BIOCONTENANTS AUTO-ASSEMBLES, A MICRO-MOTIFS ET PROTEGES DES FREQUENCES RADIO (RF) ET LEURS UTILISATIONS POUR UNE DISTRIBUTION A DISTANCE DE COMPOSES CHIMIQUES CONTROLEE SPATIALEMENT

SELF-ASSEMBLED, MICROPATTERNED, AND RADIO FREQUENCY (RF) SHIELDED BIOCONTAINERS AND THEIR USES FOR REMOTE SPATIALLY CONTROLLED CHEMICAL DELIVERY

Abrégé français

La présente invention concerne une particule à échelle nanométrique ou micrométrique pour l'encapsulation et la distribution de matériaux ou substances, y compris mais non inclusivement: des cellules, des médicaments, des tissus, des gels et des polymères contenus dans la particule, suivies de la libération des substances thérapeutiques in situ. L'invention concerne également des procédés de préparation de la particule par repliement d'un précurseur 2D dans la particule 3D, et l'utilisation de la particule pour des applications in vivo ou in vitro. La particule peut avoir n'importe quelle forme polyédrique et ses surfaces peuvent ne présenter aucune perforation ou présenter des perforations à échelle nanométrique ou micrométrique. La particule est revêtue d'une couche biocompatible métallique, par exemple l'or, ou un polymère, par exemple, le parylène, et les surfaces et charnières de la particule sont constituées d'une quelconque combinaison de métaux ou de polymères.

Abrégé anglais

The present invention relates to a nanoscale or microscale particle for encapsulation and delivery of materials or substances, including, but not limited to, cells, drugs, tissue, gels and polymers contained within the particle, with subsequent release of the therapeutic materials in situ, methods of fabricating the particle by folding a 2D precursor into the 3D particle, and the use of the particle in in-vivo or in-vitro applications The particle can be in any polyhedral shape and its surfaces can have either no perforations or nano/microscale perforations The particle is coated with a biocompatible metal, e g gold, or polymer. e g parvlene, layer and the surfaces and hinges of the particle are made of any metal or polymer combinations.

Revendications

58
What is claimed is:
1. A three-dimensional particle comprising a plurality of two-dimensional
faces
capable of self-folding to form a hollow interior, wherein a size of the
particle is
microscale or nanoscale.
1a. A particle of claim 1, wherein the size of the particle ranges from 1 nm
to 2
mm.
2. The particle of claim 1, wherein the particle further comprises at least
one hinge.
3. The particle of claim 2, wherein the hinge comprises any liquifiable
material.
4. The particle of claim 2, wherein the hinge is selected from the group
consisting of a
polymer, a gel, glass or a metal.
5. The particle of claim 1, wherein the particle has surfaces forming a
polyhedral
shape.
6. The particle of claim 5, wherein the shape is a cube.
7. The particle according to claim 1, wherein the two-dimensional faces are
patterned with perforations or pores.
8. The particle according to claim 7, wherein the perforations or pores are
created
photolithographically, electrolytically, or by using electron beam
lithography.
9. The particle according to claim 7, wherein the perforations or pores have a
size
from about 0.1 nm to about 1 cm.
10. The particle of claim 9, wherein the perforations or pores have a size
from
about 10 nm to about 1 cm.

59
11. The particle according to claim 1, wherein the particle is fabricated from
at
least one material selected from the group consisting of a metal, a polymer, a
glass, a
semiconductor, an insulator, and combinations thereof.
12. The particle according to claim 1, further comprising active electronic or
semiconductor components including transistors, sensors, actuators, light
emitting
diodes, photodiodes and solar cells.
13. The particle according to claim 11, wherein the metal is copper or nickel.
14. The particle according to claim 1, wherein the particle is a Faraday cage.
15. The particle according to claim 1, wherein the particle is coated with a
biocompatible material.
16. The particle according to claim 15, wherein the particle is associated
with a
biosensor.
17. The particle according to claim 15, wherein the biocompatible material is
a
metal, a polymer, or a combination thereof.
18. The particle according to claim 1, further comprising at least one
substance
encapsulated within the particle.
19. The particle according to claim 18, wherein perforations or pores in the
two-
dimensional faces of the particle allow release of the contents of the
particle.
20. The particle of claim 18, wherein the substance is a therapeutic agent.
21. The particle according to claim 20, wherein the therapeutic agent is
selected
from the group consisting of a cell, a chemical or biological agent, a
pharmaceutical
agent, a composition, a tissue, a gel, and a polymer.
22. The particle according to claim 1, wherein the particle is administered to
a
subject and location of the particle in the subject is non-invasively tracked
by magnetic
resonance imaging (MRI) or CAT scan (CT).
23. The particle according to claim 22, wherein the particle is imaged with
negative contrast relative to background or positive contrast relative to
background.
24. The particle of claim 20, additionally comprising a radio frequency tag.

60
25. The particle of claim 24, wherein the substance may be released upon the
particle's exposure to a pre-selected frequency.
26. The particle of claim 12, wherein the substance may be released upon the
particle's exposure to electromagnetic radiation.
27. The particle of claim 26, wherein the electromagnetic radiation is
triggered
remotely.
28. The particle of claim 26, wherein the electromagnetic radiation ranges
from
1KHz to 1 Peta Hz.
29. The particle of claim 20, wherein the substance may be released upon the
particle's exposure to inductive heating.
30. The particle of claim 29, wherein the inductive heating is triggered
remotely.
31. A method of fabricating a three-dimensional particle comprising a
multitude of
two-dimensional faces that form a hollow polyhedral shape and containing a
fillable
center chamber, the method comprising the steps:
(a) fabricating a multitude of two dimensional faces;
(b) patterning the fabricated two-dimensional faces;
(c) patterning at least one hinge on the patterned two dimensional face to
form
a hinged edge;
(d) joining a hinged edge of a first patterned two dimensional face to a
hinged.
edge of a second patterned two dimensional face to form a hinged joint;
(e) repeating step (d) to form a two dimensional precursor template having
hinged joints between adjacent two dimensional faces; and
(f) liquefying the hinges of the two-dimensional template using heat to
initiate
self-folding;
thereby inducing the three-dimensional particle to self-assemble.

61
32. The method according to claim 31, wherein the hinges of step (c) comprise
a
material that can be liquefied.
33. The method according to claim 32, wherein the material is a solder, a
metallic
alloy, a polymer or a glass.
34. The method according to claim 31, step (a) further comprising the steps
(i) spinning a sacrificial film on a substrate to form a first layer;
(ii) layering a conductive second layer on the first layer; and
(iii) patterning the layered substrate by photolithography.
35. The particle according to claim 31, wherein the particle has a size that
is
microscale or nanoscale.
36. The particle according to claim 31, wherein in step (b) the two-
dimensional
faces are patterned with perforations or pores.
37. The particle according to claim 36, wherein the perforations or pores are
created photolithographically.
38. The particle according to claim 36, wherein the perforations or pores have
a
size from about 0.1 nm to about 100 microns.
39. The particle according to claim 31, wherein the particle is a Faraday
cage.
40. A method of imaging a particle according to claim 1 that has been
implanted
into a subject, the method comprising the steps of:
(i) loading the hollow interior of the particle with at least one substance to
form
a loaded particle;
(ii) administering the loaded particle to the subject, and
(iii) noninvasively tracking the particle of step (ii) in the subject by
magnetic
resonance imaging.
41. The particle according to claim 40 wherein perforations or pores in the
two-
dimensional faces of the particle allow release of the substance in the hollow
interior.
42. The particle according to claim 40, wherein the at least one substance of
step
(i) is a therapeutic agent.

62
43. The particle according to claim 42, wherein the therapeutic agent is
selected
from the group consisting of a cell, a pharmaceutical agent, a composition, a
tissue, a
gel, and a polymer.
44. A method of treating a condition introducing into an animal in need of
treatment at least one particle of claim encapsulating a composition, wherein
the
composition is released through one or more pores within the particle into the
mammal
in an amount sufficient to treat the condition.
45. The method of claim 44, wherein the pharmaceutical composition is
contained
within one or more microbeads.
46. The method of claim 44, wherein the condition is diabetes, and the
composition is
one or more insulin-secreting cells.
47. The method for imaging a particle of claim 1 that has been introduced into
a
mammal wherein the method comprises using magnetic resonance imaging.
48. A method for targeting the particle of claim 1 to a cell within a subject
comprising
the steps of:
a) attaching to the particle an antibody against specific to the cell;
b) introducing the particle into the mammal,
wherein the particle is targeted to the cell.
49. A method of delivering one or more particles of claim 21 to a subject,
wherein the
particle is programmed to remotely release one or more reagents at a specific
time and
a specific location.

63
50. A method of claim 49, wherein the particle is remotely guided and imaged
using
MRI or CT.
51. A method of claim 50, wherein the particle is capable of releasing a
content agent
or of providing contrast to allow MRI CT imaging of its contents or of
substances
within its vicinity.
52. A method of conducting non-invasive biopsy or microsurgery, comprises
directing the particles to a site within a subject using remote means,
allowing the
particle to capture one or more substances from the site, and obtaining the
substances
from the particle, thereby non-invasively conducting biopsy or microsurgery.

Description

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
1
-Se1f Assembted. Wcr0 atterned 4inti Radi0 Frec u~nc> R1F+` Shieldeci
113-raC0vitai~~ers atuc.ll `C`heirlses t`or :Remote Saat.iallj, Controlled
C:heiiii+eat
Deliverk
CROSS-REFERENCE TO RELATED APPLICATIONS
100011 Tl7B application claims the ber~efit of U.S. provisional application
Nca.
60l81'.6,t)6-3r fil.ed 1tiiic, 2:33), 2006. Additionally, this application is
a c:a,~ntintÃation-in-
part of U.S. Serial No. 11,491,829, filed Tti1y 24, 2006, which claims
priority to US.
provisional application No. 60/ 70(,903)õ filed JLi1y 12, 2005. `l".Iie
etitirc co-ntcnts of
these applica-ions ai-c incorporatezl,by i-cfei`eia.ce, hereirl.
C,Oi"Ett:NMF;N`!' RIGHTS
10tl02] This researc.la was stapported in part by tE-ie Nat:iotial Institutes
of:1~-le'-flth
(NIH P50 CA 10-3 ) 1. 75}. "I'lae gz ~'vernment of ttae Unitcd States mav have
rights to this
i~~~entic?ll.
FIELD OF THE INVENTION
10003] The prescrat invention .rc.lat.c.s Ãc) a iaiic.ro#~Ãbric~~ted nara.o-
or Micro-sca1e
particle for encapsulation and deliv~.~ry of i-naterials or substances
itic:.tticl.ing, l:aLIt 1-10t
limited to, bic31ogica1. incdia incluti.ir~g cells, pharinac.e::cabc:al
agcnts, cornpositicarIs,
dra.igz, ti;;sue, ge[s and po[y.mcrs contained vvitbiai the laci_rticle, wit1l
subsequent release
of the tkacrapeutic materials in situ, ri-iethods of aYaakitag the particle
atad arletliocis of
uszrag the part.icl.e in in. vivo or i~~ 'vitrca applications.
1;3At"11,,.GROUl~lD OF Tt-JE INVENTION
10004] Tn recent ~~~ms, advances in regenerative meclic:.-ine bave inspired
therapies
targeted at the ccllLilar lcvcl. TI-icse tlaerapies seek to implant cells or
ceilLilar clListcrs>
manipulate cellular pat~~~~ays: aia.d target the delivery of drugs. For
exwiaple, a wide
range of cell liiae;s laztvc bec:ra csicloyed wit.liii1 semipermeable iiÃid
biocornpatible

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
2
irÃ-u-nobil:izaÃion devices that control the bidirectional diffijsiorà oi rÃ-
iolectÃIes and cell
release (R. P. LaÃira, J. L. Hayes, W. L. Chick, Nat.. :BioÃechnol. 14, 1107
(1996); t3.
Drive, P. M E4errÃatÃder, A. R Gascon, M. ~~ariÃ.aa, J. L. 1?edr-az, Trends
irÃ
13iot.ec;.}Ãno1. 20, 382 (2i30'.); N. E. SÃnÃpson, S. C. Ciratit, S. J.
131~ckbalÃd. I.
Constantinidis, Bioniate:ria1s 24, 4941 (2-003.).). Concurrent advances in
microtechnology baare revolutionized medicine; as Ã~ew implaÃ~table devices,
ziÃicroarrays, biocapsÃ:ales aÃid mia;rcÃprcÃbes -a~~~ developed. "('tiese
dev.ices have
tacilit:a.ted ce[lular encapsulation, on-demand drLÃg release, and early
diagnosis of
dis~~~es, (:1_ T. Santini, M:_ J. Cima, R. Langer. Nature 397, 33-i (1999)s J.
Kost, R.
Lan4,~eÃ-, Adv, Drug DeliveÃy ~~ev. 6, 19 (~ 99t ); L .Leoni, '1'. A. Desai,
Adv, Drug
Delivery Rev. 56, 21 1 (2004}; B Ziaie, A. :[3aidi. M. Lei; Y. tFiI, R_ A.
Sieget, Adv.
Drug Delivery .(~ev. 56, I45 (2{304); A_ Desai, r! _ Wes"t,.M. Cotien, 't'.
:L3oiaÃsski, A.
Rampersaud, A.dar. Drug Deliveay :E' wv. -56, 166:( (2004); J. T. SaiitiÃii,
A. C. R.ichards,
R. Se}Ãeidt, .-N4. J. C'i~~~.-za, R. 1n.aÃigea~, Angew. C'beaYÃ. 39, 2396
(21000); L. .FireÃ~ian. E
Mahaji7as E. Broide, M. Shapiro, -[__. Fieli, A. 4terrlber; Y. Ke~lielmati. E.
Scapa, Gut
52. 390 ('U0)3~3_ In ~:Ã~ntr~.st to ~~cAi~raneric, l~~rdr~~gii, and ,cil-ge(
based prw~cesses that
have been tisÃ;ci for encapsulation and delivoryx coi Ãvet Ãtiona1 silicon
(Si) based
microfabrication has higb reproducibility, provides ÃiiechaÃiical and
cliemical stability,
a.iid allows the iiiccarpora.tiota of electrcÃiiie and optical modules
witllizl the device,
therf'.b~' facilitating wireless telemetry, remote activation and
C:omTllÃ,iTl#cat#oll, in vivo.
Howevc:r. Si based microfal~iricdttion is inIÃerently a twti dimen<i~.~tia(
(21)j process and
it is extremely diffictilt to fabricate three-dimensional OD} systeÃ-ris using
conventional
microfabrication (M. Madou, Fundamentals of Nficrofabrication (CR.C, Boca
Ratc}n,
FL, 1997)), ~ 3D medical elevicÃ; has several advantages over its 2C3
c::ounier~.~saxt; (a) :-Ã
laqger~ emerna1 stirl`acc area to volumo ratio, thereby maxiiiiizilig
ititeractions with tlie
surrounding medium, and providiÃig space to moLrtit di.tTerent diagnostic or
deliveÃ-y
modules, (b) a finite volume a1k)~,ving encapsulation of cells and dnigs, and
(c) a
gc;ometÃ)~ that reduces t(ie: chances of the device being Ã;triclesiral.~1v
lodged in the bt~~iy-

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
3
1(1{}051 .I.n orae aspect of the larescrit iriveritjon, biocontainers (i.e.,
boxes, I-iollow
particles) have becti fabricated by a strategy that conYbizies the advantages
ot tfirce-
dirr~ensiorraliiv with the desirable a5peÃ:.ts of Si-based microfabrication to
facilitate the
delivery of i:tier~apeLatic k(gents isi sitii. For ~xampie, the eoritairier-s
are loaded rith
ztricrÃ~~eads or cells e:rnbetlded in a gel, arid thus Ã:mi be used either in
coqji.rnction with
preserit dav immobilizatiori svstents Ã.ised, in cell eracapsÃ.l1atioii
technolog, ~~, or tl~e~;~ c~z~
be used zndqendently. In a:riot.lier aspect, the biocontainers also caii be
used fcar
encapsulation o1':fiunctinnal cells witlairi the por-oras containers ior in
vitro arid M vivo
release of therapeutic agents with or wit(ioLit amma,rnosuppression. For
exarnple, the
cor-ltainers caÃa be used for ei-aeapsulatiori atid deiiveryot insulirx se-
cretirxg cells for-
iniplant-ation in patients wviÃh diabetes, for placing tLsmor innocula in
ari:iriial riiodels
where, e.onstra.iruna, cells ~,viÃltin a small re~fio.it is necessary, and ior-
de.liver~, of
tuaictioraal iietÃronal ('C; l2 cells. :I rt some embodiments, tlie faces of
the container are
patterned witli riii<:,roscale per-l:or-atiozts, allÃ3wint; control over per-
#t-Lsiort and r-el~zise ol
its contents with the sug-rortndirio; rYiecliLim. `I'be advarita~~;cÃ:uy
attfibutes of the
contaiiicrs are a parallel fabricat:ior~ process with vrwrsatilit~' in sizes
and shapes; precise
and monoel.i~pea=se strÃfirc:e porosity; and the ability for renwne guidance
iisÃr~g magnetic
ield.s. In another aspect, the coiitairzer-s of ttie present invention are
easily detected
a.rid non-invasively tracked i-ising CortvenÃional rt~agrletic resonance
irnagin g (1LI~'~Iiand.
do not require the presence of a c;o-ntr-ast ~.~~ent,

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
4
S'Li M_MARY OF THE INVENTION
1000G] The present iÃiveÃit:icÃ~i provides riaÃxoscaIe or microscale particles
for
encapsulation and delivery of materials or substances, xncludiÃxg, but fic?t.
l:izniÃed. to,
cells, clrLigs, tissue, g;elc aÃ-id polymers cotitairied within the particle,
with stibse~.~LÃeÃ-lt
re:lea.qe of the therapeutic materials in situ, methods of #ab.ricatÃÃx~~; the
particle by
foldfiu-) a''D oreceÃ.rsor iÃito tkÃe ;3D parÃicle> arid tiae tÃse of the
particle in in-vivo or in-
vitro applica.tioris. In oÃ~~ ~~iibodi~iieÃit of the preserit iriventicÃtÃ; a
three-di.metÃsiort-Ãat
particle cÃ:Ãmlarises a mLiltitLide of Ã~~vowditnensioÃÃa1 faces itlat form a
hollow,
polyhedral shape a.iid containing a IillabIe center chanibe.r, whereÃÃl a.
size of tlle
particle is niicrosc.ale or tiwiost.alc;. In wÃotlÃcr emliodimcnt the two-
+:iim.ensiona1 faces
of the particle are l.^~attemed wit.li 1.~eÃfibrations or pcares. .1n another
embodiment, the
perforations or pores are created photolitliograpliically . 1Ãx another
embodiment, the
perttarat.ions or pc?res have a. size from abotit tl. 'I nm to aboi~t 100
mic::rcÃns. 1:n another
embcadim.entr the particle is fabricated troni at least one material selected
I'rom. the
group consistiÃig of a metal: a. polymer, a glass, a semiconductor, a11
insulator, and
combinations t:}ie~~eof In another embodiii-aeÃat, the metal is copper or
nickel. tn
a.Ãiot.}ier embodiment, the particle is a Faraday t.a~,Te. In aiiother
onihod:inient the
particle is coated wit[i abiocompatible materia[. In arivlhgr embodiment, IEle
biocompatible material is a metal, a polyÃxier, or a combiriat:icari thereot.
iÃ~ another
iriil7odiriic:iit, the filla:ble: center chamber of the pa.rliclo is filled
with at least one
sÃ.ebstance comprising coiitents of the pa.rticle. In another einb~.~diinÃ;nt,
perforations or
poÃ-," in the iwo-dimeniioÃia1 faces of'the Pa.Ã-tsc.le allow re'lea.se of the
contents of the
particle. In another embc3dimentM at least one substance is ~a therapeutic
ageÃit. In
another embodiment, the the.rapeutic ageait is selected froÃn the {;~rULÃP
C0D;;iShÃ16Jr OI' a
cell, a plÃannac.etaic.al. agent, a, c:ora-:tpos-ticÃn, a tiwstÃe, a. gel,
arÃd a polyrnt,r. In another
embodiment, t}ie particle is admlfl-iistered to a sutject and location of'ihe
parfic.le HI the
S1.1bjecà is non-invasively tracked by i31agi1ek rOSC1T1ai1t:-C i111a<q?ing.
I17. iÃ.I7.C3t.l7.Gr
erÃabc.>dirnent, ttae particle is iniaged with negative coÃ-it.ra4t retiÃtive
to back,F{rcawid or
positive cLiÃ:ftl"cÃ.St relative to background.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
100071 '~(`hÃ: present invention also provides a niethod of .fa,f3.rie~~Iti~~~
~1 il~~Oe-
dinieÃ7sional particle comlarising a Ãiiiilt:it~ide of t~.vowdiaiensional
faces that l`orÃ~~ a
hollow polyhedral shape aiid containing a. fillable cetite-r= chamber, the
method
cornprÃsing tlie steps: (~~) fabricating a rnultatuele of Ãe.vo dimensionat
faces; #.b1
patterning the fabricated ÃwoTMd'amerasioÃia1 faces; (e) patterning at least
one hin4}e (ari
the pattemed two ciimetisional face to tori-n a Iiiriged ed(;e; (d) J'oinitig
a hiÃigecl ed~.~e
of a first laatterried two dÃ'iiicrisiozial face to a hinged edgge of a second
patterned t"i-o
dimensional #ace, to torin a IiiÃia;ed JoiÃat; (e) repeating step (d) to form
a two
dimensiona.l precursor template having hinged joiiits between ad~acent two
~imensioÃ-tal taces; (t") lÃiluefiying, the ti'txiges of the two-dimensional
temp1a.te using
heat; <i.Ã7Ã1 {~} selt:r-asceiiif}ling the threercd:iÃvierisaonaI ~~t-ticie_
In another eÃrtbodi~~~~i-it,
tfic hinges ol=_ ~tep (e) of the method comprise a material fhat can be
liquefied. .(.ti
a.iiot:lier ciiilaodimejit, the material is a. sold.er;. a. metallic allov, a
polymer or a glass. In
anot:h.er emlaodzmeiit, step (a) of the Ãiiei:liod 1~irÃber coÃTrprises the
steps (:i) spiÃiriiiig a
sacrificial fili-ii ori a substrate to 1 riYi a tirst Iaver, (ii) ~averÃng a
conductive secoriÃl
layer on the first layer; aaid (iii) patterning the layered st7bstrat~ by
photolithography.
l:ii another embodiment, the particle has a size that is microscale or
nanoscale. I~
another embodimeiiÃ, in ste~., (b) of the iiiethodt the ti.vti-dimensicrna1
faces are
patterned witb pertoratioiis or pores. The perforations or pores are created
photolithtagr;:tphica11~,. In another e;t7~~od:imentõ the perforations or
pores have a size
frtani about C).'1 iiin tca about 100 microns. In anotber einb~.~di~nent, the
particle is a
Faraday 100081 The presetit invention tt.irther provides a method of
imagita.gy a tliree-
dimcansi.ontil pat-tic:lc comprising a MtlltitLac9e of two-dimenwi~~ial fiaces
that form a
la~.~ilow polylicdra1 s[ial.~e aiicl coiitairiigzg a filla.blo center c~amber
that Eias beon
implanted into ~subjject, t~~e method, comprisiaxg, the steps nf` (i) loading
the fi1:la61e
center chamber of the paa~ticle with at least one substance to form a loaded
particle; (ii)
admiaiste:ring the loaded particle to the su~ae.ct; aiid (iii) noninvasively
tracking the
particle of step (ii) in the srIbAjec:t by magnetsc resonance imaging. In
an.offier

