HK1203367B - Medical organogel processes and compositions - Google Patents

Description

Medical organogel methods and compositions

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Serial No. 61/566,768, filed December 5, 2011, which is hereby incorporated by reference herein.

Technical Field

The technical field relates generally to the controlled release of drugs and includes the delivery of proteins from small particles.

Background Art

Therapeutic agents require an effective delivery method. Drug delivery involves administering a pharmaceutical compound to a human or animal to achieve a therapeutic effect. It is useful to provide a delivery mechanism that releases the agent over time. To improve product efficacy and safety, as well as patient convenience and compliance benefits, drug delivery technologies can help alter drug release profiles, absorption, distribution, or drug elimination.

Summary of the Invention

Despite extensive research in these areas, the effectiveness and success of therapeutics using biological agents remains quite limited due to the poor stability of biological agents, including proteins, in the body. Despite common knowledge that proteins should not be exposed to organic solvents in pharmaceutical processing techniques, it has been observed that many solvents can be used. Methods of using such solvents are described, including embodiments for a dual solvent delivery system, wherein the first solvent is an organic solvent in processing and the second solvent is a physiological fluid in the body.

One embodiment of the present invention is a xerogel comprising a protein powder or other water-soluble biological agent (biologic) powder dispersed within a matrix of the xerogel. The xerogel can be hydrated at the point of use and placed into tissue, where it controllably releases the protein over time. This embodiment, among others, is described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A depicts the formation of biomaterials;

FIG1B depicts the microstructure of the biomaterial of FIG1A ;

FIG1C depicts the microstructure of an alternative embodiment of a biomaterial;

FIG2A is a graph showing HPLC data of the release of ovalbumin in physiological solution at 37° C. over time;

FIG2B is a graph of the data of FIG2A normalized to protein levels at complete dissolution of the hydrogel;

Figure 3 is a graph showing HPLC data for the release of ovalbumin over time in physiological solution at pH 8.5 and 37° C. and in physiological solution at pH 7.4 and 37° C. The data are normalized to the protein level at complete dissolution;

FIG4 is a graph showing HPLC data of IgG release over time in physiological solution at 37° C.;

FIG5 is a graph of the data of FIG4 normalized to protein levels at complete dissolution of the hydrogel;

FIG6 is a graph depicting the calculated release profile of albumin from a combination of hydrogel vehicles;

FIG7 is a graph depicting the calculated release profile of albumin from a combination of hydrogel excipients;

FIG8 is a diagram of various sites at or near the eye for applying biomaterials;

FIG9A is a diagram of a method for placing a biomaterial in an eye and depicts the process of inserting a needle into the eye; and

FIG. 9B depicts various examples of sites in the eye of FIG. 9A for receiving biomaterial.

DETAILED DESCRIPTION

Embodiments of the present invention are xerogels comprising protein powder or other water-soluble biological reagent powders dispersed in the matrix of the xerogel. The xerogel can be hydrated and placed in tissue when about to be used, where it controllably releases the protein over time. The powder contains fine particles of protein. The xerogel matrix is a hydrogel made from a cross-linked matrix when hydrated. The protein is in solid phase and is essentially insoluble until the matrix begins to erode (erode), thereby allowing the protein to enter the solution. The matrix protects the protein from the influence of cells, enzymatic denaturation, and unwanted local reactions. The protein is in solid phase essentially until released by gradual solvation (dissolution), and is therefore protected from the influence of denaturation, autohydrolysis, proteolysis, and local chemical reactions, which can result in loss of effectiveness or produce antigenicity.

FIG1A depicts an embodiment of the method, which begins with protein particles 100, which are prepared by conventional means to preserve the secondary structure of the protein and, if present, the tertiary or quaternary structure. These are combined with precursors 102, 104 in an organic solvent 106. The mixture is processed to achieve the desired shape of the biomaterial, for example by casting 108, as rods 110, as particles and/or spheres 112, and molded into a shape 114. The solvent is stripped from the shape and the material forms a hydrogel when exposed to water. The entire process up to the point of actual use of the xerogel for a patient can be carried out in the absence of water and/or in the absence of a hydrophobic material. FIG1B depicts the microstructure of a biomaterial 120 produced by this method. The structure represents the materials across its manufacturing and use processes: organogel, xerogel, and then hydrogel. A cross-linked matrix is made from precursors 124 that have covalently reacted with each other. Particles 124 of a water-soluble biological agent are dispersed within the matrix. The matrix is the continuous phase, and the particles are spread out within it and are the discontinuous phase, also called the dispersed phase.

An alternative embodiment involves the use of a block copolymer precursor that is physically cross-linked by the formation of hydrophobic domains (domains), as depicted in Figure 1C. Biomaterial 130 has bioreagent particles 132 dispersed in a matrix. The precursor has a hydrophilic block 134 and a hydrophobic block 136. The hydrophobic blocks 136 self-assemble to form hydrophobic domains 138, which produce physical crosslinks between the precursors. The term physical crosslinking refers to non-covalent crosslinking. The hydrophobic domain is one such example, as well as the hard and soft segments of polyurethane or other multi-block copolymers. Ionic crosslinking is another example. The term crosslinking is fully understood by the skilled person, who will immediately be able to distinguish covalent crosslinking from physical crosslinking, as well as subtypes of physical crosslinking such as ionic, hydrophobic, and crystalline domains.

Other drug delivery approaches have used, for example, liposomes or micelles to encapsulate proteins, or to create nanoparticles using polymers or other agents in their production. Protein delivery in hydrogels typically involves isolating the protein from the hydrogel: for example, by placing the hydrogel in liposomes, micelles, or in a mixture with a binder such as a polymer. Other approaches involve adsorbing materials directly onto the proteins to inhibit their dissolution. Another approach is to precipitate the protein during delivery, as disclosed in U.S. Publication No. 2008/0187568. Other approaches use hydrogels in which soluble proteins are dispersed throughout the hydrogel, with the hydrogel being subjected to erosion-controlled release.

Despite all these efforts, the effectiveness and success of sustained-release therapies using biological agents, including proteins, remains limited because the stability of such agents in vivo tends to be poor. And loss of conformation can not only lead to loss of efficacy, but it can also be detrimental by causing unwanted effects or eliciting an immune response. Despite numerous efforts, there are no generally applicable solutions that are effective enough to have real-world clinical value, as reviewed in Wu and Jin, AAPS PhamSciTech 9(4):1218-1229 (2008).

However, surprisingly, the embodiments provided herein show that the solubility of proteins or other biological agents and the release from the matrix can be controlled by placing the biological agent as solid phase particles in a suitable matrix so that these other approaches involving polymers, encapsulating agents, binders, etc. are not required. In addition, the biological agent resists denaturation even in an aqueous in vivo environment. The particles in the matrix are water-soluble, but, despite not having any coatings, still dissolve slowly, and their dissolution in physiological fluids (which is usually measured in minutes or hours) can be extended to several days, weeks, or months. Moreover, another unexpected and surprising result has been observed: that is, the biological agents do not tend to aggregate, even if they must be present in the matrix at very high concentrations. It appears that the biological agent leaves the particles very slowly. The first operating principle (to which the present invention is not limited) is that the molecular chains (molecular strands) of a matrix made of a highly mobile polymer—for example, a polymer such as polyethylene glycol (PEG) or polyethyleneimine—form an exclusion volume around themselves that limits the solubility of any other macromolecules in the immediate vicinity. This structural property not only confines the protein to the solid phase by physical entrapment within the matrix, but also restricts the dissolution of the macromolecules, preventing the protein particles from moving into solution. When the particles and protein begin to swell through solvation with water, they are confined by the matrix until the matrix is at least partially dissolved. Thus, as the crosslink density decreases and the molecular chains move further apart, the gradual dissolution of the entrapped macromolecular particles is promoted. These processes thus provide an unexpected and surprising result: the biological agent remains in the solid phase until it is close to the time of its release from the matrix. As a result, the protein or other biological agent is stable because it does not suffer the deleterious effects of prolonged solubility. Release is also limited by the diffusion of the macromolecule out of the matrix and is influenced by the molecular weight of the macromolecule and the properties of the polymer forming the matrix. The second working principle is complementary to the first working principle and is also not a mechanism to which the present invention is limited: the molecular chains of the matrix associate with water molecules near the protein, making the protein insoluble. This second principle is applicable to polymers with highly mobile, hydrophilic straight chains, such as PEG. In addition to PEG, other water-soluble polymers or copolymers that exhibit an exclusion volume effect with the selected protein can also be selected. For example, polymers such as polyacrylic acid, polyvinyl alcohol, polyvinyl pyrrolidone (PVP) and polyhydroxyethyl methacrylate (PHEMA) will generally have such an effect. Some polysaccharides also have these effects. PEG and/or these other polymers can also be introduced into the organogel as solids. They will dissolve (solubilize) in the presence of water, i.e., in the hydrogel. Moreover, non-crosslinked PEG and/or PEG copolymers, such as PLURONIC, are additives that can be trapped in the hydrogel together with the protein to enhance the exclusion volume effect, thereby maintaining the protein in a solid state.

Aspects of the systems disclosed herein relate to significant improvements in the control of release over time by placing protein particles in a hydratable xerogel. Examples 1-2 detail methods for forming xerogels containing particles of water-soluble biological agents. The proteins albumin and immunoglobulin (IgG) were used as models for water-soluble therapeutic proteins. Powders of these proteins were prepared. The powder particles were combined with a hydrogel precursor in an organic solvent to form an organogel. Tables 1-5 of Example 1 set forth examples of organogels comprising dispersed protein powders. The organogels were crushed and sieved into collections of particles, which were evacuated of the organic solvent to form a xerogel. Working Example 2 documents the release of proteins from the xerogel.

As illustrated in Figures 2-5, the proteins were fully (completely) released; surprisingly, there was no detectable reaction of the organogel precursor with the proteins, which would prevent them from being dissolved as the matrix degraded. Indeed, these proteins, and proteins in general, contain amine and thiol functional groups that are potentially very reactive toward strong electrophiles such as the electrophilic precursors used. Although reactions with these electrophilic functional groups would be expected, the lack of reaction suggests that these reactions were prevented by keeping the proteins in an undissolved or essentially solid phase prior to gelation, while the gel-forming precursors were in a liquid phase. The release profiles showed good control over the release rate, ranging from rapid release over a few hours to release over several months.

Moreover, the rate and kinetics of release can be further controlled by combining multiple groups of particles with each other, as illustrated in Figures 6 and 7. These demonstrate essentially zero-order release, which is the ability to deliver a drug at a rate that is independent of time and at a desired concentration within the dosage form. A zero-order release mechanism ensures that a steady amount of drug is released over time, minimizing potential peak/trough fluctuations and side effects, while maximizing the amount of time that drug concentrations remain within the therapeutic window (efficacy).

Methods and materials for preparing organogel-hydrogel, dual-solvent delivery systems for water-soluble biological agents

A first embodiment involves forming a covalently cross-linked matrix. A fine powder of a water-soluble biological agent is prepared and suspended in an organic solvent that does not solvate the water-soluble biological agent, such as a protein. The term powder is used broadly herein to refer to a collection of dry particles. The term particle is broad and includes spheres, teardrop shapes, small rods, and other irregular shapes. Typically, the powder is processed to provide a controlled particle composition with a known size, shape, and distribution (difference from the average or mean). Protein powders typically contain stabilizing sugars such as sucrose or trehalose. These sugars are generally water-soluble and not organically soluble. It has been found that throughout the process, these sugars will remain with the protein until hydration to form a hydrogel. Matrix precursors are prepared that have the ability to form a cross-linked organogel by reacting with each other in an organic solvent. The precursors are selected to be soluble in the organic solvent. The precursors and water-soluble biological agent powder are mixed in the organic solvent so that the water-soluble biological agent particles are dispersed throughout the matrix formed when covalent bonds are formed between the precursors. The matrix formed in the organic solvent is called an organogel. The solvent is removed to form a xerogel. When hydrated in water, the matrix forms an internally covalently cross-linked hydrogel. This method is a serial two-solvent method because the organic solvent must be effective and removable (i.e., removable without leaving pharmaceutically unacceptable residues) for the biological agent and precursor, but the precursor must be effective in the aqueous environment in vivo. The protein is never exposed to both the organic phase and the aqueous phase. It is believed that exposure of proteins in aqueous solution to interfaces such as with organic liquids or solids or air bubbles contributes to protein adsorption and denaturation. The sequential method of organogel to xerogel to hydrogel eliminates the possibility of interfacial exposure, that is, embodiments include methods as described herein performed without exposing the water-soluble biological agent to an interface between any combination of: air, gas, water, organic solvent.

Another embodiment is to form a covalently cross-linked gel (also referred to herein as a pseudo-organogel) by employing a liquid reactive polymer as a matrix precursor. A matrix precursor is prepared that has the ability to form a cross-linked organogel by reacting with each other in the absence of an organic solvent, for example, when in a molten state. The precursor and a water-soluble bioagent powder are mixed at a temperature high enough to liquefy the precursor polymer, but low enough to maintain protein stability. Examples of such temperatures are from about 10°C to about 75°C, or up to about 60°C, or up to about 75°C; those skilled in the art will immediately understand that all values and ranges between the explicitly stated values are contemplated and incorporated herein as if fully set forth. Mixing conditions are employed such that the water-soluble bioagent particles are dispersed throughout the matrix formed when covalent bonds are formed between the precursors. The reaction is thus carried out in a melt of the polymer, the term melt meaning the absence of solvent. However, other materials, such as bioagents, sugars, proteins, buffers, may be present in the melt. Embodiments include materials and methods of making medical materials comprising forming a gel around a powder of a water-soluble biological agent, wherein the powder is dispersed in the gel, wherein forming the gel comprises preparing a melt of one or more precursors and covalently crosslinking the precursors. A majority of the volume of the gel, e.g., about 30% to about 95% v/v, can be occupied by the biological agent or other solids (e.g., sugars, buffer salts); the skilled artisan will immediately understand that all values and ranges between the explicitly stated values are contemplated and incorporated herein as if specifically written, e.g., at least 30% v/v or about 40% to about 75%.

Another embodiment of the sequential dual-solvent method involves forming a cross-linked material with physical crosslinks. One such embodiment uses a block copolymer as a precursor. The precursor has a lyophilic (solvent-loving) block and a lyophobic (solvent-hating) block. These precursors are added to an organic solvent and form a physically cross-linked matrix. The blocks (also called segments) that precipitate in a particular organic solvent to form the organogel may or may not be the same segments that precipitate in water to form the hydrogel. After removing the solvent, the resulting xerogel forms a hydrogel in aqueous solution because one or more blocks or segments are hydrophobic and one or more blocks or segments are hydrophilic. A related embodiment uses two organic solvents: a block copolymer precursor is dissolved in a first organic solvent. The copolymer solution is then mixed with a second organic solvent that is miscible with the first solvent but is a non-solvent for at least one of the copolymer's segments. The lyophobic domains form the organogel. Another embodiment uses the first and second organic solvents and also uses the second organic solvent to precipitate the biological agent, so that the organogel and biological agent particles are formed in the same step.

Another embodiment of the sequential two-solvent method involves thermal gelation. A precursor that transforms from a solution to an organogel in an organic solvent at a temperature ranging from about -20°C to about 70°C is placed in the organic solvent along with a biological agent at a temperature at which the precursor is dissolved. The solution is then cooled to a second temperature below the gelation point, and the precursor forms an organogel. Thus, the method for producing the organogel involves heating the solvent to dissolve the copolymer, then cooling the solution to precipitate at least one of the copolymer segments. The solvent is then removed to produce a xerogel. The precursor is selected so that the xerogel is a hydrogel at physiological temperatures.

