The design and functionalization of porous materials have

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1 ARTCLES PUBLSHED ONLNE: 26 AUGUST 22 DO:.38/NMAT344 Mroporous nnowire nnoeletroni sffolds for sntheti tissues Bozhi Tin,2,3, Ji Liu, Tl Dvir 2,4, Lihu Jin, Jonthn H. Tsui 2, Qun Qing, Zhigng Suo, Roert Lnger 3,4, Dniel S. Kohne 2 * nd Chrles M. Lieer, * The development of three-dimensionl (3D) sntheti iomterils s struturl nd iotive sffolds is entrl to fields rnging from ellulr iophsis to regenertive mediine. As of et, these sffolds nnot eletrill proe the phsiohemil nd iologil miroenvironments throughout their 3D nd mroporous interior, lthough this pilit ould hve mrked impt in oth eletronis nd iomterils. Here, we ddress this hllenge using mroporous, fleile nd free-stnding nnowire nnoeletroni sffolds (nnoes), nd their hrids with sntheti or nturl iomterils. 3D mroporous nnoes mimi the struture of nturl tissue sffolds, nd the were formed self-orgniztion of oplnr retiulr networks with uilt-in strin nd mnipultion of 2D mesh mtries. NnoES ehiited roust eletroni properties nd hve een used lone or omined with other iomterils s ioomptile etrellulr sffolds for 3D ulture of neurons, rdiomotes nd smooth musle ells. Furthermore, we show the integrted sensor pilit of the nnoes rel-time monitoring of the lol eletril tivit within 3D nnoes/rdiomote onstruts, the response of 3D-nnoES-sed neurl nd rdi tissue models to drugs, nd distint ph hnges inside nd outside tuulr vsulr smooth musle onstruts. The design nd funtionliztion of porous mterils hve een tivel pursued to enle new mteril properties nd pplitions 3. n prtiulr, the development of sntheti 3D mroporous iomterils s etrellulr mtries (ECMs) represents ke re euse funtionlized 3D iomterils llow for studies of ell/tissue development in the presene of sptiotemporl iohemil stimulnts 3 6, nd the understnding of the phrmologil response of ells within sntheti tissues is epeted to provide more roust link to in vivo disese tretment thn tht from 2D ell ultures 6 8. Advning further suh iomterils requires pilities for monitoring ells throughout the 3D miro-environment 6. Although eletril sensors re ttrtive tools, it hs not een possile to integrte suh elements with porous 3D sffolds for lolized rel-time monitoring of ellulr tivities nd phsiohemil hnge; suh pilit ould led to new l-on--hip phrmologil pltforms 9, nd hrid 3D eletronis tissue mterils for sntheti iolog,2. Reentl, there hve een severl reports desriing the oupling of eletronis nd tissues using fleile nd/or strethle plnr devies 3 7 tht onform to nturl tissue surfes. These plnr devies hve een used to proe eletril tivities ner surfes of the hert 3, rin 6 nd skin 7. So fr, semless 3D integrtion of eletronis with iomterils nd sntheti tissues hs not een hieved. Ke points tht must e ddressed to hieve this gol inlude: the eletroni strutures must e mroporous, not plnr, to enle 3D interpenetrtion with iomterils; the eletroni network should hve nnometre to mirometre sle fetures omprle to iomteril sffolds; nd the eletroni network must hve 3D interonnetivit nd mehnil properties similr to iomterils. Here we introdue oneptull new pproh tht meets this hllenge integrting nnoeletronis throughout iomterils nd sntheti tissues in three dimensions using mroporous nnoeletroni sffolds. We use silion nnowire field-effet trnsistor (FET)-sed nnoeletroni iomterils, given their pilit for reording oth etrellulr nd intrellulr signls with suellulr resolution 8 2. FET detetors respond to vritions in potentil t the surfe of the trnsistor hnnel region, nd the re tpill lled tive detetors 2. Metl eletrode 22,23 - or ron nnotue/nnofire 24,2 -sed pssive detetors re not onsidered in our work euse impedne limittions (tht is, signl/noise nd temporl resolution degrde s the re of the metl or ron eletrodes is deresed) mke it diffiult to redue the size of individul eletrodes to the suellulr level 2 23, size regime neessr to hieve non-invsive 3D interfe of eletronis with ells in tissue. Our pproh (Fig. ) involved stepwise inorportion of iomimeti nd iologil elements into nnoeletroni networks ross nnometre to entimetre size sles. First, hemill snthesized kinked 8 or uniform silion nnowires were deposited either rndoml or in regulr ptterns for single-nnowire FETs the nnoeletroni sensor elements of the hrid iomterils (step A, Fig. ). Seond, individul nnowire FET devies were lithogrphill ptterned nd integrted into free-stnding mroporous sffolds (step B, Fig. ), the nnoes. The nnoes were designed to mimi ECM strutures, nd speifill, to e 3D, to hve nnometre to mirometre fetures with high (>99%) porosit Deprtment of Chemistr nd Chemil Biolog, Hrvrd Universit, Cmridge, Msshusetts 238, USA, 2 Deprtment of Anesthesiolog, Division of Critil Cre Mediine, Children s Hospitl Boston, Hrvrd Medil Shool, Boston, Msshusetts 2, USA, 3 Dvid H. Koh nstitute for ntegrtive Cner Reserh, Msshusetts nstitute of Tehnolog, Cmridge, Msshusetts 239, USA, 4 Deprtment of Chemil Engineering, Msshusetts nstitute of Tehnolog, Cmridge, Msshusetts 239, USA, Shool of Engineering nd Applied Sienes, Hrvrd Universit, Cmridge, Msshusetts 238, USA. These uthors ontriuted equll to this work. *e-mil: Dniel.Kohne@hildrens.hrvrd.edu; ml@mliris.hrvrd.edu. NATURE MATERALS ADVANCE ONLNE PUBLCATON 22 Mmilln Pulishers Limited. All rights reserved.