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
6
erribc>dinicia.Ã, perforations or pores :ir.a the bi-v'o-c.linieiiSional Eaces
of the particle allow
release (yl"tkae subcÃ.a.tac.e in the fillable c.criter ch.a.iTibcr. i~~
another etaiboditaaetai:. the at
least on.e sub,,;iance of step (i) is ~thea~apeutic agerit. In atiotl~~~~
emliodymet5t,, the
therapeutic a.gcnl is selected from the 4~rOa:ap corlsisting of a ce11, a
lsharÃnaceLstical
agent, a composition, a tMue, a <=,el, and a polya~er.
BRIEF DESCR[PTION OF THE FIGU RES
100091 Figure 1 is asclaeraaatic diagraiYi of the process flow used
tot.a.bricate the
:')D containers o1'tlae precetit invenÃz011.
100101 Figure 2: (A) Optical inaage showing a collection of containers. (B-DI)
Optical and Scanning c(c;c:trosi microscopy (SE.NNI) images o:l'micrc~pattemed
cuntailiers
at ditTererct st~~~s of the fabrication proeess; (B) tlie 2L3 prectirsor witli
electrodeposited 1`aces: (0 the precursor wiala faces and lainges, and (D) the
folded
container.
1Ã1011 1 -Figure 3: (A) ~ENNI iniage of a laolltiw; opGiiaf-tced coiitairier.
(B) SEM
image cif a cojataiia.er loaded u;itla glass ixaicroboads. (C) Optical image
of a
biocoÃataiiier loaded with MDAMB-~? j I. breast cancer cells embeclclecl than
extra-
c.cl:lular matrix ÃEC.N-11 gel. fl_7;y Fielea.se of the cells by immersioti of
the container in
war~i-i cell culture medium. 4E;1 Optical image of a container loaded with a
ceI1-E~."M.-
ag,-arose srispetision stained with the tltflorescent cell viability staiÃa>
CalceÃn-A:M. (F)
Release of the viable cells from the container on itarnc.rsdon in warm cell
culttire
incr~ium.
10012.1 Figure 4: i4I_RI im~~~~~s of an ~peti faced (A;) noii-magnotÃc: CLi
c.oiitainer
a~id (B) ferromagnetic Ni coÃataiiicr. (C-D) 1~'itiitc e1~~~-nent simia(atÃon
rewLilt; of the
Mar rnagnet.ic field in the region of a L"Lgcontainer, iii the (C) xy aTid (D)
yz ceia.t.ral
planes. Ttic excitation cÃariaprised a linear polar.izecl 500 Ml-lz plane wave
ofl V.'ryi,
with tlic E. atid H fields in the z and y direction re;;pecrively. Tlie
magnetic field
cl.istoTtioias aaad the shielding effect caLaserl by t1-le wire frame are
eviclent..
100131 Figure 5: M:I;, trta.ck.irag of a coniainer in a flLiiclic chaiiiie;t.
MR inlag
ge;;; cal
the container at tliftert.nt tinae points taken tÃ:iicler pressure drivc:n
flow of'the fl.uitl.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
7
1 (1{}l41 Figure 6: Optical a~id Sl=:~M i.7nzr_ges of the tl-iree steps rrsed
to fabricate
mic.ropattemed boxes. "i'he boxes sliowÃi iiavc approxiaratc dirTletrsioris of
200
rxricr- ns. Frc~i-ti left to Ã=iglit- (a) The faces wer=e patterned using..-
photolithography atid
e:Iectrodepoaition; (b) Solder- hinges were ~.Ii~Y.t~ed rel~.tive to the faces
usirx4;
phoÃolitho,graphy, etching and eIectrodepo~ifion; and (c) the 2D precursor was-
lifted
off Ã1~e wafer upori dissolutiari of a sacrificial (aver. When t.}ie 2D
precr.rà Sor was heated
above the riie;,titig poirit of the solder, the siructr:rrc folded ilito a
3:13 cubic box [B.
CJimi et a1. voi. 7, p. 341r343, 200-5_I.
1001.51 Figure 7: Images of sorne defect modes observed: tt the solder
}lei(Yht is
not optiaiiiz~ed (A) uÃ-aderfolded or (B) overfolded boxes are obser-ved. (C)
Incomplete
etc.:hints of the seed laver usriallv results in faces that cannot fold
(i841"),, because they
are fu~ed together witii the seed Iaycr-
100161 Figure 8: SEN1 arid optical images of boxes tilled wit~ (A & B)
I?iuroziÃc
hydrogel aiid (:') MUA-'-9:13-23 l breast cazieer cells embedded in
e:xtracellular matrix
(ECM) ge1. (D) =1'he cells could be rcleased from the box b;: pulsatile
z~~~:itation in cell
tt-redia.
100171 FirÃit=e 9: (A&B) Optical ii ~agos of 2D c(iÃl; fabricated tisir7g
photolithography. By passing ct.rrrent thr-or,rgh tiie coils it is possible to
generate a
magrietic field. (C) The microbr~~ is placed alorig t..fae central axis of the
coil in order to
i.nduc:iivclv heat the bc3x,
100(8] FigÃire 10: Release of dye from a lo-idc;d. box r.apon heating.
1.00i91 Figtic=e 11: a) A scaaining electrori microscope irnage of an empty
container. The containers were three-dimensional (31)) porous cLrbeS with a
length of
approximately 200 mm and a voILrmc of 8 nL. b) Ari optical microscope image of
a
coritai~ier loaded with a dye-soa-k-cd pl~roriic ge1, cf A scliematic diagram
of t.lie
~Nperimental set-Ã.rp used to facilitate wireless micr~~ascaIe chemical
engineering (Ãlot
drawri to scale). Ccgr7tainers were manipuIfrted uSing a magnetic stylus (not
Shwvn) and.
the contents of specific c~titaizie;rs were released by directiiig an RF
source tciwardS the
container. In tbc; schc;matic representation, chemical Y is releia ;d from a
specific

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
8
conttzine.r; c;liciia:ical Ytl.aeri reac:ts wi~~~i chenaical N. in t.lic 4urro-
und.ing r~-iediui-ii f~ca t`orni
pt-nd uct I.
100201 Figtire 12: Optical imkges showing the reriiote controlled, spatially
]ocalized microkabgication witliin a cap:illary_ 'I'wo i-iiaci-owi.r~s (l and
21) were
embedded within a macrnl='abric,atecl c.apallw-y (ca. l mm in diameter and 1.5
cm in
length) aiid the capillar,v was aligned on. top of a ZD microc.oil, a, b)
First, a container
filled wit:li piriroiiic and soaked with the elicmÃcai sensitizer was guided
iiito the
cap:i11a.ry to t}ie site of the gap within wai-c 1usant~~~ a magnetic stylus,
c) "l'~ie, cllemical
wnsftizer was released bv re~inotefv heatiax, the sensitizer-soaked pluroÃiic
gel that was
encapsulated WithÃn the coiit.~~iier. '1'-(iis heating was achieved witb the
2D R:l?=' coil.
After sensitizing the gap, the first c.otii:aiÃier was removed, ~~econd
colit.aii-icÃ- lvas
guided to the same gap in microwire I. -i.tad t:fae actMator WIS released by
heating the
pluronic gel renioÃely, d) Atter activation, ttie second container was also
removed. e}
The capillary was tlicii filuslicd with a commercial electrn1eSs copper-
plating Solutiori;
chernica1 reduction (bubbles o1'thc: hydro4,en gas, a byproduct iti tt~e
reaction, cari be
seen) of copper sLilt atc to metallic copper, occtirred at the gap within
microwire I. t)
Coppe.r was deposi.ted otily in tl-iz gap $.~etwe~~i mic.rowire I, ~io
c(il?per was depositc d.
in the ga~., f niiiierowire 2.
10021] Figure 13: Cell-viability asSe~smeiat by five/deacl fluorescent imaging
of
ca1c;eiai AM and ethidium homodimer-1, both released rei-note1~~ from the
containers. a,
b) Confocal iiua.gcs of the local release of the live/dead st-~n tci L929
inouse tibroblast
cells. No red cells were observed, thus indicating tio nec.rkotic cell death
during the
aelease_ a) Transmitted light differential interference contrast (DIC) images
sho-wing
both the cells and the coiit-ainer. l;i) F(~~oresc:enr image showing on13=
localized cell
sÃa.ining,
100*2
:1 Figtic=e 14: (A) Optical image of the color c.hang;e observed on a
temperature indicator label placed under a nanoliter container, expos'ed to RF
raciiation. The color c l}agige occurs only under the container ~~owing that
the heating

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
9
is local. (.B).A plot oI'the temperature rneastared using t.lic coior-
indicator Iabcl vs. the
i ticicicnt RF power.
100:231 Figure 15: f_'csnfoca1 tiric:roseopy ir7rage of a tiatioliier
c:orrtairicr loaded
witNÃl?Nl:l?;1.i-zr gel soaked w.ith 1.1LEIDEAD<"~ assaw. 'i`1-le crpcrin-lent
iotr?wed the test
proceclÃÃres, v~=ith tlrc exception that the RF was not turned oti. --t'1Ãc
absence of cell
stainin4,~ surrounding the container (corÃxpare witb Figure 13) demcynstr-atcs
no
dzsccrr7a.lal.e c}Ãcrriica.1 release in the abscrrce of the RF radiation
trigger.
fUtl24J Figure 16=. C 'orrrparison of finite simÃilaiio rand cxpcrimerri.al
results for
the self-assembly process, (A)'I~op view (drawrr to scale) wittr dimensions of
the faces
~~id gap widt.}rs of ti-ae'?`D template used t.oselt-assemble t.lre cube. (B)
Side v:ie-%.~~ of
two ac.jaccrrt faces nf the crucÃt`orÃir (as tabricai:cd),with variables Ãased
in tl-ic liriitc
dement sirxiulatiorr. (C) Side view ol'at~iacent tacesat the oriyct o#-rci~~Nv
r>f'tlie
toldin }ainge. (L)-0 Firiite element snapshots sbowirrg (~~) urrcle~~folded.
(E) right-
atigIe 1o1ded, aiid (F) overfolded fiaces. (t -I) Optical nricroscope iÃ-
zrages of
cxpcrirne.artall;; fabricated 200P.m ciibes exlribifing the Lsrlclcrfoided,
right-angle
folded, and overt olded faces. Note: Fig t.B-F are not drawn to scale in order
to
illustrate important (iiz-ncti5iosis.
100251 Fi~~tre 17: SimÃÃ1aÃiogi results of ti~e depciiclc~ice of the fold
angle on solder
vc-i1uriie The restilts deÃ~~~~iistrate that folding angle ca:ir be precisely
eri;;irrecred by
controlling the s(?Ider- vvIÃimc at the hinge.
100261 FigÃire 18; Normalized total cncqgy curves (finite c(cniciit
simulations)
plotted as a fiirictiorr of fold ang;fe for faces with Ieragt.hs r-arrgin:;
frorn 6mrn to 50 n ni.
The curves show that folding is spontaneous at sma.ll size scales with stable
rafflirna.
As the scale increases, gravitational forces increase and folding is no longer
sporrtanc~.~us (iiiitial slope cliariges t'rorrz riegative to positive) and
there is rro minima
preserit a:t 6 mm.
10027] Figure 19: (A) An optical image showing tiee standing polyhedra
fabricated (c.xpc.rimeiita( rc.sÃilts) witl~ a. wide range of sizes all the
~~~av from '~~im to
(B) 15 tÃni, and with ditTt.re;nt shapes e.g. (C) A ;;tlr.tare pyraniid.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
100281 Figure 20: (A) Optical ii-iizt~,se of cubes With a .ratigÃ; Of StZCti
I`zsrTtic-ld in
lar~.=ertunilaers. {'1::3} lonri-ied in iniage of the otitlitied re~~iorl in
(rl) feai.Ãtt=in~~ I00 pm
cubes siitÃtic, oti top of and anion~..9 500 1mi ecgbes.
f00291Figure 21: Sync3pa.iS ol'toiding process.
100301 Figure 22: l~) Optical atid SE'~~~ images s17o-,.-ving the dÃtl'eretit
steps (tlie
photolitho4,~ral~l~.iÃ:..aJ1~' fabricated '?l) template, Ãe~;ist:r-~; of
solder ItiÃ~tges and the folded
-:D s[:rttcttire} in the fabrication of acttbic. container with one t7peÃi
face. SEM images
3
ot a(b) ct:tbic cotitainer ~vitb all opeti flaces, (e) pyrannidal fr-uStut-ti,
(d) sqttarc pyramid
with ati open face oii the bottom, (e-g) Optical image of multiple containerS
of
dltTereait shapes demo:istra:tinia tl-ae paraltel tabrication strategy> {h=h.}
SEM itx~ages of
cÃtbic cotitaitiers witl-i monodisperse pore sizes of (h, j) 5 atic.t=ons atid
(;r; k) 3 m:icrÃ?tls.
10031] Figure 23: Optical images of chcralcal release from containers ( a)
Spatially isotropic release of a dye from a container witb iclentiÃ:,a.l
I?Ãarositv, oÃi all faces
(la) AniSotropic release of a dve I'rozii a cÃ3ntaztaer- with aÃ-tisot-ropie
porosiiy (five faces
with ati array of 5 iiiic.roÃ-i porcs; ilic stxih face has a 160 i-tiicr-oti
sized pore). (c) An
e\arnplc of a remotely guided spatially controlled chetnical rÃ:;tction. The
lettÃ:r G (for
the Gracias Lab) was for-nie.t.i by the direct writirig of pheaiolphtlialein
in an alkaline
water---glycerÃ.~[ meclit.tm.
10032] Figure 24: Spatially cotitrolled cliemicai reac.tions. between multiple
containers. (a-c) Reaction of copper sulfate and potas;ait,tm hydroxide in. an
aqueous
inÃ;Ãlit.tm resulting in the tortZ~ation of copper hydroxide along the central
llrie between
the Ã:..ontainers. (d-f) The reactlon of phetiolplatlialeiti (diffusing caÃtt
of the two bottom
containers) and potassium hydroxide (diffusing ottt of t.lie top
cÃ}ta.ta:iner) in an aqt.tec=t.ts
medi utii.
DETAILED DESCRIPTION OF TRE INVENTI+EON'
100331 The terms "hollow pat-trt:Ie." "box," ::contasner" and
s.b1C}coI7tcltl7et`õ zEre used it1tf'.rcl1ai7~~eably f3ereii7 to i3lLili3 a
three-dimensional C1bjjGct,
i.c.., a rec:c;ptacle, with a hc.3i1mv inter'i.or- or an ititt:rlor capable of
containirl_g sttbate~i-ic;e;;.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
11
100341 I`lÃe term <;colloid'R or .:colioida(" as rase(i herein re::f-er-s to a
substance tiÃrÃde
Lap of asy5Ãem of partic[es dispersed in a c01Ãtia-ILiOr.aS r-freeliÃ:rm.
100351 Materials carà react quite difTeÃ-eÃitly iÃi the presence oI'aÃ-i
extemal nia:grÃe't.Ãe
tield. 'I'kiei.r reactic~ri is depe.rident orà a rart.inber of factors,
including, but rioi: liÃYiited to,
the ntaterial',, molecular structure, its, ator-rtie strLactÃ.rÃ-e, aÃid the
Ãtet mao;Ãietic field
associated witli the at.oms, Most materialS can be classified as
fot`romagnetie,
diamagnetic, or paraiii agri eti C.
1U03~"iJ The lerÃ-ii "diaÃnagnetic"' as used laer-ein refers to rYiate.rials
having a very
weak l'cirtti of magnetism exbibited only in t.lae presence of aÃx external
magnetic field,
which is tl-le result of chaÃtges irà the orbital motion ol electrons due to
the exterrÃal
rnagnetic t:ield. itidticed nia";ÃleÃ:ic r'laomerat. ÃÃi a diamagnetic mai:er-
ial is very small
and in a directioti opposite to that of t:lae applied lield_ 1-Nartipies of
diama~.t1dic
materials include, but are Ãiot limited, Ão, ct~laper, silver and gold.
1.0037:1 The terrn "1earornkgrteÃzc.,, refers to tnaÃeria.ls t iavirlg large
and pca5itive
Susceptibility to aii external riia(sÃietie field. Fer-romag-rietic rim:teÃ-
izt.ls liave soÃxÃe
ÃÃnpairec.l electrons so their atoms have a net ri-aagÃaitie moment. They
e<bibit a Stront),
attraction to t~~agnÃ;tic iÃ;lds and are able to retain their magnetic
properties after the
external field Iias beeri removed. Examples of firrr~.~magrietic rnateria.ls
includel but are
ntit 1'imited to, iroti: riickel and cz ~balt.
10038.1 The ter-rai "param~~~r-ictic: refers to materials lravi~ig &Ã small
and positive
sÃ.ÃSc:c;ptil.~ility to ni.a.gnetic fields, wliicIÃ are slightly att.racted by
a rnagneiic fiel&
]'a:ratnawictic materials do not retaiii magnetic pr-opeÃties when the
ext~.rÃtal tzeld ~is
r~~noved. These paramagnetic properties are, clÃ.7e to the presence of sor~e
unpaired
electrons and the realignment of the electron orbits Carr.secl by t1ie extemÃ.-
r~ magnetic
field. ExaÃzit.~lcs of'paramagnet:ic rriaterialS inclrik, t~~Ã.Ãt are Ãi~.~t
limited to, magnesium,
molybdenum, and litlaia.rm,
10039] The terÃai "Faraday cag&' as t.r~ed hereirr refc.;.rS to a:ri enclosure
designed to
block the e#Tects of an ele.eiric field, wliile a11~),~ving free passage to
magnetic fields.
(Sef E. M. PtÃreel1, Electricity and Magnetism, Berkele-y Physics CoÃ.ir ;
VolLÃ~ie 22

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
12
(N-l:cCfr-aw 1:=[ill, MA, 1985)B. SÃic}i an enclosure also is called a
Fa.raday shield,
Faraday Shie:[ding, Faraday ccrecsi; Faraday electrostatic sliie1dõ or
shielded room.
100401 The tet=iii "gel" as used licreii-i refers to an apparently solid,
jellvtike
materia.l fnrnied :fi:rÃ~~~i a colloidal solution. By weight, 4e(s are niostly
licli.-Eid. yet they
behave like Snlirls, I"_[ie tenii "solution" refers to a homogeneous riiixture
of one or
more substances (tlie soleites) diss1 ~(ved in another substance (the
Sc~lverit).
100411 '~('lic term "izidriÃ;tivc heating" as used bereiti refers to Ãtle
process of heating
a znetal object by electrognkgnetic iÃiduc.t-iori, where eddy currents ar-e
gei~erated within
the metal and resistance leads tc) Joule }ioatiiaz ~~t tbe metal. An induction
heater (far
~~~ v proc.ess) coÃ-asists of an electrcima<wnet., throÃi<wh whiÃ:.1~ a hiwh-
frecluency Alternating
Current (AC) is passed. Heat mav also be (5enerated bw magnetic I-iyst:eresis
losses.
10042] The teÃ'Ãn "magnetic field" as: L&scd Iicr~~ii r~~ers, tc) the re~ioti
lri 5pac=~
surrounding a magnetic bodv or erÃtit:y; stÃc.l~ as a pertrianent magnet or a
ccyiiduc-tar
carrying a ctirreÃit:, w(iere ati appreciable magnetic force is preSCtiÃ.
Suc~~ a field is
rcpresezit:cd bvinagriclic lisies of force. In an ciectromagnctÃc field, for
exaiYipte, the
magnetic field is perpendicular to the e(ectaacal field.
100431 The tÃ;rni "z~~~iielic field strength" or "magnetic field inten,ityõ
(<:ff')
refers ÃÃ.~ tlie iiiteiisity of a ina.gnetic field at a given pÃ.~iiit. Mag-
netÃc field streti:;t}i is, a
vector qtÃatitity usually expressed in amperes per meter or in oersteds.
10044.1 The term "magnetic reso-nat7c:c imaging'. or "MRI.", refers to a
noninvasive
imaging technique that ~~ses the interaction bet~vee.n radio frequency pulses,
a strong
magnetic field, and an subject to construct images iti slices/planes from the
nuclear
magnetie resonance (NTMR) signal obtained fic}m the hydrogen atc}inS inside
the
suNect. The priiicip(Ã; behind all MRI is the resonance eqttatson#
100451 t1=:; B+,
( ~quation 1)
100461 which shows that the resonance frequency u of a spin is proportional to
the
n~~~~~~ietic field B~; it is experiencing, w~iere y is the gyromagnciic ratio.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
13
100471 As used bc.rei.rr, the tÃ;rni <;mic:ro;Ã,a:lc" refers to par`tiÃ,lcs -
tliat rrreaswret`rcarn
about Ijinr or I x i C)'"; meters to about 999 l.irn in at least orrc
dimerrsiorr. As used
beÃ-c:iti the ter-iii .:.nanoscale" refers to particles tkiat measure trort-i
i.l,oÃrt 1 rianorrietcr- 0r=
1x 1 F)`9 zrrcte.a s to about 999 rraÃroinctcrs.
100481 The term "ma
gÃretic field ~radient" refers to a variation in the ma4}rletic
field with respect to position, A one-dimensional mag-neiic field gradient is
a variation
with r-espect tci oric direÃ,tioÃi, while a two-dimcnsiozral ggradiezrt is a
variation witli
respect to two dircctions. `I'1-ic rr-iosi Lisct=ul type of g;radieÃrt in
rnapetic resonance
imatyirrg; is
s a c~~ii~- dimensional liaxea:i- magnctic t~eld gÃ-ad.ient. A one-dimensional
magnetic field gradierrt along the x axis 'Lrr a rxragrretic field, B,,
indicates that the
zrragrretic field is increasing in the x direction. I'lie symbols for a
rnag;neÃic field
gradient in t}ie x, v, aiid z directiorts are G:;, G and G.
100491 In physics, the term "riiagnctic rr~omerit" or .<dipole moment" refers
to the
pole str-ength. cit a arragrictic sour-ce mttltiplied by the clistarice
betweerr the poles
(p pd), arrd is a measure of the sÃ.reÃatYth of the niagnctic source. The
rriao:rietic
moment in a magnetic field is a mea.stire of the magnetic t1ux set LÃp by
gyration of an
clectroii chrirge in a mr~ggnz.tgc ticld.
100501 The terrii "micrÃ.~paÃtern" or .`r7ticropatterrreel" 'ati tÃsed lrerein
refers to ar1y
arbitr ar-v, two-dimensional pattern haviri<w microscale feat.tiÃ-es. The term
nawpatternõ
or "nanopatterned" as uscd herein refers tc4 ~nv. arbitrary nsro-dimeÃisÃonal
pattern
l`Ãaving microscale teattzrc;s. Akcordirig to the present invcnti<Tn, the
particles are
paÃtemec1 with 1~erforatioÃrs c~~- pores r-aargirrg in size from abntit 0.1
niri to about 1.00
ml ct'ons.
1Ã1051 I The term "oscillatig~g magmetic field" or "oscillatory maynetsc
field" r~~fers
to a magrzetic field that pe-i-iod-ica11y increases arid decreases its
irrtenwity, rlr, or wlricli
othenvise varies over time.
10052] The particles of t~ic present invention may be in any polybÃ:drai
shape. The
term "polyhedral" as tisc;d lie.reiri refers to of or relating to or
resembling a l,olyhc._dron.
The ierin "pulyhedron" refers tc) a three dimensional ol~ject boLindcÃ~ by
plane