Suitable block copolymers for use in these methods include many PEG copolymers. PEG is hydrophilic and lyophilic for many organic solvents. Other hydrophilic polymers and polymer-type segments are polyvinyl alcohol, polyacrylic acid, polymaleic anhydride, PVP, PHEMA, polysaccharides, polyethyleneimine, polyvinylamine, polyacrylamide, etc. Other blocks are selected to be hydrophobic and lyophobic for organic solvents. Examples of these other blocks are: polybutylene terephthalate (PBT), polylactic acid, polyglycolic acid, polytrimethylene carbonate, polydioxanone, and polyalkyl ethers such as polypropylene oxide (PLURONICS, POLOXAMERS). The copolymer can have one or more of each block type.

These methods can be performed so that the water-soluble biological agent never contacts water from the time it is initially prepared until it is placed in the body. The water-soluble biological agent can be further processed so that once it is obtained in a purified form at a source or manufacturing location, it is then insoluble in water and/or never exposed to water during the gel manufacturing process. Exposure to water can lead to various problems. One problem is that the protein will undergo hydrolysis over time, causing it to slowly degrade. Another problem is that the protein, once it is in a dissolved state, can rearrange or form quasi-stable aggregates such as dimers or trimers.

Embodiments of the present invention include these methods performed in the absence of a hydrophobic polymer and/or a hydrophobic solvent. Embodiments requiring a hydrophobic block polymer cannot be performed in a hydrophobic-free method, but a skilled artisan can readily discern which methods are applicable. One embodiment provides for covalent crosslinking of hydrophilic precursors in an organic solvent in the presence of bioreagent particles and in the absence of a hydrophobic material at both the organogel step and subsequent steps. In some embodiments, a hydrophobic solvent may be present without detriment, depending on the solvent, and thus embodiments include the absence of a hydrophobic material other than the solvent; and/or the absence of a hydrophobic polymer; and/or the absence of a hydrophobic polymer segment.

Common sense teaches that organic solvents generally denature proteins. Some life science processes can tolerate a certain degree of denaturation, for example, in diagnostic or analytical devices. However, in the medical field, even a small degree of denaturation is undesirable. Denatured proteins can exhibit a wide range of properties, from loss of solubility to communal aggregation. Communal aggregation involves the aggregation of hydrophobic proteins closer to each other to reduce the total area exposed to water. The reduction in distance can lead to permanent or quasi-stable associations. When a protein is denatured, its secondary and tertiary structures change, but the peptide bonds of the primary structure between amino acids generally remain intact.

Surprisingly, however, it has been found that proteins remaining in the solid phase can be exposed to some organic solvents without substantial denaturation. Completely anhydrous organic solvents processed under anhydrous conditions are preferred. Denaturation by exposure to organic solvents can occur when the protein is already in an aqueous solution and/or if the organic solvent or organic/aqueous mixed solvent (e.g., ethanol/water) has a tendency to dissolve or even swell the protein particles in a limited manner. Protein-solvent compatibility can be experimentally established by exposure followed by characterization tests to determine whether the protein has been denatured and/or has undergone replacement or change of one or more chemical groups. Organic solvent compatibility can be simply tested by immersing the test protein in the test solvent for an appropriate period of time, removing the protein, e.g., by filtration and vacuum drying, and then testing the protein for recovery by HPLC or other suitable analytical methods. The solvent that is most likely to leave the protein unharmed is anhydrous and hydrophobic, but must also be a good solvent for the precursor molecules that form the gel. In the case of polyethylene glycol (PEG) precursors, solvents such as dichloromethane and dimethyl carbonate have been employed. Other solvents such as acetone (or acetone/water), ethyl acetate, tetrahydrofuran may also be useful. Supercritical fluid solvents such as carbon dioxide may also be useful for forming organogels.

Precursors are described in detail elsewhere herein. Many useful precursors can be utilized as multiple precursors. A first precursor is added to a solvent-protein mixture, followed by a second precursor that is reactive with the first precursor to form a crosslink. The first precursor can be selected to have only those functional groups that are non-reactive for forming a covalent bond with the protein in the absence of further chemical components. Proteins have amines and thiols that can be used to react with some electrophilic functional groups to form a covalent bond, as well as carboxyls and hydroxyls that can be utilized for other chemical reactions. The precursor can therefore be selected to be non-reactive to these functional groups. For example, the precursor can have an amine and/or thiol and/or hydroxyl and/or carboxyl and be non-reactive to protein. Therefore, embodiments of the present invention relate to adding a protein-non-reactive first precursor to a protein-organic solvent mixture, followed by adding a second precursor that is reactive to the first precursor.

The water-soluble biological agent particles may be free of one or more of the following: binders, fatty acids, hydrophobic materials, surfactants, fats, phospholipids, oils, waxes, micelles, liposomes, and nanocapsules. The organogel or xerogel comprising the water-soluble biological agent particles may also be free of one or more of the foregoing. The protein or other water-soluble biological agent in the xerogel may be entirely in the solid phase and may be entirely crystalline, partially crystalline, or substantially free of crystals (meaning greater than 90% free of crystals, weight/weight; the skilled artisan will immediately understand that all ranges and values within the explicitly stated ranges are contemplated).

Xerogels - water-soluble bioreagent materials can be formed into desired shapes. One method is to react the precursor in a mold having the desired shape. The shape is removed from the mold before or after solvent removal. The material can also be broken into particles, as described in more detail elsewhere herein.

After forming the matrix in the organic solvent, the solvent can be removed to form a xerogel. Possible methods include, for example, precipitation using a non-solvent, nitrogen purge drying, vacuum drying, freeze drying, a combination of heat and vacuum, and lyophilization.

If a molten precursor is used in the absence of a tertiary solvent, no solvent removal process is required. Upon cooling, the material forms a rubbery solid (if above Tm), a semi-rigid semi-crystalline material (if below Tm), or a rigid glassy solid (if below Tg). These materials are denser than xerogels formed from organic solvents. When filled with particles of other materials, such as therapeutic agents, buffer salts, visualization agents, they can be highly porous because the solid particles create and fill the pores.

All of these methods can be performed in the absence of water-soluble biological agents. The materials (including particles) are useful for many applications in the absence of biological agents. Uses include, for example, tissue augmentation, fillers, and tissue separation in radiation therapy.

Furthermore, all of these methods can be performed with additional agents instead of or in addition to the biological agents. Such additional agents include visualization agents visible to the naked eye and radiopaque agents or materials.

Particle preparation

The organogel can be formed and then broken down into particles, which are then treated to remove the organic solvent to form a xerogel. For injectable forms, the organogel can be macerated, homogenized, extruded, screened, minced, diced, or otherwise broken down into particulate form. Alternatively, the organogel can be formed as a molded article or droplet containing suspended protein particles.

One method for making organogel particles involves creating a matrix that is then broken down to produce the organogel particles. Thus, the matrix is made from a precursor as described herein and then broken down. One technique involves preparing the organogel with the protein particles and grinding them, for example, in a ball mill or using a mortar and pestle. The matrix can be chopped or diced with a knife or wire. Alternatively, the matrix can be chopped in a blender or homogenizer. Another method involves forcing the organogel through a mesh, collecting the fragments, and passing them through the same mesh or another mesh until the desired size is reached.

Before being dispersed into the organogel, water-soluble biological agents, such as proteins, are prepared as particles. A variety of protein particulation techniques, such as spray drying or precipitation, exist and can be employed, provided that the protein of interest is compatible with such processing. Embodiments of particle preparation involve receiving biological agents that are not substantially (significantly) denatured, such as from a supplier or animal or recombinant source. For proteins, the solid phase is a stable form. The protein is lyophilized or concentrated or used as received. The protein is then prepared as a fine powder without denaturation by treating the protein in a solid state and avoiding high temperature, moisture, and optionally in an oxygen-free environment. Powders can be prepared, for example, by grinding, ball milling, cryomilling, microfluidization, or grinding with a mortar and pestle, followed by sieving the solid protein. Proteins can also be processed in a compatible anhydrous organic solvent while maintaining the protein in solid form, in which the protein in question is insoluble. Reduction of particle size to the desired range can be achieved, for example, by grinding, ball milling, jet milling of a solid protein suspension in a compatible organic solvent. High shear rate processing, high pressures, and sudden temperature changes should be minimized because they lead to protein instability. Therefore, care must be taken to handle proteins or other water-soluble biological agents in a manner that avoids damage, and the use of conventional methods for making particles should not be considered appropriate and would not be expected to be useful without appropriate redesign and testing of the results.

The term protein powder refers to a powder made from one or more proteins. Similarly, the powder of a water-soluble biological agent is a powder with particles made from one or more water-soluble biological agents. The protein in the protein particles or the biological agent in the biological agent particles associate with each other to provide mechanical integrity and structure to the dry particles, even in the absence of a binding agent or encapsulating agent. These powders are different from protein or biological agent delivery using encapsulation or, for example, the approach of liposomes, micelles, or nanocapsules, other technologies of encapsulating proteins or biological agents. Powders and/or the xerogels or hydrogels comprising them can be free of encapsulating materials and free of one or more of liposomes, micelles, or nanocapsules. In addition, protein particles or water-soluble biological agent particles free of one or more of the following can be produced: binding agents, non-peptidic polymers, surfactants, oils, fats, waxes, hydrophobic polymers, polymers comprising alkyl chains longer than 4 _CH2 groups, phospholipids, polymers forming micelles, compositions forming micelles, amphiphilic molecules, polysaccharides, polysaccharides of three or more sugars, fatty acids, and lipids. Lyophilized, spray-dried, or otherwise processed proteins are often formulated with sugars such as trehalose to stabilize the protein through the lyophilization or other methods used to prepare the protein. These sugars can be allowed to persist in the particles throughout the organogel/xerogel process. Particles can be made to include from about 20% to about 100% (dry, weight/weight) protein; the skilled artisan will immediately understand that all ranges and values within the explicitly stated ranges are contemplated, for example, from about 50% to about 80% or at least 90% or at least about 99%.

Particles of biological agents or particles of organogels or particles of xerogels can be separated into aggregates having a desired size range and size distribution by a variety of methods. Very fine control of sizing is available, with sizes ranging from 1 micron to several mm, and wherein for narrow distributions, the mean and range of particle sizes are controllable. The skilled person will immediately understand that all ranges and values within the explicitly stated ranges are designed, for example, about 1 to about 10 μm or about 1 to about 30 μm. About 1 to about 500 microns is another such useful range, wherein the size falls within the entire stated range and has an average size at a value within the stated range, and a standard deviation centered around the mean, for example, about 1% to about 100%. A simple method for sizing particles involves using a custom or standardized sieve mesh size. In addition to standard U.S. and Tyler mesh sizes, sieves of market grade, mill grade, and tensile bolting cloth are also commonly used. The material forced through the mesh may exhibit deformation such that the particle size does not exactly match the mesh size; however, the mesh size can be selected to achieve a desired particle size range. Particle size analyzers are typically used, wherein the protein particles are dispersed in an organic or oil phase. Microscopic methods are also typically used to determine particle size. A spherical particle is a particle in which the longest central axis (a straight line through the geometric center of the particle) does not exceed about twice the length of the other central axes, wherein the particle is literally globe-shaped or has an irregular shape. A rod-shaped particle is a particle in which the longitudinal central axis exceeds about twice the length of the shortest central axis. Embodiments include: producing a plurality of particle assemblies, wherein the assemblies have different in vivo degradation rates, and mixing the assemblies to produce a biomaterial having desired degradation properties.

Deliver water-soluble biological agents without denaturation

These processes can be performed using proteins or other water-soluble biological agents. These include peptides and proteins. As used herein, the term protein refers to peptides of at least about 5000 daltons. As used herein, the term peptide refers to peptides of any size. The term oligopeptide refers to peptides having a mass of up to about 5000 daltons. Peptides include therapeutic proteins and peptides, antibodies, antibody fragments, short chain variable regions (fragments) (scFv), growth factors, angiogenic factors, and insulin. Other water-soluble biological agents are carbohydrates, polysaccharides, nucleic acids, antisense nucleic acids, RNA, DNA, small interfering RNA (siRNA), and aptamers. The description herein is often set forth in terms of proteins, but the methods are generally applicable to other water-soluble biological agents.

Proteins are easily denatured. However, as described herein, proteins can be delivered substantially without denaturation, including situations where no binder, lipophilic material, surfactant, or other preventive component is used. The term substantially without denaturation refers to a protein that is processed into particles without a change in the chemical structure of the protein (without the addition of chemical groups or changes in existing chemical groups) and without a change in the conformation, i.e., secondary and/or tertiary and/or quaternary structure, of the protein. In this context, the term substantially means that no significant difference (p value < 0.05) was observed between the processed protein and the control protein for an averaged test group tested under conventional conditions by an enzyme-linked immunosorbent assay (ELISA) for epitope (antigenic determinant) denaturation and by isoelectric focusing (IEF) for a shift exceeding 0.2 in terms of isoelectric point (pI), see U.S. Serial No. 13/234,428, which is hereby incorporated by reference herein for testing or protein stability and for all purposes; in the event of a conflict, this specification governs. Primary protein structure refers to the amino acid sequence. In order to be able to perform their biological functions, proteins fold into one or more special spatial conformations, which are driven by a number of non-covalent interactions such as hydrogen bonding, ionic interactions, van der Waals forces and hydrophobic packing. The term secondary structure refers to local protein structure, such as local folding. Tertiary structure refers to a specific three-dimensional conformation, including folding. Proteins with secondary and/or tertiary structure therefore exhibit local and overall structural organization. In contrast, linear peptides that do not have a specific conformation do not have secondary and/or tertiary structure. The term native means as found naturally in vivo (in fact, in nature), so that the protein can be processed into particles and released in its native conformation.

Protein denaturation can be tested by a variety of techniques, including enzyme-linked immunosorbent assay (ELISA), isoelectric focusing (IEF), size exclusion chromatography (SEC), high pressure liquid chromatography (HPLC), circular dichroism (CD), and Fourier transform infrared spectroscopy (FTIR). These tests report parameters such as changes in molecular weight, changes in end groups, changes in bonds, changes in hydrophobicity or size exclusion, and exposure/hiding of antigenic sites (antigenic groups). Generally, tests by IEF and ELISA can be designed that are sufficient to show the native conformation after processing, although other tests and test combinations can be used instead.

Experiments have shown that many factors that contribute to the ability to process and deliver proteins without denaturation can be controlled. Proteins can be prepared as powders, with the powder particle size selected based on the size of the final organogel. All organic solvents for proteins can be selected so that the protein is not solvated by the organic solvent and is compatible with the protein. Another factor is oxygen, and its elimination is useful in processing to avoid denaturation. Another factor is chemical reactions. These can be avoided by keeping the protein in a solid phase and without a solvent that dissolves the protein until such time as the protein is implanted.

One embodiment of particle preparation involves receiving a protein without substantial (significant) denaturation, such as from a supplier or animal or recombinant source. The protein is lyophilized, spray-dried, or concentrated or used as received. The protein is then prepared as a fine powder without denaturation by treating it in a solid state and avoiding high temperatures, moisture, and optionally in an oxygen-free environment. Powders can be prepared by, for example, grinding, ball milling, or grinding the solid protein with a mortar and pestle.

Making protein reagents or other water-soluble biological reagents into particles can be a useful first step for delivering the reagents from a solid phase. However, it is not a sufficient step for achieving well-controlled release or effective release from a matrix over an extended period of time. However, upon implantation, when water contacts the particles and solvates the reagent, the particles will tend to dissolve rapidly. In the case of particles in a hydrogel, for example, water permeates the hydrogel and contacts the particles. However, unexpectedly, it is possible to prevent the water-soluble biological reagents in the particles in the hydrogel from dissolving. Some mechanisms for doing so are described in this article, but will not be used to limit the present invention to a specific theory of action. One mechanism apparently involves using a matrix that prevents the reagent from moving away from the particles. Moreover, even if the molecules of the reagent dissolve, they are maintained at a local (local) location and will saturate the local location to prevent further solvation of other reagent molecules. Another mechanism involves the solvation of the matrix, which competes with the potentially soluble reagent for water, wherein the matrix has a size exclusion effect to interfere with the solvation of the reagent.

