CA2222595A1 - Therapeutic microdevices and methods of making and using same - Google Patents

Abstract

A suspension of microfabricated microdevices for use in therapeutic applications is disclosed. The microdevices have a selected shape, and uniform dimensions preferably in the 100 nm to 10 Am range. Also disclosed are microfabrication methods for making such microdevices.

Description

CA 02222~9~ 1997-11-27 TE~ EUTIC MICRODEVICES
AND METHODS OF MAKING AND USING SAME

FIEID OF ~E INVENTION
The present invention relates to microstructural devices, and more particularly to iclu~L~Ilctural devices for use in drug delivery to a body site.

BACKGROUND OF ~E INVENTION
A variety of self-assembling particles have been used or proposed for drug delivery 10 to a target site in a body. Lipid micellar particles formed of a surfactant provide certain advantages in solubilizing and delivering hydrophobic drugs, e.g., for intravenous drug delivery.
Liposome delivery systems have been employed for delivery of drugs to tissues and organs, e.g., liver and spleen, rich iri llla~ilv~hages of the reticuloendothelial (RES), for 15 depot drug release in the bloodstream, and for site specific zl~cllmnl~tion and drug release in solid tumors and sites of infecti--n Liposomes offer a number of advantages over simpler micellar particles. The lipid bilayer shell in a liposomes provides an internal aqueous CO~ ,dlL,IIcllL that is Pc~nti~lly isolated from bulk phase aqueous m~ m, allowing hydl~uhilic drugs to be se.~ d at 20 high concc;llLIdlion within liposomes, and further p~ . ."i~ .g loading of ionizable drugs by use of ion gradients across the liposomal membrane. Liposomes can also be process to have selected sized in the 30-200 nm size range for intravenous drug delivery. Finally, the outer lipsome surfaces may be coated with a hydl~hilic polymer, such as polyethylene glycol, to extend blood circulation time of the particles, and/or may be d~signPd to carry 25 surface-bound ~ntilig~nf1 molecules for selective binding to target cells cont~ining cell-specific surface ligands.
The use of synthetic polymers in drug delivery devices has focused on "smart polymers" a term given to polymers that form gels that have the ability to expand or contract in response to a specific stim~ c, such as light, lelll~eldlul~; or pH. Typically, 30 such polymers will pl~cil~ildL~ in solution or collapse with collcolllil~lL expulsion of gel pore contents. Synthetic polymers may be based on a number of types of IllOIlOIII.,.iC units, inrlu-ling vinyl ",ono",~.~, N-alkyl substituted acrylamides and the like. Copolymers have also been utilized in an attempt to combine or m~ re the ~tim--lus responsive properties of one or more known smart polymers. Polymer particles formed of negatively charged 35 polymers have been ~IP~ign~d for (i) rapid c~n-lPn~tion (for drug ellLld~lllenL) and CA 02222~9~ 1997-11-27 ~l.oc~n-lçn.c~tion (for drug release) or controlled-rate swelling and drug release, and (ii) high drug t~ d~lllellL by an ion exchange m~ h~ ."
The collct;~L~ of smart polymer particles and lipid-bilayer vesicles have been COlll'L h1ed in drug-delivery particles of the type having a contl~n.ced-phase polymer core S encased in a lipid-bilayer lllcllll~lal1e, giving advantages of both types of drug-delivery systems. In particular, the polymer core of the particle can be loaded to high drug concell~ldlion, for rapid release by particle decon-l~nc~ti-)n, and the lipid coating on the particles can be dçsign~d for targeting and/or for a target-specific triggering event which can in turn leads to rapid particle decon-l~nc~tion.
It is evident that it is possible to design self-a~ssembling lipid and/or polymer particles having various drug loading, targeting and triggering capabilities. Non~th.olçss, self-assembling particles of this type present two ~ignific~nt limitations. First, since the particles are typically spherical, the physical-contact area between the particles and target-site cells, for example, for particle binding to the cell, is more limited than would be the 15 case with a particle having more planar surfaces. Secondly, and more importantly, the functioning of self-assembling particles, in terms of Idl~t;lh~g and drug release, are limited in terms of (i) the particle materials (and Lh~;lcrvle material properties) that can be employed, (ii) the types of drug storage and drug release ll.~ that can be realized, (iii) the number of Idl~C:Lillg and therapeutic functions that can be built into the particles, 20 and (iv) the ability to place dirr~lc;llL functions at discrete locations on the particles.
It is the purpose of the present invention to provide microdevices that signifi~ntly expand the shape, materials, and functions versatility and capabilities over self-assembling drug-delivery particles.

25 SU~ARY OF THE INVENTION
The invention includes ~.u~el~ion of microdevices for use in a~h~ g a therapeutic agent to a selected target site in a subject. The microdevices have a selected non-spherical shape and uniform ~lim~nci~ns and contain the thcldpcuLic agent in a form in which the activity of the therapeutic agent is ~ essed by exposure of the microdevice to 30 the biochemical enviiul~lllclll of the target site after ~lhll;lli~ldlion to the subject. The microdevices may have surface-bound, marker-binding molecules effective to bind to a marker carried on the surface of cells at such target site.
Where the suspension is used for targeting selected cells or tissue via the bloodstream, the microdevices may be coated with a hydrophilic polymer, such as CA 02222~9~ 1997-11-27 polyethylene glycol or glycocalyx polymer, effective to enhance m~ e..~l~ee of the microdevices in suspension. The hydrophilic polymer may be conjugated to vesicle-forming lipids, where the microdevices are coated with a lipid film containing such vesicle-forming lipids. The microdevices may contain a marker-binding molecule bound to the free ends of 5 at least a portion of the polymer, this molecules being effective to bind to a marker carried on the surface of such target cells or tissue.
Where the suspension is used in pa~ Aminictration to a subject, the microdevices may have a selected m~ximnm dimension in the range between 0.1 and 3 microns. For solid-tumor ~lgt:~hlg such microdevices preferably have ~ m~imnm 10 dimension less than about 150 nm.
Where the suspension is used in delivering a theld~uLic cu~ uulld to the interctiti~l space of a target region characterized by a target-specific marker on the b~c~
bl~1e forming the v~cc~ h~re of the target region, the microdevices may contain surface-bound marker-binding molecule effective to bind to such marker, and an enzyme 15 effective to lyse the b~c~...~.l lllc:lll'~l~ule. One p~fell~1 enzyme is a type IV collagenase.
The enzyme may be covalently attached to a surface region of the microdevices, or may be contained in the microdevices in rel~c~hle forrn, e.g., upon erosion of the microdevices.
In one general embodiment, the microdevices are formed of a material designed toerode in body fluid at a selected bioerosion rate. As examples, the microdevices may be 20 formed of iron, tit:lnillm~ gold, silver, platinurn, copper, and alloys and oxides thereof.
Alternatively the microdevices may be formed of a biodegradable polymer material.
In one embodiment, the microdevices are composed of a conAPnced-phase polymer material effective to AeConAe~l~e7 at a selected rate, when exposed to plasma.
In another embodiment, the microdevices are ~ lly disc-shaped, and have a 25 l~min~ted struchure colll~illillg two or more layers, where ~Aj~rent layers are formed of dirre,~ materials. As example, the microdevices may have a tril~min~te structurecomposed of an interior layer sandwiched between a pair of exterior coating layers, where the coating layers have a slower rate of bioerosion than the interior layer, and the thcla~ uLic compound is embedded in said interm~oAi~te layer, for release as the interm.oAi~tf-30 layer is eroded.
In still another general embodiment, the microdevices have shielded wall surfacesthat are ,ub~ ulLially in~( ctDcci't)le to biological cells. These wall surface may be coated with therapeutic agents, such as therapeutic antibodies or the like. Exemplary microdevices of this type include ring shaped and cup-shaped devices.