2 ARTCLES Eletroni sstem Nnosle Bottom-up Mrosle A Nnowires Nnowire FET B Biomimetis Nnoeletroni sffold C Biologil sstem Nnoeletronis tissue hrid onstrut Figure ntegrting nnoeletronis with ells nd tissue. Conventionl ulk eletronis re distint from iologil sstems in omposition, struturl hierrh, mehnis nd funtion. Their eletril oupling t the tissue/orgn level is usull limited to the tissue surfe, where onl oundr or glol informtion n e glened unless invsive pprohes re used. We hve introdued new onept reting n integrted sstem from disrete eletroni nd iologil uilding loks (for emple, semiondutor nnowires, moleulr preursors of polmers nd single ells). Three iomimeti nd ottom-up steps hve een designed: step A, ptterning, metlliztion nd epo pssivtion for single-nnowire FETs; step B, forming 3D nnowire FET mtries (nnoeletri sffolds) selfor mnul orgniztion nd hridiztion with trditionl ECMs; step C, inorportion of ells nd growth of sntheti tissue through iologil proesses. Yellow dots: nnowire omponents; lue rions: metl nd epo interonnets; green rions: trditionl ECMs; pink: ells. nd to e highl fleile nd ioomptile. NnoES were then omined with sntheti or nturl mroporous ECMs providing ECMs with eletril sensor funtion nd nnoes with iohemil environments suitle for tissue ulture. Finll, ells were ultured within the nnoes (step C, Fig. ) to ield 3D hrid nnoeletronis tissue onstruts. The emphsis on nnosle nd iomimeti ottom-up pthw llows minimll invsive integrtion of eletroni devies with ells nd ECM omponents t the suellulr level in three dimensions. The nnoes re distint from onventionl 2D multi-eletrode rrs 23, ron nnotue/nnofire rrs 24,2, implntle miro-eletrodes 23 nd fleile/strethle eletrodes 3 7 in tht the sensors re nnosle semiondutors, nd ritill, tht the sensor network is fleile, mroporous nd 3D. As result, nnoes re suitle for 3D ell ultures tht re known to resemle the struture, funtion or phsiolog of living tissues. We hve designed two nnoes (Fig. 2) tht re free-stnding, fleile nd ontin similr omponents. Both were frited on srifiil lers, whih were susequentl removed, ielding free-stnding nnoes (Methods nd Supplementr Figs S nd S2). n rief, ler of negtive resist (SU-8) ws oted on nikel srifiil ler, solution with kinked or stright nnowires ws deposited onto the SU-8 ler nd llowed to evporte, nd then SU-8 ws ptterned lithogrph to immoilize nnowires nd to provide the si frmework for nnoes. Etr nnowires were wshed w during the development proess of the SU-8 struture. Metl ontts were ptterned lithogrph nd deposition. Finll, ler of SU-8 ws deposited nd lithogrphill defined s the upper pssivtion ler on the interonnets. Retiulr nnoes were mde eletron em lithogrph (EBL). Self-orgniztion (tht is, folding ording to the predefined lout of ending elements) reted rndom or regulr NATURE MATERALS DO:.38/NMAT344 network of 3D fetures tht mimi the size sle nd morpholog of sumiron ECM fetures, suh s the firous meshwork of rin ECM (ref. 26). Open mesh nnoes were mde photolithogrph with regulr struture, similr to the ECM of the ventriulr mordium 27,28. 3D sffolds were then relized in strightforwrd mnner direted mesh mnipultion. The plnr design nd initil frition of these 3D nnoes use eisting pilities developed for onventionl plnr nnoeletronis, nd ould enle integrtion of dditionl devie omponents (for emple, memories nd logi gtes) 29,3 nd sustntil inreses in devie numer nd overll sffold size. The 2D struture of the retiulr sffold ws designed so tht metl interonnets were stressed 8,3. Removl of the srifiil ler prompted self-orgniztion into three dimensions. Reonstruted 3D onfol fluoresene imges of tpil retiulr sffolds viewed long the nd es (Fig. 2 nd respetivel) showed tht the frmework ws 3D with highl urviliner nd interonneted struture. The porosit (lulted from the initil plnr devie design nd the finl 3D onstrut volume) ws >99.8%, omprle to tht of hdrogel iomterils 6 8. Nnowire FET devies (Fig. 2) within the sffold spnned seprtions of μm in three dimensions (Supplementr Fig. S3), nd the retiulr sffold heights were less thn 3 μm for our present frition onditions. Devies n e mde loser together (for emple, <. μm) depositing nnowires more densel on the sustrte 3 to improve the sptil resolution of nnoeletroni sensors; the spn of devie seprtions nd sffold heights n e inresed sustntill using lrger field lithogrph (see elow). Snning eletron mirosop (SEM) of the retiulr nnoes (Fig. 2) reveled kinked nnowires (out 8 nm dimeter), nd metlli interonnets (out.7 μm width) ontined within the SU-8 kone (out μm width). The feture sizes re omprle to those of sntheti nd nturl ECMs (refs 3, 6), nd re severl orders of mgnitude smller thn those for eletroni strutures 23 penetrting tissue in three dimensions. The performne of devies ws evluted through wter-gte mesurements for the nnowire FET elements in the 3D sffolds in queous medium (Supplementr nformtion). The results show devie ields ( 8%), ondutnes (.2 ±.6 μs; men ± s.d.) nd sensitivities (8.7±2.92 μsv ) omprle to mesurements from plnr devies using similr nnowires 8. 3D mesh nnoes were relized folding nd rolling freestnding devie rrs. Mesh strutures (Fig. 2) were frited suh tht the nnoes mintined n pproimtel plnr onfigurtion following relief from the sustrte. A tpil 3. m. m 2 μm mesh nnoes, ws pproimtel plnr with 6 nnowire FET devies in regulr rr with 2D open porosit of 7% (Fig. 2d). This mesh porosit is omprle to tht of honeom-like sntheti ECM engineered for rdi tissue ulture 28. n ddition, the nnowires (Fig. 2d), metl interonnets (Fig. 2d2) nd SU-8 struturl elements (Fig. 2d3) hd n rel mss densit of <6 μgm 2, the lowest vlue reported so fr for fleile eletronis, whih reflets our mroporous rhiteture. The mesh nnoes ws fleile nd n e mnull rolled into tuulr onstruts with inner dimeters t lest s smll s. mm (Fig. 2e), nd folded. Mroporous strutures of the open mesh nnoes were formed either loosel stking djent mesh lers (Fig. 2f) or shping it with other iomterils. These pilities were onsistent with the estimted ultrlow effetive ending stiffness (Supplementr nformtion), whih ws tuned etween.6 nd.3 nn m for this mesh nd is omprle to reent plnr epiderml eletronis 7. The eletril trnsport hrteristis of the mesh nnoes were evluted in phosphte uffered sline. The tpil devie ield is 9 97%, with verge devie ondutne 3 μs nd sensitivit 2 NATURE MATERALS ADVANCE ONLNE PUBLCATON 22 Mmilln Pulishers Limited. All rights reserved.

