacterial eradication during the soaking period.

Example 10

Fibroblast Biocompatibility

Human dermal fibroblasts derived from neonatal foreskin were grown to confluency at 37° C. in a CO₂ incubation chamber, in essential media supplemented with 10% FBS and pen/strep and split in a 1:10 dilution. All studies were performed with passage four cells. Plasma treated and controls samples were prepared using 6 mm skin biopsy cores to create test samples. The samples were soaked in 70% EtOH for 30 minutes and then placed into essential media for an overnight soak for at least 16 hours. The soak media was aspirated and the samples were placed individually into wells of a 24-well plate and seeded with 10,000 fibroblasts per sample delivered in 50 μL of complete media. After 30 minutes, an additional 500 μL of complete media was added to each well. Cells were assayed for metabolic activity at 1 hour and 24 hours after post-seeding using the TACS MTT assay (Trevigen, Md., USA) according to the manufacturer's instructions. Standard curves were seeded 24 hours prior to performing the MTT assay for the 24 hour samples. For the 1 hour samples, the standard curve was prepared at the time of test article preparation, which may not have allowed for metabolic activity to resume. For each time point, n=4 treated and control samples were tested. In addition four treated and control samples were measured for MTT activity without being cell seeded.

Readings from these samples were not different between treated and untreated test samples. Therefore, the dermis background to be subtracted from the cell seeding values was determined by averaging these 8 samples together. The MTT activity for the 1 hour samples was determined by assaying the dermis and media samples together, however, it was observed that significant portion of the formazin product was sequestered in the dermis. Therefore, for the 24 hour study, the media and dermis were assayed separately and background form control dermis was also assayed separately.

The results of the fibroblast assay are shown in FIG. 8. There was no significant different between the treated and control samples at either time point.

Example 11

Cell Viability

Cell viability was assessed at 1 and 4 days of exposure to the dermal matrices. For each timepoint, eight samples from each test group were placed with the epidermal surface facing up into the wells of a 96 well TCPS plate. The samples were washed with 100 μL of complete media for 5 minutes followed by aspiration of the media and placement of either cells or fresh media without cells onto the samples. For the cell-seeded samples, 10,000 human dermal fibroblasts were seeded onto 4 of the 8 human dermis test samples in 100 μL of complete media.

At the time of initial test sample cell-seeding for the 1 day cell viability samples, a standard curve was prepared in duplicate using the human dermal fibroblasts. To produce the standard curve, 25,000, 12,500, and 6,250 cells were seeded directly onto the TCPS wells. For the 4 day study, the standard curve was prepared three days post cell seeding. The media was exchanged every 24 hours during the study and just prior to the termination of the assay.

The CellTiter 96 Aqueous One Solution Assay (Promega, USA) was used to assess cell viability at 1 and 4 days post cell seeding. The kit was used according to the manufacturer's instruction with a few minor modifications made to accommodate use with human dermis tissue. To each well of the standard curve and the test samples, 20 μL of MTS solution was added. After 90 minutes, the dermis was gently transferred to clean wells leaving the media behind. The colorimetric intensity of each well was measured at 490 nm in both the dermis and media containing wells. The non-cell seeded blanks were averaged together for both the media and dermis and subtracted from the cell-seeded samples to obtain normalized readings. It was necessary to use separate blanks for each of the treatment because it was observed that modifications made to the tissue could change the background color levels of both the media and dermis. The total normalized color observed in the media and dermis was added together and the cell number was determined using the standard curve.

FIG. 9 depicts the results using the MTS assay. Relative cell number was extrapolated from a standard curve produced from dermal fibroblasts grown on TCPS. There was no significant difference between control and plasma treated samples and controls samples at either point in time.

Example 12

Gentamicin Quantification

The amount of gentamicin loaded into the human dermis was measured in samples prepared as described in Example 11. Three separate dermis samples from the control and plasma groups were weighed just prior to gentamicin determination. The samples were placed into 5 mL of 0.25M HCl in borosilicate test tubes covered with aluminum foil. The samples were autoclaved at 121° C. with a 1 hour cycle in a Steris autoclave (SG-120 Scientific Gravity Sterilizer) to degrade and solubilize the collagen. In earlier studies, the stability of gentamicin in 0.25M HCl and under autoclave conditions was assessed. No significant loss of gentamicin detection was observed.

Following the autoclave step, the gentamicin was measured using a commercially available ELISA kit (BioO, USA) according to the instruction included in the kit. Human dermis samples were handled according to the protocol as if they were milk samples. Using anticipated drug loads determined from earlier zone of inhibition studies, the autoclaved samples were diluted into 1× sample extraction buffer 1:40. Standards provided with the kit were used to create a standard curve and subsequently to calculate total gentamicin in each sample. The gentamicin load was expressed as parts per million (ppm) by dividing the weight of gentamicin by the weight of the dermis sample.

FIG. 10 depicts the results of the assay. Plasma treated samples contained an average of 24 ppm of residual gentamcin. The control tissue, which did not contain any gentamicin, showed an ELISA value of 0.1 ppm, which represents the baseline background level inherent to the assay.