These mechanisms involve achieving spacing between the molecular chains of a dense matrix. The crosslink density of the organogel matrix (and therefore the xerogel and hydrogel matrix) is controlled by the total molecular weight of the precursor used as the crosslinking agent and other precursors, as well as the number of functional groups available per precursor molecule. A lower molecular weight, such as 500, between crosslinks will provide a much higher crosslink density than a higher molecular weight, such as 10,000, between crosslinks. The crosslink density can also be controlled by the total solids percentage of the crosslinking agent and the functional polymer solution. Another method for controlling the crosslink density is by adjusting the stoichiometry of nucleophilic functional groups to electrophilic functional groups. A one-to-one ratio results in the highest crosslink density. Precursors with longer distances between crosslinkable sites form gels that are generally softer, more complex, and more elastic. Therefore, the increased length of water-soluble segments, such as polyethylene glycol, tends to increase elasticity to produce desirable physical properties. Thus, some embodiments involve precursors having water-soluble segments with molecular weights in the range of 2,000-100,000; the skilled artisan will immediately understand that all ranges and values within the explicitly stated ranges are contemplated, e.g., 10,000-35,000. The solids content of the hydrogel can affect its mechanical properties and biocompatibility and reflect a balance between competing requirements. Relatively low solids contents are useful, e.g., from about 2.5% to about 20%, including all ranges and values therebetween, e.g., from about 2.5% to about 10%, from about 5% to about 15%, or less than about 15%. The skilled artisan will understand that the same material can be used to make matrices with a wide range of structures that will have very different mechanical properties and performance, such that the achievement of specific properties should not be assumed based solely on the general type of precursor involved.

Delivery of water-soluble biological agents and other therapeutic agents

Various water-soluble biological agents and/or other therapeutic agents can be delivered using the systems described herein. Xerogel particles containing protein powder can be used to deliver water-soluble biological agents and/or other therapeutic agents. The particles can be applied inside the xerogel. The xerogel can be a preformed structure, for example, having a volume of at least 2 cm ³ (the skilled person will immediately understand that all ranges and values within the clearly stated ranges are designed, for example, from about 2 to about 20 cm ³ ), or an aggregate of particles. Alternatively, the xerogel particles can be applied directly or with a pharmaceutically acceptable binder or carrier. Other materials can include the xerogel particles. Water-soluble agents are a class of agents that can be delivered as a powder inside the xerogel. Other drugs, such as hydrophobic agents or small molecule drugs (water-soluble or hydrophobic), can also be mixed into or with the xerogel.

Proteins are a class of water-soluble agents. Xerogel particles can be processed so that proteins are introduced and released without substantial denaturation and/or in their native conformation. Some anti-vascular endothelial growth factor (anti-VEGF) agents are therapeutic proteins. Anti-VEGF therapies are important in the treatment of some cancers and in age-related macular degeneration. They can involve monoclonal antibodies such as bevacizumab (AVASTIN), antibody derivatives such as ranibizumab (LUCENTIS), or small molecules that inhibit tyrosine kinases stimulated (stimulated) by VEGF: lapatinib (TYKERB), sunitinib (SUTENT), sorafenib (NEXAVAR), axitinib, and pazopanib. (Some of these therapies target VEGF receptors opposite to VEGF).

Some conventional eye drug delivery systems deliver drugs with topical (external) eye drops. For example, after cataract and vitreoretinal surgery, antibiotics are applied dropwise every few hours for several days. In addition, other drugs such as non-steroidal anti-inflammatory drugs (NSAIDS) may also need to be frequently provided. Some of these eye drops, such as RESTASIS (Allergan), also have the stinging and burning sensations associated with their application. RESTASIS is indicated for dry eyes and must be used several times a day by the patient. Similarly, for other eye diseases such as cystoid macular edema, diabetic macular edema (DME) and diabetic retinopathy, it is also necessary to administer steroidal or NSAID drugs. Some vascular proliferative diseases such as macular degeneration are treated with intravitreal injections of VEGF inhibitors. These include drugs such as LUCENTIS and AVASTIN (Genentech) and MACUGEN (OSI). Such drugs can be delivered using the hydrogel-and-particle systems described herein, wherein repeated dosing steps are avoided; for example, no new drug applications are made daily, weekly, or monthly, or no topical eye drops are used to administer the drug.

A variety of drug delivery systems are known. These various other systems typically include intravitreal implant reservoir-type systems, biodegradable depot systems, or (non-erodible) implants that require removal. The state of the art in this regard has been described in textbooks such as "Intraocular Drug Delivery" (Jaffe et al., published by Taylor & Francis, 2006). However, most of these implants need to be removed upon expiration, can be detached from their target site, can cause visual impairment at the back of the eye, or can themselves be inflammatory due to the release of significant amounts of acidic degradation products. These implants are therefore manufactured to be very small, with very high drug concentrations. Even though they are small, they still require deployment with a needle larger than 25G (25 gauge), or require a surgical approach delivery system to be implanted or removed when needed. Typically, these are local injections of drug solutions into the vitreous or intravitreal implants, using either a biodegradable approach or a removable reservoir approach. For example, local injections delivered into the vitreous include the anti-VEGF agents LUCENTIS or AVASTIN. POSURDEX (Allergan) is a biodegradable implant used for diabetic macular edema (DME) or retinal vein occlusion, using a 22-gauge syringe delivery system for delivery into the vitreous cavity; these are powerful drugs in a short drug delivery duration setting. The therapeutic agent is dexamethasone with a polylactic/polyglycolic polymer matrix. Trials using POSURDEX for diabetic retinopathy are ongoing. And, for example, the MEDIDURE implant (PSIVIDA) is used for the DME indication. The therapeutic agent for this implant is fluocinolone acetonide and has a nominal delivery life of 18 months or 36 months (two forms). Vitreous removable implants containing triamcinolone acetonide are being tested. Its nominal delivery life is approximately two years and requires surgical implantation. Its indication is for DME.

In contrast to these conventional systems, these and other therapeutic agents can be delivered using an assembly of xerogel particles or a system comprising such particles. The xerogel particles comprise the agent. Upon exposure to physiological fluids, the xerogel absorbs the fluid to form a hydrogel that is biocompatible with the eye, an environment distinct from other environments. The use of minimally inflammatory materials avoids angiogenesis, which in many cases is harmful in the eye. Biocompatible ocular materials thus avoid unintended angiogenesis; in some aspects, this goal is achieved by avoiding acidic degradation products. Furthermore, by using hydrogels and hydrophilic materials (components with a solubility of at least 1 gram/liter in water, such as polyethylene glycol/oxide), the influx of inflammatory cells is minimized; this process is in contrast to the conventional use of non-hydrogel or rigid reservoir-based ocular implants. Furthermore, some proteins can be avoided to enhance biocompatibility; collagen or fibrin glues, for example, tend to promote inflammation or unwanted cellular responses because they release signals that promote biological activity upon degradation. Instead, synthetic materials or peptide sequences not typically found in nature are used. In addition, biodegradable materials can be used to avoid a chronic foreign body reaction, such as, for example, non-degradable thermally formed gels. Furthermore, soft materials or materials that are manufactured in situ to conform to the shape of surrounding tissue can minimize ocular distortion, and low-swelling materials can be used to eliminate visual distortion caused by swelling. High or low pH materials can be avoided during the formation, introduction, or degradation stages.

Xerogels can be prepared and used to deliver a variety of drugs (as well as drugs for local and systemic delivery to other parts of the body), including steroids, nonsteroidal anti-inflammatory drugs (NSAIDS), anticancer drugs, antibiotics, etc. Xerogels can be used to deliver drugs and therapeutic agents, such as anti-inflammatory drugs (e.g., Diclofenac), analgesics (e.g., Bupivacaine), calcium channel blockers (e.g., Nifedipine), antibiotics (e.g., Ciprofloxacin), cell cycle inhibitors (e.g., Simvastatin), proteins (e.g., insulin). The particles can be used to deliver a variety of drugs, including, for example, steroids, NSAIDS, antibiotics, analgesics, vascular endothelial growth factor (VEGF) inhibitors, chemotherapy, antiviral drugs. Examples of NSAIDS are ibuprofen, meclofenamic acid sodium, mefanamic acid, salsalate, sulindac, tolmetin sodium, ketoprofen, diflunisal, piroxicam, naproxen, etodolac, flurbiprofen, fenoprofen calcium, indomethacin, celoxib, ketrolac and nepafenac. The drug itself can be a small molecule, protein, RNA fragment, protein, glycosaminoglycan (glucosaminoglycan), carbohydrate, nucleic acid, inorganic and organic biologically active compounds, where specific biologically active agents include but are not limited to: enzymes, antibiotics, anti-tumor agents, local anesthetics, hormones, angiogenic agents, anti-angiogenic agents, growth factors, antibodies, neurotransmitters, psychotropic drugs, anti-cancer drugs, chemotherapeutic drugs, drugs affecting genitals, genes, and oligonucleotides, or other configurations.

These xerogel particles or other xerogel structures can be used to deliver a variety of drugs or other therapeutic agents. A list of examples of agents or series of drugs and their indications is provided. The agents can also be used to treat the indicated conditions or as part of a method for treating the indicated conditions. For example, AZOPT (brinzolamide ophthalmic suspension) can be used to treat elevated intraocular pressure in patients with ocular hypertension or open-angle glaucoma. BETADINE in povidone-iodine ophthalmic solution can be used for preparation of the periocular area and irrigation of the ocular surface. BETOPTIC (betaxolol HCl) can be used to reduce intraocular pressure or for chronic open-angle glaucoma and/or ocular hypertension. CILOXAN (ciprofloxacin HCl ophthalmic solution) can be used to treat infections caused by susceptible strains of microorganisms. NATACYN (natamycin ophthalmic suspension) can be used to treat fungal blepharitis, conjunctivitis, and keratitis. NEVANAC (nepanfenac ophthalmic suspension) is used to treat inflammation and pain associated with cataract surgery. TRAVATAN (travoprost ophthalmic solution) is used to reduce elevated intraocular pressure—open-angle glaucoma or ocular hypertension. FML FORTE (fluorometholone ophthalmic suspension) is used to treat corticosteroid-responsive inflammation of the palpebral and bulbar conjunctiva, cornea, and anterior segment of the eye. LUMIGAN (bimatoprost ophthalmic solution) is used to reduce elevated intraocular pressure—open-angle glaucoma or ocular hypertension. PRED FORTE (prednisolone acetate) is used to treat steroid-responsive inflammation of the palpebral and bulbar conjunctiva, cornea, and anterior segment of the eye. PROPINE (dipivefrin hydrochloride) is used to control intraocular pressure in chronic open-angle glaucoma. RESTASIS (cyclosporine ophthalmic emulsion) is used to increase tear production in patients, such as those with eye inflammation associated with keratoconjunctivitis sicca. ALREX (loteprednol etabonate ophthalmic suspension) is used for the temporary relief of seasonal allergic conjunctivitis. LOTEMAX (loteprednol etabonate ophthalmic suspension) is used to treat steroid-responsive inflammation of the palpebral and bulbar conjunctiva, cornea, and anterior segment of the eye. MACUGEN (pegaptanib sodium injection) is used to treat neovascular (wet) age-related macular degeneration. OPTIVAR (azelastine hydrochloride) is used to treat ocular itching associated with allergic conjunctivitis. XALATAN (latanoprost ophthalmic solution) is used to reduce elevated intraocular pressure in patients (e.g., with open-angle glaucoma or ocular hypertension). BETIMOL (Timolol ophthalmic solution) is used to treat elevated intraocular pressure in patients with ocular hypertension or open-angle glaucoma. Latanoprost is a prodrug in the free acid form that is a prostanoid-selective FP receptor agonist. Latanoprost reduces intraocular pressure in glaucoma patients with few side effects. Latanoprost has relatively low solubility in aqueous solutions but is readily soluble in organic solvents typically used for microsphere manufacturing using solvent evaporation.

Further embodiments of agents for delivery include those that specifically bind to the target peptide in vivo to prevent interaction of the target peptide with its natural receptor or other ligand. AVASTIN, for example, is an antibody that binds to VEGF. And AFLIBERCEPT is a fusion protein that includes a portion of the VEGF receptor for capturing VEGF. IL-1 traps (IL-1trap) that utilize the extracellular domain of the IL-1 receptor are also known; the trap blocks IL-1 from binding to and activating receptors on the cell surface. Embodiments of agents for delivery include nucleic acids, such as aptamers. MACUGEN, for example, is a pegylated anti-VEGF aptamer. An advantage of particle-and-hydrogel delivery methods is that aptamers are protected from the in vivo environment until they are released. Further embodiments of agents for delivery include macromolecular drugs, a term referring to drugs that are significantly larger than classic small molecule drugs (i.e., drugs such as oligonucleotides (aptamers, antisense, RNAi), ribozymes, gene therapy nucleic acids, recombinant peptides, and antibodies).

One embodiment includes the extended release of a drug for allergic conjunctivitis. For example, ketotifen (a kind of antihistamine and mast cell stabilizer) can be provided in particles and released into the eyes in an effective amount for treating allergic conjunctivitis as described herein. Seasonal allergic conjunctivitis (SAC) and perennial allergic conjunctivitis (PAC) are allergic conjunctival disorders (illnesses). Symptoms include itching and pink to reddish eyes. These two eye conditions are mediated by mast cells. Non-specific measures to improve symptoms routinely include: cold compresses, eyewashes with tear substitutes, and avoiding allergens. Treatment routinely consists of antihistamine mast cell stabilizers, dual mechanism antiallergens, or topical antihistamines. Corticosteroids can be effective, but due to side effects, they are reserved for more severe forms of allergic conjunctivitis such as vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC).

Moxifloxacin is the active ingredient in VIGAMOX, a fluoroquinolone approved for the treatment or prevention of bacterial infections of the eye. The dosage is typically one drop of a 0.5% solution applied three times a day for a week or longer.

VKC and AKC are chronic allergic diseases in which eosinophils, conjunctival fibroblasts, epithelial cells, mast cells, and/or TH2 lymphocytes worsen the biochemistry and histology of the conjunctiva. VKC and AKC can be treated with drugs used to combat allergic conjunctivitis.

Penetrants are agents and may also be included in gels, hydrogels, organogels, xerogels, and biomaterials as described herein. These are agents that help the drug penetrate into the desired tissue. The penetrant can be selected as needed for the tissue, for example, a penetrant for the skin, a penetrant for the tympanic membrane, a penetrant for the eye.

Xerogel particle blends and aggregates

An assembly of particles (powder particles of an agent and/or xerogel/hydrogel particles) may comprise a set of particles. The term xerogel/hydrogel refers to a xerogel and/or a xerogel hydrated to a hydrogel. For example, an assembly may comprise some xerogel particles containing a radiopaque agent, wherein those particles form a set within the assembly. Other sets may relate to particle sizes, wherein the sets have different shapes or size distributions. As discussed, particles can be manufactured in well-controlled sizes and can therefore be manufactured and divided into multiple sets for combination into an assembly.

Some populations are made of particles (xerogels/hydrogels) with specific degradability. One embodiment relates to a plurality of populations each having a different degradability profile. Different degradation rates provide different release profiles. Combinations of different particle populations can be made to achieve the desired profile, as demonstrated in Figures 6 and 7, with reference to Example 2. Degradation times include 3-1000 days; the skilled person will immediately understand that all ranges and values within the explicitly stated ranges are designed. For example, a first population may have a median degradation time of about 5 to about 8 days, a second population may have a median time of about 30 to about 90 days, and a third population may have a median time of about 180 to about 360 days.