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
14
po1ygoni or faces. T}.ae terna "polygon" refers to a mtiitisiticti
<;cornet.ric figure that is
bnui7ci bv matiy straight iiÃ-ics; such as a triangle, a sriuare, a
pcrita~~~ri, a ~ie:~a~~~ri, a
i~epta4ron., an octagon, atic{ the liike. FoÃ- cxatnpic, the particles oi't~ie-
pr~s-etit i~~~~~~itiori
mav be a cLibe or a tetra.liedrai.
10053] The te:rin "radio freqLa~iic.y" as used beaeiii refers to a
fietitieiicy or interval
of trequeiicies witiaiii the elect:roaxa~gnetic spect:runa Lised fibr
com.munÃcatÃons, usually
dcfiiicci as :~paiiriiiig fi-oiii aboui 3 k.Hz to about 300 t.i=HZ, w1licfi
c~~tTesptlrtlds to
wavclenubs of atjotit: 100 km to about I mÃii rcspectively,
[0054] The term "radio trequeiicy tag" as tised herein includes radio
frequency
icÃeait'tfj cation (IR~~ID) tag:=. Radio-frequency identification (RFID) is
aii azitomatic
identification method, relying oii storing and rcmotely retrievi~ig data
Easin;,~ devices
called RF:(:D t~w=s, Ati :ft_[tID tag c.ati be attached to or incorporated
into an object i=or
the pur-pose of id~iiÃificatioii Lisiiig i-adit-~ waves. RFID ta~.s come in
three geÃ~,erai
1,)wsrre, serrri -pwssive (also known as or crctive, Passive
tags rec{u:irc iio iriter~ia( power soiirc.e, whereas s~i-iiiwpassive and
active tags require a
pt~~vea source, usually a small battery.
[0055] The tenii. "resistance" refers to a measure of the ~ie(zrec to wi`iicb
an object
opposes the passage of aii eicctric cLirrent as re~.~resclited by tile
equation, RmmVs/I,
where R is the resistance of the object (usuaIly measured in ca}iins,
equivalent to J
s1~"}; V is the potential differe;nc~ across t.iic c3bjjec:tx usually measured
in volts, and I is
the curreiit passing thr.c~~igii the objjcc:t, usually measurc;d. in amperes).
1.005G1 The presence of a~iv sLibsta.iice in a inagnetic field alters that
field to sc~~iie
cxtent. The temi "susceptibility ettecx" rcfirrs to the degree to which a
sLibstance't
inherent magnetic moment prodtacc s polarization wheii placed in a ma:,rtctic
tieid.
100571 ft'Iic tcrins "two-dimensional" or ::2I)'.' are Lised interchangeably
herein to
refer to a figure, ol~j ect or area that }ias i1eight and width, but no depth,
atid is therefore
t7at or planar.
10058_1 The terms "tbrcc-dimc.nsionaI;~ or "3D" are used interchangeably
herein to
refer to a tigtire, cgb_jtact or area that bas height, width, and ~lepth.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
100591 I`lrÃ: particles of the prc5eait i.7averi-tiÃxn are fabricated using
aÃ. least caric
material selected :firÃ~ni tkie M,rotip consisting of a rTlctal 0-neaning aÃ-r
clenicnt that is
solid, lias a rxtet.aIlic lÃa-,terõ is malleable and dtrc.til~, aticl conducts
both liei:t arid
e1cctricity), a poI;;iiier, a glass (meaning a brit-tle transparent solid
witfl iÃreplar
atomic structure), a Seniicoridr.rctor (meaning ari e:leriieritõ ~swc1i as
silicon, that is
inÃerrnediate in eleÃ:.trical conductivity bet.weeri cor-rdLrctorS and
insr.rlat(yrs, through
whicli cozidrictiori takes place by mear~s of lioles atid electroris), and
ari: irlsLilatcar
(meaning a rxiaÃerial that is a poor conductor of 1~eat energy and
electric.ity). They
were designed as rnitaiat.Ãrre Faraday ca.<weS in order to facilitate
detect:ian in :~::tR:l. The
particles shield (r~~eariirig protect; screexi; block, absorb, avoid, or
Ã?themise prevent
the e1fec.tS of) tlie oscillating niagnet7c fields in MNM that arise fi"ori-r
radio frequcricy
(l~F) ~~a~lses aÃid t~~~.~wt~.et:ic ~iclcl ;.~r~.die.i~ts: in an itz~~.~~ir~.
; sequence. rl"~l~is sl~ieldirx4~;
occurs as a result of eddy ctgrretZts (meaning circulating currents induced
izl a
coiiductor aiioved tlia-nugh a ma>r~etic tÃelcl, or ~~l~icii is subjected to a
war-virig
ma#.~rietic field) generated in the -l:raÃiie cyf tE-ie partic.lc that induce
a local magnetic
field; which interferes dest.nic:t:ivcly witli the exxerrral mag.rret:ic
1=ie1d.
100601 In one aspect, the present invention describes the self-assembly of 3D
metallic particles frorn 2D ~.~hoÃo[ithogra:pli ically or
eleetrtilithogral.~hica.lly
micropatterned precLrrsors. The terms "ph~~to1ithography", .:pbotÃa-
1ithography", ar
"photolithographic process" refer to a. lit~ographic techiiique in which
precise piatterns
are created ori, substrates, sucb as mctaIs or rcsiris, through the use of
photograpbically6
prnducecl masks. Typically, a SÃrbstrate is coated with a photoresist tri.m,
wliich is
dn'ed or hardened, and theti exposed through irradiation by light, strch as
ultraviolet
light, shiriiaig thruÃrgh tl-ic photomask.Thc urYprotecrecl areas then arÃ;
removed,
tastrally through ctching, which leaves tlic desired pat:terriS. Electror~
beam lithography
may also be used to create the per-f'arat:ions or pores.
10051] Tl7c. particles of tlic prcScrit invention are. Self-ti}lding and self-
assembling.
The at least one hinge of these Stnactr:rres coriiprises a ma.terÃa.l,
inclLr+:lir~g btit not
limited to, a solder (meaning an a1lo~= formulated to have a specific melting
point for

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
16
u:+c J17 ]C1n1t7E_~', i'Ãletal;;), a 11#;tall9c alloy (mcs311Ã3g a
Ã311)S.tui'e ['o31t'c713iÃ3.~w, twC? or 31C]'e
nieiall:ic eieriÃcrÃts or gÃ-icta.llic aid nc3rlrneta.ii:ic clenicnts usually
liÃsed together or
di4so1v-in~,r irÃto each otkÃer .wlieti molten), apc~lymer or a. glass thai
cai be tiqr.Ãetyed.
The surfiac:.e tension nl" the liquid hinge provides the force necessary to
fold tlle "D
template iritn the 3 I) particles.
1.0062:1 In another aspect, after se(faassemti1y, the fiilable ceÃiter chamber
of the
particles of tiie present iiveitiori is available as a vessel for
encapsulation caf
t(-ic.rapeutic agents. As tised E-iereiii; the ÃerrYi 'the.ra.peutic agent"
refers, to any
pharma~cuÃical agent, composition, gene, prot.caÃi, cell, molecule, or
subst.ance that can
be Lised to treat, control or preverxt a disease, xiiedica.l cc?xditioxi or
disorder. The term
"cornpnsitit:Ãn" refers to a rnia.Ãu.re of arÃgredients. The t:erri-i "
pharrriaceutic.at
composition," as Lasc:d hereiri; reters to a c.oinposition, which has under
gone federal
aegulatory= review, "I'lie t.errii "treat" or "treating" iicltÃr;ies
abrogating, substantially
inhibitin~;, slowing or reversing the ~.~~c~t~essinrt of.' a coriditiori
5ubsÃantially
arÃieI:ioratino clirÃic:al or syniptoa~is of a condition, and subyt:arÃtia.lly
pr-eventirxgthe
appearance of clinical or symptt~nis of a c:c}adition. The amount of a
therapeutic agent
that resÃ.rl.i in a thz.rapeutic or beneficial efTect following its
admiriÃstration to a aub~jc ct,
iÃicludirig humans, is a `'therapetirÃic amoÃ.ant" or "pharmaceutically
effective arnouÃ1t".
The thera~etrÃic or beÃ~eficial etl'ect can be c.a.rriÃxg, rxiiriimizrrig,
preventing ar
ameliorating a disease or tlisordcr, or may have any. other iherapOrÃtiC or
pharÃnac.c>trtical beneficial e6c;t,t. The terÃi "disease" tiÃ
">dis~.~Ãdr:*.Ã=," as r~sed herein,
refers to ai impairment of healtli or a condition of abnormal fr.nct:icming.
"I'}ie term
:.syrr.drorne," as ti:~~~i herein, ret'ers to a patternof symptoms indicative
of some disease
or c;ondit'itgn. The term "injur-v," as usc ki hereiii. refers to d~-nage or
barrÃi to :-l
sÃmeturc or tianction of the body calÃsed by an outside agerit or force, which
may be
physical or chemical. The temi "condition," as a.rsed herein, refers to avar-
ieq= of
health states and is meant to include disorders, diseases, or injuries ca.usCd
by any
underlying mechanism or tfisordcr, arict includes the prcir~iotiori of h"l thy
tissues and
cargaÃis.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
17
100631 In so.me era-ibod.irnents, the fillabie center cl3arribcr of the
particles can be
i-ascd to encapsulate sLaclz therapeutic ag;ents as _pliarr-fraceratical
agents or drugs, liairx4;
iis4ue. 4rels and polymers, tvliicli subsequently are rc:ieased in sitr.i. As
used liereiri,, the
t.er.an "pnlyrncr4J refers to aÃ-iatri.r-al. or- syr-iÃhciic compound
consisting of long, repeated
and somcti:mcs branched chains, built rip from small suburlits c.a.iled
morlor7lcr.s.
Nattrr-al polymers incla.ide proteiris (polymer of a:riiiiio acids) &
ceI(Lilose (polymer of
s~igar riiolecules}. I'here are mariy cxariiples of synthetic po(ymcrs,
f00641In so.rnc embodimcrits, #i:znctional'==: cells +(c.~~ ;panc.rcatic
is.let cells, ncurcanal
:l't 122 cells) can be ericapsuiated t~~r in vib-o and in vivo release witli
or without
immunosuppa-ession. Sr.wh par-ticles cati be administered to a subject in
xieed tl-rereofi
by iiiic,roir~jcction, eithcr as a siÃ-igle biocoritairicr or as :1. ;rou~~
of"biocontairlers aÃid are
useful for imaging, diagnostics-, and t:hera.peutics.
100651 For exaniple, in oae embodimerit, f1ie iritericars of a multitude ~f
particles
were filled with cells that were cziibedded in agcl. I'hese cells could be
released by
imrrie.a=sing the biocontainer in ari ap}irc3pfiatc so(verat. `I'he
rYiagrictic resoi-ratic,c (MR)
images of the pailiclcs embedded in tlr.lidic media srr~gest RF shielding and
a
sr.e4ceptibility effect, prcg-v-iding tharac.tera ;tic 1-aypointc:nsitv
(+:larknc ss) wiihin tbc
particle, thereby alk-nving the particles to be easi ly detccted, 'I'lii-,
dcmoÃistraticrl is the
first st:ep toward the design ~~f "ID, micr~~laatterncd, rioriairiva.sively
trackable,
encapsulation and delivery deyricca,
100661 fi'he present invcntiozi provides a three-dimensional particle
i:omprising a
plurality ot ovo-dimensional 1`a.ces capable of self-folding to form a holimv
Ãnteriar,
wherein asi~e of the particle is mic.rosc.a(e or nanc}sc.ale. The particle
preferably ranges
in size from 1 nna to 2 mm.
100671 The part:iclc flrrtltcr comprises at lea-st oric hinge, whÃcli may be
ccrriipriscd
of any liquifiable mat.erial. For example, the hirage mav be apolvriier, a
gel, glass ar a
m etal .
1Ã1068_1 The parÃicle of the present inverit:iori Iias any shape, but
prefirablv has
ster:fac.es fc?r'ming apolyhtadra( shape, sr.rch as a crrbc. The particle's
two-dimensional

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
18
iaces are pat_temed with perf1wations oi` pores. These:: perforations or pores
may be
created phntolÃt:howrapl-iÃcally, electroiithog;raphÃcally or using electron
beam
lithography. These perforations or pores 11az~e a size ranging frorn 1bOÃJt
0.1 tirl-l tÃa
about 1crn. :('ret:erably, these perforationS or pores have a size frori-i
about 1.0 rti.m to
abaui :l cm.
1.0069:1 '1'he liai-ticte (container) of the preseiit invention may be
fabricated from
aiiir trtaÃerial, laut preferably at least c~iie material selected 1=roiti the
group consisting caf
a i-zietal, a polymer, a glass, a SemicondÃtci:or, an ii-isÃalator, and
co.mbinations ttiereof.
'I'lie paa-ticle n~ay also comprise active electronic or sernicondiÃctor
ecyiiipcyrietits suc1i as
transistors, sensors: aetuator:=, lig}ii emitting, diodes, photodiÃ?des and
solar cells. If the
particle is metal, such metal iiiay [~e copper oà rric_h..el. In otie
embodiment, t~~e particle
is a. Faraday cage. In another einbodi.itient; the parti~~e a~av !~e coated
with a
bioeompatible material, such as a metal, a pol~iner, or a Ã:,onibination
thereof The
panic1~ ~iia~~ further be associated wil(i abiowrtsor.
1[)070] '~(`he particle may#iirtl-acr comprise at least oÃ~~ substance, sue~l
as a
therapeutic agent, eiicapsulated within the paÃticis;.. The therapeutic agent
nlay be a
cell, zt chemical or biological a~~~.ait, a pharmaceutical a.gent, a
c:oml?ositi(iri, a tisstÃ.e, a
gel, aiid a polymer. In certaiii embodimerits, perforations or pores in the
two-
dimensional faces of the particle allow release of the contents of-the
particle.
10071.1 The particle of the present invention may, be adminis"tered to a
subject. 111
such ~~~i etnbodiiiient, the loca.tio~i of the pw-ticle in the subject may be
non.ainvasively
tracked bv magnetic resoÃiaÃice imag-ing or CAI, scar~ (Crl"). 'l`~e particle
may be
imaged with iieS.sat:ive contrast relative to back<wr~~~~nd or positive
contrast relative to
ba[:kgÃ"Z713t7d.
100721 liz aiiotlier ~rnbcdiment of the invention, the particle additioÃially
con1~.~rises
a radio frequency tag, wherein ttie sa.ibstata~~e may be released upoÃ~ the
particle's
expoSÃirÃ: to a pro-Selected frequency.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
19
100731 In a t:r.rr't~~~r embodi.F~ient of the partic:le Ã.xf the rnvel-
1t.i011, the substance rr~ay
be released Lipnri the particle's exposure to electrori-ia(Si-ietic radiation,
wI-rich r~iav be
tri~.rger ed r-etrioiely. The eleetrcsr~~agcic:iic radiation i-a7av r=arigc
from lK I-iz to 1Peta Uiz
1U074J In a:fiÃrrÃber cgnboclirnent of the particle of the .invent-iorl, the
srrbstarice may
be released tilaort the particle's exposure to indLactive 1~eat:ir~~~;_ Sueli
iriductive heating
niav be taig*..~ereci reriiotely.
100751 '~('l~e preserzt irivcritioÃi also provides a incttlod. o-t.'
fabricating a tklree_
dinier7sional partic[c cÃ3rzilarising a Ãiiiilt:itLide of Ã~.vowdiinensional
faces that )`or-r-fi a
hollow l,olyhedaal sliape a:rid contairiirig a tillable center chantber. This
txiethod
cor-aprises, the steps, (a) fabricating a multitude of two dimensional faces;
(Y)
patterning the fabricated t~~~Ã~-dir~ieri~:ir~i~.al #ac.es; {~.) paÃt:er-
riiri~.~~ at least Ã~rie I-rirt~~e ~ari
tfic patterned two dimensional fiace to form a hiÃi4>ed edge> (d) joinint; a
hir~~,~ed edge of
a first pattemrrd two dimensioÃia.1 face to a birx(;ed edge of a secand
patterried two
diriierisioiia( t:ace to torrii a kiirigedjoizit, (e) r~~~~eatirig step (d) to
fori~i a tNvo
dimensior7al precursor teÃiiplate 1~avirig hing;ed joii-iÃs bctweeri adjjacent
t,~~Fo
dimensional faces; and (f) lic:ir.rel=='ying the hinges of the t.~vc>-
dimertsion;rl tr~niplate usint;
heat to iiiiiizite selrr#'-fulding. This method a.(1~)~,vs the pal tic.leio
self-assemble.
100761 In one, embodiment of this niethod.t the hinges of step (e) comprise a
mateCiaJ that can be liqa.Ãetied. The mat.ctial rnay be a solder, a inetallic
a.llo`, a
polyn:rer or a glass.
10077] In mothcr e.m$.~odimerit of this motl=aod, step (a.) ftirther comprises
tl rc. ste.ps:
(i ) spimiing a sacrificial film ori a substrate to form a first (aver; (Ã.i)
layering a
conductive second layer cgri the first iayen, and (iii) patterning the layered
substrate by
phoCo1 i ihtagr;:tphy.
10078] liz tlicse met1iods, thc particle has a size that is riiicroscale or
rraÃlowcaIe and
may have two-dimensional faces patt.cnacd Nv it.la perf~~arations or pores,
whictt may be
created p}totolithcggraphically, and may vary in size from about 0. 1 nrn. to
about 100
niic.roiis. The particles of tliese r~ietliods may be a Faraday cagÃ;.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
100791 '~1'1Ãe invention further cumpz.ises a m~tht)d of irÃ-iagiÃig a.
particle of the
iÃiveÃa:iorà that }ias beet-i igi-iplaÃited it-iÃeà a Sr:abiect coÃ~ipi-ising;
the steps of: (i) Irladirig the
hollow irÃterior of the particle -witlà at least one substance tE3 form a
loaded part7cle; (it)
adrzÃ.ir7:ist.ering the loaded par-ticle to the su~ject; and (iii)
rionrcinvasively tracking the
particle of Ste}i (ii) in the SubjJect $?v magnetic t'esoriariee :irnag-ing.
1n one ernbodirlient,
the particle has pertoratiotiS or pores in its two-dimeiisioria.l faces that
allow release of
the sulasÃance W tfie hollow iriterÃor. In orie eÃiibodiÃiierit: at least oÃ-
re substance of st~-p
(i ):is a therapeutic agerii. 1'he therapeutic ;Ã.oerat may be a cell, a
pharmaceutical agerÃt.,
a coÃtiposiÃion, a tiqsue; age[, arid a p~~fymer.
~00S0:1 The meti-aocls of the invention also comprise a met}Ãod of treating a
condition co181p1"ÃSltl%M introducing 9.ilto aii a.t1.tI71al til I1eed of
tl"ea:l'rtlellt at least. C)ile
particle of the .iriveritioti encapsulaÃ:iracy :Ã. 001Ã1posit:io11, wlier-eitr
t1le composition iS
released through orie or more pores witlain the par-t:icie into the inammal in
an amount
sufficient tc) treat tlhe coridition. 'I'l~e pharmaceutical coÃ-zipÃaSitÃoÃi
rii~~~ be contained
within one or Ã-iiore m:icg-e~~~eads. In one embodiment ot'thi~ inethod, the
coridition is
diabetes, ar7d the composition is one or more irlsr.llin-Sec:rctirr~ cells.
1Ã}041.] The invenfioÃ-i further provides a method for imq ~ing a ~,~tii;1 of
f the
iÃiveÃiÃion that has been introduced into a ma.nimal cor-ri~.~rises using
ma~,~netic
resonance ima-ging.
10082.1 The iriver-itivr~ further provides a method for= rargeting the
particle of claim
1 to a. cell within a sLÃ~jec t eomprisirig the steps of: a) a.ttaching t(i
the particle an
antibody aga:inst aii atitigeÃ~ specific to t(ie cell; and b) introducing the
particle into the
mammal, whereizi the particle is targeted to the cell,
10083_1 1n aÃiother aspect, cells withiii or proximal to implanted putsc:le;
of the
present invention can be irnaged by MRI to evaltaate tlÃC efficacy of the
implant and
the condition of the encapstÃlated cells,
10084] The invention also provides a ii-acthocl of dc.Iiver-ing orte or more
particles of
the invention to a suL~je:et, wIie;reirà the particle is pro<Trami ~cd to
remotely rel e.-Ãse, one
or more reagents at a.n~= specitit, time and at any specific spatial location.
(n. one

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
21
Ã:rribodinienà of this niethot{, the particle is i`eiiaotely guided a~id
Una{,~ed tisir~g 1A.RI car
C"1`.
100851 Also pr~oviclecl is a method czf'reieasirtg a Ã:.ontrast~~,',ent from
the pi.rticlc: of
t(-ic iriventioti or of pg-Ã~vidisig contrast to allow ?,~I:IU or C,.l'
iniat;int; of its coÃiterits or of
stabstattces`Niit_[iin its vicitiiÃy_
l.00S6:1 A metliod is also provided for conductir~Ig non-invasive biopsy c?f
znic;rosurgery, coiiiprisiÃig dÃrectin~; the particles to a site within a
si:il~jec-Ã using reÃiiÃitc
r~~eaÃ7S, allowing the particle to captiire i~i-ic or ÃTloÃ-c stabstances
i=re~i-ii itle site, aild
obtaining ttie substance from the part:icle.
1.0057:1 Where a range of values is provided, it is iixiderstÃ?od that eacli
intervening
v<ilue, to ttie tcÃit}i of the tjÃiit cyl' ÃbC lower IiÃviit Ã.MlesS the
context clearly dictates
other.:vise, between the ~ipper and lo-~vcr fa~iiit of that ra.rig~ and any
ot~~er stated or
iÃiten,cning value in that stated range is encompassed witliin the invention.
Tlie ÃrF~-per
atid 1ower liiiiit: of these SÃ~ialler raiiges, wlaich may iiidep~iideiit:ly
be included iri ttie
snialler rariges, is also encompassed wit&i:iÃa the :iÃivent:ion, subject to
ariy specifically
excluded limit in the stated range. Where the stated range includes orte or
both of the
liniits; ratiges Ã;\c:luding either both of tliose iiic.luded Iiinits arÃ;
also incltid.ed in the
i ~iveiiÃion.
10088] Uii1ess defined othet-w-ise, all technical and scieÃitifte ternis Lised
hereiii
have the same meaning as comtiioÃaly understood by. one of ordinall= skill in
the art to
whic:li this inventioi} belongs. Although any methods anci materials similar
or
ecluivalerit to tliose described fierciii can als~~ be LÃscel in the practice
or testing of tIle
preseiit inventiozi, ttie preferred methods and materials are now deSc:rÃbed.
All
pUb(ic;atior~s mentioa-ied herein are incorporated hcrÃ;ir~ by reference to
disclose ~i-ici
described the met1iods andiÃ.~r matci'ia[s in connection witli wliic.li thc
pÃÃblicatialis are
clt:ed.
10089] lt must be noted that a.s Lased, herein and in the appended c:lairris,
t.llc
singular forms .<a', .zagid"> and "tlae" inc(ucle p(ara1 referearc.e;; Linlesw
t1ic context

