FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NIH grant number GM073626. Accordingly, the Government has certain rights in this invention.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/186,870, filed June 14, 2009, entitled "Microgel Compositions and Methods," by Hoare et al., which is incorporated herein by reference. FIELD OF INVENTION

The present invention generally relates to microgel compositions and methods and, in some cases, to compositions and methods for drug release and/or scavenging as well as related implants and analytical techniques.

BACKGROUND Typical approaches to treating toxic doses of a material involve filtering a patient's blood using membranes or ion-exchange resins located outside the body to remove the material. These treatments may be too slow to protect patient health, for example, in the case of anesthetic overdose since many local anesthetics are severely neurotoxic at concentrations only slightly higher than the clinical anesthesia dose.

SUMMARY OF THE INVENTION

Microgel compositions and methods are described. The subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or compositions. In some aspects the invention is a method for capturing a biological agent in vivo. The method involves identifying a subject having a biological agent in a tissue and administering to the subject an effective amount of a microgel having a capture reagent to capture the biological agent in vivo. A subject having a biological agent in a tissue includes a subject at risk of having a biological agent in a tissue, i.e. suspected of having the biological agent or overdoses thereof as well as those confirmed to have the biological agent or overdoses thereof. In some embodiments the biological agent is a drug or exogenous or endogenous substance in an amount that causes toxic effect to the subject and wherein the microgel having a capture reagent that attenuates the toxic activity.

In other aspects the invention is a method for delivering a biological agent to a subject, by administering to the subject an effective amount of a functionalized microgel carrying the biological agent, wherein the functionalized microgel carrying the biological agent is used in a concentration of l-20wt%.

The invention in other aspects is a method for performing ion exchange chromatography using a functionalized thermosensitive microgel. In other aspects the invention is a composition of a functionalized thermosensitive microgel carrying a biological agent, wherein the functionalized microgel carrying the biological agent is in a concentration of 0.5-1 mg/ml.

In yet other aspects, the invention is a biocompatible injectable tissue implant composition comprising a sterile functionalized thermosensitive microgel in a concentration of 1-20 wt%. In some embodiments the biocompatible injectable tissue implant further includes cells in the sterile functionalized thermosensitive microgel.

In other embodiments the sterile functionalized thermosensitive microgel is present in an amount sufficient for a cosmetic implant such that, when implanted under the surface the skin, the microgel lifts and supports the tissue above it. In yet other embodiments the implant is formed into a matrix in situ.

A method for applying a tissue implant to a subject is provided according to other aspects of the invention. The method involves administering a sterile functionalized thermosensitive microgel in a concentration of 1-20 wt% to a subject, wherein the functionalized thermosensitive microgel forms an aggregated matrix at the tissue site in order to form a temporary tissue matrix at the site of the tissue.

In some embodiments the functionalized thermosensitive microgel is injectable and wherein the functionalized thermosensitive microgel is injected into a tissue of a subject.

In other embodiments the method is a method for repairing a tissue and wherein the functionalized thermosensitive microgel is administered to the subject at a tissue site in need of repair in order to repair the tissue. The functionalized thermosensitive microgel may be injected into the tissue site.

In yet other embodiments the functionalized thermosensitive microgel is seeded with cells. In some embodiments the functionalized thermosensitive microgel includes a biological agent.

In other embodiments the functionalized thermosensitive microgel is used as a cosmetic implant in the subject such that, when implanted under the surface the skin, the microgel lifts and supports the tissue above it. The functionalized thermosensitive microgel may be an implant of any one of the compositions described herein in some embodiments.

In some embodiments the capture reagent is anionic, cationic, or hydrophobic. In other embodiments the microgel is a thermosensitive microgel. For instance the microgel may be comprised of poly(N-isopropylacrylamide) (PNIPAM). The biological agent may be any type of compound that can be removed from the body or delivered to the body. For instance the biological agent may be an anesthetic, such as propofol, etomidate, ketamine, thiopental, a benzodiazepine, a barbiturate, an opioid, haloperidol, droperidol, phencyclidine, bupivacaine, lidocaine, ropivacaine, cocaine or mepivacaine. The biological agent may be a heparin. In other embodiments the biological agent is an anti-cancer or antineoplastic agent, a antidiabetic agent, an autoimmune disease therapeutic, an antipsychotic agent, an antineurological disorder agent, an immunomodulator, a benzodiazepine, an opiate, a central venous system depressant, a respiratory depressant, a cardiovascular depressant, a psychomotor stimulant, a psychotropic, a sedative, a hypnotic, a muscle relaxant, an organophosphate, an antipsychotic, an antidepressant, an antirheumatic, a microorganism, or an immunomodulator. In some embodiments the biological agent is an antineoplastic agent and wherein the antineoplastic agent is administered to the site of a tumor in the subject and the microgel having a capture reagent is administered to the blood to capture excess antineoplastic agent. In other embodiments the biological agent is a nucleic acid such as antisense, RNAi, and TLR ligands. In other embodiments the biological agent is a chemical warfare agent such as sulfur mustards, nitrogen mustards, nerve agents of G and V type, lewisite and adamsite.

In one embodiment the method of administering is performed by injection.

In other embodiments the microgel is administered to the blood. The microgel has particles having a variable average particle size. In some embodiments the particle size ranges from 10 nm to 1000 nm in size. In other embodiments the microgel has particles having an average particle size ranging from 50 nm to 500 nm in size. In yet other embodiments the microgel has particles having an average particle size ranging from 50 nm to 200 nm in size. The microgel may be functionalized. In some embodiments the microgel is less than about 35% functionalized. In other embodiments the microgel is 1 -20% functionalized. The microgel may be a bulk functionalized microgel or a surface-functionalized microgel.

The concentration of the microgel is also variable. In some embodiments the microgel is used in a concentration of 0.5-30 mg/mL. In other embodiments the microgel is used in a concentration of 0.5-20 mg/mL.

In some embodiments, the functionalized microgel is used in a concentration of 2- 10wt%. In other embodiments, the functionalized microgel is used in a concentration of 5- 10wt%. In yet other embodiments, the functionalized microgel is used in a concentration of 8wt%. In some embodiments, the functionalized microgel is immobilized on a support.

In some embodiments the step of identifying identifies the biological agent as an anionic biological agent. In other embodiments the step of identifying identifies the biological agent as an cationic biological agent or a hydrophilic biological agent.

In some embodiments the microgel is a functionalized thermosensitive microgel, wherein the functionalized microgel is in a concentration of 0.5-2 mg/mL.

Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two or more applications incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the later-filed application shall control.

BRIEF DESCRIPTION OF THE DRAWINGS Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For the purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures:

FIG. 1 shows the thermo sensitivity of various microgel properties in accordance with an embodiment of the invention;

FIG. 2 shows a plot of aggregation temperature as a function of microgel concentration in accordance with another embodiment of the invention;

FIG. 3 shows a table of microgel recipes in accordance with yet another embodiment of the invention;

FIG. 4 shows a table of microgel properties in accordance with still another embodiment of the invention; FIG. 5 shows a plot of cell viability as a function of bupivacaine concentration in accordance with another embodiment of the invention;

FIG. 6 shows microscope images of cells treated with various doses of bupivacaine in the presence or absence of microgels in accordance with yet another embodiment of the invention; FIG. 7 shows a plot of cell viability as a function of bupivacaine concentration in accordance with still another embodiment of the invention;

FIG. 8 shows a plot of cell viability as a function of bupivacaine concentration in accordance with an embodiment of the invention;

FIG. 9 shows a plot of cell viability as a function of bupivacaine concentration in accordance with yet another embodiment of the invention; FIG. 10 shows a plot of cell viability as a function of bupivacaine concentration in accordance with still another embodiment of the invention;

FIG. 11 shows a bar graph of cell viability of various cell types in the presence of various microgels in accordance with another embodiment of the invention; FIGs. 12A and 12B show bar graphs of cell viability of various cell types in the presence of various microgels in accordance with yet another embodiment of the invention;

FIGs. 13A-13C show photographs of tissue response to microgels in accordance with still another embodiment of the invention;

FIG. 14 shows photographs of tissue response to microgels in accordance with another embodiment of the invention;

FIG. 15 shows a table of nerve block times in accordance with still another embodiment of the invention;

FIG. 16 shows a table of nerve block times in accordance with yet another embodiment of the invention; FIG. 17 shows a plot of nerve block times as a function of microgel concentration in accordance with another embodiment of the invention;

FIG. 18 shows histological images of tissue and residual microgel in accordance with another embodiment of the invention; and

FIG. 19 shows a plot of cell viability as a function of microgel concentration in accordance with yet another embodiment of the invention.

DETAILED DESCRIPTION

The present invention generally relates to microgel compositions and methods and, in some cases, to compositions and methods for drug release and/or scavenging as well as tissue engineering and analytical procedures such as ion exchange chromatography. One aspect of the invention is generally directed to a composition comprising a microgel. The composition may be capable of binding and/or releasing a drug or other material. The composition may be used, in some embodiments, to reduce the amount of a material in a solution. In other embodiments, the composition may be used to release a material (i.e., a drug). In another aspect, the invention is directed to microgel aggregates. In some embodiments, the aggregates may form in response to a change in temperature. An aggregate may be used, for example, to immobilize microgel particles, entrap cells, encapsulate a material, and the like. Other aspects of the invention are directed to systems and methods of making or using such compositions, e.g., by implanting the composition within a subject, methods of treatment involving such compositions, kits including such compositions, or the like.

One aspect of the present invention is generally directed to compositions comprising a microgel. As used herein, "microgels" refer to colloidal particles comprising a network and a liquid. A microgel may be stable in suspension or may be aggregated, as described in more detail below. It should be understood that "microgel" may refer to an individual particle or a collection of particles (i.e., an aggregate). A microgel may also swell and/or shrink, exhibit a change in electrophoretic mobility, show a change in turbidity or refractive index, etc. in response to a change in environment (i.e., pH, temperature, ionic strength, etc.). A non-limiting example of how properties such as those listed above vary as a function of temperature is shown in FIG. 1. Microgels generally have diameters 5 microns or less. In some embodiments the diameter of a microgel is 1 micron or less. On a microscopic level, microgels have a three- dimensional structure, they are crosslinked in some fashion (such as covalent crosslinking), and they swell or deswell according to the quality of the solvent. On a macroscopic level, microgels have high surface areas, respond rapidly to changes in their environment, have a spherical shape, and can be flocculated by salt, dues to their colloidal particle properties. There are several distinguishing features of a microgel versus a hydrogel, such as, for instance, (1) size (<5 micron for microgel, any size for hydrogel); (2) speed of swelling response (microgels swell much faster than hydrogels); (3) shape (hydrogels fit container they are made in, microgels are spherical); and (4) ability to engineer responses (high surface area of microgels make local heterogeneities in composition much more influential than they are in hydrogels, in which inhomogeneities are averaged out over the whole bulk of the hydrogel).

A network in a microgel may be a material formed using covalent bonds (i.e., a chemically crosslinked network) and/or non-covalent bonds such as hydrophobic interactions, ionic interactions, hydrogen bonds, etc. In some embodiments, the network may comprise a polymer (i.e., a thermosensitive polymer). Examples of thermosensitive polymers include, but are not limited to, poly(N-isopropylacrylamide) or other poly(N-alkyacrylamide)s or poly(N- alkylmethacrylamide)s such as poly(7V-ethylacrylamide), poly(N-/-butylacrylamide), poly(N- methylacrylamide), poly(N-isopropylmethacrylamide), etc. Other examples of thermosensitive polymers include poloxamer 407, poloxamer 188, Pluronic® F 127, Pluronic® F68, poly(methyl vinyl ether), poly(iV-vinylcaprolactam), or poly(organophosphazenes). In some instances, the network may be formed from non-polymeric materials.

It should be understood that other components may be used in place of thermosensitive polymers or to alter the sensitivity of the thermosensitive polymers to changes in temperature, for instance, added as a copolymer component, and/or as a separate component. Examples include (but are not limited to) acrylic acid, methacrylic acid, N-vinylpyrrolidone, N,N- dimethyl aminoethylmethacrylate, oxazoline, butylmethacrylate, acrylamide, or any other vinyl or acrylic monomer which can be copolymerized with the thermosensitive monomers. Block copolymers comprising one or more hydrophilic block and/or one or more hydrophobic block may also be used in some cases. For example, block copolymers of poly(ethylene glycol) with polylactide, polyglycolide, poly(lactide-co-glycolide) (PLGA), or poly(methyl methacrylate) may be used. In some cases, the thermosensitive polymer may be present with other polymers, for example, polymers for providing a structural matrix. Examples of such polymers include, but are not limited to, poly(ethylene glycol), polylactide, polyglycolide, poly(methyl methacrylate), or the like. For instance, the two polymers may be present as a polymer blend, a co-polymer, or as interpenetrating polymers.