Xerogel/hydrogel particles can be blended to achieve a desired protein release profile.Gels (such as hydrogels) with different degradation rates can be combined to provide a constant or nearly constant release that compensates for the inherent non-linear release profile of a single gel.

An assembly of xerogel/hydrogel particles can include populations of agents. For example, some particles can be made to contain a first therapeutic agent, where those particles form a population within the assembly. And other populations can contain another agent. Examples of agents are water-soluble biological agents, proteins, peptides, nucleic acids, small molecule drugs, and hydrophobic agents. Other populations can involve particle size, where the populations have different shapes or size distributions. As discussed, particles can be manufactured at well-controlled sizes and divided into multiple populations for assembly into the assembly. These multiple populations can be freely mixed and matched in combinations and subcombinations of, for example, size, degradability, therapeutic agent, and visualization agent.

The xerogel/hydrogel may further include an agent that is not in powder form. The agent may be disposed within the xerogel/hydrogel or mixed with a solution of another excipient for use with the xerogel/hydrogel. For example, the collection of xerogel particles may be hydrated to form a hydrogel immediately prior to use by adding saline or water, which may further include a drug solution. Such a drug or agent may be the same as the agent in powder form within the xerogel/hydrogel to provide an initial burst of release, or may be used for secondary therapy or visualization.

Lubricity

The aggregate is manufactured to a size and lubricity suitable for manual injection through a small-gauge needle. The hydrophilic hydrogel, crushed into spherical particles of approximately 40 to 100 microns in diameter, is small enough to be manually injected through a 30-gauge needle. Hydrophilic hydrogel particles have been observed to have difficulty passing through small-gauge needles/catheters, as reported in U.S. Publication No. 2011/0142936, which is hereby incorporated herein for all purposes; in the event of conflict, this specification controls. Particle size contributes to drag, as well as the viscosity of the solution. Particles tend to clog the needle. Drag is proportional to the viscosity of the fluid, with more viscous fluids requiring more force to push through a small opening.

As reported in U.S. Publication No. 2011/0142936, it was unexpectedly discovered that increasing the viscosity of the solvent used for the particles can reduce the resistance to passage through the catheter and/or needle. This reduction can be attributed to the use of solvents with high osmolarity (molar osmotic pressure concentration). Without being bound by a particular theory, the addition of these agents to improve syringeability is caused by: particle shrinkage, increased free water between the particles that reduces the particle-to-particle contribution to viscosity, and the increased viscosity of the free water, which helps pull the particles in and out of the syringe, preventing strain and clogging. The use of linear polymers can further contribute to thixotropic properties, which are useful for preventing particle sedimentation and promoting the movement of particles with the solvent, but exhibit shear thinning when forced out of a small opening. It has also been observed that this approach solves another problem, namely the difficulty in moving particles from a solution through a needle/catheter, as particles tend to settle and otherwise escape pickup. Expulsion of solutions of particles in aqueous (aqueous) solvents through small orifice openings has been observed; the solvent tends to preferentially migrate from the applicator, leaving behind excess particles that cannot be removed from the applicator, or that clog the applicator, or in some cases can be removed (but only by applying unsuitably high force for a typical user unsuitable for operating a handheld syringe). However, the addition of penetrants contributes to viscosity and/or thixotropic behavior, which aids in emptying the particles from the applicator.

Embodiments of the present invention include adding an osmotic agent to a plurality of xerogel/hydrogel particles. Examples of such agents include salts and polymers. Embodiments include polymers, linear polymers, and hydrophilic polymers, or combinations thereof. Embodiments include polymers having a molecular weight of about 500 to about 100,000; a skilled artisan will immediately understand that all ranges and values within the explicitly stated ranges are contemplated, e.g., about 5000 to about 50,000 molecular weight. Embodiments include osmotic agent concentrations of, for example, about 1% to about 50% weight/weight; a skilled artisan will immediately understand that all ranges and values within the explicitly stated ranges are contemplated, e.g., 10% to 30%. The agents and hydrogels can be introduced into a patient and can be part of a kit for use therewith.

Precursor

Matrices can be prepared and used to contain particles of water-soluble biological agents. Thus, embodiments are provided herein for making implantable matrices. Such matrices include matrices having a porosity greater than about 20% volume/volume; skilled artisans will immediately understand that all ranges and values within the explicitly stated ranges are contemplated. Precursors can be dissolved in an organic solvent to make an organogel. An organogel is a non-crystalline, non-glassy solid material composed of a liquid organic phase trapped in a three-dimensional cross-linked network. The liquid can be, for example, an organic solvent, mineral oil, or vegetable oil. The solubility and size of the solvent are important characteristics for the elastic properties and stability of the organogel. Alternatively, the precursor molecules themselves may be able to form their own organic matrix, eliminating the need for a third organic solvent. The term precursor refers to a component that becomes part of the cross-linked matrix. A polymer that becomes cross-linked into the matrix is a precursor, while a salt or protein that is merely present in the matrix is not a precursor.

Removing the solvent (if used) from the organogel provides a xerogel, i.e., a dried gel. Xerogels formed, for example, by freeze drying, can have a high porosity (at least about 20%), a large surface area, and a small pore size. Xerogels made from hydrophilic materials form hydrogels when exposed to aqueous solutions. Xerogels with high porosity hydrate faster than denser xerogels. Hydrogels are materials that are insoluble in water and retain a substantial fraction (over 20%) of water within their structure. In fact, water contents exceeding 90% are often known. Hydrogels can be formed by crosslinking water-soluble molecules to form networks of essentially infinite molecular weight. Hydrogels with high water contents are typically soft, pliable materials. Hydrogels and drug delivery systems as described in U.S. Publication Nos. 2009/0017097, 2011/0142936, and 2012/0071865 can be adapted for use with the materials and methods herein by following the guidance provided herein; these references are hereby incorporated by reference herein for all purposes, and in case of conflict, the present specification controls.

Organogels and hydrogels can be formed from natural, synthetic or biosynthetic polymers. Natural polymers can include glycosaminoglycans, polysaccharides and proteins. Some examples of glycosaminoglycans include dermatan sulfate, hyaluronic acid, chondroitin sulfate, chitin, heparin, keratan sulfate, keratin sulfate and its derivatives. Typically, glycosaminoglycans are extracted from natural sources and purified and derived. However, they can also be synthetically manufactured or synthesized by modifying microorganisms such as bacteria. These materials can be synthetically changed from a naturally soluble state to a partially soluble or water-swellable or hydrogel state. This change can be achieved by a variety of well-known techniques, for example, by conjugation or replacement of ionizable or hydrogen-bonded functional groups such as carboxyl and/or hydroxyl or amine groups with other more hydrophobic groups.

In some embodiments, the hyaluronic acid of the present invention can be used as the hydrogel of the present invention.For example, the carboxyl group on hyaluronic acid can be esterified with alcohol to reduce the solubility of hyaluronic acid. Such method is used to produce sheet (sheet), fiber and fabric based on hyaluronic acid by multiple manufacturers (for example Genzyme Corp., Cambridge, MA) of hyaluronic acid product, and it forms hydrogel.Other natural polysaccharides such as carboxymethyl cellulose or the regenerated cellulose of oxidation, natural gum, agar, agarose, sodium alginate, carrageenan, fucoidan, furcellaran (furcellaran), laminarin, hypnea (hypnea), eucheuma (eucheuma), gum arabic, ghatti gum, karaya gum, tragacanth gum, locust bean gum, arabinoglactan (arbinoglactan), pectin, amylopectin, gelatin, hydrophilic colloid are such as with polyhydric alcohol such as cross-linked carboxymethyl cellulose gum or the alginate gum such as propylene glycol, also form hydrogel when contacting with aqueous environment.

Synthetic organogels or hydrogels can be biostable or biodegradable. Examples of biostable hydrophilic polymeric materials are poly(hydroxyalkyl methacrylates), poly(electrolyte complexes), poly(vinyl acetate) cross-linked with hydrolyzable or otherwise degradable bonds, and water-swellable N-vinyl lactams. Other hydrogels include so-called hydrophilic hydrogels, acidic carboxy polymers (Carbomer resins are high molecular weight, allyl pentaerythritol-crosslinked acrylic acid-based polymers modified with C10-C30 alkyl acrylates), polyacrylamides, polyacrylic acid, starch graft copolymers, acrylate polymers, and ester-crosslinked polyglucans. Such hydrogels are described, for example, in U.S. Patent No. 3,640,741 to Etes, U.S. Patent No. 3,865,108 to Hartop, U.S. Patent No. 3,992,562 to Denzinger et al., U.S. Patent No. 4,002,173 to Manning et al., U.S. Patent No. 4,014,335 to Arnold, and U.S. Patent No. 4,207,893 to Michaels, all of which are incorporated herein by reference, where in case of conflict, the present specification controls.

Hydrogels and organogels can be made from precursors. Precursors are not hydrogels/organogels, but rather crosslink with each other to form a hydrogel/organogel. Crosslinks can be formed by covalent bonds or physical bonds. Examples of physical bonds are ionic bonds, hydrophobic associations of precursor molecular segments, and crystallization of precursor molecular segments. Precursors can be triggered to react to form a crosslinked hydrogel. Precursors can be polymerizable and include a crosslinking agent, which is often, but not always, a polymerizable precursor. Polymerizable precursors are thus precursors that have functional groups that react with each other to form a polymer and/or matrix made of repeating units. Precursors can be polymers.

Some precursors therefore react by chain growth polymerization (also known as addition polymerization), and involve crosslinking together monomers that introduce double or triple chemical bonds. These unsaturated monomers have additional internal bonds that can break and connect to other monomers to form repeating chains. A monomer is a polymerizable molecule having at least one group that reacts with other groups to form a polymer. A macromonomer (or macromolecular monomer) is a polymer or oligomer having at least one reactive group, often at the end, that enables it to act as a monomer; each macromonomer molecule is connected to the polymer by the reaction of the reactive group. Therefore, macromonomers with two or more monomers or other functional groups tend to form covalent crosslinks. For example, addition polymerization is related to the manufacture of polypropylene or polyvinyl chloride. One type of addition polymerization is living polymerization.

Some precursors therefore undergo condensation polymerization reactions that occur when monomers are bonded together through condensation reactions. Typically, these reactions can be achieved by reacting molecules that incorporate alcohol, amine, or carboxylic acid (or other carboxyl derivative) functional groups. When an amine reacts with a carboxylic acid, an amide or peptide bond is formed, and water is released. Some condensation reactions follow nucleophilic acyl substitution, for example, as described in U.S. Patent No. 6,958,212, which is hereby incorporated by reference in its entirety to the extent it does not conflict with the disclosure herein.

Some precursors react via a chain growth mechanism. A chain growth polymer is defined as a polymer formed by the reaction of a monomer or macromonomer with a reactive center. A reactive center is a specific location within a compound that is the initiator of the reaction in which the chemistry is involved. In chain growth polymer chemistry, this is also the point of propagation for the growing chain. Reactive centers are typically free radical, anionic, or cationic in nature, but can take other forms as well. Chain growth systems involve free radical polymerization, which involves the processes of initiation, propagation, and termination. Initiation is the generation of free radicals necessary for propagation, such as produced by a free radical initiator, such as an organic peroxide molecule. Termination occurs when the free radicals react in a way that prevents further propagation. The most common method of termination is through coupling, where two free radical species react with each other to form a single molecule.

Some precursors react by a step-growth mechanism and are polymers formed by step-wise reactions between the functional groups of the monomers. Most step-growth polymers are also classified as condensation polymers, but not all step-growth polymers release condensates.

Monomers can be polymers or small molecules. Polymers are high molecular weight molecules formed by combining many small molecules (monomers) in a regular pattern. Oligomers are polymers with fewer than about 20 monomer repeating units. Small molecules generally refer to molecules with a molecular weight of less than about 2000 Daltons.

The precursor may thus be a small molecule such as acrylic acid or vinyl caprolactam, a larger molecule containing a polymerizable group such as acrylate-terminated polyethylene glycol (PEG-diacrylate), or other polymers containing ethylenically unsaturated groups such as those described in U.S. Pat. No. 4,938,763 to Dunn et al., U.S. Pat. Nos. 5,100,992 and 4,826,945 to Cohn et al., or U.S. Pat. Nos. 4,741,872 and 5,160,745 to DeLuca et al., each of which is hereby incorporated by reference in its entirety to the extent it does not conflict with the explicit disclosure herein.

In order to form a covalently cross-linked hydrogel, precursors must be covalently cross-linked together. Generally, a polymeric precursor is a polymer that will be bonded to other polymeric precursors at two or more points, wherein each point is a connection (linkage) to the same or different polymers. Precursors with at least two reactive centers (e.g., in free radical polymerization) can be used as cross-linking agents because each reactive group can participate in the formation of different growing polymer chains. In the case of functional groups without reactive centers, three or more such functional groups are required on at least one of the precursor types for cross-linking. For example, many electrophilic-nucleophilic reactions consume electrophilic and nucleophilic functional groups, so that a third functional group is required for the precursor to form a cross-link. Such precursors can therefore have three or more functional groups and can be cross-linked by precursors with two or more functional groups. Cross-linked molecules can be cross-linked via ions or covalent bonds, physical forces, or other attractive forces. However, covalent cross-linking will typically provide stability and predictability in the reaction product structure.

In some embodiments, each precursor is multifunctional, meaning that it includes two or more electrophilic or nucleophilic functional groups, such that a nucleophilic functional group on one precursor can react with an electrophilic functional group on another precursor to form a covalent bond. At least one of the precursors includes more than two functional groups, such that as a result of the electrophilic-nucleophilic reaction, the precursors combine to form a cross-linked polymeric product.

Precursor can have biologically inert and hydrophilic part, for example, core.In the case of branched polymers, core refers to the adjacent part of the molecule that is attached to the arm extending from the core, and wherein arm has functional group, and it is often in the end of branch.Hydrophilic precursor or precursor part have the solubility of at least 1g/100mL in aqueous solution.Hydrophilic part can be for example polyether, for example polyoxyalkylene such as polyethylene glycol (PEG), polyethylene oxide (PEO), polyethylene oxide-to-polypropylene oxide (PPO), copolyethylene oxide block or random copolymer, and polyvinyl alcohol (PVA), poly (vinyl pyrrolidone) (PVP), polyamino acid, dextran, or protein.Described precursor can have polyalkylene glycol part and can be based on polyethylene glycol, wherein at least about 80 % by weight or 90 % by weight of said polymer include polyethylene oxide repeats.Described polyether and more particularly poly (oxyalkylene) or poly (ethylene glycol) or polyethylene glycol are hydrophilic normally. As is customary in these arts, the term PEG is used to refer to PEO with or without hydroxyl end groups.

The precursor may also be a macromolecule (or macromonomer), which is a molecule with a molecular weight ranging from one thousand to many million. However, in some embodiments, at least one of the precursors is a small molecule of about 1000 Da or less. The macromolecule, when reacted in combination with a small molecule of about 1000 Da or less, is preferably at least five to fifty times larger than the small molecule in terms of molecular weight and preferably less than about 60,000 Da; the skilled person will immediately understand that all ranges and values within the explicitly stated ranges are designed. A more preferred range is a macromolecule having a molecular weight of about seven to about thirty times that of the cross-linking agent, and a most preferred range is a difference of about ten to twenty times the molecular weight. In addition, a macromolecular molecular weight of 5,000-50,000 is useful, as is a molecular weight of 7,000-40,000 or a molecular weight of 10,000-20,000.

Some macromer precursors are cross-linkable, biodegradable, water-soluble macromers described in U.S. Patent No. 5,410,016 to Hubbell et al., which is hereby incorporated by reference in its entirety to the extent it does not conflict with the explicit disclosure. These macromers are characterized by having at least two polymerizable groups separated by at least one degradable region.