Example 13

Antimicrobial Activity

The experimental procedure described in the quantitative method of ASTM 2180 was modified and used to evaluate the antimicrobial effectiveness of gentamicin bound to treated and untreated human dermis. The surface area of the test samples was reduced from 3×3 cm squares to 6 mm diameter round dermal biopsy cores. The slurry inoculum volume was reduced from 0.5-1.0 to 6 μL, which provided the required 1mm depth of slurry across the sample. The neutralizing broth volume was therefore reduced to 600 μL. Finally, the duration of the experiment was increased from 24 hours to 48 hours of exposure of the inoculum to the test surfaces. Consequently, the moisture level of the dermis samples had to be controlled by placing the samples on hydrated squares of sterile filter paper moistened with 200 μL sterile PBS in order to recover viable cells from the untreated samples at 2 days post-slurry inoculation. The filter paper squares were checked form moisture level daily and approximately 200 μL of PBS was added each day.

Briefly, 18 hour bacterial cultures of S. aureus (ATCC 25923 and 10390), E. coli (ATCC 25404), or P. aeruginosa (ATCC 27853) were grown and diluted to OD₆₀₀=1.0, 0.5, 0.5, or 0.5, respectively, in an agar slurry containing 0.85% NaCl and 0.3% agar which was sterilized and equilibrated in a water bath to 45° C. Treated and untreated (control) dermal biopsy cores (in triplicate for each time point) were placed into sterile 35×10 mm petri dishes containing 2.4 cm² sterile filter papers, and 6 μL of inoculated slurry was pipetted onto each sample. The samples were allowed to gel for 10 minutes at room temperature before 200 μL sterile PBS was pipetted onto the filter paper squares to keep the cores hydrated. The petri dishes were placed into the 37° C. incubator for a specified contact time (1 hour, 1 day, or 2 days). Following the specified contact time, the untreated and treated samples were collected in 600 μl neutralizing broth (TSB for S. aureus and P. aeruginosa; LB for E. coli) to form a 1:100 dilution of the initial inoculum. The samples were sonicated for 1 min in a non-cavitating sonic bath, followed by 1 min of vigorous mechanical vortexing to release the agar slurry from the samples. Serial dilutions were performed with the neutralizing broth, plated (TSA for S. aureus and P. aeruginosa; LB for E. coli), and incubated overnight at 37° C. Percent reduction was calculated by comparing the CFU recovered from the untreated versus treated samples.

K. pneumoniae, C. perfringens and P. mirabilis were tested using almost identical conditions with a 24 hour contact time at Gibraltar Labs in Fairfield, N.J. For their studies, all bacteria were cultured for 18 hours and used at OD 0.5 in the agar slurry. The only other difference was that 1 ml was used as the volume for the neutralizing broth in the Gibraltar studies.

The internal ASTM 2180 experiments examined the antimicrobial effects of plasma treated tissue at three different exposure times (1 hour, 1 day, and 2 days) against E. coli, P. aeruginosa, and S. aureus 25923 and 10390. The percent killing and log bacterial reduction for the plasma treated samples are shown below in Table 1, while the percent killing and standard deviation for the plasma treated sample data set are shown in FIG. 11.

TABLE 1
Percent killing and log bacterial reduction for plasma treated samples
Bacterial	Percent Killing	Log Reduction
Species	1 Hour	1 Day	2 Days	1 Hour	1 Day	2 Days
E. coli 25404	99.94	100	100	3.19	4.73	5.34
P. aeruginosa	100	100	100	4.75	6.13	6.64
27853
S. aureus	96	99.99	99.99	1.36	4.67	4.64
25923
S. aureus	81.3	99.7	99.9	0.72	2.49	2.84
10390

The external ASTM 2180 experiments performed at Gibraltar Labs in Fairfield, N.J. utilized a single 24 hour (1 day) exposure time of the bacterial slurry on the tissue and three bacterial strains: Klebsiellapenumoniae, Proteus mirabilis, and Clostridium perfringens (vegetative cells). They tested the antimicrobial activity of plasma treated dermis. The percent killing and log bacterial reduction for these treated samples can be found in Table 2 below. Both of the treated samples resulted in 99.999% killing with a 5-log reduction of K. pneumonia, 99.99% killing with a 4.34-log reduction of Proteus mirabilis, and 99.999% killing with a 5.61-log reduction of C. perfringens. Both treated tissue samples were effective surface antimicrobial against all three of these bacterial species with a 24 hour contact time.

TABLE 2
Percent killing and log bacterial reduction in plasma treated dermis
against three bacterial strains through external ASTM 2180 testing.
	Bacterial Species	Percent Killing	Log Reduction
	Klebsiella pneumoniae	99.999	5
	Proteus mirabilis	99.99	4.34
	Clostridium perfringens	99.999	5.61
	(vegetative cells)

Example 14

Bone Tissue Treatment Process

Bone samples were soaked in PBS or 0.9% saline solution at 37° C. for three days. Bone samples were then immersed in 100% ethanol with gentle shaking for 15 minutes. This immersion step was repeated until a total of three cycles had been completed. Finally the bone samples were vacuum dried at room temperature for 2 hours.