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
22
clearIv dictates othe~~,vise. All techniual cmd sciei-it.ific tc::ri7is
Ãi.:aed herein have the
sarne Ãrl
eaninu;.
100901 The publications discussed het-ei~~ are provided solely for theÃr
discloswre
piior to the filing date of the present applacataoÃa. Notfiii-i4,, here:i11 is
to be construed as
an aclmiss:iozi that the p:res~rià :iÃiveÃitioii is iiot entitled to antedate
such publication by
virtue of prior invention. Fua~ther; the dates of publication provided inay be
different
froti7 the actual priblicatioÃi dates which may need to be irideperideritly
corllirmed.
EXAMPLES
10091_1 The follo'wing c;xaniples are ptit forth to provide those of ordinary
skill in
t:tie art avÃth a complete disclosLzt'e and d~script:ioii of ~~ow to make and
Lise ttie preseiit
i~~~~enÃion, ~iid are Ãiot iiiteÃided to liniit the scope ol:'what. the
inventors regard as their
i~ive tition nor are they, intoÃided to repre;;ent that the experiments bÃ;1aw
are all or t1le
only exl:rerimcnts pcrtormeci. Efforts I~ave been made to ensiir~ accuracy
with respect
to numbers used (e.g. amz ~a.Ãnts, temperature, etc.) but some experimental
errors and
~~eviat:ions :~hoL~ld be accounted for, Unless indicated otherwise, parts are
parts by
weight, ancs(Ã;cula.r weight is weight average molecular weight, temperature
is in
degrees Centigrade, and pressLwe is at or r~ear atiiiospheric.
E-varirpk- I. Tabric:irtio~n r,+af.thc C.:orataM~~~s (Particles)
1.0092.1 Fi ;uae i is a schematic dia~;;raan of the process flow used to
fabricate tlle
~ C3 containers of the present inyrt.ntic3t7.1110931 First, a 5pAm thick
sacrificial laver of poIymetlivl methaerylaÃc. (1'MNtA,
MW=996 K) Nvas speiÃi on a silicon substrate. The term "spinning" as i.ised
liereil)
refers to a process whereby a fltÃid is dropped on a rotating substratc. A '1
? in-n laver
of chrtarnium (Cr) and a 100 nii-i thick layer of copper Wt~) were evaporated
on top of
the PMMA coated wafer. 'rhe ('r layer functions as an adhesive promoter while
tile
Cu layer fiiiictiotis as a cotiductive seed laver for subsequent
electrodeposition. Since
it is necessary to etch tl-ic Cr and Cti later in t.lic process, it is nec:.-
es&iry to minimize

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
23
t}icli` thickness to achieve a i`apid etÃ;(a.. l-Iowea=er, c) minimize the
electrical resistance
of the film across the wafer dLrrÃ.raig eleeÃ.rodepositÃon, tlae rzaaterial
itaickraess has to be
increased. A thickness of 125 tir-ii was cieei-a7erl optitnal fior the prewnt
application.
After t}iiri film Ãiepositiora, the sLibstr-ate was patter-raed using pI-
rotofiihÃ?gral,lay. 'f .he
photoresist Shipley SPR220 (Rolarri and Haas, www.rolarialaias.coni;l was
first spun (ara
the wafer substrate, the thickness ~~f the p(aotcaresist was ccaratrolled by
changing the
spiti. speed aiid the titarzaber of coats. After a soft bake, the resist was
exposed to tJV
light using a aiaask: alig.raer. `I'1ae photo.mask used to pat-terta the
resisà was a
transparency mask witli six 200 Ãarxt squares spaced 20 pm apart. After
exposure, the
wafer was developed and tlic thickness ol'the resist was xneasuredusing an
Alpba-Step
p.r=rs#ilomete.a=. 'I'lieai; e[ectrc3deposit7c3ri was used to biai_ld pattern
ttae metallic faces of
tfic container in the photoresist Ãiiofd up to a taeio;ht oi' 7r15 p.m,
usir~~v; eortlftxerci~l
elect:rolytic solut:ioais ("I~eclanic: Iaxc: Nvww.teclanic,corai) containing
the metal ions of
c(ioic;e. (:`u was electrodeposited -followed by a -kliaii layer (about l. pm)
of gold (Au) to
fo.a=ri7 r7oaa-~~iagaactic coraiait-iers at-id a tliiÃi laver- (abOr.It 1 pi-
ail) of nicJ<el (Ni) to fabricate
magnetic cc4r7tainers. The Atr was Lased, to protect the CLr sL7r~acr~ from
subsequent
etr;liiiag steps witl r:Ã;riÃlÃ;r it itiert.
J0it941 A secorid round of ~.,hoÃo[ithogr-a.pl~~~ Nvas performed in order to
pattern the
hinges. A second layer of ;ST'.R200 was spLira on t.lae substrate and a hinge
photonlask
was rÃsed to pattern the hinges. The hinge mask consisted of two 1;Ãnwis
of'hÃr~ges (50 x
160 (a.tni"aÃaÃl 25 x 160 pna). The wider hsnges were at the interfaces of
ad~lacerat fa.cez
w l~ile the narrower }a~r~-es were at the ed~;es of tlie taces. AligrarneÃrt
marks were used
to ensure perfect aligrimerit of tlie 1~ingles to the faces of the 2D
pr~ectrrsor=. Prior to
hiÃagÃ,~ elcctrociepoiitiorr, the exposed t_~`tr arrcl Cr in the area of the
hlÃages were e c:heci
rasiiag commercial etclaarats (APS-100 for Cu arad CRE-473 for Cr,
'I'ec}ariic, IÃie>
~~~~~iv.teÃ:.hnic.Ã:..Ã-araa). Alt[ioLagla tlae etchants 1iave a high
selectivity of Cu or Cr ~~,ith
respect to Ni or Atr, the etch tlri-ac was c}ptimizc.d, to mirrirniz~ ~~~~~age
to the Ni or
Cu/A.u frame of the c:~~~taiiier. Pa,tre; tlnl.m.p. 232 -`Qor tira/(Ã;ar.l
1Sn/Pb: na,t.~. 183 'C3
solder was then electroplated in the hinge regioris. The lreiglit of the
hinges was

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
24
approxiiiiate[y 5Lim to about. I -5 pr.a-i depending on tlic face pattern aDd
ilie type cYl
metal Laaeci (wettinM, or iioÃircweÃting). .A_#:ter electa=odep sition, the
origirial seed laver
was etched ~iid the 2D precursor ten7plaie was itt~tnersed iri asolution of N-
Methyl
Py.a=.a=ol:icio.aic (N-MI', which dissc~~~~es, the sacrificial PMMA la;:erw)
to release the
precursors fro:rtti the wafer. Approximately 50 precursors then were scattered
in a
sinal( ca-vstallizin4,~ disli LasiÃig a pipette. A very ii~in laver of RMA-2
flux, (lzidiLim
Corporation, www.irzdium.Ã:,oÃrt, used tt) dissolve any oxide it7rttted c~ii
the solder) lvas
p(yured into tE-ie diSli. The dish was theÃi heated to 100 'C t'or abotit
?Ãrliri to about 3
mfn and tlieÃi rainped rgp tc) about `.2'50 'C t~~ about-300 'C for 20
SeÃ:,onds. BeeaÃise of
the low voltiÃaie of fltix, the agitaticin, was sufficxettt to correct for
defects in the fiolding
but iiot large eriotigb to cat~se, the crosses to ct)(lide iÃit:e~ each
ot:lier atid becoi~~~ fi-iserl.
The n7olÃen solder generated ttie l=o.i-ce needed to fold the 2D l,recur~ors
irito 3D
containers. 011 Ã:..ooliiig: ttie cotataiÃiers were perÃ~anciitly }ield
to(;et.her by solid solder
rti iiges.
1[)095] Dia.magnetic copper (Cu) containers were fab.ricated witli linear dii-
iieÃiSioÃis
of about 200 pli-a (rvhcrL one picometer isff'M ii-aeterj. As compared to
smaller or
lar.p,er sized biocapsules, the 200 pm size provides tlic maxi~~~~~~
crÃcapsula.tion volumc
while still allowiÃig the ditTtiSiott of oxy<deÃ~ and nutrients to the cells.
It is kt~ow-11 that
if cells are more than about 150 liÃri to a1~oLit 2100 ~tm away frortl the
nearest blood
vessel, the environment becomes hypoxic rR.H. Thc3n-iliiison wid L:.R Gray~
Brit. J.
C'anc:c;r. Dec. 9, 539 ( 1t355)i. In princip(e, the fabrication strategy desci
ibed hereiii also
would work oÃi. smaller or larg,-er size scales in. the design of containerS
for otIier
a.p}:slications. The 1Ãnea:r diitaenGioii of the container was orderS of
magnitude t~-naller
than the wavelength of the oscillating ~~agnÃ;tic field at 500 MH-z, which is
the hÃ~~heSt
operating frequency in our magnetic rosoriag7cc (ANI-R) scariiiers, Het1ce:
ttie size of the
perforatiotis oti ttie faces of ttie container }iacl no cietrinientaI effect
on the shielding
characteristics of the container. The thickness of flie ~aces of the container
was
clc;.signc;d to be larger thati the conductor skiri de:ptli at the frÃ;quÃ;ncy
ot'th<; racliation.
T}ie term "skin depth" refers to a measure of tlie average depth of
penetratic~i-i of an

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
c.lec.tÃ'omtzgnc:tic: ti.eld.irato a rnat.eÃ-i.a(. lt. is defined as i.he
depth at whic;li the priniary
electromagnetic (EM) t:Ãeld is attenuated byrclcc,re:lses to (lle) of the
field at the
sÃar#"ace, or to appi-rxxi~~iatciy 37 f.~ of it, -vaiue at t:i-ie scÃrf"a.cc
of the shield (A.
'1'sai.iovtch; I:Iectromatgltefic JVtr=e1esa .,41)1.Ãlicctfions
(Muwer Acatlertiiic Publishers, M.A, I ~99"),)_ A tl7:icker container also has
lov~er
conductor resistance, ensa.ÃrÃng t(iat the eddy currents persist long enough
to maintain
shielding during the time of image acclttisiÃzoÃi, I'lic sk.iÃ~ ~eptli of Cu
at 500 M:E~-lz is
about 2.9 ptt-i (C. Kittel, Intr(xlaretton (Wiley, New York, ed,, at
7(1995 )): }ieÃtce, containers were designed to ~~ve, frames with thickness
ranging from
about 7 pÃnto about 1.5 pm.
1[)096] :[terrornagnetic Ã-iickel (N:i} containers in aclditioti to the
cliarnagneÃic t'l-Ã
containers described above were fabricated to investigate tlic effect of
mag.ti~
susceptibility ori the MR images of the contaiÃaer. Map-netic field
distcÃrtions
laut nat IiÃiiited to, shape, aiiiplit~ide az~d phase clisioÃ-iiotis,
resulting from the
cliffererÃces in inagtÃetic susceptibility betvkeen an ob~-jeci and its
surrouncling Ã-iiec~ium
cause a loss of phase coherence in the magnetization of the sample. Since the
magnetic susceptibility ot'Cti is comparable to that of -~,vateÃ~, while tliat
of Ni is orders
of mallnitude higher than that of waÃeÃ-, a more pron~.~Ãunced distcartitÃÃ1
was expected for
Ni containers in ~queoLas media (L.W. Bartels, et aI., J. l EUc. lntei-v=
licOiol. 1 ~~: _3 )65
(2001)).
100971 fi'he strateg~., tise.d to fabricate both the. Cri and. Ni containers
ià Ãvralved the
auto-folcling, of 2L) metallic precursors using capillary forces. "Capillary
ac:tion''',
capillarity ' or c:apÃllaR, motion, wliicll are used interc~han1geably lierein
to refer to the
abilitv of a narrow ttzl,~e to draw a liquid upwards ~.~.~ainst the force of
gravity, occurs
when the adhesivc iÃitcmiolecular forces between tEic ligL-id aricl a solid
are stralt.;cr
t1iaai the cohesive interrnolecula:r forces within the liquid. '-['he same
effect is what
cauSr S porous materials to 5oak. L7p liclt7icls. Previous demonstrations of
acÃto-lolding
include the actuation of rÃiicroziicter size t.ompoÃaeÃats and the assembly of
~}D c~niplex
stnÃc:turtas (E. Smtala,et al, S'r:ieÃrce 268: 1735 0995)7 P.W. Brec.ti. et
al., J.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
26
<~~r~:rcr- lc~~~tr~r3rrrt~~:lr. .st 4: 170 (I.~1~~:~) K.F. Harsh ~.t~ :;~l.R
At'ftÃ~tfÃ~f.>~~ .4 ~: '?:>7
(1 E399)l;_.. [_:. Htii et a1., If;t~.fsl .a' ~`} IÃrt. [.YrartjÃ; On X-fict'o
.f.kc:ir'c~ Nfec:hcrÃfiec:zl t~.xttts,
602 (2000), D. I=4 (Ft-aeias, ei: al Adv. A-iÃrler . -l 4 ; 23 '? (2002)).
[Ut~98J .'4ccni-dirig to oiic aspect of the preseiii i~~~~erition, _>D. I-
ioIIow, peÃ-)`ora[ed
containers were fabricated from ~.D precursors. 'riie process used to
fabricate the 243
precursors, which is an extension of the process described in Exa.inple 1, and
reqÃiired
severzil. additive lavcrs, two photolithex;ra.py steps, tNvo
electzodepositikiti steps, and a.
precise sequence of subiracÃ.ive processes. Briefly, the process i~ivo_lved
i3ati:criiirig the
metallic 27D faces rflsi~~g pbotolithography and electrodeposition on top of a
sacri.ficiai
Iaver. The versatiiitv of the strateg
y was demonstrated bv fabricating precursors
wNinse faces c,orrt:aiiied two different patterÃis-----c~~~e pattern comprised
a square ti-ar~~e
with open faces, while t}ie otl~er consisted of a m:icroscale cross shaped
patterii iti the
center of each face. In asecoiid laver of pIiotoresist, hinges patterned on
the
edges of the frames. `I'Iie width oi'the lai~ige between t-~~o a.djacezit. -
t.'aces wa.s t~-vice ttie
widÃlr of the Niinge at tl-ie edges so tl-iat aCl Iiirigcci ,joia-iÃs I-iad
equal solder volume i:~pw~
foidin9: the solder vo1~ime was critical to eaast7re a folding angle of 90'
(R.R.A. Syms,
et al...r .I. I'?; 387 (2003)). After the 1iÃziges were t.,a.tterried,
the 213 precttrsors were lifted cff tt-ic wafer by dissoltitiori of the
sactiticial layer, '1`he
containers avere sel.tmassembled by 1~eating the precursors abc~ve, the
rnelting point of
the solder, wherein ti-ic liqa,tid solder with bigh surface teiision generated
t1l.e :i=or-oe
rc:c;tiired to f-o(ci ad~ja-cc:nt faces of the precursor.
1.00991 Fi,,g. 2A sbows aii optical ima<we of a c.oilect.icari of containers
that were
fabricated tising tt7e process outlined above. The fabrication ttrategy allows
a large
trUmb~,'r of containers to be constructed in a single process riin. The
primary yield-
linziting factor was the error in estiiiiatirig tiic voltitiic of the solder
to be
electr~~~eposited at eacb iiiiige. The spaciiag between t~~e adjacent taces
was also
critical-ysrhen the gap between faces was either too I'''.argx or when the
faces were
ft~sed, the yield of folding was greatly $iniited. Fig. 2B=22D show optical
and SEM
imagges ot'the mit;respattemed containers at different stages of the
fabricatÃon process:

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
27
the 21) pr-ecÃir5oi- with e1eÃ;trodeposited fiiceti, ilic preÃ;urtior witli
i.~tc:es arid h:ir~geS, aTici
t (-ie folded container.
1001001 Although a~~ opeti-Caced c.oa7taiticr B, iaot ideal for= ari
enc.ipsuilation device,
since it is corisiderablyleaky, opcn-1'aced containers were filled for easy
visualizatiO11
of their cont:entS. For M vivo applicatioris it .rtiay be de5irabIe to use the
described
strategy to construct containers wiili selectivelv sea(ed or micro/nano
perfcyrated faces,
and :(=a:1?ricate r~iore c.oztiplcti; polylicdral containers with r-oLi.rtded
vertices. A.11 open-
faced container (Fig. 3A) was loaded ~Nvith rnic.r=o"bea.ds since niany
cellular dcliaeo.,
techniques use microbeads witti cells adhered to t.faeir surtace. In order to
load the
corrtainer with microbeads, a susperision of tl-w beads irt ethatio1 ,.va=
pipetted orito the
car7iaitie.r. '1':1-le suspension entered tl-ie container as a r-csult of
capill~~~~ forces. When
tft.c ethanol evaporated, the beads were lield together by weak van der Waals
forces
4at~e~~ir~~ the weak intermolecular Rorces that arise frorxi the t:ra:risieiit
polarization of a
giveit. rtiolcctile ixito a dipole) (Fig. 3:B); the glass beads cnÃ.rid (ae
released by agptatiorl
of t Nic cor7 ta'a tie.r.
1.001011 In order to dcnionstrate cellular encapsulation, MD.~ A,1.13-23t
breast cancer
cel:ls in an extracull.trlar= matrix (EC"N't) suspÃ;i-asiori at 4 'C, were
~~~~~ied in t1w
contairiers (Fisn). 3Q, As tzSed 1-rcreitt, the terrii extracelltt[ar rt~atrix
refers to the
complex staÃrÃ:.tÃrra1 entity sÃÃrroi-rrid~~ig aaid, sÃrpportirig cells that
a:re touzid wÃtliiri
mamma1ian, tissues, as well as one or more of its constituciits incltrwiÃng,
bÃ7t not IÃmiteci
to, collagen, elastin, fibronectin arid Iwninin. MDA-4\4Ba22s1 cells are
representative
of rapidl'y proliferating cells and irximortalizcd cells, sLrch as P'I'O
cells, used in
diabetes therapy, and stem cells used M rerwenerratiorr. On incubation at 37
'C for 5
miai, the ECM sÃrStrenaic3n gellÃ;d: the cells were retained in the
biocontainer= and c:or.rld
be released by immersing tttc corita-incr in wairii cell ct.rltÃirc mcdiÃrrn
(Fig. 3D), It was
also possible to load the biocontainers witli a celI-ln,C:N-1 suspension
withiÃi an agarc?s~
~avity. lrr this casc:, a suspensiori of 5% agarose gel was tirst
micropipetted ('60 prn
tip) into the c:ontrziticr trsi~g a stcrcotac:tic: manipulator. The gel
adhered ta,~ the sides tif
t}ie container thereby 5ea(ing the faces ancl Icai>iÃi~,= a void in the center
of t1x

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
28
container. 'J`hc cell-l:C"M strsperisiozt was t.lrt:.ra rngcroirrjectcd into
this void, w}ricli w-as
then sealed with a microdrop of awY.t.roSe M~~~ci.
1001021 To demonsrat.e that ttie c.ell, were viable in the bio-ccamaincr arld
orl
release, the cells ~~~>crc stained with the fluorescerYt: dyc, C'a(ceir-i :'tM
(Sigr-fia-Aldrich),
which stains positively for live cells. Fig. :'IE sltmvS calcein-staÃr-ied
cells witltiri ttie
biocontainer and Fig. -3F s}iows release of live cclls from the container on
immersÃozt
in warii7 cell cÃi1ttare ÃiicdiÃarti. 'I'lte frames of the biocoriÃaiziers t-
tsed iri tliis
dcrnotistr-ation kiad a tkiiÃ-i gold or platiritirYi coating orr the interior
faces i=br
biocoÃ~~~atilailrÃy, qiric.e gold ~iid platinum are, inert or tÃnreactivc
materials. Pure tilt
aitd tMr`Iea.d based solders were used to fold the cotitaixrcrs. It may be
necessary to use
ot:l7cr solders cnntainirro; iricr-t lncta(s Sucli as silver arid 4old for
errliarrced
biocompatilaility. :(t: is also possible to increase ttie biocompat:ibility of
tlie containers,
bv coatirtg, the entire folded container witlt a layer of an inert metal (by
electrodeposition) or with polytiier5 (by ianrgtersion or vapor coaÃirig).
1001031 -NnnHinvasivc dctcci:ion ol'the c.oriiainers was dcri-iotist:ratcd by
cr'Ylbecldirlg
the containers in 5% agarose gel aÃid imaging them with tiIRI in a 500 X1.Hz
vertical
btire Brttker Avance micr+,3irnas~i~~ ~ystz.m. For tho images Shm? ir Irc rc,
a. s3D FLASH
ScqtrciiGC Ivith tlre cch~.~ tilne (TE) irt the range of 4-6 ms; a repetitiorl
time (TR) of 50
ms, flip-angle of -10', and a spatial resolution of 25 P111 X 215 ~ÃÃn X 20-
lun was rised.
The containers also were i~~~agetl using a standard spin echo wetluetice
(meaning a
pttlse> seclttcrice tiScci in magnetic re.soirancc irraagin<.~ based oti the
detection of a st.=ain. or
Hahn echo, w}iicla uses 90' radio.frequeaxcy pa.iIses to excite the ÃnagnetiSm
and ozte or
more 180' pt.Ã1ses to retocus the spiitS to generate signal echoes named "spin
eeboeS),
with sianilÃtr reaEÃ(ts. Fig. 4 shows MR images of a 900 }tm cli<t~-noter
c:at.~sill~ry
corrtairiirzg a CLi (Fig. 4A) and a Ni (fig. 4B) container embedded in agarose
.;cl. A
characteristic signature was ol-iSen~ed for both ttie Cu a:ttd the Ni
corttainers--tltere is a
pronounced darkness in the region of each container. These hypointense (dar~.)
signatures 1~ave bcc.i} observed before in N/1-Rl of laq4cr centimeter scale
metallic coils
(A. Shcnhav. H. Azhari, 3V1cr~,Fw. Rras-on. 52: 1465 (2004)). While the region
of