CA 02222~9~ 1997-ll-27 For use in directing the microdevices to a target site by a m~gn--tic field, or retrieving the devices from a site by a m~gn~tic field, the microdevices contain a m~gn~ti~
material.
Also disclosed is a microfabrication method for producing microdevices of the type 5 described. The method includes exposing a sheet of microdevice material to a photoablating light source through a pht~tl~m~k, to form a reticular lattice pattern on the sheet corresponding to the desired microdevice size and shape. The photoablating is colllhlucd until the desired degree of ablation is aclli~v.,d.
These and other ob ects and features of the invention will become more fully 10 alJpdl~lll when the following detailed description is read in conjullclion with the ~comr~nying dldwillgs. ' BRIEF DESCRIPrION OF THE DRAWINGS
Figs. 1-7 illustrate various embodiments of microstructures and microdevices 15 constructed in accordance with the invention, shown in pel~pe.;li~1e views (Figs. lA-7A), and cross-sectional views (.~ i..i.,g figures);
Fig. 8 is an enlarged section of the disk-shaped microstructure in Fig. 7C, showing surface bound antibody and therapeutic agents on the inner wall surface thereof;Figs. 9A-9E illustrate steps in the photolithographic fabrication of Illi~lu~lluctures of the type illustrated generally in Fig. 2A;
Figs. lOA-lOD illustrate steps in the photolithographic fabrication of microstructures of the type illu~llaled generally in Fig. SA;
Figs. llA-llD illustrate steps in the fabrication by laser ablation of microstructures of the type illustrated generally in Fig. 3A;
Fig. 12 is a sectional view of a coated microdevice.
Figs. 13A and 13B are section~l views of a lipid coated microdevice having a surface coating of hydlophilic polymers, where in Fig. 13B, a portion of the polymers have antibodies ~tt~h~d to their distal ends;
Fig. 14 is a sectional view of a microdevice having an upper surface coating of antibodies and a hydrophilic polymer;
Fig. 15 is a sectional view of a microdevice of the type shown in Fig. 6A, illu~lldlhlg how the device structure shields antibodies carried on the device from contact with an immune-response cell;

CA 02222~9~ 1997~ 27 Figs. 16A and 16B are sectional views of a microdevice of the type illustrated in Fig. 3A, showing more rapid erosion of a middle drug-carrying layer in the microdevice;
Figs. 17A and 17B illustrate the greater surface contact between a drug-deliveryparticle or capillary wall surface in a disc shaped particle constructed in acco~ ce with the 5 invention (17A) than in a conventional self-assembly type spherical drug-delivery particle (17B);
Figs. 18A-18C are embodiments of the invention suitable for I~ LIllell~ of hl~ ial tissue.
Figs. l9A and 19B illustrates ste?s in the movement of a microdevice co~ d inaccordance with one embodiment of the invention across an epithelial ~ Ialle; and Fig. 20 is a cross section view of a microdevice cont~ining a m~gnlotic layer.

DETAILED DESCRI~rlON OF THE INVENTION
I. DeflnitiOnS
Unless in-lir~trd otherwise, the terms below have the following mr~ning.
"Microstructures" or "microparticles" or "microfabricated structures" or "microfabricated particles" are particles formed by microfabrication methods.
"Microdevices" or "microfabricated devices" are microstructures that have been additional prepared to include biological agents as coatings and/or therapeutic agents;
"Microfabrication mPthorl.c" refer methods employing photom~cking or pa~el.led beam irradiation of a substrate to produce desired surface pattern features in the substrate;
The "biorh~mir~l ~"lvhulllllent of the target site" refers to one or more intrinsic physiological conditions at the target site, such as pH, salt conditions, t~ ;;ldlule, or the presence of target-specific cell surface markers or enzymes, effective to initiate and promote released of a therapeutic agent from the microdevices of the invention.
The target site may be a specific cell, tissue or organ type, the hllel~liliulll of a tissue of organ, a vascular site, or blood or plasma.
"Bioerodable" refers to a materials, such as an erodable metal, that is dissolvable in physiological mP~inm, or a biocolllL~d~ible polymeric material that can be degraded under physiological conditions, by physiological enzymes and/or chrrnic~l conditions, e.g., in a reducing or reduced-pH environment.

CA 02222~9~ 1997-11-27 WO 96/41236 PCTtUS96/09614 II. Microdevices The present invention provides microdevices that are useful therapeutically in avariety of in vitro, in vivo and ~x vivo applications, in particular, intravascular applications.
The microdevices have a selected non-spherical shape, uniform ~limrnci~ns and contain a 5 Lll~ldp~ ic agent in a form where the activity of the agent is expressed by exposure of the microdevice to the biorhPmir~l en~ilol--ll~llL of a selected target site.

A. Representative Embodiments The shape, size and composition of a microdev.ce of the present invention depend10 on the selected application. For example, devices designPd to be used in typical d~,ds~ ar applications are preferably ~ ls~ lly disk-shaped, cup-shaped, or ring-shaped. Exemplary embodiments of such disk-, cup- or ring-shaped devices are illustrated in Figures 1-7, in top perspective views (Figs. lA-7A) and cross-sectional views (Figs.. lB-7B and lC-7C).
Figs. lA and lB show a disk-shaped microstructure 20 composed of a thin disk with did l.~:Lel D between about 50 nm to about 3 microns and a thil~knPcc T between about 10 nm and about 1 microns. The disk is formed of a single material 22 which may contain the therapeutic agent (e.g., a drug in a polymer matrix). Although non-erodable materials may be used, particularly for ex vivo applications, the plert~ d devices are formed of 20 bioerodable materials, as described below.
The device may further be coated partially or completely with a coating 24 of hydrophilic polymer chains, such as chains 26, to form a coated microdevice 27. Typically these chains are added after formation of the microstructures groups. Preferred hydrophilic chains are polyethylene glycol (PEG) chains and synthetic glycocalyx, which are int~Pn~lPd to 25 either enhance the solubility of the device or, in the case of PEG, to extend blood circulation of the device. Methods of derivatizing a variety of metal surface with polymer chains are well known. For example, chains conldillillg thiol end groups at one chain end can be reacted with a metal surface under conditions effective to form thioethe} linkages.
Similarly, where the disk material is a polymer, a variety of surface ~tt~rhmPnt rhPrni.ctrie 30 e.g., involving carboxyl groups in the disk material and amine or hydroxyl groups in the polymer chains, are available for covalent linkage to the disk material. Other approaches to ~tt~rhing l-ydlol)hilic polymer chains to microstructures are described below.
Microstructures of the present invention may be formed of two or more layers of dirre~e,lL materials. For example, Figs. 2A and 2B show a microstructure 28 formed of two CA 02222~9~ 1997-11-27 layers 30, 32. As indicated in Fig 2C, the structure may have dirrclcll~ biological coatings on the two dirr~ L-material surfaces, forming a coated microdevice. For example, the upper surface of the device may be coated with molecules, such as molecules 34, of a thc.dpcu~ic agent, and the lower surface, with a molecules, such as molecules 36, of an 5 antibody, forming a microdevice 37. The antibodies illustrated are intt n~'ed for targeting the microdevices to selected target sites, e.g., selected blood, tissue or organ cells.
Methods for ~tt~rhin~ antibody or other polymer binding agents to an inorganic or polymeric support are d~t~ile(l, for example, in Taylor, R., ed., Protein Immobilization F..l,d~."~ and Ap~lications (1991), at 109-110.
Figs. 3A and 3B show a disk-shaped tril~min~te microstructure 38 having outer layers 40, 42, and an center layer 44. In the embodiment shown, the outer layers are composed of one material, while the center layer consists of a dirrclclll material, for example, a material that gives faster bioerosion than the outer layers. Other embodi nents .
may contain ~ ition~l layers. As indicated in Fig. 3C, the microstructure may be coating 15 with a film 48, such as a corrosion delay film, forming a microdevice 48. The corrosion delay layer is typically made of a material that gradually dissolves in the biochP~ni en~dlolllllcllL of the target. Examples of such coatings include tit~ninm~ gold, silver, platinum, copper, and alloys and oxides thereof.
The thic~kn~c~ of the corrosion delay layer may be selected to, for example, provide the 20 desired lifetime of the device in the bloodstream, or to allow the device to bind to its target before therapeutic agent is released. These layers may be applied by standard metal deposition procedures.
It will be d~.lc-;iaLed that the surface coatings and films in the microdevices can have any of a number of dirrclcllL function. For example they can serve as structural 25 elements of the device itself, e.g., an anticorrosive coating; as the therapeutic moiety, e.g., in the case of antibodies directed against a foreign antigen, as the targeting moiety (e.g., tumor-specific antibodies); as an element which confers other selected properties on the microdevice, e.g., glycocalyx coating; or a thc.dl~culic agent ~l~sign-od to erode over time.
It will be recognized that various p~,.llluLaLions of l~min~te structures and coatings, such as 30 those illustrated in Figs. 1-3, are collLelll~lated.
Figs. 4A and 4B show a microstructure 50 constructed according to another general embodiment of the invention. This structure includes a generally cup-shaped body 52 having a cavity 54. Referring to Fig. 4C, the cavity can be filled with one or more materials, such as material 56, which forrn the "core" of the microdevice, and may contain, CA 02222~9~ 1997-11-27 W O 96/41236 PCT/U'.,G~614 for example, the therapeutic agent, forming a microdevice 58. The device may also contain coating(s) dirr~;lcll- from coating(s) on other parts of the device, such as the coating of antibodies, such as antibodies 60, for targeting the device to a selected body site.
Figs. 5A and 5B show a similar type of cup-shaped microstructure 62. but here 5 co~ osed of a ring-shaped wall member 64 formed of one material and a planar bottom member 66, forming an internal cavity 68. This construction may facilitate, for example, the ~tt~rhmPnt of dirr~ moieties to the top and bottom portions of the device, as intlir~ted by ~tt~-~hmrnt of target-specific antibodies, such as at 68, to form a microdevice 70 shown in Fig. 5C. The cavity in the microdevice is filled with a selected material 70, as 10 above.
A similar type of cup-shaped miclo~ u~e is shown at 74 in Figs. 6A and 6B. In this mi~lo~ ;lulG, the bottom portion is formed of a l~min~te 76 composed of twodirr~,~.ll-material layers 78,80. For example, layer 80 may be formed of a relative low-density erodable polymer, to impart an overall density to the particle close to that of an aqueous ~ e--~ion m~ m, to achieve a more stable ~.u~ .ion of microparticles fortherapeutic applications. The other layer may be, for example, an easily erodable material and/or contain an ~ ed therapeutic colll~uulld, and/or be formed of a ferr~-m~gn--tir material.
As shown in Fig. 6C, the internal cavity of the structure, intlir~trd at 78, may be 20 constructed to contain a wall coating of biological molecules, such as antibodies 81, to form a microdevice 82. According to an hll~olldlll feature of the invention, and as well be ~lr~rrj'~ ed below, the antibodies are effectively ~hirl~lrd sequestered from a body's immune-le~ onse system, thus F ~ g the antibodies to carry out desired binding functions, e.g., removal of toxins, viral particles, cholesterol-colllah~ g particles, without the antibodies 25 themselves provoking an immune le..~ol~.e in the host OL~;dlli lll.
Figs. 7A and 7B illustrate a ring-shaped microstructure 84 having an internal core 86 defined by an annular ~LIll~;Luldl member 88. The core can contain, for example, a coating of a thtla~ t;ulic agent or mixture of agents, as in-lir~trd at 90, forming a microdevice 92. An exemplary coating is shown in enlarged view in Fig. 8, and includes a 30 colllbhldlion of antibodies, such as antibody molecules 94, and enzymes, such as enzyme molecules 96. In a particular embodiment, the antibodies are directed against a lipoprotein particle, e.g., Lp(a), and the enzyme is an esterase or protease enzyme capable of at least partially degrading the particles, once bound within the core of the microdevice.