As used herein, an "interpenetrating polymer network" or an "IPΝ" is a polymeric material comprising two or more networks of two or more polymers (including copolymers) which are at least partially interlaced on a molecular scale, but not covalently bonded to each other and cannot be separated, even theoretically, unless chemical bonds are broken. Thus, a mixture of two or more pre-formed polymer networks (e.g., a mixture or a blend) is not an interpenetrating polymer network. Specific non-limiting examples of an interpenetrating network include [net-poly(styrene-stat-butadiene)]-ipn-[net-poly(ethyl acrylate)]. Those of ordinary skill in the art are able to form IPΝs, for example, by blending different polymer precursors which have the ability under set conditions to react to form two or more different interpenetrating polymers that do not bind to each other, by forming a first polymer and allowing a precursor of a second polymer to diffuse into the first polymer in an interpenetrating manner and to react to form the second polymer under conditions that do not promote binding between the first and second polymer, by blending two or more linear or branched polymers with at least one polymer having pendant reactant groups and subsequently adding a chain extender to cross-link each of the polymers into separate networks, and/or by proceeding with a multi-stage polymerization process including a first polymer network that is partially polymerized to allow for high swellability and/or easy diffusion of a second polymer precursor, allowing the second polymer precursor to penetrate the first polymer network, and thereafter polymerizing both polymer networks, etc. In some embodiments, a microgel may have functional groups that impart a property to the microgel. For instance, a microgel may have ionic functional groups (i.e., positively- charged or negatively-charged). Examples of ionic functional groups include oxyanions such as carboxylates, sulfonates, sulfinates, phosphates, and the like, thiolates, protonated nitrogen- containing groups such as amines, guanidines, pyridines, and the like, etc. The ionic functional groups may be covalently attached to the microgel material (i.e., a negatively-charged polymer or positively-charged polymer). Other functional groups may impart a hydrophobic nature on the microgel. The functional groups added to the microgel are referred to herein as capture reagents. Thus, capture reagents can be, for instance, ionic, cationic, or hydrophobic. The functional groups may also be introduced using non-polymeric materials. For instance, an ionic non-polymeric material may be associated with a microgel by non-covalent interactions and/or may be entrapped in a microgel during or after formation of the microgel. In some instances such microgels are referred to as functionalized microgels or functionalized thermosensitive microgels if they include thermosensitive polymers. A microgel may be at least about 1% functionalized, at least about 5% functionalized, at least about 10% functionalized, at least about 20% functionalized, at least about 30% functionalized, or any integer range therebetween. The microgels may be bulk functionalized or surface functionalized, both of which can be achieved using the methods described herein as well as those known in the art.

A microgel particle may be a formed in a variety of sizes. For example, a microgel particle may be less than about 100 microns in diameter, less than about 10 microns in diameter, less than about 1 micron in diameter, less than about 100 nm in diameter, less than about 10 nm in diameter, etc. A microgel may have a diameter between about 10 nm and about 100 nm, between about 50 nm and about 200 nm, between about 100 nm and about 1000 nm, between about 200 nm and 800 nm, between about 500 nm and about 10 microns, 10-lOOOnm or any integer range therebetween. A preferred formulation has a particle size on the order of 50-200nm for biocompatibility.

The concentration of network in a microgel particle may be varied. For example, the concentration of network may be less than about 50%, less than about 20%, less than about 10%, less than about 5%, less than about 1%, less than about 0.1%, less than about 0.01%, or any integer range therebetween. It should be understood that the concentration can change depending on the degree of swelling of the microgel. For example, the concentration of network in a microgel may change in response to a change in environment that causes the microgel to swell or shrink.

A microgel may be provided dry or wet (i.e., at least partially swollen with a liquid). The liquid component of a microgel generally may be aqueous. An "aqueous solution," as used herein, is one which is miscible in pure water. Examples include, but are not limited to, ethanol, water containing a salt, a buffer, a surfactant, organic molecules, macromolecules (i.e. soluble polymers, nucleic acids, polypeptides, polysaccharides, etc.), an emulsifier, or pure water itself. In one aspect, microgel particles may aggregate resulting, in some instances, in the formation of a gel. Aggregation may occur in response to a change in environment such as in pH, ionic strength, temperature, etc.

In some embodiments, properties such as bulk and/or surface chemistry of microgel particles can be tuned, for example, by including an appropriate functional group in the microgel network. In some instances, such modifications can be used to alter the critical transition temperature and the physical responses observed at the transition temperature of the microgels. The colloidal stability of the microgels may be tuned by adjusting, for example, the surface chemistry, particle concentration, particle size, etc. Tuning such properties can allow controlled aggregation within different in vivo environments. As a non-limiting example, in FIG. 2 the mol% of acrylic acid in acrylic acid/poly(N-isopropylacrylamide) microgels is shown to alter the aggregation temperature. In some cases, the temperature at which the thermosensitive polymeric gel swells can be tuned by copolymerizing a thermosensitive polymer with other monomers. For instance, comonomers having different hydrophilicities compared to the thermosensitive polymer can be used to tune the transition temperature; for example, more hydrophilic comonomers result in higher transition temperatures while more hydrophobic comonomers result in lower transition temperatures. In other cases, comonomers with stiffer backbones (i.e., methacrylamide-based monomers) can be used to increase the phase transition temperature of the thermosensitive polymer, e.g., by restricting the mobility of the hydrophobic segments to aggregate as the temperature increases. An example of this behavior is discussed in the examples, below. In some instances, microgel particle size may influence gelation (i.e., formation of an aggregate). Without wishing to be bound by any theory, reducing particle size may increase the rate or degree of gelation since maintaining high total surface area maximizes the thermodynamic driving force for gelation via particle aggregation as the temperature is increased and the loosely-crosslinked, near-surface polymer chains, which sterically stabilize the surface at low temperatures, undergo a thermal phase transition.

The concentration of microgel particles may be varied. For example, the concentration of particles may be less than about 0.05 mg/mL, less than about 0.1 mg/mL, less than about 0.15 mg/mL, less than about 0.20 mg/mL, less than about 0.5 mg/mL, less than about 1 mg/mL, less than about 2 mg/mL, less than about 5 mg/mL, less than about 10 mg/mL, , less than about 20 mg/mL, etc. The concentration of particles may also be between about 0.5 mg/mL about 1 mg/mL, between about 0.1 mg/mL and about 0.5 mg/mL, between about 1 mg/mL and about 5 mg/mL, etc. It should be understood that the concentration can change depending on the degree of swelling of the microgel.

The particular concentration of microgel used in the methods of the invention will vary depending on the method as well as other factors involved in the methods. Based on the guidance provided herein, including the Examples, the skilled artisan would be capable of identifying the appropriate specific concentration appropriate for a specific method. For instance, in general, for methods involving capturing a biological agent in vivo (i.e., scavenging), it is desirable to have the microgels remain in solution rather than thermally gel in the body, such that they can circulate and capture the agent. Examples of concentrations useful for these applications are on the order of 0.5-2 mg/mL blood. However, for drug delivery and tissue implants, a higher concentration of microgel is desirable in order to promote thermal gelling upon introduction into the body. For instance, concentrations ranging from 1 wt% to 20 wt% and any integer range therebetween are useful for these types of methods. As shown in the Examples injections of 8 wt% suspensions provided good levels of drug release.

In one aspect of the invention, methods are provided for capturing a material in solution. A microgel may be able to bind a biological agent, such as a drug or endogenous or exogenous substance, for instance that is toxic, thereby reducing the amount of the material in solution. Without wishing to be bound by any theory, the microgel may bind a material through ionic interactions. For instance, a microgel having positively-charged functional groups may bind a negatively-charged material. Alternatively, a microgel having negatively- charged functional groups may bind a positively-charged material. Again without wishing to be bound by any theory, a microgel may bind a material as result of affinity partitioning. For example, the octanol-water partition coefficient logP can be used as an indicator of the relative hydrophobicity of a first and second material. In some embodiments, a microgel having a logP value less than that of a material may bind the material. In some cases, the affinity partitioning of a material into a microgel may occur even when the material and the microgel have similar charge characteristics (i.e., the material and microgel are both positively-charged or both negatively charged). In some embodiments, capturing a material may be useful, for example, for reducing the toxic effect of a material in a subject. Microgels capable of binding the material may be implanted in the subject by methods described below. For example, a microgel may be a colloidal suspension prior to injection but may aggregate after injection thereby becoming substantially immobilized. The microgel particles may be removed (e.g., surgically) after a period of time sufficient to reduce the amount of material in the organism.

One use for the methods of capturing, also referred to as scavenging, biological agents in vivo is during or following a drug overdose. An overdose is any amount over a recommended threshold for safety. Drug overdoses can be due to illicit drug use, ingestion or a controlled or uncontrolled substance that has toxic effects above threshold levels, application of a therapeutic in a clinical setting at an improper dose, such as an anesthetic or a heparin. Anesthetics include, for instance, propofol, etomidate, ketamine, thiopental, a benzodiazepine, a barbiturate, an opioid, haloperidol, droperidol, phencyclidine, bupivacaine, lidocaine, ropivacaine, cocaine and mepivacaine. As used herein the term "heparin" refers to polysaccharides having heparin-like structural and functional properties. Heparin includes, but is not limited to, native heparin, low molecular weight heparin (LMWH), heparin, biotechnologically prepared heparin, chemically modified heparin, synthetic heparin, and heparan sulfate. The term "biotechnological heparin " or "biotechnologically prepared heparin " encompasses heparin that is prepared from natural sources of polysaccharides which have been chemically modified and is described in Razi et al., Bioche. J. 1995 JuI. 15; 309 (Pt 2): 465-72. Chemically modified heparin is described in Yates et al., Carbohydrate Res (1996) November 20; 294:15-27, and is known to those of skill in the art. Synthetic heparin is well known to those of skill in the art and is described in Petitou, M. et al., Bioorg Med Chem Lett. (1999) April 19; 9(8): 1161-6. Native heparin is heparin derived from a natural source (such as porcine intestinal mucosa). In some instances, the biological agent to be scavenged is an antineoplastic agent. The skilled artisan may use the scavenging methods of the invention in the instance that an overdose of antineoplastic agent has been administered. Alternatively the scavenging or capture methods of the invention may be used as a therapeutic adjunct to a cancer therapy. For instance when an antineoplastic agent is administered to the site of a tumor in the subject but systemic effects are undesirable, the microgel may be administered to the blood of the subject to capture excess antineoplastic agent, while not interfering with local levels of antineoplastic agent at the tumor site. Anti-neoplastic agents, also referred to as anti-cancer agents are described in more detail below.

Nucleic acid biological agents may also be captured using the methods of the invention. For instance, nucleic acids such as gene therapy reagents, antisense, RNAi, and TLR ligands are all administered to subjects for therapeutic regimens. An overdose of such reagents could be detrimental in the absence of the invention. The toxic effects of such an overdose could be mitigated using the capture methods of the invention.

The capture methods of the invention are also useful for reducing the harmful effects of a chemical warfare agent. Over the past several decades, various highly toxic chemical agents including chemical warfare agents have been developed and stockpiled by several nations. The effects of such agents on a recipient subject are detrimental. The ability to remove chemical warfare agents immediately from the circulation is beneficial. The major chemical warfare agents fall into three main classes: sulfur mustards (HD and N), nitrogen mustards (HNl, HN2 and HN3), and organophosphorous nerve agents (acetylcholinesterase inhibitors) of the G (GA, GB, GD, GE, GF) and V (VX, VE, VG, VM) type. Additionally, lewisite (L) and adamsite have been produced in significant quantities.

Other biological agents that can be scavenged using the methods of the invention include but are not limited to a benzodiazepine, an opiate, a central venous system depressant, a respiratory depressant, a cardiovascular depressant, a psychomotor stimulant, a psychotropic, a sedative, a hypnotic, a muscle relaxant, an organophosphate, an antipsychotic, an antidepressant, an antirheumatic, a microorganism such as a virus, bacteria, parasite or fungus, and an immunomodulator. Detoxification of the many toxins and mutagens that enter the body daily, such as heavy metals, mutagens, toxins, nicotine, dioxins, sodium and cholesterol is also desirable and can be achieved using the methods of the invention.

Without wishing to be bound by any theory, the affinity of a microgel for a material can allow high-capacity loading of a material into the microgel phase via ion exchange and hydrophobic partitioning and retard the release of the material from the microgel phase via diffusion, making microgels suitable for drug delivery or general release of a material. The releasable material may be at least partially contained within a microgel, and/or contained within an aggregated group of microgels. For instance, at least some releasable material may be external to individual microgels but may be entrapped within the microgel aggregation upon formation of the aggregate. In one embodiment, the transport of the releasable species from within a microgel or aggregate to outside of the microgel or aggregate is altered upon heating of the microgels. For example, the diffusion coefficient of the releasable material from within a microgel or aggregate may be may be altered. In another embodiment, an aggregate may contain pores defined by microgel particles, and the microgel particles may be controlled by controlling the temperature of the microgel particles (i.e., when the microgels comprise a thermosensitive material). In such embodiments, the releasable material may be contained within the pores themselves and/or within an enclosure of the aggregate such that the drug can be transported through the pores (e.g., via diffusion through the pores) for release.

In one embodiment, microgels may be used to administer an anesthetic for a nerve block. The microgels may be controlled to release the anesthetic a rate suitable for a nerve block duration greater than about 1 hour, greater than about 2 hours, greater than about 5 hour, greater than about 10 hours, etc. Other uses as a controlled release device are described below. In some cases, the microgels may be used in non-medical or industrial applications such as bioseparation, purification, filtration, medical diagnostics, or the like. For instance, positively-charged and negatively-charged microgel particles may be used as an ion-exchange resin. As discussed above, microgel particles may also bind materials through non-ionic ways and may be used in this manner in purification processes.

In another aspect of the invention, microgel particles may be used in tissue engineering applications. As discussed above, microgel particles may form an aggregate. In some cases, an aggregate may be used as a support for cells (i.e., a scaffold). In some embodiments, cells may be mixed with a colloidal suspension of microgel particles, which may form an aggregate at a later time point thereby entrapping the cells within a gel-like structure. The aggregate may be formed outside of an organism or inside an organism (i.e., by injection).