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
29
hypoiiaÃc.ÃÃsity (darkr.ae5a) in the .14IR iÃ-iizÃ~,se was coÃnpar4able -to
the size of the Ãa.oTi-
raaag.aaei:ic Or container, it ~,vas rnucla: larger for the feÃ=.rornzagnct:ic
Ni contairaer due to a
pronounced s-uscc:[Aibi1it~~ effect (L.H. Bc:ÃÃÃaett., et aI.,.l. AI)I)1.
l'h4:s. 79: 4712 (1996).
BA. Schueler, et al., .1: Xlcigrr. Resrarr, knq~,i~~~g, 9: 596 (1999)). '1 fae
iÃYaages of
containers :Ãraadc of a ,aveia material were similar for botli opeaa faced
c.ontairaerS as
well as cross taceci containers, shou;iiag t}aat the pattern of the faces had
fittle bearing
oza the MR si graaÃure at ttzi s size scalc.
fUO 1.041 RF shielding w.a.s simulated in a non-magnetic coaitaiÃ-ier with a {-
:iaÃite
eleÃ~~ent model for a 200 Sp scatc wire t`raÃxac that was excited by a linear
polarized
electÃ-orÃra4~netic wave. Fig. 4C-4D are simulation ro-SÃilts showing
rÃiagnetic field
d1st:o.a=t:innS in the vicita.itv of the cotataiÃ-ie.r :Ã.tad reduced field ma-
aait-ude in the irtaÃcri(ar
of the coÃlt ai t1 er.
() 1055 ] For maziv biomedical applications it is Ãaecessary toÃaon-
ÃÃavasive:ly track an
eÃicza.pSÃ.alatioÃi devic.e. 'I'lic (:Ã:a container of the present invention
could be t-r-acl;:ed
spatially aÃid teÃ-iip~.~rally with in flow thÃ=oia"tl aÃ-i S-shaped 500 }sÃ-
ia diameter
fluidic c.}iaÃiiicl_ The chaainel was fabricated by molding poly di~nethyl
siioxane
(PT~NIS) in an SU-8 pbotoresflst ÃiaoId that was patterned Ã.Ãtiing pt Ãotf-
Ait1aography. The
channel was seated with a. s~cc)Ãad, t'iat, oxygen plasma treated. PDNIS
la.yer,
Polyethylene tubes were conaaecÃed to ttae inlet and otÃtJet ports of ttae
cllaziÃie1, the
channel was flEished with silicone c3i1, and the container wÃ:Ãs' inrrodiiced
into the
channel. Under prcwsure driveÃr flow, the container moved withira the
clyanÃie1 andwaS
imaged at ditTererat positions; the secli-ieaxce of N~I:R[ images is Shcawra
ira Fig. 5. This
ready trackability wit~~ TNIRI. at a>erv short echo times, without the need
for a contrast
agent, highlig}its a ma~jor advantage of the 3D metaIlic: bioc:oniainers or
the present
invention as coniparcd to manly other erica.psLÃlati~.~n systems.
Exanip1e ,27, MMuMtican caf Near Alc~qtxetic H~.}l~~V in th~.~ rerg;ion r_?/`
tla~:f
container:
1001061 To denaonstrate an RF `hicldirm effect, tbe near m.~gneti.c> field
response in
the iric..ilÃity Ã3t" t(ae coaztaiÃaeà was siztÃulat.eti usiÃa.g a fin.ite
c.lcnient electroanergrÃet.ic

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
siniultztion ptacktage, F11K{_} (EM Softwarc. & Sytitenis-SA Ltd.,
wwts.ic:ko.Infoi'). A
t:i-all-wavc i-iieiiiod of Ã-iioÃ-iicaits approach ~~ias used to simulate
t:lae.taear mag-netic field
iri t}le region of a 100 Eam wirc: fracrie wii}t wire segrnents of 8 tiiYa
radius', as,'uanrng
perfect electric conductors coated witli copper (coÃidÃac.tivitv ___ 5.8 13 x
10'7 `?.na`'). 'f .lae
,imulation of the cubical wire fiariie model was performed witli a linear
polarized
platae wave excitationat 500 ?1't~Hz; we used an excitaticati sotirce of i V/m
incident oza
the wire fraiia.e, with E in the z dizectioia aiid H in ttae v directioti
(Fig. 4('). The
copper wia-c fraria.c was assigned a relative permcability, of I. thereby
yimulatiÃag only
the RF shielding etTect and n~~t the susceptibility etfects. Fig. 4C shows the
near
magnetic fi.~eld a-espoÃ-ase in bot.ii the x-v aaid the y-z central planes.
1001071 l.aa cnnclusioÃa, the dcscrAbed sÃaate;,~~can be tased to
flibric.za.ie 3D, arbitrarily
trackable, biocorat~l ners that allow perfusion bctcveen
the caÃatcnts of tlie biocotataiÃaer~ ~iid the si-irroÃiÃadiÃig medium.
'I'laese biocontainers
are eracapstilaÃzozi devices that do iaot lose their detectability wlaezi
loaded ~vitb
biological coritcta.Ã. Due to their Stre.ta~;t:la and iiigii porosity; stacla
m"eÃallic
biocontaiiic~~~ axc: uscftÃi as basic eiet1~entS of a scatfE}ld to gt7idc the
gror~~Ih of cells in
_3C3. Since the fabrication strategy described. bcre is cora~patil_ale with
conventional 2D
nzicrofa.bricaÃion, it also may be possible tÃ.~ add electromechanical modules
for remote
activation, -,vireless coraamLaiiicatioai: sigaaaJ processing, a:iad
biosensing to the faces of
the bicscotitai~iers,. to enable medical diag-nostics and therapeutics". The
present
invention also otivisioz}a that sucl~ ~'I%D cotitaiiie.rs, whicla fttracti~n
as small Faraday
cages, avill iiid Latility in z ~tiaer applications requiring electromagnetic
s(aieldiÃig in
small o>olumes.
E.~~~~~~e 3: Aficrqf4bt~ication c~~id Se?f-Avs~.anzblV qf 3d Aflcrobox~-y,v
fi)r
Biot~~edical AlyWcations
~r ~:1~?t=p~~t:~1 t r:~t~:
1Ã10:1.O81 The process t~sed to ttibricate the boxes consists of
micrcrlabriÃ;at7on arid
Sttr~ace tension driveia sclt-asscml.~Iy, [h.F. Harsh, V.M. Bright, & Y.C. I
ee. S~n.s.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
31
Aciwricai=s A, vol. 77, 23 7-244, 1999 ; E..[.:. .[-lui, R. T. 1-;*owÃ,, & M.
S. Rodgers, in
13th .Irrt. C70.rf on pp. 602tt607; R.R.A. Syms,
EM YeatÃnan, V'.M. Bi-iglii, & GM Whiiesides,,I vol.
12, pp. 387.417, ~'~(}0? 1 io fabricate atad fold a 2I3 precursor :into a 3:D
hollow
,Ãauct.ure. I"l~e fabrication process involved tfiree steps: (1) patterning
the faces oti tlie
2D precursor (2) patternin(; solder hiiiges bemNreeta ttie faces, aild (3)
selt=assembly of
the 2D precursor (:(tig. 6) `I'be boxes self-assembled wtieii the precursors
were heated
above the meltiiig poiÃ-it of t}ie solder, where:in the liquid solder with
high surfiace
tension geiierated the force reqtgired to assemble adjacetxt sÃ.lrtaces. The
fabrication
strateg"y allows a large raainber of boxes to be constructed in a single
process nin.
Copper (C:.`u) atid t-iic.kcl (NÃ) boxes have beeti fabricated with and
without gold (Au)
coated SurfiaceS (to it3crea~e baoiitettne"'a).
Defet:t Modes:
tOO1091 Several defect axiodes iverc observed (Ci4F. 7); hoo~~~ever tvhen the
process
was optimized yic:lÃi` as high as 90% froz~i a single wafer wer-e obt~Ã_irled.
Apa.rt f:rc.~rn
obvioais defect modes Sucki as over electrodeposition that merges t:lle taccs.
;nmcnt cst'the Ia~~igcs i.vitb respect -to the f-iices, atid over or Ãs.i-ider
etciiing of the
sc;ed laver, the lar_gÃ;st defect gtc~ was ttae hei<:~~t of st~ld~:r`
Ã~l~:Ã;t~Ã~t~Ãi~.~~t~;;it~:d at the
~es. I:t: too Ãliticii of- too little solder is cieÃ;trodepoSited the
structure OVer Or Li.rtider
folds=. Ã11 order to deien-iiiiie opÃiiv~~~ira solder beaght ~'or 90"
.tiolding;. published design
rules [R.R..A. Syms. C.M. Yea#.man. V.M. Briglat, & G.M. Whit:Ã.sidc.s, 3,
<t~r~:rc~~~Ic~trr3r~rt ~:#rc~rric r.r1 ~~ ~., 'vol. 12, pp. :347-4:17, 200:~1
were ÃiSed. Additionally, iTi
order to iiicrease the ei-ror toleraÃ-ice, liir~~~s were desi,,:ried t3etweeti
adjacerit faces to
be tzvice t(ic widtli of kateral solder re(;iot~s patterned along the edges of
the faces.
During electrodeposition, due to the elevated temperature (200 : CY) the
precursors ,rere
agitated (due to convective tlow Ãn the flLaid in which the boxes were
seIfwaSsembIed);
this agitation aided in correcting metastat~~e mininia (errors) aiid helped
the box fold to
the thermodynamic mirziÃ~iuÃii,

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
32
L0atiiiw:
1001101 To demonstrate that the boxes could function as encapsulation
deviÃ:es,
they were loaded with a variety of medically relevant constituents
inclt,tciÃ~~ gels,
beads, (i.qÃaidsr and cells (Fig. 8). For easy Visiralization, boxes w-ith all
opei1 tat.:..es wt;re.
tased. f=1owea=ei', .iÃa real applications, boxes witli oiily one c~~~eTt face
fcir loading, w-iÃb
the otkaer faces close(l or pi.grcÃLis-, wc3Ã71Ãi be Ã7;cd.
IÃ30.1.l11 The byclrogel PltarorÃic; F127 (201NO ;soltttion) e\hibits a thern-
ioreversible
transition from a l:iqÃa.ici solution at l~,,,v temperature (e.g. 4 "C} to an
ordered micellar
ctibic phase at roniÃi tetrzperattare_ This pi-operiy ra-iakes ii: vet)5
attractive in tttc storage
and releast. iia datag cielgoe.ty. The 1Ãvtizoge( consisted ot':;t 20% w!"w rÃ-
lixttare of1?ttaroDic.
I. l d7 l,pt:Yly(etlav1eiÃe oxide,)-blot:.k-lacaly (propylene oxide)block-
poly(ethylene oxide)
capolvzrÃer, (BASF C 'c~rp. -www.bas1'.t;om) in wat:er. ~l'';'tie saiaip_le
was staakera tisila.4,) a
vortexer to speedup the atixin.g pÃ`ocess and stored Ãtt 4 `'C be-tbÃ'e usage.
In order to
load the hydrogel in the bo\, a drop of the liKltticl solution was placed sati
the box. ;l7ta~
to the b;.droplailic: si~~~v~--alls t.~-1`tkie ri~~tal(ic boxq the
solt.itit.~ta r-eadily eÃatered the box.
'E3t~s.a.es were also loaded witlà "~=1==;DA11!fl:B-'' 31 breast cancer cells
era-ibedded ir1
exÃ.a-itcel:ltalar rnaÃ.t-.ix 41:C:1~1~ gel. (NV1:C3A-=k-1B-23 i cells a.rt-.
representative of rapidly
proliferating or .iÃa-irÃ-Ãc3rtta:lired. cells such as fiTC'3 cells tased in
diabetes therapy, faTid
stern cells Lased in regeÃ-acrat:ioti}. FÃg;_ 8C shows: a box loaded wit:li
cancer ~ell~ that
were briefly suspended in ECM glel at 4 'C. 'I'lie suspextsic?tx was
introduced into the
lanx arÃd was kelat at 37 "C for l:5 r2aÃ.ta to allow the ECM gel to
polyraa.erire. The cells
were stable in ttie box, ;Ã.tid could be released (Fig. SD), by pulsatile
agitation of the
box. ':I'}aes~ experiments clemottst:rate that it is relatively stt-
a.ightl<cyruard to load tla,e
1?o.x.~s wiÃli a variety of coristitLietiÃs.
lnterartion with RF Fiellds:
lÃ3O 1.121 Sirice tl.ae boxes ai`e rnet.aligc tl-iev iÃÃtei-at:.t witl-i :RF
fields and belifa-ve its
Faraday cages. This fÃsa.ittrtr tta~ bet;n Ãtsetl Lo ctet~.~ct. Ãtrtd track.
the boxes rt.n.totelrr tÃ:;ino.
magnetic reswaztrÃce gnaaging (N'l:RI). A t}.aaÃacteristicsi<:ÃÃattare was
sabserv~.acl fot bcitb

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
33
the Cii and the ^+;:i boxes-there was a pronotanced darkness iTi the region of
each box.
This hypointense siwt-lature t`ac.iliÃated .re-ady trackabiiity wit:li M:Ett.,
at siroÃ-t ecl-io tiriies.,
without itie need for a contrast agent, arid highlighted a major advantage of
these
encapsulation devices as eoÃ-irpared to existing polyme.r:ie systeais.
1001131 Since the boxes iriÃeract witlr RF fields, this firatLai-e su,= ests
the possibility
of inductively hea:ting the box using a remote RF field generated by passing
azl
aItezzrati~ii), ctirr-erit tkrre~uggh a coil [:E. J. W. `I'er Maten and I B.
M. N-Ielissen; vol. '28,
nn. 2, pp. I 287-1 `?9i3, 1992; C. K. C'boLi, in -5r`!r C "orrf
i=trmr=zrtrzentalroa,r trml
<,WeasurfAmc?n/ Iechõ 1988; pp. 69a7 r, J S. Curran and A. N-1. Featherstone,
J.. vol. 2, rio. -1: pp, 157-160, 1988; K. Hamad-4chifferlx. J.J. Schwartz,
t.T Sa:zitos, S.
Zhang arid IXII. J~cobson, Ndirrric , vol. 41 5, pp. ! 321-15-5. 20021. 13oxes
coiYiposed of
dia.a~~~gnetic t.CLi, Au) and ferromag.Iletic (Ni) metalc were fiabri^ai:ed.
W~~~ii a box iS
placed in a coil throu,,,,,h wliicft aii AC curreat is passed, aii
electromagnetic force is
induced. According to Faraday and :L.:eriz-'s Law, E =-N" aIr` , (t ), where E
is
eIectromotive fii}r~~~ (Ei~I:F) indueed :in the box, tp is Ã-nagrret:ic f'l~ix
generated in theRF
coil. and N'is the number of the coil tums. The i'=.ndt7ced E:~~~1~' caLiscs a
cLirTent to flow
in the box which can cause Iie.atii-ag, TIic heat generated can be calculated
a.41' = .~-~,'` R.
(2)Y. wlier'e 11 is the heating power ger~~rated by the cur-r-errts, aiid R is
tlre resistance of
t:1le sample.
1001141 The alternating current i-n the box is stibjec t to the skin-dept.~
~hono-nienon,
i.c. tlic c::trr.re;nt den.si.tv decreases with deptli. Sirice the thickness
of the surtkes of flw
boxes can be controlled with a raii9c of tbicknesses limited only by the
photolithographic as~ect ratio LÃsed to patt.em the 21D precursor, bka:~es may
be
fabricated with wall thickness con~para.b(e to ttie skiii depth to mitiimize
the e1eotrical
resisÃaire.c. Additionally, if the box is ferromkgg7etic (e.g, Ni}~ t.he. h
eatirrg is iarcreased
due to magnetic hysteresis. As the prima~y purpose of irrduction heating is to
maximize the heat oncrgy generated in the box: the apert.tirc of the inductive
heating
coil is designed to be as small as possible wid the box needs to be fabricaied
w~~~ith a
matena1 that i'eattires low resistance and high pc;rnieabilitv.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
34
1 (1{}l 151 '('wo kinds of configurations were d.eincmst.rated. [n s~iie case
tl-ie btsxes
were iritrntluced iiit~ a v:ial arouiid wliieli is wrappecl a e.Or~ coil
th.roi.E4,~li which AC
current is passed (200 M'E4z tc) I C31-1:r, 0 i tcz I Watt). In order to itite-
grate heating of
t(-ic boxes wiili '?D Ixzic.rofluÃd.ics, and to maximire inductive coupling,
21:3 coils were
also flabr.ieaÃed. "I'he 2I) coils ar~ fa~iicatecl photolitEio4>-rap}iically
(Fi,x 9) aiid cari be
made with a varieÃv of ta.irns a.iid s~acrng. 'l`he box is placed along, the
central axis of
the coil in order to maximize iÃiduetive heatirtg. A:liliougb the ziuÃiibez"
of turnsin the
'?D coil is less tliarz tE-iat: o1'the 3D co:il, the eavÃti, of'the ?D co:il
is eorriparable to the
..,ize ot-the box to maximize liiduetive coupling, hiduct:ive heatino
characteristics of tlle
bnNes aÃ-ld the coils are 1ieijag measured.
R.eleasiti; a chemical froni the bcax upon hÃ:atln ; ~;
1[)01161 :I.n order to demonstrate Ãhat a cliemicaJ cotald be released irnrII
t:l~e b nx-
upoÃl k~eat1lig, ilie l?c~.a was loaded with a l7ydrr3gel that was dyed red.
lnyÃially, one
g.a-ain (.>C l'(ti.a-ÃsiaiÃ:. F88 (Molecular weggftt.: 1. 1.400.meIting
pÃ:aini.: 54 'C, obÃ_ainecl t:rom
BASF) was c{issoived:ir.a 1.0 anl. of aÃ;elonc:, '('lle sainple o-vas t1~eTi
heated aTid sonicated
to aid disso[ui:inn. A few drops of the dye erythrosine were added to the
sÃ?lutlori. Ari
open fkiced box xvas loaded with. tlae dyed lavdroge1 solution usi:ti- a
sy.rin~e and the
box. was allowed to sit until the aÃ;etor~e evaporated. Sr~-icÃ; the hydrogel
dissolves M
water, the loaded box was iÃiimersed in dÃ.~dec,iaie (hydroc'el does Ãiot
dissolve i11
dodecane) and placed on a glass sd.ide Tl~~ slide was heated tÃ, 70 'C' on a
hot plate
and optical. photographs were taketi at 3, 7 and 10 m.ii3ÃitÃ:s. 'l`lae ge1
softened t~iid the
dve was released icito the dodecane solution ffig. .10). The release of
chemicals fircam
other l7Nrdre~.o,els in water, as ~~~ell as c~l~tirvliri~i~; inductive RF
t~eati~~u~ of the loaded
boxes, is Ã:.Lirr~iitly being ~~ivestigated. For in-vivo applicatioxis it will
likely be
necessary to Eiea:t tlie box approxirnateiy 10 "C' above the temperature of
the human
bÃ`~dy .

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
CONC: LU~SiONS
1001171 Tia starni-nary, a azew erac.ap sÃa.IÃttic.iia device platform that
cÃ.~iaibiriÃ;s ttrc
tavtarab1e aspects of three dimensionality with Si microfabrication has beerr
dÃ,'rt-itansÃrafed. Development of devices rrsiÃb 7aanopt3rous faces fclr cell
encapsulation
therapy (without imr-ntarac3stri~press.ion) and ciesiizÃairig boxes with
optinlired RF freat.ira_g
profiles for reriaote relea;~~ of chemicals as in prr.3g:ress,
E.Ya~~~L)le 4: R~.pnirt~.t
.~ Radio-lT~rUt u~,pn. c ? Controll~.ad Nano1her Ch~-yntistr v
arrd Cizeitir.c=y l I)ef.,avc~.rr, (rn .S'y bgrat~.~s,
1[){1118] Containers }rave been tab.r:icated Or.rt Of ta-leta.i, wh:icli
a_lioNved tl-iem to be
rer-zirst:e1v coupled to electromagnetic sorarces. I'lY:iS ~eatrare was used
to eraable wireless
control over btrt.}a the spatial guidance (using magÃaefic containers) as well
as the
delivery of naraolitr,r vo(uziir,s of chemical reagents. `I'i~e containers
cati be gr:iidrwd in
spatial p,a:ttems tE-iat: are aiot lii-raited by flow proffles.iÃa
eotivetatiorial aiierofluidics, that
is, downstream fto:m a channel iiiiet. Tt7e remote-controIled iiaric?liter
containers
enha:ncet~e capabilities of present-day xAcÃ-oflÃrid:ics by enabling spatially
cÃ~iitrolted
c(icziiica1 ~~eactioÃiS, miÃ:rofiabricakiian N.~iiÃbin capillaries, aald ola-
dezYiatid localized
de1iverd~ of chemicals to crxrlÃu.red ce(ls_
~
100119_1 A combination of conventional mier~~fibriÃ:,atiran and self-assembly
[1-'. G.
f,..eorrg: Z. Gu, T. Ko[i, D. H, Gracxas, J. Am, Ã;laem. 4oc. 2006, 128:
113"36-11 337; &
CJimi, T. LeoÃag, Z. Grxa, M. Yang, D_ AÃ-teÃiiov, I. N-1. L3tlr.riwa11a, D.
H. Gracias,
Biomed. Microdevices 2005, 7, 341-34,57] were used tÃ) fabricate g0ld-coated,
nickel
naÃaoliier contairicr5 (FigLrrÃ; 1I a). To facilitate chemica1 delivÃ;ry, the
c..orltairier-s were
filled with a gel that was soaked in ti-re chemical reagent to be released
(Fi.;tare 11 b).
'B.N~o gels were r_.rsed~ pluroraic.151 for general dry-release exprwr-iÃnentS
and po1y(Ne
isopr~~py1acry1amide) (P1~IPAna) [T. Hirokawa, T. Tanaka, J. Cheni. Phys.
1984, 81,
63i9-6;>80; N7:. E lsl.am. A. INI6~~saved, Z. DogiÃ. J. Zbang. T. C.
Luberi41cy, A. G
YocJii. Phys, Rev. Lett. 2004, 9`21. 088303] for cheriaicai delivery in
aqÃreoÃrs solutions
aiid to living cells. P1r.aroÃaic is a water-soluble block copoIvraier
bydrogel that scatteras