3 NATURE MATERALS DO:.38/NMAT344 ARTCLES 2 3 z z d e f 3 2 g h i Condutne (μs) Count ~.7 mm. mm Count Sensitivit (μs V ) Sensitivit (μs V ) Numer of turns Condutne (μs) ΔG/G, ΔS/S (%) Devie inde Figure 2 Mroporous nd fleile nnowire nnoes., Devie frition shemtis. () Retiulr nnowire FET devies. () Mesh nnowire FET devies. Light lue: silion oide sustrtes; lue: nikel srifiil lers; green: nnoes; ellow dots: individul nnowire FETs., 3D reonstruted onfol fluoresene mirogrphs of retiulr nnoes viewed long the () nd () es. The sffold ws lelled with rhodmine 6G. The overll size of the struture, z = μm. Solid nd dshed open mgent squres indite two nnowire FET devies loted on different plnes long the is. Sle rs, 2 μm., SEM imge of single-kinked-nnowire FET within retiulr sffold, showing () the kinked nnowire, (2) metlli interonnets (dshed mgent lines) nd (3) the SU-8 kone. Sle r, 2 μm. d, Photogrph of mesh devie, showing () nnowires, (2) metl interonnets nd (3) SU-8 struturl elements. The irle indites the position of single-nnowire FET. Sle r, 2 mm. e, Photogrph of prtill rolled-up mesh devie. Sle r, mm. f, SEM imge of loosel pked mesh nnoes, showing the mroporous struture. Sle r, μm. g, Histogrms of nnowire FET ondutne nd sensitivit in one tpil mesh nnoes. The ondutne nd sensitivit were mesured in the wter-gte onfigurtion without rolling. The devie ield for this mesh nnoes is 9%. h, Wter-gte sensitivit nd ondutne of nnowire FET in mesh devie during the rolling proess. Upper pnel, shemti of the nnowire FET position (ellow dot) during the rolling proess; 6 denote the numer of turns. i, Reltive hnge in ondutne nd sensitivit of 4 nnowire FETs evenl distriuted throughout full rolled-up mesh devie. Upper pnel, shemti of the nnowire FET position (ellow dots). n h,i the thiknesses of the tuulr strutures hve een eggerted for shemti lrit. 7 μsv (Fig. 2g). Representtive ondutne (G) dt (Fig. 2h) from single-nnowire FET (Fig. 2h, ellow dots, upper pnel) during the rolling proess showed <.7 μs ondutne hnge ( G) or <2.3% totl hnge for 6 revolutions. Devie sensitivit (S) remined stle with mimum hnge ( S)of.3 μsv, or.% vrition. The stle performne during rolling n e eplined the low estimted strins of metl (<.%) nd SU-8 (<.27%) in this tuulr onstrut (Supplementr nformtion), nd showed tht the properties were pproimtel independent of lotion. Furthermore, 4 devies evenl distriuted on si lers of rolled-up sffold (Fig. 2i) showed mimum G = 6.8% nd S = 6.9% versus the unrolled stte, demonstrting devie roustness. Repetitive rolling nd reltion to the flt stte did not degrde the nnowire FET performne. These findings suggest the potentil for relile sensing/reording of dnmi nd deformle sstems. NATURE MATERALS ADVANCE ONLNE PUBLCATON Mmilln Pulishers Limited. All rights reserved.

