FIELD OF THE INVENTION

This invention is in the area of hydrogels or organogels of cross-linked poly(organophosphazene) polymers which contain entrapped enzymes, other biologically active materials, or diagnostic imaging agents and methods for their use.

BACKGROUND OF THE INVENTION

The immobilization of biologically active materials or imaging agents in polymeric systems has many advantages in pharmaceutical and other types of controlled delivery devices, analysis, biomedical engineering, as well as in industrial applications.

Immobilized biologically active materials in polymeric systems are specifically of great interest for pharmaceutical controlled delivery devices. A number of polymers have been used for this purpose. Synthetic polymers are preferred over natural polymers for their reproducibility and ease of manufacture. Examples of biodegradable polymers include poly(anhydrides) , poly(orthoesters) , and polydactic acid). Examples of "non-degradable" polymers include ethylene vinyl acetate and poly(acrylic acid) . U.S. Patent No. 4,880,622 to Allcock, et al. discloses controlled release articles of pharmaceuticals, pesticides, herbicides, plant growth regulators, and fertilizers in a poly(organophosphazene) matrix that has alkoxyether, polyether, imine, polyimine, sulfide, polysulfide, ester, and polyester substituent

groups. The '622 patent does not specifically address the preparation or design of biodegradable polyphosphazenes for the controlled delivery of substances. For many applications, biodegradable delivery devices are preferred for biological applications because they do not have to be surgically removed.

A number of enzymes and other biologically active agents have been immobilized in a variety of support materials with retention of biological activity. Immobilization can provide an improvement in the stability of an enzyme. The enzyme-support combination is usually easily separated from the reaction medium. Using this technology, a single aliquot of the immobilized enzyme can sometimes be used repeatedly to achieve more catalytic cycles than may be obtained from the same amount of free enzyme.

Four general methods have been used to immobilize enzymes and other biologically active molecules: (1) covalent binding to solid polymeric surfaces; (2) immobilization by physical adsorption onto solid surfaces; (3) microencapsulation; and (4) entrapment within cross-linked gels, films or fibers. The most widely used method involves the covalent binding of an enzyme to an activated support. An illustration of the covalent binding of enzymes to a poly(organophosphazene) polymer is provided by Allcock, H.R., Kwon, S., Ma.crom.ol . 1986, 9-, 1502, which discloses the covalent binding of glucose-6-phosphate dehydrogenase and trypsin to a poly[bis (aryloxy)phosphazene) ] . The aryl group was modified by reaction with nitric acid and then reduced to form the aminophenoxy group on the polyphosphazene. The aminophenoxy groups were then activated with cyanogen bromide, nitrous acid or glutaric dialdehyde which reacts to

form a covalent bond with trypsin or glucose-6- phosphate dehydrogenase.

Kokufuta, E., et al. , J. Chem . Soc . Chem. Commun . , 1992, 416 discloses that 1,4-cκ-D- glucosidase can be entrapped in poly(vinyl methyl ether) ("PVME") which has been cross-linked to form a gel by exposure to gamma irradiation. The entrapment creates temperature control over the reaction catalyzed by the immobilized l,4-c.-D- glucosidase, as the reaction ceases above 37°C when catalyzed by the entrapped enzyme, due to contraction of the PVME hydrogel. A comparison between the free enzyme and the immobilized enzyme showed that the entrapped enzyme had only about 20% of the activity of the free enzyme. Accordingly, it would be desirable to provide an immobilized enzyme system which retains a higher amount of its activity. Another disadvantage of this system is that the organic backbone of the polymer is subject to homolysis on exposure to gamma irradiation.

Diagnostic ultrasound imaging is another area in which "loaded" polymeric systems can be very useful. Diagnostic imaging is a powerful, non- invasive tool that can be used to obtain information on the internal organs of the body.

The advent of grey scale imaging and color Doppler have greatly advanced the scope and resolution of the technique. Although techniques for carrying out diagnostic ultrasound have improved significantly, there is still a need to enhance the resolution of the imaging for: (i) cardiac, solid organ, and vascular anatomic conduits (for example, the imaging of macrophage activity) ; (ii) solid organ perfusion; and (iii) Doppler signals of blood velocity and flow direction during real-time imaging.

Traditional, simple ultrasonic echograms reveal blood vessel walls and other echo-producing structures. However, since echoes from blood normally are not recorded, identifying which echoes are from which blood vessels is usually difficult. For example, echoes from the far wall of one blood vessel can be confused with the near wall of an adjacent blood vessel, and vice versa.

Ultrasonic contrast agents can be used to increase the amount of ultrasound reflected back to a detector. Ultrasonic contrast mediums fill the entire intraluminal space with echoes and readily permit identification of the correct pair of echoes corresponding to the walls of a particular blood vessel.

Ultrasonic contrast agents are primarily used in high-flow systems in which the contrast enhancement can be quickly evanescent. For echocardiography, a full display of bubble agents, ranging in size from two μm to 12 μm, and persisting from two or three to 30 seconds, has been used. For other applications, such as neurosonography, hysterosalpingography, and diagnostic procedures on solid organs, the agent must have a lifetime of more than a few circulation times and concentrate in organ systems other than the vascular tree into which it is injected. It must also be small enough to pass through the pulmonary capillary bed (less than eight microns) . Aqueous suspensions of air microbubbles are the preferred echo contrast agents due to the large differences in acoustic impedance between air and the surrounding aqueous medium. After injection into the blood stream, the air bubbles should survive at least for the duration of examination. The bubbles should be injectable intravenously and

small enough to pass through the capillaries of the lungs.

Air-filled particles with a polymeric shell should exhibit a longer persistence after injection than a nonpolyτneric microbubble, and may be suitable not only for cardiology but also for organ and peripheral vein imaging. A variety of natural and synthetic polymers have been used to encapsulate imaging contrast agents, such as air. Research efforts in this area have to date primarily focused on agarose and alginate as the encapsulating polymers.

European Patent Application No. 91810366.4 by Sintetica S.A. (0 458 745 Al) discloses air or gas microballoons bounded by an interfacially deposited polymer membrane that can be dispersed in an aqueous carrier for injection into a host animal or for oral, rectal, or urethral administration, for therapeutic or diagnostic purposes. The microballoons are prepared by the steps of: emulsifying a hydrophobic organic phase into a water phase to obtain an oil-in-water emulsion; adding to the emulsion at least one polymer in a volatile organic solvent that is insoluble in the water phase; evaporating the volatile solvent so that the polymer deposits by interfacial precipitation around the hydrophobic phase in the water suspension; and subjecting the suspension to reduced pressure to remove the hydrophobic phase and the water phase in a manner that replaces air or gas with the hydrophobic phase. There are two major disadvantages of this process. First, only polymers that have very specific solubility profiles can be used to prepare the microbubbles, i.e., they must be "interfacially depositable" on a hydrophobic phase in an aqueous medium, and soluble in a volatile organic solvent that is water-

insoluble. Second, the process requires the use of organic solvents, which may be hard to completely remove from the microbubble and which may be injurious to the patient's health. In light of the important and very diverse uses for polymeric systems that contain immobilized materials, it would be of interest to provide new materials for a range of applications.

It is therefore an object of the present invention to provide a polymer matrix which contains an entrapped enzyme or other biologically active material that retains a significant portion of its biological activity on entrapment.'

It is a further object of the present invention to provide a new polymeric system for the controlled delivery of substances that is biodegradable.

It is yet another object of the present invention to provide microcapsules made from synthetic polymers that contain a diagnostic imaging agent.

SUMMARY OF THE INVENTION

In one embodiment, a controlled delivery device is provided that includes a substance to be delivered, including but not limited to an enzyme or other biologically active molecule or drug, or a diagnostic imaging agent, in a poly(organophosphazene) matrix, wherein the poly(organophosphazene) contains at least (i) a substituent group that can be radiation crosslinked; and (ii) a substituent group that is susceptible to hydrolysis under the conditions of use, to impart biodegradability to the polymer. Suitable hydrolyzable groups include, but are not limited to, for example, chlorine, amino acid,

amino acid ester, imidazole, glycerol, and glucosyl. Any ratio of radiation-crosslinkable substituents to hydrolyzable substituents can be used that provides a desired product. The poly(organophosphazenes) can optionally also contain ionic substituent groups that can be crosslinked or hardened using ions of opposite charge, as disclosed in U.S. Patent Nos. 5,308,701 and 5,149,543, incorporated herein by reference. Further, the poly(organophosphazene) can contain one or more substituent groups that are not radiation crosslinkable or hydrolyzable under the conditions of use, to tailor the polymer exhibit specific physical properties. The radiation crosslinkable substituent groups are typically those that contain an aliphatic C-H, C-Cl, or C-Br bonds, however, they can alternatively or also contain alkenyl substituents that can be crosslinked on exposure to radiation. The polymers can be formed into virtually any shape or size, depending upon the physiological environment of use, although microspheres or nanospheres are preferred. The polymer can be shaped and sized, for example, for buccal, oral, vaginal, intrauterine, ocular, and anal insertion or for parenteral insertion or intravenous, subcutaneous, or other type of injection. In the latter instance, the polymers should be in the form of particles small enough to fit through a syringe tip, generally less than a few hundred microns.

The photosensitive polyphosphazenes can also be used for the preparation of gas-filled polymeric microbubbles, which are useful in the process of diagnostic ultrasound imaging, and can be prepared in micron and submicron sizes that are injectable and that are capable of passing through the pulmonary capillary bed.

The delivery devices can be targeted to specific regions of the body by covalently binding to the polymer a targeting molecule. The targeting molecule can be, for example, a protein or peptide (such as a hormone, antibody or antibody fragment such as the Fab or Fab ₂ antibody fragments, or a specific cell surface receptor ligand) , lipid, polysaccharide, nucleic acid, carbohydrate, a combination thereof, or other molecule, including a synthetic molecule, that identifies and localizes at the target material.

The devices can also be designed to minimize tissue adhesion by covalently binding a poly(alkylene glycol) moiety to the surface of the microcapsule. The surface poly(alkylene glycol) moieties have a high affinity for water that reduces protein adsorption onto the surface of the particle. The recognition and uptake of the microcapsule by the reticulo-endothelial system (RES) is therefore reduced.

In one embodiment, the terminal hydroxyl group of the poly(alkylene glycol) can be used to covalently attach biologically active molecules, or molecules affecting the charge, lipophilicity or hydrophilicity of the particle, onto the surface of the microcapsule. The biologically active molecule can be a protein, carbohydrate or polysaccharide, nucleic acid, lipid, a combination thereof, or a synthetic molecule, including organic and inorganic materials.

In another embodiment, a cross-linked poly(organophosphazene) polymer which has an enzyme or other biologically active molecule entrapped therein is provided. The polymeric material can be used as a structural material that exhibits the activity of the entrapped substance for analytical, biomedical engineering, or industrial applications.

The physical characteristics of the polymeric material can be determined by the appropriate selection and ratio of the substituent groups on the poly(organo)phosphazene. The cross-linked enzyme-containing poly(organophosphazene) is prepared by mixing the polymer with the enzyme, and then cross-linking the polymer by exposure to radiation (for example, ultraviolet, gamma, electron beam or x-ray) in a manner that entraps the substance in the cross-linked matrix. The polymer is then mixed in or swollen with an aqueous solution.

Entrapped enzymes maintains a significant proportion of its activity on entrapment, and are typically active for at least five enzyme cycles. The aqueous solution can include any number of other components that do not adversely affect the performance of the enzyme, including cofactors, substrates, and pH adjusters, including buffers. For example, a urease enzyme can be entrapped in cross-linked poly[bis ( (methoxyethoxy)ethoxy) - phosphazene] (hereinafter referred to as "MEEP"), which is converted to a hydrogel on soaking in an aqueous solution. The urease hydrogel was able to convert urea to ammonia over at least five cycles. In an alternative embodiment, an organogel polymeric material can be prepared that contains an enzyme or other biologically active material entrapped in a polymer that is mixed or swollen with an organic solvent or a mixture of water and an organic solvent. This embodiment is appropriate for those enzymes that can catalyze reactions in organic or mixed aqueous/organic mediums.

The polymeric systems can also be used to immobilize other substances, including drugs, pesticides, herbicides, plant growth regulators, or fertilizers.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph of the increase in pH of the solution over time (hours) following the addition of 0.01 mole of urea to 0.08 g of urease immobilized within a cross-linked MEEP hydrogel. Figure 2 is a graph of the reproducibility of the increase in pH over time (hours) during repeated treatment of urease immobilized in a MEEP hydrogel to urea. Figure 3 is a graph of the increase in pH over time (hours) after five successive additions of 0.01 mol of urea to 0.08 g of urease immobilized in a MEEP hydrogel .

Figure 4 is a graph of the increase in pH over time (hours) after the addition of 0.01 mole of urea to 0.08 g of urease dissolved in pH 7 buffer. Figure 5 is a graph showing a comparison of the increase in pH over time (hours) , after the addition of 0.01 mol of urea to: non-irradiated free urease; free urease irradiated with 0.5 Mrad of gamma radiation; and urease immobilized in a MEEP hydrogel .

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions A. General Definitions

The term amino acid, as used herein, refers to both natural arid synthetic amino acids, and includes, but is not limited to alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaoyl, lysinyl, argininyl, and histidinyl.

The term amino acid ester refers to the aliphatic, aryl or heteroaromatic carboxylic acid ester of a natural or synthetic amino acid. The term alkyl, as used herein, refers to a saturated straight, branched, or cyclic hydrocarbon, or a combination thereof, typically of C, to C ₂₀ , and specifically includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, 3-methylpentyl,

2,2-dimethylbutyl, 2,3-dimethylbutyl, heptyl, octyl, nonyl, and decyl.

The term (alkyl or dialkyl)amino refers to an amino group that has one or two alkyl substituents, respectively.

The terms alkenyl and alkynyl, as used herein, refers to a C ₂ to C ₂₀ straight or branched hydrocarbon with at least one double or triple bond, respectively. The term aryl or aromatic, as used herein, refers to phenyl or substituted phenyl, wherein the substituent is halo, alkyl, alkoxy, alkylthio, haloalkyl, hydroxyalkyl, alkoxyalkyl, methylenedioxy, cyano, C(O) (alkyl), -C0 ₂ H, -OS0 ₂ H, -S0 ₃ H, -P0 ₃ H, -C0 ₂ alkyl, amide, amino, alkylamino and dialkylamino, and wherein the aryl group can have up to 3 substituents.

The term aliphatic refers to a hydrocarbon, typically of Cj to C ₂₀ , that can contain one or a combination of alkyl, alkenyl, or alkynyl moieties, and which can be straight, branched, or cyclic, or a combination thereof.

The term halo, as used herein, includes fluoro, chloro, bromo, and iodo. The term aralkyl refers to an aryl group with an alkyl substituent.

The term alkaryl refers to an alkyl group that has an aryl substituent, including benzyl, substituted benzyl, phenethyl or substituted phenethyl, wherein the substituents are as defined above for aryl groups.

The term heteroaryl or heteroaromatic, as used herein, refers to an aromatic moiety that includes at least one sulfur, oxygen, or nitrogen in the aromatic ring, and that can be optionally substituted as described above for aryl groups.

Nonlimiting examples are furyl, pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl, benzimidazolyl, purinyl, carbozolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, isooxazolyl, pyrrolyl, pyrazolyl, quinazolinyl, pyridazinyl, pyrazinyl, cinnolinyl, phthalazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, pteridinyl,

5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolσpyrimidinyl, and pyrazolopyrimidinyl.

The term heteroalkyl, as used herein, refers to an alkyl group that includes a heteroatom such as oxygen, sulfur, or nitrogen (with valence completed by hydrogen or oxygen) in the carbon chain or terminating the carbon chain.

The term poly(organophosphazene) , as used herein, refers to a polyphosphazene in which one or more of the pendant groups contain carbon.

The term substrate, as used herein, refers to a material which is susceptible to a reaction catalyzed by an enzyme which is entrapped by the poly(organophosphazene) hydrogel.

The term hydrogel, as used herein, refers to a polymeric material which swells in, but does not dissolve in, an aqueous media.

The term organogel, as used herein, refers to a polymeric material which swells in, but does not dissolve in, an organic solvent.

The term biologically active molecule or material as used herein refers to an organic molecule including a drug, a protein, polysaccharide, nucleoprotein, lipoprotein, synthetic polypeptide, or a small molecule linked to a protein, carbohydrate, glycoprotein, steroid, nucleic acid, nucleotide, nucleoside, oligonucleotides (including antisense oligonucleotides) , cDNA, nucleic acids, genes, vitamins, including vitamin C and vitamin E, lipid, cell or cell line or combination thereof, that causes a biological effect when administered in vivo to an animal, including but not limited to birds and mammals, including humans.

The term drug, as used herein, refers to any substance used internally or externally as a medicine for the treatment, cure, prevention or diagnosis of a disease or disorder, and includes but is not limited to immunosuppressants, antioxidants, anesthetics, chemotherapeutic agents, steroids (including retinoids) , hormones, antibiotics, antivirals, antifungals, antiproliferatives, antihistamines, anticoagulants, antiphotoaging agents, melanotropic peptides, nonsteroidal and steroidal anti-inflammatory compounds, and radiopharmaceuticals.

The term biodegradable polymer refers to a polymer that degrades within a period that is acceptable in the desired application, usually less than several weeks or months and typically less than a year, when exposed to a physiological

solution of pH between 6 and 8 having a temperature of between about 25°C and 37°C.

As used herein, the term nanoparticle or nanosphere typically refers to a particle, usually a solid particle (as opposed to a capsule) , of size ranging from 10 to 1000 nm. In a preferred embodiment, the nanoparticle is biodegradable, biocompatible, has a size of less than 200 nm and has a rigid biodegradable core that has incorporated in it the substance to be delivered.

The term "microparticle, " as used herein, refers to a particle, usually a solid particle, of size ranging from greater than one micron to 1000 microns. B. Poly(organophosphazenes)

Poly(organophosphazenes) are a class of polymers which have been reported to exhibit a number of interesting properties. The photochemical behavior and stability of poly(aryloxyphosphazenes) have been described previously. See, for example,

Allcock, et al . , Macromol. , 1979, 12. _ι 108; and Gleria et al. , Macromol.. 1987, 2Q_, 1766.

Poly(organophosphazenes) have the general formula:

wherein n is an integer.

Non-limiting examples of R (which can vary independently on the polymer) include but are not limited to the following groups: aliphatic, aryl, aralkyl, alkaryl, amino acid, amino acid ester, carboxylic acid, heteroaromatic, carbohydrate,

including glucose, heteroalkyl, halogen, (aliphatic)amino- including alkylamino-, heteroaralkyl, di (aliphatic)amino- including dialkylamino-, arylamino-, diarylamino-, and alkylarylamino-, -oxyaryl including but not limited to -oxyphenyl-p-methyl, -oxyphenylC0 ₂ H, -oxyphenylS0 ₃ H, -oxyphenylhydroxyl and -oxyphenylP0 ₃ H; -oxyaliphatic including -oxyalkyl, -oxy(aliphatic) C0 ₂ H, -oxy(aliphatic)S0 ₃ H, -oxy(aliphatic)P0 ₃ H, and -oxy(aliphatic)hydroxyl, including -oxy(alkyl)hydroxyl; -oxyalkaryl, -oxyaralkyl, -thioaryl, -thioaliphatic including -thioalkyl, -thioalkaryl, and -thioaralkyl, organosilicon, including but not limited to - (alkyl) -Si(alkyl) ₄ , including -CH ₂ Si(CH ₃ ) ₄ ;

-NHC(0)0- (aryl or aliphatic), -0- [ (alkyl)0] _x - CH ₂ ) _y NH ₂ , wherein the alkyl group can vary within the moiety, including -0- [(CH ₂ ) _x 0] _y -CH ₂ ) _X NH ₂ ; -0- [(CH ₂ ) _x O] _y CH ₂ ) _x NH(CH ₂ ) _x S0 ₃ H, and -0- [ (alkyl) -0] _y - (aryl) or aliphatic) , wherein the alkyl group can vary within the moiety, including -0- [ (CH ₂ ) _x 0] _y - (aryl or aliphatic) , wherein x is 1-8 (which can vary within the moiety) and y is an integer of 1 to 40. The groups can be bonded to the phosphorous atom through, for example, an oxygen, sulfur, nitrogen, or carbon atom.

These poly(organophosphazenes) are easily synthesized from poly(dichlorophosphazene) by replacement of the highly reactive chlorine atoms with the desired organic side chains. The properties of the resulting poly(organophos- hazenes) can be controlled with appropriate selection of R, which may be the same or different. Further, the R groups may be different substituents within a single polymer.

Poly(organophosphazenes) can be formed with two or more types of pendant R groups by reacting

poly(dichlorophosphazene) with two or more nucleophiles in a desired ratio. In general, when the poly(organophosphazene) has more than one type of pendant group, the groups will vary randomly throughout the polymer. Thus, the poly(organo- hosphazene) will contain phosphorous atoms which are bound to two like groups or two different groups. The resulting ratio of the two or more pendant groups in the poly(organophosphazene) will be determined by a number of factors, including the ratio of starting materials used to produce the polymer, the temperature at which the nucleophilic substitution reaction is carried out, and the solvent system used. While it is very difficult to determine the exact substitution pattern of the groups in the resulting polymer, the ratio of groups in the polymer can be easily determined by one skilled in the art.

The properties of the poly(organophosphazenes) such as its degree of hardness, Tg, hydrophilicity, hydrogel or organogel character, acidity, and film forming ability can be controlled through proper selection of the R groups.

Examples of poly(organophosphazenes) , and methods for their synthesis include those described in U.S. Patent No. 4,440,921, which discloses that biologically active molecules containing a carboxylic acid residue can be covalently attached to a polyphosphazene via condensation with a pendant amino group on the polyphosphazene. (see also Allcock, H.R. ; Hymer, W.C.; Austin, P.E. Macromolecules 1983, 16, 1401) . U.S. Patent No. 4,880,622 discloses novel poly(organophosphazene) polymers that are useful for the controlled delivery of pharmaceuticals, pesticides, herbicides, plant growth regulators, and fertilizers. U.S. Patent No. 5,053,451 discloses

that poly ⁽ carboxylato-phenoxy)phosphazene can be ionically cross-linked to form a hydrogel. U.S. Patent No. 5,149,543 discloses a composition that includes a biological material such as a liposome, virus, procaryotic cell, or eucaryotic cell encapsulated in an ionically cross-linked poly(organophosphazene) or other polyelectrolyte.

1. Radiation Crosslinkable Poly(organophosphazenes)

Poly(organophosphazenes) bearing an aliphatic C-H, a C-Cl or C-Br bond can be cross-linked by exposure to various types of irradiation, including but not limited to gamma, ultraviolet, x-ray, or electron beam radiation. Allcock, H.R. et al. , Biomaterials, 1988, 9_, 509; Allcock, H.R. et al., Aacromol., 1992, 25., 5573. Aromatic C-H bonds are typically less reactive than C-H, C-Cl, and C-Br bonds on exposure to radiation, and therefore, poly(organophosphazenes) in which the R group contains aromatic C-H bonds only or other bonds that do not undergo homolysis in the presence of gamma, ultraviolet, or other high energy radiation may not be useful, or may be less useful, for the preparation of the poly(organophosphazenes) described herein. Poly(organophosphazenes) can be used that contain a combination of R groups that contain aromatic C-H bonds or other bonds that do not undergo homolysis in the presence of irradiation, and those that contain groups that contain bonds which undergo homolysis. Water soluble poly(organophosphazenes) which are cross-linked have been reported to form hydrogels. Allcock, H.R. et al. , Biomaterials, 1988, 1, 509; Allcock, H.R. et al. , Macromol . , 1992, .25., 5573 disclose the formation of hydrogels of poly[bis(methoxyethoxyethoxy)phosphazene] ; poly[bis (methoxy-ethoxy)phosphazene] ;

poly [bis (aminoethoxy) ethoxyphosphazene] ; poly [bis (butoxyethoxy) ethoxyphosphazene] ; and poly [bis (ethoxy-ethoxy) ethoxyphosphazene] .

In one embodiment of the present invention, the R groups are selected such that the crosslinked poly (organophosphazene) is capable of forming a hydrogel in water in the cross-linked state. The poly (organophosphazene) can be cross-linked using any method to cross-link poly (organophosphazenes) which does not significantly adversely impact the substance to be entrapped, for example, by exposure to radiation, including but not limited to gamma, x-ray, electron beam, or ultraviolet (UV) radiation. In a preferred embodiment, n is greater than 4, for example, between 10 and 30,000, and more usually between 1000 and 20,000.

Non- limiting examples of specific polymers which form hydrogels include: [NP (0CH ₂ CH ₂ 0CH ₂ CH ₂ 0CH ₃ ) ₂ ] _n ; [NP (0CH ₂ CH ₂ 0CH ₂ CH ₂ NH ₂ ) ₂ ] _n ; [NP (0CH ₂ CH ₂ 0CH ₂ CH ₂ NH ₂ ) _x (MEE) ₂ . _x ] _n ; [NP ( OCH ₂ CH ₂ NH ₂ ) ₂ ] _n ; [NP ( 0CH ₂ CH ₂ NH ₃ ) _x ( MEE ) ₂ . _n ;

[NP ( OCH ₂ CH ₂ OCH ₂ CH ₂ OH ) ₂ ] _n ; [NP ( OCH ₂ CH ₂ OCH ₂ CH ₂ OH ) _x ( MEE ) ₂ . _x ] _n ;

[NP (OCH ₂ CH ₂ OH) ₂ ] _n ; [NP (OCH ₂ CH ₂ OH) _x (MEE) ₂ . _x ] _n ;

[NP(NHCH ₃ ) ₂ ] _n ; [NP(CH ₃ ) ₂ ] _n ; [NP (O (CH ₂ CH ₂ 0) _m CH ₃ ) ₂ ] _n ; [NP(PEGME av Mw 350) ₂ ] _n ; [NP(PEGME 350) _x (MEE) ₂ . _x ] _n ;

[NP(PEGME av Mw 550) ₂ ] _n ; [NP(PEGME 550) _x (MEE) ₂ . _x ] _n ;

[NP(PEGME av Mw 750) ₂ ] _n ; [NP(PEGME 750) _x (MEE) ₂ . _x ] _n ,

[NP(PEGME 350) _x (PEGME 550) _2-x ] _n ; [NP(PEGME 350) _x

(PEGME 750) ₂ . _x ] _n ; [NP (PEGME 550) _x (PEGME 750) ₂ . _x ] _n ; [NP(Brij ^® 30) _x (MEE) ₂ . _x ] _n ; [NP (Bri j ^® 30) _x (PEGME 350) ₂ . _x ] _n ; [NP(Brij ^® 30) _x (PEGME 550) ₂ . _x ] _n ; [NP(Brij ^® 30) _x

(PEGME 750) ₂ . _x ] _n ; [NP (OCH ₂ CH ₂ OCH ₃ ) ₂ ] _n ;

[NP (0CH ₂ CH ₂ 0CH ₃ ) _x (0CH ₂ CF ₃ ) ₂ . _x ] _n ) ; [NP (MEE) _x (OCH ₂ CF ₃ ) ₂ . _x ] _n ;

[NP ( OCH ₂ CH ₂ OCH ₃ ) _x ( MEE ) _2-x ] _n ; [NP ( OCH ₂ CH ₂ OCH ₃ ) _x ( PEGME 350, 550 or 750) ₂ . _x ] _n ; [NP (NHCH ₂ CH ₃ ) ₂ • HCl] _n ;

[NP(NHCH ₂ CH ₂ CH ₃ ) ₂ • HCl] _n ; [NP (NHCH ₂ CH ₂ CH ₂ CH ₃ ) ₂ • HCl] _n ;

X is a number between 0 and 2 and represents the ratio of substituents in a random copolymer. For example, if x is 0.3, 2-x is 1.7. As used herein, MEE refers to methoxyethoxyethoxy and Brij ^® 30 refers to polyoxyethylene (4) lauryl ether

(C ₁₂ H ₂₅ (OCH ₂ CH ₂ ) ₄ OH, sold, for example, by Aldrich Chemical Company. PEGME is poly(ethylene glycol)methyl ether, which is sold as Carboxylyx by Union Carbide. In one embodiment a poly(organophosphazene) is used that contains at least approximately 20 percent, and preferably approximately 25 percent or more, of a moiety of the formula -O- [ (alkyl) -0] _y - (R) , wherein R is an aliphatic group, and preferably -0- [ (alkyl) -0] _y - (alkyl) , (wherein alkyl can vary within the polymer) , and more preferably, -0- [ (CH ₂ ) _x 0] _y - (alkyl) , wherein x is 1-8, and preferably 2-3 (which can vary within the moiety) and y is an integer of 1 to 40. In one embodiment, the poly(organophosphazene) s can contain oxy-poly(alkylene oxide) subgroups in combination with other groups in an appropriate ratio to achieve a desired property, such as the glass transition temperature of the polymer. Examples of these other pendant groups include aromatic sulfonic acid and aromatic carboxylic acid moieties, alkoxy, including but not limited to ethoxy, propoxy, butoxy, pentoxy, and oxyalkaryl, including benzyloxy. Particularly preferred poly(organophosphazenes) include those that contain at least one R selected from the group consisting of methoxyethoxy, (methoxyethoxy)ethoxy, (aminoethoxy)ethoxy, (butoxyethoxy) ethoxy, (ethoxyethoxy) ethoxy. A particularly preferred polymer is poly[bis ( (methoxyethoxy) ethoxy) -phosphazene] (also referred to as "MEEP") .

MEEP is a preferred polymer for the enzyme immobilized hydrogels of the present invention due to its unusual combination of solid state and hydrogel properties, and its ease of radiation cross-linking by gamma-rays or ultraviolet light. Allcock, H.R. In Ring-Opening Polymerization : Mechanism, Catalysis, Structure, and Utili ty; Brunnelle, D., Ed.; Hanser: Munich, Germany, 1992. The radiation cross-linking of this polymer results in an increase in cross-link density that eliminates viscous flow and increases structural stability, but decreases the amount of water that can be imbibed. The amount of cross-linking can be controlled easily by variations in the irradiation dose. The rate of cross-link formation is approximately 1 cross-link/1000 repeat ^• units/Mrad. Hydrogels derived from this polymer provide a facile pathway for small molecule diffusion (Allcock, H.R.; Gebura, M. ; Kwon, S.; Neenan, T.X. Biomaterials 1988, 19, 500; Allcock, H.R. ; Kwon, S.; Riding, G.H. ; Fitzpatrick, R.J.; Bennett, J.L. Biomaterials 1988, 19, 509) , an effect that can be attributed to the ease of main-chain and side-group reorientation in this polymer, and the high ratio of water to polymer in the hydrogels, even when the degree of cross-linking is as high as 1 cross¬ link/25 repeating units.

C. Poly(organophosphazenes) with Alkenyl Substituent Groups A poly(organophosphazene) can be used that has an alkenyl substituent that is capable of crosslinking on exposure to radiation.

Non-limiting examples of unsaturated monomers that can be bound to polyphosphazene substituent groups include: cinnamic acid, cinnamyl alcohol, cinnamonitrile, cinnamaldehyde, other cinnamic derivatives, chalcone, 2-acetamido acrylic acid, 2-

(acetoxyacetoxy)ethyl methacrylate, 1-acetoxy-l,3- butadiene, 2-acetoxy-3-butenenitrile, 4- acetoxystyrene, acrolein, acrolein diethyl acetal, acrolein dimethyl acetal, acrylamide, 2- acrylamidoglycolic acid, 2-acrylamido-2-methyl propane sulfonic acid, acrylic acid, acrylic anhydride, acrylonitrile, acryloyl chloride, ct- acryloxy-β,β' -dimethyl-g-butyrolactone, N-acryloxy succinimide, N-acryloxytris- (hydroxymethyl) aminomethane, N-acryloyl chloride, N-acryloyl pyrrolidinone, N-acryloyl-tris(hydroxymethyl)amino methane, 2-amino ethyl methacrylate, N-(3- aminopropyDmethacrylamide, m-aminostyrene, o- aminostyrene, p-aminostyrene, t-amyl methacrylate, 2- (1-aziridinyl)ethyl methacrylate, 2,2'-azobis- (2-amidinopropane) , 2,2' -azobisisobutyronitrile, 4,4' -azobis- (4-cyanovaleric acid), l,l'-azobis- (cyclohexanecarbonitrile) , 2,2' -azobis- (2,4- dimethylvaleronitrile) , 4-benzyloxy-3- methoxystyrene, 2-bromoacrylic acid, 4-bromo-l- butene, 3-bromo-3,3-difluoropropene, 6-bromo-l- hexene, 3-bromo-2-methacrylonitrile, 2- (bromomethyl)acrylic acid, 8-bromo-l-octene, 5- bromo-1-pentene, cis-1-bromo-l-propene, β- bromostyrene, p-bromostyrene, bromotrifluoro- ethylene, 3-buten-2-ol, 1,3-butadiene, 1,3- butadiene-l,4-dicarboxylic acid, 3-butenal diethyl acetal, 3-buten-2-ol, 3-butenyl chloroformate, 2-butylacrolein, N-t-butylacrylamide, butyl methacrylate, o-bromostyrene, m-bromostyrene, carvone, carvyl acetate, cis 3-chloroacrylic acid, 2-chloroacrylonitrile, 2-chloroethyl vinyl ether, 2-chloromethyl-3-1rimethylsilyl-1-propene, 3- chloro-1-butene, 3-chloro-2-chloromethyl-1-propene, 3-chloro-2-methyl propene, 2,2-bis (4-chlorophenyl) - 1,1-dichloroethylene, 3-chloro-1-phenyl-1-propene, m-chlorostyrene, o-chlorostyrene, p-chlorostyrene,

1-cyanovinyl acetate, 1-cyclopropyl-l- (trimethylsiloxy)ethylene, 2,3-dichloro-1-propene, 2,6-dichlorostyrene, 1,3-dichloropropene, 2,4- diethyl-2,6-heptadienal, 1,9-decadiene, 1,2- dibromoethylene, 1,l-dichloro-2,2-difluoroethylene, 1, 1-dichloropropene, 2,6-difluorostyrene, dihydrocarveol, dihydrocarvone, dihydrocarvyl acetate, 3,3-dimethylacrylaldehyde, N,N'- dimethylacrylamide, 3,3-dimethylacrylic acid, 3,3- dimethylacryloyl chloride, 2-dimethyl aminoethyl methacrylate, 2,4-dimethyl-2,6-heptadien-l-ol, 2,4- dimethyl-2,6-heptadienal, 2,5-dimethyl-l,5- hexadiene, 2,4-dimethyl-l,3-pentadiene, 2,2- dimethyl-4-pentenal, divinyl benzene, 1,3- divinyltetramethyl disiloxane, 8,13-divinyl- 3,7,12,17-tetramethyl-21H,23H-porphine, 8,13- divinyl-3,7,12,17-tetramethyl-21H,23H-propionic acid, 8,13-divinyl-3,7,12,17-tetramethyl-21H,23H- propionic acid, 3,9-divinyl-2,4,8,10- tetraoraspiro[5,5]undecane, divinyl tin dichloride,

1-dodecene, 3,4-epoxy-l-butene, 2-ethyl acrolein, ethyl acrylate, 2-ethyl-l-butene, 2-ethylhexyl acrylate, 2-ethylhexyl methacrylate, 2-ethyl-2- (hydroxymethyl) -1,3-propanediol triacrylate, 2- ethyl-2- (hydroxymethyl) -1,3-propanediol trimethacrylate, ethyl vinyl ether, ethyl vinyl ketone, ethyl vinyl sulfone, (l-ethylvinyl)tributyl tin, m-fluorostyrene, o-fluorostyrene, p- fluorostyrene, glycidyl acrylate, glycidyl methacrylate, glycol methacrylate, 1,6-heptadiene,

1,6-heptadienoic acid, 1,6-heptadien-4-ol, 1-hexen- 3-ol, hexafluoropropene, 1,6-hexanediol diacrylate, 1,5-hexadien-3,4-diol, 1,4-hexadiene, 1,5-hexadien- 3-ol, 1,3,5-hexatriene, 5-hexen-l,2-diol, 5-hexen- l-ol, hydroxypropyl acrylate, 3-hydroxy-3,7,11- trimethyl-1,6,10-dodecatriene, isoamyl methacrylate, isoprene, 2-isopropenylaniline,

isopropenyl chloroformate, 4,4' -isopropylidene dimethacrylate, 3-isopropyl-a-a-dimethylbenzene isocyanate, isopulegol, itaconyl acid, itaconyl chloride, lead (II) acrylate, linalool, linalyl acetate, p-mentha-1,8-diene, p-mentha-6,8-dien-2- ol, methyleneamino acetonitrile, methacrolein, [3- (methacryloylamino) -propyl] trimethyl ammonium chloride, methacrylamide, methacrylic acid, methaσrylic anhydride, methacrylonitrile, methacryloyl chloride, 2- (methacryloyloxy)ethyl acetoacetate, (3-methacryloxypropyl) -trimethoxy silane, 2- ( ethacryloxy)ethyl trimethyl ammonium methosulfate, 2-methoxy propene, methyl-2- (bromomethyl)acrylate, 5-methyl-5-hexen-2-one, N,N' -methylene bisacrylamide, 2-methylene glutaronitrile, 2-methylene-1,3-propanediol, 3-methyl-l,2-butadiene, 3-methyl-l-buten-l-ol, 2-methyl-l-buten-3-yne, 2-methyl-l,5-heptadiene, 3-methyl-1,3-pentadiene, 2-methyl-1,4-pentadiene, 3-methyl-l-penten-3-ol, methyl vinyl ether, methyl vinyl ketone, methyl-2-vinyloxirane, 4-methylstyrene, methyl vinyl sulfone, 4-methyl-5- vinylthiazole, myrcene, t-β-nitrostyrene, 3-nitrostyrene, 1,8-nonadiene, 1,7-pctadiene, 7-octene-l,2-diol, l-octen-3-ol, l-penten-3-ol, t-2,4-pentenoic acid, 1,3-pentadiene, 1,4- pentadiene, 1,4-pentadien-3-ol, 4-penten-l-ol, 4-penten-2-ol, 4-phenyl-1-butene, phenyl vinyl sulfide, phenyl vinyl sulfonate, 2-propene-1- sulfonic acid, phenyl vinyl sulfoxide, 1-phenyl-l- (trimethylsiloxy)ethylene, safrole, styrene, 4-styrene sulfonic acid, styrene sulfonyl chloride, 3-sulfopropyl acrylate, 3-sulfopropyl methacrylate, tetrachloroethylene, tetracyano- ethylene, tetramethyldivinyl siloxane, trans 3-chloroacrylic acid, 2-trifluoromethyl propene, 2- (trifluoro¬ methyl)propenoic acid, 2,4,4' -trimethyl-1-pentene,

3,5-bis (trifluoromethyl) -styrene, 2,3- bis (trimethylsiloxy) -1,3-butadiene, vinyl acetate, vinyl acetic acid, 4-vinyl anisole, 9-vinyl anthracene, vinyl behenate, vinyl benzoate, 4-vinyl benzoic acid, vinyl benzyl acetate, vinyl benzyl alcohol, 3-vinyl benzyl chloride, 3- (vinyl benzyl) - 2-chloroethyl sulfone, 4- (vinyl benzyl)2- chloroethyl sulfone, N- (p-vinyl benzyl) -N,N' - dimethyl amine, 4-vinyl biphenyl, vinyl bromide, vinyl butyl ether, 9-vinyl carbazole, vinyl carbinale, vinyl cetyl ether, vinyl chloroacetate, vinyl chloroformate, vinyl crotanoate, 4-vinyl-1- cyclohexene, 4-vinylcyclohexene dioxide, vinyl cyclopentene, vinyl dimethylchlorosilane, vinyl dimethylethoxysilane, vinyl diphenylphosphine, vinyl 2-ethyl hexanoate, vinyl 2-ethylhexyl ether, vinyl ether ketone, vinyl ethylene, vinyl ethylene iron tricarbonyl, vinyl ferrocene, vinyl formate, vinyl hexadecyl ether, vinylidene fluoride, 1-vinyl imidizole, vinyl iodide, vinyl laurate, vinyl magnesium bromide, vinyl mesitylene, vinyl 2- methoxy ethyl ether, vinyl methyl dichlorosilane, vinyl methyl ether, vinyl methyl ketone, 2-vinyl naphthalene, 5-vinyl-2-norbornene, vinyl pelargonate, vinyl phenyl acetate, vinyl phosphonic acid, bis(2-chloroethyl)ester, vinyl propionate, 4-vinyl pyridine, 2-vinyl pyridine, 1-vinyl-2- pyrrolidinone, 2-vinyl quinoline, 1-vinyl silatrane, vinyl sulfone, vinyl sulfone (divinylsulfone) , vinyl sulfonic acid sodium salt, o-vinyl toluene, p-vinyl toluene, vinyl triacetoxysilane, vinyl tributyl tin, vinyl trichloride, vinyl trichlorosilane, vinyl trichlorosilane (trichlorovinylsilane) , vinyl triethoxysilane, vinyl triethylsilane, vinyl trifluoroacetate, vinyl trimethoxy silane, vinyl trimethyl nonylether, vinyl trimethyl silane, vinyl

triphenylphosphonium bromide, vinyl tris(2- methoxyethoxy)silane, and vinyl 2-valerate.

3. Hydrolyzable Substituents

In one embodiment of the invention, a poly(organophosphazene) is provided that contains at least one substituent group that is susceptible to hydrolysis under the conditions of use, to impart biodegradability to the polymer. Suitable hydrolyzable groups include, but are not limited to, for example, chlorine, amino acid, amino acid ester, imidazole, glycerol, and glucosyl. Any ratio of radiation-crosslinkable substituents to hydrolyzable substituents can be used that provides a desired product. The degree of hydrolytic degradability of the polymer will be a- function of the percentage of pendant groups susceptible to hydrolysis and the rate of hydrolysis of the hydrolyzable groups. The hydrolyzable groups are replaced by hydroxyl groups in aqueous environments to provide P-OH bonds that impart hydrolytic instability to the polymer.

It should be understood that certain groups, such as heteroaromatic groups other than imidazole, hydrolyze at an extremely slow rate under neutral aqueous conditions, such as that found in the blood, and therefore are typically considered nonhydrolyzable groups for purposes herein. However, under certain conditions, for example, low pH, as found, for example, in the stomach, the rate of hydrolysis of normally nonhydrolyzable groups

(such as heteroaromatics other than imidazole) can increase to the point that the biodegradation properties of the polymer can be affected. One of ordinary skill in the art using well known techniques can easily determine whether pendant groups hydrolyze at a significant rate under the

conditions of use. One of ordinary skill in the art can also determine the rate of hydrolysis of the polyphosphazenes of diverse structures as described herein, and will be able to select that polyphosphazene that provides the desired biodegradation profile for the targeted use.

4. Substituents with Ionizable Groups

In an alternative embodiment of the present invention, the poly(organophosphazene) includes an ionizable substituent group that can cross-link using multivalent ion, as described in U.S. Patent Nos. 5,053,451 and 5,149,543, incorporated herein by reference.

II. Enzymes Enzymes suitable for entrapment in the poly(organophosphazene) hydrogel include any enzyme which can be immobilized within a poly(organophosphazene) upon cross-linking and which does not lose a significant portion of its activity during immobilization. An enzyme has retained a substantial amount of its activity on entrapment is typically one that has an activity which is at least approximately 25% that of the free enzyme on the first cycle of catalysis after entrapment. However, there are enzymes that are so active that they continue to perform adequately at less than 25% capacity, and perhaps at 0.1, 1, 5, or 10% capacity, and those applications that do not require high enzyme activity (for example, an assay to detect urea as opposed to a method to convert all urea to ammonia) . For those applications, the meaning of a "significant amount of enzyme activity" must be considered in light of the enzyme and or the application. Preferably, the entrapped enzyme has an activity which is at least 50 or 60%

of the free enzyme on the first cycle of catalysis after entrapment. Optimally, the enzyme exhibits 70% or more of the activity of the free enzyme under these conditions. Categories of enzymes which are suitable for entrapment include, but are not limited to, proteases, ureases, esterases, nucleases, collagenases, dehydrogenases, decarboxylases, deoxyribonucleases, isomerases, phospholipases, peroxidases, phosphatases, phosphodiesterases, glucosidases, glucuronidases, oxidoreductases, transferases, hydroxylases, lysases, ligases, kinases, phosphorylases, DNA polymerases, DNA gyrase, DNA ligase and ribonucleases . Specific members of these classes which are suitable for entrapment are set forth below. These examples are non-limiting and those skilled in the art will be aware of other enzymes which are suitable for entrapment. These additional enzymes are also considered to be within the scope of the present invention. Unless otherwise noted, the identification of an enzyme as, for example, "Type VI-A (from horseradish) " is the identification of the relevant enzyme from the 1994 Sigma Chemical Company catalog. Where a source for an enzyme is indicated, for example, from a certain food or organism, it should be understood that the enzyme or substantially the same enzyme obtained from other sources can used in place therof. Coenzymes and enzyme co-factors, including but not limited to vitamins, vitamin derivatives and acetyl coenzyme A can be immobilized in the poly(organophosphazene) .

Nonlimiting examples of ureases include: Type III (from Jack Beans) , Type IV (from Jack beans) ; Type VI (from Jack Beans) ; Type VII-A (from Jack Beans) ; Type C-3 (from Jack Beans) ; Type II-C (from

Jack Beans) ; Type IX (from Jack Beans) ; Type X (from Bacillus pasteurii) ; and urease (ATP- hydrolyzing) .

Nonlimiting examples of proteases which are suitable for entrapment include: bromelain, chymopapain, chymotrypsin, including - chymotrypsin, clostripain, collagenase, elastase, ficin, kallikrein, metalloendopeptidase, papain, pepsin, peptidase, proteinase A, proteinase K, trypsin, Type I (from bovine pancreas) ; Type II (from Aspergillus Oryzae) ; Type IV (from Streptomyces caespitosus) ; Type VIII (subtilopeptidase A, from Bacillus licheniformis) ; Type VIII-A (from Bacillus licheniformis) ; Type IX (from Bacillus polymyxa) ; Type X (thermolysin, from Bacillus thermoproteolyticus rokko) ; Type X-A (from Bacillus thermoproteolyticus rokko) ; Type XIII (Aspergillopeptidase molsin, from Aspergillus saitoi) ; Type XIV (pronase E, from Streptomyces griseus) ; Type XV (from Bacillus polymyxa) ; Type

XVI (from a strain of Bacillus subtilis) ; Type

XVII-B (from Staphylococcus aureus strain V8) ; Type

XVII (from Staphylococcus aureus strain V8) ; Type

XVIII (newlase, from rhizopus species) ; Type XIX (from Aspergillus sojae) ; Type XX (endoproteinase arg-C, from mouse submaxillary gland) ; Type XX-S (endoproteinase arg-C, from mouse submaxillary gland) ; Type XXI (from Streptomyces griseus) ; Type

XXIII (from Aspergillus oryzae) ; Type XXIV-A-1 (from P 8038) ; Type XXVI (serratiopeptidase, from serratia species) ; Type XXVII (nagarase) ; Type XXXI (from a strain of Bacillus licheniformis) ; endoproteinase Lys-C; HIV-1 (expressed in E. coli) ;

Type VIII-AB (from Bacillus licheniformis) ; proteinase A (endopeptidase, from bakers yeast) ; proteinase K (from Tritirachium album) ; metalloendopeptidase (from grifola frondosa) ;

carboxypeptidase Y; endoproteinases, including endoproteinase arg-C, endoproteinase asp-N, endoproteinase glu-C, endoproteinase lys-C; leucine aminopeptidase; and trypsin. Nonlimiting examples of esterases which are suitable for entrapment by the polyphosphazenes include: carboxyl esterase, carboxylic-ester hydrolase, porcine pancreatic esterase, substilin, and pig liver esterase. Nonlimiting examples of viral enzymes include inverse transcriptase.

Nonlimiting examples of nucleases which are suitable for entrapment by the polyphosphazene polymers include: nuclease (from mung bean sprouts) ; micrococcal nuclease (micrococcal endonuclease, from Staphylococcus aureus, foggi strain) ; nuclease Pj (from Penicillium citrinum) ; and nuclease S _t (from aspergillus oryzae) .

Nonlimiting examples of collagenases suitable for entrapment include: sigma blend collagenase (clostridiopeptidase A, from Clostridium histolyticum) ; Type F; Type H; Type L; Type N; Type IA; Type I; Type VIII; Type II; Type IV; Type V; Type XI; Type VII; Type III; collagenase (from Achromobacter iophagus) ; collagenase/dispase (from Achromobacter iophagus/Bacillus polymyxa) ; and collagenolytic proteinase (from Paralithodes kamtshatica hepatopancreas) .

Nonlimiting examples of dehydrogenases include the following varieties of glucose-6-phosphate dehydrogenase: Type V (from bakers yeast); Type VII (from bakers yeast) ; Type IX (from bakers yeast) ; Type XV (from bakers yeast) ; Type XV-B (from bakers yeast) ; Type XI (from torula yeast) ; Type XII (from torula yeast) ; Type XXV (from

Bacillus stearothermophilus) ; Type XIX (from bovine adrenals) ; Type XXVI (from human erythrocytes) ;

Type XXIII (from leuconostoc esenteroides) ; Type XXIV, L-glutamic dehydrogenase, including Type I (from bovine liver) ; Type II (from bovine liver) ; Type III (from bovine liver) ; Type IV (from bovine liver) ; Type VI (from bovine liver) ; Type VIII (from rat liver) ; Type VIII (from sheep liver) ; glutamic dehydrogenase (NADP) ; and malate dehydrogenase.

Nonlimiting examples of decarboxylases include: L-arginine decarboxylase, L-glutamic decarboxylase, L-histidine decarboxylase, L-lysine decarboxylase, L-ornithine decarboxylase, oxalate decarboxylate, orotidine-5' -monophosphate decarboxylase, L- phenylalanine decarboxylase, L-tyrosine decarboxylase, and L-tyrosine decarboxylase apoenzyme.

Nonlimiting examples of deoxyribonucleases include: deooxyribonuclease I including Type II (from bovine pancreas) , Type II-S (from bovine pancreas) ; (Type IV (from bovine pancreas) ; DN-25

(from bovine pancreas) ; DN-EP (from bovine pancreas) ; deoxyribonuclease II including Type IV (from porcine spleen) ; Type V (from bovine spleen) ; Type VI (from bovine spleen) ; Type VII (from bovine spleen) ; Type VIII (from porcine spleen) ; and ATP dependent deoxyribonuclease (exonuclease V; exodeoxyribonuclease V) .

Nonlimiting examples of isomerases include: of-glycerophosphate dehydrogenase-triosephosphate isomerase; phosphohexose isomerase; phosphoglucose isomerase; phosphomannose isomerase; and trisephosphate.

Nonlimiting examples of phospholipases include: phospholipase A ₂ ; phospholipase A ₂ ; isoenzymes including phospholipase A ₂ -I, phospholipase A ₂ -III, and phospholipase A _j -IV; phospholipase B; phospholipase C including Type I (from C.

perfringens) , Type I-S, Type IX (from C. perfringens) , Type XIV (from C. perfringens) , Type

III (from B. cereus) , Type IV (from B. cereus) , Type V (from B. cereus) , Type XI (from B. cereus) ; phosphatidylinositol specific phospholipase C; phospholipase D including Type I (from cabbage) , Type II (from peanut) , Type III (from peanut) , Type

IV (from cabbage) , Type VI (from Streptomyces chromofuscus) , and Type VII (from streptomyces species) .

Nonlimiting examples of peroxidases which can be entrapped include: Type VI-A (from horseradish) ; Type VI (from horseradish) ; Type XII (from horseradish) ; Type I (from horseradish) ; Type II (from horseradish) ; Type X (from horseradish) ; peroxidase isoenzymes, Type VII (from horseradish) ; Type VIII (from horseradish) ; Type IX (from horseradish) ; Type XI (from horseradish) ; NADH peroxidase (from Streptococcus faecalis) ; microperoxidase, including microperoxidases MP-8, MP-9 and MP-11 (all from equine heart cytochrome C) ; and lactoperoxidase.

Nonlimiting examples of phosphatases include both acidic and alkaline phosphatases. The acid phosphatases include: Type I (from wheat germ) ;

Type II (from potato) ; Type III (from potato) ; Type IV-S (from potato) ; Type V (from bovine milk) ; Type VII (from white potato) ; Type X-A (from sweet potato) ; prostatic acid phosphatase. Alkaline phosphatases include: Type VII-S (from bovine intestinal mucosa) ; Type VII-SA (from bovine intestinal mucosa) ; Type VII-N (from bovine intestinal mucosa) ; Type VII-NA (from bovine intestinal mucosa) ; Type XX (from bovine intestinal mucosa) ; Type XXX (from calf intestinal mucosa) ;

Type XXX-A (from calf intestinal mucosa) ; Type VII- T (from bovine intestinal mucosa) ; Type VII-TA

(from bovine intestinal mucosa) ; Type VII-NT (from bovine intestinal mucosa) ; Type VII-NTA (from bovine intestinal mucosa) ; Type XXX-T (from calf intestinal mucosa) ; Type XXX-TA (from calf intestinal mucosa) ; Type I-S; Type VII-L; Type VII- LA; Type VII-NL; Type VII-NLA; Type XXX-L; Type XXX-LA (all of the preceding from bovine or calf intestinal mucosa) ; Type IV (from porcine intestinal mucosa) ; Type V (from chicken intestine) ; Type VIII (from rabbit intestine) ; Type X (from dog intestine) ; Type XI (from horse intestine) ; Type XII (from sheep intestine) ; Type XIII (from pigeon intestine) ; Type XVIII (from porcine intestine) ; Type XIX (from eel intestine) ; Type XXIII (from trout intestine) ; Type XXV (from guinea pig intestine) ; Type XXVI (from cat intestine) ; Type III (from E. coli) ; Type III-L (from E. coli) ; Type III-S (E. coli) ; Type IX (from bovine liver) ; Type XV (from bovine placenta) ; Type XVI (from porcine placenta) ; Type XVII (from human placenta) ; Type XXI (from bovine milk) ; Type XIV (from human placenta) ; Type XXXII (from bovine kidney) ; Type XXXIII (from bovine liver) ; and alkaline-biotinamidocaproyl phosphatase. Nonlimiting examples of phosphodiesterases include phosphodiesterase I including: Type V (from bothrops atrox) ; Type II (from crotalus adamanteus venom) ; Type VI (from crotalus adamanteus crude dried venom) ; Type VII (from crotalus atrox venom) ; Type IV (from crotalus atrox crude dried venom) ; Type VIII-S (from crotalus durissus terrificus venom) ; and Type VIII (from crotalus durissus terrificus venom) ; phosphodiesterase II, including Type I-SA (from bovine spleen) ; Type I (from bovine spleen) ; 2' :3'- cyclic nucleotide 3 ' -phosphodiesterase; 3' :5'-

cyclic nucleotide phosphodiesterase; and 3' :5'- cyclic nucleotide-activator phosphodiesterase.

Nonlimiting examples of glucosidases include: cϋ-glucosidase, including Type I (from bakers yeast) ; Type III (from yeast suspension) ; Type IV (from brewers yeast) ; Type V (from rice) ; Type VI (from brewers yeast) ; Type VII (from yeast solution) ; 3-glucosidase; maltase; isomaltase.

Nonlimiting examples of glucuronidases include: jS-glucuronidase, including Type IX-A, Type VII-A,

Type VIII-A, Type X-A, Type H-l, Type HP-2, Type H- 2, Type HP-2S, Type H3, Type H3AF, Type HA-4, Type H-5, Type L-II, Type S-I, Type B-l, and Type B-3, Type B-10.

III. Substances for Diagnostic Imaging

Any desired inert gas can be incorporated into the polymeric materials at the time of hydrogel or organogel formation, including air, argon, nitrogen, carbon dioxide, nitrogen dioxide, methane, helium, neon, and oxygen to form a diagnostic imaging product. Sterilized air or oxygen is a preferred imaging contrast agent. The ratio of polymer to gas is determined based on the gas that is to be encapsulated, for example, as required to produce a particle size small enough to be injected.

Other contrast agents can be incorporated in place of the gas, or in combination with gas, using the same methods. These are useful in imaging using the more common techniques such as ultrasound, magnetic resonance imaging (MRI) , computer tomography (CT) , x-ray, as well as the less common positron emission tomography (PET) and single photon emission computerized tomography (PET) .

Examples of suitable materials for MRI include the gataliniu chelates currently available, such as diethylene triamine pentacetic acid (DTPA) and Gatopentotate dimeglumine, as well as iron, magnesium, manganese, copper and chromium. These are typically administered in a dosage equivalent to 14 ml for a 70 kg person of a 0.5 M/liter solution.

Examples of materials useful for CT and x-rays include iodine based materials for intravenous administration such as ionic monomers typified by Diatrizoate and iothalamate (administered at a dosage of 2.2 ml of a 30 mg/ml solution), non-ionic monomers typified by iopamidol, isohexol, and ioversol (administered at a dosage of 2.2 ml 150-

300 mg/ml) , non-ionic dimers typified by iotrol and iodixanol, and ionic dimers, for example, ioxagalte. Other useful materials include barium for oral use.

IV. Method of Preparation of the Hydrogel

The polymer hydrogels can be prepared by preparing a mixture of a poly(organophosphazene) , such as MEEP, and the desired substance to be delivered, such as an enzyme, and cross-linking the poly(organophosphazene) . One method of cross- linking the poly(organophosphazene) is by exposing the mixture of poly(organophosphazene) polymer and enzyme to gamma, ultra-violet, or other high energy radiation (for example x-ray or electron beam) in an amount and for a time period sufficient to cause cross-linking of the poly(organophosphazene) .

The optimal time period and dosage of radiation can be easily determined for the desired poly(organophosphazene) . When using gamma radiation, a dosage of between approximately 0.1

Mrad and 30 Mrad, and preferably, between 0.2 Mrad

and 0.5 Mrad, for a period of time ranging from approximately one second to ninety days is sufficient. Sources of gamma radiation include a ^Co, nuclear reactor, and a linear accelerator or synchrotron.

The length of time necessary to complete the crosslinking is a function of the strength of the source, the substituent groups on the polymer, and the degree of crosslinking desired. In general, crosslinking with a source of 0.25 Mr/hour often takes between approximately 1 and 20 hours, whereas crosslinking with as source of lOMr/hour takes only a short time.

In an alternative embodiment, the poly(polyorganophosphazene) can be cross-linked using ultraviolet radiation. Ultraviolet cross¬ linking is usually accomplished in a time period of one second to thirty minutes, depending on the UV source intensity. For the purpose of convenience in the discussion and examples below, poly[bis ( (methoxyethoxy) ethoxy)phosphazene] (polymer 1, also referred to as "MEEP") is used as the illustrative poly(organophosphazene) for production of the enzyme immobilized hydrogel. The invention, however, is not limited to the use of this poly(organophosphazene) as the hydrogel polymer, and is instead a general technique.

Gamm -ray-induced cleavage of the C-H bonds can generate free radicals at any of the five carbon atoms in each side group. Subsequent intermolecular radical combination leads to cross¬ linking. In most radiation-induced cross-linking reactions the side-group coupling processes are accompanied by backbone cleavage. The inorganic backbone structure of poly(organophosphazenes) generally and MEEP in particular is less sensitive

to radiation damage and cleavage than is the backbone in classical organic polymers, even at high radiation doses. This free radical cross¬ linking mechanism offers the opportunity for covalent binding of the polymer chains to any substrate that bears surface carbon-hydrogen or carbon-chlorine bonds and especially to those that have aliphatic residues at the surface.

The method and compositions of the present invention will be further understood with reference to the following non-limiting examples.

Example 1: Synthesis of Poly[bis ( (methoxyethoxy) ethoxy) -phosphazene] (1)

(a) General procedures and materials. Materials. Most of the compounds were obtained and purified as described in Allcock, H.R.; Rutt, J.S.; Fitzpatrick, R.J. Chem. Mater. 1991, 3 , 442, and Allcock, H.R.; Fitzpatrick, R.J. Chem. Mater. 1991, 3 , 450. The polymer syntheses were protected by an atmosphere of dry nitrogen using standard Schlenk line techniques. Tetrahydrofuran (THF, Omnisolv) was distilled from sodium benzophenone ketal under a dry nitrogen atmosphere. 2- (2- Methoxyethoxy) ethanol was obtained from Aldrich Chemical Company and was distilled from barium oxide and stored over molecular sieves (4 A) . Sodium (Aldrich) , urease (Sigma) , urea (Sigma) , and the buffers (pH 4, pH 7, pH 10, Fluka) were used as received. Hexachlorocyclotriphosphazene (Ethyl Corp.) was obtained from a trimer/tetramer mixture by two fractional sublimations (30 oC, 0.1 mm Hg) . Poly(dichlorophosphazene) was obtained by the thermal ring opening polymerization at 250 oC in a sealed evacuated Pyrex tube, as described in Allcock et al . , J. Am. Chem. Soc. , 1965, .87, 4216. pH measurements were taken with a Beckman 40 pH

meter. All measurements were obtained at 25oc, and the electrode was standardized with pH 10 and pH 4 buffer solutions.

(b) Preparation of [NP (OCHjCHjOCHjCH _j OCH _j ) _; ,] „ (1) . Uncross-linked poly[bis ( (methoxyethoxy) - ethoxy)phosphazene] (1) was prepared as described in Allcock, H.R.; Austin, P.E.; Neenan, T.X. ; Sisko, J.T.; Blonsky, P.M.; Shriver, D.F. Macromol ecules 1986, 19, 1508, and Blonsky, P.M.; Shriver, D.F.; Austin, P.E.; Allcock, H.R. J. Am.

Chem. Soc . 1984, 106, 6854. The synthesis is based on the replacement of chlorine atoms in poly(dichlorophosphazene) by treatment with the sodium salt of (methoxyethoxy) ethanol in tetrahydrofuran solution.

Poly(dichlorophosphazene) (12.0 g, 0.104 mol) was dissolved in dry THF (750 mL) . Sodium (9.52 g, 0.412 mol) was allowed to react with 2- (2- methoxyethoxy)ethanol (78.48 g, 0.62 mol) in dry THF (350 mL) . The alkoxide solution was added to the polymer solution. The reaction was allowed to proceed for 72 hours at room temperature. The reaction solution was concentrated, and the polymer was precipitated into hexane. The polymer was then dissolved in deionized water and was placed in cellulose dialysis tubing (12-14,000 molecular weight cutoff) for 72 hours and was then dialyzed against methanol for 48 hours. The polymer solution was dried under vacuum to yield pure polymer 1.

Example 2 reparation of MEEP Hydrogel- Immobilized Urease.

Poly[di (methoxyethoxy) -ethoxy]phosphazene 1

(10.0 g, 0.035 mol) was dissolved in deionized water (200 mL) . Urease (0.8 g) was added to the solution of polymer 1. After the mixture of polymer 1 and urease became homogenous, a film was

cast onto a Teflon sheet. After evacuation of water, the film was further dried under a vacuum at 55°C. The resulting film was sectioned into two halves which were sealed in a vacuum. The films were cross-linked by exposure to ^Co gamma radiation from the Brezeale Nuclear Reactor Facility on the campus of the Pennsylvania State University. One film was cross-linked by irradiation with 0.2 Mrad of °°Co gamma radiation, while the other was cross-linked by irradiation with 0.5 Mrad of ^Co gamma radiation. The films were then divided into subsections (approximately 1.1 g per section) and were washed with and swelled in pH 7 buffer for 24 hours to remove enzyme adsorbed at the surface of the hydrogel. The gels were retrieved from the buffer solution and were dried in vacuo at 35°C for 48 hours.

Example 3 Measurement of the effectiveness of enzyme immobilization The cross-linked poly(organophosphazenes) containing the entrapped enzymes were tested to determine whether the urease was indeed immobilized in the polymer system. The MEEP/enzyme conjugate was placed in an aqueous media and allowed to swell. The swollen MEEP/enzyme conjugate was left in the aqueous media for several weeks. Upon removal of the gel from the aqueous media, a urea solution was added to the media and the pH of the gel-free aqueous media was monitored. After 24 hours, no significant change was detected in the pH of the solution. This indicates the absence of free urease. Changes in the degree of cross¬ linking using either 0.2 or 0.5 Mrad of gamma radiation did not change this result.

Example 4 Measurement of the activity of MEEP hydrogel-immobilized urease.

The MEEP/urease conjugate formed by exposure of the MEEP to 0.5 Mrad of gamma radiation was placed in a pH 7 buffer solution (5 mL) and allowed to swell for 30 minutes. A solution of urea (1.0 M solution, 10 mL, 0.01 mol urea, 0.60 g urea) was then added to the swollen hydrogel and supernatant solution. The pH of the resulting mixture was monitored over a 24 hour period. The experiment was repeated five times with the same gel, as well as with different gels exposed to the same radiation dose to ensure reproducible results. Figure 1 provides the average results of the four experiments. In Figure 1, (O) represents the increase in pH over time for urease immobilized in a MEEP gel which was cross-linked by exposure to 0.2 Mrad gamma radiation; (D) represents the increase in pH over time for urease immobilized in a MEEP gel which was cross-linked by exposure to 0.5 Mrad gamma radiation; (•) represents the increase in pH over time for a MEEP gel which contains no immobilized urease which was cross¬ linked by exposure to 0.2 Mrad gamma radiation; and (■) represents the increase in pH over time for a MEEP gel which contains no immobilized urease and which was cross-linked by exposure to 0.5 Mrad gamma radiation. The addition of urea to the mixture resulted in a rapid pH rise for the MEEP/urease composites swollen in the solution, indicating the conversion of the urea to ammonia by the hydrogel. In contrast, the MEEP hydrogels which contained no immobilized urease had no effect on the pH of the solution. Figure 2 shows the results of repeated treatments with urea on the same polymer hydrogel . The same swollen gel was treated sequentially with five different urea solutions. In each case, the

enzyme trapped within the MEEP hydrogel generated the same initial increase in the pH of the solution and the same leveling off time for the pH (two hours) when recycled. The hydrogel used for this was that disclosed in Example 3. In Figure 2, (O) represents the data from the first experiment; (•) represents the data from the second experiment; (D) represents the data from the third experiment; (■) represents the data from the fourth experiment; and (Δ) represents the data from the fifth experiment. Figure 3 shows the results of the addition of sequential portions of a single urea solution to a MEEP hydrogel containing immobilized urease. Five successive portions containing urea (0.01 mol) were added to the MEEP hydrogel. As shown in Figure 3, the first addition of urea caused the largest increase in the pH of the aqueous medium. Addition of the second through fifth portions of urea increased the pH of the media only slightly. In Figure 3, (O) represents the data from the first addition; (•) represents the data from the second addition; (D) represents the data from the third addition; (■) represents the data from the fourth addition; and (Δ) represents the data from the fifth addition.

Example 5 Measurement of the Activity of

Irradiated and Non-irradiated Free Urease

Two solid samples of urease (1.5 g each) were irradiated with 0.2 Mrad or 0.5 Mrad of gamma radiation from a ^Co source. Samples of the irradiated urease (0.08 g) were placed in 5 mL of a pH 7 buffer solution. After the mixtures became homogenous, a solution of urea (1.0 M solution, 10 mL, 0.01 mol of urea, 0.60 g of urea) was added to the homogenous mixtures. The pH of the mixtures was monitored for 24 hours. Each experiment was

conducted four times to ensure reproducible results.

Figure 4 provides the average results of the four experiments. In Figure 4, (•) represents the increase in pH over time for non-irradiated free urease; (■) represents the increase in pH over time for free urease irradiated at 0.2 Mrad; and (A) represents the increase in pH over time for free urease irradiated at 0.5 Mrad. The data shown in Figure 4 indicate that the irradiated free urea samples generated a final pH value which was approximately 10% lower than that obtained for the native (non-irradiated) free urease.

Figure 5 compares the results of the free irradiated urease and non-irradiated urease disclosed in comparative example 1, with the results for the urease immobilized in the MEEP hydrogel, disclosed in Example 3. In Figure 5, (•) represents the increase in pH over time for non- irradiated free urease; (■) represents the increase in pH over time for free urease irradiated at 0.5 Mrad; and (A) represents the increase in pH over time for urease immobilized in a MEEP hydrogel which was cross-linked by exposure to irradiation at 0.5 Mrad. Figure 5 shows that the urease immobilized in the MEEP hydrogel is approximately 10% less than that obtained for the free, irradiated urease and about 20% less than that obtained for the free, non-irradiated urease. Thus, a considerable amount of the enzyme is retained after immobilization, and the MEEP-enzyme conjugate allows facile cycling and recovery of the active species.

V. Methods of Making Microcapsules. The method of preparing the microparticles should be selected to provide a microparticle

having the desired size for the intended use. In a preferred embodiment for the preparation of injectable microcapsules capable of passing through the pulmonary capillary bed, the microcapsules should have a diameter of between approximately one and seven microns. Larger microcapsules may clog the pulmonary bed, and smaller microcapsules may not provide sufficient echogenicity. Larger microcapsules may be useful for administration routes other than injection, for example oral (for evaluation of the gastrointestinal tract) or by inhalation.

The polymers disclosed herein can be fabricated into loaded microparticles or nanoparticles using any appropriate method known to those skilled in the art.

In one embodiment, the polymer is mixed with the substance to be delivered, and the mixture is dried and then frozen to between approximately -90 and - 100 degrees Celcius. The frozen material is then chopped to a powder, using, for example, a sophisticated Waring Blender. The chopped material is then warmed and swollen in the desired solvent for use. This is a useful procedure for the preparation of microparticles.

A. Preparation of a polymer solution. In one embodiment, a concentrated solution of the poly(organophosphazene) is extruded as a particle of desired shape into a nonsolvent and then collected, dried, and radiation crosslinked. If the polymer is water soluble or swellable, the nonsolvent would typically be a hydrocarbon such as heptane or pentane.

Described below are two methods for the preparation of injectable particles that contain gas. In one method, a jet head is used that allows the co-extrusion of a solution of polymer and air

to produce nascent microencapsulated air bubbles which fall into a hardening solution of counterions. A second method employs ultrasound to introduce cavitation-induced bubbles into the polymer before capsule formation by spraying. To incorporate gases other than air, a solution of the desired polymer is placed in an atmosphere of the desired gas and sonicated for a sufficient amount of time before crosslinking to ensure that gas bubbles are dispersed throughout the microparticulates. In either case, the determining factors on size of resulting microcapsules will be the selection and concentration of polymer and solvent, and size of droplets formed by the atomizer.

Alternatively, a substance to be delivered can be mixed into the polymer solution.

1. Preparation of one to ten micron Microcapsules An example of an air-atomizing device is a

Turbotak, from Turbotak, Inc., Waterloo, Ontario. A Turbotak is a hollow stainless steel cylinder, 2.64 cm. wide x 4 cm. long. Liquid is fed into the Turbotak from the top and pressurized air is fed from the side. The pressurized air mixes with the liquid, forcing tiny liquid droplets out through the orifice of the nozzle. The air pressure can be set to between 50 and 80 psig. The distance between the orifice of the Turbotak and the pan containing the crosslinking ions is fixed at between about one to two feet. The size of the nozzle orifice is 1 to 2 mm in diameter.

Air can be pressurized with a syringe pump such as a Razel pump, having a flow rate in the range of between 5 ml/hr and 30 ml/hr or a Sage pump, having a flow rate in the range of between 0.02 ml/min and 126 ml/min.

Mixing pressurized air with a polymer solution aerates the polymer solution and produces a high yield of air-encapsulated polymeric microcapsules.

Even without sonicating the polymer solution, microcapsules produced using the Turbotak nozzle have entrapped air, as seen by light microscopy.

2. Method for the Preparation of larger Microcapsules

Larger microcapsules can be prepared using a droplet-forming apparatus by spraying an aqueous solution of polymer containing the gas or other substance of interest through an apparatus such as a plastic syringe, where the polymer solution is extruded through a needle located inside a tube through which air flows at a controlled rate.

The rate of polymer extrusion is controlled, for example, by a syringe pump. Droplets forming at the needle tip are forced off by the coaxial air stream and collected in the nonsolvent. The shape and size of these microcapsules depend on the polymer and parameters such as the polymer extrusion rate, air flow, and needle diameters used in the microencapsulation procedure.

Macrospheres with millimeter diameters can be prepared by extruding the polymer through pasteur pipets or their equivalent.

VI. Detecting gas-encapsulating microcapsules

The relatively homogenous population of spherical gel microcapsules, filled with air bubbles, can be seen by an inverted microscope. Most microcapsules produced by the first method are smaller than seven microns in diameter. Particle size analysis can be performed on a Coulter counter.

Due to their in vivo stability their potential application is extended beyond vascular imaging to liver and renal diseases, fallopian tube diseases, detecting and characterizing tumor masses and tissues, and measuring peripheral blood velocity. The microcapsules can optionally be linked with ligands that minimize tissue adhesion or that target the microcapsules to specific regions. The method for imaging by detection of gas bubbles in a patient uses a transducer which produces pulses, illustrative of ultrasonic acoustic energy, having predetermined frequency characteristics. A first pulse has an increasing frequency with time, and a second pulse has a decreasing frequency with time. Imaging arrangements produce images of the region within the specimen after exposure to the first and second pulses.

The conventional technique for determining the presence of bubbles in the blood stream uses a Doppler shift in the frequency of the ultrasonic acoustic energy which is reflected by the blood. The amplitude of the Doppler bubble signal increases nearly proportionally with increases in the radius of the bubble. The human hearing mechanism is considered the most accurate processor for recognizing whether bubble signals are present or absent. For this reason, it is preferable to

have a skilled operator to obtain satisfactory results using Doppler blood flow monitoring equipment .

To determine whether the air-filled microcapsules are useful for in vivo imaging, the following in vi tro method, described in more detail in the following examples, can be used.

Microparticles prepared by the above methods are suspended in a capped tissue culture tube. For ultrasound imaging, the tubes are placed on top of a pad covered with coupling medium above the transducer. The transducer is held in place at roughly a 90° angle of incidence to minimize any motion artifacts. The transducer acts as a transmitter and also receives ultrasound radiation scattered back from the tube. B-mode and Doppler images are established for tubes filled with polymeric microcapsules and the resulting images are compared with a control consisting of an image from a tube containing buffer alone. The B-mode of display gives a two dimensional image of a slice through the scanned tube.

This method was used to obtain in vi tro results on the microparticles in the working examples described below. These results correlated well with the in vivo results, as shown by Doppler imaging techniques (described below) . Since the in vi tro and in vivo data showed a high degree of correlation in the working examples, this test is reasonably predictive of the in vivo stability of microparticles.

Modifications and variations of the present invention will be obvious to those skilled in the art from the foregoing detailed description of the invention. Such modifications and variations are intended to come within the scope of the appended claims .