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
36
at 52''C and is c:ompatiblc witl-i aiv.idc ran;Fe of chemicals [R
A1exa.ndridis, `I`. A.
Hatton, C:`oiloid SLart. ~i-ivsicoclieÃii. l;no;. Aspect. 1995, 96, 1-46].
Hydr-ogels based on
PNIPAmIfi are thermoresprxrisive materials tliat are widely crsed in drLÃg
delivery,
because Ã1~ev ut-idcr~~o a structural tra.rasÃtion racar- the temperature rai-
r,,~ of tl-re I-ILtmarti
body [-H. Yu, D. W. Grainger, J. C'ontrolled. Release 1995, 34, ilittl?i; K S.
Soppimath, `i'. M. f"triiiriabliavi, A. M. Dave, S. G. Kurnbar, W. E. J-
',LrdzÃraski, Drug
Dev. Ir7d. Pba.rm. 2002, 28, 957-974]. This transition temperature, as well as
the
collapse kinetics ni":Ã'N1PAm, cati be altered by addirtg co-moticri-rers aild
cliarigirig the
degree of caoss-linkino.- [(~_ A. Stile.; W. R. Burghardt, K. E. Healy,
Macromolecules
1999, 32, '7370-::379]_ Hence, PNIPArn i5 ati ideal candida:te for remÃaÃe-con
trÃ?lled
release to living cells at-id in liquid ri-iedÃ'a.
100 1.201 Once loaded, a container was placed iri tlic react.ion vessel of
elioice arid
could. be guided in ariv spatial tr,~jectory Ã.isi~ig a magnetic stylus. After
guidaÃice to the
desired 1oca.t:iori, a radio-firequericyr (RF) #Ãcld gcrierated by a 243 micÃ-
ocoil wa.s
directed towards t:lic coriiait-ie.r_ The pov,~er in the RIF field coupled
iriduc[iaelN? Ã:o the
metallzc container, thereby proclLrcing eddy cLÃrrcnts in the frame and
heating it up by a
Joule effect. It is possible to heat c;vÃ;i-a ~onma.gnetic metallic containers
by indtictivc
c.otiplirz.g, ari~.~l tlie heating meclia:riisrri is clitTererrt from tl`rat.
used to heat ~.~Ã~~lymeric
magrietic microspberes. ;Sirice the coratairxers were inic.rnfibdÃ:,atcd, the
electr'rcal
characteristics cou(d be made reproÃiÃrc.ibIe, and the temperature coÃ71c1 be
precisely
controlled by changing t1ie irZciderit power. This rc;prodticibility shou(d be
cÃ,`~l rtxa.sted
with t1~e pozver needed for release trorrx polymeric magrretic microspheres,
wliich cazi
vary greatly ~ecatÃse of pol~'dispcrsiviN, in sizes and inhomogeneous
distribution of
magnetic particles within clrffererit macrosl?hc res.
1001211 Blv heating thc corita:incr, the gel encapsulated ztiitl`riri it
sotteried (crr
collapsed) and released the c.bemical at tlic targreted spatial location
~Figure 1 Ic.i. `l'he
metallic cc>mainers are essential to obtain heating at the power and frequency
settings
tisc.d. No r:clc.ase was observed from tlic gel in control experiments (on
expa,~sLirc to thc.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
37
R.F radiation, btià :iÃà tlic absence of the container) bccatrsc of
ric{,~Ii~ibIe dielectric
heaiin_g at tE-ie kÃcqucricy arid power sirtttÃ.irgs tÃscd (see the
Stal,po.it:ing Iriiormati(~n).
1Ã101221 The rettrote-coritrolled c:oa7talirei-s, rna:14_e it pi5ssib1e- to do
cherrris-tr~, witli
unprecedented spatial cotitÃ-ol .in 1-aardrtc3ri-eac.li reiorrs. 'I'o
hÃg;hfigtrt this feaÃtare, Vve
repaired a break oap in o:ire of two aqjaceÃit microwires embedded within a
capillary;
the capilla), was accessible orily by hipt.lt aiid ~~t.ttpttt ports (Figure
12), The gap witliizi
microwire 1wa.s repaired by remotely gtaidirig cntitaiziers to that site iti
air Wi~;i.irc 1"
a,b3 and reirioÃely releas:ing first a chemical scÃisitizei= atid tltei-i an
activator (t.tsitrg two
wparate containers) locally at the site of the gap (Figure 12c). "1'he
sensitizer and
activator were titi ai-ad palladium catalysts, respectively, which facilitated
the
electroless deposition of copper. After scrisita.r:itrg atid activating the
spatial rcgiwl
withiii the
gap ofmicro-wire lorÃIy (t i,gure 2d), tlrc eiiÃ:irc ca.p.illaiy was flushed
Nvit}Ã a
commercial soltÃtiott of copper su(fate (1~igtÃ:re 2e), <'11ttioi.ig}i both i-
rucrow.ires and tlie
wa1ls of the capillary wiere exposed to the copper su(1=aie solution,
rtieÃaffic copper
deposited oifly at the c}icmÃc.ally scns:itized atid activated gap in t-
iricrowii=e I (t- igurc
12 f.). Electrical resistivity measurements c:crtifirmcd electrical continuity
ol'microwire
tac:ross, the gap. This result demonstrates the a,ttfllity of the
c(irit~nor=.w for 1crc:ali,zcd
clierzrical delivery and chemistry within capillaries arrd ~.~tEier small
spaces. (iI
comparison to alrea.dv e5;.istinO; Ãiietliods of Ãnlcr~~affibrication in
capilla:ries. J. C.
?~=1cDonald, ~ M. W(ritesidÃ;s. Acc. (::lrem, R.cs. 2002, 35, 491-=199;
MiaclotÃ,
Ft.tnclanic.Ãita(s of Microfabrication, CRC, New Yorls. 1997: 13. J. A. Kenis,
R.
lst~~~gflov, G. M. Whitesides: ;ScieÃicc 1999, ?85, 83a85]', this itxventioWs
ziiethod is Ãiot
limited bv . the geometry of tlie c:al,iIlaai= or laminar flow profileS.
1001231 ~secoÃic! demtÃnstÃ'atiotr. highlights the titility, o1't.tre
rÃarÃo(iic.r containers in
the r-cizioÃe-con t-rollecl, localized dirlivcry of sub-nanoliter volumes of
chciiiicalw to
specitic cells cttltuaed ori strl-istrates, Containers were loaded with
I'NIPAÃn, soaked in a
liv~.~:dead ('gaecnr`rcd) two-color fltÃcsresc.eticc viability stain [Irt-
vÃt.t-ogen liv(-Aead stain
product giiide to stalÃi cells 1ocally in a crjlttÃri dish aircl
to t<t.~rify that Ãio Ãiet;i-i_stic cell death cscctÃrrc;cl dLariÃau cltet-
nical release as a consequence

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
38
of the heat.iÃÃws [S. Cc3r-On, S. Boexcb, C'. Maneschg, Ã.'. RacirÃ-layr, G.
.(~arfsc.~~, R
Klocker, ELar. Uro1. 20430, ;' ) 7, 499-5,04] or exposure to the R-E
Ã'a.diatit-11.t1_
1001241 The L929 mouse fibroblast cells were cLÃ:(ture-d irt -35,-mtià well-
plates wiih
glass itilays and growrz to c.c~~ifltic:ÃÃcy. At the start of the
rernotercreleas~ ex~erimcÃit,
the ~prnwt:li media was removed and the cells were rinsed witli phosphate-
buffered
saJine to dilute tlie sertÃm esterase activity, tbere-by rriiriimizirig backg-
round
fluorescence. '~('o eÃiable reÃiiote release of the staiia, ati RF coil was
placed below the
plate directly tjÃider a coiitaitier, aÃid the coil was powered i.Ãp at '-2r3
W for tÃ-iiiÃltlt:e to
collapse the. encapstÃlated PNIPAm aÃid refease the stain. Fliiorescent images
were
obtained 30-60 minutes after Ã-elease, toa.Iiowsufficient time for uptake of
the stain. It
is c.1eaa :i:rÃÃni the coiii"ocal fluorescence images (Figure 13) that the
stairi was released
loca11a<~.z~~~ ~~~ithiii a.Ã~.~.d.iuc c~l`-iess than 50~~ lt.i-Ã~. from the
center c~#the c~~r~tal~~er. (t: cati
~
also be seeÃi that the cells exposed to the stain ha.d green fltÃcarescence,
thtÃ;s indicating
that they were alive, and Ãio red fluorescing or dead cells were obsened.
"('he results
iÃidicat.e that neither the temperature used to collapse the encapsulated
(''N:I:('':1m ri(ar
the RF radiation c,a.used necrotic cell death. It should be noted that no
lcakaye or
spontaneous rÃ;1eas~. (that is, Ãio cell staining) was observed fÃ-uni the
containers in
experiments where Ã~~ RF field was a.pplied. Biocompatihility studies show Ã1o
necrotic cell death occurs in the presence of the containers over 48 h.
1001251 Tn CcglIClLÃSitÃn, the metallic, self-assembled nanoIiic.r containers
cwi. be
titilized for rcniote>-coÃ}trol(ed rÃiic.rofabricatic~ti and cheniica1
cic:livery in hard to reach
spaces. 'i'he containers will I~e useful in tabiicatiÃig complex and
reconfigurable
microarialvtacal, microflÃ.iidic, and micro-elecxromechartieai tvsterns. The
localized
rcnicitc: dc:1iven, of chemicals to cells establishes a methodology for
r~.motely
manipLÃlatiÃig thc chcmicai ajid biological micrv-eÃivirvnmerit for
applications in cell
engineering, tissue en4,~ineering; a:iid drug developÃiieÃit. Fina1Iv> the
containers pro4.,ide
aii attractive platform for the intogra.tioÃa of addit:ionai features of
wireless devices (for
emiÃnplc, frc.qÃ.eeÃic:v-sclective remote coÃitroI and remote coriÃmunication)
with the
delivery of tiancs(iitar volLÃmes of c.licmicals.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
39
~XPER1MENT-AL 17E'll~,UL'-';
F'fkbr=iraÃic~~~ of the ilicroc0titainers:
1001.261 Brieflv, the fabrication process involved t.lre self asSemt7ly of a
two
dimeÃ-rsiorral (":D) template irrto the 3D cii.bic cnratainer. First, '?D
met.allic templates
consisting of six square pc3r(yuS fiac.cs were pbÃ~to(ittlograpliica(ly
patterned. A Secorrd
layer of photolithography was used to pattern solder hinges ~ii the Ãxute:~-
edges arid irI
beÃwcerr faces. `('lie '?D template spontaneously fotdeel iritÃa t.}re 3D
cubic contaiÃler
wIreii it was }ieated 6rr a 17.Ãiiel) above the meltirig point of.'ttie solder
liirlge5, wbereiri
t(-ic surface tension of the ri-iolteri solder provided ttle 1`orce to drive
Selircassemb1y.'f lrc
final size a:trd porosity of t[ie 31) container was varied by patterning the
21) template
alapro-pri.ately. In this experimerxt; containers were fabricated from nickel
(Ni), a
zrragrretic matcr-ial, to crrable rernotc tYuida:ncc. I'tie oiiter and ir-
irrer surffices of the
containers .:vcre coated witl-i gold (Au) to irac:rcasc E?iocoarpatib.ilit;:
ai-id decrease
electrical resistance (low electrical reSistaiic.e, iracrcases t.lre skin
depth for penetratiari
of electrornagnÃ>tic: waves). The fabÃ-iUation process was highly parallel and
large
nLrml~~rs. of coritainers could be 1'a.bricated in a cost-effective manner.
PREPARATION OF TIJE C;ELS:
1'Ã301.271 PIr.rronic&: The gel ivas made by coa7rbirrirr : O.5 g of F68
Plrarcarrict
(BASF) with 0.5 mI. of water. The mixture was sonicated for 10 mirrtF es to
c2isurc
complete mixing. Gelation c3c.cLirred after excess water evaporarecl.
1Ã101281 PNIPArii: Tl-ie Ã''Nl;Ã''Am gel was made I-roiir two stock
solLitions, A and &
So1r.iiirYn A. consisted c3f.' I..6701. ~,s N-i.soprola?ilaca-ylarr-fide
(1?Ni:l?Arrr), 0.0083 g N, 'ti'..
meth.vIerreb:Fsacr-ylaa:r}idÃ; (BIS), and 15 n-aI_, rarater. Solution B
consisted of 0.0129 ~ of
amnionitini persult'ate (APS) and 15 mC. of water. Both solutions were
vorte:~ed r.indl
the solute d.i`solved. Gelation was <ac(i.leved t?t' mixing equal volurr-i~s
of cacl~ soluficXi
togetlrcr witlr 0.4 r~ (,v`v.) of Nv N, N`, N'yteta-
aun~.~drvlenctbyl~.~rrediai-trirre (TMG[3) a.rrd
oc.cr,trre(l w~ithin 5 niinErtÃ;s.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
REMOTE dC-ti1DAN-(.`E;
1001291 Remote gtiiciaÃice was aclaaeved by inanipula:tir~~ a ~~~ag~ietic
stylus below
the reaction vesse(, ln ortter to redLÃ.ce t.tae f'ric.t.ioti between the
comaÃnE~~~~ ~i-ici the
su.r~ac.c of the vessÃ;1, the stylus was rotated alÃai1~,r the base of the
vessel causing the
Ã:Ãantainers to tumbl.e along the surface.
'i'HE 2D lf~U..N.11CROCt)IL SE'1'-UP:
[U0l.3()1 A ":D gniczoco.il was fabricated using phot:olithc~~~aptay on a f3r-
irlted circuit
boazd (PC~) as the I-',:F source. r1"be mici-ocoil was placed either below or
above tlie
contaiiiers at separation distances of approxiiitately 1-5 Ãnan. A c,lÃrrent
at 800 MHz
(RF) was passed t}irougli the coil to geiierate ar~ afteriiating zY~agÃletic.,
field iti a
directiora perpendicular to the surflace of the c.oai; ata incident power ita
ttae ran,)e of 1r7
Watts was usecl. The sufface of the coil was air cooled to remove any JoÃiIe
heat
gerteratec1Ãn the coil.
REMOTE R.EPAIR. OF A MICROWIRE WITHIN A CAPILLARY:
1.00131 1 M.icrow<i res (100 ~tm thick copper wi''.res, spaced 2 iiint apart)
with break
gaps of 50 ttm were fabricated ~~~i a glass slide using photolithography. Tlte
capillary
was formed usii}g po(yci.imethyI siloxane (RDTVt:S) walls that w~c;re scale:c~
against the
glass slide (containing the wires) a:iad ariofliefl glass slide (the i=caot of
ttae capi11ary ).
Seal'in4,~ was a.chie-ved bv plasma sa.Ãrface modi.f:ic:atioit of the :l'DMS}.
The capilia.t-y
with the eanbc:cl.dc:d 3vire:s (f:=igÃarÃ; f?.A), wa.s approxiÃ-nazely l mm in
widtla. and 1 .5 ci-n
in length a.tid was ~.~taly accessible by the iiapLat a.nd ~.3titpuit ports it
itti two eaids.
1001321 The sensitizer soaked E'la.Ãrotiic~gD gel was prepared by
Ã:,ca.mbining 0.5 ztiL of
sc;nait.izing soIÃiiic3a~ (Transene) witla 0.5 g of Plitronrc F68 (BASF). The
activator
soÃiked ) ~'1u.i Ãsni e ~ ~ gel was
prepared by 7ng x,i.ta g 0.5 ml., of activating ;;c~l tati o~.ra
(Transene) 0.5 g of pIar~.gnac F68. Prior to loading, eacii. mixttire was
sonicated for
5 minÃites to enstire complete mixing. A 1 ~tL drop of eac.h of the mixtitre.s
was pIaced
on two separate Ã:ontai.riers ar.ad ttae solutions weÃ-c allowed to gel
Ã.gvernight. (_14
hours). The containers were [bera cut out of the gel, to eiasca.r~.~ that the
gel reniaiiaed
only withiaa the cotit-ainer.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
41
1 (1{}1331 After w*uid.ir.ag tI-ic coaztaiÃiers to the gap Mitbirl
IÃUUr:arN~ire 1, WC ~t.iral applied
4 Wat-ts of power to tE-ie 2D coil until the gel softened and released the
sensitizer
solution from ilte container. T1Ãerà the power was t-edticed to _3.0 Watts -
Lor l n-IiÃlLtt.e to
allow sa.il='==:ti~cietit tizite for dift'us.ion Of tltC sOlÃ.atiOÃ-I through
f1ic gel and to the surface ol
the gap. 'I'he cl'aatine1 was tl'aeÃi flÃ.rs}iecl w:it}i water to reÃiiove the
container aÃi~ excess
geI. 'I`his process was then repeated, fk-.)r the secoÃid coritainer ti(led
with the activaÃor
solutzorl .
1001.341 .'4.ai electroless copper solution was Ã-iiade by rnixirig
eomtiiercial solutions,
PC clecÃreiless copper solution A arid PC eiectr~~less copper solirtion B
(both from
']'raÃiserÃe), irà equal voitÃriies. A .yz-int~e, wit.là a diameter of 0.9 mm
was fitted with 0.9
znrri 11) ta:ibirig, 1'he (Ylkzer end of the tiibe was placed iri oÃ-ie
openingof the channel. A
syzir7ge pLartip ((tAZF<.1.) was used to flow the platirig sc)Iution irito the
channel aÃid
over the 13rokeÃi microwires, A pulsatile flow was used to taciiitat.e the
platirig reanon
by maintaining a high local eoriceritratzori of copper ioÃis while <iilowirtg
sufficient
time t`nr deposition. During tE-ie e:x.perAiiaerat., the copper- plating
solution was kept at.
45 C ,
REMOTE CONTROLLED DELIVERY TO LIV7.Nt. CELLS:
11101351 CoritaiÃiers werG loaded with PNIl'`'AÃti and allowed to sit
overnigIlt, O1l the
day of the cxpcr-ir.Ãient. the L.ive;/~~adO two-color fltrc3rescence stain
(1:nvÃtrogen.) w:-Ãs
prepared at concentrations of 0.5 cÃM Calcein AM aÃZd 1.0 ~iM EtlÃidiÃ,rrii
hoÃnodiÃnor-l..
The PNIPAiÃi filled containers wei-c sa.ahr.a:ierg,,ed in tlzest.aiaÃ
st}lLrtion for 3.5 ilciLar-s prior
to beginning the expei-iii:aerat to allow the PNIPAm r~t-n~le time C.k)
rehydrate and absorb
the liv(-~Idead stain.
10{ll361 :t.,9d~-~ mouse fibroblast cells (Sigma) w~,ere cultured and
rrÃaiÃitairÃed
tollowin;standaa=d c;ell. culture prc3toccais. The cells were cultured in 75
crn2 culture
flask in 85% MiaiimEÃm Essential '~~Ã;ditrm Ek(gles containing L-<;~lutt.amÃne
and sodir.rni
bicarbonate with (0% laor;~~~ sera.trii mid s-Grpplementec1 witlà N1_EM non-
Ã;sserltial ainirÃo
acids and ;aÃ3ciitirÃi. pyÃ-taa=ate The cells Ã~iaiiÃtaiaied in ari incubator
set to 37"C w.i-tii
a. wa.ter-saturatec~ 5% C02 at--ai~.~sphere, 1_,929 cells were
sul~sicultr7:red 2-3 times per

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
42
week utilizing tr-yp;,in-1'11')TA arid seeding tEic Ãiew flask at a cieÃ-isity
of =x 104
cells.?rn1;. 1'he seeding deÃ-isity was veritied by rerÃao-,>irig a sample of
tl-re tiypsinized
cells, stairring the cells Wittà t.Ã-y11aÃi b11ie aiÃd cÃsitia..- a bemacy-
t.ometer to cociÃit tlÃe
nr:irÃilaer of viable ce[ls.
1001371 :Kernote release experÃr-rietits were conducted in 35 rnrii well-
plates witli
glass inlays (I G1assabott.omsdislies inc.) for optinauni contoca1 microscopy.
Brietly,
cells were seeded at a dezisiÃy of 250 tL of 2 x 105 cells:rÃi1:, directly
orito the glass
iÃ-flav <i.r7d allowed to recà .fioÃ~ 30 rYtin to prorYtoi:e adtlesiotÃ. 'i`wo
rYii_: of g;Ã-owth ÃxÃedia
was then added ajid the cells were irlctgbated for 48 hnurs to achieve a
confluent
monolaveÃ-.
1[){1138] After rerÃiote release, the cells were iÃ-iiaged usiny, a Carl Zeiss
contbcat
microscope. Br=ietl;;, the.Ãnicroscol~e was setup witb lasers and filt~rs,
i~ecorni-fiended in
the Live!Deacl,,x assay protocol. C;alceiri AM was excited at 488 Ãar~~ and
ethidium
horÃ-zodix~~er-i was excited at 543 nan. D-Ye up-ta:l5e was detected witli
filter- eÃ-rbes of 1:31;'
505.5;3 )t] (for calce:iri in live cells) arÃd I.P 650 (for etlÃieliu-rÃ-r
homodi.iiler iti dead cells).
CONTROL EXPERIMENTS
HEATING CHw'~RAf.:TERllS'li'ZCS<
1Ã10:1.391 To demonstrate control oa ei- heating of the nanoliter containers
with tl-ic
incident po3ver of tl-ifa magnetic field generator, a tc;mperatiive control
experiment was
condÃ.tct.ed, The sc:ttÃp of the experiment was similar to otir other RF
controlled release
experirnents. TIw nrÃl_y di1l'er=erÃc~e was the p(aceÃ-nent of a
Norirc:(teversible
C)Nl:l:GA:I:,Al=;iEi.: Label (Ouiega `I 1:,-S series)urader- the nanoliter
corÃ'[air7er so that it
ccirel(l be heated witl-i good thermal contact. The i_ernlaeÃ`ai_ri.re of the
CODÃrÃiTier sUÃ1~We
cari be dedLÃc:ed color changes in the labels th,it occÃir at 38"C, 49'CY
60"C' arid 7VC
respectively. 'I'1-ic iiÃcideÃ-ià power c3t`Ãhe rnagrieÃ:ic field was iricr-
eased u-rÃtil tlie specific
label cliar34ed color (after Wai.t.ing .fo.r approximately .10 seconds).
1"'iw_ 14(a) shows the
col.or- c:?ÃarÃ-~t:: in the Iabe1. oser` whicb the containeà w;Ãs lala~ed Fig.
14(b) is a plcÃt of

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
43
the Ãc:.rnpertatÃarc measured b~:r t1~~.1~il~eis vs ihac iai~.~iehe~~t
pÃ:g~n~c:r'. b~' c;l~at1;~irr~ t~hre
ita.cident power, the heating could be precisely coaYtro_lhed. t;t slaoÃald be
rroted t:liat the
exact depends oia t-lre sh~eei#ie coa7taiiaer used and the experit-iieÃit
(i.e. dry, or The color
c(a.a.aau;e occurs on.[v Lirzcier the eorrtaiaier showiaY, tE-iat: the
heafita4,~ is wet release,
urrouÃadiÃag local. (B) A pIot of the temperature meastared usirag the color
inclicat(yr
s s
label environment) vs the iracicleia:t RF power
&Ilfri:I SI' A'1"lAi.: AND -NIATI:R-AI.T SEI..f:.C-1'lVITY OF REitlO'I'.I:s
.It.I:si_EASI. FROM
TIiE CONTAINERS
1001401 To deÃrioÃrstrat.e tlic material arid spatial selectivities, the
fo(lowiirg cc?ntrot
expeririierrt was performed. 'I'wo eorrtaialers loaded wit:hr (''lurotridk:
4~;e1 (soaked ~.vith
focsd coloring) were placed 3 ri-iraa a}iart: fro.in each other in a Petri
disha. Aalotlier
isolated piece c~fge1 (ia.ot eÃacapsÃ.ilated witbiax a ~~ontairrer) was also
placed in the disla.
The Petri dis[i was aligned over a 2D xriierocoii at a distance of ? nirYr:
such Ãhrat in the
plarae of tlie disla, the isolated piece of gel was aligned directly over the
celater of tlle
coil: one of the containers was aligned within the c:hrcumference of the coil
bcÃt off'set
from the cc:titeÃ~ by 300 ag-iel the second container was nri;;a(ignÃ;(i and
lay outside
the 21L) coil, ft'I-ie coil was hsc),wered up at 800 MHz. Only the gel
wit.hila the container
aligÃred avithrira the circumferetace of t(ae coil lieated LÃp aiid scafteraed
at a power of 4.7
Watts. Even when the power was increased to 7 Watts, the isolatÃ;ci piece of
gel placed
in the region of highest field as well as the gel in the raiisa(ignÃ;d
container remained
unchanged. This coÃatrol experhrraeÃat demonstrates that the inclticti~~e
beathÃ~g had }aigla
spatial and material selectÃviN,. The ex~eriixaerat also shows that the metal
used to
tahsric;atc: the nanoliter cvirt-ainer is c ssetitial to enable the inductive
he<atiÃag. It shoiald
be noted that a Ãnar?ttetic matei'ial is g7ot Ã-eqtaired to fiacilit.at~
remote heating (the
magraetic property is used morelv for spatial gÃiidance). Also demonstrated
was the
release from containers wihha iio Ni, i.e. c:oratiposÃ;d of copper / gold.
NO DIFFU'SIt)N OF CHEMICALS IN THE ABSENCE OF RF R.ND1AT.ION.
1001411 A control experiment was pt;.rtt}rmed to clem.onstr.,a[e abseric:e of
dit-lb:;is.an
of ihic I.1'':C /DEA:D' assay (i.e. dea7ron;t.ratC 110 tiPODUÃrrCOUs leakage
of Ã;herTiic;als}