4 ARTCLES NATURE MATERALS DO:.38/NMAT344 4 μm 6 μm A μm A Free-stnding prt 6 μm B 7 μm fied prt Height (μm) 3 2 B SU-8 3 nm SU-8 3 nm Cr nm Pd 7 nm SU-8 3 nm Projeted length (μm) d e f Figure 3 Geometr ontrol design in nnoes.,, Bsi design nd struturl suunit for simultion., Top-down view of the entire suunit. Blue rions re stressed metl lines with SU-8 pssivtion. Red lines re single SU-8 rions without residul stress., Cross-setionl views of those two ke struturl elements used for simultion., Plot of projeted (on the plne) length versus height (in the z diretion) for the vertil lue rion in s determined from the simultion. Open red squres with error rs re eperimentl dt (mens ±s.d.) reorded in ir for point A nd B in.the simultion of the ending of the suunit model for the retiulr struture ws rried out using the ommeril finite element softwre ABAQUS. The inset shows 3D view of the simulted struture, nd the sle r shows different heights in the z diretion. d, Shemti showing the integrtion of periodi retiulr-devie domins (light lue) into fleile mesh (green). n individul retiulr domins, the 3D devie positions reltive to the glol fleile mesh n e ontrolled their geometr designs ( ). e,f, Design ptterns () nd eperimentl dt () for two retiulr units. SU-8, metl nd nnowires re shown in lue, pink nd ellow in e. Chnging the struture of the onneting feture (white rrows) etween djent devie units during pttern design () ields ontrolled vritions in the 3D positioning of the nnowire FETs, whih n e further tuned the stress in the metl onnetions. n these eperiments, the devie positions re 4 μm (e) nd 23 μm (f) ove the mesh plne. Sle rs in e,f, 2μm. We hve rried out simultions of suunit of the self-orgnizing retiulr struture (Fig. 3 ). Mesurements of ending for the orresponding eperimentl strutures (Fig. 3, open red squres) re onsistent with the simultion (Fig. 3). Additionll, hnges in struturl prmeters (for emple, the totl length of the suunit nd thiknesses of SU-8 or metls) ield preditle hnges in the ending ngle of the suunit (Supplementr Fig. S4). n this w, ordered 3D nnowire FET rrs n e designed nd frited using retiulr- or mesh-like strutures tht inorporte multi-ler metl interonnets with uilt-in stress to self-orgnize (roll-up) the sffold (Supplementr Fig. S4). Finll, we hve designed retiulr domins in mesh-like strutures (Fig. 3d). mges of retiulr domins (Fig. 3e,f) show tht regulr nnowire FET devies with distint devie positions n e relized vring the struturl prmeters of individul elements. Overll, this pproh ields hierrhil 3D nnoes with sumirometre to mirometre sle ontrol in retiulr domins nd millimetre to entimetre sle in the mesh mtri folding or rolling s shown ove (Fig. 2). The retiulr nd mesh nnoes were lso merged with onventionl mroporous iomterils. Speifill, gel sting, lophiliztion nd eletrospinning were used to deposit nd onstrut mroporous ollgen (Fig. 4), lginte (Fig. 4) nd pol(lti-o-gloli id) (PLGA; Fig. 4), respetivel, round nnoes. A onfol fluoresene mirogrph of hrid retiulr nnoes/ollgen sffold (Fig. 4) shows lerl tht the ollgen nnofires (green) re full entngled with the nnoes, with no evidene of phse seprtion. SEM imges of the open mesh nnoes/lginte hrid sffold produed lophiliztion (Fig. 4) show tht the fleile nnoes mesh is intimtel nhored to the lginte frmework, whih hs similr pore struture s the pure lginte sffold prepred under similr onditions. Finll, optil mirogrphs of multilered mesh nnoes/plga sffold (Fig. 4), whih ws prepred eletrospinning PLGA fires on oth sides of the nnoes nd susequent folding of the hrid struture, highlight the intimte ontt etween nnoes mesh nd PLGA fires. The hrid nnoes/iomteril 3D sffolds retin the originl nnowire FET devie hrteristis. For emple, mesurements in phosphte uffered sline solution showed tht G/G nd S/S were less thn ±9% for the mesh nnoes/plga omposite versus re nnoes. Hrid nnoes were stle under ell ulture onditions. For emple, nnowire FET devies in the hrid retiulr nnoes/mtrigel sffold in neuron ulture medi (Fig. 4d) hd S/S < ±% over nine-week period, suggesting pilit for long-term ulture nd monitoring with the nnoes. These results show tht nnoes n e omined with onventionl iomterils to produe hrid sffolds tht now provide nnosle eletril sensor omponents distriuted in three dimensions. 4 NATURE MATERALS ADVANCE ONLNE PUBLCATON 22 Mmilln Pulishers Limited. All rights reserved.

5 NATURE MATERALS DO:.38/NMAT344 ARTCLES d 2 ΔS/S (%) Time (week) Figure 4 Hrid mroporous nnoeletroni sffolds., Confol fluoresene mirogrph of hrid retiulr nnoes/ollgen mtri. Green (fluoresein isothionte): ollgen tpe-; ornge (rhodmine 6G): epo rions. The white rrow mrks the position of the nnowire. Sle r, μm., SEM imges of mesh nnoes/lginte sffold, top () nd side () views. The epo rions from nnoes re flse-oloured in rown for lrit. Sle rs, 2 μm () nd μm ()., A right-field optil mirogrph of the folded sffold, showing multilered strutures of PLGA nd nnoeletroni interonnets. The inset shows photogrph of the hrid sheet efore folding. A sheet of PLGA fires with dimeters of 3 μm ws deposited on oth sides of the devie. No dmge or redution of devie ield ws oserved following this deposition. Sle rs, 2 μm nd mm (inset). d, Reltive hnges in nnowire FET sensitivit over time in ulture (37 C; % CO 2, supplemented neurosl medium). n = ; dt re mens ±s.d. The hrid nnoes were evluted in 3D ulture 32,33 for severl ell tpes. Emroni rt hippompl neurons were ultured in the retiulr nnoes/mtrigel for 7 2 ds (Supplementr Fig. S). Reonstruted 3D onfol mirogrphs from two-week ulture (Fig., nd Supplementr Fig. S6) showed neurons with high densit of sptill interonneted neurites tht penetrted the retiulr nnoes (Fig. ), often pssing through the ring strutures supporting individul nnowire FETs (Fig. nd Supplementr Fig. S6). Notl, the widths of the sffold elements (pssivted metl interonnets nd struturl rions) were similr to those of the neurite projetions, demonstrting the omintion of eletronis with iologil sstems t n unpreedented similrit in sle. 3D nnoeletroni rdi ulture ws hieved from hrid mesh nnoes/plga sffolds (Supplementr Figs S7 S9). Confol fluoresene mirosop of rdi 3D ulture (Fig. ) reveled high densit of rdiomotes in lose ontt with nnoes omponents. Epifluoresene