Synthetic precursors can be used. Synthetic refers to molecules not found in nature or not generally found in humans. Some synthetic precursors do not contain amino acids that occur in nature or do not contain amino acid sequences that occur in nature. Some synthetic precursors are polypeptides that are not found in nature or not generally found in humans, for example, dimerization, trimerization, or tetramerization of lysine. Some synthetic molecules have amino acid residues, but only one, two, or three adjacent amino acids, wherein the amino acids or clusters thereof are separated by non-natural polymers or groups. Polysaccharides or their derivatives are therefore not synthetic.

Alternatively, natural protein or polysaccharide may be suitable for use with these methods, such as collagen, fibrinogen, albumin, alginate, hyaluronic acid and heparin. These natural molecules may further include chemical derivatization, such as, synthetic polymer decoration (decoration). The natural molecule may be cross-linked via its natural nucleophile or after it is derivatized with a functional group, such as, as in U.S. Patent No. 5,304,595, 5,324,775, 6,371,975 and 7,129,210, each of which is hereby incorporated by reference into the degree to which it is not conflicted with the content clearly disclosed herein. Natural refers to the molecule found in nature. Natural polymers, such as proteins or glycosaminoglycans such as collagen, fibrinogen, albumin and fibrin, can be cross-linked using reactive precursor substances with electrophilic functional groups. The natural polymers conventionally found in health are degraded by the protease proteolytically present in health. Such polymers can be reacted or derivatized with activatable functional groups via functional groups on their amino acids, such as amines, thiols, or carboxyl groups. Although natural polymers can be used in hydrogels, their gelation time and ultimate mechanical properties must be controlled by appropriately introducing additional functional groups and selecting appropriate reaction conditions, such as pH.

Precursors can be manufactured with hydrophilic moieties provided that the resulting hydrogel retains a desired amount of water, for example, at least about 20%. In some cases, the precursor is still soluble in water because it also has a hydrophilic moiety. In other cases, the precursor is dispersed (suspended) in water but is still reactive to form a cross-linked material. Some hydrophobic moieties may include multiple alkyl groups, polypropylene, alkyl chains, or other groups. Some precursors with hydrophobic moieties are sold under the trade names PLURONIC F68, JEFFAMINE, or TECTRONIC. The hydrophobic moiety of a hydrophobic molecule or copolymer is such a hydrophobic molecule or hydrophobic moiety: it is sufficiently hydrophobic to cause molecules (e.g., polymers or copolymers) to aggregate to form micelles or microphases comprising hydrophobic domains in an aqueous continuous phase; or it is sufficiently hydrophobic to precipitate from an aqueous solution of water at a pH of about 7 to about 7.5 at a temperature of about 30 to about 50 degrees Celsius, or otherwise change phase while in the aqueous solution.

The precursor may have, for example, 2-100 arms, each arm having a terminal end, keeping in mind that some precursors may be dendrimers or other highly branched materials. An arm on a hydrogel precursor refers to a linear chain of chemical groups that connects a crosslinkable functional group to a polymer core. Some embodiments are precursors having 3-300 arms; the skilled artisan will immediately understand that all ranges and values within the explicitly stated ranges are contemplated, for example, 4-16, 8-100, or at least 6 arms.

Thus, a hydrogel can be made, for example, from a multi-arm precursor having a first set of functional groups and a low molecular weight precursor having a second set of functional groups. For example, a six-arm or eight-arm precursor can have hydrophilic arms, such as polyethylene glycol terminated with a primary amine, wherein the arms have a molecular weight of about 1,000 to about 40,000; the skilled artisan will immediately understand that all ranges and values within the explicitly stated ranges are contemplated. Such precursors can be mixed with relatively small precursors, such as molecules having a molecular weight of about 100 to about 5000, or no more than about 800, 1000, 2000, or 5000, having at least about three functional groups, or about 3 to about 16 functional groups; the skilled artisan will understand that all ranges and values between these explicitly stated values are contemplated. Such small molecules can be polymeric or non-polymeric and can be natural or synthetic.

Precursors that are not dendrimers can be used. Dendrimers are highly branched, radially symmetrical polymers in which atoms are arranged in many arms and sub-arms radiating outward from a central core. Dendrimers are characterized by their structural perfection (e.g., based on evaluations of both symmetry and polydispersity) and require specific chemical methods for synthesis. Thus, technicians can easily distinguish between dendrimer precursors and non-dendrimer precursors. Dendrimers have a shape that typically depends on the shape of the solubility of their component polymers in a given environment and can change significantly depending on the solvent and solutes around them, such as temperature, pH, or ion content.

The precursor can be a dendrimer, for example, as disclosed in U.S. Publication Nos. 2004/0086479 and 2004/0131582 and PCT Publication Nos. WO07005249, WO07001926, and WO06031358, or their U.S. equivalents; dendrimers can also be useful as multifunctional precursors, for example, as disclosed in U.S. Publication Nos. 2004/0131582 and 2004/0086479 and PCT Publication Nos. WO06031388 and WO06031388; each of said U.S. and PCT applications is hereby incorporated by reference in its entirety to the extent that it does not contradict the disclosure herein. Dendrimers are highly ordered, have a high surface area to volume ratio, and exhibit numerous end groups for potential functionalization. Embodiments include multifunctional precursors that are not dendrimers.

Some embodiments include precursors consisting essentially of oligopeptide sequences of no more than five residues, such as amino acids comprising at least one amine, thiol, carboxyl, or hydroxyl side chain. The residues are amino acids, such as those found in nature or derived therefrom. The backbone of such oligopeptides may be natural or synthetic. In some embodiments, peptides of two or more amino acids are combined with synthetic backbones to produce the precursors; some embodiments of such precursors have a molecular weight in the range of about 100 to about 10,000 or about 300 to about 500. The skilled artisan will immediately understand that all ranges and values between these explicitly stated ranges are contemplated.

In some embodiments, the precursor can be prepared into the amino acid sequence that does not contain the enzyme cleavable by introducing the site, comprises the sequence that does not contain easily attached by metalloproteinase and/or collagenase.In addition, precursor can be manufactured into and does not contain all amino acid, or does not contain and exceeds about 50,30,20,10,9,8,7,6,5,4,3,2 or 1 amino acid whose amino acid sequence.Precursor can be non-protein, means that they are not naturally occurring protein and can not make and can not make by adding synthetic material to protein by making naturally occurring protein cracking.Precursor can be non-collagenous, non-fibrin, non-fibrinogen and non-albumin, means that they are not one of these proteins and are not the chemical derivative of one of these proteins. The use of non-protein precursor and the limited use of amino acid sequence can be useful for avoiding immunoreaction, avoiding unwanted cell recognition and avoiding the danger relevant with using the protein that is derived from natural source. The precursor may also be non-sugar (containing no sugars) or substantially non-sugar (containing no sugars exceeding about 5% weight/weight of the precursor's molecular weight. Thus, for example, the precursor may exclude hyaluronic acid, heparin, or gellan. The precursor may also be both non-protein and non-sugar.

Peptides can be used as precursors. Generally, peptides with less than about 10 residues are preferred, although larger sequences (such as proteins) can be used. The technician will immediately understand that each range and value included in these clear and definite ranges, such as 1-10, 2-9, 3-10, 1, 2, 3, 4, 5, 6 or 7. Some amino acids have nucleophilic groups (such as primary amines or thiols) or can be derived to introduce nucleophilic groups or electrophilic groups (such as carboxyl or hydroxyl) groups when necessary. If the synthetic polyamino acid polymers are not found in nature and are engineered to be different from naturally occurring biomolecules, they are generally considered to be synthetic.

Some organogels and hydrogels are made with precursors containing polyethylene glycol. Polyethylene glycol (PEG, also known as polyethylene oxide when present at high molecular weight) refers to a polymer having a repeating group ( _CH2CH2O ) _n where n is at least 3. A polymeric precursor having _polyethylene glycol therefore has at least three of these repeating groups connected to one another in a straight line. The polyethylene glycol content of a polymer or arm is calculated by summing up all the polyethylene glycol groups on the polymer or arm, even if they are interrupted by other groups. Thus, an arm having at least 1000 MW of polyethylene glycol has enough CH2CH2O groups to total at least 1000 MW. As is customary in these fields, a polyethylene glycol polymer does not necessarily refer to a molecule terminated in a hydroxyl group. The symbol k is used to abbreviate molecular weight in thousands, for example, 15K means 15,000 molecular weight, i.e., 15,000 daltons. SG refers to succinimidyl glutarate. SS refers to succinimidyl succinate. SAP refers to succinimidyl adipate. SAZ refers to succinimidyl azelate. SS, SG, SAP and SAZ are succinimidyl esters having an ester group that degrades by hydrolysis in water. Hydrolytically degradable therefore refers to a material that degrades spontaneously in vitro in excess water in the absence of any enzymes or cells to mediate degradation. Degradation time refers to the effective disappearance of the material as judged by the naked eye. Trilysine (also abbreviated as LLL) is a synthetic tripeptide. PEG and/or hydrogels, and compositions comprising the same, can be provided in a pharmaceutically acceptable form, meaning that they are highly purified and free of contaminants such as pyrogens.

functional groups

Precursors for covalent crosslinking have functional groups that react with each other outside the patient or in situ to form materials. The functional groups typically have polymerizable groups for polymerization or react with each other in electrophile-nucleophile reactions or are configured to participate in other polymerization reactions. Aspects of polymerization reactions are discussed in the precursor section herein.

Thus, in some embodiments, the precursor has a polymerizable group that is activated, for example, by photoinitiation or redox systems as used in the field of polymerization, or an electrophilic functional group that is a carbodiimidazole, a sulfonyl chloride, a chlorocarbonate, an n-hydroxysuccinimidyl ester, a succinimidyl ester, or a sulfasuccinimidyl ester, or as described in U.S. Patent Nos. 5,410,016 or 6,149,931, each of which is hereby incorporated by reference in its entirety to the extent that it does not conflict with the explicit disclosure herein. Nucleophilic functional groups can be, for example, amines, hydroxyls, carboxyls, and thiols. Another class of electrophiles is acyl groups, for example, as described in U.S. Patent No. 6,958,212, which describes, inter alia, a Michael addition scheme for reacting polymers.

Some functional groups, such as alcohols or carboxylic acids, generally do not react with other functional groups, such as amines, under physiological conditions (e.g., pH 7.2-11.0, 37° C.). However, such functional groups can be made more reactive by using activating groups, such as N-hydroxysuccinimide. Some activating groups include carbonyldiimidazole, sulfonyl chloride, aryl halide, sulfosuccinimidyl ester, N-hydroxysuccinimidyl ester, succinimidyl ester, epoxide, aldehyde, maleimide, imidate, etc. N-hydroxysuccinimide ester or N-hydroxysulfosuccinimide (NHS) group is a useful group for cross-linking proteins or amine-containing polymers, such as amino-terminated polyethylene glycol. The advantage of the NHS-amine reaction is that the reaction kinetics are favorable, but the gelation rate can be adjusted by pH or concentration. The NHS-amine cross-linking reaction results in the formation of N-hydroxysuccinimide as a by-product. Sulfonated or ethoxylated forms of N-hydroxysuccinimide have relatively increased solubility in water and therefore their rapid clearance from the body. The NHS-amine cross-linking reaction can be carried out in an aqueous solution and in the presence of a buffer such as a phosphate buffer (pH 5.0-7.5), a triethanolamine buffer (pH 7.5-9.0), or a borate buffer (pH 9.0-12), or a sodium bicarbonate buffer (pH 9.0-10.0). Due to the reaction of the NHS group with water, it is preferred to prepare an aqueous solution of the NHS-based cross-linker and the functional polymer just prior to the cross-linking reaction. The reaction rate of these groups can be slowed by maintaining these solutions at a relatively low pH (pH 4-7). A buffer may also be included in the hydrogel introduced into the body.

In some embodiments, each precursor comprises only nucleophilic functional groups or only electrophilic functional groups, as long as both nucleophilic and electrophilic precursors are used in the cross-linking reaction. Thus, for example, if the cross-linking agent has a nucleophilic functional group such as an amine, the functional polymer may have an electrophilic functional group such as N-hydroxysuccinimide. On the other hand, if the cross-linking agent has an electrophilic functional group such as sulfosuccinimide, the functional polymer may have a nucleophilic functional group such as an amine or a thiol. Thus, functional polymers such as proteins, poly (allylamine) or amine-terminated di- or multifunctional poly (ethylene glycol) can be used.

One embodiment has reactive precursor species each possessing 3-16 nucleophilic functional groups and reactive precursor species each possessing 2-12 electrophilic functional groups; the skilled artisan will immediately understand that all ranges and values within the explicitly stated ranges are contemplated.

The functional group can be, for example, an electrophile that can react with a nucleophile, a group that can react with a specific nucleophile such as a primary amine, a group that forms an amide bond with a material in a biological fluid, a group that forms an amide bond with a carboxyl group, an activated acid functional group, or a combination thereof. The functional group can be, for example, a strong electrophilic functional group, meaning an electrophilic functional group that effectively forms a covalent bond with a primary amine in an aqueous solution at pH 9.0 at room temperature and pressure and/or an electrophilic group that reacts via a Michael-type reaction. Strong electrophiles can be of a type that does not participate in Michael-type reactions or of a type that participates in Michael-type reactions.

Michael-type reactions refer to 1,4 addition reactions of nucleophiles on conjugated unsaturated systems. The addition mechanism can be purely polar, or proceed through a free radical-like intermediate state; Lewis acids or appropriately designed hydrogen-bonding species can act as catalysts. The term conjugated can refer to the alternation of carbon-carbon, carbon-heteroatom, heteroatom-heteroatom multiple bonds and single bonds, or the attachment of functional groups to macromolecules such as synthetic polymers or proteins. Michael-type reactions are discussed in detail in U.S. Patent No. 6,958,212, which is hereby incorporated by reference in its entirety for all purposes to the extent that it does not conflict with the explicit disclosure herein.

Examples of strong electrophiles that do not participate in Michael-type reactions are: succinimide, succinimidyl esters, or NHS-esters. Examples of Michael-type electrophiles are acrylates, methacrylates, methyl methacrylate, and other unsaturated polymerizable groups.

Initiation system

Some precursors use initiator reactions. The initiator group is a chemical group that can initiate a free radical polymerization reaction. For example, it can exist as an independent component or as a pendant group on the precursor. The initiator group includes thermal initiators, photoactivatable initiators and oxidation-reduction (redox) systems. Long-wave UV and visible light photoactivatable initiators include, for example, ethyl eosin groups, 2,2-dimethoxy-2-phenylacetophenyl groups, other acetophenone derivatives, thioxanthone groups, benzophenone groups, and camphorquinone groups. Examples of thermally reactive initiators include 4,4'azobis(4-cyanovaleric acid) groups and analogs of benzoyl peroxide groups. Several commercially available low-temperature free radical initiators, such as V-044 available from Wako Chemicals USA, Inc., Richmond, Va., can be used to initiate free radical crosslinking reactions at body temperature to form a hydrogel coating with the aforementioned monomers.

Metal ions can be used as oxidizing agents or reducing agents in redox initiation systems. For example, ferrous ions can be used in combination with peroxides or hydroperoxides to initiate polymerization, or as part of a polymerization system. In this case, ferrous ions will serve as reducing agents. Alternatively, metal ions can be used as oxidizing agents. For example, ceric ions (the 4+ valence state of cerium) interact with various organic groups (including carboxylic acids and carbamates) to transfer electrons to the metal ion and leave initiating free radicals on the organic group. In such systems, metal ions act as oxidizing agents. Potentially suitable metal ions for either role are any transition metal ions, lanthanides, and actinides having at least two readily accessible oxidation states. Particularly useful metal ions have at least two states separated by only one difference in charge. Among these, the most commonly used are ferric iron/ferrous iron; cupric/cuprous; ceric/ceric; cobalt/cobalt; vanadates V to IV; permanganates; and manganese/manganese. Peroxygen-containing compounds such as peroxides and hydroperoxides, including hydrogen peroxide, t-butyl hydroperoxide, t-butyl peroxide, benzoyl peroxide, cumyl peroxide, can be used.

