AU2008202119A1 - Electroprocessing in drug delivery and cell encapsulation - Google Patents

Description

14/05 '08 12:06 FAX 613 8618 4199 FB RICE CO.

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AUSTRALIA

1i j- oil rn s a ii Hi A i M>ii -ii, tl-L .n i I FB RICE CO Patent and Trade Mark Attorneys Patents Act 1990 VIRGINIA COMMONWEALTH UNIVERSITY INTELLECTUAL PROPERTY FOUNDATION COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Electroprocessing in drug delivery and cell encapsulation The following statement is a full description of this invention including the best method of performing it known to us:- COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:06 FAX 613 8618 4199 FB.R-ICE CO, 0004 00 o la ELECTROPROCESSING IN DRUG DELIVERY AND CELL

ENCAPSULATION

This application is a divisional application of Australian Patent Application No.

2002224387, (Acceptance No. 20022243 87) the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION o The present invention relates to novel compositions comprising electroprocessed 00 materials with substances, and methods of making and using these compositions in 0 delivery of substances.

BACKGROUND OF THE INVENTION Numerous methods exist for delivering substances to desired locations in vivo or in vitro. One such method uses devices or objects that contain a substance and will release the substance within a desired location. One desirable application for such methods is the administration of such objects to a location within the body of an organism, followed by the subsequent release of the desired substance into the body. In these examples, the implant often contains the substance and a carier. After implantation, the substance is released by a variety of means including, for example, diffusion from an implant or dissolution or other degradation of a capsule coat.

Bioco mpatability is a desirable attribute in compositions designed for substance delivery. With surgical and subdermal implants, for example, the substance to be delivered is often contained in a matrix comprised of synthetic polymers. Where natural products are used in making bandages, the products typically comprise wood products such as cellulose or other materials that are not readily absorbed by the body of the recipient Accordingly, such bandages must eventually be removed. Implants compressed from natural materials that may be absorbed by the body are one way to improve biocompatability and is one area in which improvements are desired.

There is also a continuing need for greater versatility and flexibility in substance delivery technology. Additional techniques for controlling release kinetics and spatial patters of release or delivery are examples of developments that can improve substance delivery. Implants in which there is refined control of structure at the microscopic or molecular level and overall implant shape are also desired. Such methods could allow, for example, frller refinements in control of pore size or other attributes that affect diffusion in and out of a matrix, or more refined control of the distribution of a COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 08 12:07 FAX 613 8618 4199 FB RICE CO. o1005 00 0 o 2 substance within a matrix. New methods that allow encapsulation of living cells within a matrix are especially desired. Such methods would allow implants to contain, for example, cells that produced desired substances, cells that promote tissue growth, or cells that serve both of these functions.

SUMMARY

SThe present invention provides compositions comprising an electroprocessed _material and a substance. The substance may be the material itself, or another substance which may be delivered with the electroprocessed material to a desired site.

Sometimes the compositions comprising an electroprocessed material and a substance 0 10 are in the form of a matrix. The electroprocessed materials may include any natural or Snon-natural materials or blends thereof. The substance is released from the composition or causes the release of molecules or compounds from the composition.

Substance release can occur in vitro, in vivo, or both.

The present invention also includes a method for delivery of substances to a location using the present compositions comprising an electroprocessed material and a substance. The locations can be in vitro, in vivo, or both. The invention also includes methods for making the compositions of the present invention.

The compositions of the present invention include an electroprocessed material and a substance. The material can include naturally occurring materials, synthetically manufactured materials, or combinations thereof. Naturally occurring materials include natural organic or inorganic materials, genetically engineered materials and include synthetic alterations thereof. Synthetic materials include materials prepared through any method of artificial synthesis, processing, or manufacture. The invention includes materials that degrade and can absorbed by the body, or will persist in whole or in part and become portions of an extracellular tissue matrix. The compositions may be made using any electroprocessing technique, including but not limited to electrospinning, electroaerosol, electrospraying or electrosputtering techniques, or any combination thereof. Accordingly, electroprocessed droplets, particles, fibers, fibrils, or combinations thereof are all included in the compositions of the present invention. In a preferred embodiment, the electroprocessed materials form a matrix, and in some cases are similar to an extracellular matrix. Matrices may also be formed from materials than can combine to form another material, such are precursor materials. For example, fibrinogen, when combined with thrombin, will form fibrin.

Any material that may be electroprocessed may be used to form an electroprocessed material to be combined, either before, during or after electroprocessing, with COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:08 FAX 613 8618 4199 FB RICE CO.

006 00 0 o 3 a substance, to form the compositions of the present invention. The compositions of the present invention contain one or more substances, The substance includes any type of substance desired, with examples including molecules, cells, objects, or combinations thereof. In some cases, the substance is the electroprocessed material itself. Molecules can be any size, complexity, or type, including both organic or inorganic molecules as well as any combination of molecules. Molecules include naturally occurring and synthetic molecules. Examples of molecules include, but are C not limited to therapeutics, cosmetics, nutraceuticals, vitamins, minerals, humectants, 0C molecules produced by cells, including normal cells, abnormal cells, genetically 00 O 10 engineered cells and cells modified through any other process. Both eukaryotic and O prokaryotic cells are included in the category of substances. Substances also include, without limitation, antigens, antimicrobials, antifungals, molecules that can cause a cellular or physiological response, metals, gases, minerals, ions, and electrically, magnetically and electromagnetically light) reactive materials. Cells are derived from natural sources or are cultured in vitro. Combinations of different types or categories of cells can be used. Examples of objects include, but are not limited to cell fragments, cell debris, organelles and other cellular components, tablets, viruses, vesicles, liposomes, capsules, and other structures that serve as an enclosure for molecules. It is to be understood that the composition of the present invention comprises at least one substance. Accordingly, numerous substances or combinations of similar or different substances may be combined with the electroprocessed materials.

The substances may be combined with the electroprocessed material through electroprocessing techniques or through other techniques. The invention also includes embodiments in which the composition comprises electroprocessed matrix materials without an additional substance. In that embodiment, the electroprocessed matrix materials may act as a substance.

The invention provides numerous uses for the compositions of the present invention. One possible use is the delivery of substances. Substance delivery from the compositions of the present invention can occur in vivo, for example upon or within the body of a human or animal. Substance delivery can also occur in vitro, for example within a cell culture apparatus or well. Substances delivered include those substances contained within the compositions, other substances produced by the substance contained in the composition, or both. For example, a substance may be a cell contained within the electroprocessed material, and the cell may synthesize and release one or more molecules. Cells may release molecules in response to signals, so that the molecules are released in a specific desired circumstance. For example, an inducible COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:08 FAX 613 8618 4199 FB RICE CO. @1007 00 0 0 4 promoter in an engineered cell within an electroprocessed material may be used to stimulate the expression and or release of a growth factor.

The compositions of the present invention are versatile with respect to control of substance release from the compositions. Release kinetics of substances can be controlled by manipulating a wide variety of matrix parameters. In various embodiments, the release rate, onset of release of more than one compound either at the _same or different times, creation of gradients of release and spatial patters of release C, may be manipulated. Compositions that contain electrical or magnetic materials can be 0, influenced to move, cause motion, or produce a biological activity by applying an 00 10 electric current or a magnetic field to the composition located on or within a body, or in vitro. Electroprocessed compositions that contain light sensitive components may be designed. These compositions may move or be induced to release or bind substances in response to specific wavelengths of light. Compositions containing nucleic acids or genetically engineered cells, for example, can be used in gene therapy. Other examples include embodiments used in wound care, tissue organ replacements, and prostheses.

In some embodiments, the electroprocessed material itself contains desired properties of substances, and acts as a substance without addition of another substance. The invention may therefore include a wide variety of methods of using the compositions of the present invention in medical, veterinary, agricultural, research and other applications. The compositions of the present invention may provide safer and more predictable release of substances and may provide a major advance in the field of substance delivery, especially drug delivery.

The invention also includes methods for making the compositions of the present invention wherein any type of electroprocessing technique, combination of electroprocessing techniques, or a combination of an electroprocessing technique and another technique, such as aerosol techniques may be used. The method may include streaming, spraying, dropping or projecting one or more solutions, fibers, or suspensions comprising the materials to be electroprocessed toward a target under conditions effective to deposit the materials on a substrate. The substances to be combined with the electroprocessed materials may be electroprocessed toward the target either before, during or after electroprocessing the material. In this manner, the substance may be incorporated within the electroprocessed material during formation, or may coat the electroprocessed material. Accordingly, one or a plurality of sources of materials and substances may be used to provide the ingredients for the electroprocessed composition of the present invention. For example, collagen and a COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:09 FAX 613 8618 4199 .FB RICE CO.

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polymer such as poly glycolic acid may be electroprocessed through any combination of electrospinning and electrospraying from two sources. At the same time or at selected times thereafter, substances may be provided from other sources: for example, Sa third source provides a growth factor, a fourth source provides an anti-angiogenic factor, and a fifth source provides genetically altered fibroblasts. These sources of substances may provide the substances through one or more electroprocessing techniques, such as electrospin, electrospray, electroaerosol, electrosputter or any C, combination thereof. These sources may also provide the substances to the 0, electroprocessed material through non-electroprocessing techniques, such as aerosol 00 10 delivery, dripping, coating, soaking or other techniques.

o In one embodiment, the compositions of the present invention may comprise one or more electroprocessed materials that form a matrix combined with at least one substance. Either the source or target is charged, and the other is grounded. The substrate upon which electrodeposition occurs can be the target itself or another object of any shape or type. For example, the substrate can be an object disposed between the orifice and the target. In one embodiment, the substrate is a location on or within an organism, such as a tissue, a wound site, a desired location for substance delivery, or a surgical field in which the composition is to be applied. By manipulating process parameters, compositions of the present invention can be manufactured with a predetermined shape, for example, for depositing the material onto or into a molded substrate. Substrate shape can be manipulated to achieve a specific three-dimensional structure. Targets can also be rotated or otherwise moved or manipulated during electroprocessing to control distribution of the electroprocessed material and, in embodiments involving electroprocessed fibers, the orientation of the fibers.

Substances included in the composition can be combined with the matrix material by any means before, during, and/or after electrodeposition.

The electroprocessed compositions may be formed into any desired shape. For purpose of substance delivery, the desired shape is dictated by the application. Nonlimiting examples include the following: in the form of a patch for application to the skin; in the form of a wafer or tablet for ingestion; in the form of a wafer for application to a site of removal of glioma; in the form of a wrap to surround a tumor; in a particulate form for spraying on a surgical site; and in particulate form for delivery of substances through inhalation.

COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:10 FAX 613 8618 4199 FB RICE CO.

[009 00 S6 Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic drawing of an embodiment of an electroprocessing device C including the electroprocessing equipment and a rotating wall bioreactor.

00 10 Figure 2 is a schematic drawing of an embodiment of an electroprocessing device o including the electroprocessing equipment and a rotating wall bioreactor.

Figure 3 is a graph showing the release profile of vascular endothelial growth factor (VEGF) from one embodiment of the present invention obtained by electrospinning a solution comprising collagen, polylactic acid (PLA), polyglycolic acid (PGA), and

VEGF.

Figure 4 is a graph showing the release profile of VEGF from an embodiment of the present invention obtained by electrospinning a solution comprising collagen, polylactic acid, and polyglycolic acid, and VEGF and subsequently cross-linking the electroprocessed material by exposure to glutaraldehyde vapor.

Figure 5 is a graph showing the release profile of the tetracycline from several embodiments of the present invention obtained by electrospinning solutions containing tetracycline along with PLA poly(ethylene-co-vinyl acetate) or a combination of PLA and poly(ethylene-co-vinyl acetate).

Figure 6 is a graph comparing the release profile of tetracycline from an embodiment of the present invention and several other compositions. The embodiment of the present invention was obtained by electrospinning a solution containing tetracycline with poly(ethylene-co-vinyl COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 *08 12:10 FAX 613 8618 4199 FB RICE CO. ~010 00 (Nacetate)_. The- other compositiona were periodontal fibers containing Z2vfl% tetracy-cline hydrOehlorirja and films containing teftcycline with polylactic; acid, pe*(ethylceeco-vny1 acetate) or a combbnetion ofpolylacdfc acid and pojy(etyl c~vinyl acetilte).

5 Figmre 7 is9 a graph Showing the release projie of tetracycline from several embodimuents of the preent invenition obtained by electrospiuning solution containing tetracline With poly(ethylenea-co..vinyj acetate).

Figure 8 is a schematic drawing ofanother ernbodhrnst of an electroprooessing device including o 10 the elecrroprocessfrtg equipment and a rotating wall bioreactor.

00 DETAILED DES CRTPTTON OF ThE lPE"RRD

EM&BODIMENTS

Tho term "sbstan-.6" shell be ised throughout this application in its broadest deftnition.

ci The term substance includes one or more mnolecules, objecis, or cells of any type or size, or ofnbioiajions thFreof- Substances can be in any form. including, but not limjited to solid, semisolid, wet or dry mbdnre, gas5, soluion, suspension combinations thareat Substaces include molecules of mny size and in any combination. Cells include aUl cell types ofpmkasyotic and eukaryotic cells, whether it' natural state or altered by genetc engineerng or any other process. Cells carn his ft=n a natural soiwc or cultired in vitro and can be living or dead- Combinations of different -types of cells can be used_ Objects can be of any size, shape, and composition that may be combined withi or coupled to an electroprocessed. material Exampples of.

objecs inclUde, but re- not limited to, cell fragments, cell debris, fragments of~oefl walls, fragments of viral walls, organelles and other cell1 conponeaNt, tablets, viruse, vesicles, lipasoines, capsule;, nmaparticulatms and othwr structures that serve as an enclosure fi~r molecules. The compositions of the present in-vexltion may comprise one substance or any combiatloa of substances.

The terms aeetorcaig nd "eleckrodepoaitionr shal bts de~fined broadly to include, all methods of cletrospinbI clectrosprayig, clectroacrosoling, and electrosrputtoring of natrials, combinatigns of two or more such methods, aud any other method whcrein material: are streamed sprayed, sputtered or dripped across an electric field and toward a target The electroprocessed matexis can be eleefroprocessed frm one or more grounded iteervoirs in the direction of a charged substrate or from charged reserviEs toward a grounded target 'Tectrospinring" means a process in which fibers are formed from a solution or melt by streaming sn electricafly charged solution. or meit trough an orifice. 'Tezoeooigmean a process in wich~ droplets are formed firom a solution or melt by streming an eleecay cbur~ed polymer solution or melt through an orifice. The term electroprocessjag is not limite to the xpecific exkmplos set forth herin 1 axid it inacjhdn %ny moans of using arm electrical field for depositing a material on a target 7 COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05''08 12:11 FAX 613 8618 4199 FE RIPZ CO. 01 00 The term 'tateriaV" rfers to any eompoimd, molecule, subusane, or growp or combination thereof that forms any type of structure or group of strurcturs during or after elcctroprooessin, Materials include natural materials, synthietic materials, or eontiuatious thereof. Nai~ly occurring organic muirrial. include any substances naturally fond in te body of plant or other organismso, regiardless of whether those materials bare or can be produced or altered synthetically. Synthetic materials include any matedaals prepared ftrough any method of artificial synthesis, processing, or mianufacture. Preferably the mterials axr biologically comphatibls matmials.

One Wlass of syathetiG materials, preferably biologically compatible synthectic, materials, comprises polymers. Such polymers Imnde but are not limited to the ;Following- 00 poly(urethanes), poly(siloanes) or silicones, poly(ethylene), poly(vinyl pysidone), poly(2o ihycbuxy ethyl 2tietacrylate), poly(N-vinyl pyrrolidone), poly(metl methacrylate), poly(vinyl 0 alcMrohol), poly(acrylic acid), polyacnryLamidt, poly(ethiylene-co-vinyl acetate). ply~etln Poc61mn glycol), poly(mehaczylir, acid). polylactides (FLA), potyglycoides (IPGA), -poly(Iactide-ooglycolides) (PLGA), pulymnhydxride, =Wd polyortboesters or any odier sinihir synthetic polymers that may be developed that are biologically compatible The ter= 'biologically compatible, synthetic polymnerl" shall also include copalymers and blends, and any other combinations of the forgoing either together or with other polymers generally. The wse of these polymers will depend on given applications mnd sperifications inquired. A more detailed discussion of these polymers and types of polymers is set forth in Brannon-Beppas, Lisa, Polyzners in Controlled Drag Duliveq," Medical Plastics and Bioinateials. November 1997, which is incorporated by -refereree as if set £brth fully herein.

"Materials" also include eleetroprocessed materials that are capable of changing into differnt materials during or alter eletrprocessing. For example, the protein fibrinogen, when -comnbined wit thxrmbin, fbrmu fibrin. Fibrinogen. or tinorbin that are eleciropracessed as well as the fibrin that later forms ar included 'within Ithe definition of materials. SimillAy, procollaglen wil form collagen when combined with procoflagen peptidase. Procollagen, procollagen petidase, and collagcn =r anl within the definition.

In -a preferred enubodimonmt, the elecloprocessed materials form a =atrix. ThD termn "naatt refers to any structure comprised of electroprocesed Muaterias Matices are comprised of fibers, or droplats. of materials or bliands of fibers end dr-oplets of any size or shupe. Matrices amC single sflqbzms or groups of structure and can be formed through one or moro electirxprocessiag metb~ods using onie or more matozials. Matrices an engineered to possess specific porosities. Substance =ay be deposited witbia,, or anchored to or placed on matrices Cells. a substances which may b e deposited wiithin or on matrices.

One prefered clas of mateials for eleetroprocessing to make the compositions of the present invenflon comprise proteins. ExtraO6Ihlar matlix proteins -are a preferred class of proteins in the priesent invantiom Examples include but are not limited to collagen fibrin, clastiz, lamninin, and fibnonectim- Additional prefeared materials amc other components of the 8 COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:12 FAX 613 8618 4199_ FB RICE CO.

I012 00 0 0 eXtraoellular matrix, for example pzoteoglyonu. In each case, those names are used throughout the present application in their broadest definition. There are nultiple types of each of these proteins that are naturally-occurring as well as types that can be or are synthetically manufactured or produced by genetic engineering. Foar example, collagen occurs in many forms and types. All of these types and subsets are encompassed in the use of the proteins named herein. The tern protein frther includes, but is not limited to, friagments, analogs, conservative amino acid substitutions, and substitutions with non-naturally occrming amino acids with respect to each named protein. The term "residue"' is used herein to refer to an amino acid (D or L) or an amino acid mimetic that is incorporated into a protein by an smide bond. As such, the ammo acid may be a naturaldly occunrring amino acid or, unless otherwise limited, may encompass 00 known analogs of natural amino acids that function in a marmr similar t the naturally occurring o amino acids anino acid mimetics). Moreover, a amide bond mimetic includes peptide backbone modifications well known to those sdiled in the at.

Fmtheanorc, one of skill will recognize that, as mentioned above, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than more typically less than in an encodcd sequence are conservatively modified variations where the ahlterntiaons result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitations for one another: 1) Alanine Serine Thronine 2) Aspartic acid Clutamic acid 3) Aspaxagine Glntamina 4) Arginine Lysiue (IC); 5) Isoleucine Leucie Methionina Valine and 6) Phenylalanine Tyrosine Tryptophan It is to be understood that the term protein, polypeptide or peptide firther includes fragments that may be VO to 95% of the entire amino acid sequence, and also tensions to the entire amino acid sequence that are 5% to 10% longer than the amino acid sequence of the protein. polypeptide orpeptide.

When peptides are relatively short in length less than about 50 amino acids), they are often synthesized using standard chemical peptide synthesis techniques. Solid phase synthesis in which the C terminal amino acid of the sequence is attached to an insoluble support folowed by sequential addition of the remaining amino acids in the sequence is a preferred method for the chemical synthesis of the antigenic epitopes described herein. Techniques for solid phase synthesis are mown to those skilled in the art.

Aitenatively, the proteins orpeptides that may be elootrocooessed are synthesized using recombinant nucleic acidmethodology. Geneally, this involves creating a nucleic acid sequence that encodes the peptide or protein, placing the nucleic acid in an expression cassette under the 9 COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:12 FAX 63884199 FB RICE MO [a013 00 ciconitrol of -a particular promoter, eXPresaiU the ME&td Or Protein in a host, isolat thc.

expressed peptide or protein and, if requfiired, reunIng the peptide orpotn.Tciqe sumtcient to guide, one of skill through such procedurs are found in the literature.

WbeM several desired proin fi-agmets or Pptpides amc encodcd in tfie nucleotide sequence incorporated itto a vsector, one Of skil in thZeart will appreiate: that the protein fragments or peptides mnay be -separated by a spacer molecule such as, for example, a. peptde, Consisting of one or more amino ads Generaly, the spacer wil have no speci biological activty other than to join the desred protei fragments or peptides togethr, rt sresm minimum distance Or Other spatial relations*i between them2. However, the cisuenc am Ci 10 acids of the spacer may be selected to ifluence some property of the nMomec such as the ci folding, net charge, or hydrophobicity. Nucleotdea sequences encdg for the production, of 00 r~esdus which MY be uSeti in Purification of the m-pessd recombinat protin may be built o ~~itAo the Vecor. Such ser'anos Mr known in the Mrt For exam.Eple, a nnc~eorid~ uec encoding9 for a poly histidine sequence may be added to a vector to facilitate purification of the expresed I combznunt protein on a nickel clb=n.