CA 02222~9~ 1997-11-27 wo 96/41236 PCT/US96/09614 The optimal dimensions of microdevices of the present invention similarly dependon the application. The ,.,~xi~.llll dimension of the devices (the diameter of the disk in the case of disk-shaped devices) is typically in the range between 0.05 and 5 microns. For example, in applications where the device is ~sign~d to extravasate through pores lining S the walls of capillaries supplying a tumor, the m~ximllm ~lim~n~ion of the device is preferably less than about 150 nm, to allow passive movement of the device through the v~cnl~hlre. In applications where the devices are f~ n~d to circulate or to bind to a target without extravasation, they may be larger. It is recognized, however, that particles larger than 200400 nm may be rapidly cleared from the bloodsLIcdlll and in addition, 10 cannot be sterilized by filter sterilization.
The Illil~;llll~lll dimensions of the microdevices are constrained only by the microfabrication process itself. Certain layers and coating which may be contained in a device such as described above (e.g., a layer of targeting antibodies) can be as thin as a single layer of molecules. The ,ni"i",..~" size again depends on the application. For 15 example, in the case of devices made from biodegradable materials, the smaller the device, the faster it will dissolve. The stability of device of the present invention in a particular application may be readily ~ ?cl by one of skill in the art using tagged (e.g., fluorescent or radiolabelled) devices in a model system.
Another important property of microstructures and microdevices is the 20 bioerodability of the material employed in making the microstructure. Some metals, such as iron, are rapidly dissolved in serum, whereas others, such as gold, are much more slowly eroded. Therefore, to achieve a desired rate of erosion, metals may be mixed in alloy.
A variety of bioerodable polymers, inrl~ ing polyglycolic, polylactic, polyu~ dlle, 25 celluloses, and derivatized celluloses may be sPlecteA~ and a variety of charged polymers, such as heparin-like polysulfated or polycarboxylated polymers are suitable in forming one or more of the mi~au~llu~;Lule layers. The latter polymers have the advantage of being swellable and shrinkable under selected ionic conditions, and can be loaded to high drug conr~ntr~tion by ion-exchange effects, for drug release under physiological conditions.
30 Such polymers are described in PCT application WO/US94/01924 for "Condensed-Phase Microparticle Composition and Method" which is hlcoll~oldled by herein by reference.
Any of the above-described microstructure materials may be selected to enhance the detectability of the microdevices, e.g., as tli~gnt~r,tic microdevices or for purposes of tracking the biodi~ il)uLion of therapeutic microdevices. For example, the microdevice may CA 02222~9~ 1997-ll-27 WO 96/41236 PCTtUS96/09614 be fabricated with X-ray or MRI-resolvable material. X-ray-resolvable materials include iron, silicon, gold and gadolinium. MRI-resolvable materials include gadolinium and iron.
Further, the microdevices can be tagged so as to allow detection or vi~ li7~tion.
For example, microdevices are rendered radioactive by implantation or surface atr~rhmt~nt of radioactive isotopes such as I-123, I-125, I-131, In-111 and Tc-99m. Radioactive devices cullc~ làl~d at a site of disease can be i~ ntifiPd by a radiation detectors such as the ~y-ray cameras ~;u~ llly used in scintigraphy (bone scans), resulting in j-lPntifir~ti~n and localization of such regions. Microdevices can also be tagged with fluol~,sc~llL molecules or dyes, such that a conc~ alion of microdevices can be detected visually.
The ~LIucLuldl material used in forrning the micl.)sLI~l~;Lule is selected to achieve desired erodability and drug release properties. In the case of drug release, the structural material may be one or more biodegradable polymers. Classes of biodegradable polymers include polyorthoesters, polyanhydrides, polyamides, polyalkylcyanoacrylates, polyphosl)}laLclles, and polyesters. Exemplary biodegr~ ble polymers are described, for 15 example, in U.S. Patent Nos. 4,933,185, 4,888,176, and 5,010,167. Specific examples of such biodegradable polymer materials include, for example, poly(lactic acid), polyglycolic acid, polycaprolactone, polyhydroxybutyrate, poly(N-palmitoyl-trans~-hydroxy-L-proline ester) and poly(DTH callJollal~).
As in~ tPd above, the polymer matrix may also contain a hydrogel, such as a 20 cross-linked polymer of hydroxyethyl methacrylate and ethylene (~im~-th~rylate or a cellulose ether-type hydrogel (Doelker, in Hydrogels in Medicine and Pharmacy, Vol. Il, Peppas, N., Ed., CRC Press, Boca Rotan, 1987, pp. 115-154). Hydrogels are typically water-swellable matrices cullL~illillg dispersed or dissolved drug. Hydrogels are preferably used in combination with water-insoluble drugs, such as steroids (Zentner, G. M., et al., J.
Pharm. Sci, 68, 970 (1979)), and high molecular weight drugs such as insulin (Davis, B.
K., Experientia 28, 348 (1972)), enzymes (Torchilin, V.P., et al., J. Biomed. Mater. Res.
11, 223 (1976)), and vaccine antigens (Bernfield, P., et al., Science 142, 678 (1963)).
The solubility of an active agent in a particular polymer can be P,~ ed by the use of solubility parameters (Hildebrandt, J. H., et al., THE SOLUBILITIES OF
NoNELEcrRoLrrEs; Reinhold: New York (1950)), where close solubility paldlllcLel values O
between an active agent and polymer tend to favor compatibility and solubility. The active agent will typically be di~ ed or dissolved in the polymer matrix. Alternatively, the active agent may be covalently att~h~d to the polymer backbone through reactive pendant chains that can be cleaved, typically by hydrolysis. In utilizing this approach, the device CA 02222~9~ l997-ll-27 wo 96/41236 PCT/US96/09614 will also contain a means for providing controlled release of active agent from the polymer backbone, such as by cleavage of the covalent ;.~ h",~"~ The diffusion or release properties of the polymer matrix can be modified by various terhniq--Ps, such as cross-linking, ch~mir~l structure motlific~fion, blending of two or more polymers, or by the 5 addition of pl~tiri7Prs (e.g., butylb~l~yl~ te, trioctylphosphate, dio~;lyl~,~.ll.AI~te7 glycerol, polyethylene glycol, and polypropylene glycol) or solvents. The matrix may also contain additional colll~o~ , such as pl~,selvàliv-es or bacteriostatic agents, e.g., methyl hydro~yl,~l~.,aLe, chlorocresol, benzalkonium chlorides, and the like.
Typically the therapeutic agent is attached to or incorporated ir~to the polymer layer 10 after microfabrication, to prevent exposure of the agent to the rh.omic~l etching procedures .li~;..~5ed in the section below.