An example of an initiating system is a combination of a peroxy compound in one solution and a reactive ion, such as a transition metal, in another solution. In this case, when the moieties containing two complementary reactive functional groups interact at the site of application, no external polymerization initiator is required and the polymerization proceeds spontaneously without the application of external energy or the use of an external energy source.

Visualization reagents

Visualizing agents can be used as powders in xerogels/hydrogels that reflect or emit light at wavelengths detectable by the human eye, allowing a user applying the hydrogel to visualize the object when it contains an effective amount of the agent. Agents that require mechanical assistance for imaging are referred to herein as imaging agents, and examples include radiopaque contrast agents and ultrasound contrast agents.

Some biocompatible visualization agents are FD&C BLUE #1, FD&C BLUE #2, and methylene blue. These agents are preferably present in the final electrophilic-nucleophilic reactive precursor mixture at a concentration exceeding 0.05 mg/ml and preferably in a concentration range of at least 0.1 to about 12 mg/ml, and more preferably in a range of 0.1 to 4.0 mg/ml, although greater concentrations can potentially be used, up to the solubility limit of the visualization agent. The visualization agent can be covalently linked to the molecular network of the xerogel/hydrogel, thus maintaining visualization after application to the patient until the hydrogel hydrolyzes to dissolve it.

The visualization agent can be selected from any of a variety of non-toxic coloring substances suitable for use in medical implantable medical devices, such as FD&C BLUE dyes 3 and 6, eosin, methylene blue, indocyanine green, or coloring dyes commonly found in synthetic surgical sutures. Reactive visualization agents such as NHS-fluorescein can be used to introduce visualization agents into the molecular network of the xerogel/hydrogel. The visualization agent can be present together with a reactive precursor substance such as a cross-linking agent or a functional polymer solution. The preferred coloring substance may or may not become chemically bound to the hydrogel. The visualization agent can be used in small amounts, such as 1% weight/volume, more preferably less than 0.01% weight/volume, and most preferably less than 0.001% weight/volume; the skilled person will immediately understand that all ranges and values within the explicitly stated ranges are contemplated. The agent tends to mark the location of the particles and provide an indication of their presence and dissolution rate.

Biodegradation

Xerogels can be formed from organogels so that when hydrated in physiological fluids, they form hydrogels that are water-degradable, as measured by the hydrolytic degradation of the hydrodegradable groups in vitro, which loses its mechanical strength and eventually dissipates in excess water. This test predicts hydrolysis-driven dissolution in vivo, a process that is the opposite of cell- or protease-driven degradation. However, it is worth noting that polyanhydrides or other conventionally used degradable materials that degrade to acidic components tend to cause inflammation in tissues. However, hydrogels can exclude such materials and may not contain polyanhydrides, anhydride bonds, or precursors that degrade to acids or diacids. The term degradation by solvation in water, also known as dissolution in water, refers to the process by which the matrix gradually dissolves, a process that does not occur for covalently cross-linked materials and materials that are insoluble in water.

For example, electrophilic groups such as SG (N-hydroxysuccinimidyl glutarate), SS (N-hydroxysuccinimidyl succinate), SC (N-hydroxysuccinimidyl carbonate), SAP (N-hydroxysuccinimidyl adipate), or SAZ (N-hydroxysuccinimidyl azelate) can be used with hydrolytically unstable ester linkages. More linear hydrophobic linkages such as pimelate, suberate, azelate, or sebacate linkages can also be used, where these linkages are less hydrolyzable than succinate, glutarate, or adipate linkages. Branched, cyclic, or other hydrophobic linkages can also be used. Polyethylene glycol and other precursors can be prepared using these groups. When water-degradable materials are used, crosslinked hydrogel degradation can proceed by water-driven hydrolysis of biodegradable segments. Polymers containing ester linkages can also be included to provide a desired degradation rate, where groups are added or subtracted near the esters to increase or decrease the degradation rate. Thus, degradable segments can be used to construct hydrogels with desired degradation profiles ranging from a few days to many months. For example, if polyglycolate is used as a biodegradable segment, the crosslinked polymer can be made to degrade in about 1 to about 30 days, depending on the crosslink density of the network. Similarly, a crosslinked network based on polycaprolactone can be made to degrade in about 1 to about 8 months. Degradation times generally vary depending on the type of degradable segment used, in the following order: polyglycolate < polylactic acid < polytrimethylene carbonate < polycaprolactone. Thus, degradable segments can be used to construct hydrogels with desired degradation profiles ranging from a few days to many months.

The biodegradable linkages in the organogel and/or xerogel and/or hydrogel and/or precursor can be water-degradable or enzymatically degradable. Illustrative water-degradable biodegradable linkages include polymers, copolymers, and oligomers of glycolide, dl-lactide, 1-lactide, dioxanone, esters, carbonates, and trimethylene carbonate. Illustrative enzymatically biodegradable linkages include peptide linkages that are cleavable by metalloproteinases and collagenases. Examples of biodegradable linkages include the following polymers and copolymers: poly(hydroxy acids), poly(orthocarbonates), poly(anhydrides), poly(lactones), poly(amino acids), poly(carbonates), and poly(phosphonates).

If it is desired that the biocompatible cross-linked matrix be biodegradable or absorbable, one or more precursors having biodegradable linkages between functional groups may be used. The biodegradable linkages may also optionally serve as one or more water-soluble cores of the precursors used to make the matrix. For each approach, the biodegradable linkages may be selected so that the resulting biodegradable, biocompatible cross-linked polymer will degrade or be absorbed within a desired period of time.

Can select matrix material, make degradation product be absorbed into circulatory system and be removed from health substantially via kidney filtration.Matrix material can be hydrogel in physiological solution.One method is to select precursor that does not decompose in the body, wherein the connection between precursor degrades to return to precursor or precursor with the little change caused by covalent crosslinking process.This approach is opposite with selecting biological matrix material destroyed by enzymatic process and/or material that is removed by macrophage or causes the material of by-product that is not actually water-soluble.The material removed from health by kidney filtration can use technology mark known to technician and detect in urine.Although there can be at least some theoretical loss of these materials to other body systems, the common outcome of material is kidney clearance process.Therefore, term removes substantially and refers to the material that usually removes by kidney.

Application

Administration of the xerogel can be performed directly into the site of interest. For example, a xerogel lenticule can be applied to the cornea, or a membrane can be applied to the dermis or epidermis. Xerogel particles can be administered by inhalation. Furthermore, powder delivery systems can be used to inject xerogel powder directly into tissue.

Administration of the xerogel may also involve hydration near or just before the time of use. The xerogel is exposed to an aqueous solution, such as saline, and allowed to absorb water to form a hydrogel. The hydrogel is implanted directly, surgically, or via injection through a syringe or catheter.

Embodiments of the present invention include administration at or near the eye. The structure of the mammalian eye can be divided into three main layers or membranes: the tunica fibrosus, the tunica vascularis, and the tunica neuropil. The tunica fibrosus, also known as the tunica fibrosus of the eye, is the outer layer of the eyeball composed of the cornea and the sclera. The sclera is the supporting wall of the eye and gives the eye most of its white color. It extends from the cornea (the transparent front part of the eye) to the optic nerve at the back of the eye. The sclera is a fibrous, elastic and protective tissue composed of tightly packed collagen fibrils and contains about 70% water.

Overlying the fibrous membrane is the conjunctiva. The conjunctiva is a membrane that covers the sclera (the white part of the eye) and lines the inside of the eyelid. It helps lubricate the eye by producing mucus and tears, although it produces a smaller volume of tears than the tear glands. The conjunctiva is typically divided into three parts: (a) the palpebral or tarsal conjunctiva, which is the conjunctiva that lines the eyelid; the palpebral conjunctiva folds back at the upper and lower fornixes to become the bulbar conjunctiva, (b) the forniceal conjunctiva: the conjunctiva where the inner part of the eyelid meets the eyeball, and (c) the bulbar conjunctiva: the conjunctiva that covers the eyeball above the sclera. This area of the conjunctiva is tightly (firmly) bound and moves with the movement of the eyeball. The conjunctiva effectively surrounds, covers, and is attached to the sclera. It has cells and connective tissue, is slightly elastic, and can be removed, cut off, or otherwise taken off to expose the surface area of the sclera.

The tunica vascularis, also known as the ocular tunica, is the middle vascularized layer that includes the iris, ciliary body, and choroid. The choroid contains blood vessels that supply oxygen to the retinal cells and remove waste products of respiration. The tunica neuritrina, also known as the optic nerve membrane, is the inner sensory organ that includes the retina. The retina contains light-sensitive rods and cones and associated neurons. The retina is a relatively smooth (but curved) layer. It has two points where it is distinct: the fovea and the optic disc. The fovea is a depression in the retina just opposite the lens that is densely packed with cones. The fovea is part of the central area of the retina (macula). The fovea is largely responsible for a person's color vision and enables the high acuity that is necessary for reading. The optic disc is the point on the retina where the optic nerve penetrates the retina to connect to the nerve cells within it.

The mammalian eye can also be divided into two main segments: the anterior segment and the posterior segment. The anterior segment consists of the anterior chamber and the posterior chamber. The anterior chamber is located in front of the iris and behind the corneal endothelium and includes the pupil, iris, ciliary body, and aqueous humor. The posterior chamber is located behind the iris and in front of the vitreous humor, with the lens and zonular fibers located between the anterior and posterior capsules in a watery (aqueous) environment.

The cornea and lens help to converge light to focus on the retina. The lens behind the iris is a convex, elastic disk that focuses light on the retina through a second body fluid. It is attached to the ciliary body via a ring of suspensory ligaments called the zonule of Zinn. The ciliary muscle is relaxed to focus on distant objects, which stretches the fibers connecting it and the lens, thereby flattening the lens. Light enters the eye, passes through the cornea, and enters the first of the two body fluids (aqueous humor). About two-thirds of the total refractive power of the eye comes from the cornea with a fixed curvature. Aqueous humor is a transparent substance that connects the cornea of the eye to the lens, helps maintain the convex shape of the cornea (necessary for the convergence of light at the lens) and provides nutrients to the corneal endothelium.

The posterior segment is located behind the lens and in front of the retina. It occupies about two-thirds of the eye and includes the anterior vitreous membrane and all the structures behind it: the vitreous humor, retina, iris, and optic nerve. On the other side of the lens is the second humor (vitreous humor), which is bounded on all sides by the lens, ciliary body, zonules, and retina. It allows light to pass through without refraction, helps maintain the shape of the eye, and suspends the delicate lens.

FIG8 shows some delivery points at or near the eye 200. The eye 200 includes the sclera 212, iris 214, cornea 222, vitreous 232, zonular space 242, fovea 236, retina 238, and optic nerve 225. One area for delivery is locally at 260, where area 260 is indicated by a point on the surface of the eye 200. Another area is within the vitreous, as indicated by numeral 262, or transscleral, as indicated by numeral 264. In use, the xerogel (or gel or hydrogel or precursor thereof) is delivered, optionally through a needle 268, into the eye, either within the vitreous, as at 262, or periocularly, as at 272, using, for example, a syringe 266, a catheter (not shown), or other instrument. Another area is subconjunctival (not shown), below the conjunctiva 211 and above the sclera 212. The drug or other therapeutic agent is released into the intraocular space. In the case of posterior ocular diseases, drugs can be targeted to a general target area 274 via periocular or intravitreal routes, where they interact with biological features to achieve treatment. Embodiments include placing the xerogel in contact with the retina 238 or near the retina 238 without contacting it. For example, xerogels, hydrogels, and/or particles (or rods, microspheres, single materials, beads, or other shapes described herein) can be delivered to a location adjacent to or on the retina 238. Hydrogels are advantageously anchored in the vitreous gel and do not allow for diffusion of particles. In contrast, other systems using rods or slippery microspheres do not provide anchoring and diffusion or migration in response to movement or friction of the eye. Placing the reservoir at or near the retina (or other location) allows for high concentrations to be achieved at the desired site, where small particles can be used to deliver the drug for effective treatment. In contrast, spheres, rods, or other shapes that are too large to diffuse or migrate have a volume/surface area ratio that is unfavorable for effective controlled release. Another area for placement of xerogels, hydrogels, and/or particles, or other materials comprising such particles, is in the punctum (not shown), for example, by placing the particles in a punctal plug (silicone, polysaccharide, hydrogel, or other material) that is inserted into the punctum of the eye.

Sites where a drug delivery depot can be formed in or near the eye include, among others, the anterior chamber, the vitreous (intravitreal placement), extrascleral, in the posterior subtenon space (inferior fornix), subconjunctival, on the surface of the cornea or conjunctiva. Periocular drug delivery using subconjunctival, retrobulbar, or subtenon placement of ocular hydrogel implants has the potential to provide a safer and improved drug delivery system to the retina compared to topical and systemic routes.

An example of in situ placement is illustrated in FIG9A for intravitreal implantation. In FIG9A , a xerogel implant is intravitreally injected approximately 2.5 mm posterior to the limbus through a pars plana incision 390 using a subretinal cannula 392, as depicted by a magnifying glass 394 positioned to visualize the incision 390 in the eye 310. This can be performed after dissecting or otherwise clearing the conjunctiva, if desired. Subretinal cannula 392 (or other suitable cannula) is then inserted through incision 390 and positioned within the eye at the desired target site, e.g., at least one of sites 396, 398, 300 ( FIG9B ), where the xerogel is introduced and subsequently forms a hydrogel in situ. The xerogel forms an absorbable gel 302, 304, and/or 306 that adheres to the desired target site. Particles containing a therapeutic agent may be included in one or more of the gels. Notably, the precursor can be placed using a 9-gauge needle. Embodiments include placement using a 25-gauge needle. Additional embodiments include using needles with diameters smaller than 25 gauge, such as 26, 27, 30, 31, 32 gauge.

Intravitreal in situ implant embodiments can improve the efficacy and pharmacokinetics of effective therapeutic agents in the treatment of eye diseases and minimize patient side effects in several ways. First, the implant can be placed at the specific disease site in the vitreous cavity, bypassing local or systemic pathways and thereby increasing drug bioavailability. Second, the implant maintains local therapeutic concentrations at the specific target tissue site over an extended period of time. Third, the number of intravitreal injections will be significantly reduced during the 12-month treatment regimen, thereby reducing the patient's risk of infection, retinal detachment, and transient visual impairment (white spots floating in the vitreous), which can occur until the drug in the vitreous migrates downward toward the inferior wall of the eye and away from the central vitreous or mitral retina.

A xerogel or a xerogel hydrated into a hydrogel (xerogel/hydrogel) can be placed on scleral tissue with or without the presence of conjunctiva. The xerogel/hydrogel can be attached to the sclera or other tissues near the sclera to promote drug diffusion through the desired tissue or provide a stable reservoir to guide the therapeutic agent as needed. A hydrogel adhesive such as a sealant can be used as an adhesion aid. In some embodiments, the conjunctiva of the eye can be removed, macerated, cut away, or cut away so that the tissue can be lifted away from the sclera to access a specific area of the sclera for implantation or injection of the xerogel/hydrogel. The xerogel/hydrogel is placed to create a layer on the surface and adhere to the surface. The conjunctiva can be allowed to contact the tissue if it still exists or maintains sufficient mechanical integrity to do so. In some embodiments, the xerogel/hydrogel is composed of at least 50%, 75%, 80%, 90% or 99% water-soluble precursors (calculated by measuring the weight of the hydrophilic precursor and dividing it by the weight of all precursors, so as to ignore the weight of water or solvent or non-hydrogel components) to enhance the non-adhesive properties of the hydrogel. In some embodiments, such hydrophilic precursors substantially (substantially) comprise PEO. In some embodiments, drugs for reducing tissue adhesion mediated by biological mechanisms (including cell mitosis, cell migration, or macrophage migration or activation) are included, such as anti-inflammatory drugs, anti-mitotic agents, antibiotics, paclitaxel (PACLITAXEL), mitomycin (MITOMYCIN), or taxol.