The compositions described herein can be used as implants, for instance, cosmetic implants or tissue engineering or repair implants. The implants can be thermo gelled outside of the body and delivered mechanically to the body, or in some instances, more prefereably can be injected as a liquid and allowed to gel within the body at or near the site that the material is delivered to the body. Cosmetic implants include for instance, dermal fillers, joint or tissue spacers, and breast or other tissue augmentation or reconstruction. The microgel implants can be loaded with drugs, such as antibiotics, antiproliferatives and the like, to help prevent infection and mitigate tissue reaction.

A dermal implant or filler is an implant of the material described herein that lessens (and possibly removes altogether) the appearance of one or more wrinkles or other topological defects on the skin. The cosmetic implant can be injected under one or more wrinkles or other topological defects. Once implanted under the wrinkle or defect, the microgel forms an aggregated structure, providing enhanced support under the skin, thereby lifting and contouring the skin. Such lifting lessens (and possibly removes altogether) the appearance of the wrinkle. The cosmetic implant can also be implanted under folds and/or sagging skin to lift the skin as desired. Likewise, regions of the face, such as cheeks, nose, eyes, and ears (soft tissue) can be reconstructively augmented or enhanced using the invention. A joint spacer can be used to keep the components of joints spaced apart, such as in the knee or in vertebrae. The joint spacer may be used as an intervening layer as needed, such as when an individual is awaiting knee or back surgery. For example, if cartilage is degraded, the microgel may be used in its place. Further, if a meniscus that caps a joint is damaged or degraded, the microgel may be used as a replacement. The microgel can be considered an artificial disc, when vertebrae are damaged or degraded. The advantage of the microgel in this use is that it is injectable, moldable, and ultimately removable. Thus, if an individual is awaiting surgery, such as knee replacement surgery, the microgel can be injected in a minimally invasive manner and removed once the replacement joint is ready, or the surgery is complete. The microgel can be used in a similar manner on any tissues requiring spaced proximity to each other. For example, injury or trauma to the eye may benefit from use of the microgel.

For breast or other tissue augmentation or reconstruction, the microgel can be used as an alternative to silicone or saline as fillers of breast implant, and advantageously can achieve a high viscosity once the gel is thermally formed. Augmentation or reconstruction of other body areas also falls within the scope of the invention.

The microgels can also be used for forming new tissues by implantation such that a matrix forms (or is formed prior to transplantation) optionally with the addition of appropriate cells for regenerating tissue, for instance construction of new or repair of existing tendons and ligaments. Tissues connecting bones and muscles are referred to herein as "connective tissue". Tendons are tissues which attach muscles to bones; aponeuroses are sheet-like tendons connecting one muscle with another or with bones; ligaments hold bones together at joints. Tendons and ligaments are elongated, cylindric structures formed of dense connective tissue, adapted for tension in one direction, with fibers having an orderly, parallel arrangement. The most common variety of dense regularly arranged connective tissue has a predominance of collagenous (white) fibers arranged in bundles. Fibroblasts are placed in rows between the bundles. The tissue is silvery white, tough, yet somewhat pliable. The collagen bundles of the tendons aggregate into larger bundles that are enveloped by loose connective tissue containing blood vessels and nerves. Externally, the tendon is surrounded by a sheath of dense connective tissue. If the microgels are implanted with cells, a variety of cells can be used to form tissue. In general, the cells should be viable when encapsulated within microgels. In some embodiments, cells that can be encapsulated within microgels in accordance with the present invention include, but are not limited to, mammalian cells (e.g. human cells, primate cells, mammalian cells, rodent cells, etc.), avian cells, fish cells, insect cells, plant cells, fungal cells, bacterial cells, and hybrid cells. In some embodiments, exemplary cells that can be encapsulated within microgels include stem cells, totipotent cells, pluripotent cells, and/or embryonic stem cells. In some embodiments, exemplary cells that can be encapsulated within microgels in accordance with the present invention include, but are not limited to, primary cells and/or cell lines from any tissue. For instance tenocytes and ligamentum cells can be used to form connective tissue. Fibroblasts differentiate to form collagen and can also be used. For example, cardiomyocytes, myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells (e.g. monocytes, neutrophils, macrophages, etc.), ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, sperm cells, egg cells, liver cells, epithelial cells from lung, epithelial cells from gut, epithelial cells from intestine, liver, epithelial cells from skin, etc., and/or hybrids thereof, may be encapsulated within microgels in accordance with the present invention.

Exemplary mammalian cells that can be encapsulated within microgels in accordance with the present invention include, but are not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, Madin-Darby canine kidney (MDCK) cells, baby hamster kidney (BHK cells), NSO cells, MCF-7 cells, MDA-MB-438 cells, U87 cells, Al 72 cells, HL60 cells, A549 cells, SP 10 cells, DOX cells, DG44 cells, HEK 293 cells, SHS Y5 Y, Jurkat cells, BCP-I cells, COS cells, Vero cells, GH3 cells, 9L cells, 3T3 cells, MC3T3 cells, C3H-10T1/2 cells, NIH-3T3 cells, and C6/36 cells.

Autologous cells obtained by a biopsy are preferred in some instances. For instance cells may be isolated from autologous tendon or ligament by excision of tissue, then either enzymatic digestion of cells to yield dissociated cells or mincing of tissue to form explants which are grown in cell culture to yield cells for seeding onto microgels. To obtain cells, the area to be biopsied can be locally anesthetized with a small amount of lidocaine injected subcutaneously. Alternatively, a small patch of lidocaine jelly can be applied over the area to be biopsied and left in place for a period of 5 to 20 minutes, prior to obtaining biopsy specimen. The biopsy can be obtained with the use of a biopsy needle, a rapid action needle which makes the procedure extremely simple and almost painless. This small biopsy core of tissue can then be transferred to media consisting of phosphate buffered saline, divided into very small pieces which are adhered to a culture plate, and serum containing media added. Cells are dissociated using standard techniques, such as treatment with collagenase or trypsin. Alternatively, the tissue biopsy can be minced and the cells dispersed in a culture plate with any of the routinely used medias. After cell expansion within the culture plate, the cells can be passaged utilizing the usual technique until an adequate number of cells is achieved. The cells can be maintained and/or proliferated in culture until implanted, either in standard cell culture dishes or after seeding onto matrices, as described below. Alternatively, cells can be seeded into and onto the microgel at the time of implantation.

Any of a variety of cell culture media, including complex media and/or serum-free culture media, that are capable of supporting growth of the one or more cell types or cell lines may be used to grow and/or maintain cells in accordance with the present invention. Typically, a cell culture medium contains a buffer, salts, energy source, amino acids (e.g., natural amino acids, non-natural amino acids, etc.), vitamins, and/or trace elements. Cell culture media may optionally contain a variety of other ingredients, including but not limited to, carbon sources (e.g., natural sugars, non-natural sugars, etc.), cofactors, lipids, sugars, nucleosides, animal- derived components, hydrolysates, hormones, growth factors, surfactants, indicators, minerals, activators of specific enzymes, activators inhibitors of specific enzymes, enzymes, organics, and/or small molecule metabolites. Cell culture media suitable for use in accordance with the present invention are commercially available from a variety of sources, e.g., ATCC (Manassas, Va.). In certain embodiments, one or more of the following media are used to grow cells: RPMI- 1640 Medium, Dulbecco's Modified Eagle's Medium, Minimum Essential Medium Eagle, F-12K Medium, Iscove's Modified Dulbecco's Medium. The microgels may be used for tissue engineering applications in order to replace, repair, and/or regenerate tissue and/or organ function or to create artificial tissues and organs for transplantation. In some embodiments, microgels can be used for many tissue-engineering applications, including growth of bone, cartilage, vascular tissues cardiac tissues, endocrine glands, liver, renal tissue, lymph nodes, pancreas, and other tissues. In some embodiments, microgels can be used to deliver signals to cells, act as support structures for cell growth and function, and provide space filling. In specific embodiments, tissue engineering can be utilized to provide a potential method of generating a sufficient supply of cardiac tissues for transplantation. The microgels described herein can be formed in situ through aggregation, enabling the microgels to conform to the shape of the implantation site. Multiple injections of microgel material containing the same or different cells and/or the same or different drugs can be used to create complex tissues with controlled architecture and spatial distribution of cells.

The microgels of the invention also can be used as a temporary sealant in surgical procedures, for example as an option to severing or cauterizing blood vessels. A blood vessel may be sealed by injection or insertion of the implant within the lumen of the vessel or by covering an area of bleeding tissue.

Properties such as the size of microgels may cause different interactions between cells and the microgels. For instance, microgels may interact differently with macrophages or be partitioned within the body by a different pathway depending on the size of the microgels. In one example, as the microgel particle size increases, persistance of microgel at a site of implantation may generally increase. Conversely, decreasing the size of microgel particles may, in some embodiments, allow the microgels to be cleared from an implantaion site more quickly. In some embodiments, a microgel may persist at an implantation site for less than about 2 months, less than about 1 month, less than about 14 days, less than about 7 days, less than about 4 days, less than about 2 days, etc. A microgel may also persist at an implantation site for greater than about 2 months, greater than about 6 months, etc. In some cases, microgels may be cleared from an implantation site with no residual inflammation.

As used herein, a "thermosensitive material" is a material that alters its size (linearly) by at least about 0.004% in response to a change in temperature by at least about 0.5 ⁰ C or at least 1 ⁰ C. The thermosensitive material may increase or decrease in size, depending on the type of material. In some cases, the alteration may be at least about 0.01%, at least about 0.03%, at least about 0.1%, at least about 0.3%, or at least about 1%, and in some cases, this change is measured under physiologically-relevant conditions (e.g., at a temperature of 37 ⁰ C). For example, in some cases, a size change may result from a change in the affinity of the polymers for water as the temperature increases, causing the absorption of expulsion of water from the polymer network.

The thermosensitive material, upon heating, may cause or stop the release, or otherwise cause a change in the release rate, of a drug or other releasable species from the composition. For example, the composition may begin releasing a releasable species, or stop the release of a releasable species, or the composition may exhibit a change in the rate of release of the releasable species from the composition. As non-limiting examples, the composition may exhibit an increase of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 100%, at least about 300%, at least about 500%, at least about 1000%, at least about 2000%, at least about 3000%, etc., in the release of releasable species from the composition, relative to the amount of release of the releasable species from the composition in the absence of heat.

As discussed, microgels may be used to control release of a biological agent such as a drug or other releasable material. The drug or other releasable material may be present within the microgels in any form, e.g., as a solid, as a liquid, contained within an aqueous or an organic solution, or the like. In one set of embodiments, the drug or other releasable material (i.e., species) may be present as a controlled release formulation that can release drug over an extended period of time (e.g., at least over 24 hours, and often over a week or more, even when exposed to a pure water environment). The releasable material may be contained within an enclosure (if one is present), and/or contained within the thermosensitive material, e.g., as a component of the thermosensitive material and/or contained within pores within the thermosensitive material. In one set of embodiments, the releasable species is a drug or other compound where the control of release from a microgel is desired. For example, the drug may be a small molecule (e.g., having a molecular weight of less than about 1000 Da), a protein or a peptide, a nucleic acid, a hormone, a vitamin, or the like. In some cases, the releasable species may be present as particles, such as nanoparticles. For example, the particles may have an average diameter of less than about 1 micrometer, less than about 500 nm, less than about 400 run, less than about 300 nm, less than about 100 nm, less than about 50 nm, less than about 30 nm, less than about 10 nm, etc.

As another example, the microgel may be used for the controlled release of a drug or other releasable species to a subject. The term "controlled release" generally refers to compositions, e.g., pharmaceutically acceptable carriers, for controlling the release of an active agent or drug incorporated therein, typically by slowing the release of the active agent or drug in order to prevent immediate release. Such controlled release compositions and/or carriers can be used herein to prolong or sustain the release of an active agent or drug incorporated, e.g., a chemotherapeutic or an anesthetic. Thus, the terms "controlled release" and "sustained release" are generally used interchangeably throughout this document unless otherwise indicated. The releasable species may be a drug such as a therapeutic, diagnostic, or prophylactic agent. Releasable species include, for instance, small molecules, organometallic compounds, nucleic acids (e.g., DNA, RNA, RNAi, antisense), TLR ligands, proteins, peptides, metals, an isotopically labeled chemical compounds, vaccines, immunological agents, etc. It should be understood that releasable species may also be capturable species.

In one embodiment, the releasable species are organic compounds with pharmaceutical activity, such as, for instance, a clinically used drug. Examples of releasable species include an antibiotic, anti-viral agent, anesthetic, steroidal agent, anti-inflammatory agent, anti-neoplastic agent, antigen, vaccine, antibody, decongestant, antihypertensive, sedative, birth control agent, progestational agent, anti-cholinergic, analgesic, anti-depressant, anti-psychotic, psychotropic β-adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, non- steroidal anti-inflammatory agent, nutritional agent, etc. In one embodiment, the drug is an anesthetic, such as propofol, etomidate, ketamine, thiopental, a benzodiazepine, a barbiturate, an opioid, haloperidol, droperidol, phencyclidine, cocaine and their salts and prodrugs. An anesthetic may also be an amino amide anesthetic selected from the group comprising bupivacaine, levobupivacaine, lidocaine, mepivacaine, ropivacaine, tetracaine, prilocaine, ropivacaine, articaine, trimecaine and their salts and prodrugs. Other non-limiting examples of anesthetics include tetrodotoxin, saxitoxin, or similar compounds (e.g., site 1 sodium channel blockers). The drug may be used to treat any condition, such as cancer (e.g., as a chemotherapeutic agent), a chronic disease (not necessarily cancer, e.g., epilepsy, a neurodegenerative disease, a cardiovascular disease, an autoimmune disease, diabetes, etc.), etc.