B. Microfabrication of the Microstructures The structural portion or layer (i.e., microstructure) of the microdevices of the 15 present invention may be microfabricated using any suitable Illi~ rablication method, such as the photolithography and photoablation methods detailed below.
Figs 9A-9E illustrate the steps in forming a disk-shaped microstructure 100 (Fig.
9E) by photolithographic t~rhniq~les As shown, the structure includes two layers 102, 104, which will be formed of two i-l~ntir~lly llu~-lbel~d layers (102 and 104) forming a planar 20 expanse 106. This l~min~te expanse is formed according to conventional methods for deposition of metal layers, e.g., rhPmir~l vapor deposition, ~ull.,lhlg or the like, and/or mrth()tlc for producing thin polymer sheet material.
As a first step in the process, the l~min~te expanse is attached or otherwise bonded to a sacrificial layer 108, such as pho~l,holou~ doped silicon dioxide, deposited by rhrmir~l 25 vapor deposition, and the top of the l~min~te is coated with a photoresist layer 110.
Suitable negaliv~- or positive-resist material are well known, e.g., "Introduction to Microlilho~-a~lly", Thompson, L.F., et al., eds, ACS Symposium Series, Washington D.C
(1983). Additional details on microfabrication methods useful in the .. i.. ri.. l.. e of devices according to the present invention are described in, e.g., co-owned PCT patent publications 30 WO 95/24261, WO 95/24472 and WO 95/24736.
The coated l~min~te is irradiated through a photomask 112 having a series of circular openings, such as opening 116, corresponding in size to the desired size of the microstructures, e.g., 50-200 mn tli~m~t.or. Methods for forming photomasks having desired phol~ k patterns are well known.

CA 02222~9~ 1997-11-27 WO 96/41236 PCT/U~.~G/~0~14 In the embodiment shown in Figs. 9A-9D, the photoresist is a negative resist, m~ninE that exposure of the resist to a selected wavelength, e.g., UV, light produces a chPmic~l change (in~ t~d by cross h~trhing) that renders that altered resist resistant to etching by a suitable etchant. The dl~pe~u~lce of the coated l..,.i,..l~ after ph-~tt)m~c'~
5 irradiation UV and etching is shown in Fig. 9B. As seen, I~ i"~t~ 106 is now covered by a plurality of discrete disk-shaped resist ~ m~-nt~, such as elements 118, col.c~.~olldi.lg in size to the planar dimensions of the desired Illicl~ uctures.
The l~min~t~ is now treated with a second etchant material effective to dissolve the l~min~t~ in the exposed areas of the l~min~te In the case of a l~",i"~l.o metal layer, ~he 10 etchant may be a suitable acid solution; in the case of a l~min~t-- biodegradable polymer layer, the etchant could be an enzyme solution, an aqueous solution having a pH effective to break down the polymer, or an organic solvent known to dissolve the particular polymer. It will be al!~lccidlcd that in the case of l~."i".l~c, more than one etchant solution may be required. The l~min~t~, after complete etching, has the appearance of Fig. 9C, which lS shows a series of disk-like, resist-coated ~lPmPnt~ on the sacrificial layer.In the final plc~JdlaLion steps, the resist is removed by suitable rhPmi~l Llcd~ lL
(Fig. 9D), and then the sacrificial layer is removed, again by conventional ch~omi~
callllclll, leaving the individual microstructures, such as microstructure 100.
Figs. lOA-lOE illustrate further photolithographic processing effective to produce 20 cup-shaped Illi.,lo.l-uctures, such as shown at 122 in Fig. lOE. In this processing, the etched l~min~t~/sacrificial layer structure or ~ul~.lldlc shown in Fig. 9D is further coated with a positive resist material 124, as shown in Fig. lOA. The coated l~min~te is then irradiated through a photomask 126 having a series of circular openings, such as opening 128, whose ~ lllrlr~:~ co~c~olld to the desired "internal" ~ m.ot~rs of the microdevices.
25 The mask is aligned with the ~L~. Lldl~, as shown, so that the mask openings are in registry with the already formed discs in the snbstr~
Irradiation of the ~ub~ lldlc through the photomask causes photo-induced changes in the resist (in~ ttod by cross h~t~hing) that render the irradiated regions susceptible to a selected etchant. The d~ea~dllce of the coated l~min~te after photomask irradiation UV and 30 etching is shown in Fig. lOB. As seen, this l.caLlllclll has produced a cylill(llical opening, such as opening 130, in the center of each Illicl~Ll~lcLule 100 in the ~.ub~ dLe.
The l~min~t~- is now treated with a second etchant material effective to dissolve the upper l~min~t~ layer in the exposed areas of the l~min~te, producing the series of CA 02222~9~ l997-ll-27 microstructures 122 seen in Fig. 10C. Removal of the photoresist (Fig. 10D) and sacrificial layers produces the free microstructures 1~ shown in Fig. 10E.
It will be a~lccidlcd that the mi-;lu~Lluclulcs formed as just described may be further treated by standard photolithographic technirll-ec to produce other desired surface S features and or layers. Further, inflf-nt~rif~ns or cavities may be filled with a material dirr~ from the microstructure material by known methods. Thus, for example, U.S.Patent No. 5,200,051 describes photolithographic methods for depositing perm-selective and plvlei~.~reuus layers on a planar ~ub~tlate. These same technifluf-s can be applied to the present invention, to h.coll,olatc such layeAs in the microdevices of the invention.
In another general a~ oach, the microdevices are p,.~ llf-d from a s~lbstr~t~f- by excimer laser photoablation t~orhniq~lf-~ Methods of laser micrù,~ ;,,;,,g or dry etching have been described, e.g., U.S. Patent Nos. 5,368,430, 4,994,639, 5,018,164, 4,478,677, 5,236,551, and 5,313,043. This method is most suited to a polymeric ~ulJ~ldl~, because of the ease with which a laser beam can photoablate polymer structures.
Figs. 11A-llD illustrate the method as applied to producing a disk-shaped tril~min~tP microstructure such as shown at 134 in Fig. 1 lD. In this illustration, a tril~minzlte expanse 136, formed on layers 138, 140, 142, is formed on a sacrificial layer 144. The expanse is then irradiated with a focused excimer laser beam, such as in-lic~trd at 146, which is p~tternf-d (by u~Llcalll photu",~ki"~ and imaging) to give a beam that is 20 effective to photoablate the l~min~tP in the areas corresponding to spaces between desired-size circular flemrnt~ on the l~min~te, as in-lir~tf-d in Fig. 11B. This ablating is continued until the beam has cut through the entire l~min~tto depth, as shown in Fig. 1 lC, forrning the disc shaped mi-,lu~lluclulcs on the sacrificial layer. Removal of the sacrificial gives the plurality of structures 134.
C. Microstructure Surface Slluclulcs The term "molecular coating" is used herein to describe a coating which is bound to the surface (outer or interior) of a microstructure. The molecular coating may be bound directly to the surface of the device or bound to a lipid or a resin such as an electron 30 donating group, e.g. -NH2, OH or the like derivatized onto or associated with the surface of a structural layer of the device. The molecular coating may cncullllJass all or a portion of the surface area of the device. Molecular coatings that can confer various f h~ . islics or impart selected functional properties are described below.