mirogrphs of rdi ells on the surfe of the nnoes rdi pth showed stritions hrteristi of rdi tissue 28,32 (Fig. d nd Supplementr Figs S8 nd S9). n ddition, the in vitro totoiit of nnoes in 3D neurl nd rdi ulture ws evluted (Fig. e,f). Differenes etween hippompl neurons in retiulr nnoes/mtrigel versus Mtrigel over 2 ds, ssessed with stndrd LVE/DEAD ell ss 33 (Fig. e), nd etween rdi ells in hrid mesh nnoes/mtrigel/plga nd Mtrigel/PLGA from 2 to 2 ds, mesured with metoli tivit ss (Fig. f), were miniml. These studies show tht on the 2 3 week timesle, the nnoes omponent of the sffolds hs little effet on the ell viilit, nd thus n e eploited for numer of in vitro studies, inluding drug sreening sss with these sntheti neurl nd rdi tissues. Etended studies will e needed to evlute the nnoes for longerterm implnts, lthough the min omponent of nnoes, SU-8, hs demonstrted long-term hroni ioomptiilit suitle for in vivo reording 34,3. The monitoring pilities of the nnoes were first demonstrted in 3D rdiomote mesh onstrut (Fig. g). The output reorded from single-nnowire FET (Fig. g) 2 μm elow the onstrut surfe showed regulrl sped spikes with frequen of Hz, lirted potentil hnge of 2 3 mv, signl/noise 3 nd 2 ms width. The pek mplitude, shpe nd width re onsistent with etrellulr reordings from rdiomotes 2. The potentil of the nnoes-sed 3D rdi ulture to monitor pproprite phrmologil response ws investigted dosing the 3D rdiomote mesh onstrut with nordrenline (lso known s norepinephrine), drug tht stimultes rdi ontrtion through β -drenergi reeptors 36. Mesurements from the sme nnowire FET devie showed twofold inrese in the ontrtion frequen following drug pplition. nterestingl, reordings from two nnowire FETs from the rdi pth on nordrenline pplition showed sumilliseond nd milliseond level, heterogeneous ellulr responses to the drug (Supplementr Fig. S). Additionll, multipleing mesurements mde with retiulr nnoes/neurl onstrut (Supplementr Fig. S) showed tht the 3D response of glutmte tivtion ould e monitored. Together these eperiments suggest nnoes onstruts n monitor in vitro the response to drugs from 3D tissue models, nd thus hve potentil s pltform for in vitro phrmologil studies 9,. Lst, simultneous reordings from four nnowire FETs with seprtions up to 6.8 mm in nnoes/rdi onstrut (Fig. h) demonstrted multipleed sensing NATURE MATERALS ADVANCE ONLNE PUBLCATON 22 Mmilln Pulishers Limited. All rights reserved.

6 PLGA ARTCLES NATURE MATERALS DO:.38/NMAT344 z z d SU-8 Metl e Vile ell perentge (%) Neuron Smple Control f Metoli tivit (.u.)... Crdiomote Smple Control Time (d) Time (d) g h Condutne 2 ns + nordrenline Condutne ns 2 s Time Time (ms) Figure 3D ell ulture nd eletril sensing in nnoes.,, 3D reonstruted onfol imges of rt hippompl neurons fter two-week ulture in Mtrigel on retiulr nnoes. Red (Ale Fluor 46): neuronl β-tuulin; ellow (rhodmine 6G): epo rions. The metl interonnets re flse-oloured in lue, nd re imged in the refleted light mode. The white rrow highlights neurite pssing through ring-like struture supporting nnowire FET. Dimensions in, : 37 μm; : 37 μm; z: μm; in, : 27μm; : 27μm; z: 68μm., Confol fluoresene mirogrphs of sntheti rdi pth. ( nd ), Zoomed-in view of the upper nd lower dshed regions in, showing metl interonnets, the SU-8 sffold (rrows in ) nd eletrospun PLGA fires (rrows in ). Sle r, 4 μm. d, Epifluoresene mirogrph of the surfe of the rdi pth. Green (Ale Fluor 488): α-tin; lue (Hoehst 348): ell nulei. The position of the soure drin eletrodes is outlined with dshed lines. Sle r, 4 μm. e, Perentge of vile hippompl neurons ultured in nnoes/mtrigel versus Mtrigel. Cell viilit ws evluted with LVE/DEAD totoiit ss. Cells were ounted from 3D reonstruted onfol fluoresene mirogrphs. n = 6; dt re mens ±s.d. Differenes etween groups were ver smll lthough sttistill signifint (p <.). f, Metoli tivit of rdiomotes evluted using the MTS ss. n = 6; dt re mens ±s.d. Differenes etween groups were ver smll lthough sttistill signifint (p <.). g, Condutne versus time tres reorded from single-nnowire FET efore (lk) nd fter (lue) ppling nordrenline. h, Multiple eletril reording of etrellulr field potentils from four nnowire FETs in mesh nnoes. Dt re ondutne versus time tres of single spike reorded t eh nnowire FET. of oherentl eting rdi pth, with sumilliseond time resolution. Our urrent devie design ields reltivel sprse devie distriution with 6 devies over n re of out 3..m 2. nreses in nnowire FET densit, the use of ross-r iruits nd implementing multipleing/demultipleing for ddressing 3 ould llow the nnoes sffolds to mp rdi nd other sntheti 6 NATURE MATERALS ADVANCE ONLNE PUBLCATON 22 Mmilln Pulishers Limited. All rights reserved.

7 2 ns NATURE MATERALS DO:.38/NMAT344 ARTCLES Lumen d e Lumen Lumen f Condutne nner tuing 6.9 Outer tuing Vsulr onstrut Ag/AgCl PDMS Outer nner, Time (s), 2, 2, Figure 6 Sntheti vsulr onstrut enled for sensing., Shemti of the snthesis of smooth musle nnoes. The upper pnels re side views, nd the lower ones re either top views ( nd ) or zoom-in view (). Gre: mesh nnoes; lue fires: ollgenous mtri sereted HASMCs; ellow dots: nnowire FETs; pink: HASMCs., () Photogrph of single HASMC sheet ultured with sodium L-sorte on nnoes. () Zoomed-in view of the dshed re in, showing metlli interonnets mrosopill integrted with ellulr sheet. Sle r, mm., Photogrph of the vsulr onstrut fter rolling into tue nd mturtion in ulture hmer for three weeks. Sle r, mm. d () Miro-omputed tomogrph of tuulr onstrut segment. () Zoomed-in view of the re outlined in. The rrows mrk the individul nnowire FET-ontining lers of the rolled onstrut. Sle r, mm. e, Hemtolin Eosin- () nd Msson-Trihrome- (; ollgen is lue) stined setions ( 6 μm thik) ut perpendiulr to the tue is; lumen regions re lelled. The rrows mrk the positions of SU-8 rions of the nnoes. Sle rs, μm. f, Chnges in ondutne over time for two nnowire FET devies loted in the outermost (red) nd innermost (lue) lers. The inset shows shemti of the eperimentl set-up. Outer tuing delivered thing solutions with vring ph (red dshed lines nd rrows); inner tuing delivered solutions with fied ph (lue dshed lines nd rrows). NATURE MATERALS ADVANCE ONLNE PUBLCATON Mmilln Pulishers Limited. All rights reserved.