0nce expressed, recmbinant peptides, polype-ptides and prOteins cant be purifed according to standard procedures knovm to ome of ordinary skil in the art; including imnoniumm sulfate precipitation, affinity coluna c'olumnA Ckomatogr~phy,, gel eleofrophoresis and the like.

Substantiany Pure CoQmpositions of about 50 to 99% hoogeneity ere5 preatrred, and So to!95% or greater hoymogeneity pro nLst pnfgred for u§;0 as therapeutic agents.

Also, m1Oleoe Capable of tbnning some ofite named poteins 0Mn be mixed with other polymer during electroprocessing to Obtain desired properties for uses of the formled prOteina i33 thec matrix.

Throughout thI's applicaffon the term "solu1tion" is used to describe the liquid in the reservoirs of the eleotroprocessing metho. The term is defined broadly to include any liquids that contain materials to be eeclrorooessd. It is to be understood that any solutions capable of foring a material during electroproce.ssi ame included withint the scope of thke present invoatkmoz in Jrtis 4pplioatior the teat "!solution also refe= to susp~isions or emul sn the mateial or anytingz to be elecfrdpoitod. Solutios" =ar be in orgaic or bioocaly comPatible fo0n9. This broad definition is appropriate in view o.f the lage number Of solvents or other liquids and carrier molecules such As polyethylene glycol (PEG), that can be used in the many variations of elecfroprocess4g In this applicatiog the term '-solution- also refers to me-lt, hydrated gels and suspensions cobtaining the Materals, rbbstances or anyothn to be elecrodeposhitrd solvents Any solvent flint wjill allows dejIvery of the Material or substance to the orfice or tip o'f a sYringe under such conditions that the muaterial Or ubstnce will be: processed as desired may be used fo dissolving Or suPending the material Or ther substance to be electropmocesse& Solvents COMS ID No: ARCs-19o46o Received by IP Australia: Time 11:45 Date 2008-05-14 14,'05 '08 12:13 FAX 613 8*18 4199 ta ICE CO. 0014 00 CA ~useful fOr diSsolving Or FSpendin a maerial or a substance will depend on the material or substance. Eleefrospinig techniques often require 'nero spociWec solvent conditions. For example, non1 uros-Inked Dibrinomer can be clectrodeposited or elecwospun from solvents such as nrea,4 Monooffioroacetic acid,. water, 2 .2 2-h-fluiorocta, or l,l,1,3,3,3-hmffioro-2 S proPanol (also known as he fluoriSO.prOpNolo r HTIP). Collagen can be eloefrodeposited as a Solution Or suspensin in water, 2,,-dloehnl or BflP. E-lastin ca~n be electrodeposited as- a Solution Or suspension in water, 2 1 2 2 -t boluethanol, isopropancj, or HkTP. Other lowe order aloohnol, especiafly balogenated alcohol, may be use&. Proteins and peptides associated with meumbranes =r often hydrophobic and thus cannot dissolve in aqusous solutions. Such o 10 proteins cam be dissolved ini organic solvets such as maethanol, chiorolbun, and tifluoivethanol ciMiE). Any other solvents 1mmvu to one of skill in the protei chemcal art may be Uled, fxor 00 examrple solmt.s ascEt in chromatography, especialy high perfonnance liqvid chrornaiogapbjy.

Proteins and peptides are also soluble, for example, in ElFIP, hexafluoroaceon;, abloroalcohiols in conjugation with aqueous -solutions of milneral acids, dinaethylacetaaniide containing litbinnin chloride, in veiy dilute acids sucb as acetic acid and fbrmic acid- N-methyl morpholine- NX-oxide, is another solvent that c= be used with many polypeptides.

In functional tai=., solvents wed for a ectroprocessing li ha, pricilrole of creating a mixture with a polymer, or polymers, such that electroprocessing is feasible. Vic concennaflon of a given solvent is often an important considEration in determining the type of electroprocessin that will occur. For wxam*pe in cectrsprayijig, the solvent should assist in the dispersion of droplets of alectropomsed material so that the initial jet at'liquid disintegrates into droplets. Accord&iny, solvents used in eleetrospraying should not creat forces that will stabilize an unconfined liquid oobmn lI eleotrospinning, interacions between molecules of eleclroproee. wateria stabilize the jet, leading to fiber forroation. Acordingl, for alectrosun emnbodimen the solvent should sufficiently dissolve or disperse the polymer to prevent the jet from disintegraftng into droplets and should thereby allow formation of a stable jet in the form of a fiber. In some embodinents, the- tratisition from electrospraying to electrospirining can be deteninined by exaning Brookfleld viscosity measurerneg for polymer solutions as a ftnon of concentraton. Brookfield viscosity increases as cloflowtraion of a polymer or other material to be electroprocessed bizcreses- Above a critial oncentraion associated -with cxtens{vo chain entanglementa of xnaterials, howeverr the Batolfic-d viscosity will increase more rapidly with concenrxaim, as opposod to a more gradual, linear rise with concentraion at lower eoncentrMios ]For example, the Brook~eld Agosity of a poly(lactlde) sample obtained from Alkermes dissolved in chloroform shows srx uptur in the IBrookftield viscoity/conc~tfrgjo plot at approxbvately 7-8 w/v. A sample of poly(vthylene-co-vinyl acetate) from Duipont (ELVAX 40W) show, an upturn at 14-15 wNv. In bath cases, these deparures torn linearity approximatly coincide with the transiton from electrospmaying to electrospining& COMS ID No: ARCS-i 90460 Received by IP Australia: lime 11:45 Date 2008-05-14 14/05..08.12:o4 Fa±zucAXJ613 8618 4199 PR D T rV 9- 00 One Crnnpoent Of the comupositions of the present invention is the eleotroprocessed material. As defined aove, the eiecroproce~ed material of the present invention cau include '4 5 nmatural nhaterials, synthetc mateaias, or eombinatjons thereof Examples include but are not burnaied to aninio acids, peptides, denatumed peptides such as Weafin floma denatured collagen, polypeptide;, protein;s carbohydrate;. lipids, nucleic, acids, glycoproteins, liporteins, FJlYcolipds, glvcosaininogiycans and prteoglycmz. PPDeu Some preferred matetials are naturally, occuninxg exflacellalar matrix materials and I0 blds Of naWTuraly occuring extracellular mnatrix materials, including but not limited to ~collagen, librin elastin, larininj fibionectin, hyaluronic acid, chondroitin 4 -sulfate, chondrofrin 00 6-sulfate, dennatn sulfate, heparin sulfate, henparin, and Icoratan sulfato, and proteoglycans.

o ~These moaterials may be Lsolate fi-am humns or other aimale or cells or synthetcally marzufaci~en Some especially preferd naturl matrix nnaterials are collagen and ffbrin and flbronectjt. Also included are crude extracts of tissue, airacellular matix material, extrats of non-natira2 tissue, or extracelhLnv matit materials exracts of cancerous tissue), alone or in combination. Extracts of biological material; including but not limited to cells, tissues, organs,arnd tumors may also be lettvprocessed. Collagen has been eleefrospun to produce a repea-fing, banded pattern observed wvit electron 'ninropy- This batnded patmer is typical of Collagen fibrils produced by natural processes banded plattern is observed in collagen when it is Produced by cells). In some embodimenlts, collagen is eleetrospun such that it has a 65 rmn banding patern it is to be undorwua tat these electroprocessej materials may be combined withi othc= materials and/or substances in foning the compositions of the present invention. For eransaplc, an electfroroped peptide may be combined with an adava-ut to enkane immvnogenicity When irqplantet s utaneon~y. As anothe example, an electroprocessed collagen matix, coutaining cells, may be combined with En Oeetmprooesse4 biologicaly compatile polymer and growth IA.otors to stimuilato trowth and division of the calls kn the collagenmahrx Synthetc mateials include any matrials prepared through any method of artifcial syntsis, processin& or mvanufacture The *,nthetc matedja are preferbly biologically compatible for adminisftatin in vivo or in viva. Such polymers include biat mr not limited to the IbiloWin~g: poly(urethaes), PO]Y(siloxsues) or silicone; poly(ethylene) poly(vinyl pyroidonu), poly( hydroxy ethyl MethaCryaite), pOly( vinyl prroliaone), paly(inethyl maIbacaylwate). poly(vigyl alcohol), pcly(anxyuie acid), polyaeylammde, poiy(ehye.cADxiY acetate), poty(etbylerae glycol), poly(merbatiyjic acid) polylacfic- acid (PLA), polyglycolic acids; (PGA). polYlfctlde-co-glycojjdes) (PLGA), nylons, polyainides, polyanbydrides, poly~cthylene,co-vinyl alcohol) (BVOIj), polycaprolactone. poly(vinyi acetae) CPVA), polyvin~ylhydxoxide, polY~ethyinne oxide) (IEO) and polyoxthoestersor any other Similar synthetic, polymers that may be developed that arc biologically compatible. Some prefentd synthotic matrix materials 12 COMS ID No: ARCS-i 90460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:14 FAX 613 8618 4199 -FB RICE CO.

00 include PLA PGA. opolymema Of -PA and PGA, polycaprolaetone, poly(ethylene-co-vinyl1 acetate), (E-VOII), PVA, and P33O. Matices can 'be formed of electrospun fibers, electroacrosol, electrosprryed, or electrosputtered droplvls, or a combination of ilie foregoing.

In embodlimentzi in which natural materials am used, those materials cmn be derived ftrm Sa natiral source synthetically maufactured, or manufactured by genetically enagineered cells.

For eampe, getcally enginere proe~zjs cmabs prepared with specific desired sequences of amnino acids that dir from the ahral. pzoteimh& In one illustrative embodiment, deslitble sequences that form binding s;ites on a collagea protein Ibr cells or peptides can be included in higher amts than found in natural collagen.

o 10 By selecting different materias, or combinations thereol many characteristics of the, (N electroproessed material can be; manipulated. The, properties of the matrix comprised of 00 o electroprucessed material and a substance may be adjustecL As discussed in greater detal o below, electsoproeessed materials themelves Qan provide a therapeutio effect When applied. In addition, selection of matrix materias can affect the pennaneacy of an implanited. naafrk For esample, matrices made of fibrin will degrade more rspily while matrices inude of collagen are more durable and synthetic matrix materials art; moro diuable still- Use of matrices made of natural materials such as proteins also minize rejection or iminunlogiool response to an iaplanted matrx Accordingly selection of materials for elecuroprocessing and use in substance detivay is inthienced by the desired uise. In one embodiment, a skin patch of electroprocessed fibrin or collagn combined with healizg promoter and lati-rejection substances may -be applied to the skin and may subsequently dissolve into the skim n. anotheor emubodiment, ani implant for delivery to bone may be constructed of materials nseth for promoting bone growth, osreoblass and hydroxyap atit;, and may be designed to endre for a prolonged period of time.

Synthetic componezits, suvh as biaommatible substances can be used to modulate the* release of mnserials taom an electroprocessed composition For amnpl; a drag, or series of drugs or other wmerials to be released in a controfled fashion c-an be- electropro ceased into a series of layer. One laer is composed of PGA pis a drug, the nexct layer PLA plus a drug, a thiird layer is composed of polyr-aprulactone plus a drug. The layered construct can be; implanted. =d as the sucessive layers dissolve or breakdown, the drag (or drugs) is releasd in turn as ofeh successive layer erodes. Unlayered structares can also be used and release is controlled by the relative stability of each ccmponcnA of the construcL Another advantage of the synthetic materials is that different solvens can. be used. This can be important for Vae deivery or some ma~terials. For example, a drug may be soluble in some orgatos, and using synthetics increases the number of erials that can be electroprocesed. The breakdowvn of these synthetic, ZnzatenaIS can be tailored anid regulaed in ways that re not available to natural materials. The synthetics am usually not subject to enzymatic breakedown, and =mny spontaneously undergo hydrolysis. ln addition to these chactedsies, substanes can be: released from eeeotroprocesed materials in response to electrical, ma~eic and light based signals- Polymes tha are sensitive to Such signals can be- used, or the polymers may be derivatized. i a way to provide such 13 COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:15 FAX 613 8618 4199 FB RICE CO. 0 (17 00 sensitivity. These properties provide flatiblity in makting and using eoltroproccssed materias dfesigned to deliver various substances, ia vivo and in vitro.

In some embocbmezrt of the present invention, the electroprocessed material itself provides a therpoutic. effect. For example, in soine embodiments electiuprocessed edll1agen promUotes cellular inffilration and differentiation, so Mn elotropi-oessed collagen matrix alone assists with healing, The P-i5 site, a 15 amnino acid sequencr. withini the collagen molecule, promotes osteoblasms to produce and to Becete hydroxyapatijt, a courponent of bonr, Anothier example of specitc sites and sequences 'within collagen molecules that can be manipulated and processd in a simila fa~shion includes the RGI) binding sites of the intogrin molecule. The It/SD site is a sequente of three amino acids (Arg-Gly-Aap) present in many mnatrix materials (N that serves as a binding size for cell adhesion. It is recognised and bound, tbr example, by 00 o integrins. In addition, elec'rroproce materials can be entriched with specic desired 0 ~~sequences before, duning, or after elcrpoesn.Sequences can, be added in linear or other forms. In some embodiments, the RGJJ sequemces mre arranged in a cyclic toxi rgferred to as An electroprocessed ruatediat such as a uMatix, can also be composed of specific subdomiains of a maffix constituent and can be prepared with a synthetic backbone that c=nbe, denivatzzed. For example, the RGD peptide sequence,. and/or a heparin binding domain and/or Other Sequencms, can be cheicrally coupled to Synthetic materials. The syntetic polymer -with the afttached sequence or sequences can be electroprocessred into a construc This produces; a matrix with unique properies- In these exarnples the RGD site provides a site. for cells to bind to tad interct with the mnatrix. The hepadn-bix,4lng 8ite provides a site for the anchiorage of peptide towrh factor to the synthetic backbone. Angiogailc peptides, genetic material, growth bctur, cytokies, enzymes and drngo are other non-limiting cniplea; of snbstanes that can be altached 2S to the baoldbone of ani elctiroprocessed mateial to provide eiconality, Peptide side chins May also be used to attach Molecules to LiUetiOnrW groups on polymeric; backbones. Molecules and otherr substanes can be attached to a matediaj to be electroprocesed by any technque knowu in the arm -Another embodiment of matrix muaterials th-at have a thMrapetic effect is ceetimrpm=ed ff1izFibri Fji matrix material asists in arest of bleeding. Filirin is a c-omponent of the provisional matrix that is laid down during the early stages of healing and may also promote the growth Of taSCUlabree in adjacent regions, and in many other ways is a natural healing promoter, Fiklinogen as an eleCtroprocessed material cam alsOassist in healing. When pilaced in contact With a wocd, hor example, fibinogen will react with tbnin preset in the blood plasma from the womnA and form lbriz, thereby providing the same healing properdes of a fibrin material.

14 COMS ID No: ARCS-19046o Received by IP Australia: Time 11:45 Date 2008-O5-14 14/05...2 P '08 12:16 FAX 6 13 ;flA 419 FB RICE CO, 1 00 CA Substancay As discussed abo-ve, the word 'substaner in the present trwcntionl is used in its broadest defintion. hI emabodiments in whiei tecrmp ositions of the present invention comprise one or moret substances, substances can include any type or size of molecules, cells, objects or combinations thereof. The compositions offthe preet rnvenflon may comprise one substance or any lcombination of substances.

In erabodiUments in which the substances are molecules, any maolecule mu be used- Molecules may, for example, be organic. or inorganic and maybe in a solid, semisolid, liquiid, or gas phase. Molecules may be present in combinations or mixtures with other mblecujes an! my be in soluton, suspension, or any other ftnm- Examnples of classes of molecules that may be 00 used include human or veterinary therapeutics, cosmetics, untracenticals, agricalturals such as o herbicides, pesticides and fertilizers, vitamins, amino acids, peptides, polypeptides, proteins 0carbohydrats. lipid;, nucleirc acids, glycoproteina, lipoproteang, glycolipids, gly-osaminoglyc-ans, proteoglycaus, growth factrs hormones, newornuitters, pheromnes, blhalones, pfostaglnidins, ininunoglobuiim, monoldnes and other cy-tokines, humeclain, metals, mnerals, ions, telcically and miagnetically rcactva materials, light scestive materials, autz-caidants, taolecules that may be motaboaizd as a sour=e of cellular ergyW, atigens, =nd any mnolecules that can cause a cellular or physiological response. Any combination of molecules can be'hsed as well as agonists or aritagonists.

Sever'al preferred embodiments 'use therpeutic moleculas include use of any thorapeutic molle including, without imitation, any phurmacentleal or drag. E3xamplesi of pharrnaeuticals include, but me not limited to, anesthetics, hypnotics, svdatives and sleep inducers, antipsyohoties, entidepresapxts, Antiallergies, ;mtiangiuals, aatiartHitica, antiastbanaiics, SfltldiabctIo3, antidiairhes dmaga, anticonvulani, antigout drugs, sntlaistanni, antipruxitics, ieiios, antieinetics, aufispasraridies, appetit muppresauntrs, neuroactive substances, nftrOtransmtter agonits, antagans, receptor blockers and reuptaire modulators, betaadrenergic blockers calcium channel blockers, disulfaim and disulfarim-like drugs, muscle relaxants, aualgesics, antipyretios-, stimulants, aafichflnestase agnat, ptrasympathonimetie Agents, hormones, Anticaaganml, anitbroxnbodics, fhrombolyties, inimtrnoglobuxms, immunospprss~u honunne agnn~nanit vitami, h1 n tinizrbial agents, antineoplastics, antacids, digestants, laaies, cathadcs,, antiseptics, diuretic;, diinfftctns frag1icd, Octoparasiiides, andparasilics, heavy metals, heavy metal antagonists, chelating agents, gases and vapors, alkaloids, Salt, ions, antacoidls, digitis, cardiac glycoSides, antiaxrbYtlznic2s anhypertensives, vasodilators, vasoconsiator, adiuscairrics, gaugioril stimulating agets ganglionje blocking agents, nenromiuar blocing agents, adrnngic nerve inhibitors, nti-oxidlants, vitamins, coranilc, wntinflmannstxies, wound care products, snhtoinbogegjc- amets, ardtumoral agents, anthombogenic agents, antisegioganic agwts, anesthetic, antigenio agwnt, wound healing agents, piant extracts,. growth faUtas emollients, biMietantS, zee=tion/ati.rcjcction drug, spernivjdes, conditioners, anibacterial ageun COMS IDNo: ARcS-19046o Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:16 FAX 613 8618 4199ESRC &COIh 9 FB RICE CO. Z o 1'q 00 antitussives, histaMins-cilcing drugs nionomanie oxidase inhibitor. All snbstas listed by the Plianmazxpeia. am also included within the substances of the present invention- Antibiotics useful in t&e present invention include, but axe not bniitod to, aooxicil SaMPllttacibl, ampicfilh, bacitracin, beclomotbaSon; bwzodne, betainethasone, biax cephalospOrins, chloratphenicol, ciprofloxacin, clotiimazole cyclosporin, docycline enoxacin, ezytbromycin, gentamycinl, miceonazoe, ziemyo4n norfdioxacin, nystatin, oflonei, pefloxacin p4=1mil, pentbfyrlline, phenaxymethylpenaicll, polymixi nfiampici tetracycline, tobnnyin tne~losan, vaztomyoin, zitbitmax, derivatives, metabolites, and mixtures thereof, or o 10 compounds having sinall antinmobjal ativty.