CA 02222~9~ 1997-11-27 Figure 12 illustrates a general embodiment of a coated microdevice 150. A central layer 152 in the device is coated with a corrosion delay layer 154, such as gold or nickel, which is in turn coated with a layer of various molecules 156 that comprise the biorhf mi interface to the blood. The lower side of the device in the figure that will bind to the cell 5 or tissue target (e.g., a capillary wall) is coated with ligand molecules 158, such as IgG
antibodies, that are effective to specifically bind to the marker molecules on the target (e.g., tumor capillaries).
In one general embodiment, the molecular coating is a marker-binding agent, typically an antibody of antibody fragment, for targeting the microdevice to a specific cell 10 type, e.g., tumor cell. A number of monoclonal antibodies to tumor-specific antigens are known to the art. See, for example, pages 301-323 of Cancer, 3d Ed., edited by V. T De Vita, S. Hrllm~nn, and S.A. Rosemberg. Among the specific anti-tumor antibodies known are those that bind to pallcal~;h~u-lla antigens (PCAs), an example of which is the tumor-associated glycùplotein antigen known as TAG-72. Such antibodies thus bind to a broad 15 spectrum of cancers, and to difr~ cellular mutations in the mf~ct~tir cascade. Examples of antibodies of this type are B-72.3 and the CC49 antibodies. Microdevices coated with anti-PCA antibodies are particularly useful in the diagnosis and treatment of dineoplasms.
To f~rilit~tr tracking of a I~ dllt;uLic microdevice of the present invention, and/or 20 to enable the use of ~ gn~stic devices, one of the structural or coating elements of the microdevice may be decign~d to be detectable using, for example, X-radiation, scintigraphy, nuclear m~gn~tic. resonance, optical inspection (e.g., color, fluorescence), or ultrasound, as noted above.
A molecular coating may also be used to prevent or inhibit ,~ r-~l of serum 25 opsonins to the devices, to extend the blood circulation lifetime of the devices. As described below, such hydlu,ullilic polymers may be conjugated to vesicle-forming lipids, and the microdevices can be coated with a lipid film COIIIdillillg such vesicle-forming lipids.
The microdevices may also be coated with a natural or synthetic glycocalyx to enhance its solubility. Other hydrophilic natural polymers, such as glycogen or polyserine, 30 are also useful for this purpose, as are biocrmr~tible detergents such as non-ionic poly~ li,ed oxyethylene ethers, e.g., "TRITON-X 100".
In one embodiment of a microdevice 160, shown in Figure 13A, hydrophilic polymer chains, such as chains 162, are conjugated to vesicle-forming lipids, such as lipids 162 (see for example, U.S. Patents 5,013,556 and 5,213,804), which in turn are anchored CA 02222~9~ 1997-ll-27 in a lipid film 164 formed on the exterior surface of the microstructure. Methods for forming lipid films, such as lipid bilayer films, on particles are detailed for example, in above cited WO/US94/01924.
Examples of hydlo~ ilic molecules suitable for conjugating to vesicle-forming 5 lipids include polyethylene glycol chains having molecular weights between about 1,000 and 10,000 daltons. Other suitable hydrophilic molecules are polyvillylpy"olidone (PVP), poly~ Lhyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, poly",~ll,a~l.ylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethy!cellulose.Referring to Figure 13B, a portion of the hydrophilic polymer chains may additionally contain a marker-binding molecule 166 (i.e., a ligand) at their free end. Such marker-binding molecules may be conjugated to activated free ends of the hydrophilic molecules using known methods.
Figure 14 shows how a gradient of hl.;l~l.7illg hydrophobicity can be established by 15 de~..,ash.g the number of hydluphobic groups, such as groups 170, 172, and hl-,l.,a~ lg the number of hydrophilic groups, such as groups 174, per unit volume as a function of inclcasillg distance from the surface of a device, here in~lic~ted at 176. A synthetic glycocalyx can thus be tailored to provide the functional coat desired for a particular device.
A hydrophobic ~llvilol--llcillL can be ~ in-~d near the surface of the device to harbor 20 mobile hydrophobic groups, while the entire device is kept in suspension in an aqueous envi-u-~-llt;~-l by hydlol)hilic groups on long chains.

D. The,~,u~ulic Agents Microdevices of the present invention consist of microfabricated structural çl~mPn 25 (microstructures) in association with a therapeutic agent and any other functionality-e.lhdllch~g agent or coating. The therapeutic agent may be (i) incorporated into the microdevice during microfabrication (either as a structural ~.ub~.Ll~le or as a coating), or (ii) associated with the device after it is mi~;lu~ r;~rtl~red (e.g., by coating or conjugating it onto the device). As is described below, therapeutic agents such as drugs can be30 incorporated into matrices (e.g., polymer matrices) which may be deposited into wells or pits m~mlf~rtmed into the devices.
Thclaptulic agents associated with the microdevice after it is microfabricated include coatings of therapeutic biomolecules, such as Ihe-~ulic antibodies (e.g., antibodies directed against LDL or a viral antigen).

CA 02222~9~ 1997-ll-27 The activity of the therapeutic agent is ~ cssed by exposure of the microdevice to the biorhpm~ enviiulllllGIlL of the target site. The target site can be a particular location in the body, or the site of ~ .ation. For example, in the case where the target is a set of molecules circulating in the bloodstream, the bio~hPmir~l ell~ilulllllell~ of the target site 5 is the blood. In cases where the target is a non-ch.;uldlil,g tissue, such as a tumor, the bioçhP~nir~l envilulllll.,.lL of the target site is the envhulllllt;llL in proximity to the tumor.
The lh~ldl)tuLic agent cont~inPd in the th~.dp~ulic devices of the present invention may be a releasable agent or an immobilized agent. A releasable agent is a therapeutic col~lpuulld, such as a drug, that is designPd to be released at a selected target in order to 10 exert its therapeutic efficacy. An immobilized therapeutic agent is one that pelroll~ls its therapeutic function while immobilized on the microdevice. For example, microdevices cont~ining therapeutic antibodies as described above are an example of an immobilized the.d~)~ulic agent.
Microdevices of the present invention may be fashioned to deliver a selected 15 therapeutic agent or ligand to a selected target in a form where the Llleld~ulic agent is shielded from recognition by the subject's immune system. In one such embodiment, the therapeutic agent (e.g., an antibody directed against LDL or against a viral antigen) is attached to the bottom portion of a cup-shaped microdevice, as exemplified in Figure 15.
The microdevice shown, inflir~tPd at 180, has a tri-l~min~te structure consi-,~illg of two 20 bottom layers 182, 184, and an upper layer 186, which defines an internal cavity 188. The exposed surface of layer 184, which forms the inside bottom of the cup-shaped microdevice, has a surface layer of covalently bound antibodies, such as antibodies 190.
The material forming the device is selected for surface specific ~tt~hmPnt of antibodies to the upper surface of layer 184. The exposed surfaces of layer 186 can be optionally coated 25 with hydlu,ullilic molecules, as inflic~te(~
The d;~ . D of such a device is small enough (e.g., less than about 150 nm) and the thirknpcs of layer 102 large enough (e.g., greater than about 30 nm) so that the cell m~,lll,~dlles of an immune-response cell, such as in~ ted at 192, that may contact the device while it is in circulation cannot contact the layer of antibodies on the exposed surface 30 of layer 184. By preventing such contact, the host immune response directed against therapeutic antibodies 128, while they are associated with the microdevice, will be decrease or eli...i. ~ed .
CA 02222~9~ 1997-11-27 E. Microdevice Suspension The invention includes a suspension of microdevices of the tyye described above for use in ~lmini~rering a therapeutic agent to a selected target site in a subject. To form the sllcllPn~ion7 microdevices as described above are sllcrenrlPd in an aqueous medium at a 5 selected c<J,lcc~ alion. The optimal col-r~ rion will depend on the ~;hdldc~ lics (e.g., solubilization properties) of the microdevice, type of therapeutic application and mode of a.l",i"i~/,alion. For example, c~ posilions for oral ~h~ dlion can be relativelyviscous, and may Illelcrole contain a high Collcclllldlion (e.g., >50%~ of the microdevices.
Solutions for bolus injections ~crc.~bly contain a relatively c.l~-r~ od suspension of 10 microdevices (e.g., 10-50%), but not so collcel.l~dled that it has an ~yp~l,ciably higher viscosity than saline (to ",i"i",i~.~ need for large-bore needles). Solution used for continuous intravenous infusion typically contain a relatively low cunrr'.l.dlion (e.g., 2-10%
su~ycl~ion) of microdevices, due to the relatively large volumes of fluid that are ~llllilli~l~ lcd.
~ 15 The microdevices can be su~pPn~lPd in any suitable aqueous carrier vehicle. A
suitable ph~"~rc~.lir~l carrier is one that is non-toxic to the recipient at the dosages and Collc~illllalions employed and is compatible with other ingredients in the formulation.
Examples of suitable carrier vehicles include but are not limited to water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Suspensions for use in injectable formulations are preferably isotonic with the subject's blood. Generally, the carrier can contain minor amounts of additives such as suhst~nrPc that enhance isol~llicily and rh~mic~l stability, e.g., buffers and preservatives, as well as low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohy-lldles including glucose or dPl~tr~n~, chPI~ting agents such as EDTA, or other excipients.
Prior to ~ dlion to a subject, the suspension of microdevices is sterilized by asuitable sterilization method. Heat-stable microdevices can be heat-sterilized, e.g., using an autoclave. Alternatively, microdevices that are not heat-stable may be sterilized by passage through a cull,lllcl-;ially-available sterilization filter, e.g., a 0.2 ~m filter. Of course, filtration may be used only in cases where the microdevice is smaller than the pores of the sterilizing filter.

III. Applications Microdevices of the present invention can be ~lmini~Pred to a subject in need oftherapeutic intervention via any suitable ~lmini~tration method. The particular method CA 02222~9~ l997-ll-27 employed for a specific application is cl~ ".i.,rd by the att~n-ling physician. Typically, the microdevices will be ~.I.,.i"i.~l~ .ed by one of the following routes: topical, palellL~;lal, inhalation, oral, vaginal and anal.
As ~ cu~ed above, microdevices of the present invention are particularly useful in S Lll,a~ ,.ll of m~lign~nt tumors. In these applications, the type of tumor inflllPn~es the mode of ~ dlion. For example, skin cancer may be treated by topical application of a preferably viscous suspension; lung cancer may be treated by inh~l~ti~m of an aerosolized aqueous microdevice ~"~ ion; cervical cancer may be treated by vaginal ..-l",i,-i~l,d~ion of a microdevice ~u~l~ension; and colon cancer may be treated by rectal a.l"~ l,.lion of 10 such a suspension.
The majority of thc.dl~eulic applications involve some type of pal~llLt;ldl .dLion, which includes intravenous (i.v.), hlll,..l"lc~ r (i.m.) and sub-c~t~nf ouc (s.c.) injection. Microdevices suitable for parenteral a.l"~ l,dlion preferably have a selected m~ximllm dimension in the range between 0.1 and 3 microns. Specific examples 15 of devices useful for pal~ le..dl ~ dlion~ particularly intravascular ~.l",i";:~il,alion, are described below.
Further, the ~-l",i"i,.l~alion can be systemic or local. The non-pdl~,.ll.,.dl examples of ~.llllilli.~l,dlion recited above, as well as i.m. and s.c. injections, are examples of local ~.l",i"i~ lion. Intravascular ~imini~tration can be either local or systemic. Local 20 illllavds~;ular delivery can be used to bring a therapeutic ~ e to the vicinity of a known lesion by use of guided catheter system, such as a CAT-scan guided catheter.
General injection, such as a bolus i.v. injection or contimlon~/trickle-feed i.v. infusion are typically systemic.
In a pl~rtlled embodiment, the microdevices of the present invention are injected 25 into the blood stream and allowed to circulate and localize to their target. Exemplary targets include ~ ,.ll types of freely-circulating molecules, cells and/or tissues accessible via the circulatory system. The effectiveness of the microdevices in these applications is potentially affected by several factors, in~ln-ling the subject's immune response directed against the microdevices, and the solubility of the devices in circulation.
A. Targetin~ Circulatin~ Molecules and Viruses In one general application of the present invention, the microdevices described herein are dc;livaLi~ed to contain an antibody or ligand e~fective to bind to a circulating blood molecule, and are used Lll~ ;r~lly to reduce the co~ lion of the circulating CA 02222~9~ 1997-11-27 blood molecule. The microdevice used for such an application p~ere:lably contains the antibody or ligand in a manner that protects the antibody or ligand from attack by the subject's immune system. An example of such a configuration is shown in Figure 15.
By way of example, such microdevices may be derivatized to contain antibodies 5 directed against very low density li~u~Loleills (VLDLs) and/or low density lip~lo~l;hls (LDLs), elevated levels of which are associated with increased risk of coronaly heart disease in hurnans. The therapeutic agents in such microdevices are the anti-LDL and anti-VLDL antibodies. The activity of the thc~a~,c.lLic agents (i.e., antibodies) is expressed by e~û~ul~ of the microdevice to the bioch~mic~l environment of the target site, i.e., upon 10 injection into the bloodstream. The expression of thelap~uLic activity is lllallir~ d in this example by the antibodies' binding of VLDLs and LDLs in the bloodstream. Such antibody-containing microdevices are pl~r~lably microfabricated using structuralcompositions that biodegrade after a selected period of time, e.g., several hours to several days. Once the structural portions of the microdevices have degraded, the LDL-antibody 15 and/or VLDL-antibody complexes are released and degraded by lllaclvl,hages.

B. Tar~etin~ Cells and/or Tissues Microdevices of the present invention may be used to target and deliver thela~t;uLic compound(s) to selected cellular or tissue targets. The cells may be circulating cells, such 20 as monoml~ t~d blood cells (MBCs), bacterial cells in a systemic infection, etc., or non-circulating cells, such as cells Cv~ g a fixed tumor mass, or the epithelial cells forming the lining of vessel or capillaries. Any cell-specific molecular marker may be detected by the m.othn-ls of the invention so long as the device is fabricated with a marker-specific ligand or antibody. ln a pl~,r~llc;d embodiment, the molecular markers to be i-lPnti~led are 25 tumor-specific antigens. In selecting specific antibodies for in vivo applications, an efficient strategy is to use human antibodies specific for the COIlllllOll generic molecules that are relatively more plentiful in target diseased tissue cell lllelllblalles than in healthy cell S.
An exemplary microdevice 194 useful for targeting a tissue in need of exposure to a 30 therapeutic agent is shown in Figs. 16A and 16B. The device has a tril~min~t~ structure cvl1si~ g of a Lhela~ulic-agent-~;-lltaillillg layer 196 sandwiched between two targeting/support layers 198, 200. The exposed surfaces of the support layers can be coated with, e.g., (i) hydrophilic molecules, such as molecules 202, to improve the su~pPn~ion or CA 02222~9~ 1997-ll-27 WO 96/41236 PCT/U'.~C~ 14 circulation characteristics of the device, and (ii) binding molecules, such as antibody molecules 204, directed against a marker on the target cell or tissue.
In one embodiment, the therapeutic-c~ aillillg layer erodes at a faster rate than the outer, targeting/support layers. This feature, illustrated by the partially-eroded middle layer S in Figure 16B, allows the microdevice to remain securely ~ttArhrd to the target even as the therapeutic layer dissolves away. A related embodiment has a bilaminar structure coll7i,lillg of one therapeutic layer and one targeting/support layer as described above, where the therapeutic layer dissolves at a faster rate than the targeting/support layer.
When targeting n; ncirculating cells inside capillaries (e.g., the f-mloth.oliAl lining), 10 the binding between the device and the molecular marker should be snfficiently strong to overcome the drag force exerted by the flowing blood. This objective can be ~Aticfi~d by having a relatively large, relatively planar surface area for specific binding and a relatively low profile in the capillary's blood-flow space.
Referring to Figure 17A, a disk-shaped microdevice 202 iS attArh~-d to capillary15 wall 204 via interactions between marker molecules 206 on the capillary wall and ligand molecules 208 on the device's surface. Epithelial cell markers which are specific for certain pathologies, e.g., tumors, have been itl.ontifird The total shear strength, IG~les~,l.led by arrow 210 in the figure, is due to the total binding interaction at the interface between the device and the capillary wall. As seen, this 20 shear force is greater than the sum of the blood flow forces, represented by arrow 212, acting to move the device in a dowlL~7ll~,dlll direction (toward the right in the figure). This favorable state is due to the high ratio of total area of device contact with the capillary wall to the device's profile in the region of blood flow.
By C~JIl.~d.;soll, Figure 17B shows a cross section of a spherical microparticle 214 25 bound by ligand-specific interactions on a capillary wall 216. Here the ratio of area of device contact with the capillary wall to the device's profile in the region of blood flow is quite low, as can be d~le-,ialGd. Accoldill~;ly the shear force holding the particle to the capillary wall, in-1ir~t~d by arrow 218, iS smaller than the flow force on the particle, in~licAtPd by arrow 220, giving an unstable binding condition.
The same factors, particularly the high surface area of contact, produces ~.~hAnred binding between microdevices of the invention having ~ ,1A~ 11Y planar surfaces, and cell surfaces.

CA 02222~9~ 1997-ll-27 C. Tr~n~lumin:~l Tar~etin~
The invention further provides a therapeutic method in which a microdevice crosses the vascular ~ lllI,ldne to a preselected site for delivery of a Ihel~p~llLic agent. In one embodiment, the microdevice is coated with a ligand to the endothelial or b~
5 I~ llblane of a neoplastic cell. Examples of ligands l~ ,.hlg the b~çmrnt Ill~l..I,.d,-e are an anti-collagen type I, IV, V, anti-rlbloneclill, anti-proteoglycans and anti-laminin antibodies. In a pl~r~ll~ e.llbodi~ , the ligand is anti-collagen IV antibody. The device is designed so that, upon binding to the target cell, it migr~teC into the underlying tissue and delivers the Ih~ld~c;uLic agent.
For the illustration of these uses, l~fe.ellce is made to a microdevice 224 in Figure 18A, composed of a microstructure 226 and a core 228. A cross-sectional view of the device is given in Figure 18B. The device may be protected with an anticorrosion layer, such as layer 230, which may be selected to have dirr~.e~l- thi~ l.Rcsçs in its .l.i.;.~L-~Icture and core surface areas. Also as shown, the outer surface may be coated with hydrophilic 15 polymers, such as described above, and antibody molecules, such as molecules 232, for capillary-wall targeting.
Another embodiment of the microdevice, in~ir~ted generally at 234, is shown in Figure 18C, with an anti-corrosion coating 236 present only on the inferior surface, hydrophilic polymer chains only on the lateral surface, and targeting antibodies, such as at 20 238, only on upper portions of the device, as shown.
Figs. l9A and l9B illustrate the method by which the microdevices are able to cross a vascular-wall Ill~.llbldllC or wall, such as in-lir~trd at 245. The illn~tr~tion is with respect to microdevice 234 shown in Fig. 18A, and co~t~h h~g anti-collagen IV antibodies on its upper surface in that figure. After i.v. a~ll.,illi~l,~Lion, and migration to the target capillary 25 wall surface, the anti-collagen IV antibodies adhere to the subendothelial b~r~
-b~ c (Fig l9A). The en~loth~ l cells tend to retract when contacted from their luminal surface, and s~~bseq~~~ntly to migrate over bodies co..~;li..g the contin---~us b~rmt?nt lllt;lllblane~ so as to separate such bodies from the circulation. These processes underlie well-known biological actions such as the repair of cuts in the vessels walls, and 30 the extravasation of tumor cells from both venules and arterioles to initiate ,.,~ ic c~r~-lrc. The time required for the reaching of a configuration such as shown in Figure l9A is about 24 hours. The microdevice may be coated with an anti-corrosion layer ~lesignrd to allow for such a time interval, with an a~p.op.iate safety factor. The CA 02222~9~ l997-ll-27 microdevice may be complexed on its passive (open) side by fibrin 240, platelets 242, and immune cells 244, as shown.
Different functions may be assigned to the device's core, in~lir~tlqd at 246. The first of these is Iysis or dissolution of the b~clom~nt membrane. For this purpose, the core may 5 consist exclusively or partially of ~h~ lre5 or biological entities that degrade such Ill~,llI)ldnes. Examples are matrix-degrading enzymes among serine prulcinases, neutral metallu~ ehlascs, and cysteine protch~ases. In a plerellcd embodiment, microdevice core contains type IV collagenase, a metall~)~loleillase that is specific to collagen type IV, and thus particularly efficient for the Iysis of the b~c~mPrt ~llclllblane After binding of the microdevice to the walls of the target v~cc~ tllre, the membrane-degrading agent within the core is released, and gradually dissolves the bacr",~
.,lbldlle. The device thus pcncLldles the cell stroma, while mcl~ ldlle lccon~ ction takes place by natural processes. A configuration such as shown in Figure l9B is reached, with the microdevice stably lodged within the intcl~Liliulll. The extravasation process 15 requires about 2-12 hours.
At any time following binding, Illcllll~ldne Iysis, and/or tr~nchlmin~l migration, other functions may be performed, in~hl~ling the delivery from the microdevice of thl,lapculic agents d~plu~lidle to the pathology being treated. The diffusion of such therapeutic agents to the targeted tissue in indicated by arrows 248 in Figure l9B. For the 20 treatment of neopla,cms, preferred thc~dp~.llic agents include anti-cancer drugs, including ~"li",~ l~holites, alkylating agents, plant alkaloids, and ~ntitllm~r antibiotics; biological agents, inrlll~ling monoclonal antibodies, hll~,~rclons, and interleukins; çh~""~s~
i.e., rh~-mic~l.c that decrease the ~ re of cells to drugs, such as misonidazol and its analogs; and compounds which enhance the scl~ilivily of tumor cells to radiation, e.g., 25 halogenated pyrimi~linrc~ and compounds that radios~ hypoxic cells, e.g., nilloillladizole compounds.

D. Tumor Tar~eting In one embodiment, microdevices are clesignPd for the in vivo lledllllcll~ or detection 30 of tumors by extravasation. This targeting occurs by circulation of the microparticles through the bloodstream, over an extended time period, and passive extravasation of the devices through colllpl~lllised regions of the vaccnl~tl-re, which tend to correspond to vascular regions servicing a solid tumor The microdevices therefore have a preferred maximum dimension of less than about 150-200 nm, to allow for passive transit through the CA 02222~9~ 1997-11-27 W O 96/41236 PCT/U5~CI'0~614 cu~ ised vacc.ll~ture, and a hydrophilic surface coating to ensure prolonged blood circulation lifetime.

E. Tar~etin,e bY External Ma~netic Field In another embodiment of the invention, the microdevices are fabricated to include a strong m~gnPti~ subdomain. An example is illustrated in Fig. 20 showing a microdevice 250 having a core which in this case contains two dirr~,~e..~ core layers, such as layers 252, 254, and a m~gnPtic layer 256 by which the particles can be guided in the body by an external rnagnetic field.
Magnetic removal of particles from biological fluids for in vitro applications is taught in U.S. Patent Nos. 4,018,886 and 3,970,518.
When a~~ .iale, the devices may be limited to circulate within localized diseased areas by application of localized m~gnPtic fields. For example, a device containing a m~gnPti~ core as well as d~.iale therapeutic agent(s) is a.l."i,.i~l~red locally or 15 ~y~Lt:ically, and a local magnetic field applied to the ~iicç~ced region. This is a useful procedure for the Llt.d~ of some lesions for cases in which surgical excision is not desirable, or possible. Such cases include melanoma with multiple superficial mPt~ct~Cçs and penile cancer. The concentration of radioactive tags with magnetic particles in vitro is taught in U.S. Patent No.3,993,997.
For rhpnnothprapeutic therapies, localized m~gnPtic fields can be used as described above for collcc;llLIdlillg radiotherapeutic microdevices in known tlicç~.ced sites. This is a useful procedure for the Ll~aLIll~.ll of superficial lesions for cases in which surgical excision is not desirable, or possible.
.
F. Tar~eetin~ IntraoperativelY
The device of the invention may be used for intra-operative diagnosis and intra-operative Ll~dL---enL of mPt~ct~ti- d--m~inc. For this application, the device carries antibodies to target antigens expressed on the surface of neoplastic tissue. Examples of such antibodies include anti-collagen IV antibodies, for the cases of breast and colon cancers, as well as Iymph node mPt~ct~cPc of the former. The device core can contain any desired combination of therapeutic agents, such as ~nfimpt~bolites~ alkylating agents, plant alkaloids, antihlmor antibiotics, monoclonal antibodies, h~le~r~lolls, interleukins, chemo-s~ , and the like. In the course of surgery directed to the removal of an ~ Pntifipd large lesion, a suspension of microdevices in physiological fluid is applied to a region CA 02222~9~ 1997-11-27 W O 96/41236 PCT~US96/09614 adjacent or related to the main lesion, if such area is ~u.~e~;~cd of ,.,P~ ;c infiltration.
The microdevices that do not bind to the tumor antigens in the area of exposure are removed, e.g., by washing, suction, or m~gn~tir~lly, as described above. The bound devices identify the exi~tenre of Illr~ lic domains, which the surgeon may elect to 5 remove. However, even if these are not removed, therapeutic action against them is provided by the LhcldpcuLiC agents loaded within the microdevices. The limits or rlimin~tes the necessity for follow-up or adjuvant chemotherapeutic or radio~llcldl.cu~ic ~lcd~ enl of mrt~ct~tir colonies.

It will be dlJplecidled from the foregoing that the present invention provides anumber of advantages in therapeutic delivery- composition over prior art particle compositlons.
The microdevice particles employed can be design~od with selected shapes, preferably having at least one planar surface, for enh~nred ligand-specific binding to target cells. Further the particle sizes and shapes are effectively uniform, and can be made as small as existing types of "nanoparticles".
The devices can be ~ signPd with a number of discrete functio~ls, include surface binding, ~u~ nsion and RES-evasion functions, and one or more dirr~c~l therapeutic agents contained in separate Cu~ alllllcllL for dirr~.c.lL release rates.
The particles may be constructed of a variety of dirr~cllt materials, to achieveoptimal and dirrclelllial drug release at a target site. One of these materials may be a radiopaque material, such as a metal for vi~ li7ing the biodistribution of the particles, or a ferrom~gn-otic material to allow m~gn-otir field particle guidance.
In one embodiment, the microdevice has a core in which foreign lhcld~cuLic agents, such as antibodies and/or cl~yllles can be shielded from the host's immune system, allowing a variety of novel applications for clearing undesired serum components.
In still another embodiment, the microparticles are designed for either passive e~lld~asdlion into solid tumors, or active tr~n~lllmin~l movement through a culllbi-ldlion of capillary-specific binding and partial Illclllb~dlle degradation.
Although the invention has been described with respect to specific embodiments and applications, it will be appreciated that a variety of changes and modifications may be made without departing from the invention as rl~imr(l

Claims (24)

IT IS CLAIMED:

1. A suspension of microdevices for use in administering a therapeutic agent to a selected target site in a subject, comprising a suspension of microdevices in an aqueous medium, said microdevices (i) having a selected non-spherical shape and uniform dimensions and (ii) containing said therapeutic agent in a form where the activity of the therapeutic agent is expressed by exposure of the microdevice to the biochemical environment of the target site after administration to the subject.

2. The suspension of claim 1, wherein said microdevices have surface-bound, marker-binding molecules effective to bind to a marker carried on the surface of cells at such target site.

3. The suspension of claim 1 or 2, for use in targeting selected cells or tissue via the bloodstream, wherein said microdevices are coated with a hydrophilic polymer effective to enhance maintenance of the microdevices in suspension.

4. The suspension of claim 3, wherein said hydrophilic polymer is a natural or synthetic glycocalyx.

5. The suspension of claim 3, wherein said hydrophilic polymer is conjugated to vesicle-forming lipids, and said microdevices are coated with a lipid film containing such vesicle-forming lipids.

6. The suspension of claim 3, wherein said microdevices contain a marker-bindingmolecule (i) bound to the free ends of at least a portion of said polymer, and (ii) effective to bind to a marker carried on the surface of such target cells or tissue.

7. The suspension of any of claims 1-6, for use in parenteral administration to a subject, wherein said microdevices have a selected maximum dimension in the range between 0.1 and 3 microns.

8. The suspension of claim 7, for use in solid-tumor targeting, wherein said microdevices have a maximum dimension less than about 150 nm.

9. The suspension of claim 7, for use in delivering a therapeutic compound to the interstitial space of a target region characterized by a target-specific marker on the basement membrane forming the vasculature of the target region, wherein said microdevices contain surface-bound marker-binding molecule effective to bind to such marker, and an enzyme effective to lyse the basement membrane.

10. The suspension of claim 9, wherein said enzyme is a type IV collagenase.

11. The suspension of claim 9, wherein said enzyme is covalently attached to a surface region of the microdevices.

12. The suspension of claim 9, wherein said enzyme is contained in said microdevice in releasable form.

13. The suspension of claim 12, wherein the enzyme is released from said microdevices upon microdevice bioerosion.

14. The suspension of any of claims 1-13, wherein said microdevices are formed of a material designed to erode in body fluid at a selected bioerosion rate.

15. The suspension of claim 14, wherein said microdevices are formed of a material selected from the group consisting of iron, titanium, gold, silver, platinum, copper, and alloys and oxides thereof.

16. The suspension of claim 14, wherein said microdevices are formed of a biodegradable polymer material.

17. The suspension of any of claims 1-16, wherein said microdevices are composedof a condensed-phase polymer material effective to decondense, at a selected rate, when exposed to plasma.

18. The suspension of any of claims 1-17, wherein the microdevices are substantially disc-shaped, and have a laminated structure containing first and second disc-shaped layers formed of first and second different materials, respectively.

19. The suspension of claim 18, wherein the microdevices have a trilaminate structure composed of an interior layer sandwiched between a pair of exterior coating layers, and the coating layers have a slower rate of bioerosion than the interior layer.

20. The suspension of claim 19, wherein said therapeutic compound is embedded insaid intermediate layer, for release as the intermediate layer is eroded.

21. The suspension of any of claims 1-20, wherein the microdevices are substantially disk-shaped, and have a radially-disposed center region which contains said therapeutic agent.

22. The suspension of any of claims 1-21, wherein the microdevices hold the therapeutic agent such that the agent is shielded from direct contact with the subject's lymphocytes.

23. The suspension of any of claims 1-22, wherein the microdevices contain a magnetic material.

24. A microfabrication method for producing microdevices for use in the claim 1 suspension, comprising exposing a sheet of microdevice material to a photoablating light source through a photomask, by said exposing, forming a reticular lattice pattern on said sheet corresponding to the desired microdevice size and shape, and continuing said exposure until the desired microdevices are formed.