8 ARTCLES tissue eletril tivities over the entire onstruts t high densit in three dimensions. We hve lso etended our pproh towrds the development of rtifiil tissue with emedded nnoeletroni sensor pilities. Speifill, vsulr nnoes onstruts were prepred proesses nlogous to those used for tissue-engineered utologous lood vessels 37,38 eept for the ddition of the nnoes (Fig. 6 nd Supplementr Fig. S2). Humn orti smooth musle ells (HASMCs) were ultured on 2D mesh nnoes with sodium sorte to promote the deposition of nturl ECM. The hrid nnoes/hasmc sheets (Fig. 6) were rolled into multi-ler 3D tuulr strutures nd mtured (Supplementr nformtion) without mrosopi delmintion or desqumtion (Fig. 6), nd nlses showed tht the ells epressed smooth musle α-tin (Supplementr Fig. S3), the ke ontrtile protein in smooth musle 37. The distriution of nnoes in the tuulr onstrut ws visulized miro-omputed tomogrph (μct). A projetion of the reonstruted 3D μct dt (Fig. 6d) reveled regulrl sped metl interonnets with t lest four revolutions (rrows, Fig. 6d), onsistent with the nnowire FET mesh nd tissue rolling. Anlses of hemotolin Eosin- nd Msson-trihrome-stined setions (Fig. 6e) reveled smooth musle tissue 2 μm thik, with elongted ells nd ollgenous nnofires, nd emedded SU-8 rions from the nnoes (Fig. 6e). These findings onfirm the 3D integrtion of nnowire FET nnoeletronis with helth smooth musle. The potentil of this vsulr onstrut to funtion s iomedil devie ws demonstrted 3D ph sensing (Fig. 6f, inset). As the etrvsulr ph ws vried stepwise with luminl ph fied, simultneous reordings from p-tpe nnowire FETs in the outermost ler showed stepwise ondutne dereses with sensitivit of 32 mv per unit of ph. nnowire FETs in the innermost ler (losest to luminl) showed minor seline flututions. This ilit to resolve etrvsulr ph hnges mkes possile the detetion of inflmmtion, ishemi, tumour miro-environments or other forms of metoli idosis due to overprodution of orgni ids or impired renl idifition 39,4, lthough we stress tht the implnttion of these nnoes-sed vsulr nd other nnoes-emedded onstruts for in vivo studies will require sustntil future work. The nnoes onept nd implementtions desried here represent new diretion in merging nnoeletronis with iologil sstems euse we hve demonstrted 3D mroporous mteril/devie pltform tht is distint from either engineered tissue 6,4 or fleile eletronis 3 7, Looking forwrd, there re severl res to develop. Cell intertions with nnoes ould e tuned modifition of the nnoes with growth determinnts 6,32. n ddition, the elements in the nnoes ould e epnded to inorporte nnosle stimultors nd strethle designs to provide eletril nd mehnil stimultion to enhne ell ulture. Methods Kinked nd uniform silion nnowires were snthesized the nnoluster-tlsed methods desried previousl 8. Devies were frited on silion sustrtes (Nov Eletroni Mterils, n-tpe. V m) with 6 nm SiO 2 or nm SiO 2 /2 nm Si 3 N 4 t the surfe. EBL nd photolithogrph on nikel relief lers were used to define the metl ontts to the nnowires nd the prinipl fetures of the sffolds. The ke steps used in the frition of the retiulr nnoes were s follows. First, nm nikel metl ws ptterned nd deposited, nd served s the relief ler for the free-stnding sffolds. Net, 3 nm ler of SU-8 photoresist (2., MiroChem, Newton) ws deposited over the entire hip (Supplementr Fig. S) followed pre-king t 6 C nd 9 C for 2 nd 4 min, respetivel; then n isopropnol solution of n + n n + kinked nnowires ws deposited onto the SU-8 ler. After identifing nnowire positions optil imging (Olmpus BX) nd designing the interonnet nd SU-8 ptterns in GOR Pro (WveMetris) nd DesignCAD, EBL ws used to pttern the overll SU-8 sffold struture round hosen nnowires, whih ws post-ked (6 C nd 9 C for 2 nd 4 min, respetivel) nd ured (8 C, 2 min) to ield the fleile struturl support for metl interonnets. NATURE MATERALS DO:.38/NMAT344 The silion sustrte ws then oted with methl methrlte nd pol(methl methrlte) doule-ler resist, the resist ws ptterned over the hosen SU-8 rions nd then non-smmetril Cr/Pd/Cr (./ 8/ 8 nm) metls were sequentill deposited followed metl lift-off in etone to form the nnowire interonnets. The non-smmetril Cr/Pd/Cr ler struture ields uilt-in stress, whih drove 3D self-orgniztion when the struture ws relieved from