00 Some specific examples offpharmaceudcal agents thiat are useful as substances include, o but are not lncitcd to, quiholones. such as oxolinic acd norfioxaci, and nalidizic acid, 0 sulfonamides, nonoxynol 9, Thsidic acd cophalospozius, cyolosporine. acebuitolol, aoetylcysteine acetylsaliyli ac iaqclavfr aiprazolam,4 alacaleidoDl allatin,0 allpurinAl ambroxol amilcAcn arnilarid;, arainoacotic acid, amisodarone aindtdptylie, almlodipine, ascorbic acid, aspxrtame, asternole, atenolol, benserazide, beuzalAunim hydroclilorido, benzoic acid, bezafibrate, biotin, bipeddon, bisoprolol, bromazepain, bromliexine, brunmocriptine, badesonidle, bnfehaznac, butlomuedil, buspbmoe, caffeine, camphor, captopd]l, =abainaepine, carbidopa, cmrbopLatin, cefacblor, cefaexi, cefhtroxfl, cefazolin, caftxime, cefotaxire. ceflazidime, ceftriaxoze, cefurxime, selegiline, ebloramphenicol, cblorbeninrn obloraidone, choline, cilastatin, Cimelidini, ensapnida cisplaldin, clarithromycin, clavulanie acid, clomnmian, clotapin; clonsapain, elondin; codeine, cbolesYntwin;. cromaoglycic acid, cyanocobalainiii cypiote-rone, desogesftrel, dexametasone, dexpenhcnol, dsxfrometbwphan, dcxWroropo~dpha, diazepaza. diciofenec, digoxin, dihycbcodein;, dlhyds-aergotamine, dihydroergotaxhi diltiazem, diphezihydamine, dipyridamole, dIPYroe, disopyzamide, domperidom, dopamine, dcnccycline, enalsprit ephwlrin;, epinzephrine, ergocalcifrL, ergut=aein erytinamycin, estradiol, etbinylcutiadiol, *tuposide, Eucalyptusglobulus, fainotidine, felodipio fenoflbnt;, fenoturol, fetanyl, flx 'iu mnonouncleotide, tluconazole, flnnarizhxe, flnorourail ftoxeine, iluntiprofen, faxoscraida, gulpamfi, geinfibrozil, Gingico bia glbenolaeide, glipizlde, Glyeyirrza glabia, grapefruit Seed extract; grape seed extract, gcisecoftlvin, gafenesin halopeiidal, heparto, )iyauronic acid, hydrochiorotbia2:ide, hydrocodonae, bydrocortson;, bydromorphone, ipratropim hydroxide, I buProfen, inipenern, indomethacin, iohexol, iopamidol, isosorbide dintifrate, isrosorbide xlMonontrax, isofretinin, ketotifeu, keaoanazolo, Icetoprofen, icetorolac, labealol, lactnose, lecitii, leVOcaniinc, levodapa, levagutamide, 1evonorgetreL levothyroxine, lidocaine, lipase, impranine,, lisinopril, loperamid;, lomepam, lovastain, medroxyprogsterone, menthlL aethotrc~rate, mothyldopa, me-tbyiednisoloue, metcolopramide,. metoprolol, nieonazle, 11id=azolin mznocycn ijordj iipradl, morpine, N-methylephedrin;, nattiofrtry, naProxen, nicardipine, nicergoline, Micotitaid, nicotinle, nicotinic acid, nifedipine, nhnodihi, 16 COMS ID No: ARCS-i 90460 Received by IP Australia: Time 11:45 Date 2008-05-14 14,'05 '08 12:17 FAX 613 861i8 4199 FB RICE CO. Iiz 020 00 fl11traztjam, nitrendipne, niihs northnisterone,, norfoxanin norgestxel, moittiptylin omepraole, uudanston, pancreatin panthencl, pantothenic acid, paracetamol, phenobarbital derivatives, mietabolites, and other such compounds have similar activty Sonic preferred drugs or compounds iualad;, but are not limited to, estrogen, androgen, cortisone, and cyclosporim- GCrowth factors usefal in the present invention include, but axe not imtited to, transforng growth factorjcs tasforming growth factor-a tGtLW'), plateletderived rowth Thctors M'PGMD, fibroblast growth faeiMr F"rx including FGF acidic ON Isoforma 1 and 2, FOP basic form 2 and FGP 4. 8, 9 and 10, nerve gruwth. actos VNOF' including NOF 2Z5s, NGF 7.Qs and beta. NGP and xwnrofropbins, brai dered neurotrophic factor, cartilage derived factor, bone growth factors (BUY), basic fibroblast growith factor; inmlkt-a~e growth factor vascular endothelial growth -factor (VEG$ 1 granulcyte colony 00 stimulating factor (G-CSP), insulin lik growth facrtor I and A~ hepafocyre growth factor, o gli9al aenofrophic growth factor (GDNF), stewn call hcLtor Icertiaocyte growth factor CA traisforruig growth factors (TUE), inoudig TV-Ps alpha, beta, betal, heta2, beta3, Skeletal growth factor, bone matrix derivea growth factors, and bone derived growth factors and mnixture thereof.

Cytokcines useM~ in the present invention include. but are net 1inftred to, cardjotropbin strwnaj cell derived factor, Luacrophage dezived ch=ayjue WMC), mnelanoma growthi stimulatory actvty (XOSA), macropbage inflaniaory proteins 1 alpha (M-lalpha), 2, 3 alpha, 3 beta, 4 and 5, IL-I, 11-2, IL-S, IL-4, IL-5, IL-6, IL-7, IL-8, 11-9, I1-10, IL-1I, 11-12, IL- 13. TNP-a, end TNP-0. i nnoglobulias useU in the present invention include, but =r not limalted to, Igo, IgA, 1WM, IgD, IAB and mixtures thereof- Someo preifeired growth factors Include VEaI (vascular endothelial growth factor), NO~s (nerve growth factors), PDC3P-A.A, PDGF-BB1, PDCF-AB, FOTb, FO.Fa, and BGP.

Othier molecules useful as substances in the present invention include but ame not limuited to growth hormones, leptin, 1eul=Wna iuhbMioY fActor (LI19, tumor necrsis factor alpha and beta, endostatin, dirombospondin, osteogenic protein-I, bone morphogeuetic proteins 2 and 7, 'nteonactzn soxAatomedin-Iifr peptie, osteocalcin interferon alpha. interferon alpha A, interferon beta, interferon gamma, interferwi I alpha. and inIterlekios 2,3, 4,5 6,7,9,y9. 10, 11, 12,13, 15, 16, l7 and 18.

&ibodiants involving amno adds, pep-tides-, polyp eptides, aud proteins may include any type or combinations of such mmol=Wues of any size and coznplexjty. Examples include, but Ame not binted to strudtual proteins, enzymes, and paptide, honnonesi Thes compounds can serve a variety of Iuotions. In some embodiments, &s matix may contain peptides containing a sequence that suppresses enzyme activity through competition for the active site. i other applications antigenio agents that promote an immune response and invoke imuninity can be incorporated into a constrt In substanes such as nucleic acids, any nucleic add can be present. Examples include, but =e ]lot limited to deoxrlbonuclsi acid (DNA), eut-DtA, and ribonueleic acid (RNA).

17 COMS IDNo: ARCS-i 90460 Received by IP Australia: Time 11:45 Date 2008-05-14 14'0-3 12:18 FAX 613-3618 4199 FB RJCT k, rf 002 0 ci Bmbodimcexts invol-ving DNA Include,, but are not limited to, cDNA sequenew, natural DNIA sequenc-es from any Inure; and sense or anti-sense oigonucleotideis. For example, DNA can be naked U.S. Patent -NTOS. 5,580,9-59; 5,910,488) or complexed or encapsulated 11.

Patent Nos. 5,908,777; 5,787,5 67). DNA can be presen in vectors of an~y kinmd, tbtexanpic in a 5 viral Or plasmid t'eclOr. In some embodiments, nucleic acids used will serv to promote or to itNibit the eXPression Of genes in Cells inside and/or outside the electroprocssd 'natix. The nucleic acids cam be in any that is effective to enhance its uptake into cells.

Cells ax a Substnzea Ln out odizoents in which cells arc a substnce, anY cell ca be Used. Cells; that can be used include, but are not limaited to stemn cells, committed stem cells, and differentiated cells.

0 o marro~w stem cells and( umibilical cord stem cell Other wacamples of celia used in various emnbodiments include but are not limited to: Osteoblanls, myoblasts, neuroblasts, afrxblast, glioblasts, germ cells, bepatocyres, chondrocytes, kexatinocyes smonth miscle cels cardiac muscle cells, Connective tissue cells, epitholial clls, endothelial Cells, hormone-se~creting cells, cells Of the- imne: system, and neurons& ln some embodiments it is unnecessary to pro-select the type Of Stemn cell. that is to be used, because manny types of' stern cells can be induced to differenthe in anx organ specific pattern once delivered to a given organ. For example, a stem cellU delivered to taxe: liver can be h~uced -to become a liver cell simply by placing the: stem rCli witina the biochemical envijonert oft.le liver. Cells in the matrix cau servo; the purpose of providing scafbldimg or seeding, producing certain compound, or both.

E-mbodiments in which the substance comprises cells include cells that can be cultared in rib-ao, derived from a natural sorce, or produced by any other means. Any natoral soweeo of prokazyotie or euktaatic cells may be used. Embodfiments in which the matrix is implanted in an Organism can use cella froim the recipient, cells from a conspecific donor or a donor fum a diffieent specks, or bactria or micurobrial cili. Cells harvested from a source and cultured prior to use arc also included.

Some embodiments use cells tha haye been genetically engineered. The engineering involves Programmnug the cell to express one or morm Scnus, repressing the expression of one or more geres, or both. One, example of genedeally engineered cells usefl in tie. present invention is a geneticaly engineered cell that makes and secretes owe or more desired molecules, Wen electsoprocesed mnatriceos comprising genetically engineered cell are implanted in an Organism, Molecules pxoduced can produce a local effec or a systemic effect, and can include; the molecules identified above as possible substsriea Cells can also produce autigurde mnaterials in esabodimmentim which one of the purposes of the matrix is to produce an inunnie response.

Cells may produce Substances to aid in the following non-inclusive list of purposes: itibit or stimulate inflammation; hzflitate healing; reals imnwniorejeetion; provide hormone replacement; replace llenrotansiuiters; inhibit or destroy cancer cells; prTomote cell rnw@b 18 COMS ID No: ARCS-19o46o Received by IP Australia: Time (I-tm) 11:45 Date 2008-05-14 14u5 W-08 12:18 FAX 613 8618 4199 FB RPE CO, 0O22 00 (Ninhbit Or Stimulate formiaion of blood vessels; anginent tissue; and to SUpplement or replace the §3llO'wing tissue, neUrons, skin,, syrovial flud,. tendon;, cartilage, g ensboiuce, organ;, dura blood vessels, bozie manow, and eximoellular matrix.boe Genetic engineeing can involve, for example, adding or removing genetc maeria to or frolm a ceL. altering existing1 geneti material, or both. Embodimnents in whlich cells are cranafected Or otherwiae tagineeres to. express a gene can use, tansitly or pennanenfly transfwcted genes Or hot Gene seqauces may be full or partial Iengt, cloned or na=ually Subgtanous in thea electrorwsed compositions of the present invention also comnprise objeews. Examples of objects include, but are not liumited to, Cell fragments, cell debr-is, (N ~organelles and other cell omnponents, tablets, and viruses as well as vesicles; liposoines, o0 capsules, flanopatiolps; arid other stuctures that serve as an nlsmfroece-Inoe 0 embodimelats, the objects constitute vesiclee, liposornes, capsules, or other enclosures tha contain compotuxis that aru releaed at a time after Clectroprocessing such as at the time of implantation or upon later stimulation or ineraction. In one illustrative embodiment transfeton agents such as lipinsm contain desired 'mcleotide sequences to be incorporated into cells that ar located in ciron the elecfraprocessti maial or matrix. InL other embodimnts, cell fragments or cell debris are incoiporatedI into the mik The: pnesenct of cell -fragments is knownu to promote healing in some tissues.

Magnetically or elecincally reactive mataials am also examples of substances that are optionally included within couxpositirm,, of the present invention. Examples of magnetically aetivq materials include but =r not limited to ciaron black or graphite, carbon nanotbs, fenrofluids (colloidal suspensioris of magnetic particles), and various dispermions of eleerdcallyconducting palyrs. Fenrofluids COnTabiin Particles approximnately 10 fnz in clarneter, Polynaer-enapsbded magnetic particles about 1-2 microns in diameter, anid polymers with a glass transition teeabum below room teapft~ are particularxy usefl. Examples of Electrically active polymers include, but are not limitedi to, electrically conducting polymers such as polymnilines, polypyroics and tonically conduvting polymers sucha as sulfonated Polyhxcrylamides are related materials.

In other embodiments, some; substances i the eleclrorocessed material or matrix SUPplement or augment th Ganto of other substances. For exaapec, when the composition wompnses cells that express a specifit gene 4 the composition can contain oligonnomleotides that are taken up by the cells and affect gene cxpression in the cels. bronectin is optionally incorporated into the matrix to lnvreaso cellular -uptake oftoligonuclcutide 1 by pinocycosis.

As discused in detal above, the electanproecawl matorial itself can provide a thenzpeue efteot. The invention t9= includes embodiments involvingz methods of causing a therauic effect through delivery of an eletapmoessed material to a location without incorporating additional subgtaces in the eleefroprocened materiaL Bmbadimivnts in which the 19 COMS ID No:ARCS-19046o Received by IP Australia: Time 11:45 Date 2008-05-14 j-4X05 '08 12:19 P.Al 613 8618 4199 FE RICE CO.

Z023 00 Cl ~mafrix ToatreWi~ alone is delivered as well as tbosB in whicht other substances are iincludod in the matix are within the scope of the present invention.

Methods of~falong the Compordfton The method of main.g the compostiris includes eleciroprocessing the materals and optionally olcrpcsigthe substances. As defined above, one or maore electroprocessing teichnliques, such as elecrospim, electrospray, electroarol, clectrasputter or any combination thereof may be employed to make, the electroprocesed materials and matrices ina the o 1 coinposdOn of the present ir'eiion. In the most tidamenWa sense, the electroprocessing Cl apparaitus for electmoprocessing material includes a eleetrodeposftkng mechanism and aL target o0 substriate. The electrodeposidung mecchamism includa a reservoir or reservoirs to hold the one or o more, solutions that are to be electroprocessed or elecirodeposita The reservoir or reservoirs have at least one orifice or nozzle to allow the, uleamihg of the solution from the reservoirs. One or a plurality of nozzles may be configured4 in an alecu processing apparatus. If there are multiple nozzles, each nozzle is ttrached to one or more reservoirs contaixing the sama or diffitent solutions. Siwilarly, there can be a single nozzle that is concoted to mnultiple reservoirs containing the ame or diffmrent solutons, Multiple nozzles may be connected to a single reaeroir DecauSe differeni emboajments involve mangic or multiple nozzleasendor reservoirs, any referwces, herein to one or nozzles or reservoirs should bpe considered as referring to emnbocinenbg involvig single nozzles, reservoirs, and related equipment as well as embodiment involving plual nozzles, rtservoirs, and related equipruet. The size of the nozzles can be vazed -to provide for ineased or decreased flow of solutions out of the nozzles One or more pumps used in connection wit the reservoirs can be used to control the; flow of solution strzeaming from the reservoir through the nozzle or nozzles. The pump can be prograaimd To increase or decrease the flow at different points during electroprocessiag, In ths invention paumps are wot necessary hurt provide a usefh methiod to control the rate at which material is deliverd to the electric field for processing. Merial cmn be actively delivered to the eleotnio lieNd as a prefonned aerosol using devices such as air brushes, thereby incrcasing the rate of elecwrodeipoeion and providing novel coinbinadons of materials. Nozzles may be Programmned to deliver material simltaneously or ia sequence.

The elcrpossgocur due to the preace of a charge in eithier the orifices or the target, While the other is grounded. In some einbodinats, the nozzle or orifice is charged and the target is shown to be grounne& Those of skill in the eleetroproeess-Izg arm~ wil recognize that the nzzle and solntiwj can be grunded arid the target can be electrically charged. lbsa creation of the electdicl field. and the efibt of the electrical field on the eletoprocessed materials or substanes that will I bun the elecfropmcessed. composition.

The target substratc can also be -used as a variable feature in the electroccssing of *natenula wsed to make tic eiectroproccsscd, cornpositiox. Specelly, the taget can be the COMS ID No:ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14Au5 08 12:20 FAX 613 8618 4199FBRC&CO02 FB RICE M 0024 00 actual substadte for the mateals used to make electoprocewed matix, or etectcspwcaesse matrix itself is deposited, Altemnatively, a substrate can be disposa between the target and th nozzle& For instance, a petr dish can. be disposed between a novlem and a target, and a watri can be fbrmed. in the dish. Other variations include but are: not limited to non-stick surface between the nozzles and target and placing tisuies or surgical elds between the tart a-u nonles. The target can also be specifically charged -or wounided dong a preselected. pattern s' that the solution stretamled from the odtce is direcd into specific directions, The electric fli can be controlled. by a nieroprocessor to create an electoroce aiM matrix having a desire( geomety. The tazget and the nozzle; or nozzles can be engineered to be movable with respect tc each other thereby allowing additional conrol over the geomety of the eleclroprocessed. mialr to bt Conned. The entire process can be controlled by a miczcpr Oo that is programmed Witt specific parameters that 'will obtain a specific preselected clecr cesed. mat. It is to In mnderstood that any electropmcessing technique may be used, &one or in combiationa Witi tflthwr electroproces sing technique, to make the compositions of tLepresent invention.

Any material that cm be clectroprocessed is Wiffin the me aod of the present invention- Forms of electoprocesed collagen include b-ut are not lilmited topreprocessedt collagen in a liquid suspensaion cr solution, gelatin, particulate suspension, or hdrated ge- An exaple fbi fibrin is at prefonned gel electroprocessed by subjecig it topresur=, for example by using a syriage or airrsh apparatus with a pressure head behind it to extd the fibrin gel into the clectrical field- ik geml, when producing fibers using alectrcp essing techniqvica, especial eleotrospinming, it is preferble to use tias monomer of thie polymc fiber to be fanned, In some emrbodiiments it is desirable to Use monomers to produce fner Mtent&. In other embodimccnts, it is desirable to include partial fibers to add inatrda strengto the matrix and to provide additional sites fur incorporating substancoes. MatrixK rnateials uL as collagen in a gelatin form may be used to imrove the ability of the material to issolve, olid extraction method ca be used in prepaiing such gels to maintain the structure of the man ei subuimits. Units can ten be treated with enzymes. to altar the stutura of the monomers-.

In embodimeas in which two materials combine to funrmi third, material, the solutions containing these components esm be mixed together imediatelybefore they ae stramed from an orifice in the electroprocessing procedure, It this way, the. third matelrial omu literally as thme nod~fiber or microdroplets aze fknued in the eleckrospinning mroess. Alternalively, such matrices can be formed by eleofrospraying a moeule that can folm matrix mterials into a Taoist or otherwise controlled aftosrpher of other molecules nec tow alow formation of the matrx to form famets within the electric field. For example, fi ~dogen ma be spmyed into a atmospher of throinbin. Materials such as ibrinogertThat are capable of forming other materials such as fibrin can also be elenirasprayed onto a target ththas tbrowbin. Alternatively thromibin can also be, slectsosprayed onto a target that has firinoge In embodiments in which two or more matrix materials area combined to formn a tbird (for ~cample, combining tibrinogen and thromlbin to £orm fibdA) he matrix materials can be; ci e dC COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 1405 os0 12: 20) FAX 613 8618 4199 FB RICE CO.

0025 00 0 0 electroprocessed in conjunction with or sparately from each other, typically under conditions that do not allow the two molecules to form the third until the desired time. This can be accomplished several waye. Using fibrinogen and thrombin as an example, the two matrix materials can be electroprocessed fi-rm a solvent that does not allow thrumbin to function.

Alternatively, the fibrinogern or thrombin can be packaged in a carrier material. In this application the fibrinogen is electroprocessed onto the target from one solution source (by itself or with a carrier), and the thrombin is deposited in an electroarosol manir from a separate source. Thelbrombin can be encapsulated and sprayed as a fine mist ofparticles. Altematively, Ci tbrombin and fibrinogen can be mixed with a canter, such as PEG, or other synthetic or natural 0 10 polymers such as collagen The canrrier acts to hold the reamotants in place until they are initiated.

00 These methods are not limited to thrombia and fibrinogen and also are used with embodiments 0 Sinvolving other combinations of matix materials that combine to form a third materialt The C entire product is preferably stored under dry conditions to prevent the reaction of the two materials. When the material is placed in a moist environment, the materials are able to combine and the product matrix material is fanned.

As stated above, it is to be understood that canrriers can be used in conjunction with matrix materials. Different materials, such as extracellular matrix proteins, and or substances, can be mixed With PEG or other known carriers that fon filaments. For example, fibrinogan and collagen can be mixed with PEG or other known cariers that forn ilsmrnts. This produces "hairy filaments'" with the hair being fibrin. The "hair?' cross-link the surrounding matrix carrier into a gel, or provide reactive sites for cells to interact with the substance within the matrix carrier, such as immunoglobulins. This approach can be used for forming a matrix or gelling molecules that do not normally gel. For example, in embodiments In which a specific type of matrix material will not form filaments, then the matrix material can be combined with flba and PEG and electroprayed to form an electroprocessed fibrin-containing matrix. Once fibrin fonrmation begins, a gel ofthe matrix material and fibrin together is produced.

Altematively, the material can be sputtered with another molecule that forms a sheet.

Examples of molecules that fom sheets include PGA, PLA, a copolymer of PGA and PLA, collagen, and fibroneotin. I some embodiments, a sheet is formed with two or mor materials that can combine to form a third material. This sheet can be placed in a wet environment to allow conversion to the third material.