In other embodiments, the sclera is substantially free of conjunctiva. The conjunctiva is a significant tissue mass (mass) covering much or all of the sclera. The conjunctiva can be punctured or penetrated with a needle, catheter, or trocar and the precursor introduced into the gap between the sclera and conjunctiva. This placement of the implant is referred to as subconjunctival placement. In some cases, the conjunctiva can be punctured to access the natural potential gap between the tissues to be filled by the precursor. In other cases, a potential or actual gap is mechanically created using a trocar, stent, or the like, which disrupts the adhesion between the sclera and conjunctiva, allowing the introduction of the precursor. The conjunctiva has sufficient elasticity to allow the introduction of a useful amount of xerogel or to force it into such a natural or created gap. Therefore, in some cases, the xerogel/hydrogel volume is about 0.25 to about 5 ml; the skilled artisan will immediately understand that all ranges and values within the explicitly stated ranges are contemplated, for example, about 1 ml or from 0.5 ml to about 1.5 ml.

Furthermore, removal of the xerogel, which has formed into a hydrogel, whether present in the eye or periocular area, is also readily accomplished using a vitrectomy cutter (if the implant is located in the vitreous cavity) or a manual I/A syringe and cannula (if the implant is located on the scleral surface) or an irrigation/aspiration handpiece. This contrasts with the major surgical procedures required to remove some conventional non-absorbable implants.

In a further embodiment, the xerogel/hydrogel material can be placed in the patient, for example, in a tissue or organ, including subcutaneous, intramuscular, intraperitoneal, in a potential gap in the body, or in a natural cavity or opening. The material provides a reservoir for the release of the agent over time. Embodiments therefore include a volume of about 0.5 to about 500 ml (referred to as the total volume in the case of a delivered particle assembly) for placement; the skilled person will immediately understand that all ranges and values within the clearly stated ranges are designed, such as 1-10 ml or 5-50 ml. Intraperitoneal or intramuscular injection, for example, is a useful area for extended controlled release of the agent over a period of hours, days, or weeks.

The material described herein can be used for delivering medicine or other therapeutic agents (such as imaging agents or markers). A kind of application mode is to apply the mixture of xerogel/hydrogel particles and other materials (such as therapeutic agents, buffer, accelerators, initiators) to the site by needle, cannula, catheter or hollow wire. The mixture can be delivered, for example, using a manually controlled syringe or a mechanically controlled syringe such as a syringe pump. Alternatively, a double syringe or multi-tube syringe or multi-lumen system can be used to mix xerogel/hydrogel particles at the site or near the site with hydration fluid and/or other reagents.

The xerogel can be provided to the site in a flowable form, for example, as flowable particles. The xerogel can be suspended in a liquid and applied to the site. The xerogel particles can be made to have a maximum diameter sufficient to be manually ejected from a syringe through a 3 to 5 French catheter or a 10-30 gauge needle. Those skilled in the art will readily appreciate that all ranges and values within the explicitly stated ranges are contemplated, for example, 25-30 gauge. The use of a small needle is particularly advantageous in the eye, a sensitive organ. Application to other organs is also advantageous, for example, to control bleeding or other lesions. The particles can be formed by generating a hydrogel and then breaking it into smaller pieces. The material can be ground, for example, in a ball mill or with a mortar and pestle, or chopped or diced with a knife or wire. Alternatively, the material can be chopped in a blender. Another method involves forcing the material from the organogel or gel step through a mesh, collecting the fragments, and passing them through the same mesh or another mesh until the desired size is reached, followed by the production of the xerogel. The xerogel/hydrogel can contain particles loaded with a therapeutic agent. Some or all of the hydrogel particles can contain particles loaded with a therapeutic agent. In some embodiments, a first population of therapeutic agent-loaded particles loaded with a first therapeutic agent is contained within a first population of xerogel particles, and a second population of therapeutic agent-loaded particles loaded with a second therapeutic agent is contained within a second population of xerogel particles. In this manner, multiple agents can be released from a single implant. Embodiments of particles include those having a specific shape, such as a sphere, rod, or disk.

Embodiments include placement of a plurality of xerogel/hydrogel particles. The xerogel/hydrogel particles can include a therapeutic agent, such as a protein such as anti-VEGF. The particles can be sized to be manually passed through a 27-gauge or smaller diameter needle. Pressure can be manually applied to force the particles through the needle.

An alternative for delivery of particles is to preform the gel into shaped particles and then introduce the material into the body. For example, the xerogel/hydrogel can be formed into spheres, rods, cylinders, or other shapes. Embodiments include solid rods of xerogel/hydrogel for subcutaneous implantation and delivery of one or more agents.

Xerogels/hydrogels as described herein can be used for tissue augmentation. The use of collagen for skin augmentation is well known. Xerogels/hydrogels, for example, particles, can be used as skin fillers or for tissue augmentation. Embodiments include injecting or otherwise placing multiple particles into tissue, or forming a hydrogel in situ. The material can be injected or otherwise placed at the desired site.

As described herein, xerogels/hydrogels can be used to separate tissues to reduce the radiation dose received by one of the tissues. As described in U.S. Patent No. 7,744,913 (which is hereby incorporated by reference herein for all purposes, wherein in the event of a conflict, this specification governs), a spacer material can be placed in a patient. Some embodiments include a method comprising introducing a spacer into a position between a first tissue location and a second tissue location to increase the distance between the first tissue location and the second tissue location. In addition, there may be a step of administering a dose of radiation to at least the first tissue location or the second tissue location. For example, the method is to deliver a therapeutic dose of radiation to the patient, comprising introducing a biocompatible, biodegradable granular xerogel, such as an aggregate of particles optionally having radiopaque contents, between the first tissue location and the second tissue location to increase the distance between the first tissue location and the second tissue location, and treating the second tissue location with a therapeutic dose of radiation such that the presence of the filler device results in the first tissue location receiving less radiation dose than the amount of radiation dose that the first tissue location would receive in the absence of the spacer. The spacer can be introduced as a xerogel that forms a hydrogel in the patient, which is removed by biodegradation of the spacer-hydrogel in the patient. An example is a case where the first tissue location is associated with the rectum and the second tissue location is associated with the prostate. The amount of radiation reduction can vary. Embodiments include at least about 10% to about 90%; a skilled artisan will readily appreciate that all ranges and values within the explicitly stated ranges are contemplated, for example, at least about 50%. Radiation can alternatively be directed toward a third tissue, such that, as a result of its separation from the other tissues, the first or second tissue receives a lower amount of radiation. The first and second tissues can be adjacent to each other in the body or separated from each other by other tissues. The volume of the spacer used to separate the tissues depends on the configuration of the tissues to be treated and the tissues to be separated from each other. In many cases, a volume of approximately 20 cubic centimeters (cc or ml) is suitable. In other embodiments, as little as about 1 cc may be required. Other volumes range from about 5 to 1000 cc; a skilled artisan will readily appreciate that all ranges and values within the explicitly stated ranges are contemplated, for example, 10 to 30 cc. In some embodiments, the spacer is administered in two doses at different times to allow the tissue to expand and accommodate the spacer and thereby receive a larger volume of the spacer than would otherwise be readily possible. The tissue to be separated by the spacer includes, for example, at least one of the rectum, prostate, and breast, or portions thereof. For example, a first portion of the breast can be separated from a second portion.

Reagent test kit

A kit or system for making a hydrogel from a xerogel can be prepared so that the xerogel is stored in the kit and made into a hydrogel when needed for use on a patient. In addition, a kit can be made for applying the xerogel in the form of a xerogel. An applicator can be used in combination with the xerogel and/or hydrogel. The kit is manufactured using medically acceptable conditions and contains components with sterility, purity, and pharmaceutically acceptable preparations. The kit may contain an applicator (when appropriate) and instructions. The xerogel particles comprising the therapeutic agent may be available for mixing with a solution provided in the kit or separately. The xerogel components may be provided as one or more containers of xerogel that form a hydrogel, wherein the xerogel is in the form of multiple particles to be placed in a patient, or as a whole implant. The solvent/solution may be provided in the kit or provided separately, or the components may be premixed with the solvent. The kit may include a syringe and/or needle for mixing and/or delivery. The kit or system may include the components described herein.

Some embodiments provide a single applicator, e.g., a syringe, that contains the xerogel particles for delivery, wherein an aqueous solution is added to the applicator for hydration, and the syringe is then used to place the material in the patient. The xerogel particle solvent can be substantially aqueous, meaning that the solvent is approximately 99% water by volume, with salts or buffers present as needed. Other safe and biocompatible solvents, such as dimethyl sulfoxide, can be used. The xerogel particles can further include powders of proteins and/or other reagents.

The packaging of the precursor and/or the entire kit is preferably carried out under dry conditions in the absence of oxygen. The precursor and/or kit components can be placed in an airtight sealed container such as a glass or metal (foil) container that is impermeable to moisture or oxygen.

At the end of the implantable material manufacturing process, the xerogel containing protein powder or other solid phase, water-soluble biological reagent can be subjected to gamma sterilization. Alternatively or further, before and/or after the assembly and sealing of the test kit, a sterilization process can be present. In this technology, low moisture conditions are often useful. It has been observed that the aggregate formation and cross-linking of the solid phase dispersed powder resistance under gamma radiation. This result is unexpected and surprising because gamma radiation sterilization is generally considered to damage protein or peptide biological reagents. Not subject to the constraint of a specific working principle, it is believed that the non-existence of small particle size and moisture is unfavorable for these undesirable reactions.

Further description

(1) A first embodiment of the present invention relates to a method for manufacturing a medical material, comprising forming an organogel around a powder of a water-soluble biological agent, wherein the powder is dispersed in the organogel. (2) A second embodiment of the present invention relates to a method for manufacturing a medical material, comprising forming a gel around a powder of a water-soluble biological agent, wherein the powder is dispersed in the gel, wherein forming the gel comprises preparing a melt of one or more precursors and covalently crosslinking the precursors. (3) A third embodiment of the present invention relates to a method for manufacturing a medical material, comprising forming an organogel around particles of a powder of a biological agent, wherein the particles are dispersed in the organogel, and removing a solvent from the organogel to thereby form a xerogel, wherein the method is carried out in the absence of water. (4) A fourth embodiment of the present invention relates to a method for manufacturing a medical material, comprising forming an organogel or gel from a melt, manufacturing a xerogel from the (organo)gel, and providing the xerogel as an aggregate of particles, wherein the xerogel is a hydrogel when exposed to an aqueous solution. (5) A fifth embodiment of the present invention relates to a pharmaceutically acceptable material as in any one of embodiments I to IV. (6) A sixth embodiment of the present invention relates to a medical material comprising a pharmaceutically acceptable biodegradable xerogel comprising dispersed protein particles, the protein being a therapeutic agent and having secondary and/or tertiary structure. In addition, the protein can be released from the particles in an aqueous solution in a substantially non-denatured conformation. (7) A seventh embodiment of the present invention relates to a (pharmaceutically acceptable) biomaterial for controlled release of a therapeutic water-soluble biological agent comprising a pharmaceutically acceptable xerogel comprising solid particles of the biological agent dispersed therein (optionally, wherein the xerogel does not contain a hydrophobic material) and wherein when exposed to water, the xerogel is a hydrogel. (8) An eighth embodiment is a method of making any of the materials of embodiments VI or VII.