Further non-limiting examples of drugs or other releasable species that may be used include antimicrobial agents, analgesics, antinflammatory agents, central venous system depressants; respiratory depressants; cardiovascular depressants; organophosphates; psychomotor stimulant; counterirritants, coagulation modifying agents, diuretics, sympathomimetics, anorexics, antacids and other gastrointestinal agents; antiparasitics, antidepressants, antihypertensives, anticholinergics, stimulants, antihormones, central and respiratory stimulants, drug antagonists, lipid-regulating agents, uricosurics, cardiac glycosides, electrolytes, ergot and derivatives thereof, expectorants, hypnotics and sedatives, antidiabetic agents, dopaminergic agents, antiemetics, muscle relaxants, parasympathomimetics, anticonvulsants, antihistamines, beta-blockers, purgatives, antiarrhythmics, contrast materials, radiopharmaceuticals, antiallergic agents, tranquilizers, vasodilators, antiviral agents, and antineoplastic or cytostatic agents or other agents with anticancer properties, or combinations thereof. Additional therapeutic agents which may be administered in accordance with the present invention include, without limitation: antiinfectives such as antibiotics and antiviral agents; analgesics and analgesic combinations; anorexics; antiheimintics; antiarthritics; antiasthmatic agents; anticonvulsants; antidepressants; antidiuretic agents; antidiarrleals; antihistamines; antiinflammatory agents; antimigraine preparations; antinauseants; antineoplastics; antiparkinsonism drugs; antipruritics; antipsychotics; antipyretics, antispasmodics; anticholinergics; antirheumatics; sympathomimetics; xanthine derivatives; cardiovascular preparations including calcium channel blockers and beta-blockers such as pindolol and antiarrhythmics; antihypertensives; diuretics; vasodilators including general coronary, peripheral and cerebral; central nervous system stimulants; cough and cold preparations, including decongestants; hormones such as estradiol and other steroids, including corticosteroids; hypnotics; immunosuppressives; muscle relaxants; parasympatholytics; psychostimulants; sedatives; and tranquilizers; and naturally derived or genetically engineered proteins, polysaccharides, glycoproteins, or lipoproteins.

Specific non-limiting examples include acebutolol, acetaminophen, acetohydoxamic acid, acetophenazine, acyclovir, adrenocorticoids, allopurinol, alprazolam, aluminum hydroxide, amantadine, ambenonium, amiloride, aminobenzoate potassium, amobarbital, amoxicillin, amphetamine, ampicillin, androgens, anesthetics, anticoagulants (e.g., heparin), anticonvulsants-dione type, antithyroid medicine, appetite suppressants, aspirin, atenolol, atropine, azatadine, bacampicillin, baclofen, beclomethasone, belladonna, bendroflumethiazide, benzoyl peroxide, benzthiazide, benztropine, betamethasone, betha nechol, biperiden, bisacodyl, bromocriptine, bromodiphenhydramine, brompheniramine, buclizine, bumetanide, busulfan, butabarbital, butaperazine, caffeine, calcium carbonate, captopril, carbamazepine, carbenicillin, carbidopa & levodopa, carbinoxamine inhibitors, carbonic anhydsase, carisoprodol, caφhenazine, cascara, cefaclor, cefadroxil, cephalexin, cephradine, chlophedianol, chloral hydrate, chlorambucil, chloramphenicol, chlordiazepoxide, chloroquine, chlorothiazide, chlorotrianisene, chlorpheniramine, chlorpromazine, chlorpropamide, chlorprothixene, chlorthalidone, chlorzoxazone, cholestyramine, cimetidine, cinoxacin, clemastine, clidinium, clindamycin, clofibrate, clomiphere, clonidine, clorazepate, cloxacillin, colochicine, coloestipol, conjugated estrogen, contraceptives, cortisone, cromolyn, cyclacillin, cyclandelate, cyclizine, cyclobenzaprine, cyclophosphamide, cyclothiazide, cycrimine, cyproheptadine, danazol, danthron, dantrolene, dapsone, dextroamphetamine, dexamethasone, dexchlorpheniramine, dextromethorphan, diazepan, dicloxacillin, dicyclomine, diethylstilbestrol, diflunisal, digitalis, diltiazen, dimenhydrinate, dimethindene, diphenhydramine, diphenidol, diphenoxylate & atrophive, diphenylopyraline, dipyradamole, disopyramide, disulfiram, divalporex, docusate calcium, docusate potassium, docusate sodium, doxorubicin, doxyloamine, dronabinol ephedrine, epinephrine, epirubicin, ergoloidmesylates, ergonovine, ergotamine, erythromycins, esterified estrogens, estradiol, estrogen, estrone, estropipute, etharynic acid, ethchlorvynol, ethinyl estradiol, ethopropazine, ethosaximide, ethotoin, fenoprofen, ferrous fumarate, ferrous gluconate, ferrous sulfate, flavoxate, flecainide, fluphenazine, fluprednisolone, flurazepam, folic acid, furosemide, gemfibrozil, glipizide, glyburide, glycopyrrolate, gold compounds, griseofiwin, guaifenesin, guanabenz, guanadrel, guanethidine, halazepam, haloperidol, hetacillin, hexobarbital, hydralazine, hydrochlorothiazide, hydrocortisone (Cortisol), hydroflunethiazide, hydroxychloroquine, hydroxyzine, hyoscyamine, ibuprofen, indapamide, indomethacin, insulin, iofoquinol, iron- polysaccharide, isoetharine, isoniazid, isopropamide isoproterenol, isotretinoin, isoxsuprine, kaolin, pectin, ketoconazole, lactulose, levodopa, lincomycin liothyronine, liotrix, lithium, loperamide, lorazepam, magnesium hydroxide, magnesium sulfate, magnesium trisilicate, maprotiline, meclizine, meclofenamate, medroxyproyesterone, melenamic acid, melphalan, mephenytoin, mephobarbital, meprobamate, mercaptopurine, mesoridazine, metaproterenol, metaxalone, methamphetamine, methaqualone, metharbital, methenamine, methicillin, methocarbamol, methotrexate, methsuximide, methyclothinzide, methylcellulos, methyidopa, methylergonovine, methylphenidate, methylprednisolone, methysergide, metoclopramide, matolazone, metoprolol, metronidazole, minoxidil, mitotane, monamine oxidase inhibitors, nadolol, nafcillin, nalidixic acid, naproxen, narcotic analgesics, neomycin, neostigmine, niacin, nicotine, nifedipine, nitrates, nitrofurantoin, nomifensine, norethindrone, norethindrone acetate, norgestrel, nylidrin, nystatin, orphenadrine, oxacillin, oxazepam, oxprenolol, oxymetazoline, oxyphenbutazone, pancrelipase, pantothenic acid, papaverine, para-aminosalicylic acid, paramethasone, paregoric, pemoline, penicillamine, penicillin, penicillin-v, pentobarbital, perphenazine, phenacetin, phenazopyridine, pheniramine, phenobarbital, phenolphthalein, phenprocoumon, phensuximide, phenylbutazone, phenylephrine, phenylpropanolamine, phenyl toloxamine, phenytoin, pilocarpine, pindolol, piper acetazine, piroxicam, poloxamer, polycarbophil calcium, polythiazide, potassium supplements, pruzepam, prazosin, prednisolone, prednisone, primidone, probenecid, probucol, procainamide, procarbazine, prochlorperazine, procyclidine, promazine, promethazine, propantheline, propranolol, pseudoephedrine, psoralens, syllium, pyridostigmine, pyrodoxine, pyrilamine, pyrvinium, quinestrol, quinethazone, uinidine, quinine, ranitidine, rauwolfia alkaloids, riboflavin, rifampin, ritodrine, alicylates, scopolamine, secobarbital, senna, sannosides a & b, simethicone, sodium bicarbonate, sodium phosphate, sodium fluoride, spironolactone, sucrulfate, sulfacytine, sulfamethoxazole, sulfasalazine, sulfinpyrazone, sulfisoxazole, sulindac, talbutal, tamazepam, terbutaline, terfenadine, terphinhydrate, teracyclines, thiabendazole, thiamine, thioridazine, thiothixene, thyroblobulin, thyroid, thyroxine, ticarcillin, timolol, tocainide, tolazamide, tolbutamide, tolmetin trazodone, tretinoin, triamcinolone, trianterene, triazolam, trichlormethiazide, tricyclic antidepressants, tridhexethyl, trifluoperazine, triflupromazine, trihexyphenidyl, trimeprazine, trimethobenzamine, trimethoprim, tripclennamine, triprolidine, valproic acid, verapamil, vitamin A, vitamin B ₁₂ , vitamin C, vitamin D, vitamin E, vitamin K, xanthine, and the like.

Diagnostic agents include gases; commercially available imaging agents used in positron emissions tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRJ); and contrast agents. Examples of suitable materials for use as contrast agents in MRI include, but are not limited to, gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium. Non-limiting examples of materials useful for CAT and x-ray imaging include iodine-based materials.

Prophylactic agents include, for instance, vaccines, nutritional compounds, such as vitamins, antioxidants etc. The releasable species may be delivered as a mixture in some cases, e.g., a mixture of pharmaceutically active releasable species. For instance, one or more releasable species may be present in a single composition. Alternatively, a composition may include multiple components, each housing a single releasable species, but where more than one type of releasable species is present within the composition. For example, a local anesthetic may be delivered in combination with an anti-inflammatory agent such as a steroid in the same or separate compositions. An antibiotic may be combined with an inhibitor of the enzyme commonly produced by bacteria to inactivate the antibiotic (e.g., penicillin and clavulanic acid).

As discussed, the composition may be implanted into a subject, such as a human, according to one aspect of the invention. The composition may be implanted in any suitable location within the subject, e.g., in an area where localized delivery of a drug or other releasable species from the composition is needed, or in an area providing ready access to the bloodstream or to the brain, depending on the application. For instance, the composition may be implanted subcutaneously, on or proximate a nerve or an organ, etc., or the composition may be positioned on the surface of the skin in some cases. It should be understood, however, that the invention is not limited only to implant applications. For instance, the pharmaceutical compositions may be administered to an individual via any route known in the art. These include, but are not limited to, oral, sublingual, nasal, intradermal, subcutaneous, intramuscular, rectal, vaginal, intravenous, intraarterial, and inhalational administration. When administered to a site other than the intended site of therapy the compositions of the invention, may be modified to include targeting agents to target the composition to a particular cell, collection of cells, or tissue. A variety of targeting agents that direct pharmaceutical compositions to particular cells are known in the art (see, for example, Cotton, et al. Methods Enzym. 217:618, 1993; incorporated herein by reference). The targeting agents may be included throughout the particle or may be only on the surface. The targeting agent may be a protein, peptide, carbohydrate, glycoprotein, lipid, small molecule, etc. The targeting agent may be used to target specific cells or tissues or may be used to promote endocytosis or phagocytosis of the particle. Examples of targeting agents include, but are not limited to, antibodies, fragments of antibodies, low-density lipoproteins (LDLs), transferrin, asialycoproteins, gpl20 envelope protein of the human immunodeficiency virus (HIV), carbohydrates, receptor ligands, sialic acid, etc.

As used herein, a "subject," means a human or non-human animal. Examples of subjects include, but are not limited to, a mammal such as a dog, a cat, a horse, a rabbit, a pig, a sheep, a rat, a mouse, a primate (e.g., a monkey, a chimpanzee, a baboon, an ape, a gorilla, etc.), or the like. The implantable composition may thus contain one or more biocompatible materials.

As used herein, "biocompatible" is given its ordinary meaning in the art. For instance, a biocompatible material is one that is suitable for implantation into a subject without adverse consequences, for example, without substantial acute or chronic inflammatory response and/or acute rejection of the fabric material by the immune system, for instance, via a T-cell response. It will be recognized, of course, that "biocompatibility" is a relative term, and some degree of inflammatory and/or immune response is to be expected even for materials that are highly compatible with living tissue. However, non-biocompatible materials are typically those materials that are highly inflammatory and/or are acutely rejected by the immune system, i.e., a non-biocompatible material implanted into a subject may provoke an immune response in the subject that is severe enough such that the rejection of the material by the immune system cannot be adequately controlled, in some cases even with the use of immunosuppressant drugs, and often can be of a degree such that the material must be removed from the subject. In some cases, even if the material is not removed, the immune response by the subject is of such a degree that the material ceases to function; for example, the inflammatory and/or the immune response of the subject may create a fibrous "capsule" surrounding the material that effectively isolates it from the rest of the subject's body and thereby prevents proper release of the releasable species from the composition; materials eliciting such a reaction would also not be considered as "biocompatible materials" as used herein. The compositions of the invention may be used to deliver a drug to the subject in an effective amount for treating disorders such as cancer and chronic disorders such as neurological disorders, diabetes, cardiovascular disorders, autoimmune disease and pain. An "effective amount," for instance, is an amount necessary or sufficient to realize a desired biologic effect. An "effective amount for treating cancer," for instance, could be that amount necessary to (i) prevent further cancer cell proliferation, survival and/or growth and/or (ii) arresting or slowing cancer cell proliferation, survival and/or growth with respect to cancer cell proliferation, survival and/or growth in the absence of the therapy. According to some embodiments of the invention, an effective amount is that amount of a compound of the invention alone or in combination with another medicament, which when combined or co- administered or administered alone, results in a therapeutic response to the disease, either in the prevention or the treatment of the disease. The biological effect may be the amelioration and or absolute elimination of symptoms resulting from the disease. In another embodiment, the biological effect is the complete abrogation of the disease, as evidenced, for example, by the absence of a symptom of the disease. As used herein, the term "treating" and "treatment" refers to modulating certain tissues so that the subject has an improvement in the disease, for example, beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. One of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease.