the sustrte. The silion sustrte ws then oted with uniform 3 4 nm ler of SU-8, nd EBL of SU-8 followed uring (8 C, 2 min) ws used to define the SU-8 pssivtion ler over the deposited metl interonnets. The retiulr nnoes, inluding the interonneted kinked nnowire FET devies, were relesed from the sustrte ething of the nikel ler (Nikel Ethnt TFB, Trnsene Compn, Dnvers) for 6 2 min t 2 C. Lst, the free-stnding nnoes were dried using ritil point drer (Autosmdri 8 Series A, Tousimis) nd stored in the dr stte efore use in tissue ulture. Eh EBL step will tke min 2 h depending on the writing speed nd re, feture size nd ompleit, nd eletron em dosge (for emple, the tpil re dosges for SU-8 nd pol(methl methrlte) EBL re 3 8 μcm 2 nd, μcm 2 t 2 kv, respetivel). The entire frition took 2 ds depending on the durtion of the individul steps. A similr pproh ws used in the frition of the mesh nnoes (Supplementr nformtion), eept tht p-tpe nnowires nd photolithogrph were used nd the entire proess took 2 3 ds. NnoES/ollgen(Mtrigel) hrid mtries were mde sting 2, μl ollgen or Mtrigel solution onto the edge of (retiulr nnoes) or diretl ove (mesh nnoes) the nnoes sffolds, nd t 4 C. The solutions were llowed to form gels round nnoes under onditions of 37 C nd % CO 2 for t lest 2 min. The 3D nnoes/lginte sffolds were prepred from phrmeutil-grde lginte (Protnl LF/6, FMC Biopolmers) lium gluonte rosslinking nd susequent lophiliztion to produe sponge-like sffold ( mm 2 mm, d h). To prepre NnoES/PLGA hrid sffolds, sheet of PLGA fires with dimeters of 3 μm ws deposited on oth sides of the mesh nnoes. The hrid sffold n e folded to inrese the thikness. Emroni Sprgue/Dwle rt hippompl ells, neontl Sprgue/Dwle rt rdiomtes nd HASMCs were ultured in nnoes using estlished protools (Supplementr nformtion). Optil mirogrphs of immunohistohemill nd histologill stined smples were reorded using either Olmpus Fluoview FV or Olmpus FSX sstems. The strutures of nnoes were hrterized with Zeiss Ultr/SuprVP field-emission SEMs or the HMXST miro-omputed tomogrph X-r imging sstem (model: HMXST22, X-Tek). The in vitro totoiit of nnoes ws evluted using the stndrd LVE/DEAD Viilit/Ctotoiit Kit (Moleulr Proes, nvitrogen) nd the CellTiter 96 AQueous One Solution Cell Prolifertion Ass (Promeg Corportion). Crdiomote reordings were rried out in Trode solution with mv d.. soure for the nnowire FETs. The urrent ws mplified with multi-hnnel premplifier, filtered with 3 khz low-pss filter (CerAmp 38) nd digitized t khz smpling rte 8 2 (Aon Digi44A). n etrvsulr ph sensing eperiments, single poldimethlsilone mirofluidi hmer ws used to deliver two flows of phosphte uffer solution, where inner nd outer tuings were used to deliver solutions with fied nd vried ph, respetivel. The eletril mesurements were onduted using lok-in mplifier with modultion frequen of 79 nd 39 Hz, time onstnt of 3 ms nd n mplitude of 3 mv; the d.. soure drin potentil is zero. Ag/AgCl referene eletrodes were used in ll reording nd sensing eperiments. The lirted potentil vlues (in millivolts) reorded from nnowire FETs were otined s the rtios etween devie ondutne hnges (in nnosiemens) nd the sensitivities (in mirosiemens per volt or nnosiemens per volt) tht re determined individull in wter-gte eperiments. Reeived 27 Ferur 22; epted 9 Jul 22; pulished online 26 August 22 Referenes. N, K. et l. Direting zeolite strutures into hierrhill nnoporous rhitetures. Siene 333, (2). 2. Shedler, T. A. et l. Ultrlight metlli miroltties. Siene 334, (2). 3. Ple, E. S., George, J. H., Willims, C. K. & Stevens, M. M. Sntheti polmer sffolds for tissue engineering. Chem. So. Rev. 38, 39 (29). 4. Wlie, R. G. et l. Sptill ontrolled simultneous ptterning of multiple growth ftors in three-dimensionl hdrogels. Nture Mter., (2).. Kloin, A. M., Ksko, A. M., Slins, C. N. & Anseth, K. S. Photodegrdle hdrogels for dnmi tuning of phsil nd hemil properties. Siene 324, 9 63 (29). 6. Dvir, T., Timko, B. P., Kohne, D. S. & Lnger, R. Nnotehnologil strtegies for engineering omple tissues. Nture Nnoteh. 6, 3 22 (2). 7. Krehenuehl, T. P., Lnger, R. & Ferreir, L. Three-dimensionl iomterils for the stud of humn pluripotent stem ells. Nture Methods 8, (2). 8 NATURE MATERALS ADVANCE ONLNE PUBLCATON 22 Mmilln Pulishers Limited. All rights reserved.