Further embodiments are: (9) As in any of 1-8, wherein the (water-soluble) biological agent is a protein. (10) As in any of 1-9, wherein the protein has a molecular weight of at least about 10,000 Daltons and a sugar is associated with the protein. (11) As in any of 1-10, wherein the powder is used and is a first powder, wherein the method further comprises a second powder comprising a second water-soluble biological agent, wherein the first powder and the second powder are dispersed throughout the organogel. (12) As in any of 1-11, wherein the powder is used and has an average particle size of about 1 μm to about 10 μm. (13) As in any of 1-12, wherein the organogel is formed in the absence of an aqueous solution. (14) As in any of 1-13, comprising removing the solvent from the organogel, if desired, to thereby form a xerogel. (15) As in any of 1-14, comprising removing the solvent by a method selected from vacuum removal, lyophilization, and freezing followed by application of a vacuum. (16) As in any of items 1-15, comprising the xerogel, wherein the xerogel is a hydrogel when exposed to an aqueous solution. (17) As in any of items 1-15, comprising the powder, wherein the (water-soluble) biological agent is substantially retained in the powder in a solid phase when the hydrogel is formed, and slowly dissolves over a period of time when the hydrogel is exposed to a physiological fluid in a mammalian body. (18) As in 17, wherein the dissolution over a period of time is in the range of about 1 week to about 52 weeks. (19) As in any of items 1-18, wherein the biological agent in the gel is a protein having secondary and/or tertiary structure, wherein the protein is released in a substantially non-denatured conformation, as can be measured by, for example, enzyme-linked immunosorbent assay and isoelectric focusing. (20) As in any of items 1-19, wherein the gel or organogel or xerogel comprises a covalently cross-linked hydrophilic polymer. (21) As in any of items 1-20, wherein the gel organogel or xerogel organogel comprises a covalently cross-linked hydrophilic polymer selected from the group consisting of polyethylene oxide, polyvinyl pyrrolidone, hyaluronic acid, polyhydroxyethyl methacrylate, and block copolymers thereof. (22) As in any of items 1-21, wherein, when the hydrogel is present, the hydrogel is biodegradable by spontaneous hydrolysis through hydrolytically degradable linkages selected from esters, carbonates, anhydrides, and orthocarbonates. (23) As in any of items 1-22, wherein, when the organogel is present, the organogel comprises a block copolymer that forms the organogel and, after removal of the solvent to form the xerogel, forms a hydrogel upon exposure to an aqueous solution. (24) As in any of items 1-23, wherein, when the organogel is present, the organogel comprises a polymer wherein the organogel (and the hydrogel) comprises an ionically cross-linked polymer. (25) As in any of items 1-24, wherein, when the organogel is present, the organogel comprises a member selected from alginate, gellan, collagen, and polysaccharide. (25) As in any of items 1-24, comprising forming a plurality of particles from: (a) the gel, (b) the organogel, (c) a xerogel made from the gel or the organogel, or (d) a hydrogel made from the gel or the organogel. (26) As in any of items 1-25, wherein, when the organogel is present, the organogel is formed from a precursor in an organic solvent, wherein the precursor chemically reacts to form covalent bonds to thereby form the organogel, wherein the organogel is covalently cross-linked. (27) As in any of items 1-26, wherein the precursor reacts by free radical polymerization to form the organogel. (28) As in any one of items 1-27, wherein the precursor is a first precursor comprising a first functional group and further comprises a second precursor comprising a second functional group, wherein the first functional group and the second functional group are reactive in the organic solvent to form a covalent bond. (29) As in 28, wherein the first functional group and the second functional group are each selected from an electrophile and a nucleophile, and the reaction between the first functional group and the second functional group is an electrophilic-nucleophilic reaction to form a covalent bond. (30) As in 28 or 29, wherein the electrophilic group comprises succinimide, succinimide ester, n-hydroxysuccinimide, maleimide, succinate, nitrobenzene carbonate, aldehyde, vinyl sulfone, azide, hydrazide, isocyanate, diisocyanate, tosyl, tresyl, or carbonyldiimidazole. (31) As in any one of items 28-30, wherein the nucleophilic group comprises a primary amine or a primary thiol. (32) As in any of items 28-31, wherein the first precursor and the second precursor are water-soluble. (33) As in any of items 28-32, wherein at least one of the first precursor and the second precursor comprises a synthetic polymer. (34) As in any of items 28-33, wherein the first precursor comprises a polymer selected from the group consisting of polyethylene glycol, polyacrylic acid, polyvinyl pyrrolidone, and block copolymers thereof. (35) As in any of items 1-34, comprising the organogel, comprising preparing the organogel into a structure selected from the group consisting of rods, sheets, particles, spheres, and aggregates of at least one thereof. (36) As in any of items 1-35, comprising, or further comprising, a therapeutic agent, wherein the therapeutic agent comprises a fluoroquinolone, moxifloxacin, travoprost, dexamethasone, an antibiotic, or vestibulotoxin. (37) As in 36, wherein the organogel further comprises a permeation enhancer. (38) As in any of items 1-8, wherein the organogel is physically crosslinked by domain formation, the method further comprising forming the organogel from a precursor in an organic solvent, wherein the precursor is a block copolymer comprising a first block and a second block. (39) As in 38, comprising heating the mixture of the precursor and the organic solvent and allowing the solution to cool, thereby precipitating at least the first block of the copolymer precursor, wherein the domain comprises at least the first block. (40) As in 38 or 39, comprising mixing the precursor in a first organic solvent in which the copolymer precursor is dissolved, wherein all blocks of the copolymer precursor are soluble in the first organic solvent, and adding a second organic solvent miscible with the first organic solvent, wherein the first block of the copolymer precursor is insoluble in the second organic solvent, wherein the second solvent is effective for forming the domains, wherein the domains comprise the first block of the copolymer. (41) As in any of items 38-40, wherein the copolymer precursor comprises a block selected from polyethylene glycol. (42) As in any of items 38-41, wherein the copolymer precursor further comprises a second block selected from the group consisting of polylactic acid, polyglycolic acid, polytrimethylene carbonate, polydioxanone, polyalkylene glycol, polybutylene terephthalate, and polylysine. (43) As in any of items 1-37, wherein the organogel is free of hydrophobic material; or free of hydrophobic polymer, or free of all hydrophobic materials except the solvent (which may be slightly hydrophobic). (44) As in any of items 1-43, comprising preparing a powder of the biological agent according to a method that avoids denaturation of the biological agent, and once the powder has been prepared, preventing the powder from being exposed to water. (45) As in any of items 1-44, wherein the biological agent is a therapeutic protein having a secondary and/or tertiary structure. (46) As in any of items 1-45, comprising a xerogel, wherein the xerogel is a hydrogel after exposure to water. (47) As in any of items 1-46, wherein the hydrogel, or the hydrogel made from the gel/organogel/xerogel, is biodegradable. (48) As in any of items 1-47, comprising the xerogel, wherein the cumulative release of the agent reaches 90% weight/weight of the agent over a period of about 1 month to about 6 months after the hydrogel and particles are placed in a saline solution. (49) As in any of items 1-48. (50) As in any of items 1-49, wherein the xerogel comprises a covalently cross-linked hydrophilic polymer. (51) As in any of items 1-50, wherein the water-soluble biological agent is a protein having a secondary and/or tertiary structure. (52) As in any of items 1-51, wherein the water-soluble biological agent is substantially retained in a solid phase when the hydrogel is formed and slowly dissolves over a period of time when the hydrogel is exposed to a physiological fluid in a mammalian body. (53) A biomaterial as in any one of 1-52, comprising the organogel, wherein the organogel comprises a covalently cross-linked hydrophilic polymer. (54) A biomaterial as in 53, wherein the polymer comprises a member selected from the group consisting of polyethylene oxide, polyvinyl pyrrolidone, hyaluronic acid, polyhydroxyethyl methacrylate, and block copolymers thereof. (54) A biomaterial as in any one of 1-53, wherein the material is a structure selected from the group consisting of rods, sheets, particles, spheres, and aggregates thereof. (55) Any one of 1-54, comprising the xerogel, or a method of providing the xerogel as an aggregate of particles, for example, by a method selected from the group consisting of: (a) manufacturing the organogel and crushing it to form particles for the aggregate, (b) manufacturing the xerogel and crushing the xerogel to form particles for the aggregate, and (c) manufacturing the organogel into a plurality of particles for the aggregate, wherein the particles are freed of an organic solvent to manufacture the xerogel. (56) A method as in 55, comprising producing a plurality of particle aggregates, wherein the aggregates have different in vivo degradation rates, and mixing the aggregates to produce a biomaterial having desired degradation properties.

These embodiments 1-56 can further be prepared as a kit having a polymer, a biological agent or protein, and an applicator, wherein the kit is in a sterile container. These embodiments 1-56 can further be practiced by placing the material, or a material made by one of the methods, in contact with a patient's tissue. Examples of tissues are the intraperitoneal space, muscle, dermis, epidermis, natural cavities or spaces, the abdominal cavity, the prostate, the rectum, a location between the prostate and the rectum, the chest, tissue between the radiation target and healthy tissue, and the vasculature.

Example

Example 1. Preparation of organogels and xerogels containing protein particles

Polyethylene glycol (PEG) compounds

PEG compounds are obtained using the following structures:

Table 1 PEG esters

Preparation of PEG solution

Weigh out the PEG powder as shown in the table below and place it in a 10 ml graduated cylinder:

Table 2 Preparation of PEG ester solutions in dichloromethane

Example PEG esters (g) 1A-1 8a15kSS 0.86 1B-1 4a20kSG 1.33 1C-1 8a15kSG 0.86 1D-1 4a20kSAP 1.33 1E-1 4a20kSGA 1.33

Table 3 Preparation of PEG amine solution in dichloromethane

Example PEG amine (g) 1A-2 8a20KNH2 1.14 1B-2 8a20KNH2 0.67 1C-2 8a20KNH2 1.14 1D-2 8a20KNH2 0.67 1E-2 8a20KNH2 0.67

Once the PEG was dissolved, dichloromethane was added to the 10 mL mark.

Preparation of ground egg albumin

In a nitrogen-filled glove bag, egg albumin (Worthington Biochemical Corporation; LS003048) was ground using a mortar and pestle and sieved through a stainless steel sieve to particles less than 20 μm.

Preparation of ovalbumin organogel

The ground ovalbumin was weighed into a polyethylene female Luer-Lok syringe. The PEG amine solution was mixed with the ovalbumin to form a suspension. The PEG ester solution was placed into a male polyethylene Luer-Lok syringe. The syringes were paired and the solutions were mixed syringe to syringe for 10 seconds and allowed to sit in the male syringe for 10 minutes, during which time a gel containing the protein formed. The syringes were cut open and the gel-protein cylinder was removed. The gel was placed under vacuum overnight to dry. The following table summarizes the samples prepared in this manner.

Table 4 Preparation of albumin organogel

Preparation of ovalbumin-PEG xerogel

The syringe containing the ovalbumin organogel was cut open and the gel-protein cylinder was removed. The gel was placed under vacuum overnight to dry. The dried xerogel was stored at 5° C. under a nitrogen headspace.

Preparation of ground rabbit IgG

Rabbit IgG (IgG from rabbit serum; Sigma; >95%) was manually ground using a mortar and pestle in a nitrogen-filled glove bag and sieved through a stainless steel sieve to less than 20 μm.

Preparation of Rabbit IgG Organogel

Ground rabbit IgG was weighed into a polyethylene female Luer-Lock syringe. The PEG amine solution was mixed with the ovalbumin to form a suspension. The PEG ester solution was placed into a polyethylene male Luer-Lock syringe. The syringes were paired and the solutions were mixed syringe to syringe for 10 seconds and allowed to rest in the male syringe for 10 minutes to form a gel containing the protein. The syringes were cut open and the gel-protein cylinder was removed. The gel was placed under vacuum overnight to dry. The following table summarizes the samples prepared in this manner. The following table summarizes the samples prepared in this manner.

Table 5 Preparation of rabbit IgG organogel

Preparation of rabbit IgG-PEG xerogel

The syringe containing the rabbit IgG organogel was cut open and the gel-protein cylinder was removed. The gel was placed under vacuum overnight to dry. The dried xerogel was stored at 5° C. under a nitrogen headspace.

Example 2. In vitro release of proteins from hydrogels

Protein stability in buffer solutions

Ovalbumin (Worthington Biochemical Corporation; LS003048) and rabbit IgG (IgG from rabbit serum; Sigma; >95%) were dissolved in TRIS buffer at 0.065 mg/ml. An initial sample was taken for baseline and at various time points to determine protein stability in the buffer. The samples were analyzed for protein content by HPLC and ELISA. The results are summarized in the table below.

Table 6 HPLC protein stability study (50 mL Tris buffer, pH 8.5, shaking at 50 rpm)

Elapsed time (hours) Collected egg albumin IgG collected 0.00 100.0% 100.0% 2.00 98.3% 97.1% 6.00 97.2% 99.5% 24.00 95.5% 97.7% 48.00 95.0% 98.3% 96.00 94.1% 93.3%

Table 7 ELISA protein stability study (50 mL Tris buffer, pH 8.5, shaking at 50 rpm, 37°C)

Elapsed time (hours) Collected egg albumin IgG collected 5 minutes 97.7% 109.5% 2 hours 99.5% 87.1% 6 hours 98.4% 85.5% 24 hours 91.3% 76.0% 48 hours 99.9% 78.8% 96 hours 70.1% 83.8%

The results showed that the protein was sufficiently stable for use in accelerated in vitro protein release testing.

In vitro sustained protein release studies

The xerogel sample from Example 1 was cut, weighed and added to 50 ml of TRIS buffer in a 50 mL centrifuge tube. A stainless steel dissolution cage was used to hold the sample in the lower half of the centrifuge tube. The tube was immersed in a shaking water bath at 37°C and 50 RPM.

Table 8 Accelerated and real-time in vitro protein release studies

Buffer medium samples were taken at 2 hours, 4 hours, and 8 hours and every 8 hours thereafter until gel degradation. The buffer medium was completely exchanged at each time point. The collected samples were analyzed by HPLC and ELISA. The results are shown graphically in Figures 2-5.

Customized drug release profile

Combinations of various excipients can be used to tailor the release rate of the therapeutic agent. The release rates for each particle are combined and the composite total release rate is calculated, as depicted in Figures 6 and 7. Figure 6 depicts essentially zero-order release kinetics from about 10 to about 60 hours. Figure 7 depicts a fine-tuned system. There is a first-order release that provides an initial burst for the first 24 hours, followed by an additional zero-order release from about 24 to about 100 hours. Zero-order release continues until the final dissolution of the material.

Example 3. Formation of a cross-linked gel from a melt of a precursor

0.86 g of an approximately 15,000 dalton 8-arm branched PEG (8a15KSS) terminated with SS was melted at 50°C. 1.14 g of an approximately 20,000 dalton 8-arm branched PEG (8a20KNH2) terminated with a primary amine was weighed into a 10 ml syringe along with 0.5 g of bovine serum albumin (BSA) powder and then immersed in a 60°C water bath to melt the 8a20KNH2. A drop of 8a15KSS melt was placed next to a drop of 8a20KNH2 melt/BSA on a 50°C hot plate. The drops rapidly mixed to form a gel in less than 2 seconds. As observed by microscopy, the resulting gel contained solid BSA particles.

The formed gel was transferred to a scintillation vial filled with Tris-buffered saline (TBS) pH 8.5 buffer to allow for rapid hydrolysis of the polymer and release of BSA.

After gel degradation, the resulting TBS release medium was noted to be transparent, indicating solubility of BSA in TBS and showing no effect of the treatment on protein solubility in terms of aggregation or denaturation.

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The patents, patent applications, patent publications, and references cited herein are hereby incorporated by reference for all purposes; in case of conflict, the present specification controls.

Claims (23)

1. A method for manufacturing medical materials, including An organic gel is formed around a powder containing a water-soluble protein, wherein one or more hydrophilic precursors react in an organic solvent to form covalent bonds in the presence of the powder to form the organic gel, wherein the one or more precursors are soluble in the organic solvent prior to crosslinking and at least one of the hydrophilic precursors comprises at least 80 wt% polyethylene oxide (PEO) repeatings, the powder being at least 50 wt% protein particles, wherein the powder is dispersed in the organic gel, and The organic solvent is removed from the organic gel to form a dry gel without exposing the protein to the interface between water and the organic solvent, wherein the dry gel forms a hydrogel upon exposure to an aqueous solution. 2. The method of claim 1, wherein the protein has a molecular weight of at least 10,000 Daltons and the sugar is associated with the protein. 3. The method of claim 1 or 2, wherein the powder is a first powder, wherein the method further comprises a second powder comprising a further water-soluble biological reagent, wherein the first powder and the second powder are dispersed throughout the organic gel. 4. The method of claim 1 or 2, wherein the organic gel is formed in the absence of an aqueous solution. 5. The method of claim 1, wherein the solvent is removed by a method selected from: vacuum removal, lyophilization, and freezing followed by vacuum. 6. The method of claim 1, wherein the protein is held in the powder in a solid phase when the hydrogel is formed, and slowly dissolves over a period of time when the hydrogel is exposed to a physiological solution in a mammal. 7. The method of claim 1, wherein the protein has a secondary and/or tertiary structure, wherein the protein is released in a non-denatured conformation that can be measured by enzyme-linked immunosorbent assay (ELISA) and isoelectric focusing. 8. The method of claim 1 or 2, wherein the hydrogel is biodegradable by spontaneous hydrolysis of a linker selected from the group consisting of esters and acid anhydrides. 9. The method of claim 1 or 2, further comprising forming a plurality of particles by: the organic gel, a dry gel made from the organic gel, or a hydrogel made from the organic gel. 10. The method of claim 1, wherein the precursor is reacted by free radical polymerization to form the organogel. 11. The method of claim 1, wherein the one or more hydrophilic precursors comprise a first precursor containing the PEO repeat and a first functional group and a second precursor containing a second functional group, wherein the first functional group and the second functional group are reactive in the organic solvent to form covalent bonds. 12. The method of claim 11, wherein the first functional group and the second functional group are each selected from an electrophile and a nucleophile, and the reaction between the first functional group and the second functional group is an electrophilic-nucleophilic reaction that forms a covalent bond. 13. The method of claim 12, wherein the electrophilic group comprises succinimide, succinimide ester, n-hydroxysuccinimide, maleimide, succinate, nitrobenzene carbonate, aldehyde, vinyl sulfone, azide, acylhydrazine, isocyanate, diisocyanate, toluenesulfonyl, trifluoroethanesulfonyl, or carbonyldiimidazole. 14. The method of claim 12, wherein the nucleophilic group comprises a primary amine or a primary thiol. 15. The method of claim 11, wherein the first precursor and the second precursor comprise synthetic polymers. 16. The method of claim 11, wherein the first precursor comprises a polymer selected from polyethylene glycol, polyacrylic acid, polyvinylpyrrolidone, and block copolymers thereof. 17. The method of claim 1 or 2, wherein the organic gel is free of hydrophobic materials. 18. The method of claim 1 or 2, comprising preventing the powder from being exposed to water until the medical material is used on a patient once the powder has been prepared. 19. Biomaterials manufactured by any one of claims 1-18. 20. The method of claim 1, wherein the protein is free of binders, lipophilic materials and surfactants. 21. The method of claim 1 or 2, wherein the organic solvent comprises dichloromethane and/or dimethyl carbonate. 22. The method of claim 1 or 2, wherein the organic solvent comprises one or more of dichloromethane, dimethyl carbonate, acetone, ethyl acetate, and tetrahydrofuran. 23. The method of claim 1 or 2, wherein the one or more precursors comprise at least three PEO repeats.