In some embodiments, the present invention provides a method of treating a cancer comprising administering to a subject in whom such treatment is desired a therapeutically effective amount of a composition of the invention. A composition of the invention may, for example, be used as a first, second, third or fourth line cancer treatment. In some embodiments, the invention provides methods for treating a cancer (including ameliorating a symptom thereof) in a subject refractory to one or more conventional therapies for such a cancer, said methods comprising administering to said subject a therapeutically effective amount of a composition of the invention having one or more anti-cancer drugs therein. A cancer may be determined to be refractory to a therapy when at least some significant portion of the cancer cells are not killed or their cell division is not arrested in response to the therapy. Such a determination can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of treatment on cancer cells, using the art-accepted meanings of "refractory" in such a context. In a specific embodiment, a cancer is refractory where the number of cancer cells has not been significantly reduced, or has increased.

The invention also provides methods for treating cancer by administering a composition of the invention in combination with any other anti-cancer treatment (e.g., radiation therapy, chemotherapy or surgery) to a patient. Cancers that can be treated by the methods encompassed by the invention include, but are not limited to, neoplasms, malignant tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth such that it would be considered cancerous. The cancer may be a primary or metastatic cancer. Specific cancers that can be treated according to the present invention include, but are not limited to, those listed below (for a review of such disorders, see Fishman, et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia).

Specific cancers include, but are not limited to, biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS-associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms including Bowen's disease and Paget' s disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Kaposi's sarcoma, basocellular cancer, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma, teratomas, choriocarcinomas; stromal tumors and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms' tumor. Commonly encountered cancers include breast, prostate, lung, ovarian, colorectal, and brain cancer.

The compositions of the invention also can be administered to prevent progression to a neoplastic or malignant state. Such prophylactic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79.). Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. Endometrial hyperplasia often precedes endometrial cancer. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. A typical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.

The prophylactic use of the compositions of the invention is also indicated in some viral infections that may lead to cancer. For example, human papilloma virus can lead to cervical cancer (see, e.g., Hernandez-Avila et al., Archives of Medical Research (1997) 28: 265-271), Epstein-Barr virus (EBV) can lead to lymphoma (see, e.g., Herrmann et al., J. Pathol. (2003) 199(2): 140-5), hepatitis B or C virus can lead to liver carcinoma (see, e.g., El-Serag, J. Clin. Gastroenterol (2002) 35(5 Suppl 2): S72-8), human T cell leukemia virus (HTLV)-I can lead to T-cell leukemia (see e.g., Mortreux et al., Leukemia (2003) 17(1): 26-38), and human herpesvirus-8 infection can lead to Kaposi's sarcoma (see, e.g., Kadow et al., Curr. Opin. Investig. Drugs (2002) 3(11): 1574-9).

Examples of conventional anti -cancer agents which can be incorporated in the compositions of the invention include methotrexate, trimetrexate, adriamycin, taxotere, doxorubicin, 5-flurouracil, vincristine, vinblastine, pamidronate disodium, anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab, megestrol, tamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate, etc.

Another form of anti-cancer therapy involves administering an antibody specific for a cell surface antigen of, for example, a cancer cell. In one embodiment, the antibody incorporated in the composition of the invention may be selected from the group consisting of Ributaxin, Herceptin, Rituximab, Quadramet, Panorex, IDEC- Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22, OV103, 3622W94, anti- VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-I, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD- 72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART IDlO Ab, SMART ABL 364 Ab and ImmuRAIT-CEA. Other antibodies include but are not limited to anti-CD20 antibodies, anti-CD40 antibodies, anti- CD^ antibodies, anti-CD22 antibodies, anti-HLA-DR antibodies, anti-CD80 antibodies, anti- CD86 antibodies, anti-CD54 antibodies, and anti-CD69 antibodies. These antibodies are available from commercial sources or may be synthesized de novo.

Examples of anti-cancer agents include, but are not limited to, DNA-interactive agents including, but not limited to, the alkylating agents (for example, nitrogen mustards, e.g. Chlorambucil, Cyclophosphamide, Isofamide, Mechlorethamine, Melphalan, Uracil mustard; Aziridine such as Thiotepa; methanesulphonate esters such as Busulfan; nitroso ureas, such as Carmustine, Lomustine, Streptozocin; platinum complexes, such as Cisplatin, Carboplatin; bioreductive alkylator, such as Mitomycin, and Procarbazine, Dacarbazine and Altretamine); the DNA strand-breakage agents, e.g., Bleomycin; the intercalating topoisomerase II inhibitors, e.g., Intercalators, such as Amsacrine, Dactinomycin, Daunorubicin, Doxorubicin, Idarubicin, Mitoxantrone, and nonintercalators, such as Etoposide and Teniposide; the nonintercalating topoisomerase II inhibitors, e.g., Etoposide and Teniposde; and the DNA minor groove binder, e.g., Plicamydin; the antimetabolites including, but not limited to, folate antagonists such as Methotrexate and trimetrexate; pyrimidine antagonists, such as Fluorouracil, Fluorodeoxyuridine, CB3717, Azacitidine and Floxuridine; purine antagonists such as Mercaptopurine, 6-Thioguanine, Pentostatin; sugar modified analogs such as Cytarabine and Fludarabine; and ribonucleotide reductase inhibitors such as hydroxyurea; tubulin interactive agents including, but not limited to, colchicine, Vincristine and Vinblastine, both alkaloids and Paclitaxel and Cytoxan; hormonal agents including, but note limited to, estrogens, conjugated estrogens and Ethinyl Estradiol and Diethylstilbesterol, Chlortrianisen and Idenestrol; progestins such as Hydroxyprogesterone caproate, Medroxyprogesterone, and Megestrol; and androgens such as testosterone, testosterone propionate; fiuoxymesterone, methyltestosterone; adrenal corticosteroid, e.g., Prednisone, Dexamethasone, Methylprednisolone, and Prednisolone; leutinizing hormone releasing hormone agents or gonadotropin-releasing hormone antagonists, e.g., leuprolide acetate and goserelin acetate; antihormonal antigens including, but not limited to, antiestrogenic agents such as Tamoxifen, antiandrogen agents such as Flutamide; and antiadrenal agents such as Mitotane and Aminoglutethimide; cytokines including, but not limited to, IL-I α, IL-I β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12, IL-13, IL-18, TGF-β, GM-CSF, M-CSF, G-CSF, TNF-α, TNF-β, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-.γ, and Uteroglobins (U.S. Pat. No. 5,696,092); anti-angiogenics including, but not limited to, agents that inhibit VEGF (e.g. , other neutralizing antibodies (Kim et al. , 1992; Presta et al, 1997;

Sioussat et al, 1993; Kondo et al, 1993; Asano et al, 1995, U.S. Pat. No. 5,520,914), soluble receptor constructs (Kendall and Thomas, 1993; Aiello et al, 1995; Lin et al, 1998; Millauer et al, 1996), tyrosine kinase inhibitors (Siemeister et al, 1998, U.S. Pat. Nos. 5,639,757, and 5,792,771), antisense strategies, RNA aptamers and ribozymes against VEGF or VEGF receptors (Saleh et al, 1996; Cheng et al, 1996; Ke et al, 1998; Parry et al, 1999); variants of VEGF with antagonistic properties as described in WO 98/16551; compounds of other chemical classes, e.g., steroids such as the angiostatic 4,9(1 l)-steroids and C21 -oxygenated steroids, as described in U.S. Pat. No. 5,972,922; thalidomide and related compounds, precursors, analogs, metabolites and hydrolysis products, as described in U.S. Pat. Nos. 5,712,291 and 5,593,990; Thrombospondin (TSP-I) and platelet factor 4 (PF4); interferons and metalloproteinsase inhibitors; tissue inhibitors of metalloproteinases (TIMPs); anti-Invasive Factor, retinoic acids and paclitaxel (U.S. Pat. No. 5,716,981); AGM- 1470 (Ingber et al, 1990); shark cartilage extract (U.S. Pat. No. 5,618,925); anionic polyamide or polyurea oligomers (U.S. Pat. No. 5,593,664); oxindole derivatives (U.S. Pat. No. 5,576,330); estradiol derivatives (U.S. Pat. No. 5,504,074); thiazolopyrimidine derivatives (U.S. Pat. No. 5,599,813); and LM609 (U.S. Pat. No. 5,753,230); apoptosis-inducing agents including, but not limited to, bcr-abl, bcl-2 (distinct from bcl-1, cyclin Dl; GenBank accession numbers M14745, X06487; U.S. Pat. Nos. 5,650,491; and 5,539,094) and family members including Bcl-xl, McI-I, Bak, Al, A20, and antisense nucleotide sequences (U.S. Pat. Nos. 5,650,491 ; 5,539,094; and 5,583,034); Immunotoxins and coaguligands, tumor vaccines, and antibodies.

Cancer therapies and their dosages, and recommended usage are known in the art and have been described in such literature as the Physician 's Desk Reference (56 ^th ed., 2002), which is incorporated by reference.

The term "neurological disorder" as used in this invention includes neurological diseases, neurodegenerative diseases, and neuropsychiatric disorders. A neurological disorder is a condition having as a component a central or peripheral nervous system malfunction. Neurological disorders may cause a disturbance in the structure or function of the nervous system resulting from developmental abnormalities, disease, genetic defects, injury or toxin. These disorders may affect the central nervous system (e.g., the brain, brainstem and cerebellum), the peripheral nervous system (e.g., the cranial nerves, spinal nerves, and sympathetic and parasympathetic nervous systems) and/or the autonomic nervous system (e.g., the part of the nervous system that regulates involuntary action and that is divided into the sympathetic and parasympathetic nervous systems).

As used herein the term "neurodegenerative disease" implies any disorder that might be reversed, deterred, managed, treated, improved, or eliminated with agents that stimulate the generation of new neurons. Examples of neurodegenerative disorders include: (i) chronic neurodegenerative diseases such as familial and sporadic amyotrophic lateral sclerosis (FALS and ALS, respectively), familial and sporadic Parkinson's disease, Huntington's disease, familial and sporadic Alzheimer's disease, multiple sclerosis, olivopontocerebellar atrophy, multiple system atrophy, progressive supranuclear palsy, diffuse Lewy body disease, corticodentatonigral degeneration, progressive familial myoclonic epilepsy, strionigral degeneration, torsion dystonia, familial tremor, Down's Syndrome, Gilles de Ia Tourette syndrome, Hallervorden-Spatz disease, diabetic peripheral neuropathy, dementia pugilistica, AIDS Dementia, age related dementia, age associated memory impairment, and amyloidosis-related neurodegenerative diseases such as those caused by the prion protein (PrP) which is associated with transmissible spongiform encephalopathy (Creutzfeldt- Jakob disease, Gerstmann-Straussler-Scheinker syndrome, scrapie, and kuru), and those caused by excess cystatin C accumulation (hereditary cystatin C angiopathy); and (ii) acute neurodegenerative disorders such as traumatic brain injury (e.g., surgery-related brain injury), cerebral edema, peripheral nerve damage, spinal cord injury, Leigh's disease, Guillain-Barre syndrome, lysosomal storage disorders such as lipofuscinosis, Alper's disease, vertigo as result of CNS degeneration; pathologies arising with chronic alcohol or drug abuse including, for example, the degeneration of neurons in locus coeruleus and cerebellum; pathologies arising with aging including degeneration of cerebellar neurons and cortical neurons leading to cognitive and motor impairments; and pathologies arising with chronic amphetamine abuse including degeneration of basal ganglia neurons leading to motor impairments; pathological changes resulting from focal trauma such as stroke, focal ischemia, vascular insufficiency, hypoxic- ischemic encephalopathy, hyperglycemia, hypoglycemia or direct trauma; pathologies arising as a negative side-effect of therapeutic drugs and treatments (e.g., degeneration of cingulate and entorhinal cortex neurons in response to anticonvulsant doses of antagonists of the NMDA class of glutamate receptor), and Wernicke-Korsakoff's related dementia. Neurodegenerative diseases affecting sensory neurons include Friedreich's ataxia, diabetes, peripheral neuropathy, and retinal neuronal degeneration. Other neurodegenerative diseases include nerve injury or trauma associated with spinal cord injury. Neurodegenerative diseases of limbic and cortical systems include cerebral amyloidosis, Pick's atrophy, and Retts syndrome. The foregoing examples are not meant to be comprehensive but serve merely as an illustration of the term "neurodegenerative disorder."

Parkinson's disease is a disturbance of voluntary movement in which muscles become stiff and sluggish. Symptoms of the disease include difficult and uncontrollable rhythmic twitching of groups of muscles that produces shaking or tremors. Currently, the disease is caused by degeneration of pre-synaptic dopaminergic neurons in the brain and specifically in the brain stem. As a result of the degeneration, an inadequate release of the chemical transmitter dopamine occurs during neuronal activity.

Currently, Parkinson's disease is treated with several different compounds and combinations. Levodopa (L-dopa), which is converted into dopamine in the brain, is often given to restore muscle control. Perindopril, an ACE inhibitor that crosses the blood-brain barrier, is used to improve patients' motor responses to L-dopa. Carbidopa is administered with L-dopa in order to delay the conversion of L-dopa to dopamine until it reaches the brain, and it also lessens the side effects of L-dopa. Other drugs used in Parkinson's disease treatment include dopamine mimickers Mirapex (pramipexole dihydrochloride) and Requip (ropinirole hydrochloride), and Tasmar (tolcapone), a COMT inhibitor that blocks a key enzyme responsible for breaking down levodopa before it reaches the brain.