The dried bone samples were them treated with oxygen plasma for 30 seconds. Immediately thereafter, the bones were immersed in 1,1′-carbonyldiimidazole solution for 30 minutes, and then immersed in a 10 mg/mL gentamicin aqueous solution for 2 hours followed by washing with deionized water. Finally, the bone samples were soaked in PBS for three days to remove any unbound gentamicin.

The present invention is not to be limited in scope by the specific embodiments disclosed in the examples which are intended as illustrations of a few aspects of the invention and any embodiments that are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.

Claims

1. A composition comprising:

a biological material activated by oxygen plasma and

a ligand bound to a surface of the biological material.

2. The composition of claim 1, wherein the biological material is collagen.

3. The composition of claim 1, wherein the biological material is tissue.

4. The composition of claim 1, wherein the biological material is bone.

5. The composition of claim 3, wherein the tissue is dermal tissue.

6. The composition of claim 5, wherein the dermal tissue is acellular.

7. (canceled)

8. (canceled)

9. (canceled)

10. The composition of claim 8, wherein the dermal tissue is derived from a human cadaver.

11. The composition of claim 1, wherein the biological material is dehydrated.

12. (canceled)

13. The composition of claim 11, wherein the biological material is dehydrated by a solvent exchange process.

14. (canceled)

15. (canceled)

16. The composition of claim 13, wherein the solvent exchange process comprises soaking the biological material in a hydrophilic solvent followed by soaking the biological material in an organic solvent followed by placing the biological material under vacuum.

17. (canceled)

18. (canceled)

19. (canceled)

20. The composition of claim 1, wherein the ligand is a coupling agent.

21. The composition of claim 20, wherein the coupling agent is selected from the group consisting of 1,1′-carbonyldiimidazole, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, N,N′-disuccinimidyl carbonate, N-hydroxysuccimimidyl chloroformate, isocyanate, salts thereof, and a combination thereof.

22. The composition of claim 1, further comprising a second ligand bonded to the composition.

23. The composition of claim 22, wherein the second ligand is a pharmacological agent.

24. (canceled)

25. (canceled)

26. The composition of claim 23, wherein the pharmacological agent is an antimicrobial agent.

27. The composition of claim 26, wherein the antimicrobial agent is an antibiotic.

28. The composition of claim 27, wherein the antibiotic has an available nucleophilic group.

29. The composition of claim 26, wherein the antimicrobial agent is selected from the group consisting of amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, geldanamycin, herbimycin, loracarbef, ertapenem, doripenem, imipenem/cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, ticarcillin, amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanate, bacitracin, colistin, polymyxin b, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfonamidochrysoidine, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, linezolid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, rifaximin, thiamphenicol, tigecycline, tinidazole, pharmaceutically acceptable salts thereof, and a combination thereof.

30. The composition of claim 26, wherein the antimicrobial agent is selected from the group consisting of the list of chlorhexidine, biguanides, quaternary ammonium compounds, pharmaceutically acceptable salts thereof, and a combination thereof.

31. The composition of claim 1, wherein the activated biological material has surface hydroxyl groups, surface carboxyl groups, or a combination thereof.

32. The composition of claim 1, wherein the source of oxygen plasma is O2.

33. The composition of claim 1, wherein the source of oxygen plasma is air.

34. (canceled)

35. The composition of claim 23, wherein from about 5% to about 95% of the pharmacological agent is maintained in the composition after soaking in an infinite sink in phosphate buffer saline for 24 hours.

36. The composition of claim 23, wherein from about 50% to about 95% of the pharmacological agent is maintained in the composition after soaking in an infinite sink in phosphate buffer saline for 24 hours.

37. (canceled)

38. (canceled)

39. (canceled)

40. (canceled)

41. The composition of claim 23, wherein from about 5% to about 95% of the pharmacological agent is maintained in the composition after soaking in an infinite sink in phosphate buffer saline for 14 days hours.

42. The composition of claim 23, wherein from about 50% to about 95% of the pharmacological agent is maintained in the composition after soaking in an infinite sink in phosphate buffer saline for 14 hours.

43. (canceled)

44. (canceled)

45. (canceled)

46. (canceled)

47. (canceled)

48. (canceled)

49. A composition comprising a biological material activated by oxygen plasma.

50. A composition comprising:

acellular tissue;

a coupling agent bound to the acellular tissue; and

a pharmacological agent bound to the coupling agent.

51. A method of treating biological material comprising: contacting biological material with oxygen plasma to form activated biological material.

52.-98. (canceled)

99. A method of performing reconstructive surgery in a patient in need thereof comprising implanting or grafting a composition of claim 1 in a patient in need thereof.

100. A method of administering a drug to a patient in need thereof comprising implanting or grafting a composition of claim 1 in a patient in need thereof.

101. A method of dehydrating a biological material comprising soaking the biological material in a hydrophilic solvent followed by soaking the biological material in an organic solvent.

102-105. (canceled)