In addition to the multiple equipment variations and modifications that can be made to obtain desired results, similarly the electroprocessed solution can be Thried to obtain diffeent results. For instance, any solvent or liquid in which the material in dissolved, suspended, or otherwise combined without deleterious effect on the process or the safe use ofthe matrix can be used. Mvaterials or the compounds that forin materials can be mixed with other molecules, monomers or polymers to obtained desired results. In some embodiments, polymers are added to modify the viscosity of the solution. In still a further variation, when multiple reservoirs are used, thc ingredients in those reservoirs are electrosprayed separately or joined at the nozzle so 22 COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14-,05 '08 12:21 FA-1 613 8618 4199 VM D T U4'0 08 122 AM63026i~ uatn S(iz 00 (N that the ingedients in the various reseavoit~s can react withi each other simultaneously with the streaming Of the solutionz into the elec-tri fild- Also, when multiple reservoirs mre used, th~e aifurnt ingredients in different reservoirs can be phaseld in temporally during the processing period. These ingredents my jinclu substances, S EIFmbodinwts involving alterdous to the electroprocesed materials thwnselvesar within the scope of the present invention. Same materials can be directly altnc4 for exawple by altering their carbohydruju profile. Also, other materials can be attached to the matrix matrias before, durn or atae eloctoprooessing using known teolmiques such as c'hemical cross-linkcing or through specific biding interactions PDG7 binds to collagen at a specific biudhDg site). Furthetr, the temperature snd other physical properties of the process cau be (N ~modffied to obtain differet resuts. The matrix may be compressed. or stretched to pxaduce novel 00 o floatanal properties.

o Still further chemical variations are possible- IFibrin fur example, is fanned in different ways. Building an sleofmrocessed matrix comprised of ibrin, therefore, involves different ways Of bringing the molecules capable of fominfg fibrin, such as fibrinogen and tombiD, together through elecircpmcessig methods. Electroprocessed materials and Matrices can also be manipulated after they are formed 'with the electropmocessing methods.

A matrix of eletoprogessed tibers, in accordance with the present invention, can be prduced as described below-. La the case of elactrpun fibrin, while any molecules capable Of touing fibrin an be used it is preferable to tlectroprocea Eibrinogen or tiroibin to make.

f i bi fiber.

Elecfropoessftlg using multiple jets of different polymer svolution and/or the same solutons with diffcnvu± types and amounts of substwice growth factors) can be used to prepare libraries of biomateuials for rapid screening. Sncb libraries ane desired by those in the 2$ Jdrarrnacoulical, advnoed mnaterials and catalyst indutres using combinatorial synthiesis tecbniques for the rapid prqadon of large -number libraries) of compotmnja that can be screenedL Per exat!ple, the uilmum mriount of gro-with factor to be relesed and the optimal release rat fromn a fibrous polymer scaffold to promote theo differentiation of a certain type: of cell can. be Investigated using thm composgitions and methods of the present invention.- Other variables inolude fber diameter and fiber compositon. Electroprocessing permits access to. an ay of Samples on which cells can be cultured in parallel and studied to determine selected cmpositions which serve s promising cell growth substrates.

Various effective condifions, can be used to eteciroprocess a maiux. While the following is a desciptiot of a pre*zed mlethod other protocols can be followed to achieve the same result fltksring to Figwem 1 in electrospinning fibers, Micropipette's 10 are filled with materials and suspended above a grounded target 11, for instance, a metal pround screen placed insidc; the centlal cylinder of the RCCS bioreactor. Although this embodiment involvos two micropipettes; acting as sources Of MaMnit, the preent iavention include-s ewbodiments involving onily one source or more than two Soures A B=n wire 12 is placed in. the solution to charge the solution 23 COMS ID No: ARCS-19046o Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:22 FAX 613 8618 4199FBRC &CO FB RICE CO.

0~j027 00 in each pipette tip 13 to a high voltage, At a specific voltage detemiined for each solution. alid SPps=mtu arngemenl t, oa solulion saspended in each pipette tip is dirtd toywards the grunzded tawge This stream 14 of matttial may fonn, a contignous filamnt, tbr example -when Collagen is the material, that upon reaching the grounded targe 7 collects and dries to form a r=dtnmwsicrA, Ultra tlan, interconeted matrix of eleotroipronessed collagen fibers.

fleperadig Upon reaction conditions a siqgle continuous filament may be formed and deposited in a non-woven. matix.

As noted aboye combinations of electroproessing techrliques and substances are used in some embodiments. Referring now to Pigure 2, micropipette tips 13 =r each coinnected to o 10 mnicropippetwas 10 tha contain differet materials or substances. The mieropipettes are 00 suspended abov0 a grunded target 11l. Again, flne 'wires 12 are used to charge the solutions.

o O~6ne micrpipette produces a stream of cdllagmt Ilbei 14. Mother niTopipette produces a 0 ~steam of electrespun PLA fiber 16, A tbirdniaopipetta produces =n eeotacsol of cells 17.

A fourth nxieropipette produces=si electrspray of)PLA droplets; 18. Although the mioropipettes; are attachied to the same voltage supply 1:5, PIA is eleefosprayed rather than electrospun fi-r the g'urh micropipette due to variation in the concenfration of PTA- in the relations, Alternatively, separate voltage supplies (not shown) can be attached to each ruticropipette to allow vying eeiorus in ethods to bo used through application of different voltage potentials.

Similarly, referring now to Figuro S8 tho same material can be applied as eleotrospun.fibers 1.9 fronm on= of the two 'loropipettes and eleclrospra-yod droplet 20 fronm the other znicrapipett disposed at a diffren augles with respect to the groundod substrate 11. Again, the mioropipette tips 13 ame attahed to inicropipetteo 10 that contain vaying cocentfraioxxs of materials and thus produce: diffrent types of electroprocessed streams despite using the s;ame voltage supply 15 through fine wire, 12.

Minimal electrical cnxrent: is involved in thig process, and, Therefore, electroprocessing, in this case, electrospinning, does not d6iuature the materials t&at form the electroprocessed miaterial;. because thec current causes little or nio texnpenxure inceae in the soluttions during the procedue. Th melt electrospining, there is some temperature ira~se associated with the n~inig of the matbrial. In such embodiments, care is exercised to assue that t&a materials or substance are not exposed tn (emiieatures that will- denature or otherwvise damage or inure them, An eleofroacrosojing process can he; used to produce a dense, matte-like matrix of aleotroprocessed droplets of matrial The electroamerolizig process is a InodificIZ on of the electrospinning process in that the eleptroarosl process utilizes a lower concentatifon ofimatrix maials or molecules that forn eloctroproecd matrals durting the procedure. Insted of Producing a splay of fibers or a siagle lM~ent at the charge tip of the nozzle, small droplets an formed, The drolets then travel from the charged tip to the grounded substrae to form k.

sponge-like mlatrix composed of fused droplets. In sonme embodiments, the: droplets ane less than 24 COMS ID Na:ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:22 FAX 613 861@ 4199ESRC &Co FB RICE CO.

0~028 00 t0 microno in diameter. fIn otherf embodiments a coustruct composed of fibijis Ath droplets, lik ~"beads Ott a string" may ha produced- Droplerts may range in size from 100 nanometers to mians depending on the polymer and tolvent, As with the oelectrospinning proces dcsciibed earler t electroaerosol proces can be carried out using various effective conlijiops. T'he same apparatus tha is used in the electro spinning process, for isanace as shown in Pig"r 1, is 'ntilzed in thea elweaersol proces. The differences from efleitrospinning include the conceniration of the materials or substances that fain matfix materials placed in solution in the micropipette reservoir and/or the voltage used to create the stream of droplets.

o 10 One of ordinary siE in the art recognizes that changes in the concentration of materials (N COT Aubstrnces ini the solutions require imodification of the specific voltages to obtain the 00 o fonnation and.5tcaming of droplzts from the tip of app&tte o T~he elcotoprocessing process cam be inaanirraated to meet the specific requirements for any given Application of the clectroprocesised compositions made wit these mnethods. In one embodiment, the micropipeutea can be mounted on a frame that moves in the x, y and a planes with respect to the grounded =bAtrate. The micropipetmns can be mounted around a grounded substrate, for instance -a tubular mandrel. In W~s way, the materials or molecules that form matezials streaed from the: snicrapipettesq can be specifically aimed or patterned. Although the micrapipettes can be moved ruanually, thej fhame onto which the mnropipettes axv nmntd is preferably controlled by a microprocessor and a motor that allow thr pattern of streaming collagen to be predetermined by a person making a specifc mattix. Such niaropmoemsso and motors are known to one of ordinary skill in the art. For instanc;, matrix fibers or droplets can be oriented in a specifio dirucfion, they can be laytzd r they can be prgammdto be completel =~dom and not oriented.

In the electrospirwing process, the stream or stras =a branch out to foun Ubers. Ile degree of braching can be varied by miny fhdor inlxung, but not limited to, voltage, ground geometry., distance from mieropipatts tip to the substrate, cinider of micropipehe lip, and concentration of materials or comnpouwis that will form the electroprocead materials& As noted, not all reaction conditions and polymers way produce a trae m=tifaaent, under some conditions a single continuous filament is produiced. Matwials and various combinations can also be delivered tot1e electjc field of the system by injecting the materials into the, fild from a device that wvill cause them to aerosol. This process can be varied by many factors including, but not limited to, voltage (for example lranging from about 0 to 30,000 volts), distance from mnicropipet Tip to thei aubstraje (for uxample from 0-40 &he relative positon of the 3S icropipottc tip and target above, below, easde etc.), and the. diameter of mieropipet tip (apprwximately 0-2mm). Severl of these varables arc well-known to those of sidill in the art of electrospiiming microfiber textile fabziw.

The greometry of the grounded tnrgt can be modified to produce a desired matix. By varying the grounid geometry for instance having a planar or linear or mullltiple points ground, COMS ID No: ARCS-i 90460 Received by IP Australia: Time 11:45 Date 2008-05-14 14'O5 '08 1221 FAT a14 o.~,o 14/0 -08-2:2 J' 6117i al fl ii RICE CO. I10029 00 (N ~the dixie-dOn Of the str earing material[s can be varied and custoniyed to a particular application For Entete, a grounded target comprising a series of parallerl lime can be used to client eerospun materials in a spedfio direction. The grouded tare can, be a cylindrical madrel whereby a tubular Meati is fanned. Most preferably, the ground is a variable surface that can be controlled by a microprocessor that dictates a. specific ground geometry that is proprmned into Rt, Alternatively, for instance, the ground can. be mounted on a frame that morves in the x, y, and z Planes With respect to a sftationaz micropipetre tip stmsining collagemi The substrate onto which the material; are streamed, sprayed or sputtered can be the grounded target itself'or it can be placed between the mierepipette tip and the grounded tarEtt.

The substralte can be specifically shiaped, for instance in the shape of a nerve guide, in patch, ci facza sheath, or a vascular gafat far subsequent use in vivo. The electroprocessed compositons 00 G= cab. Bhrd to fit a defeu or site to beuLL Eaples include asiti =which atumorhas beeAnremnoved, au injury site in tie skin (a cat, a biopsy site, a hole or other defect) and a missing or shattered piece; of bcueA The elecfroprocessed ocunpositionis may be shaped into shapes btwuh for substance dolivery, for example, a sAi patch, a lozenge for ingeston, -an infaperitoneal Jmpla4, a subdeinnal inPI~nt, tho interior lining oft& st=4t a caffiovawoulzr valve, a tendon, a ligament a dental prosthesis, a muscle implant, or a nerve guide, Elteofroprocessing allows great flexibility and allows for customizing the construct to virtually ay ahape neoded. Many mnatzices are sufficiently flexible to allow them to be foned to virtually any shape. In shaping matrices. pcations of the mtrx may be sealeol to one a:notherby, for example, hWa sealing, chemical sealing, and application of mechanical pressure or a combination thereof. Arn example of tint sealing is the use of croasltingn techniques discussed herein to Larva crozslnkfig between two portions of the matrix Sealing mnay also be used to close an opening in a shaped matrix. Suturing may also be used to attach portions of matrices to one another or to close an opening in a matrix. it has been observed thlat inclusion of synthietic poly==r enhances the ability of matrices to be heat sealed, Othr variation of eleczuopmocausing particularly electospiming and electroacrosoling include, but ameno: limited to the tllowing.

1. Using different soinlions to produce two or more differenat fibers or droplets simaultaneoUcly (fiber or droplet =ray). In this case., the singe eomponexa s~obnions can be mainrtained in separate resevoirs.

2. Usitn ied solutions (for example, materials along with substances such as cells, growth factors, or both) in the sanjg reservoir(s) to pro duce fl bexsbor drople ts compos ed o f elec~sedmaterials; as well as one or more suibstartces (fiber composition "blends').

Nonbiologieni 1but biologically compatle material can be mixed with a biologimcl molecule.

3. Utilizing multiple potetials applied for the different solutions or the same Golutions.

4. Providing two or m==r gcometrically different grounded targets smal ad large mesh 26 COMS ID No: ARCS-19o460 Received byIP Australia: Time (I-tm) 11:45 Date 2008-05-14 14/05 '08 12:24 FAX1 61' 8618 4199FBRC&CO FB RICE CO.

[a1 030 00 the roold orumandrel or other ungrounded target in front of the grounded Target.

6. Applying agents such as Teflon onto the target to facilitate the rem-oval of electroproccessed materials fromn the targer (Le. xnalce the miateria morc slippery so thiat the elecfropirocesej materials do not stick to the target), 7. Fonxdng an ceconprocessed material that includes materials applied using multple W=Le rercess*n methods. For example, elevtrospun fiber and electroacrosol drolets in thle same composition cam be benoficial for some applications deending on the particular structure desired& This combinati on of fibers and droplets can be obtained by using the camrs o0 it) icropipette and solution and varyig the olectnocal c-harge; varying the distace from the (N grounded sub.7tma; varying the polymer conceutiation in theo reservoir, using rxnlliple 00 o micropipettes, some for streaiin fibers anid others for steaming droplets; or any other 0 variations to the mehod envisioted by those of skllM in this art. The Sibers and droplets can be layered or mixed together in same layers. In applications involving multiple micropipettes, the micropipettes can be disposed in the same or differeint directions and distances with rafe-rence to the target.

S. TUsing multiple targets.

All these variations can be done senparately or in combination 'to produce a wide variety of elecfroproccsed materials and substances.

The various properties of t. clectroprocessea materials can b& adjWtd in accordance with the needs ad specifications of the calls to ba suspended and grown within them, The porosity, ft inetace, can be varied in accordance with The method of rnaig the electroprocessed materials or m~atrix. Elcrpcsia particular matrix, for iristance, can be; varied by flbr (droplet) size and density. If the cells to be grown in 11t matrix require a Dreat deal ofnuntienz flow and wasta expnlson, then a loose matrix can be created. On the other hand, if the tissue to be made requires a veay dens environment, then a dense matrix cian be designed.

Porosity can be rmipulated by mixing salts or other extractable agents. Removing the salt will leave boles of defined Sizes in the matrix.

One embodiment for appropriate; conditions for ectroprocessing Atbrin is presented 'below. For clectroproceasig ffbrin by comubining f;irnen 2tnd tiuprabin, the- appropriate approximait ranges are: voltage 0-30,000 -volts; 7.0 to 7.4; cakiuua 3 to 10 Mt tempenatau to 400Q; ionic strength 0.12 to 0,20 N4, thiroinbin 0.1 to 1.0 units per at and 5ibrinogen 5 to m/nl. For electreprocesing fibrin zmtomer, the pH starts at 5 afd finceases to 7.4 whilet the ionic strength starts above 0-3 M and decreases to 0,1M 1Te other coadilons are similar as slated witlthi s paranap& Blec-troprocesied fibrin matrices of varying properties can be engineered by abifig the pKi changing the ionic strmth, altering the calcium concentration, or adding additional polymeric substrates or cationic materials. For electroprocessing collagen, the appropriate approximate ranges are: voltage 0-30,000 volts; pH 7.0 to 8.0; teinperatrwe 20 to 4200Q and collagen D to 5 nag/mI. Electorocessed collagen miattems of varying properties can 27 COMS ID No: ARCS-19o46o Received by IP Australia: Time (I-tm) 11:45 Date 2008-05-14 14/05 '08 12:25 FAX 613 8618 4199 FB R.ICE CO. a 1031 00 be engineerd by shifting the pHi, changin the ionic, strength (egS. addition of organic salts), or adding addiiional polymei ubstrates or cationic materials.

Shapes Of-Electroprocessed Materials and Mcnricu& Electroproressed materials ca be electrodeposited iside a specifirally shaped mold.

For instgace, a particular type of organ or tissue that to be replaced has a specific shape, such as a sdapatch to Sit a blopsy &ita or alargecscalp area folowing awide armazunoved after discovering a mna]ignamf melanoma. That shape is the nproduced and create inside: a mold Cs] designed to mimic tlat shape. This mold can be, filled by elootrodopositing the material into it.

o 10 In this way, the matrix waatly mimnics the =old shape. In some embodiment, matrices that will 00 become, extracelluhar matrices and that have a specific shape are used in the creaion of a new orgm ollo andsolid organs ohn be made. Mixing cells with the. material dnjig elatropraingforc cll&wibinthematix o tat heydonot have to migrate into a mtrix Methods of Combining Substances wit Electroprocessed Materials Substances can be ccaibine4 wit the eledtroprocessed materias by a varioty of means.

In someO emboaiment, the substance comprises molecules to be released from the electroprocessed nrateri~l and is therefore added to or Inc-orporated within te matrix of electroprocessed marrial- Substances can be mixed in the solvent caz~$txs or solutions of materials for olectropromessig. In this system matemials can be mixed with various substauze&.

and directly electroprocessed. The, resulting composition comoprising ani electroprocesed matrix and substance can be topically applied 'to a specific site and the substances released fromn the material as a function of te material undergoinlg breakdown in the sbnounding envirommaet Substances may also be released from the electroprocessed compositions of the present invention through diffasion.

The state: of the electroprocessed material in relation to the incorporated substances is dictated and can be controlled by the chermistry of the system and varies based on the selection of matrix materialsl, solvent(s) used, and solubility of the matix materials in those solvents. These parmnatw cau be manipualaed. to control, the release of the substances (or o&her elomants into the sunroundiug envirornent). If substance to be incorporated into the elecsmopmcessed material wre not miscle 'with the inatejdig separate solvent resevoirs fo~r the; different components can -be used& Mioing in s5uc an embodiment ooix prior to, during, and/or after deposition on the target, or a combination thereof- It is to be understood that slbsanes 'cay be entrapped or entangled within. an electmprocessed material bonded to a material beforo the material -undergoeseekprcsig or boimd to specfi.c sites -w$tbin the matrix material.

In a variation of this embodiment the mibstanee is a. partiele or aggrgate compiising a matrix of comnpounads or polymers such as alg~inte tiwi, in tmm, contain one or more compounds tt will be Welesed from the electroprocessed milteriat Drugs can be combined wit alginate by. for examiple, combining a drug suspension or drug pafliculate in the alginate in the presence; COMS ID No: ARCS-190460 Received by IP Australia: lime 11:45 Date 2008-05-14 14/05 '08 12:25 FAX 613 8618 4199 EB RICE CO. [a 032 00 of elcivxn. Alginate is a carbohydrato that fvrm aggregrtes when exposed to ca~ciumL the aggregates can be used to trap dlrugs- The aggregates dissolve over lizzie,, releaing the trapped substances, such aa cells trapped in ulgise. Tha particles which are thenL iwNuornted witbin tie larger elee-troprocessed matrix, =r biologically compatible but relatively stable and will degrade gradually. In some circumstances the chectroprocessed materials resemble a string of peanls. This is- a physical aspect of the electroprocessing. If the polymer concentration is low, elecirospraying of beads octera As polymer concentraton increases there arc some beads and eome fibers. A further increase in polymer conceaffation leads to predominmtly or all fibers.

Ci Therefbr;, the appearance of the pearls on a string is a tansition phaseo ~10 If a. drug (for examuple, peniclli) dloes niot bind or inteact with an electrospun matrix 00 anater is., the drug can be entrapped In PGA or PLA pellets or electrozerosoled to produce pellets o in the electrospun materiaL. The pellets &r electroserosoled droplets containing the drug begin io dissolve after adrainislration to deliver the entrapped matrial. Son agents can be coupled to aynttior, or natural polymer by ccvaleat born], prior to or atkt spinnin.

In other embodiments, the substance is electtiooocesseL Substances can be elecfropro ceased from the stunt orifice as the materials or from different orifices. Substances can also be subjected to &he sae Or a dferet tjpe of cleciroprocessing as the material. A mnolecule can be bonded to the electroproenssed material directly or through linkng to a molecUle thaft has a afflniwv for the, material. An example of thin embodiment involves bonding Polypelide substances to heparin, which has an affinity fur collagen materials. This embodiment allows release relate to be controlle by coo-ing the rate of degradation of the inatenal, for 6xaruplu bY enzymatic or -hydrolytic breakdown.