One group of neuropsychiatric disorders includes disorders of thinking and cognition, such as schizophrenia and delirium. A second group of neuropsychiatric disorders includes disorders of mood, such as affective disorders and anxiety. A third group of neuropsychiatric disorders includes disorders of social behavior, such as character defects and personality disorders. And a fourth group of neuropsychiatric disorders includes disorders of learning, memory, and intelligence, such as mental retardation and dementia. Accordingly, neuropsychiatric disorders encompass schizophrenia, delirium, attention deficit disorder (ADD), schizoaffective disorder Alzheimer's disease, depression, mania, attention deficit disorders, drug addiction, dementia, agitation, apathy, anxiety, psychoses, personality disorders, bipolar disorders, unipolar affective disorder, obsessive-compulsive disorders, eating disorders, post-traumatic stress disorders, irritability, adolescent conduct disorder and disinhibition. Examples of antipsychotic drugs that may be used to treat schizophrenic patients include phenothizines, such as chlorpromazine and trifluopromazine; thioxanthenes, such as chlorprothixene; fluphenazine; butyropenones, such as haloperidol; loxapine; mesoridazine; molindone; quetiapine; thiothixene; trifluoperazine; perphenazine; thioridazine; risperidone; dibenzodiazepines, such as clozapine; and olanzapine. Benzodiazepines, which enhance the inhibitory effects of the gamma aminobutyric acid (GABA) type A receptor, are frequently used to treat anxiety. Buspirone is another effective anxiety treatment.

According to an embodiment of the invention, the methods described herein are useful in treating autoimmune disease in a subject by administering a composition of the invention to the subject. Thus, the methods are useful for such autoimmune diseases as multiple sclerosis, systemic lupus erythematosus, type 1 diabetes, viral endocarditis, viral encephalitis, rheumatoid arthritis, Graves' disease, autoimmune thyroiditis, autoimmune myositis, and discoid lupus erythematosus.

"Autoimmune Disease" refers to those diseases which are commonly associated with the nonanaphy lactic hypersensitivity reactions (Type II, Type III and/or Type IV hypersensitivity reactions) that generally result as a consequence of the subject's own humoral and/or cell-mediated immune response to one or more immunogenic substances of endogenous and/or exogenous origin. Such autoimmune diseases are distinguished from diseases associated with the anaphylactic (Type I or IgE-mediated) hypersensitivity reactions. The compositions of the invention are also useful in the treatment of diabetes. Diabetes is a chronic metabolic disorder which includes a severe form of childhood diabetes (also called juvenile, Type I or insulin-dependent diabetes). Type II Diabetes (DM II) is generally found in adults. Patients with diabetes of all types have considerable morbidity and mortality from microvascular (retinopathy, neuropathy, nephropathy) and macrovascular (heart attacks, stroke, peripheral vascular disease) pathology. Non-insulin dependent diabetes mellitus develops especially in subjects with insulin resistance and a cluster of cardiovascular risk factors such as obesity, hypertension and dyslipidemia, a syndrome which first recently has been recognized and is named "the metabolic syndrome."

Antidiabetic agents, include insulin, insulin derivatives and mimetics; insulin secretagogues such as the sulfonylureas, e.g., Glipizide, glyburide and Amaryl; insulinotropic sulfonylurea receptor ligands such as meglitinides, e.g., nateglinide and repaglinide; protein tyrosine phosphatase- 1 B (PTP-I B) inhibitors such as PTP-112; GSK3 (glycogen synthase kinase-3) inhibitors such as SB-517955, SB-4195052, SB-216763, N,N-57-05441 and N.N-57- 05445; RXR ligands such as GW-0791 and AGN-194204; sodium-dependent glucose cotransporter inhibitors such as T- 1095; glycogen phosphorylase A inhibitors such as BAY R3401; biguanides such as metformin; alpha-glucosidase inhibitors such as acarbose; GLP-I (glucagon like peptide- 1), GLP-I analogs such as Exendin-4 and GLP-I mimetics; and DPPIV (dipeptidyl peptidase IV) inhibitors such as LAF237;b) hypolipidemic agents such as 3- hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, e.g., lovastatin, pitavastatin, simvastatin, pravastatin, cerivastatin, mevastatin, velostatin, fluvastatin, dalvastatin, atorvastatin, rosuvastatin and rivastatin; squalene synthase inhibitors; FXR (farnesoid X receptor) and LXR (liver X receptor) ligands; cholestyramine; fibrates; nicotinic acid and aspirin;c) anti-obesity agents such as orlistat; and) anti-hypertensive agents, e.g., loop diuretics such as ethacrynic acid, furosemide and torsemide; angiotensin converting enzyme (ACE) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perinodopril, quinapril, ramipril and trandolapril; inhibitors of the Na-K- ATPase membrane pump such as digoxin; neutralendopeptidase (NEP) inhibitors; ACE/NEP inhibitors such as omapatrilat, sampatrilat and fasidotril; angiotensin II antagonists such as candesartan, eprosartan, irbesartan, losartan, telmisartan and valsartan, in particular valsartan; renin inhibitors such as ditekiren, zankiren, terlakiren, aliskiren, RO 66-1 132 and RO-66-1168; beta- adrenergic receptor blockers such as acebutolol, atenolol, betaxolol, bisoprolol, metoprolol, nadolol, propranolol, sotalol and timolol; inotropic agents such as digoxin, dobutamine and milrinone; calcium channel blockers such as amlodipine, bepridil, diltiazem, felodipine, nicardipine, nimodipine, nifedipine, nisoldipine and verapamil; aldosterone receptor antagonists; and aldosterone synthase inhibitors.

Cardiovascular disorders, treatable using the compositions of the invention, include but are not limited to disorders of the heart and the vascular system like congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, peripheral vascular diseases, and atherosclerosis. Heart failure is a pathophysiological state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failures such as high- output and low-output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause. Myocardial infarction (MI) is generally caused by an abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously narrowed by arteriosclerosis. MI prophylaxis (primary and secondary prevention) is included as well as the acute treatment of MI and the prevention of complications. Ischemic disease is a condition in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen, such as stable angina, unstable angina and asymptomatic ischemia. Arrhythmias include atrial and ventricular tachyarrhythmias, atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation, as well as bradycardic forms of arrhythmias. Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension, renal, endocrine, neurogenic, others. Peripheral vascular diseases are vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand and include chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders. Atherosclerosis is a cardiovascular disease in which the vessel wall is remodeled, compromising the lumen of the vessel.

In one embodiment, compositions containing an anesthetic (e.g., bupivacaine, levobupivacaine, lidocaine, mepivacaine, ropivacaine, tetracaine, prilocaine, ropivacaine, articaine, trimecaine and their salts and prodrugs) are administered in the vicinity of a nerve to provide a nerve block. Nerve blocks provide a method of anesthetizing large areas of the body without the risks associated with general anesthesia. Any nerve may be anesthetized in this manner. The compositions containing the releasable species are deposited as close to the nerve as possible without injecting directly into the nerve. Particularly preferred nerves include the sciatic nerve, the femoral nerve, inferior alveolar nerve, nerves of the brachial plexus, intercostal nerves, nerves of the cervical plexus, median nerve, ulnar nerve, and sensory cranial nerves. In an embodiment, epinephrine or another vasoactive agent may be administered along with the local anesthetic to prolong the block. The epinephrine or other agent (e.g., other vasoactive agents, steroidal compounds, non-steroidal anti-inflammatory compounds) may be encapsulated in the compositions containing the local anesthetic, encapsulated in compositions by itself, or unencapsulated. Additionally a pharmaceutically effective glucocorticosteroid is administered locally or systemically, to a patient, before any local anesthetic is administered to the patient. In this aspect, the glucocorticosteroid dose will then potentiate, e.g., prolong the duration or increase the degree of anesthesia of a later-administered local anesthetic. One of ordinary skill in this art would be able to determine the choice of anesthetic as well as the amount and concentration of anesthetic based on the nerves and types of nerve fibers to be blocked, the duration of anesthesia required, and the size and health of the patient (Hardman & Limbird, Eds., Goodman & Gilman's The Pharmacological Basis of Therapeutics Ninth Edition, Chapter 15, pp. 331-347, 1996; incorporated herein by reference).

As used herein, the term "anesthetic agent" means any drug or mixture of drugs that provides numbness and/or analgesia. Examples of anesthetic agents which can be used include bupivacaine, levobupivacaine, lidocaine, mepivacaine, ropivacaine, tetracaine, prilocaine, ropivacaine, articaine, trimecaine and their salts and prodrugs, and mixtures thereof and any other art-known pharmaceutically acceptable anesthetic. The anesthetic can be in the form of a salt, for example, the hydrochloride, bromide, acetate, citrate, carbonate or sulfate. More preferably, the anesthetic agent is in the form of a free base. The dose of anesthetic included within the composition of the invention will depend on the particular type of anesthetic as well as the objectives of the treatment. For example, when the drug included in the compositions of the present invention is bupivacaine, the formulation may include, e.g., from about 0.5 to about 2 mg/kg body weight. Since the formulations of the present invention are controlled release, it is contemplated that formulations may include much more than usual immediate release doses, e.g., as much as 450 mg/kg anesthetic or more. The effective dose of anesthetic sufficient to provide equivalent potency (i.e., equally effective doses), can range from about 1 to about 50 mg injected or inserted at each site where the release of anesthetic agent is desired.

The compositions of the invention can generally be used in any art known procedures for anesthetizing a patient. For example, they may be used for infiltration anesthesia, wherein a formulation suitable for injection is injected directly into the tissue requiring anesthesia. For example, an effective amount of the formulation in injectable form is infiltrated into a tissue area that is to be incised or otherwise requires anesthesia. In addition, the anesthetic formulations and methods according to the invention can be used for field block anesthesia, by injecting an effective amount of the formulation in injectable form in such a manner as to interrupt nerve transmission proximal to the site to be anesthetized. For instance, subcutaneous infiltration of the proximal portion of the volar surface of the forearm results in an extensive area of cutaneous anesthesia that starts 2 to 3 cm distal to the site of injection. Simply by way of example, the same effect can be achieved for the scalp, anterior abdominal wall and in the lower extremities.

Further, for even more efficient results, the local anesthetic formulations and methods according to the invention can be used for nerve block anesthesia. For example, an effective amount of the formulation in injectable form is injected into or adjacent to individual peripheral nerves or nerve plexuses. Injection of an effective amount of an anesthetic formulation according to the invention into mixed peripheral nerves and nerve plexuses can also desirably anesthetize somatic motor nerves, when required. The formulations and methods according to the invention can also be used for intravenous regional anesthesia by injecting a pharmacologically effective amount of microspheres in injectable form into a vein of an extremity that is subjected to a tourniquet to occlude arterial flow. Further still, spinal and epidural anesthesia using formulations, e.g., injectable compositions will be appreciated by the artisan to be within the scope contemplated by the present invention.

The compositions may be used alone or combined with other pharmaceutical excipients, such as a pharmaceutically acceptable excipient or carrier, to form a pharmaceutical composition. As would be appreciated by one of skill in this art, the excipients may be chosen based on the route of administration, the releasable species being delivered, the time course of delivery of the releasable species, etc. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

Implanted compositions, may be implanted directly or formulated and then implanted. If a composition is injected, the compositions may also be formulated or injected alone. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In a particularly preferred embodiment, the compositions are suspended in a carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween 80. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

If the compositions are delivered to a subject by alternative routes, they may be prepared in formulations suitable or oral, rectal, vaginal, nasal, subcutaneous, or pulmonary delivery. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredients, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compositions with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the compositions. U.S. Provisional Patent Application Serial No. 61/186,870, filed June 14, 2009, entitled

"Microgel Compositions and Methods," by Hoare et al., is incorporated herein by reference. The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

EXAMPLE 1 This example demonstrates characterization of microgels. N-isopropylacrylamide

(NIPAM, 99%), acrylic acid (AA, 99%), dimethylaminoethyl methacrylate (DMAEA, 99%), N,N-methylenebisacrylamide (BIS, 99%), sodium dodecyl sulfate (SDS, 99.5%), bupivacaine hydrochloride (BPV, 99%) were purchased from Sigma-Aldrich. Ammonium persulfate (APS, 99%) was purchased from Fluka. Sterile saline for injections was purchased from Baxter Pharmaceuticals. Phosphate buffered saline (PBS) was purchased from Invitrogen. All water used in the synthesis and purification was of Milli-Q grade. The % functional monomer content of copolymer microgels and the recipes used to prepare the microgels are shown in FIG. 3. The microgel codes are assigned to indicate the type of functional monomer used to prepare the microgel (AA or DMAEA), the relative loading of the functional groups in the microgel (expressed in terms of mol% total monomer), and the relative size of the microgel (S = small, M = medium, L = large). All monomers and surfactants were dissolved in 150 mL water inside a 500 mL round-bottom flask and heated to 70°C under a N ₂ purge and 200RPM magnetic mixing. After 30 minutes, 0.10 g of APS dissolved in 5mL water was injected into the flask to initiate polymerization. The polymerization reaction was continued for four hours, after which the microgels were cooled and decanted into a 500 kDa MWCO poly(vinylidene fluoride) dialysis membrane. Microgels were exhaustively dialyzed over 8 cycles against 4 L of Millipore water to remove residual surfactant and linear oligomers, which are by-products of microgel synthesis. The microgels were then lyophilized to dryness for storage.

The particle size of the microgels was evaluated using a Zeta Plus dynamic light scattering instrument operating at a 90° detection angle (Brookhaven Instruments Inc.) The electrophoretic mobility of the microgels was measured using the Zeta Plus instrument (Brookhaven Instruments Inc.) operating in phase analysis light scattering (PALS) mode. The particle size and electrophoretic mobilities at 25°C and 37°C (measured in PBS) of a series of microgels are shown in FIG. 4. The electrophoretic mobility is related to the zeta potential of the microgel; the higher the absolute value of the electrophoretic mobility, the higher the surface charge on the microgel.