9 NATURE MATERALS DO:.38/NMAT Hutmher, D. W. Biomterils offer ner reserh the third dimension. Nture Mter. 9, 9 93 (2). 9. Huh, D. et l. Reonstituting orgn-level lung funtions on hip. Siene 328, (2).. Bker, M. Tissue models: A living sstem on hip. Nture 47, (2).. Shwille, P. Bottom-up sntheti iolog: Engineering in Tinkerer s world. Siene 333, (2). 2. Ruder, W. C., Lu, T. & Collins, J. J. Sntheti iolog moving into the lini. Siene 333, (2). 3. Timko, B. P. et l. Eletril reording from herts with fleile nnowire devie rrs. Nno Lett. 9, (29). 4. Viventi, J. et l. A onforml, io-interfed lss of silion eletronis for mpping rdi eletrophsiolog. Si. Trnsl. Med. 2, 24r22 (2).. Kim, D-H. et l. Mterils for multifuntionl lloon theters with pilities in rdi eletrophsiologil mpping nd ltion therp. Nture Mter., (2). 6. Viventi, J. et l. Fleile, foldle, tivel multipleed, high-densit eletrode rr for mpping rin tivit in vivo. Nture Neurosi. 4, 99 6 (2). 7. Kim, D-H. et l. Epiderml eletronis. Siene 333, (2). 8. Tin, B. et l. Three-dimensionl, fleile nnosle field-effet trnsistors s lolized ioproes. Siene 329, (2). 9. Qing, Q. et l. Nnowire trnsistor rrs for mpping neurl iruits in ute rin slies. Pro. Ntl Ad. Si. USA 7, (2). 2. Cohen-Krni, T., Timko, B. P., Weiss, L. E. & Lieer, C. M. Fleile eletril reording from ells using nnowire trnsistor rrs. Pro. Ntl Ad. Si. USA 6, (29). 2. Timko, B. P., Cohen-Krni, T., Qing, Q., Tin, B. & Lieer, C. M. Design nd implementtion of funtionl nnoeletroni interfes with iomoleules, ells, nd tissue using nnowire devie rrs. EEE Trns. Nnoteh. 9, (2). 22. Prohsk, O. J., Oltug, F., Pfundner, P. & Drgun, H. Thin-film multiple eletrode proes: Possiilities nd limittions. EEE Trns. Biomed Eng. 33, (986). 23. Niolelis, M. A. L. (ed.) Methods for Neurl Ensemle Reordings 2nd edn (CRC, 28). 24. MKnight, T. E. et l. Resident neuroeletrohemil interfing using ron nnofire rrs. J. Phs. Chem. B, (26). 2. Yu, Z. et l. Vertill ligned ron nnofire rrs reord eletrophsiologil signls from hippompl slies. Nno Lett. 7, (27). 26. Dequh, J. A., Yun, S. H., Goldstein, L. S. & Christmn, K. L. Deellulrized porine rin mtri for ell ulture nd tissue engineering sffolds. Tissue Eng. A 7, (2). 27. Hnle, P. J., Young, A. A., LeGrie,. J., Edgr, S. G. & Loiselle, D. S. Three dimensionl onfigurtion of perimsil ollgen fires in rt rdi musle t resting nd etended sromere lengths. J. Phsiol. 7, (999). 28. Engelmr, G. C. Jr et l. Aordion-like honeoms for tissue engineering of rdi nisotrop. Nture Mter. 7, 3 (28). 29. Lu, W. & Lieer, C. M. Nnoeletronis from the ottom up. Nture Mter. 6, 84 8 (27). 3. Yn, H. et l. Progrmmle nnowire iruits for nnoproessor. Nture 47, (2). 3. Wng, M. F., Mleki, T. & Ziie, B. Enhned three-dimensionl folding of silion mirostrutures vi therml shrinkge of omposite orgni/inorgni iler. EEE/ASME J. Miroeletromeh. Sst. 7, (28). ARTCLES 32. Spir, Y., Krukov, O. & Cohen, S. ntegrtion of multiple ell-mtri intertions into lginte sffolds for promoting rdi tissue regenertion. Biomterils 32, (2). 33. Xu, T. et l. Eletrophsiologil hrteriztion of emroni hippompl neurons ultured in 3D ollgen hdrogel. Biomterils 3, (29). 34. Cho, S. H. et l. Bioomptile SU-8-sed miroproes for reording neurl spike signls from regenerted peripherl nerve fires. EEE Sensors J. 8, (28). 3. Voskeriin, G. et l. Bioomptiilit nd iofouling of MEMS drug deliver devies. Biomterils 24, (23). 36. Zipes, D. P. & Jlife, J. Crdi Eletrophsiolog: From Cell to Bedside th edn (Sunders, 29). 37. L Heureu, N., Pâquet, S., Lé, R., Germin, L. & Auger, F. A. A ompletel iologil tissue-engineered humn lood vessel. FASEB J. 2, 47 6 (998). 38. L Heureu, N. et l. Humn tissue-engineered lood vessels for dult rteril revsulriztion. Nture Med. 2, (26). 39. Neri, D. & Supurn, C. T. nterfering with ph regultion in tumours s therpeuti strteg. Nture Rev. Drug Disov., (2). 4. Krut, J. A. & Mdis, N. E. Metoli idosis: pthophsiolog, dignosis nd mngement. Nture Rev. Nephrol. 6, (2). 4. Dvir, T. et l. Nnowired three-dimensionl rdi pthes. Nture Nnoteh. 6, (2). 42. Sekitni, T. et l. A ruerlike strethle tive mtri using elsti ondutors. Siene 32, (28). 43. Mnnsfeld, S. C. B. et l. Highl sensitive fleile pressure sensors with miro-strutured ruer s the dieletri ler. Nture Mter. 9, (2). 44. Tkei, K. et l. Nnowire tive mtri iruitr for low-voltge mro-sle rtifiil skin. Nture Mter. 9, (2). Aknowledgements We thnk F. Kosr for help on μct imging of sntheti tissue smples nd J. L. Hung for ssistne with ulture hmer preprtion. C.M.L. knowledges support from NH Diretor s Pioneer Awrd nd MKnight Foundtion Tehnologil nnovtions in Neurosienes Awrd. D.S.K. knowledges Biotehnolog Reserh Endowment from the Deprtment of Anesthesiolog t Children s Hospitl Boston nd NH grnt GM R.S.L. knowledges NH grnts DE323 nd DE66. Author ontriutions B.T., J.L., T.D., D.S.K. nd C.M.L. designed the eperiments. B.T. nd J.L. performed eperiments. T.D., J.T. nd Q.Q. ssisted in the initil stge of the projet. L.J. nd Z.S. performed lultions nd simultions. B.T., J.L., D.S.K. nd C.M.L. wrote the pper. All uthors disussed the results nd ommented on the mnusript. Additionl informtion Supplementr informtion is ville in the online version of the pper. Reprints nd permissions informtion is ville online t Correspondene nd requests for mterils should e ddressed to D.S.K. or C.M.L. Competing finnil interests The uthors delre no ompeting finnil interests. NATURE MATERALS ADVANCE ONLNE PUBLCATON Mmilln Pulishers Limited. All rights reserved.