I0 othe-r embodiments, the, electroprocessed material can entrap substance during the electrodeposition process. This can bes accomplihed by disposing substances in the space between the so'ure of the elecfroproccassed streamn and the target for the electroprocessed material. Placing such Substances in the space between the source and tairget ca be accomphatied by a nuamber of methods, including but not lhzdtad to, s=sending in air or other gase, dring sprying or electruprocessing the substanues The substances can be present in thaut space in, for example, particuate, aersol, colloidal, or vapor form In. these embodimnent, the eleotroprnoessed material or matix: will physically entra the substances. This embodiment can also be used to encapsulte large patcles 1 such as cells, large particles, or tablets. For example, if a tablet is dropped through the matrix as hi formr, the tablet is surrounded by the maitrix. If a sua Obi eAt ea celtis dropped tbugh theomatrix asIt fbrus or placed in an aerosol within the matzix 4 the object may toe taped between filaments, within filmaents or "atce to the outside Of the filament. For exnmpi; by suspending cell in a solution or -witbin a mtix, tme c=ils cain become part of an rilecirosptm matrix during fabrication of the JIlanrins. Alternatively, encapsulation can Occur by, dropping substances onto or through a mahlix material streamn as a matrix forms. The celS thus become surrounded by a matrix of electOpocessed material. These embodiments can be used to incorpoatu within a nmdtnx 29 COMS ID No: ARCS-190460 Received by IP Australia: Time (H:ml 11:45 Date 2008-05-14 '08-i-2i26 DFL61 8618ft 4199aiB RICE

CO.

00 C] obstnmces that Ore not soluble and/ar ar-e too, large to Lo',na a sl"spesion in the solvet wed tbr the prOdUction of the material. POr ubstaucs in a mist or 'vapor Loin, conktrolling distaibutio and compoat'O." of substances in the space between the sourc and target cam be used to le th physcalandche ica pr periesOf the electroprocessed -mateial and the patter of distribiution 5 of the substances in thfe e1ecfroProcsud material. For all of the foregoing embodiments, thle substnces ca', be placed in te eleofropr=oased zutetil it OAPsules5' veicls, r othcr conltairrnuts for subsequent release, Sinc the solvent carrier often evaporates in the el1ectroprocesing9 tehxriqna as the elearoprocessecl materia forms, such as a flgmee subsltance 5 may be placed in the electrprocesed matrix =nd solvent toxicity is greatly reduced o 0 or eliminated- C]T Yn bodimente wherein the substance. comprises coill, the calls can, for example, be 00 nuspenderi in a solution Or other liqud tMat contains the material to be electraprocessed, disposed in the area between the solutions and taret, or delivered to a target or substrate from a separate soru beoe z n, orater electoproesg Cells con be dripped through thue muatrix, onto ie atrx a itdepsit onthetaret r sspededwithin an aerosol as a delivery system for the cells to the elecip oessed material. The cells can be delivered in this mannier while the matrix is being formed. As an examgple, cardiac fibroblasts were suspended in phosphate-buffereti salin~e (PBS) at a cocerutioi of apprcrimateiy one Million cell pew milliliter. The suspension of calls wag placed within a reservoir of a Psache air brush. To test the effiacy of using t. type of device to delivor cells, the cell spension was initialy sprayed onto a 100 rm cuhure dish. Sonia of the cells sunvived, attahed to the dish and spread ot over the substatm, lIn a secoind trial, the culture dish wag loed fthrther away fromr the air brash and the expeiment was repeated. Cells wer observed on the dish. They appeared to be Ilattened by the imlpact and.

Were partially spread out over the surface of theB substratum, Culture media was added to the dish and the cells were planed into an incubator. After one hour of cultpre, the clls vwe insected again, and many w=r found to have spread o=t fuirther over the substatum These reslt deinonwtrato that a simple airbrush device can be used to place cell: Into an aerosol droplet and deur thm au demazad to a =aface or site of iterest. Cell viability can be Dlproved by restricting this technique to cells that a=e resistat to the shear forces produced in the Teechnia; deveoig a cell suspension -with additfves that cushions the cells or refining the aerosolizing device to produco a momc lamin= flaw. in addition, direting the cell aerosol into mnatdic materials as the matrix is £firznjng in the spav between the target or mndrel and the source(c) of Moleoules being electnpinaessed produces the effic± of costioning the cells. While not wanting to be bound by the follo'wing statemnent, it is believed that tha cells wil be trapped in the stoa of Alus or other bodies produced by electnispinzring or electrprocssing and Pulled onto the miandrel This situation may be less traumatic to the cells tha diretl sprayi tho cells onto a solid surface.

Ir one embodimit, the cells arcr added either before or at the samne time as the materials or compunds that [orn eledrUprocessed materials are brought together. In this way, the cells COMS ID No: ARCS-19o460 Received by IP Australia: lime 11:45 Date 2008-05-14 14/05 '08 12:27 FAX 613 8618 4199 FB RICE CO.

1034 00 0 are suspended throughout the three-dimensional matrix. In embodiments in which the electroprocessed material comprises fibrin formed by combining thrombin and fibinogen, the cells are typically included in the mixture tha± contains the fibrinogen (whether it is plasma or purified fibrinogen). Whenvver materials comprise two or more separate materials that combine to form a different matcrial (such as fibrinogen and thrombin) bringing the materials together immediately prior to insertion into a mold, or immediately prior to the streaming step in the electrospinning process helps result in a good distribution of cells in suspension in the resulting extracellularmatrix, Cells can be added as the filaments are produced in the space between the targt and polymer source. This is accomplished by dripping the cells onto the target, dripping the cells into OO the matrix as it forms, aerosoling the cells into the matrix or onto the target or electrospraying the 00 cells into the matrxb as it condenses and formas near or on the grounded targiet In another embodiment, cells are sprayed or dribbled into a forming electrprocessed material or matrb and thereby trapped as the electroprocessed material crosses the air gap between the source solutions and target.

An altemative method to deliver cells to an electuprocessed material involves electroaerosol delivery of the cells. Cells can be deposited by electrostatic spraying at, for example, 8kV directly onto standard polystyrene onulture dishes, suggesting that electrostatic cell spraying is a viable approach. Cardiac fibblasts in phosphate buffered saline (PBS) bave been electroaerosoled up to a 20 ICv potential difference. In another cxample, Schwann cells (rat) were plated on a PS petri dish by conventional methods after one day. Scliwana cells were also electrosprayed onto a PS petri dish with a metal ground plate behind the dish at 10kV after one day. Both samples grew to almost conflence after one week The electroaexosol approach provides some distinct advautages. First, the shear forces produced during the delivery phase the production of the aerosol) appear to be much less traumatic to the cells. Second, the direction of the aerosol can be controlled with a high degree of idelity. In essence the cell aerosol can be painted onto the srtee of interest. This allows the cell to be targeted to specific sites. In elcctroaerosol delivery, cells are suspended in an appropriate media culture media, physiological salts, etc.) and charged to a voltage, and directed towards a grounded target. This process is very similar to that used in electroprocesiog, particnlarly electrospining. The produces a fine mist of cells trapped within the droplets as they are poduced and directed at the grounded target.

Cells cm be delivered using aerosol and electronarosd tchniques onto an electroprocessed material that is f£zming by an electrprocessing teelmique. The electroaerosol of cells can be delivered in parallel (La. alogside) the dlectprocessing material or from a separate site. The cells can be delivemed to the storm of filaments or particles produced within the air gap in the electrodeposition process or directed at the target Tbhe cells and electuprocessed material also can be delivered in an altmating sequence to the target, i.e.

electrodeposit the material aerosol the cells, electrodeposit the material, aerosol the cells. This 31 COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '0)8 12:28 FAX 613 8618 4199 FB RICE CO, ~13 0035 00 allows for the discrete layeritg of the construt in separate layers. PfiemrL, a vapor source can be provided tht di-rects water Onto the mandrel o'f target used to collect the cells. providing lbis moist=r improves cell viabiity by keeping the cells from dehydrating during proces -ng.

Cells Can be added to Thfe clectroprocessed material at any time or htan any orientation in any aerosol statzzy. Again the advantage of the process in general is that Collagen, for example, collects in a dried state on the target mandrel. Accordingly, although some solvet Thr collagwn =ay be toxic, they are last frin the systemn befor the filaments collect an t&e target Cells ca= al5o be trapped within a carier prior to producing an aerosol. For exaple, cells can he encapsnlated wvith-in a matedal W11c alginate. The encapsulated cells are physically protected from shea and trauxua during processing. Cells delivered in this form to the 0 electnmproee~m material will have higher viability When sprayed or electrostaticaly seeded.

00 An electroasrsol or othierwise elmctrproceso niaterial can also be: delivered directly to al0nxfi ie o eleorrpprocan material can be produced directly onto a skin wound, with or without suboacs such as molecualcs or cells Additional cells or mnaterials can tfe= be aerosolized onto or into the wound site. Other surgical sites can also be amnenable the delivery of materials using various electrodeposition techniques or combirlations therof of these methods.

In other embodiments, substances can be applied to the cecctropr ossed material after tnmznaion. for example by soaking The lectmpmocessed material ini thne Substance or by spraying the substance; onto the Gleelioprocessed material.

Persons skilled in the art will recognize that mome tian o method for Combining the substances with electroprocssed materials can be used in a single embodiment or application.

Combauing methods cma be especally uxeti in emabodiments iuvolving releas of more tha n ecompo'und or compou-jds intended to have complex release Inetics, although suchi coninajions are not limited to those embodinea.

Magnetcanly and eleatrically active materials can be eleetroprocess4d ineluding for exampi; preparing coadnetijig polymer ia= produceod by elc-trospinnia. In addition, conducting polymers can. be prepared in-situ in the matix by, for example, incorporation of a Muonomer (eqg.. pyrrole,) followed by freatient with polymidauion iniiaor and oxdant FeCl4) Finally, conductg polymers can be grown in the nxatruaf t electroaprocesing by using -a matrix-coated conductor as the anode lbr electrochemical synthesis 0f for example, polypyxrole orpolyanfline. Compounds that can text electroprocessed rnhrials can be added to Mn aqueous Solution of pyrrolo or aniline to create a condicting ptyntMr at the anode with the entrapped elCc oprocasseti iaterial..rmsg conipowels,, which can then be ted wit other compounds to allow fonnation of the material to occur.

Many eMbodiments of the present invention involve Theans for manipulating the pattern or distribution of eleotroprocesed materials and/or substances within an electropruowwsed 32 COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14-,05 '08 12:28 FAX 613 8618 4199FBRC &CO 06 FB RICE CO, [a 036 00 Matrial. For example, an blecoprcessing target can also be specifically charged or rounded along a preselected pattern so that elactroproesed materials streamed toward the target are directed into specific, directions or distributions on the. target or on a substrate, The elecwoi field can be controlled by a rnicroproceamor to crete a matrix having a desired geometry. The tarjg and the eleofroprocaing nozzle or nozzles can be movable with respect to each other and to the target thereby allowing additional cont~rol aver thie geornety of the electroprocessed rmteria to be irmet lit embodiments in which msbsance are elecfroprocessed thiis manipulation will also allow ontfrol of the distribution of substances within the electruprotessed mwain-jes. )For example a matrix can be preared on a mandrel, and substances from a sepaiatB reservoir can be o 10 sprayc4 dripped, electropmocmese in a speciflo pattern over the; axstinz matrix. This may also beacmcse ysxuteosyeeipaigamrxkoaoesm n sbto 00beaomlsebysutnouyelropyigamtxfrmoeolcanasusae ofrom another source- In this example the matrix saunae may be stationary and the substanee 0 sovrce is moved with respect to the target mandrel.

te Other featurs that allow establlhent of such a patler ncue fotre not limited to; elecfropiocussing methods 1 the uscmultipkc orifices with different cotnsfrelectroprocossint, adthe existence of numerovs methods for omrlbining subsltancea with the matefiaLs- For example, a gadient of substlances can be created along a oectrprocesed material. Th embodiments in whch the- matrix is shaped into a cylindrical construct, fbr example, the gradient can be prepared along the long a~cis of a. construct (leA to right) or the perpezdicular axis (inside to cw)- This configuration is used to provide a chemoatiactant gradieut to Oude the movement of celle within a specified site. Thus, for example, in Borne eanbodiments i which neovascular agents are prepared in a perpendicular gradient along a collagen-based constuct the a gents can be concentrated on the clormn sm-face of a shee% of the mateiiaL. The ventral side can be placed agains a wound and the. higher concentration of angiogenie. materials on the dorsal fttrfae of the construct will increse the mipraton of endotbielial cells through the eteerrospun material.

Again, ernbodhnezn wvith complex patterns can use a nuaupracessor progamimed with the specific parmeters to obtain a specific, preselected leoctroprocessed patter of one or more elecnroprocessea polynm-m, optionally -with one or more substances& slrfor the Compostions of the Present venion Substance dernh-y One use of the compositions of the present invention is the delivery of one ar more substance to a desired location. In some emobodiments, the electroprocessed materials are used siimply to deliver the matexials itself, In other embodiments, the electoprocessed -materias are used to deliver subsianlces that Mr contined in the eleofroprocessed materiel or that are produced or released by substancems contained in the electoprocessed material. For example, aui electroprovesspd material contaiuing Cells can be; implanted in a body and used to deliver mnolecules produced by the cells after implantation. The preent compositions can be used to 33 COMS ID No: ARCS-i 90460 Received by IP Australia: Time (I-tm) 11:45 Date 2008-05-14 14/05 A8 12:29 FAX 613 8618 4199FBRC &CO FB RICE CO, 04037 00 CA deliver substances to ant in viva location, an in Wav location or other locations- The present COmposiflons can he adminiseed to these, locations using Any maethod., ra the field Of 'substance. delivery, the compositions of the present invention have many attributes that allow delivery of substances using a 'wide variety of release profiles and release 5 kin~efics. For examnple, selection of the substance and te method by which the substance is combfied with the sectroprocessed material affects the substace, release profile. To the extent that the substances are not innubilitd by thie electrcoprocessed material, release from the electropincesseti material is a. function of diftbivn. An =~anmple of such =n embodiment is one in Wbich the substanc, is sprayed oato, the eleotroprocesd material. To the extent that the o 10 substances are izumobilized by the electxocessed mnaterial, release rate is mome closely relatetd CA to the rate at which the cleetxoprocessed marial degrades. An example of such an embodiment 00 o is one inl which the msutace is covalently bonded to the eleafropooessed maeiial For a o substance is trapped within an eleutruspim nagrae or fil-ament, release kineticS would be detenotied by the rate at which the s'unuding material degrades or disintegrates. Still. other cxaznples are subsmances that are coupled to the eletrcprocened material by a light senritive bond. Bxpotgz such a bond to ligh releases the. substance fr-on the elactropzucessed material.

Converely, iru some embodints of this invention, materials can be exposed to light to cause binding of agents in vivo or in vitro. Combijniag the comnpound with the eleciroprocessed Material in solndon rather than in suspension, will result in a differen pattern of release and thereby pro-Vide Yet another level of control tbr the process. Purther, the porosity of the eleciraprocesec material can be: regulated, which affiots the rate of release of a substanc.

Piabanced porosity facilitates release. Substance release is a"s enhance4d by fragmenting or pulverizing the eleatroprocesseci materiaL- Pulverized material cam, for example be applied to a.

Wound site, ingested or formed into another shape such as a capsule or a tablet In embodiments iin which the substance is presen in the form or a large particle such as a tablet encapsulated in the electroprocessed material or a molecule trapped inside an elecuroprocensed filament, release is dictated by a complex interplay of the rate the particles dissolve or degrade and any breakdown or degradation of the electroprocesed matrial slructlre. In embodfiments in which the substance compiseso cells tha will express one or more desired compounds, fhntars that affect the' functiom and viability of the c8ll9 and the iming& inttensiy, and duration of exrssion can all affet the release kIneica. Chemicals that affect cell function, such as oligonnolaotides, Promoters; or ihibitors; of cell adhesion hormones, and grow&h kctors, Ibr example, can be incorporated into the electroprocessa± material and the release of tbase substances from the eleftroprocessed material can provide a means of caittalling expression or ethe tmcdons of celia in the elecfropmuoned materiaL Release kinetics in some embodiments are mnipulated by cross-linking electroprocessed natrial Through any means. ln some embodimt, tos-liking will alter, for example, the raft at which the electxoprocessed material degrades or the rate at whicb a compound is released from the elob-oproccssed material by incasing stractural rigidity and delaying subsequent 34 COMS ID No:ARCS-19o46o Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:3u, FAX 613 8618 4199 TM DT,-t7 00 cidisolution of the lctroProceesod mateziat Eleeropwcuesed miaterials cant be faiTmed in the preence .Of crOne.Jbixg agents§ or can be treated 1with cross-linking agents after electlodsposfi 0 on Any technique for aross-Iinlemg materials may be used as know-n to one of ordinary skill in the art Pxau*Ies of techiniques inahluo applicaticon of crosa-lixJd agents and 5 application of cUrti cross-1hing radiations. Examples of crss-liking agents that work with onet or more protetns include but =e not limited to condensing agents such as aldehydes e.g., slatuared~yd; cnrbodibnjide EDC (1-etbyl-3g3 dimethyl arnInpropyly), photosesuilive materials that cross link upon exposur. to specific wavelengts of light osmiumr tetroxidek carbodimjide hydmory4 2 and NEXS (0-hydroxYsueiniinide) a=d Factor X~la. Ultraviollet radiation is one example of radiadot used to crosslinc mauixc aterials in some embodiments.

Natural mAttrials can be, cross-linked with other natural mnaterials. For example, collagen can be 00 Qwoss..Iizkcd and or sta~~jijun. by the aditfion gf fibronectnu and or heparin sulfate- For some polymers heat can be used to alter the matrix and cross link elements of the matrix by fulsing adjarcom ~rponents of the constrmot. Polymers may also be, partially solubilizrd to alter the 1ISstrucljn Of the Mateial, for examPle brilef 0XOpSUr of sonic Synthetics to alcohols or bases can partially dissolve: and anneal adjaccat tlmnns togethe. S~ome poiymecrs may be cross-linked being chemical Thsiom or heat funion techiques. Synthietir polymriers generally caa be crsslinkedusing high energy radiation eleetron beanw, gamma rays). These typically work by the creation of fren radicals on the polymer backbone which Iha= couple, affording cross links- Backbone free radicals can also be generated via peroxds, sac compounds, ayl ketones andother radioa producing compoumjs in the presence of heat or liht. Reduction-Oxidalion reactions that produce radicals peroxides in the presence of transition metal salts) can also be -used. In many cases, functional groups on polyr backbones or side chains um be: reacted to fom cross-links. For example, polysecohades can be: treated with diacylcblarides to fom diester cross-links. Cmoss-bnjejng may also ocon miter application of a matrix where desirable.

For exainpi; a mnatix applied to a wound may be cross-linkeod after applicatfont to enhance adhesion of the matrix to the woud, The release kinetics of the substance is also controlled by manipulating the physical abd chemical composition of the electroprocessed mateiial. Per cicerapi; =mall fibers of PGA are 3D more susceptible to hydrolysis than larger diameter fib= of PGA- An agent delivered within an electroprocesse(J material composed of smaller PGA fibers is released more quickly thu when prepared withini a materjat composed of larer diSauCterGA fibers.

In some embodiments substances such as peptides can be released in a controlled mnner ina a localized doan- 'Examples include nmbodien in whIch the substance is chemilcally or covalently bonded to the electroprocesed miaerial. The formation of peptide gradients is a critical regulatory component ofT many biological proceasc, for example in neovasculogenesks TIn surgical applications, and-vascular peptides or axutiRs oligunutJcofide can be incorporated into an elecftoproc~ed mate"ia t&at is then. wrapped around or placed within a tumor that is inaccessible to conventinal treatmnts to allow for localized relAease ad effect. Reles of the COMS ID No: ARCS-19046o Received byIp Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:30 FAX 613 8618 4199FR IC &Co I03 FB RICE M [a 039 00 CA ant=i-vascular substancues suppresses tumnor growftht Antsens oligoucleatidas lan be released fr*omE th nsiruct into the tumor and used to supress The expression gene sequences Of interesL- In another eyaznple anti-sene sequences directed agaist gene sequences that control proliferation can be deliverd within arn electroprocesed matrix coated sta The stretch 5 normally associated with tile placement of the stent initiates smooth muscle cell proliferation, and Buti-Sense sequences designed to suppress cell division reduce the deleteriorus effects of ther smooth muscle cell prdliferation. associated with the procedure. La anaother embodients fte electroprocessed material delivers sonse =al antisense Oligonuceotides to promote or to inubbit localized CelI timotion for a period oftime. For examaple, an antiseuse oligonUcleotida; is released o 10 from e lmPrOewsed materiul to auprs the expression of a deleteiionsersmei Ci wouacL- Examples of sucb enzymes ame matix metalloproteinases (lvflvPs) whe1~l are often o0 ovejp~sed i dhrcie wo s I anothe vxax'pl;, the e1ectoproocssed ma ppidt a o woundl releases plasmids that cotain niuoleotidn sequences coding for tissue. iNitors of mnexallopmteinases CllMs). Cells in the wound will express 71W;ps resltinig in local delivery of TIMPs that will ibit MMP tnction.