In this example, the particle size of the microgel increases as the amount of functional monomer used in the preparation is increased and the amount of surfactant used to prepare the microgel is decreased. For each degree of functionalization tested (i.e., 5.8%, 19.8%, 33.1% acrylic acid), microgels prepared in the absence of surfactant have a particle size on the order of 800-1000 nm, microgels prepared with 0.05 g of surfactant have a particle size on the order of 250-450 nm, and particles prepared with 0.2 g of surfactant have a particle size of 100-280 nm. The electrophoretic mobility of the microgels at 37 ⁰ C scales with the degree of acid functionalization in the microgel. AA-I microgels have mobilities in the range -0.9 > μ _e > -1.2, AA-4 microgels have mobilities in the range -1.7 > u^ > 2.3, and AA-8 microgels have mobilities in the range -2.6 > μ _« > -2.8. This result suggests that adding more acrylic acid to the microgel recipe results in the incorporation of more acidic functional groups in the microgel. It should also be noted that no correlation was observed between the electrophoretic mobility and the particle size at a given degree of functionalization, as would be expected given the same net monomer composition of each like-functionalized microgel. Based on these results, the library of microgels synthesized allowed for the independent evaluation of the biological responses and drug delivery capacities of microgels as a function of both the degree of functionalization and the particle size. The DMAEA microgel had a similar size to the "small" acrylic acid- functionalized microgels, making it a useful control to evaluate the impact of functional group content on drug delivery. It should be noted that although the DMAEA microgel has a net cationic charge in water, divalent phosphate ions in PBS bind to the cationic sites on the microgel surface to convert the net charge from negative to positive when tested in PBS.

As described in the following examples, the library of microgels synthesized permitted a detailed evaluation of the effect of such properties as the microgel concentration, the functional group content of the microgels, the type of functional group incorporated into the microgels, the size of the microgels, and the distribution of functional groups within the microgel matrix on the capacity of microgels to bind cationic drugs and scavenge excess local anesthetic in physiological media. Binding was assayed in cell growth media by testing the capacity of microgels to maintain the viability of C2C12 myotubes in the presence of moderate- to-high bupivacaine concentrations; smooth muscle cells are susceptible to bupivacaine- induced toxicity.

EXAMPLE 2

This example demonstrates effects of microgel concentration. An MTT assay was used to evaluate the biocompatibility of the microgels with mouse-derived C2C12 myoblasts (cultured in Invitrogen DMEM medium supplemented with 10% fetal bovine serum and 1 % penicillin streptomycin), mouse-derived 3T3 fibroblasts (cultured in ATCC DMEM medium supplemented with 10% calf serum albumin and 1% penicillin streptomycin), and mouse- derived J.1774 macrophage-like cells (cultured in Invitrogen DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin). Each cell line was plated in 1 mL aliquots in a 24- well plate at a density of 30000 cells/well and permitted to adhere over 24 hours. In the case of the C2C12 myoblasts, the FBS growth medium was replaced with 2% horse serum and 1 % penicillin streptomycin-supplemented DMEM media to differentiate the myoblasts into myotubes over the course of 8 days, with regular media changes every 3 days. Passages 3-35 were used for biocompatibility studies. Materials were sterilized in their dry state under a UV lamp over a period of three hours, after which 0.9% saline solution was added aseptically and the microgels were resuspended under gentle mixing at a concentration of 20mg/mL. Aliquots (O.lmL) of microgels were added to each of the wells of the multiwell plates, with O.lmL of sterile saline added to the cell-only and media-only controls to conserve volume. Four replicate wells were tested for each material, with media-only and cell-only controls (also performed in quadruplicate) also included on each 24-well plate tested. At time points of 24 hours and 4 days after material addition, both the media and the test material was removed and replaced with 1 mL of fresh media and 100 μL of MTT reagent. Solubilization solution (Promega) was added after four hours of incubation and the plates were mixed on an orbital stirrer for 24 hours. The absorbances of each of the wells were then measured in duplicate in a 96-well plate using a multi-well plate reader (Molecular Devices) operating at 570 nm. Results are baseline-corrected to eliminate the impact of media absorbance and are normalized relative to the cell-only results.

The addition of microgels to cell media had a significant impact on the cytotoxicity response of myotubes in the presence of bupivacaine, as shown in FIG. 5. FIG. 5 shows cell viability (MTT assay, relative to cell-only control) as a function of bupivacaine concentration and AA-6S microgel microgel concentration after one day cell exposure. In the absence of microgels, myotube cultures become completely non-viable when ~0.5 mg/mL bupivacaine is added to the cell media. However, the addition of as little as 0.18 mg/mL microgel results in approximately 70% cell viability in the presence of 0.5 mg/mL bupivacaine, while close to 100% cell viability is maintained at the same bupivacaine concentration when the microgel concentration is increased to 0.91 mg/mL. This protective effect of microgels against bupivacaine-induced cytotoxicity was confirmed using live/dead fluorescence staining. FIG. 6 shows both optical microscope and fluorescence live/dead imaging results for C2C12 myotubes exposed to different concentrations of bupivacaine in the presence and absence of 0.91 mg/mL AA-6M microgel after one day of incubation. In the absence of microgel, loss of confluence and cell viability is first significantly observed at 0.36 mg/mL, with complete loss of viability observed at 0.73 mg/mL. This is consistent with the MTT result shown in FIG. 5. However, in the presence of the microgel, no significant viability loss is observed at 0.36 mg/mL bupivacaine and significant viability is maintained even in the presence of 0.73 mg/mL bupivacaine, although the reduced confluence of the cell layer and more rounded shape of many "live" cells under this condition suggests that the live cells are under stress under the higher bupivacaine concentrations. Hence, the ability of microgels to protect myotubes from bupivacaine-induced toxicity is confirmed via both metabolic and cell viability assays.

EXAMPLE 3 This example demonstrates the effect of functional group concentration. FIG. 3 shows the cytotoxicity of bupivacaine in the presence of various concentrations of AA-20S microgel containing 20% acrylic acid groups (MTT assay, relative to cell-only control) as a function of bupivacaine concentration and microgel concentration after one day cell exposure. A comparison of FIG. 7 (20 mol% acrylic acid) and FIG. 5 (6 mol% acrylic acid) shows that increasing the acidic functional group content of the microgel significantly increases the capacity of the microgels to bind bupivacaine and maintain cell viability at high bupivacaine concentrations. For example, in the presence of 0.72 mg/mL bupivacaine, the addition of 0.91 mg/mL AA-6 fails to preserve any cell viability after one day of bupivacaine exposure while the addition of 0.91 mg/mL AA-20 preserves approximately 60% cell viability over the same time period. This increase in viability is likely related to the increased affinity of the more highly acid-functionalized microgel for bupivacaine, which is cationic at physiological pH. Increased microgel functionalization permits increased electrostatic binding between the anionic acid groups and the cationic bupivacaine, increasing the percentage of bupivacaine into the microgel phase and reducing the concentration of free bupivacaine in the cell media available to induce a cytotoxic response. It should be noted that high concentrations of 20 mol% acrylic acid-functionalized microgels induced some limited cytotoxicity at low bupivacaine concentrations, and a significant reduction in cell viability was observed even at non-toxic bupivacaine concentrations when >1.82 mg/mL AA-20S microgel is added to the cell suspension (p = 0.0006) under the specific conditions tested. This effect was confirmed visually by a noticeable change in the color (i.e. pH) of the cell media upon adding the microgel to the media. Thus, while microgels with higher acidities can bind more bupivacaine, under these specific conditions an upper limit existed on the microgel concentration and/or the acid fraction in the microgel in order to prevent a moderate cytotoxic response in the absence of bupivacaine. To this end, FIG. 19 shows the concentration dependence of 3T3 fibroblast, J.1774 macrophage, C2C12 myotube, and Me-T mesothelial cell viability (MTT assay, relative to cell-only control) upon the addition of different concentrations of AA-20S in the absence of bupivacaine one day cell exposure.

At microgel concentrations up to 0.5 mg/mL, none of the cell types screened exhibited a statistically significant reduction in viability in the presence of microgels, while only fibroblasts (p = 0.002) exhibited a significant reduction in cell viability at concentrations up to 1 mg/mL. However, at microgel concentrations greater than 2.5 mg/mL, all the cell types exhibited at least a minor cytotoxic response, with fibroblasts being most sensitive to the presence of the microgel.

EXAMPLE 4 This example demonstrates the effect of functional group type. FIG. 8 shows the cytotoxicity of bupivacaine on C2C12 myotubes (MTT assay, relative to non-treated controls) in the presence of microgels (0.91 mg/mL) functionalized with different mole percentages of N,N-dimethylaminoethylacrylate (DMAEA, cationic at pH 7.4) relative to microgels functionalized with acrylic acid (AA, anionic at pH 7.4) at different degrees of functionalization after one day cell exposure. Some cell-protective effect is observed upon the addition of cationic, DM AEA-functionalized microgels despite the electrostatic repulsion between the cationic DMAEA functional groups and cationic bupivacaine in physiological media. Bupivacaine loading into the microgels can be attributed to the affinity partitioning of bupivacaine into the microgel phase. The octanol-water partition coefficient logP is an indicator of the relative hydrophobicity of different chemicals; chemicals with similar logP values have similar hydrophobicities and thus have an affinity for each other in multi-phase systems. Cationic bupivacaine has a log/* value of 0.18, while a polymerized NIPAM residue has a log/* value of 0.06 indicating that bupivacaine has a high affinity for NIPAM residues. However, it should be noted that no significant change in partitioning was observed as the DMAEA concentration in the microgel increases from 6mol% to 20mol% under the conditions tested. This was anticipated given that there is no specific affinity between the DMAEA residues and bupivacaine.

In this example, acrylic acid-functionalized microgels exhibited higher bupivacaine binding than DMAEA-functionalized microgels at all degrees of functionalization tested and showed significant increases in bupivacaine binding as the degree of acrylic acid functionalization increased. Thus, both hydrophobic partitioning and electrostatic interactions play significant roles in facilitating drug loading into microgels and protecting cells from bupivacaine-induced toxicity.

EXAMPLE 5 This example demonstrates the effect of microgel size. FIG. 9 shows the cytotoxicity of bupivacaine to C2C12 myoblasts in the presence of 20 mol% acrylic acid microgels (0.91mg/mL) of different sizes after one day cell exposure (MTT assay, relative to non-treated controls). No significant difference was observed between the cell viability maintained by 20 mol% functionalized microgels of large (~815 run), medium (~345 nm), and small (~170 nm) sizes. Thus, the surface area of the microgels had no significant impact on the capacity of the microgels for drug binding, at least over the one day interval used for testing in this example. The same result was observed for 6 mol% functionalized microgels.

EXAMPLE 6 This example demonstrates the effect of functional group distributions. Previously, in vitro drug binding studies suggested that FIG. 10 shows cell viability (MTT assay, relative to non-treated controls) of C2C12 myotubes in the presence of 6 mol% acrylic acid microgels (0.45 mg/mL microgel) containing different functional group distributions after one day cell exposure and indicates microgels with functional groups localized in the core of the microgel have improved drug binding properties as compared to microgels with functional groups localized on the surface. Microgels prepared using methacrylic acid (MAA), in which functional groups are localized primarily in the microgel core, bound the greatest amount of drug, followed closely by acrylic acid (AA)-functionalized microgels which had functional groups distributed evenly throughout the gel network. By contrast, microgels functionalized with vinylacetic acid (VAA, in which functional groups are isolated on microgel surface) bound significantly less bupivacaine, whereas microgels functionalized with fumaric acid (FA, in which functional groups are paired together on the microgel surface) bound the least bupivacaine. However, the effect of changing the functional group distribution appeared to be minor compared to the effect of changing the number of functional groups in the microgel under these conditions. EXAMPLE 7 This example demonstrates the lack of significant cytotoxicity by microgels. FIG. 1 1 shows the cytotoxicity of microgels (MTT assay, relative to cell-only control) with different functional groups, different functional group concentrations, different sizes, and different functional group distributions (2mg/mL microgel concentration) after one day cell exposure. All of the 6 mol% functionalized microgels tested exhibited no significant cytotoxicity toward of macrophages, myotubes, and mesothelial cells regardless of the functional group present and the distribution of those functional groups. When the degree of functionalization was increased to 20%, only the small microgels induced no significant cytotoxic response; the larger microgels were significantly more cytotoxic than the smaller microgels, particularly toward myotubes.

EXAMPLE 8

This example demonstrates peritoneal injection of microgels. Saline-suspended microgels (1 mL total volume) were injected into the peritoneal cavity of SVl 29 mice of mass 30 g at doses up to 1250 mg/kg (n = 8). Mice were anesthetized with isoflurane prior to injection, with the outside skin sterilized with an isopropanol rub. No mortality or behavioral impairment was observed in any of the mice 1 hour, 2 hours, 6 hours, 1 day, or 3 days post- injection, suggesting that the microgels were biocompatible inside the peritoneum. Dissections after 1 day indicated the presence of some residual microgel which remained in suspension, while dissections after 3 days indicated no obvious microgel residue in any of the mice (n=4).

EXAMPLE 9

This example demonstrates cell biocompatibility of microgels. Microgel biocompatibility was assayed using 3T3 mouse fibroblasts, Jl 774 macrophage-like cells, and C2C12 mouse myoblasts in cell culture. Results are shown in FIG. 12 after 1 day and 4 days of cell incubation in the presence of 2 mg/mL microgel suspended cell media.