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
44
froiir loa(ie({ narrol.iter coratagne.rs, .ir.a the abscrrce of RF
.rad.iation. 'I"~ie e\~perinrerrt
followed t}re test procedures wit1a: the exeeptaora that the RF was raoi iur-
raec1 orta. "['Iri.s
helped to etisure that the iii-tre frarrae f~'br exposure was the 5arrie.
Confocal rnicro;~~~py
was used i:c) verif:y tEa.at: rro le.a.~~~ge, of tE-ie Live/Dead assay E-iad
occurred frorxi the
1'~1:(=f'Am in the absence of RF radiation (remote heating) over the tirn~
scale of the
exlaerÃraaerat (Fig. 1. 5).
Excrli../c, 5: Sur~ ~~c c> ?'ensiran Dravc-m Se1=FoNing Polvilcr~ra
Fabrication ca f'patternect pofj~h~.~dr=a:
1001421 "1`lae t:inst step in the process irrvolvecl t:tac fabrication of 2D
templates
c>orr-ilatased of patt.erriec{ faces and aoltier- lai:riges tlaiat woLr:(d
eventually fiald iap inÃo 117
ho11o`v polyheclra. A polymeric sacrificial Iaver rr-iade of polyr~-ietlryl
meÃIraca-ylate was
spin-coai~ed oÃito a silicon (Si) substrate to iaci(atate a{ibseqtierat
release of the 21)
tezriplate4. A r-actaiiic seed laver was thea7 evaporated orito the
sacrificial layer to create
wafe;.r-sca:(e electrical contact ftar subsequent e(ectroÃlelaersitioi-i
steps. The faces were
p,atterrred using phr.rtcalitho~,s.raphy wid fabricated Uti11a1"
e::iect_rodepzgsitio.ta. Since
conventional p}rotnlithograpE-iv was used to pattea=aY fiices, any arbitrary
patierri could
be .ine:orprarai:ed. Faces ccaFnprs:;ecl of eitlrer- copper (Cu) or rilcktl
(M) were f_abricated;
c1:roice of n:retais was determined b~,,r cost, otcli selectivlty rn<ith
respect to the sce-d lbiyer,
ease of depositi t.a5 and the need fÃsr magnetic functionality. A 5eccrzrd
ltaye.r of
pIotolftkaosMrqplay v:vas tised tt) patiem the solder }ri~ige t. rlapl3tes.
After laingge patÃerrairag>
the exposed seed layer in the la.irzge re~lori kac3Ligaded by tEa.c faces was
etcliec:i to cliscontlect
tlac wrtieriy'.irrÃ~ seed layer oxrly between tl-ac faces, wfiile retainingr
clectrical corrtit-wity Wit}r
the rest of the seed laver at tlae face cc?rners. The solder hinges weru
electr~~~eposit.ed, and
then the 2IJ terr~jfla.tw was rc1ea`es:d I`roi-tr the substra:te by etcliirqg
the reraiainirrg, seed lz7yfer-
at3d ciissolvint, tlac sa.crifrcitrl Iaves. A tt,raapl:rte conrpt?sGt1: of six
sqaarv faces, arrat-r~U~. in a
c:raiei.ftrr-rra and held together by solder lairr~~~; ,va5 tisGt1: to form a
crit)e Apart frorn the

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
solder iaA between faces, there is no other tether. Self-folding was carried
out .in a h.igh
bc]ili_iÃ~,,, lac]iz3t so[vei-at, N rneÃlaylpyrsoi.idt>iae (i1;M1?), wlaicl3
tuas heated abave the iTicltirÃet
poil-a. of the solder (-188"C) A small a_t-noua-1t of 44-5 RMA (rr)sin,
rnfldlSf activated) f-ltix
aA-us added to tlie solvi.nf to cleati and dissolve any oxide layers c~i-i
t:hesoldvr and Ã.IiEreby
erIsÃIre 4~c~c~~~ solder reflow.
13e:sigii t_onsiderati0ns:
1001431 lii tlic design, Ni was a1wavti used as the topmost surface layer of
the face
in contact wit}i tlie }iiiiges, Even for the Cta poIvliedra, the top ofthe
faces were coated
with a tltin laver of Ni prior to hinge depcAsitiÃ}aa, Solder does not wet Ni
scÃrfa~~s w-ell,
so the solder stays in the region where it is elc ctrodeposited. ancl does not
spread across
the eiitire surface of the face during ~~ldit ~~ (wliie[i occlirs wlien solder
is in contact
with Cti). When ttie Ni coa:tiiig was absent, we still observed folding,
however the
yields ,,vÃ:re pcscgr. The low yield was a resiift of the solder migrating
away ~rcim the
regions vvhe>re it was cl.cpositccf, thereby iiaaki~ig it very difficult to
control. the vol~ime
of solder in the }iinge region between faces (which ultimately determines tlle
fizial
~~ldi~~g angle).
1001441 The design of the 2D template ir.Itimafely determines, the final shape
and
porosity of the polyhedra. S1iowti in Fig. IA is a typical -2D layoÃit of the
tarcs ai1d
hinges. AÃÃtodcsh AtttcsCAD 2005 was iÃ.SeÃ.l to generate the lavotit file
L~~ed to
fat?r:ie;aÃe two pltot.omtÃsk~ (one for the fzÃces, otte for the hinges). To
fabricate a cÃÃbc,
scfuar:e.fiÃc:e:~ separated b;~ ~gap, c, of 10--1.5% of the face d.in1ension,
I.:, in Fig. IA,
were t}'t.~ie;alk= U:~etl. SÃsrne tolerance in the gap width was observed, as
the molten
solder teiicls to draw t(ic fac e;~ laterally towards ea c;h other clLiring
foldill.g. l:t shoitld
also be noted that sir.aÃ:e the ~.~a}a wiÃIt:(i. .is I0--15% cxf :[.,, it was
oftei-i tlle fniD.iITiÃ11Ti
feature size of the pbotonnask and Iithcagi-apliv pioc~.`sk, e.g. i-~.~r .15
Lti-ii cubes, tl-ie
requirred gap 3vidth of 1.5-2 ~um represented the st-nallest lithographically
patterned
feature.
1001451 :1n contrast with prior swrfzÃce tension-based seli=folÃ.ling,
ovorlti, two types cal
1ii-Yges tVCT'e ÃISetl: internal ones betd~~eeii faces (folding hinges~ and
external Ã.anes at the

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
46
periphery of the t.'aÃ;es (locking biatges). Th.ae folding hinge witll}t
(s:liÃ:AN'tt as W irt Fig.
11:3) was '?5% of L, artd the ht.ittw;e 1e2~~Ãh was 80r90`?i3 of I. If tE-ie
folding htirlge lertgtlts
were smaller the cÃrbes Ccst-a7ed were, iaot seal~d cotitplet.ely at the
cÃarrters.
Longer htirtge lengths (>90%) were ut-iÃiecessary, sirtee rleigE-lborirlg
hinges would
overlap at t1ie corners. AdditiotiaIly, hinge letigt}ts of 100% were
irto(artipatible wit}i
the tabrica:tiora proÃ:.ess: tliese liirtge patterns Ã-esa.iit.ed in the Ã:,ot-
rtp(ete removaI of the
seed IaiJer at t:Ei~ peritiieter of tEic 2D templates during the etcb stop
after
Phtotnliih7o;.~rapE-ly of tl-ic laees. "1'his .retYtoy:t.l -forÃaYerl in art
eleetrically discorttitl~torts
..,eed layer that prevented sul-iseqLient electrodeposition of t.hte hinges.
Re~o-w. of the
folding ltirtges providecl the torque to rotate a~~acerrt faces. Lockxrtg
hirtges that I-tad
the same letigtli bLat half t_E-ie width of the to1ditig hinges played a
secondary role in tlte
foldino.~ (yl: the ~:I) tetr~~~latei thev as a stabilizing stop, increased
fiartlt
tolerance in folding, atid ertsa.Ãred a 1'atiat fold angle of 9V [ Sytits, R.
R. A. J.
~~,fic~rmd
-evoorrtech. t. 1995, 4, 177-I841. Additionally, IÃsckirig hinges increased
the
t3=tcchtanictri strezt4xt~ht and se.ttli.d Ãhte edges of tf~.c
t~st~f~>13~;~clr~a when ~-~~~o htali=<:ire~i l~~clÃ.ixt~;
11in~~es tÃtsed arid formed a. sin<~=te h~i~~e cotitait~ing the equivalent
volume of a folciinQ
hinge. t=otdiÃtg was Ã:ott:tplete withzti-t seconds vdtest the 1oi<kistg
lti''. ~,=es iitei kintl itFse~~ ~~~ttr
each other. The fusiort oecut=ged as a resfalt of Ã1le tYtitiitYtizatiÃ.>tr of
interfacial free energy
beto~~eett the i-nolteÃt. locking solder bitt:;e on each face 3t-i~.-~ the
surrounding lielrtid. 0-zt
coolitig, the solder hinges solidified aiid the polyhedral st.ritc.tt.tre s~-
as locked in to place.
Finite cleraietrt sintu tal.iotr t;
1001461 T:n orcior to better understand the self=assimbly, process, we
perfo:r~~ed titiite.
e1enient sirt-tul.atiun5 crsig-tg the software prc3gr~n-i SLtrfac:e Evolver
[Surface Evolver was
developed by Kera Brakke -fi`oart tl-ie Susquehanna University Del:tartmertt
cyf
M,ithenra.tics. The latest Windows version v^.`26Ã;, Ãtpdat~.~ci.
Sehlteiitl:rtvr= .I T, 2005 was
ttsr d]. Suafa~~ Evolver determines the tt-iirtimttm energy sutfacÃ; for a
giveti. initial
sLtr:face atid a set cyC physical ~~iistraints, suc4i as gravity, detisit.y,
artcl sÃÃt~a0e tension.
The iterations for evolviazg a mininaÃa.na sÃga=f;tce are cctrttrctlled
manually by -thie user.
Scripts were devÃ:lopeti to automate the task of varyin~.F parameters and
evolving

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
47
riireltiple surfaces. Sinau[Ãrtions pe.r.{=ornaed bave :irac:irzcled only
f~~io acljjacent. SqrÃare
taces, kield to~.~ether by a siriM,le solder fo(d:irawY hing;e, since this
cal,tr.Ãr-ed itie essential
frÃrietioti of ilie folelir~~.~ tÃinges, that play the ct-iiieal r=ole iri
I~brmÃng i well-folded
str-uCtUre. (hie face was asSLari-ied to be i^'axed, while the other was
allowed to rotate
frrvely around the solder hinge; tli:iS assumption parallels what was obServed
in
eNlaeriÃiieÃits. 'I"~o dete-r-mine ttie equilibrii-rm RoId a:ragle for a given
geometry, we used
the fiiol1owitig strategs- 1.~-i~.r:~t~, K arid Lee, Y. ~ =:
~'~=~~c~c~~ec~~rr~~s t~f Sf'.~1:; Sari Jose, tJSA.
1E398]: rrÃiniÃ~ia1 energy surfaces were g;eriera:ted t`or- arigles of
rotatiori (out of the "D
plarie) between 0 (flat) and 120 (overfolded) in itiereriierital steps of 5
Tlle
equi(:ibririrn angle coÃÃesponding to the g(obal mxnimrrm erxerg ~' was then
tleterr~,iiretl
frr~sn7 the rliiriiÃ-iitiÃ~i of the surface energy trend lii-ie versus angle
plot, -for- a particular
given face dr li3 E:11tiAoI:'l.
1001471 ShowÃi in Fig= 16 (B-F) are illustrations of ttie firiite eleriieiit
simulaticyn f(yr
the foldirig process. In ilie 2D teÃnp(a:te, the fo(dzrag hinge solder- iS iti
the fc~rrYi of a "I'-
sIiaped ri~.~E-It prism. On reflow, the solder IicILie#ÃeS arYt~ forrzis a
Ã~ou-rided contour (Fi(I.
I60. DtÃc to the }iig}i interEacia1 teiisiora of the liquid solder (,..
481Ãn.1im) [White, D.
W. G. D-cxns. 1971, -~4~}t~7-3t~;~~~]. tliere is a st~'oi)g d~'ivi~~<~ fork:e
to nunimize
the e>~pased ifÃterl'aeial area bet-v~,teri tlle rYÃolteÃi solder and ttie
srirrow3diÃ-r, t~ufdic licittid,
'i'his dririÃ~F.~ force cÃatayey the solder to ball rÃp w.hicb cesuÃlts in the
rotation of atjjace at.
faces, 'T'he fold angle is primarily controlled by the solder volume. We
observed evidence
for this coeitrol M botl-a simtilatioais and experimental observations.
Different solder
volumes gerieÃ=ated underfir?(ded (Fi~. 16 D. ( i ), correctly folded (Fig. 16
E. H), or
overfolded (Fig. 16 F. 1) structures. A plot of the dependence of the fold
~iggle on solder
-vol.ttme (generated by siÃiit.rlatiotZs, Fig. 17) shows that ttie fold angle
decreases witls
increasing solder vo1urage. ~xper-iÃ-nerltally, ttie solder vo1Ã.rÃiie Ãhat
deterriliries the
equiliba=irÃÃn fold angle w.as n~anipulated by coxitroIliÃig the lleight of tl-
ie electrodeposited
solder for a given lainge geometr),.
1001481 Sitice the scaling properties of the prot.e<sti were of interest, the
gravitational
potential eneqgy of botti the solder and t(ic faces were taken into account,
in addition to

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
48
t}ÃC iilterlac;ia1 IRÃ1-faCC Cr.aes-gtr ol'the soider. it has beeÃÃ shoo-~,-n
by cÃthers and verifie;i-1 in
our simulations tl-iat: Ãbe mkgnÃtude of g;rav:itat:ioaY,t.l e1=f=ects are
esyeniiadly negligible
compared to the iÃitcÃ-facial sLÃT=Cace eneÃaj~tÃtitii si-r.e-s become large
(i.e. ttiÃià scale).
However, oa:Ãr inclusion of a o;ravitational ener(,w tet-tzi aIlowecl LÃs tc)
determine the
relative magniturles of each of the forces as the feature sizes were scaled up
or dowÃt.
The fact that stÃrfac.e farces scale favorably witlt decreasiÃig size is aÃi
attractive feature
of stirtace teiisioÃi drzveii self-assembly aztd has the potential to provide
widespread
LÃtiliiy Ha tl3e assembly of microfabracated ia-iic,i-o aiad minz~sc:Ã1e
siruct.tÃ:res.
(00149] t'Ãt order to determine the effect of size scaling ort the foldiÃlg
process,
sim.ulations for ':[) templates were performed witl-a taces sized ti`c?Ãxt the
iÃ-iz~ scale to
i}Ãc .ann scale for a fixed solder vc31ijÃiie. :(n cacb case, all diÃ-
iieiisiotis (height, widtli, arid
length) were l.ittearl~~ scaled 1~~~ tlÃc same c~~Ã~.st:~.tit i=act~~r. ri.it
~eriet=;~~~ landscape was
ol3sen,ed (Fi4,~. 18) which drives the folding process art~l that tltere are
different
energies for differerzt fold atÃgles (for a ;;ivetà geÃaÃ~~~~rY. aÃ-id solder
voluÃite). '1'he iÃiitial
slope of the energy cLag-vcs isÃdicat:es the rnag;nÃtude of'tlÃc rotatiolial
force of the faces
and dctermzjics w17ether the f.'oIdiÃag process is spontaneous or not.. A
Ãtc(;at.iYe iÃlitiai
slope (Fig. IS, 50 tim tc) '2 nini curves) restilts in a spz3ntaneous
folclirig process while a
positive iiiitial slope (Fig. I8, 4 itim to 6 Ãiim ctÃrves) indicates a non-
spontaneous
prcicess. The minima in the ctÃrves (Fig. 18, 50 tiiti to 4 mrti) around 100
are
indicative of a stable, ecltÃilibÃ-iEim folded c.onflg),:uÃ'ation. The absence
of a minimurn in
the ctÃrvc for 6 mm, faces implies the absence of any stable folded
configuration, i.e.
the two faces prefer to rema:in flat. 'I'}icse results c.ait be explained by
noting that as the
:~ize of the faces increases the weig}tt increases and gravitational forces
begin to
dominate ctÃmpaÃ-ccl to the surface tension forces in tlie mÃ-n size scale.
Hence, the
initial slope of the ciicrg
,y landscape bccoiiies positive in t.hc mm rango. and the process
becoÃiies noti-spontaÃteoa.is. At sÃxialler sizes, sa.irlace forces overcome
gra:vitatianai
forces and the process 1ieccgmes spontaneous all the way d~~~~~~~ to the
Panosca1e. lt.
should be noted that in the siÃnu(ations, bulk properties for the materials
and the solder
were asstimed and c-AYUZ.ts such as phase segregation, inrcrn-Ãetallic
formation, and.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
49
d.iffiision `vi.tliin the solder ivere i~.inored, if't}iese atiaamption,,;.:
Et.olrl, it appears t}tat the
self-folding process weatald work oÃ-a the i-iatioscale. For our stan~ard-
eonletry, material
Ã~ensities, atid solder surface [ensiOft, our SiRIiy~atatxa~~ ~~~ow tlie n1ax-
IT11t1i-11 sponNne:,e-)Us
t:c]Id.iaiÃ~ si~e to be L ;~= 1400 ~rrn_ S.iina.iia.taoais tilso slic?w that
iii aii extreme case of zi Iow,
surface energy hinge (10 Ã~yriesic.an; e.g. a la<lit.id polymer) aiirl tieavv
faces ('0 g/c.an, e-.Q.
a dei-ise iaieÃ:a.l), folding is still spontaneous for poiyi-iedca as large
3s. 165 una. This implies
that it Sliould be possible to fold structures witli taces cozripcasecl of
alniosà ~iiy solid
material ancl aYiÃla liii-ages ctsinposed. of vai-trially any liquefiable
iYaat:Ã;'ria1 up to a size scale
of ai'oL1t'2t'~ 165 pt17 for our particular ge<3J11et.[y.
Experimental results:
1001501 Experimentally, cubic poly[iedra rarigirtg in size from 15 pi7a up to
2 i7am
were folded (Fig. 19). We }aave also been able to fi-Ad polyhedra of other
sltapes. (Fig.
19C). Although we believe smaller polyhedra c:ait be fabricated, we(tave been
limited
by oter photolithographic capabilities. Below teiis of microiis, hinge gap
width$
approach the sub-micron size :scale and alternative pattemitig techniques,
such as
electron beaiit lithograplay:are aequired to fabricate the 2D templates. Our
theoretical
simulations show that folclifl-ig of smaller poI~~~odra are spontaneous dLie
to the large
nlagtlittÃde of the surface forces at small size scales. Although
silnulatioiis show tllat
the fo(cling of polvhedra with large faces, i.e. 2 mm taces, is a non-
spontaneous
process, cxpcriiiae:atfi.kdlv w~. -k-vt;re zil_ple to -folt.l 2 ttam cubes. We
rationalize tl-iis result
based oii i-wo observations. Firstly, agit~itioii ti-rie to convectic~~i
currents iri the lteat.c;d
fluid occurs experir~~entally'. This agitation can provide the ini.tia1.
cltiviaa.g force tr.Y lift
tae~.'s anaa-ginal.ly over the activation barrier for folding, Secondly, it
shoL7ld be noted
that `vh.ile ,ve proportionally scaled al1. size a=ari;rbles in the
simulatiOD;; (C.g. a 2 MITi
face was simulated wit~i a [bickness of 80 Pin}q it was tiot possible to do so
e\perhnentally. Due to restrictions on tlie height of the photoresist and
resolvable
aspect ratios, we, electrodeposited a Ãbit.l:ra~~s of onlv 12 ~im for 2 Tt~iii
ctibeti; the faces
of the experimental templates thus had Ri substantially lower weight,
i.nÃ:rea;:ing the
threshold at which foldc~~~ ~eca:tne non-spontaneous to larger sizes.
AccotFrtti~g frai= this

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
fixcd:f:ranYe tlYicl:~iess in otar si.nnuiation, .vc determined that the
(str¾~e:,t size.to.r which the
sei.f=.fioltli_11~ PFOcCss WOtIld ~vork ti~r tI-ac rnateri.t.is rt5ecl in our
process is --:7 zmn_ A1tboug"1-I
we do taOt ea:t7eCt t-O ttse ',t lithrxgrapbic prrxc~ss, to fabricate
;trttcit.tres as larye 7 miii,, the
process of seltrfoldin4x may still be refe~~~atit: ttà this ;~i.~~. scale,
~;~s~,~;~cirtJl~? in the i~ackrt~~it~~7
of elec:.t-rortic devices.
Tolerance of the prUtt'ss:
1001511 Wafcr scale patterning of the 2D te~~iplatc:s is highly parallel, O.g.
we t.~i.ck
approximately 1000 (f.-l00 ltm) aaid I00,000 (.L='i 5 ~tm) w.D crttcÃftarrtxs
(yt) a 3"
water. The fo1dÃng process is also highly parallel, and lat;;e nitmhers ot~:
2D templates
can be foltlt;ci at unce. Experimentally, the t'olding process also appears to
be
considerably fault tolerant and we have otteti been able to aciiieve yields in
excess of
90% a:tid t`alaricate large iittmbers ~~f polvhedra (Fig. 20). We lia~~e also
observed that
tc.aldin9 occurred even when hinge regibtty. was not pertectiy, centered
across adjacent
faces. Experimcntally, to increase fault tolerance, we ta.rgctecl ottt= solder
voitime to
result in a slighà overi'o1d (.._.100' of rotation trotn the hcarizortta(}.
Since we tt:sed.
locking ltiti<.~es, Ãl~is c~vert~~id etis~tred that the faces tiiet, allowing
the locki~.~; hiÃ~,9es to
ftrst.'. This increased the tolerance of the process [Svriis, RRA, J.
NIrc~roelc>c.rtrc~inec.=h.
1995, 4, 177-184] and sealed the cubes at the edges attd comers. Additionally,
convection
currents existed in the tiot scaltzticati clttriti{; the foldisig process.
J'hese c.otivection c<t.trresits
wtt;witztt4'cf the 2D tc;ni~~latkas and. Ltic:rt;ases:i fo(ding, angle
tolerance by encou.ra~.-ing the edges
of tht:. fiact:.s tc) coll.idc., this allowed the locking soldei- liinges to
fttst' and liolcf the faces
to4~ether w=ith c,ottsiclerable strert4=th. CJacobs, H.O. et al., Science
2002. 296. 323-325-~
Gractas, I3.H. et a(., Sf-zr'nf'c' 2000, 289, 11 70=1 17?1.
C[1nCluSi {)Td S:
l001521 Irt conclusion, a surface tcrzsiota based fe~ldirtd; process has
l~aectt presented
that can be utilized to t`abticate utii:ct:E-ie.red, hollow patterned
polytiedr-a wit:li a wide
range of sizes from t:lie mm icp the nm. By leveraging wcl(-t;wtablÃsht;d
lithograpbic

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
51
methods in microelectronics, this faEaric.ation process provides ',i rOtrt.C
tO irI~:.Orptsrale
precisely ~~igira.ecred monodisperse porosity, transistors, sensors, and otl-
rer information
processing devices ori the prxlylicdÃ-a to create "st-nart particles." ITsirig
sira-11.ilati011s, we
N~ave also demorasÃ.r=aicd iliat the Neling WOr.aid work e.vitla a wide range
of t=ace
mate:r.iak arid liquctial?[e, hin0es. also dernorastrat~s that the
Lrti:liaatiora of
inÃed'acial forces, which scale fa.vcaaably at small sizes, is a useful
paradigra. for raiicro
arid raaraatabri cat:i on.
E a.atn Ve 6: Spatialty Controlled Ch~.~tnist,~~~ ~ ~ing Renaotcty Galhl~.~c1
A'anof.zter GS'c=ale G~~ituhierv
100 1531 Along wit.la coraverational channel based rnic.rntllridic devices,
sev'eral
nanoliter scale claemical encapsulants have been developed, includin~. those
based f~rl
pc?lvriiers; gels, and litlr.rid drops [for example: (a) Lim, F.; Sun, A.. M.
Science. 1980,
210, 908a910. (b) Ch~ng, T. M. S. Nin Rev. Drarg Disc.ovcrv. 100", 4, '1l-235,
(c)
Langer, R. .3.cc. Clarrm, Rcs. 1993, 26, 537-42. (dyfice, J. D.; Sorig, H.;
Lyon, A. D.;
Ismagilov, R. F. Laai(ir~7uir 2003, 19, 9I27-9133.(e) Harnmer, D A.:
DiScla.er.D. E..
Ann. R.c,z=, I.Mati.r. Res, 2001, _3I, _187- 104. In cot rt:ra.st to the above
organic systems,
nzicromachinecl si1icori-based devices can have extreme prccisiori, high
rcprodtrc:ibili.tv, excellent mechanical strength, good chemical stability, as
well as the
ability to incorporate sens.ira`s, sik;nal c.ond.itionfla;F, and actuating
ttanct.iOD;; iD clOSC
prc.e:si.rni#-y oi- ori the saane substrate. H:txz~evcr, :;1:3 i-nic-
rotnac1}iiacd nanoliter scale
reservoir systems 3vith c:oritr-ollc (l porosity do not exist at the present
time dLre to the
inherent t.wta diriierasionality of t:(-rc photolithographic process tlaat is
r,rsecl in
conventional `ilicoia based r.a-lic..r-~rDachirling.
1001541 De;raaonSt.F-ated here. is the deveIopii-aent of 3D containers witla.
precisely
engineered sLer-l'ac:c porosi~, and their utility in chemical encapsulation,
gLricl~.~ci
delivery, and spatially controlled c.hcmis[r~>. Brretly, the process
irivr,>lved t1w
photolithographic fabrication of.' a 2D metallic template with solder hia~ges
(Fig. 22a).
The 2D template self-assembled into the 31) hollow polyhedron when it wa;
heated

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
52
above the ri-ic;lti~~g point of tlic solder binges, whe.reiii t.EIC
stlrftÃ.c.C tc.DSiOTi zgf tllC tsl
solder provided tl-ic force to d.r.ive Selt-asscn-ibly [(a) Syala, R.R:t.
Yea.[mall. E,M.;
Brig,lit., V.M , Whitesides., G.M. I 'NIEVIS 200-3., 12, 387 --- 417. (b) 1-
lui, E. ;1; ; I-~ower
R. =I ;Rocigers, M. R.; Uth :lnt. Conf MEMS, 2000õ 6021r607.{c} Gimi, B;
=1_eorig; rF; Gu, Z,;'r'ang, M.; Aa-teriiov, D.; 1;31~~ijwalia. Z.; Gracias,
D_ H. Biomed.
Micr-odevice"005, 7, -341-3]. Containers 1~ave been tabrica:t.e~ I.N.1itla
clit't'ereÃit shapes
aiici volurnes ran~irzo t`rc~iia 230 picoliters to 8 tia~iat~likers (Fig . 22
a-d), I'he fabricaÃ:icat~
process was also 1-iigblv pai-a11el; containers of different sba~~~~ ~~ld
Sires coulcl be
fabricated in a single process run (i.e, from a single wafer, :1~'~~ 211 e-g).
When the
process z~,as optimized yields ranged from fi0-90 o (yielcls varied fo~r
difTerent sI-~aped
caniaine.r~ depending on tE-i~ ~~~~~~~~er of fb(d:ing f=aces and ttie
syrnmei.rN) for a 3-
wa:ler, I":1le tii.~~or v.icld li.anite.rs itt the t~,br-cai:iort proc~~~
~~~ere t:lic phoioIit1iog-raphic
fidelity in the reg:isÃav ot' hinge5 with respect t.o the tices, and t:l~e
volu.me of solder if)
t1ic hziiges ~Syms :R. R. A. J. MENrtS 1999, 8. 448-455. (15) De.ri.,. 'I'.:
Wliitesides. G.
M.; Rad}iakrishiiati, M,; Zabcyw. Ãi.; PreÃitiss. NI1'':.App1. Pllys_ Lett.
2001, 78, I7r5r
1777]. Since pIaotolltbcgCFraphic mic:rc}fabaYcatrc}n is highly precise, it
was a.lso possible
to pafterii one or more faces of the containers with monodisperse pores (Fig.
?22 h-k).
'1-1ie size. of tlie pores fomied was limited by the I.~1i~.~tomaskS Lised
(wIlicli in this case
had a resolution of 3 mici-otiS). Bv controlling the porosity it was possible
to ent;Ãneer
the rcaigeni release profiles as shown in Fig, 21.
001 '55,55~ fi'(ie c:onta.inerw were loaded ~isi~ig stereotactic
Microilijecti~.~n with a
solution cif a.:a;el (or po1~~r~~ea ) at~cl the chemical to be released. When
the solvent
evaporated, the gel r~inaÃnecl within tlie containers. The cheniieals were
released by
i.nuncrsing the loaded containers in a. sOlLItiOn that softened or cliswolved
the gel (or
~.~~.~lvizicr). Siizce gds 4aiid polymers) are available wit:l~ a wide range
of aolubÃIiq= aiid
s~
c~lenfng temperatures, it was, possibte to manipulate the clieiiiica.l release
rates tiSing
different ~~~lventS an~ temperatures. The images shown i.n the paper were
obtained
tising containers, loaded with a block copolymer bydrogc.l (Pluronicnx-).
Release
experinieiits were cl.oa-ita in a. water-alcohol 1.~asecl mc;di~IM (Details in
the

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
53
Sul]plernc.nitary SecÃ.ioazl..By varyiaig the relative poros.ity oia
diffcrcntfaces of the
container it was possible to get both isotropic. (Fig. 23:a.) as well as
ataisotrol3ic. (Fi(T.
?3b) chernical release profiles. Since tlae f'abric.ation process was
compatible with a
variety of aterials, it was possible to l;al?r:icate nickel based containers
that could be
remotely guic1ed using Ãaaagtietic fields. A spatialfv controlled. (the letter
G --any
arbitra:ry trajectory is posslble) claciaa.icaJ reaction was demonstrated by
directly
releasing (~%.Titing) a p:H ÃzadÃcatoa- phezaolphÃlaalcin in a microwcll
i~illcd witla an
a1k<alinesolutinn (Fig, 23c). Direct writing was possible by iaaanipulating
tlae
plaezaolplathaleiat-pluronic loaded coiatairter usiaxga magnetic stylus that
uasmt~~~ed
tinder the micrc?well. It slaould be nÃfted: that while guided manipulation
was clone
usita.~.~ a laer~~iatietat niagnet, it is possible to use otlicr well-
developed microcoil based
raaag.aaei:lc ixaanipulation circ.uits, [Deng, T.; Whitesides, G. N1;
Racllaakriy tnait. M.;
Zabow, C~.; Prentiss, M.Appl_ Phys. Lett, 200 1, 78;. 1775- :I777]', to
reproducibly control
t:(~c moveraaeaat oi"Ãlae corztaiiiei-s and li~iic,e tEic spatial release
o~t.'tlae chemicals witli
arl}atrard. p;a.tte ta.s.
1.001561 Spatially localized chemical rcactioias were also demonstrated
between
mu.ltiplc nanoliter scale containers (Fig. 24 a-c). 4k"hen two c~.~titainers
loaded Nvit1i
copper sulfate and patassiLim hydroxide respect.ivelv were brou:;lat close to
each otEier
in an aqueoLis medium, a c1a.eataica1 reaction (to ~orixa copper laydroxÃde}
occurred only
along the cciitra( line between t(ic two diffiasing rates; tlic reaction
occurred nearer the
containers witb the slower diffttsing chemical (Fig. 24d-f). These
ex1,eriine;ntz farth.cr
demonstrate that the spatial coaatrol over chemical reactions can be extended
to more
c.ornplek rexaction frotit~ involving mt.tltiple containers.
10015,71 -1n conclusion, as opposed to all organic cncapsia(ants, the
comaÃners allow
taizpreccclciated spatial coiatrol over tlac release of cheiiaical reagents by
iTi rttic of their
versatility in slaapes and sizes; aitisotropic l`a.ces; monodisperse porosity,
and their
ability to be gmdded in micrcgl'ltÃidic channels Lasliag magnetic fields.
Additionally, the
nieta11ic containers interact witb remotc electromagnetic fields that allow
thiaii. to bc
easily d~.~ttactecl tza-id tracked (U5ing magneric resonance imaging, MRI).
ThLis, tlle

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
54
containers proa=ide an attractive laIa1-Cora7a for e:ngi.tac.e:.rirrg rei-
iiÃ.stc::ty guided, spatially
controlled clae.mica.l reactions in rYiicrc3t lLiidÃ'c. systems.
Fabrication of tlac MicrOcOnta'sncrs:
1001581 .t1. 5.5 pznwthick Sac.riticia.i 1aver ot poly (meÃlaw 1.iaaettlacrw
lza.te) (P'41:M,'L,
MW: 996K) [Sigriia-Adlr-icli was ypuii oai a silic.on wa:fer.
On top of -lre PMMA-c.c~~ted waf:ei`, a 15.nna ad}resior3-prÃ.arnÃ:at.ir3g
c}rr`crrTiiUM (Cr) l~IVer`
and a 100 rarii conductive seed copper (Cu) layer were evap~.~rci.ted. After
tl-ae tlairl fill-a}
deposition, we spin coated a layer of Shipley 41'R.220 7.0 photoresist
[;Rohiai and Haa.S,
www.ro}uYahaas.ccam J, The tkrickriesS of the photoresist vva5 c.otatrtal:lec1
via the spin
speed aÃid bv varyina, tlie number of c.oatiÃigs applied_ :1fter- a soft bake;
the resi,,;t lvas
exposed to [iV light using an t:ltra pline Series Quintel ma.sk alip,.raer
[QuiÃitel C't?rp.,
v,ww.quÃntelcorp.c.ona ] arad patterned using a ti`a.raslaarenc,v xnask. After
developing the
photnresisi; electroclepÃ3sitÃr~~i was used to ~.~row tlre rtaetallic t`ratnes
of the
znic.rocontainer-s with.ira the plaotc3resist mold to a heiglYt of 6w15 urta
tdelsendirxgon t:lae
characterÃstics reqa.Ãired by ~~arioÃ.~s applications). We used ccammercial
electrolytic
soIutions that contained the rr-aetal iÃ~iis of choice ['1"echaic, [ne.,
www:tec.bnie..cÃ-arar j tca
e:lectrodelaoait dikTereÃat Ã-iietals. For the construction of non-mag aaet:ic
coratairaer-s. Cu
was electroplated, and for ma,xietic containers, :~:i was uSed_ A second round
of
photolitho.graphy was perforÃaaed in order to pattern the laiÃiges. A laver of
SI'R'??U was
sptira Ã.~ia top of the stibstrate aiad exposed tÃ.~ tlre hinge mask. WÃdeÃ-,
irateà rral hinges
were located betweet~ aqja.cent faces, whereas the t}ainrier, external
liiriges resided at
the otiter edgeS of the frames. Alignment muks were tised to enst7xe
aligiiment of the
hinges to tlac franie>s of the -2D pre:cLirSors. After tlae. Iringe patteraa;:
were developed in
451 Developer, the exposed C:Li (seed) a:Ãad Cr (adhesion) regions Ãra between
tlle
electrodelaosited frames were etc}ied using commercial etchants Ã:AP'~-100 for
CLI and
CRE-473 for Cr jTechnic. Inc., www.tec;hnic.comj). Tiai:'lead (60:'40, m.p. -Y
18~? T)

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
solder [Tc:c:hnic:.q 1:11c,., www.tc:c;linic.c;om] was then electroplated
iTitc:s the hinge regions.
The hei~.~lit of the hinges was apprc3xiÃiiately 16 ;~~i-ii. xkt`Ãer the
solder electrocleposiÃ:iorl,
the photoresist layers were stripped off with acetotae, the remaining Cu seed
itid Cr
adhesion laverc were elclwd atid the 2Drcp.reeursor teiTipiaÃe composed of
rnetal frames
connected with qolder hinu;~s was ai-nriiersed in NwMet~iv:1 l;'vrrolidone
(N\11)} CSi~,~niar
Adlrich, www.sigma-alcirich.coarij to dissolve the saci-ificial 1''NNIltIA
layer a:tid release
the precursors frc~~ii the wa:fieÃ~, A1iproxiÃ~iaield 50 precursors izl NM:P
wer~ spread
across a s~~iall crystallizaÃion disl-a aÃid a siYiaJl aaYioÃint of 45,' RM_A
flux [1Ã1diiim
C'cirporaÃion, www.indium_com] was added to dissolve any solder oxides that
may
have.foa-med. The disli wa.s heated to 100 'C:` for 3 miiiut.es and then r~:~n-
peci zip to 250
<,C' loÃ~ approximately 9Ã3 secoÃ-ids uÃit71 the solder becai-iYe ti-iolteÃi
Durin4~: re{'lc?~v; i~- the
sol~er .:~~et t~ie top 1aver of metal ofii the 2D precursor, the fzabnoation
yields were Iloor.
Solder wet copper well but did tioà we Ni well, }ience for contaiÃxer,"Ii ith
C'i.l frames it
wa.s necessary to add a tliiii Ni laver to iriiprc~~~e yieids, WheÃi ;s6lder
reffinved, [tie
niolten solder at the Iii~iges asid generated the toÃ-qÃae to fold the 2l:3
precursors into 3.13
micrcgccgntainers. Upon cooling, the solder solidified anct permanently licld
the
container ~rwnea togethcr.
Ct~tititinei- Loading:
1001591 Two metbÃsds were tised tc? load reagents into the contaitiers,
depending o-n
the we;ttabili.ty of the c;hÃ;ia-iicaI reagent ori the c:orataines-. Wtieri
the ctic:Ãi-iia;FÃ1. wet the
c~iitairier weltq several boxc.s ivc:.re simultaneously loaded by im~~iersing
t?ien-i in a drop
ol='the chen:Ãie;al i-cagent. The solvent was removed by evaporatioii. This
lc:ft behind the
Pc?lviller [P( T011iocE~' F68, BASF, www.basif.t.oml soaked with the chemical
reagent.
10016Ã31 The 5eÃ:or.ad nic:thod utilized t,,vt~ three-a~~s Newport
micromanipulators
[Models 460A & M462, uwu.neupor-t.c;otn ] to iiidepei}dei}t1y i:caritrol tlxc
position oi'
the microc:.untainc:r and the s'=rÃ~<~~~ [World Precision Insrr~r:~~~;r~ts,
Inc:. :"~~~,~afi1T~~
Syr:irlgc;, www.wpnnt:.z.ora-ij. The syringe was ou[-fitted witli a 36-gauge ~-
icedle [WPI;l
36 f_i-augc. Needle. wwwwp.ii.nc.com] t.o facilitate Io,Ãci.i~ig af the
mic:.rOc:.ODtai~-lerS.
Cheniacal Release & ~eaclioaa Specatacs:

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
56
10016:11 lted I)ye (Fig. ~~a-b}: Containers tvere loaded witlr
a mixture cnriipnsed of 1.6 rnl, i0.26I wYl F DX:{_` Red 40 [:~~~cCo-rri-iieA
& Co., lÃic.,
www.mccorxnick _corn] arrel an aqueous po(vtireric solÃrt-iorr Ã:.orriposed of
1.0 c, of
P1a.rrozr.1c:F68 dissolved in 10 niL of water (18A ?~10) . A 2:1:2 (by
volr.rrne) mixture of
g1ycernl:ethanol:wate:r was used as ttie diffusion metl:itrrir arrd this
medium was added
to a small cliamber ccrÃrt.~~iiirig ttie loaded miÃ:.Ã-oÃ:.otxta.itier. The
diffusion profiles were
irr-rkged usizig a. stereozoom binocular rgzia;roscope.
1Utll.~"i21 caliyr-G uided 1'berro(pbt&ialei.ti-KOt=:l Reaction (Fig. 23c): 'f
.lre
indicator mixture for the pla~iiolplat(ialein-k.OH reaction was pr-epa:red by
adding 0.25
mL of p}rertotp}rthaleirr solution {0.5 h; of ptr.errc?lpht.lraleitt [Frey
Scientitie.
~~wv.v .:i=a~evscieÃrtitic .coÃ-ir] .i11 Ioorr-ii. ot ~~5%etlrarloi)toarr
afli,ÃeoLispolyri-ieÃ-icsÃ~lÃtt:i(ari
composed of :I .O g of PlÃrronic F`68 d:issc)lved in 10 ÃTiL -water= and
loaded irrto a Ãrickel-
based microcontairrea, The microc~~nÃairr~r was placed irrto a well of a
tissue ctiItuÃ-e
pIate, [;Falcozi(P)1~.1u:ltiwell`l M`l'issa:~e Culture lslate: 24 Well,
arici a 1:1:1 (by vo[Ãr.me) g;lycer-ol- water: I ?~~~ KOtl(aq} mediurrl was
iriirodÃtcecl irito
the chanil?er. The micrcgccgrit.ainer was guided a-nd, controlled usMrw a 0.35
pcÃli lb., :1 1/8"
dia~icter A_(\iCo rouaid bar magiiet [McMastea -C"arr, ww-w-.mc:ma.ster.coÃr
Ãj.
C x~~>~?ei ~ Il) St:al~`i'tt~> ~'c fr~<t~?t'~~t~ate fsl..)t~{ Rccic;ifon ff-ig
24a-ck
L'uSC.)::~aq3 + 2 KC.)H(aq) K,S(.).,(aq) + C'Ãi(OH)>4s)
1Ã)Ftl.1.~"i31 The copper sulfate reactaÃit mixture was pr~epar-ed by
dissolvirio 1.0 g of
Plu.rorriÃ, F68 irrÃ:o 10 rril. crt' 0_5 "~=1==; Cu{ii}~~~~ aqueous yolÃrt:ior
[Sit'rrra-Altl,riclr.,
z~ ~~,w.si_Qrr-ia-a1tir.ic.}i-com I arrd was loaded irrto a ri-
iierÃsc:4sit.tairrer. The potassi111,11
hydroxide reactant tiaix[us-e was larelaai-ed by dissolving :1.0 g of
P1uronit:. F68 fl-Itts 10
rrrl:, of 1.0 M KOH(~~) arrd was loaded into a second rnicroconta.irrer.
'f'1're
micr-ocoÃ-ttairae~~ were placed in close proximity into a poly (dirxretlivl
siloxane)
[PDMS> Dow C:`orrr:iÃrg Sylgard(k# 184, www_dÃ~N.veorning.con) j trrier-owelt.
'f .lr.e
znicro-~x-e:ll was fabricated by:mc~lding PDM:S against aii SU-8 p}rotoresist
Ãn. aster: The
ditfus'Mr and reaction Ãriedium was water.

CA 02656648 2008-12-23
WO 2008/108862 PCT/US2007/072029
57
1~=f3I1----Rcxcraicm (j_{g '4_ `.l'he iTtc{ic:a,tor ri-iixture for the
p1terinlp}rthaleinwkOH i-cactioti was prepared by adding 0,25 mL of
phenolphthalein
solution to an ac1UeOLIs pÃ~IyrTtcrie sO(utioti eÃ7i-a7pÃ5s-cd of 1.0 g of
Plurortic F68
dissolved in 10 txzl, water. The alkalirle ri-iixtu.re was prepared by
acldirtg 0_3 mL ol
4M :Kt7H(aq) [Sigma-Alclrich. W-w,~x.sagtnaraldrÃch.comj to an aqueoLts
polymeric
soltÃtion composed of l,O g Pluronic F68 and 1.0 iTIL water. Two containers
were
loaded wit:ti the pli~iin11~lit:EialciÃi sc~lutioia azid one vvith Ãbe KOt:-:(
solt-itiÃiti. 'The tkirec
containers were tlw.ti placed into a 1'D:MS Ãiiici-owe(l, with water as itie
diffusion atid
reaction medium. 'I'}ie reactions were also imaged uSino a Stet-eozoom
binocular
micrc?scope.
1[){1164] W1ii1e the presetit: itivetitioti E-iaS bec~i described with
re{eretice to the
specitie evabod.itx~ents t}icreof it should be understood by those skilled in
the art that
various changes may be made atid eqttivaleiits may be substitÃtted without
departing
troti-z the true spirit arzd scope of the iriveritioÃi. :In additinri< many
modificatiot~s may
be zrtade to adopt a particular sittiat:ioti; ma.t:cr-ia(, compoyitiott of
matter, process,
process step or steps, to the objective slairit and scope of the present
invention. Ali
st.tc:li modifications are intended to be witliii t the scope of the c.laims,
ap.1,e~~~ieci hcreto.

Dessin représentatif