Physical prcessing of the formed electovprocesced material is another way to manipulate release, inetics. In some embodimenrts, mechanical ibres, such as compression, applied to an electroprocessed m~aterial basten the breakdown of &oe mat by altering the crystalline stracmtre of the materiaL Sirnature of fth matyix is tbus another paraeter that can be manipulated to affect release kisics. Polynrethanes and other elasfic materials such as poly(ethylene-co -vinyi acetate), siiconea 1 and polydienes polyisoprene), polycaprolacton. polyglycolic acid and related polymers muc examples of m~aterials whose release rate ca be altered by mechanicat st-i Release Iknetics can also be controlled by preparinag laminates comprising layers of electroproessed materials with diffrut proporides and substmancs. For exazupi; layred strluctures composed of ahernatimg elsftropreceesec materials can be prepared by sequentially elecfmroocesskg diffhren materials onto a target. The outer layers cani fbr exanple, be tailored to disslve faster or slower than respect the finne layers. Multiple agents can be dcliverd by this method, optionally at different release rates& Laye=s ca be toilored to provide a complex, multiinetic relema profle of a single 1g9=j over time. Using combinations of the foregoing can provide fbr release of muldpl, substances relewad each with a complex proffie.

Suspending- a isubstance in particles that are incorporated i the elecltroprocassed material Provides mnother mean for controlling release profile, Selecton of the composition of these smaller partcle matrices provides, yet anter way to control the release of compounds A=r the electroprocessed niatexiat- The rees profle can be tailored by the compositon ofte material used in the process, Embodirmts also exist in which the substances are ontained in liposomes or other vesicles in the elcrpoesdmatrix. Vesicles are prepared that will release one or more compo'unds, when placed in fluids at a specific pH rag; temperature rnge, or ionic 36 COMS ID No: ARCS-190460 Received by P1 Australia: lime 11:45 Date 2008-05-14 14/05 '08 12:31 FAX 813 8618 4199 FB RICE CO.

0~041, 00 c-i concentioa. Methods for preparing such vesicles are kniown to persons of skill in the art The elec~oproesed material can be delivered to a site of interest immediately or is stored either dry or at a pH at which release 'will niot ocor, and then delivered to a location contairling liquids that have a pHl at which release will occur. An example of Iiis embodiment is an alectropoessed material containling vesicles that Will release a desied compouncl at the pH of blood or other fluids released from a woun&L The matrix is placed over a wond anid releases fluids upon discharge of fluids3 from the wound.

Incorporating consli aet ta ar magneticanly sensitive or electrically sensitive into the eleckropracessed mnaterial provides anoter means of Controlling thie release profile.' A magnetic or electric field can then be: subsequently applied to saome w- all of the matrix to alter the shape, c-i porosity and/or density of' the elecrropmocessed molerial. For example, a field can stimualate 00 =ovement or orzfbnnational cbuangm in the matrix due to the movement of magnetically or oeleobtcaljy sensitive Particles. Such movemnent can affect the release of compounds from tho (N eloctroprocased material. For example, altering the conkhnalaion of the material can increase or decrease the extent to which the materWa is favorable for comnpound release.

In some embodiments, magnetic or electrically sensitive constituenta that have been processed or co-processed with an elecrrnprocessec matevial can be implanted snbdeany to allow dehivexty of a drug over a long intervul of time. Bypassing a magnetic -field or an electlcal field across thie material, drug release is induced. The dlectroprocessedt material structure; is stable and does not substarntiafly charge without elecirornefie stimulation. Suich embodimt provide controJJ~d drug delivery over a long period of time, For examaple, an lmtrprocewed material that has magnae or electrical properties and insulia can be fabricated and placed subdemnzally in an inconspkmun= site- By passing a magndi field or an electrical field amros the composition, insulin release: can be indced. A similar strategy may be Used, to release compounds from a construct that hos light sensitive elements, exposing these miaterials to light will either cause the material itself to breajcon and or cane the release of substanes that are bound to the elecropmcessed mnateria by the light sensitive mo~iety.

Iti othier embodiments, the substances ooupnisc vesicles encapsulated within the electroprocessed material along with eleet4i or magnetic materials. Tho vesicls coutaka a compound to be released from the vesicles. Placing mi electrical. or magnetic field acros the eieofroprocessed materia cuses the compounds within tOw vesicles can be released by, fir example, doforming the vesicles to the point of niptue or by changing the penneAbility (in some cases reversibly) of the vesicle wall. Examuples of these enmbodimnets irutnde iransfecfion agents, such as liposomes, dhat contain uclieic acids t&a enbauce the efficiency of fhe process of gene delivery to the cell.

In other emodiments the composjtjoa comprising an electroprocesuad material and substalac is used as a f aeftnW patch far localized delivery of medication. or of a component of such a patch. In some of these embodiments, electrically conductive materials are incVorp orated into such a composition% which is then used as a component of an lontophoresis 37 COMS ID No: ARCS-i 90460 Received by IP Australia: rime 11:45 Date 2008-05-14 14/05 '08 12:32 FAX, 613 8618 4199 T.M D T 11 004 0 (N s'yste in whic-h o'Ne Or nore substance is delIiverd in response; to the passage of electric current mec t IY conductive Materials can have a direct heeling effCt on bone; irduies. For exapleplainga.Small electric curret across a fActure Site promotes healing. An elecotprooftwecj bone imeltic, that conducts or produces current can be: made and pLaced within 5 a fracture. The additioin of the cectrjcaj current Will promote healing at a rate that is faster than tho addition Of the tie tprocessed composition alone, In other embodiments) a electnprocessa omaexia or a portion thmref contaiig electrwAagnetjc Fprrtes is stimulated by exposure to a maagnet to tuove adtebytoapl or to release phy'sical pressure to IL pre ure-sensitive capsule or other enclosure that contans MOleclules to be released fromL the material. Depending on the eanbodhneur, the maovement will affect the release relate of the encapsulated maolecules.

00 oespone of toe composition toc, leatric and magnetic flelds can be regulated by tertuzes o such as the composition of the electroprocesses material size of t filaments, and the; amount of conduie atrial added. Electromechnica respoa frmr olyanilinelis the result of dopinginduced volume chalnges, wherea ion gradients leading osmotic pressure gradients are rc-sponible, for field-iduced deformation in ioniC gelb such as poiy( 2 -acryla~ldo-2metjyl Prop aoeulfonicacid). In each ca=; ion tanspoxt kinetics dominates the respons; and facile transport is observed wit the smll fibern. Gel swelling anti shriniking kinetics have been shown lo, be proponianaj to the squar of the diameter of a gel fiber Electromechanical response tines of fiber bundles of less 9=x 0.1s. are possible in the regime of typical mulscle.

Embodinmnt involving delivezy of molecules produced by cells pro-vide miay means by which rejection and immune response to cells can be avoided. Embodiments using cells from a recipient thus avoid the problems associated wit ztecio and inflannmaoy and immunologk'al reVonse to the cells. In embcdirs in whrich cells from an organism. other than the recipient M0 Used, the matrix can sequester the cells from immuneo surveillance by the rectpient's immune system Sy controlling parameters mach as the pore size of the eleviroprocessed material or mIatrix, nutritive, support to the cells tapped in the mahtr can be permitted whie the cells ar protected from detecton Cmd response by tho recipients imurne "ysem, As an exaruple.

pancreatic islet cells that mnumfacture inulin collectod from a donxor van be encap3sted in an *leciorocessed matrix and implanted in a recipient who cannot make insulin Such an implant Can be placed, for example, snbdermauy, within the liver, or Jntramsularly. For some imume responses permanent sequestration from the hos system may not be. necessary. The elecrxoprocessed mater-ial can be designed to shied the implanted matelal for a given length of time and then begin to breakdown In still othier embodiments, bacteria or other microbial agents engieered in rnanfatnn the: desired cowpound can be used. This embodiment provides the advantages of using cells that are more eaily mnanipulated than cells from the recipient or a donor. Again, the eletroprocessed material can servo to shield the bacteriat fim Immrune response in Ubs emibodianut. The advantge of using a bacteria carrier is that thms microbes an more easily manipulated to express a wide variety of products. Embodiments in which cellsar 38 COMS ID No: ARCS-19o46o Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:33 FAX 613 8618 4199FBRC &CO04 FB RICE CO, 1) 4 2 00 transientl tranitbted allow for expresion to be; limited To a defined period. Transient genetic engineering allows cells to revert to their origna state i embodiments in which Such reversion 3S desfired to minimize the risks of complications.

In somev embodiments, cells ame genetically engineered such that the epression ofa speific gene may be promocted or inhibited through various means known in the: art. For example, a tetraycline sensitive promnoter cana be engineered into a gene sepuence. That sequence is not expressed unti the tetracy7cline is presenL Cell markers or bacterial mark=n can also be used to identify the inserted inatedal. For exaplo, green fluorescent proteins placed within an engineered genetc material glow green when expressed Embodimnts using this feature allow -verifcation of 1. viability of the cells, biacteria, or gene sequences in a matrix.

00 The visibility of such a marer also assists in recovering an implanted electroprocessed composition.

Although the present invention provides versatlity in release Idneties entodixnents also Etrst in which one or morm substances are not released at all from the; electroproessed material- Substances may performa a fuintion at a- desired site, For example, In some embodiments, antibodies for a specific molecule ame immobilized on an eleeftopro ceased matrix and tha composition is placed at a desired site. In this embodiment the aTibodies acts to bind the molecules in the vicinity of the cozuposition. This embodiment is usefi for isolatig molecules that bind to an antibody. Another enrnpln'is a elewtoprocessed matrix containing iunobilized substrates that wil bind irreversbly to an undesiable enzyme and therby inactivate the enzyme.

The compositions of the present invention may be ombined with pharmacerticafly or cosmetically acceptable carriers and administered as compositons in yin-c or in vva Forms of admi-nistration include but are not limited to ir~ectiona, soluton, crams, gels, implants, pumps, ointnmnts, emulsions, suspensions, nicrosphercs, particle, uicrp articles, nanopartic-les, liposome;, pastes, patchat, tablets, transdennsi deliverty devices 1 sprays., aerosols, or other means fameiliar to one of ordinary skill in the ar. Such phannaeautionfly or cosmetically acceptable carriers are commonmly known to one of ordinay skill in the art Pharmaceutmcl trinulatioa of the present invention cern bc prepared by procedures known in tMe art using well known and readily available ingredients. For example, the compounds caa be ftinnulared with commnon exeipients, diluents or cariers and fanned into tablets, capsles, suspersions powders, and the like. Rxhaplea of excipients, dilans, and camera that are suitable for nrch formulations inchLde the following: fillers and exenders starch, sugars, mnritol, and silicie derivatives); binding agents caborymethyl cellulose ad other cellulose derataive, alginates, gelatin, and polyvinyl-yrrolidone); moisturizing agents e glycerol); disintegrating agents calciutn carbonate and sodium bicarbonate); agents for retarding dissolution parafinm); resorption accelerators quatemary anunonium compounds); surface active agents cetyl alcohol glycerol monostearate); adlsorptive carriems kaolina and bexztcmite); ernulsifiers;, preservatives, sweeteners; stabilizers; coloring agents; paibming agetnts; flavaring 39 COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:33 FAX 613 8618 4199 FBRC&CO I4 FB RICE C0, a043 00 agenKt; lutricants tab. calcium and magnesium stearte); solid polyethyl glycols; and Ct mixtures thereof.

The ter'ms "'Pharmaceutically or cosmetically acceptalble camrie or "phuaacutictily or cosmtically acceptablu vehicle" are used herein to mean, wdiout Enrtadans, any liquid, solid or Ssemni-solid, includinig but not linitra to watcr or saline, a gel crem, salie; solvent diluent, fluid ointmaent base 3 ointment, paste, implant lipo some, micelle, giant mnicellc; and the like wWh is snitable for use in contact with livintg. zni j or huma tissue without causing adverlse physiological or cosmetic responses, and which does not interwat with the other components; of the composition. in a deleterious manner, Other pbamaceuaticaly or conneticaily acceptable o 10 cariers or Vehicles known to owe of 9bil in the -art may be employed to make compositions for 00 delivering -the molecules ofthe present invention.

o The formulations can be so constated that they release the active ingredient only or 0 preforalay in a particular location, possibly over a period of time. Snch combinions provide yet a thither mlechanism for controlling release kinetios. The coatings, envelopes. and protectve mnatrices may be made, for example, froma polymeric substances or waxes.

Methods of in vivo administra-don of the compositions of thie present invention, or of foimulafiori comaprising sueh compositions and other materials such as carriers of thie present uwyentwn that are particuly suitable for various forica include, but a= not hnraitcd to, oral administration (cg. buccl or sablingrs adazixistralion), anal admiistration, rectal administration, administration as a suppository, topica application, aersol application, inhalation, intapaitoneal adruigration, intavenous adrainistraton, Irandermal Adiniistration, intaeral admiaistraton, subdennal udwinistretion, intramuscuLar administration, intrAnterine administration, vaginal admnistration, adznduation into a body cavity, surigical admoiuitaion at the location of a tumoer or internld injury, admninisttion into the lumien or parenohyma of an organ, and parenteral admilnistratian. Tecimiques useful in the various Loins of administrations above include burt are not limited to, topical application, ingestion smgical administration, bfJ acions, sprays, franuaenal delivery devices, osmotic pumps, clectrodepositing dirctly on a desired site, or othe means familiar to ono of ordintiy skill ina the art. Sits of application can be exteral such as on td= opidurwis, or intmral, for example a gastric ulcer, a surgical field, or elsewhere.

The coinpositions of the present invention oan be applied in the foun of creaz, gels, solution, suspeasons liposois. partiulms or other means known to one of sill in the art of formulation and delivery of therapent and cosmetic compounds. Ultxafie particle size; of elealroprocessed mateialh can be used for inAlation delivery of theratics. Some examples of appropriate formulaicns for subcutaneous adniinistmdfon incalude but are not limited to implanms depot, needles, capsules, and osmotic pumps. Some examples of appropriate fonnlulations for v-aginial administration inclue but =x not fimited to crems and rings. Some examples of appropriat fozulations for oral adrnistrlticn include but ame not limited to: pills, liquids, syrups, and suspensions. Some ccanples of appropate fonnulatiocs for traasdermal COMS ID No:ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:34 FAX 61.3 8618 4199 FB RICE 71044 00 Cl admnistrtion include but ame not limited to gels, creamns, pastes, patcihes, sprays, and gel&.

Sonme exaimples of appropriate delivery mechams ibr nbhcutancnw administntim include but are not limited to implants, depots, needles, aapm~lsb and osmotic pumps. Fomnuzatons suitable for parenteral administration include but are not lbirrtod to aqaeous and non-aqueous sterile inj~ecton solutions which rmay contain anti-wdodants, buffers, bacteriostats and solutes whdich render the formulation isotonic ait the, blood of the intended recipemn and aqueous and non-aqueoius sterile susensions which mzyV include ampenldixg agents and Thickening agents.

)rtsrnporaneous injection solutons anid suspensions may be prepared from sterile powders, Cl granuiles; and tablets commonly used by one of ordinary sill in the mrt.

o0 Embodiments in which din compositions of the invention are combined with, for Cl example. one or more "pharmaceutically or coumetically acceptable carrier" or exczipients may o0 oiweaiently be presented in unit dosage form and may bes prepared by conventional o pharmaceutical technfiques. Such techniques include the step of bringing into association the Cl eornpoiditons containing the active ingredient and the pharnaceutical carder(s) or excipiant(s), 1S In geneal4 the ibrnulations mr prepard by uniformly and intimately bringing into association the active ingredient with liquid carrier. Preferred unit dosage formulations are those containing a dose or unit; or an appropriate fraction thereof, of the. administered rngreidierft It -should be understood that in addition to the ingredients particulaly mentioned above, thriulations comprising the compositious of the present invention may include other agents commonly used by one of ordinary skill in the art. The voluame: of administration will vary depending on the route of adminisrton. For example, inntrnuscular injections may ringe in volume from shout 0.1lito The comrpositions of the presen inventon may be administered to pasons or animals to provide Substaces in any dose raage that will produce desired phyuiologjcal or pharmacological results. Dosage willi depend upon fth substance or substances adminitered the therapeutic endpoint desired, the desired effiective conoenlation at the site of action or in a body fluid, and the type, of administration. Tnatcm regarding appropriate doses of substanlces an known to Persons Of ordinary skill in the art andt may be iimud in rfrnces such as L-S. Goodnm and A.

Oilman, ed;, The Pharmacological Basis ofnTerapeutics, Marmillan ?miblkbing, New York, and KITZUIng Basic Clinical Phamwlogy, Appleton L=&ig Nlonvalk Coimeicut, 6 EdA 1995)- Ome desh-able dosage range is 0.01 pig to 100 mg. Another desirable dosage- range is O-d 9g to -50 nag Another desirable dosage range is 0. 1 pg to 1.0 pg.* A clinician skilled in the at of the: desired therapy may chose specific dosages and dose ranges, and fr-equenoy of adrninitation, as required by the; vhcumsianjzs arid the substances to be administerd. For example, a clinician skiled in the at of horone replacemnt therapy may chose specific dosages and dose ranges, and frequecy of administration, for a pubatance such as progestemone to 'be admnistered in combination 'wit the estroganie and estrogenic nodUory molecules as required by the ciroumntanues. For example, progesteronle, and other progestEns knWnM to one of skill in the art may be adiai~tcrd itn counts ranging from about 50 ptg to 300 nmg, preferabily 41 COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:35 FAX 613 ,618 4199FBRC&Co FB RICE CO.

I0045 00 Jig to 200o mg. more prefersbly I rug to 100 Mi. Specific dosages andi combinatins of dosages' Of estm genie and estrogenic Modlatory Molecules and progestins will depend on the route =d freqeune of admiitration and also On the condWOon to be leatecL For example, wlhen the composition is ftirnulated, 5)E ora administration, preerbly in the form of a dosage unit 911ch as a capule, ebach dosage unit may preferably contain 1 jig to 5 g9 of estrogenic and estrogenic modulatory Molecult* and 50 jig. to 300 Mzg ouf progerone.a U.S_ Patent No_ 4,900,734 provides addtional examples of acceptable dose comiations of estogenic molecules ON and progestins.

OtheOr uses involving electrically or nzagnettecauy active consuitsnts Tho mposiflons of the pxesent invention -have a number of additional uses aside from 00substance delivery. Embodimnlets exist in whith the incoporation of eloctrically or niaguctivally o ~active construnt in the electropracesstd material 4lorws the eleclroprocessed material 10 move rhythmially in response to an osciflating electric or magnetic field. Such an electroprocesed material ca be used, for exampe; ka left ventriopin assist device by providin~g a pimping action or a vennricdar massage to a heart patent Oc fla-tion an be accomplished by passive movement of a magnetic or electric field with respect to the cornctive, material, or vice vesa By mnanipuzaling material selection, the electoprocessWc material can be designed to remain in place permanently or to dissolve over time, elfiminating the need for surgery to recover the device, ocethe heart had recovered safficicnfy- Embodiments also exist in which an imanuted, eleefroprocessed material is used to convey an electric charge or cmrent to tissut- For example, electric-ally active canstimuents can be electrically stlmnlate-d to promote, nural ingrwth stem cell diffsriation, or contration of engineered muscLr) or to promote the formation of bone in orthopedic, applications in which electroprocessed mate:Wa is used as a carrier to reconifruct bone- In one emnbodimnent, for exampi;en aulectrorocessed uateria is applied to a bone ijy site =d used to apply an electric curret to the mnateal to facilitate mzd to promote healing, The appliation of a. small electric cut to an injured bone is known to accelerate. healing or promote thec healing of boune In other embodimet involyjog magnetically zective materils. a magnetic field is useXd to position an elecfroprommed matcial contai 'n substances by relatively non-invasive means for example by directing the movemnut of the mateaial witink the peritonenam. In other embodijmeis a compositon containing electicanly active compounds is-used to produce electric felAd-diven cell migration This approach accelerates the healing process and mibnize the iski of bacterial collonizatio. Th one cucapl; an orthopedic implant is coated wit a very thn 100 maicro) layer of an electrically active polymen- With a very thin electrode afttahed to the coating, upon pDst-mplantaioA an slectric field cani be applied ia an external elctfrode such that the electric field-driven cell migrationl is towards the implant Mirface. The dizectloa can be 42 COMS ID No: ARCS-i 90480 Received by IP Australia: Time (I-tm) 11:45 Date 2008-05-14 14/05 '08 12:36 FAX 613 8618 4199 FB RICE CO, 4 046 00 CA'reversed if so desired. Field orientafion depends on the geometry of the imnplant and ext.rnal elect-ode, I CgeLoeitiu of the, present invention are alo usfrl for testing anid applying various gene therapies. By 'v~orking with the compositions vitro", different types ofgene theray and manipulaton am be achieved by inert preselected DNA in suspensions of calls, material, etc. For example, nonviral techniques snuch as elecfr~opora&,vs ame used to tret cukuareci Cells prior to inseton into the mrix of the pros= invenion In other embodiments, cells are treated within the naiix bafore the comnpositon is isertd, into a recipient. In vitro gene transfer avoids ci the exposre of a recipient to viral products, reduces risk of inflasm~atjl from residual viral o0 particles and a-vids the potential for germ eel] line vira incorporation. lIt avoid& the; problem of o finding or engineerlng vfial coats large enough to accept large genes such as the one for Factor VMI (anti-heniophulic frtor). However, in vito gene therapy is accomiplished in seine emabodiment by. for example, incoxporadng MhA into the elacbrorocessed material as it is created ftrough the elcchsoprocrss-g techniques of the presnt invoution, whereby some; DNA 'will be incorporafed inate the in vivo cells in contact wit the composition after application of the composition to th~e recipient Tis is especially true of small gene sequences, such as antisense oligonbuclea-tjdes.

Use of an etectroprocessed con !poflflon as tissue or orgyan replacement The abiliy to combine cells in an electroprocessed material provides toe ability to use the compositions of the; present invnion to build tissue, organs, or organ-lik t-Ssuc. Cails includedin snob tissues or organs cart include, cells that serve a fimetion of delivering a substance, seeded cells that will pravido the beginiings of ropla-eteant tissue, or both. Many types of Cells can be Used to create tissue or organ s te cells, conunitted stem cells, and/or differentiated cells are used in various embodiments. Also, depending on the type of tissue, or organ being made, spec types of committed stem cells ame used. For instaince, myoblast cells arm used to build various iMerle stracturs nenroblasts are employed to build nerve; ad osteoblasts are chosen to build boe- Examples of stemn eells used in these emodimnents include bat am not limited to embryonic stem cell, bone marrow stemn cells ard tumbilical cord, sten cells used to make organs or oxgsn.ljk tisse such as livers, idneys, eW. Exmples of tissuie embodimnents that use differentiated cells include fibroblat in a matrix used for a patch, for example a hernia patch, endothelis cells for skin, osteoblasts fart hone, ad diferentiated ceLls lilte cadaver donor pancreatic islot cells for a delivery device to piace these cels in a specific site, for example the livev. In3 some emnbodinmsr the shape oft.e eleolrcpromsed composition helps send signals to the CellS to grow and reproduce in a specific type of dashed way. Otar nubstances (for example, differentiation iaducers) can be added to the elecroprocessed matrix to promote specific types of cell1 growth. Further, different ndxture of cell types are inorporated. into the composition in 43 COMS ID No: ARCS-19oi 460 Received by IP Australia: Time 11:45 Date 2008-05-14 14 05 08 12:36 FAX 613 8618 4199 K IE&C 4 FB RICE CO IM047 00 In certain disease states, organs arc soared to the point of being dyslimotionst. A classic example is cinhosis. In cinliosis,. norumal heparocytes are tapped in ibrons bands of scar tis~ea In one emnbodiment of tbe invention, the liver is biopisied, viable liver cells are obtained then cultured in an eleclioprocessed inatr, and re-implanted in thez patient as a bridge to or replacemnent for routine liver inansplanfatons- Mixing of conmnitte cell1 Iiiies in a tbree dimensional clectruprooossed matrix can be Used to produce strucbnes that mimic cnpier mrg=,s Vor exmple, by growing gluesgon secreting ells, insualin seeetlnig ell, somnatostatin secret culls, zad/or pamnatic polypeptide secreting cells, or combinaliune thereof., in separate cultures, and then mixing them together with electroprocessokd materials through eeoropmccssing, an artificial ptincreatic islet is create&L 00 o These structures am then placed under the akiw6 rrtropexitnneally, intrhepaically or in other 0 desirable locations, as ixuplanable, long-term treatments for diabetes.

Ini other examples, bonnone-producing cells are used, for exainpi;, to replace anterior pitoftay cells to affect synithesis and secretion of growth hormone secretion, ln3tei~iing hormone, follicle stmlatig bornmne, prol~actdn and thyroid stimulating hormne armng othaer& Gonadul cells, such as Leydig cells and follicular cells are employed to supplement testostetone or estrogen levels. Specially designed aomzbinations are neat!i in hormonae replacement therapy in, post 2a perimenopansal women, or in =en following decline in endogenous; testosterone secretion. Dopaniinc-producing neurns are uised and implanted in a matrix to supplemt defective or da=aged dopainne cells in the substantia nigm.. In. sain embodlnwma, stem cellsfro~m the reolpimntor a donor can heinmixed wit slight!)' damaged calls, for example Pancjraic inlet cells, or hgpatocytes, and placed in an clcrpoesdmatrix and later harvested to control tke differentiation of the stem cells into a desired cell .type; This procedure is Perfcormned in vitro or in Wwv. The newly formed differentiated cetlls arm introduced into the patient.

The ability to use electroproessed maerials and ratdeec to bioengineer tissue or organs creates a wide variety of binengineered tissue replacement appications. Exammples of bloengieread components include, but are not limited toD, skeletal irl cardiac, unwcle, nerve guides, brain constuct as a filler for damnaged/mrmoved areas of thie brain that wre lost duringZ accident or diseasq, a filler for other missing tissues, cartilage soaftu1dings, sheets for cosmetic repairs, sin (sheet. with cells added to make a skn equivalent), vascla grafts and components tereof, and sheets for topical applications (sin covering but no additnal cells, just a patch).

In some embodiment, such matrices ax combined with drug arid substance delivery electropro cessed matices of the present invention in ways tha will improve the ftncffon of the imnplant For example, antibiotics, anti-intimaoriea, local anesthetics or combinalions thereat can be, added to the matrix of E. biocagineered iorgaa to speed the heag process and reduce discomfort One method or prepaing lurpimits of the present iventio is use of a biorenetor. There 4.4 COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:37 FAX 613 i613 4199 FB RICE CO 01048 00 0 0 are several kinds of commercially available bioreactors, devices designed to provide a low-shear, high nutient perfusion environmuent Until recently, most of the available bioresetors maintained cells in suspension and delivered nutrients and oxygen by sparging, through the use of impellers, or other means of stirring. The RCCS bioreator (Synthecon) is a rotating wall bioreactor. It consists of a small inner cylinder, the substrate for the electrospinning process, positioned inside a larger outer cylinder. Although the electrospun or electroaerosol matrix can be fabricated on the inner cylinder, other locations within the bioreactor also can be used for plaaement of a mairix for seeding. The gap between the inner and outer cylinders serves as the culture vessel C- space for cells. Culture medium is oxygenated via an external bhydrophobic membrane. The low o 10 shear environment of the Synthecon 1UCS bioreactor promotes cell-cell and cell-extracellular 00 matrix (BCM) interactions without the damage or "washing away" of nutrients that occurs with o active stirring or sparging. Typically, the TCCS device is operated at rotation rates of B up to 0 EPM, as required to maintain cells in sspension, and at less than S RPM (preferably 2-3 RPM) for cultures immobilized along the center shaft of the vesseL The Synthecon bioreactor can be used in a standard tissue culture incubatr. These values for spin rates and other parameters can be varied depending on the specific tissue created.

Electmrprocessed materials, such as matrices, are useful in formation of prostheses. One application of the electroprocessed mataices is in the fonnation of medium and small diameter vascular prostheses. Some preferred materials for this embodiment are collagen and elastin, especially collagen type I and collagen type III. Some examples include, but are not limited to coronary vessels for bypass or graft, femoral artery, poplitcl artery, brachial artery, libial artery, radial artery or corresponding veins. The electroprocessed material is useful especially when combined with endothelial cells on the inside of the vascdlar prosthesis, and smooth muscle cells, for example a collagen tbe, and also when combined with fibroblasts on the ouwde of the collagen tube. More complicated shapes including tapered andior branched vessels can also be constructed. A different-shaped mandrel is necessary to wind the large ibeXs around or to orient the electrospumnelectroaerosol polymer.

Combination of electroprocessed matrix materials and wound polymer fibers can provide optimal growth conditions for cells. The polymer forms a basic strhuctual matrix and the electroprocessed matrix is used to deliver the cells. This facilitates cell attachment onto the structural matrix. Furtheore the stress in the polymer also orients fibers in the matrix providing further spatial caes for the cells.

In an ahernative fabrication strategy, a cylindrical construct is eletoTspin onto a suitable target, for example a cylindrical mandrel. Other shapes can be used if desirable based upon the shape of the site into which the implant will be placed. Maitrices in this embodiment are composed, for example, of electroproceased fibrinoge/fibain (for example to promote neovascularization, cellular integration and inffltration ftoma the srounmding tissue), electroprocessed collagen (to promote cell iTfltation and lend mechanical integrity), and other components, for example PGA, PLA, and PGA-PLA bleads, PEO, PVA or other blends. The COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:38 FAX 6313 86135 419,'F RC 4 FB RICE CO.

a(149 00 relative ratio of thfe different components of this constmu is tailored to specific applications (e-g.

moure fibrin, le~ss collagen for enhanced vasularization. inL a skin graft). To fkbricate a cylindrical Muscle the construct is filled with muscle or stem cells or other cal type and the dista ends of the clectrospun constructs =e suturred or sealed shut In some embodiments, cells are mfixed withL vaiu aXInfrast eulmce thir distribution-withinmtconstrncL. Foreoxwnple, the cells ea be mixed with elctroprocessed filnin or collagen prior to izisarton into the ocostra Tho objective of this stradtegy, is to provide additional reechlidal support to the congtrut and provide: the cefll wit a three dimensions] ruatix within thec construct to promote growth. This also helps to maintain the cells in an even dialibution wig-An the- ronstrut This method cmb be used to o 10 enhance the alignment of the cells within the construct This illig nmaterial c-gm be extruded 00 direcly into the cylindrical conefuct as the filling is extruded, alignment occur. Mixing o endotheial cells with the other cells inserted into the canstmrt (or othur cell types) can be done 0 to accelerate nieovascularixation. Another method to accomplish ls objective is to elactrodeposit endotjhelial. cells diretldy itto the electrolprocessed collagen-matrix that aids i fobmaton of the cylindrical sheath. The alipament of the fibers witbin the eleotroprocessed matrix that comprises the construct are optionally controlled by controlling the rejanve mnovement of the target end source soltion wit respect to one anothiet Other cell types, such as tendon ffbroblas, are optionally elecirospun into or onto the outer surface of the construct to enhance the formation of the outer connective tissue sheath that fonna the constiuct.

In another example a sheet of electroprocesed material is prepared, rolle Uni a cylinderand inserted into an eloctropirocessed cylindcn. The construct is Afld -with cells as described above, sutured shut end placed if. a bloreactor or diretly in situ. By aligning the fibrfls of the electospum sheet of material in paralel with the long axis of the outer cylinder a muscle-like, eleetropromcsd composition is produced. Cells in ontact with tha ftbrilrs that z= arrayed along the long axis of the sheet spread it parallel with the fibrils of the sheet, forming a muscle conet=ia of cells an-aed and layered in a pattern of organization siroiar to that present in vivo.

The cylindrical tissue constict is then implanted or placed within a RCCS bioreactor. Rats of rotation to maintain this type of construct in suspension range from 4-20 rpml, depmlding upon the over mass of the tissue and the specific mnaterials used to fabricate the outer cylinder.

Vasrnilhnzaiion of the engineered tissue, containing electroprocessed matrix rnatial will ocur in situ several days aftor surgery. In some embodimemse, neovasculization of =m engineerd construct contaiving electopxocessd material is enhianced by mixing endothellal cells. into thec construct during fabrication, Anofthr altemAlive for suppying imgineered tissue containing eleofroprocessod material with a vascular supply is to temporarily transplant the tissule into the omentum.L The onicatum bas on wxtensin and rich vascular supply that can be used like at living incubatar for the support of engineered tissu. The engineercd tis=u is removed from a bioreacto;, wrapped in the; omentm and supported by the diffusion of imtdies aiad oxygen fronm the sumrunding tissue in the rnentum. Alterntively, or in addition to this sppyroath, engineerd tissue is connected directly to the endogenons vascular supply of the omEutwn. A blood Vessel 46 COMS ID No: ARCS-i 90460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:38 FAX 613 86318 4199 TM 101T,-V 00 CA can be partially perforated or cut or left dissected free, of the oxnerta The engineeres tissu Contuining electroprocewsed colageo, fibdn, or other materials, depending upon the co U-uc±. is wrapped arouaid the vesseL The et1gineered tissue is supported by nutients leaking fromn the pnrborated vesseal or by the simple diffusion of nutrients if the vessel is left Jiflat. Regardles of strategy, the; engineerd tissue is sninde4 by the: omentum and its rkh vascular supply.

Tissue centalnn electroprocessed. material can be engineered With an endogenous, Vascular system. This vaacnkiu system cant be composed of artificial vessels or blood vessek excised from a donor site; on the transplant recipient The engineered tissue, containing CdCtrprooessv matix material is then assembled arounod the vesse- 35y enveloping such a vessel with the tissue during or after eassembly of the engineered tisse 1 the engineered tissue has Cl a vessel that cew be attached to thes vascular system of the recipient. In this example, a vessel in 00 the omnntuw or other tissue is cut, aud the vessel. of the enginem-ed tissuc is connected to the: o two free ends. of the orneaenl vessel Mlood passes from the omental Vessel into the vascular system of the engjneere tissue through* the tissue aad drains back into the omentwn viessel. By wrappin the tissue in the omenturn and concdnag it to an omeutal blood vessel, the engineered tissue is supported by the diffihaon of natrients from ther omentum and the vessel incorporated into the tissue dining its Thbrication. Afler a suitable period of time tho timse is removed fr-omt the ornentum and placed in thea correct site in the recipienL By using this strategy the engineered tissue containing electroprocessed material is supported in a nutrient rxich environment during the firs several days following removal from the bioresotor. The environment of the ornetm also promotes the ibradnn of new blood vels in implanted tissue., This oinn incubator stratogy can be coimbined with the othe-r strategies such as combliig angiogenic factors in the matrix naeial during eefprcsig.Several options are available. Fis the implants can be seeded 'with vugioblasts and/ar endothll~al CeOl to accelerate- the formation of vascular elemants once the enginieereI tssne is placed in situ. Second, angiogetdc peptides caa be introduced into the engineesed tfissue via an osmotic pump.. The use of an osmotic pump permits delivery of peptides or, as noted, angiogenico peptidee a growth fictors directly to the site of inrterest in a biologicnl.

1 efficient and cost-effective mmxr, VEGPF deivered to lacher, bind limbs of rabbita accelerated ospifly bed growth, increased vascular braniching and improved mrscularpfaan 0 with respect to ischnric controls. An alternative appro ach is to seed Hy diffmtreiatad tissue, cnstmecs coniaiming elecfrproc~ed muatrix mateijal with additional endotbelial ceils and or angioblasts shortly before they ate knplmnled in situ.

In some embodiments, the stei cells or other cells used to constmct the imp~lant are isolated from the subject, or other compatible donor requiring tissue reonshurtion. This providzgs the advantage of using cells ftht will not induce an immunn rtsponse, becat'se they originated wit the subject (autologons tissue) requiring thfe reconsirnctox. Rclalively sall biopsies can be used to aotin a micient nume of cells to 1constuct the implant This minimizes tbneional deficit and damage to endogenous tlss~ that serve as theo donor site for the cells.

47 COMS ID No: ARCS-19046o Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 '08 12:39 FAX- 813 8818 4199 FB RICE CO. lao51 00 AI" "foni embod-iments, the mairice of the present invention, include substances in: the 3rrari that will improve the- peformaMce Of the imlplantekd electrorocessca matrx Examples of substances that em be wsed include peptide growth factors, antibiotics, anad/or ati-rejection drugs. Altornafively, rells that are engin,=edr to vrufacnire desired canpoiins can be 5 icldd, TheD entire cOnSbnct is, for ermple, Oul Wred in a bioreactor or conventioinal culture or placed dirtly in vivaV, ]For exaple, neo-vascularization can be slinulated by angiogenic arnd ZrOwtli-promnoting factuzs, administered as peptide, proteins or as gene therapy. Angiogenic agents can be incororatcd into thoeolactroprocesed marix. Nerve gowth factors canbe eloospun into th,) mabix to promote growth or nearous into the mtix and tissue. In a desradabe mtrix, the gndnil degrdaioxAbrrdvm of the mahtc will release these ibetors ci and accelerate growth.t of desired tissues.

00 Mprce7Zrocsse matices can also, be used in connection with other matrix buiding procssw k ohewword;, an extruded tube can have 2n outside layer eleofroapun onto it wherei the different layers completment each other and provide an approprtate matrix to Promote a specific type of cell growt As au example, a vascular graft comprised primarily of a collagen tube can have an electrospun layer of both other mnateials such as collagen, or fibrin and cells, added to prTomot the acceptability of the graft in a particular recipient. A second example is an in vin-c skin preparation formed by growing fibrubWasts in oe layer, covering the first layer with elwposdcollagen, and then growing a seconud layer omnposed of epidermal cells in the fibrin matrix. This layering tevbinique, can be: used to muake a variety ofedsaue.

stabudfy and storage ofate Eiectroproeufsai Compositins The stability of thle comnpositions of the present inlvention colzptsing electroproeessed materials comnbiued 'with substances also allows forv long t=r= storage of the: compositions between formation and use. Stability Mlows greater flexibility for the user in embodfients in which a drug or other substa.-mce ig applied after formation of the electroprocessed mateial, for example by scialdug and srynyig A formed elecwoproened Mtrix can be fabricated and aured, and ten the o=ac substa compostion to be delivered to an indilvidial patient can be prepared and tailored to a specific need shortly before implantation or applicaton. This feaure, allows users greaer flexibility in both treatment Options and inventory management. Many cleotroproesd materials are dry once they are spun.~ essentially dehydrated. thereby faifitaing storage in a dry or frozen state. Furtbew, the elcctroprocesi compositions are snbstantialy sterile upon compledcn, thereby providng =n additioraJ adivantage in 0menpei anid coarnatic applieazios Storage conditions for the compositions of the present im'erttlon will depend on the elecropyozed 1 mnaterials and substances therein, In embodiments involving prottam, for eXample, it may be necesry or desirble to store the compositions at temperatures below 0CC, Under Vflcunn, or in a lyophilized condition. Othe storae oondidiona can be used, for exaple, at roomn tenpemr.e, in darkness, in vacuum or under =edce~d pressure, under iner aunospheree% 48 COMS ID No: ARCS-19046o Received by IP Australia: Time (HKm) 11:45 Date 2008-05-14 14,;05 '08 12:40- FAX 611 8613 4199 FS RICE CO, 05 [a 052 00 (Nat refrgeratmi temperature, in aqwusos or other liquid solutions, or in powdered formi. Persons .of ordinary slill in the Aft recc'gnize appropriate storagv conditions for the materials mid mibstanees; contained in the comnposition&s ad wil be able to select appropriate storage conditions.

The compositions of the present Invention and fornniltions comprising thiose S comipositionts may be sterilized trough conzweaiotal means known to one of ordiriq skil in the 4Ar. Such means include but are not linkfed to ifitration radiation, and heat The compositions the present invention mnay also be combined with baoeioststic agents, such off thiineivs4l to inhibit bacterial growth.

Forulations comrnpising the compositions of the present invention may be preented in nit-dose or multi-dose containers, for axanple, sealed amnpulee and vias, andnnay be stored insa (N freeze-dried (lyophilized) condition requiring only the addition of tihe sterile liquid carrier, for 00 eape-wtrfrinjections, inaneriately prior to use. Extemporaneous inecfion aolutions and o suspensions mazy be prepared frm sterfie powders, granules and tablets omumonly used by out of ordinary skill in the art Prefenred unit dosage lbnnulations are those contairdung a dose or unit. or an appropriate fraction thereot, of the adndinistered ingredient It should be undersood that in addition to the ingredients particularly maentioned ubov, the formulations of the present invention may include other ageuts coamonly used by one of ordinary 'kill in the art Tho comnpositions of the present invention may be packaged in a variety of ways depeding upon the method used for adminlistering the omnpositiot- Generally, an article for disiribution includes a container which containa the composition or a formulation comprising the composixion in an appropriate; form. Suitablo container are well-known to those skilled in the art and inniude materials such as bottles (plastic and glass), sachets, ampules, plastic bags, metal cylinders, and the like The container mnay also include a tamper-proof assemblage to prevent indiscreet access to the contents of the, package. In addition, the contanEr has deposited thereon a label Which descrbes the contents of the container. The label may also include appropriate warnings.

The present invention is fttffier illustrated by the tllowiuz example; which are no0t to be -construed ini any way as imposing fimitais upon the scope treof On the conins, it is to be clearly lmdcratood that -resort can be ha& to vndous other onibodiments, modifications,' anid equivaleuis therecc4 which, srfer reading the description herein, cmn suggest ffiemselves to those Wkiled in the art withmzt departing from the spirit of the present invention.

1 is Fibrobinst growth thetor (FOF, obtained ftrm Chenuiaon, Teasacud;6 CA) was dissolved in aL solution of matrix material comnprised of type I collagen PGA and PLA The, percentages re-fer to the weight of lbsc matrials with respect to oxis another. These materials were dissolved in EW at a fina concentration. of 0.08 gin per =Lk Su~cieart FOP was added to 1 Ml of solution to provide an FOP concertr-atlon of 50 ug/mI et the collagenP/PLA eleotrospirning solution. The material was eleotrospun into the: shape of a cylinder onto the 49 COMS ID No: ARCS-19o46o Received by IP Australia: rime 11:45 Date 2008-05-14 14/05 'OS 12:40 FAX 613 8618 4199 D T 00 (N outet surthc Of at grounded and spinning 16 guage needle about 25-35 mm in length- After applicaionl the, cylider was sutured stint looping a sutur around the outside of the construct and pulling tight to seal the ends- Alirmntively, a hot forceps is used to pinch the ends; together and literally heat seal the ends shut These miethods formd a hollow, enclosed contatrct The S construct -was then surgically iinplauted within the Vastus latenlis mnuscle of a rat The constract was left in place for seiwu days -and recovered for inspection. FGF in the matrix acceleratetd -MUtate formation within the electospum matiix by promoting musce formation wilhin the wall ON of the electrospum cylinder.

BXA.IvILE 2 Ci Vascular Endotbelial. growth factor (vEG?. obtainled fiom Chemicrjn, Temnecula, CA) 00 owas dissolved in a solution of matlix, xuaterial comprised of type I collagen rIGA (10%,4) o an d PLA as described in EXAMPLE 1. Th~ese materials were dissolved in HMlP at a fifLal coneerrtsion of Ct0R gm per mlt Sufficient VHG1 Was adlded to I ml of solution to pro-vide a.

VPiGP concentatmon of 50 ng/nil of the, collageu/P(IA electrospinning solutiona The material was eleotrospun to Ibun a construct and implanted into a rat musce using the same procedures set forth in Bxanmple 1. VBGTF inesed the density of fractional capillaries that wern present throughout die construct This was evidenced by the presence of capillaries coaining red blood ceLs Cs)- BXAMPIJB 3 Constructs of eleclroproeessed collage andIPGA:PFLA copolymer, with VEGP spun into the matrix were prepared uMing 80% collagen and 20% rO&PL& Thz collagen and PGA:.FLA wmdissolved in HEMP at a final combined concentration of 0.08 gmn per ral. Solutions wer prepaird in whicht different amounts of VEO (35 ere, added to 1 ml, of the solution of collagen and PGA:PLA copolymer. Sexnrte solutions we-re prepared containing 0 ng, 25 m&g S0 ng, and 100 rig ech it I maL Constructs were prepared fot echb solution by electrospinning one nal. The constructs were cut into smaller sections and placed in a phosphate buffer solution "PS).

Relese of VEOF into the P13S was meamured as a finotiou of tinat by the BLIA method. The EUSA kit for VEGP war pa chatd from Chenaicon. Juternatiou italr numiber est2l4) and the, directions provided in the, kit were followed to perform the ELIA- Samples were centriflaged, to remove particlate matter antd stored at -20 'C prior to -use, An idential construct was subjected to crosslinking by expdsing it to 'glutuldehyde vapor at room. temperature ad subjected to an identical ELTSA assay. A sample of the eletmrowsed constnact was placed in a 100 a tissue culture dish. AS35 mm tissue culture dish con1taining I ml of 50% gltaraldehyde was placed inside the 100) rn tissue culture d&h.

The lid of the 100 mm lissue culture dish was ruplaced. and the sample was aLlowed to sit for mnutes at room tewmatase The sample was rinsed in sterile waer or culture, media. The amount of VBCIE (expressed in picogrms per 1 mg of electmpun material) Lbr the non cross- COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14 051. (8 12:41 FAkX 613 8618 4199EBRC&Co FB RICE CO, U~ 0.54 00 linked =dA crnas-liuked samples was measued at dxffcret times are presented in Figure 3 and 4,respeivly In Fligure 3 and Figure 4, the open diamonds reprasent release fromt the V.bers electruspim frmM ftto solution Containing PCIAYLA copolyznex and collagen -to which no VEOF was added.

Thbe open squares represent release frome fiber electosun from the solution confaining PGAtPLA copolymer and collagen to which 25 ng of VEOF -were added, The open circles represent release from the fibers electrospum fr-om a solution containing POATELA copolymeri and collagen to which 50 ng of VEGE were added. Tma open triangles represent release froma the fibers electrosrun from a solution contairling PSA-PLA copolyMer end colagen to which 1 00ug o 10 Of VW3GF Were added. Results demonstrate not only than the matri releases VEOIF in. PBS but 00 also that cross-lining wit gliatmtidehyde slows rel-eas from the matrix.

0 0 EFrjsjpuj 4 ?olveithylene-eo-vinyl acetate) (PEVA) was a gift fromn DuPont (Elves 40, 40 vinyl act&14 PBVA pellets were soaked in ethanol for several days to remove antioxielams.

Poly~lactic acid) (100 L PEA) was a gift fom 'by Alkermes, Inc. (Medisorb@) with a number-average molecular weight, M. of 205 and polydisperity of 1.7. All solvents were analYtical grade and were used as received. This(hydroxyzuetbyl)aninoahae hydrochloride (Txinna0 HO0) and tr shydroxynethylamiae (Tzizm4)-base) were suplied by Sigma and wee wsed without Atiher purification to prepare buffer solutions of pH 7-35. Tetacclie hydroobloside was also obtained from Sigrla Acisite peiodontal fiber (0.5zrn PEVA) contaning 25wrt% teracqycline hydrochloride was a gif from Airs. Corporation (Palo Alto, CA).

~L'ecmrospinnbng was carried out using 14 wt/v solutions of 100% flBVA. I100%/ PLA or mixtures of the two in clilorofbnrt The mixtures used were 25% PEVAI7S% PLA, 50%150%1 of ecb and 75% PEVAI2S% PJAA with percentages by weighit Tetracycline hyrochloride, Which is insoluble in chloioform, was sotubilzed in a smiall amwunt of methanol and added-to the, polymer solutons prior to electospinnig. The resulting solutions were yellow but clear, indioating homnogeneous solubilization of both the polymrs= and drug.

The electrospinning set-np consisted of a glass pipette (held parallel to grcsnd or angled at 45' downward), 0.32 mom diameter silver-mcated copper wire (positive lead), a coper sheet (grund electrode) mt 30 cm from the pipete,- and a Spellmnan CZ61lDOOR high voltage suppy- A positve voltage (15 WV) was appled throgh the copper ire to the polymner solution inside the glas pipeft The solutions were delivered ija. syringe ptips to ctuntrol the mass flow rate, whih rnmged from 10G-18 nil/b. More couvenierilly, t solution can be held in a plastic syringe with the high voltage supply connected to the mneta syringes needle. The solutions were dzliyrered via syringe pumps to control the mss Rlow rat;i wbich ranged from 10-18 Mnd/o h resulting electriclly charged fibers were collected on rotatinkg matal plate to produce a sheet of non-woven fabric.

51 COMS ID No: ARCS-i 90460 Received by P1 Australia: lime (I-tm) 11:45 Date 2008-05-14 14/05 08 12:42 FX613 8618 4199 F IE&C.lhs FB RICE CO, [a 1)55 00 A lOOL PLA containing 50/ tetracycline hydrochloride (by weight) was elcfroSpun from 14% Will solution in cbiorofbrm, wtth a maw flow xr of the polymer solution between 18-21 nabb. PEVA containing S% teauyclie hydrochloride (expressed herein a by weight of total polymer) was clectrospun from 14 Wil solution with a mass flow rate of MM/h Bknds containing 5% tetracycline hydrochloride and consistig of 25% PLA and 75% PEVA were elecMtrospun at ma= flow M&tS of 13-18 ufit A 50/50 PLAIIEVA blend wt 5%1 tetrayclie hydroxide Wvas sptm at a mass flow raz bf 10-13 nil/h. A 50/5 PLA/PEVA blend with 25%4 tetracyclin hydroxide (by 1weight of total polymer weight) was spun at a mass flow rate of I1nil/ar. A blend containing 75t-5 PXLA/PBVA with 5% tetaclins hydroxidde was span. With a mass flow rate of 17 nil/h. The collected 'fabric' was used for stuadying the release of tetracycline 00 hydrochlorideo PFor comparative purposes., cast filuns wer Made, from differnt compositions of PLA and 0 PP/A. As with the fibi; films were made of 25% PEVA/75% PLA, 50 4/V50%1 of each, and PEVAJ2S% PIA and 5% tetracycline hydrochloride was added to each. The solutions in chloroform were cast onto glass petri dishes, left at room temperature until The chloroform was evaporated, then died at 25 6 C under vacuum for 3 hours. The release: of tetracycline hydrochloride fruni ACflSLTE@ (P1W/A) periodontal fiber was also roruparect Release of tetracycline hydroohlori(4 w=s determined using UV-VIS rmeasureents carried -out at Perkin-Elmer UVNIS Lambda 40 Speelophotometer The molar exciheton coefficien for tetracycline hydrochloride in Tri buffer was towed to be 15,800 from a linea Beez-Latbert. plot of absorbance at 360 urn 'vs. concentration. Release of tetacycline hydrochloride was detennined by placing a known mans of polymer and drug in tis buffer and monitoring the absorbance at 360 nm as a fincdon of ime. The lbuffer solution Was changed if the releaed drag gave absorbance higher than 2.0- Data are reported as the %4 tetracycline hykocfflorjde released based -upon the expeted amout in the smles flnn the feed coaposilion The morphology of te lecwrospn samples wute studied withi JSM-820 Scanning electaronnLroscope (JZOL Ltd.).

The release profiles of tetracycline; hydxochlorids frm electxopnn fibers and the canst Qibs are shown in Pigmes 5 and 6. In Figure 5, tho solid diainonts denote release from the fibers electrospun from the solution in which. the polymer was 50% EVA. and 50% PLA and tetracycline was added. The open chdlas denote release from the, fibers electra spun from the soluio in which the polymaer was 50% EVA and 50% FLA and 25% tetracycline was added.

The open triangles denote release. from the fibers electrospun from fit solution in which the polymer was 100% HLA and 5%6 tetracycline was added. The solid squares denote release from the fibers electrospun from, the solulion in which the polymer was 100%/ EVA and 5%1 tetracycline was added- In Figure 6, fte open diamonds dcrxtre release froam thie fibers electrospun from the Solution conteing 100%/ EVA. The open squares denote release from the. AC~rhS EV PPA) pexiodontal fiber. The open tri angles denote release froma the Mim in which the polymer was 52 COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14 14/05 08 12:42 FAX 613 861& 4199 P IE&C.~5 FB RICE CO.

Z056 00 PLA and 50% PEVA. The open cireka dimote release from the' film in which the polymer wvas 25% FLA and 75% rEVA The solidl diamonds connected by a thickR line denote release from the film conitaining 100% PLA The sold tiangles denote release, from the film in which the polymcr was 75% PLA and 25% PEVA. The solid square denot, release from the fibers eleetroepun from the solution contaiig 25% PTA and 75%9 PEVA. The solid diamxuad connected by a thin line denote release fromn fim aontaining 100% EVA.

Bleciosun EVA showed a higher releae rate than the mats derived from PLA/ErVA (50(50) or pure FIX Blecospum PEVA released 65% of its chug content -within 100 hours, ci ~wheras the electrospun 50/50 mixtur of PFJVA and PTA released about 40% over the samte o 10 time Period. Mats of PLA libers with no PEVA esbibit some instantaneous releas; with 00 negligible release over 50 ho=-t The 50:50 sample wit ZSwto tetracycline hydrochloride o releases the. drug more rapidly tha the 5% sam~pe although the releaged of the fonme 0 appraches that of the letter after 150 bra Figure 7 shows release profiles of tes electospun. ?ENA samples, two from the samer batch of mat and anottier from a diffret preparaton -under ideutical conditions. The release amouts of each sample ar denoted by solid diamonds, solid square, and open tianglms, respectively. The profiles are quite similar fidicating very good reproducibility. In general, the initial rate oftrelease or all fotmulations including ACUSrIT5® (denoted as Alm) is highL during the first 10-12 hours, mjost likely due to. ree- of drag sequestered on the sample surfaces. The total percnt released froin the cast tilna (Figur 6) wer lower than that of the electrospun mats, as would be expected. dm to the much lower surface aces of the former. The PLAJEVA 75/25 film released 30% of its teiracyclino hydrochloride in 120 brs, whereas the 51bn of 50150 'PLAIEVA showed a slightly lower percent of releae in the same period of time. Release from the- PLA filn was much lowtr only 6% released in 120 his, whereas the PBVA film showed S% release over the same period.

A mixture of ou'othrd insulixn eecaelig colls is seeded into an clekoprocesed collagen matrix to form =n electrocessed collagen-contxixiing tissue. Tho eloctrprocessed. matix contafining the insubn secreting @611s in implanted into a diabetic recipient in need of insulin.

This electropmccessed eolla~en or ftbdin-containg *ime optionally contains a vessel. The matrix is implanted into the retropritoneal space and the vessel is anastomosed into the hepga portal circulation. Thaull is released from thie insulin-containitg ccli and tranmdied to the ciruation.

The ulectroprocessed matrix containing t&e insulin secreting cells is optionally supplemented with cells that synthesize and secret ginoagon. somatostalin, pancratic polypeptide, or omrbinations thereof, in order to mimic the hormonal complement of the pancreatic islet.

53 COMS ID No:ARCS-190460 Received by IP Australia: lime 11:45 Date 2008-05-14 14/05 '08 12:43 FAX 613 8618 4199 13 RICE CO.

l057 00 ci OPtiona uy, heterologous cells (for cramp!; engineered bauteria or cells from a donor) are placed in a matrix with a POre size that will allows diflion of nutrients to thie cells but does not allow or inhabits or delays the detection of the Cclls by th~e recipient's immune system ZXANLE 6 Keradinocytes are harveste from a healfiay Site of a Patient suffering from a chronic Waound. The cells ar grown in cultura and transfeoted by elwfroporaou tof xpes VB Next, thes frmnsfected cells are mixed Or prepaed in an clectrospun Collagen matili. Antise; Ofgonoleolide for matix metaloprot&inases (MMvPs) are al5o spun into the matrx The matrix c-I'is topically applied to the sorface of the wonact The cells niear and in the implant take up dme 00 anhisense sequences, expres their transfected g=n sequences and MMEP production is reduced, o in* other applicafious the cells may 'be geneficaily enterod to secrete VEOP, thereby c-i Promoting heaiint Release of the anlisense, ollgrmunleatides suppress expression of MMws.

which are typically overepressed in a chronic woun&. Thus the wound site is repaired with an impl=n that S=Tmntaneously promotes natural healing responses. Optionally, the matrix is comprised of fibrin or a mix of' fit ad collagen. The fibrini assists in. cessation of bleeding and promotes healing.

EXAMPLE 7 Osteoblasts fonm a patient with a bane ixjiey wre cultared and incorporated into a reectop-n matrix comprising type I collagen Thbe matrbx is formed in tile shape of a cavity or defect at the injuy site. nlone. growth heor MM 11, bone morphogenic pxotein (EMP) or sequercs of ganes encoding for these proteins, are electrosptn into thie matrix are optionally icorporatad into the eleotro an matrix. The: matrix assist in growth of new b one, and the Rap or RlV jin the mimicx promnote bone growth.

Optionally, the collagen used is produced in iWtr by genetically engineered cells that, express a collagen polymer with more r-.i5 sitces dmn in normal collagen. The excess of sites Promotes osteoblasts to produce and seretes hydroxyapatite and Thithier aid bone growth.

Optionally, the matrix is further eleciroprocessed with polypyrrolez, which wre electrically active mafteials. Electrodes owe attached to each end of the implated mari.

Charged electrodes are later applied to the suzfate over the electrode. to croate a small electric curet acrosg the huplant to fher facilitate ealing of the bone ijinY.lo another embodimenit piezoeleetric elements mifay be electrospan into the matrix to produce electric discharges that prom &theadjnyr EXAMNxZ

B

In another aampl; similar to that daeribca far skeletal imuscle a cardiac parh is prepared. A sheet of clectroprocessd inateial is prepard with aligned filaments of collagen.

54 COMS ID No: ARCS-19046o Received by IP Australia: Time 11:45 Date 2008-05-14 14,05 08 12:44 FAX 613 8618 4199 FB RICE CO. Z058 00 0 0 The sheet is folded into a pleated sheet in the desired shape and or rolled into a cylinder. A second construct is electrospun in the desired shape, for example a rectangle. The pleated sheet that mimics the cellular layers of the intact heart is inserted into the clectoprocessed rectangular fozm. The construct is filled with cells, sutured shut and placed in a bioreactor or directly in situ By aligning the fibrils of the pleated electrospun sheet of material in parallel with the long axis of the outer rectangular fomn, a cardiac, muscle-like construct is obtained. Native cardiac tissue is composed of layers of cells arrayed along a common axis with adjacent cell layers slightly off axis with the overlaying and umdelying layers. This satucture is more precisely mniicked by the methods described below in which a matrix is prepared and cells are directly electroprocessed, O 10 dribbled or sprayed unto the matrix as it is prepared. Cells in contact with the fbrils that are 00 arrayed along the long axis of the sheet spread in parallel with the underlying fibrils of the sheet, O fonrming a muscle construct of cells arrayed and layered in an in vivo-like pattern of organization.

SThe-construct can be dirctly implanted or placed within a RCCS bioreactor Rateas of rotation to maintain this type of construct in suspension tange from 4-20 rpm, depending upon the mass of the tissue and the specific materials used to fabricate the outer cylinder Variations of this design include the addition of angiogenic factors in the matrix, gene sequences, and agents to suppress inflammation and/or rejection. Other cell types may be added to the construct, for example microvascular endothelial cells, to accelerate the formation of a capillary system within the construct Other variations in this design principle can be used. For example, cells may be electroprocessed into the matrix as it in deposited on the ground target. By vatying the pitch of the fibers during spixAdng and spraying, dribbling or electrprocessing cells onto the fibers as they are deposited very precisely controls the positioning ofthe cells within the construct.

All patents, publications and abstracts cited above are incorporated hereinb by reference in their entirety. It should be understood that the foregoing relates only to preferred embodiments of the present invention and that ntmerous modifications or alterations can be made therein without departing from the spirit and the scope of the present invention as defined in the following claims.

COMS ID No: ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14

Claims (9)

1. A comiposition comprising: an electroproce=sed material and a sabstanca.

2. The composition of Uaix 1, wherein the material is one or mr= fla materials, one or more syuthe&i mater ials, or a combination theref.

3. The composition of Claim 1, wheren the. materia compises a combintion o one or more naliraj materials and one or more synthetic niateiabs. o0

4. The ccnzqosifion of Claim 2, wherein the natural material comprises one or mi o amino acids, peptides. danatg-ed peptides, polypeptides, proteins, carbohydrates, lipids, nit acids, glycopfowtefs, lipoproteins, glycolipids, glycoaainoglycans, proteoglyccas, ox combination thereof S. The composition of Clain 2Z wherein the synthtfic matetial comprises one moricpolyniere.

6. The; composition of Claim 1, wherein flue substanc comprises a therapei substance, a cosmetir. substance or a combination thereof-

7- The composition of Claimn wberein thns substance comupnses one or m mnolecules, clls, obj eels, or combiuations thereof S. A composition comprising. an clectlaprocesed material wherein. elactroprocessed material provides a biologica eI~ht when delivered to a desired locatior vivo or in vitro.

9. A miethod of deliveing a substance or an electroprocesed material to a desi locationi, compiising placing any of the compositions of Claims 1 to 8 at the desired locatiom A method of manufactuing the comiposition of "Claim 1, cotpria etbemdepositing one or more electrically-charged soittions comprising a naienal or molersl capable of fanning the material onto a grounded substrate under conditions effective eleettodepesit the material or the molecules bapable of foxining the material on the 9ubstrab fbim the electroproce material; and, adding the sub stanc to the electrodeposited mater Irm the cnposition. 56 COMS ID No: ARCS-i 90460 Received by IP Australia: Time 11:45 Date 2008-05-14 14,,-5 '08 12:44 FAX 613 8618 4199 F IE&C.ts FB RICE M 0060 00

11. A method of mmifacturing the composition of Claim 8, cornpi! electrodepositing oneb or =are elevctlially-rarged solutiorns cowprisig a material or molca capable of forming the material. onto a grounded substrate -under conditions pifectivi electrodeposit the matezial or the iMoules capable of fanningS the materia on the substrat form the eloctroprocessed material.

12. Use of any of the elawoproeessed compositins of Cl-ams 1-8 for delivery ON substance or ant electroprocessed materia. o 13. The use provided in Claim IZ wherein the substance comprises a therape 00 substane' a cosmetic substance or a combinafion thereof. 0 o14. The use, pro-vided in Claim. 12, whierein the aubptance comprises one or n2 molecules, cells, objects, or combiwaion tercofi Use of any of the elecrtroprocessd compositions of Claims 1-8 for preparatio a medicament usetbi fot delivery of a substance or an electroprocessed inaicial. 57 COMS IDNo:ARCS-190460 Received by IP Australia: Time 11:45 Date 2008-05-14