Overall, microgels had only minimal impacts on cell viability, even at the relatively high concentrations used in this assay, and no significant change in biocompatibility was observed upon longer exposure times. Microgels with lower degrees of acid functionalization maintained higher cell viability than microgels with higher degrees of acid functionalization. For example, the cell viability in the presence of AA-6L was significantly higher than that of AA-20L for fibroblasts (p = 0.02), macrophages (p = 0.006), and myoblasts (p = 0.0004) despite the similar particle size of both these microgels. Microgels functionalized to the same extent with the basic co-monomer DMAEA (DMAEA-20) exhibited good biocompatibility with each cell type, with no significant loss of cell viability upon microgel addition. Particle size may have an impact on the microgel biocompatibility for certain cell types if the acid content of the microgel is sufficiently high. While AA-6L and AA-6S do not exhibit significantly different biocompatibilities with any cell type (p > 0.06), AA-20L significantly reduced the viability of myoblasts compared to AA-20S (p = 0.0008) despite the equivalent number of -COOH groups contained in both microgels. However, neither macrophages (p = 0.41) nor fibroblasts (p = 0.06) showed a significant sensitivity to particle size even with the AA-20 microgels. Thus, the acid content more strongly influenced the cell biocompatibility than the particle size.

EXAMPLE 10 This example demonstrates the tissue response to sciatic nerve injection of microgels. Young adult male Sprague-Daley rats with weights ranging between 350-450 g were purchased from Charles River Laboratories (Wilmington, MA) and housed in pairs using a 7AM-7PM light-dark cycle. Animals were cared for in compliance with protocols approved by the Animal Care and Use Committee at the Massachusetts Institute of Technology. NIH guidelines for the care and use of laboratory animals (NIH Publication #85-23 Rev. 1985) have been observed. Each rat was injected only once to ensure the blockade effect was attributable exclusively to the tested formulation.

Rats were briefly (< 2 minutes) anesthetized with isofluorane in 100% oxygen prior to injection. A 25G needle was introduced postero-medial to the greater trochanter, pointing in an anteromedial direction. Upon contact with the bone, 0.3 mL of a microgel suspended in a bupivacaine solution was injected into the left leg of the rat. Microgel concentrations ranging from 20-160 mg/mL and bupivacaine concentrations ranging from 5-15 mg/mL were evaluated, all prepared in sterile saline. The right leg was used as a control. Both sensory and motor block were evaluated at half hour time points by a blinded experimenter for the first 1.5 hours of the block and 45 minute time points for the remainder of the block. Two data points are also obtained after the completion of the nerve block, one on the next scheduled time point of the test and the other 24 hours post- injection, to ensure complete recovery of normal neurological function. Sensory blocks were evaluated using a modified hotplate test, while motor block was assessed by measuring the weight each animal could bear on a single leg.

FIGs. 13 A, 13B, and 13C shows the tissue response to the injection of non bupivacaine- loaded microgels of different sizes (AA-6L, 840 nm; AA-6M, 265 nm; AA-6S, 100 nm; respectively) at a rat sciatic nerve after four days of implantation. In FIGs. 13A-13C, the arrows point to the residual microgel deposit/inflammation complex. The inset to FIG. 13 A is the cross-section of the inflammation capsule shown in FIG. 13 A.

Injection of the large, 840 nm microgels induced a large inflammation response resulting in the formation of a thick inflammatory capsule (FIG. 13A inset) around a milky residual microgel suspension. Significant tissue matting was also observed, with score 3 adhesions noted between the inflammation complex and the surrounding tissue and significant local bleeding observed upon dissection due to increased vascularization around the inflammatory complex. In contrast, when the microgel size is reduced to 265nm, a solid gel- like deposit is recovered after four days. Considering that a syringe filled with AA-6M microgel undergoes reverse thermal gelation at 30 °C, this deposit likely consists of a mix of thermally-gelled microgels together with immune cells. Score 1 adhesions were observed between the gel deposit and the surrounding tissues, and no significant bleeding occurred upon dissection. When the microgel size was reduced to 100 nm, no microgel deposit or inflammatory response whatsoever was observed after four days. No adhesions or tissue matting were observed between the injection site and the surrounding tissues, with the tissues appearing similar to those of pristine nerves. Hence, by changing the particle size of microgels with the same total acid content, significantly different immune responses are induced. A similar trend was observed with the AA-20 microgels, as shown in FIG. 14. FIG. 14 shows the tissue response to a sciatic nerve injection of AA-20L (815 nm) and

AA-20S (170 nm) microgels after four and fourteen days of implantation. The arrows point to the residual microgel deposit/inflammation complex. AA-20S underwent reverse thermal gelation at ~30°C whereas the large AA-20L microgel did not undergo thermal gelation until above physiological temperature. Consequently, the large AA-20L microgels remained as a suspension and induced a significant immune response, forming a thick capsule around the suspended gel after 4 days. After 14 days, the inflammatory complex persists but the liquid microgel suspension observed inside the complex after four days is converted to a solid, spongy inner core, a condition which persists one month post-injection. Again, the area around the injection was highly vascularized and matted, with score 2-3 adhesions consistently observed. In contrast, the smaller AA-20S microgels underwent a thermal phase transition to precipitate on the nerve and create a small, hydrogel-like deposit which initiated only a minimal immune response after 4 days and was completely resorbed in all rats dissected after 2 weeks. No significant adhesions occurring at either time point. An identical trend was observed for the AA-33L and AA-33S microgels. Thus, the immune response of a microgel injection appears to be determined by the size of the injected microgel and the tendency of that microgel to undergo reverse thermal gelation when injected in vivo; thermally-gelling microgels induced significantly lower immune responses than non-thermally gelling microgels.

Injection of the DMAEA-functionalized microgels (particle sizes 145-245 nm at 37 ⁰ C) yielded similar results to the small AA-20 and AA-6 microgels, with zero adhesions, zero residual microgel, and no significant immune response or adhesions observerable four days post- injection. Correspondingly, all DMAEA-functionalized microgels tested underwent reverse thermal gelation at physiological temperature. Indeed, DMAEA microgel suspensions of concentrations of 80 mg/mL or greater gelled in the syringe if the syringe was held in the experimenter's hand for more than ~30s.

EXAMPLE 11

This example demonstrates the duration of nerve block. FIG. 15 shows the duration of effective nerve blocks, as measured via latency and motor neurological testing, for different microgels injected as 80 mg/mL suspensions in a 5 mg/mL bupivacaine solution. Microgels prepared with cationic monomers carrying the same charge as bupivacaine (7% DMAEA) achieved a nerve block duration which was not significantly different from a 5 mg/mL bupivacaine solution injected in the absence of microgels (p — 0.14). Microgels prepared with acidic monomers significantly increased the duration of block according to the acid content in the microgel. AA-6 microgels of all sizes achieved significantly higher durations of nerve block than bupivacaine-only injections (p = 0.005 for AA-6L, p = 0.0007 for AA-6M, and p = 0.001 for AA-6S) but significantly lower durations of block than AA-20 microgels (p = 0.002 for pair-wise comparisons between each of AA-20L and AA-6L, AA-20M and AA-6M, and AA-20S and AA-6S). Hence, the anionic charge of the microgels effectively prolonged the duration of anesthetic release, and the presence of more acidic groups promoted more and stronger interactions between the acidic microgel and the basic bupivacaine, retarding drug release. For the AA-20 microgels with higher acid contents, smaller microgels also prolonged the duration of block longer than larger microgels (p = 0.03 comparing AA-20L and AA-20S). This result was contradictory to the general expectation in particle-based drug delivery, given that smaller microgels have higher surface area-to-volume ratios and thus would be expected to release anesthetic faster than larger gels. Without wishing to be bound by any theory, this observation can be rationalized based on the ability of the small microgels to thermally gel upon injection to form a bulk, ion-exchanging hydrogel deposit on the sciatic nerve. In comparison, the large microgels remain in suspension at physiological temperature. Thus, the effective in vivo surface area for drug release was larger with the larger, more colloidally stable microgels, resulting in a faster overall drug release rate.

Contrary to the above trends, FIG. 15 indicates AA-33S facilitated a significantly lower duration of nerve block than AA-33L (p = 0.002) despite thermally gelling in vivo like the AA- 2OS microgel. It is hypothesized that this observation was related to the higher acid content (and thus high affinity for bupivacaine binding) of the AA-33 microgels which may have retarded the drug release to the point that the release rate was below the clinically-effective dose of bupivacaine. To test this hyphothesis, nerve block experiments were performed by loading different concentrations of bupivacaine into the microgels and assaying the effective increase in the duration of nerve block achieved relative to the injection of that same concentration of bupivacaine in the absence of microgels. FIG. 16 shows latency nerve block data for high concentration bupivacaine solutions in the presence and absence of 80 mg/mL AA-6S and AA-33S microgels.In the absence of microgels, injecting higher concentrations of bupivacaine increased the duration of nerve block, although non-linear increases in latency were observed. Co-injection of AA-6S prolonged the duration of nerve block by -80 minutes using 5 mg/mL bupivacaine solutions, an enhancement in nerve block duration which remained unchanged as the BPV concentration was increased. This observation suggests that the AA-6S microgel had only a minimal effect on retarding anesthetic release, consistent with the relatively smaller affinity between these low acid-functionalized microgels and bupivacaine. In contrast, co-injection of AA-33S exhibited only a -1.5 hour prolongation of nerve blockade in the presence of 5 mg/mL BPV but a -4.5 hour prolongation in nerve blockade in the presence of 15 mg/mL BPV. This result was consistent with the release rate hypothesis outlined above. The AA-33S microgel had a large number of ionic binding sites for bupivacaine, and release of non-bound bupivacaine was significantly electrostatically and electroosmotically retarded by free anionic charges in the microgel network. As a result, smaller loadings of bupivacaine were retained strongly by the gel, resulting in an extremely slow release rate and a relatively short window during which a clinically effective dose of anesthetic diffuses from the microgel. This accounted for the lower duration of nerve block of AA-33S compared to AA-20S in the presence of 5 mg/mL bupivacaine. As the amount of bupivacaine in the formulation was increased, more bupivacaine binding sites were consumed, fewer free charges persisted in the microgel network, and more free bupivacaine was present in the microgel network. Thus, although the microgel network offered the same physical resistance to diffusion, a clinically- effective dose of bupivacaine was released from the microgel over a significantly longer time, accounting for the large prolongation of the release observed at higher bupivacaine loadings. The effect of microgel concentration on the duration of effective (latency) nerve block as a function of microgel concentration and microgel acid content using 5 mg/mL bupivacaine solutions is shown in FIG. 17. At low concentrations, each microgel facilitates a longer duration of nerve block when more microgel is added, as expected given the higher number of BPV binding sites and higher volumetric gel capacity for drug partitioning. However, at higher microgel concentrations, each of the microgels showed a different correlation between duration of effective block (DEB) and the microgel concentration. For AA-33S, while both the 40 mg/mL and 80 mg/mL suspensions thermally gelled in vivo, the 40 mg/mL microgel suspension resulted in a longer block than the 80 mg/mL microgel suspension. This is consistent with the release rate hypothesis outlined previously, in which the number of BPV binding sites in highly acid-functionalized microgels becomes so large at higher microgel concentrations that the drug release rate is retarded to below a physiologically-relevant level. The AA-20L microgel exhibited similar behavior, showing a linear increase of block duration up to 80 mg/mL microgel (R ² = 0.92) and then a decrease in DEB at higher microgel concentrations. In parallel, AA-20L microgel concentrations of 80 mg/mL and less remained as suspensions in vivo while concentrations of 120 mg/mL or higher thermally gelled. As a result, the decreased block duration observed at higher concentrations of AA-20L can be attributed to both the increase in BPV binding sites at higher microgel concentrations and the formation of a bulk, self-assembled hydrogel which significantly reduces both the surface area available for diffusion and the water content (i.e. pore size) within the individual microgels upon particle aggregation. Both these effects significantly slowed bupivacaine diffusion from these microgels, shortening the amount of time during which a clinically effective dose was delivered. The duration of block for the AA-20S microgel, which thermally gelled in vivo at all microgel concentrations tested, varied linearly with the concentration of microgel used in the formulation (R ² = 0.98) up to 160 mg/mL microgel.

Rats were sacrificed one and four days post-injection. The sciatic nerve was removed together with surrounding tissues by a blinded dissector and placed immediately in Accustain formalin-free fixative. The nerve was sectioned and stained with hematoxylin-eosin to prepare histology slides using standard techniques. Slides were analyzed by an observer blinded to the nature of the material injected into the animal being observed.

Cytotoxicity data are presented as means with standard deviations. The durations of nerve blockade from in vivo neurobehavioral testing are expressed as means with standard deviations. Comparisons between sensory and motor blocks for a given group are made using a paired t-test while those between different groups are made using unpaired t-tests. Linear regressions and t-tests are performed using Microsoft Excel.

Dissected sciatic nerve sections exposed to various microgels for periods of time ranging from four days to one month were subjected to histological analysis. FIG. 18 shows representative histologies (4 days post-injection)for sciatic nerve and surrounding smooth muscle tissue around the site of a sciatic nerve injection of 80mg/mL of AA-20S microgel loaded with 5mg/mL bupivacaine. No significant cell toxicity was observed in either nerve tissue or smooth muscle tissue upon exposure to even a high (8 wt%) microgel concentration. Muscle cells remained polygonal with multiple peripheral nuclei. Large numbers of macrophages with a smaller number of neutrophils populated the small quantity of residual microgel remaining at the injection site 4 days post-injection, with a collagen barrier separating the muscle cells and the residual gel material.

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one." The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of, when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 211 1.03.

What is claimed is: