Evaluation of Iron Oxide Nanoparticles for Lymph Node Detection with Magnetomotive Ultrasound – A Pilot Study in Rats

Male Spr agu e Dawley r a ts w er e injec t ed subcut an eously with 10.7 mg F err o tr an® (n=9), 10.5 mg Mag tr ace® (n=9), or salin e (n=3) dor sally a t t he r o ot of the t ail . On eu thani z ation o f the r a ts af t er 1, 5, and 24 hour s, th e pr o ximal and dist al lymph nodes w e r e har v e s ted and an aly z ed b y his t ology [hemat o xylin/ eosin, P erls Prussian Blu e (PPB)] and inductiv ely c ouple d plasma-op tic al emission spectr oscopy . The prima ry aim w as t o evaluat e the distribu tion of th e ir on o xid e n anopar ticles thr oughou t th e lympha tic s y st em; the ge ner al heal th of the rats aft er subcu t an eo us adminis tra tion of these na nopar ticles w as also moni t o r ed . Re s u l t s A t 1 hou r aft er subcu t an eous injecti on, Ferr o tr an® and M ag tr ac e® accumulat ed in the pr oximal lymph nodes. Af t er 24 hou r s, bot h par ticles had spr ead t o dist al lymph nodes, bu t only Ferr o tr an® reache d the mesent e ric and mandibula r lymph nodes . In addi tion, F e rr o tr an® p enetrat ed t he l ymph nodes mor e deeply than M ag tr ac e® at 24 hour s. N o to xicity w as obser v ed with ei the r nanopa r ticle. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 3

Although bo th c ompou nds disseminated w ell, F e rr o tr an® accumulated better and mor e r apidly in lymph nodes than Mag tr ac e®. B ec aus e accumula tio n and time a r e impo rtant par ame t er s f or imaging , our da t a indic at e th a t F e rr o tr an® is a po t e n ti ally mor e suitable pa rticl e f or MMUS in clinical use. K eyw or ds: r ec t al cancer , na nopar ticles, magnet omo tiv e ultrasound, lymph nod e, subcut ane ous (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 4 In tr oduction Color ec t al c anc er is one of th e mos t c om mon c ancer s, with 732,000 new c as es w orldwide in 2020 [1]. Nev er thel ess, sur viv al in c olor ec t al cance r has incr eas ed in r ec ent decades due t o impr ov ed tre a tme n t , changing pa tt e rns in risk f act or s, an d bett e r scr eeni ng , with 5-y ear sur viv al r a tes reaching 67% [2,3]. The pr esenc e of lymph node met ast as es is a k ey det ermina n t of th e pr ognosis of r ec tal c ancer (and cancer s in g ener al), guiding th e dev elopm ent of an adequ a te t r e a tme n t pl an. Combined wi th c ompu t ed t omogr aph y (CT), biops y , and c olonosc opy/r ect oscopy , lymph node s t atus is g en erally assessed by magnetic r eson ance imaging (MRI) [4], F- 18 fluor odeo xy glucose posit r on emission t omogr aph y (PET) /CT , or PET /MRI [5,6]. Finally , lympha tic flow and r egion al lymph nodes c an be visualized by r adionuclide scin tigr aph y in v arious c anc er s. These methods, how ever , a r e associ at ed with sev er al dr awback s. MR I is insuf ficien t f or diagnosing lymph node st atus, be c ause i t only analy z es th e shape and siz e of lymph nodes. Larg e lymph nodes c ould be suspect e d of met ast ases an d treated as such, but they migh t r esult fr om inflammation or cons ti tu t e big g er nodes th a t ar e b enign. Con v e r sely , smaller lymph nodes will not appe ar t o be questionabl e, base d on siz e, and will thus be ov er look ed . Consequently , the diagn os tic accur acy of the se methods f or small met asta tic lymph nodes (< 5 mm) declines subs t antially . In a dditio n, scin tigr a ph y is suit able f or identifying sen tin el lymph nodes but can not de t ect the presenc e or absence of c a ncer in them [7]. In c ontr ast, PET c an dif f erentiate c ance r ous v er sus nonc anc er ous lymph nodes [8] but is e xp ensiv e t o perf orm. Thus, current methods de tect lymph node met a s tases suboptimally or hav e limit ed widesp r e ad use, necessi t ating a more tenabl e appr o a ch t o ide n tifying lymph nodes f or diagno s tic purposes . Furthe r , due t o th e una v aila bility of a suitable technique , it is challenging t o de t er mine whether a tumor has spr ead t o r egional lymph nod es, f or cing mos t pa ti ents to unde r go t o t al meso rect al e x cision (TME) by de f ault . How ev er , s tudi es ha v e shown th a t ne arly 90% of pa tie n ts with st a g e T1 who under w e n t TME (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 5 e xpe rienc ed no spr e ading of the t umor to lymph nodes and thus w ould no t ha v e neede d this ex t ensiv e sur g e ry [9]. This ov ertreatment nega tiv el y impacts quality of lif e and r aises he alth c ar e c osts, necessita ti ng mor e e f f ec tiv e t ools t o de termine lymph node sta tus in rectal c ance r and address this signific an t unmet n eed . Magnetomotiv e ul tr asou nd (MMUS) is a nov el diagnos tic imaging method th a t ha s tr emend ous potential as a pl a tf orm f or making mor e accur at e diagnos es [10]. MMUS is based on the de t ec tion of vibr a ting ir on oxide nan opar ticles in tissu e. Thr ough appli c ation of a time-v a rying magnetic field, the nanopar ticles and thus the immedi at e su rr ounding tissu e are set in motion [11,12 ]. Theor e tic ally , in this modality , c on v e n ti onal US is fir s t p erf or med t o generat e a US image. W hen an al t e rnating magnetic field is applied, th e nanop articl es within th e ti ssue begin t o vibrat e and set the tissue i n motion; th is activity is det ec t e d by US, yielding a c ontr as t ima g e a t a resolut ion of millimet e r s. Be c ause t his motion is detected by US, the low echog enici ty of nanoc om pounds c an be over c om e [13], g enera tin g bet ter imag es a nd impr oving the diagnos tic accur acy . In add ition, nan opar ticles are tr a nspor t ed p rimarily by phagocyt osing macr ophages; th ese macr oph ag es do no t ent er tumor cells. Thus, the t umor cells i n a lymph node will harbor limi t ed amo unts of nanopar ticles, r esulting in less vibr ation— a dif f e r ence t ha t can be dis tinguished by MMUS, diff erenti a ting b etw ee n lymph nodes with and withou t t umor cells, bey ond th e imaging of sen tinel lymph nodes t hat is a cc omplished by other t echn iques. Wheth er MMUS can mak e this dif f e r enti a tion in a clini c al setting r e mains t o be confirmed in clinic al studies . Furthe r , MMUS devices ha ve been d ev el oped t o be smalle r and cheap er and t o require less tr aining t han MRI, with t he goal of r ecor ding smalle r lymph nodes and metas tases down to 2 mm. T o det ect lymph node me t ast ases by MM US, ir on o xid e nanop articl es should be in ject ed i n to the tissue and spr ea d t o the lymph nodes . No t ably , the nano par ticles should ha ve suf ficien t t ime t o reach th e (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 6 lymph nodes be f or e the an aly sis is perf or med. Furt her , the nan opar ticles should n ot be t o xic a t th e adminis tered dose . T w o superp ar amagnetic iron o xide n ano particles (SPIOs) are now being e x amin e d with r eg ar d t o their suit abili ty f or MMUS in clinic al pr act ice. Ferr o tr an®, a n ultr asmall sup erparamagne tic ir on o xid e (USPIO) particle , is an e f f ective in tra v eno usly adminis t e r ed c ontr ast ag e n t f or de t ec ting lymph node met a s tases by MRI in v arious c ancer s [14-17]. F er r otran® is curr e n tly in a Phase II I s tudy f or impr oving MRI imag es in pr ost at e c ancer . Mag trace® is an SPIO (Endomag , Cambridg e, UK) that has be en a ppr ov ed by the FD A as a sen ti nel lymph node tracer in b r east c a ncer patie n ts. The dis tribu tion of intr a v e nous injection of F err o tr an® has be en s tu died using MR I f or det ec ting lymph node metas tases in v arious cancer s, inclu ding r ectal c ance r [16,18,19]. The assess men t of lymph nodes in r ectal c ance r by MMUS ent ails lo c al injection of th e tr ace r inside th e rect al linin g of the in tes t ine, ar ound th e tumor . Consequ ently , subcutaneous delivery of nanop articl es, which will r esult in a dif f e r e n t biodis tri bution than i n tr a venous injecti o n—thus nec essita ting distinct ex amin a tio n window s and doses—has po t e n ti al application in diagn osing r ectal c ance r s tag e using MMU S. Al though smaller nanopar ticles migh t b e e x pec t ed t o distri but e mo r e e f ficiently within the lymph a ti c s y s t em, th ere is no evidence th a t nan opar ticle siz e is the sol e determina n t of th e r at e of distribu tion. Further , giv en the dispar at e struc tures betw e en F e rr o tr an® and Mag tr ac e®, it w oul d be premature to assume that th e f ormer w ould cir cul a te to a gr e at er e x tent, pr ompting us t o de t ermin e which of the lat ter dissemin at es bett er f or use in MM US. The aim of this s tudy w as t o e x amin e the spr ead and di s trib ution of two ir on o xi d e nanopa rticles — F err o tr an® an d Mag trace®— t o lymph no des in biologic al tissu e, after subcutaneo us adminis tra tion in male Spr ague Dawley r a ts. This model was chosen, based o n siz e c onsiderations: their lymph nodes a r e appr o xim at ely the sam e siz e as human mesor ec t al lymph nodes , and th e r at is r ou ghly the same leng th (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 7 as the meso r ectum [16], ensuring t hat we ar e studying a r el ev ant dis tance fr om t he injection si t e t o th e lymph nodes. As a sec o nda ry aim, w e mo nit o r ed t he g e ner al h ealth of the ra ts after loc al subcu t an eous adminis tra tio n of F err o tr an® a nd Mag tra ce® t o c onfirm th e r e por t ed saf e ty of the se nanopar ticles . Thus, the ov er a r ching purpos e of this s tudy w a s t o obtain an initi al indica ti on of which tr acer is mor e sui t abl e f or diagnos tic imaging with MMUS. Ma t erials and methods Animals Male Spr agu e Dawley r a ts w er e ob t ain ed fr om Jan vier Labs (Fr ance). A t the tim e t ha t the doses were adminis tered, th e r ats w eighed appr oximat ely 200–250 g. The r ats w e r e house d in type III Eu r opea n s t and ar d cag es (3 r ats per c a g e) with Asp en w ood chip bedding (T apv ei, B r og aa r de n, Denmark). Each c ag e cont ain ed a hide an d chewing s tick f or en vir onme nt al enrichm ent. The t emp er ature w as held betw e en 20 °C and 24 ° C, and the humidi ty w as set t o 50% to 65% RH. On the da y of arriv al , the animals w e r e pi ck ed r andomly fr om the cr at es an d allocat ed successiv ely t o the t est gr oups. No randomiz ed allo ca tio n sequence w as used . B e f or e admini s tra t ion of the test c ompounds, all anim als unde rw e n t an ac clima tiza tio n period d uring which they w er e obser ved f or signs of ill health; an y animal in po or c ondi tion w as e x cluded. Ul timately , no animals w e r e e x clud ed fr om the s tudy . Af t er admini s tra ti on of the t est c o mpounds, all animals w e r e insp ected throughout th e s tudy with r eg ar d t o their general c ondi tion . All clini c al signs and beha vior al ab normali ties wer e r ecor d ed. The in v es tigat or s w e r e no t blinded t o the gr oup alloca tio n during the e xp erime n t , out come assessme n t, or dat a an aly sis. This s tudy w as c ond ucte d in acc or danc e with license numb er s 18 164-21 and M140-16 per th e Malmö/Lund Ethics Commit tee o n Animal T es ting. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 8 T es t c ompounds This s tudy assessed th e ir on o xid e nanop article solu tions Magtr ace® (Endomag , Cambridg e , UK) and F err o tr an® (f er umo xt r an; SPL Medical, Ni jmeg en, Ne therl ands). F er r ot r an® (f erum o xtr a n) is being dev eloped by SPL Medic al as an MR I c o ntr as t ag e n t f or de t ecti ng lymph node metas t as es. F e rr o tr an® c omprises 25-nm ir on o xid e nanop articl e s with a 4-nm-diamet er c o r e of F e 3 O 4 an d a de x tr an c o ating and is s t abiliz ed with sod ium citr ate. Magtr ac e®, dev elop ed by Endomag , is used f or br eas t cancer st aging by assis ting in loc al lymph node de tection a t the tumor si t e , as par t of a sen t inel lymph node biops y . Mag tr ace® is c omp osed of 60-nm ir on o x ide nanopa rticles with a 4-nm-diamet er cor e of F e 2 O 3 and Fe 3 O 4 and a c arboxy dext r an c o a ti ng and is s t abi liz ed with sodium chloride . T w o bat ches of F er r o tr an®—b at ch 1 (B3008435) and ba t ch 2 (B3008435)—c onsis ti ng of a ly ophiliz ed pow der (0.5 g of pow der), w ere rec onstit ut ed in vials with 5 ml 0.9% NaCl solution t o a c onc entr ation of 19.9 mg /ml. The vials w er e in v er ted sev e r al times to ensure th a t a homog en eous solution w as ob t aine d (no v orte xing). After rec on s ti tutio n, th e solutions of ir on oxide nan opar ticles w e r e unif ormly dark br own t o black. The 2 b at ches w e r e st o r ed a t r o om t emperature until r e c onstitu tion and injection. All injec tions of the r e c onstitu t e d tes t compound w e r e perf ormed on th e same da y . One b a t ch of Magtr ace® (1114Ph112), su pplied as a soluti on (28 mg F e/ml), w as d ilut ed in 0 .9% NaCl. T o ensur e that a homogene ous solution was obt ained , th e vial w as in v er t ed sev eral ti mes (no v ort exing) t o a c once n tra tio n of 21.2 mg F e/ml. Prior to filling the s yring es, all solu tions w e r e m ix ed by g e n tl e swirling t o ensu r e homo g en eity . Dosing (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 9 Spr ague Dawley r a ts w ere administ e r ed s ubcut an eously , dor sally at the r oot of the t ail, with Ferr o tr an® , Mag tr ace® , or saline (9 mg /ml) (s t or ed at 4 °C un til use; Timeline Bio r ese ar ch AB) (T able 1). No f ormal a priori sample siz e calculation w as pe rf or med t o sel ect th e number of r a ts pe r tre a tme n t gr o up, be c ause this quali t ativ e explorat o ry s tudy w as intended t o pr ovide ini tial insights int o th e spr ead of th e tes t c ompounds. The r a ts wer e administ e r ed th e test c om pounds under isoflur an e ane s th esia. The injection site w as sha v ed b e f or e injecti on. T o determine w hether inc r eas ed blood an d lympha tic circula tion in the a r ea af f ects the sp r ea d of the pa rticles, the inj ection si t e w as massaged g e n tly f or r oug hly 2 min appr o xim at ely 90 min aft er th e injection f or 1 r a t per tr e atment and tim e point (6 r a ts in t o t al). The actual dose v ol umes w e r e de t ermin ed by w eighing the s yring es pri or t o and after adminis tra tio n. T able 1. Doses of te s t c ompou nds. Te r m i n a t ion Ra ts we re t e rminat Num ber (n) of rat s Injection T erm ina tion (hour s) Dose v olu me (ml/r a t) Dose (mg F e/k g) Dose ( m g Fe /rat ) n=3 Contr ol 24 0.48 0 0 n=3 Ferrotran® (Batch 1) 1 0.49 44.7 ± 1.2 10.4 ± 0.4 n=3 Ferrotran® (Batch 2) 5 0.48 ± 0.01 43.4 ± 1.1 10.8 ± 0.1 n=3 Ferrotran® (Batch 1) 24 0.50 45.4 ± 1.2 10.5 ± 0.4 n=3 Magtrace® 1 0.47 ± 0.01 42.1 ± 3.0 10.4 ± 0.1 n=3 Magtrace® 5 0.48 ± 0.00 43.6 ± 2.2 10.5 ± 0.1 n=3 Magtrace® 24 0.48 ± 0.01 41.4 ± 1.8 10.4 ± 0.1 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 10 ed by C O 2 inhala ti on per the schedul e in T able 1. Af t er t e rmination, the ra ts w e r e sha v ed and photogr aphed using a mobile ph one camer a. The diame ter of the a r e a of disc olored skin ar ound th e injection site w as measured. Tissue c ollectio n and handling The r a ts wer e o pen ed and dissec t ed 1–6 hour s aft er t e rmination t o expos e the ly mph nodes. The appear a nce of the o r g ans, lymph nodes, and lympha tic v essels w as no ted. The f oll owing lymph nodes w er e t ak e n fr om both sides when possibl e: superficial inguinal , external ili ac, dee p inguinal, mesenteric, a xilla ry , and mandibul ar . L ymph nodes an d the tissue in and ar ou nd the injec tion s it e wer e diss ected with a sc alpel, ph o t ogr aphe d, and an aly z ed per T abl e 2. In some c ases, 1 lymph node (le ft or righ t) w as pr ocessed f or inductively c ouple d plasma -optic al emission spec tr omet ry (ICP-OES), and the o the r node w as fix ed f or hist ology . L ymph nodes f or ICP-OES w er e w eigh ed, fr o z en a t -20 (i7=C, and s t o r ed a t -80 (i7=C, and those f or hist ol ogy w er e fi x ed in 4% buf f ered f ormalin (see Hist ology). Some l ymph nodes c ould not be c ollec t ed f or t echni c al reasons and a re the re f or e missing. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 11 Num ber o f r a ts sa mp led per an al ysis Injection T ermination (hours) L ymph nodes for ICP-OES L ymph nodes for hist ology Injection site tissue sample for hist ology Con tr ol 24 n=2 n=2 n=0 F err otr a n® (Ba t ch 1) 1 n=0 n=3 n=1 F err otr a n® (Ba t ch 2) 5 n=2 n=2 n=0 F err otr a n® (Ba t ch 1) 24 n=0 n=3 n=1 Ma gtr ace® 1 n=2 n=3 n=1 Ma gtr ace® 5 n=2 n=2 n=0 Ma gtr ace® 24 n=2 n=2 n=1 T able 2. Sample h andling. His t ol ogy Tissues w er e d eliv e r ed t o Micr omo rph A B (Lund, S w eden) in 4% buf f ered f ormali n. The samples w e r e deh y dr at e d, clea r ed, infiltrat ed with pa r af fin in an aut om at ed TISSUE-TEK V .I.P . (M iles Scien t ific, New ark, US), and embedde d in par af fin. Sect ions (4 μm ) w er e pr ep ared and dri ed in an oven a t 37 °C ov ernight. Hematoxylin and Eosin Sections w e r e d epar af finiz ed and h y drated in dis till ed w at er , aft e r which they w er e st aine d in Ma y er´s hemat o xylin (BioOp tic a , Milano , Italy , 05-6002) f or 6 minut es, w ashed in running tap w at er f or 10 minut es, a nd w ashed in distilled wat er f o r sev er al seconds. N e x t, t hey w er e st ai ne d in eosin (BioOptica, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 12 05-11007) f or 3 minut es, w ashed in distil led w at e r f or sev er al sec o nds, and deh y d r at ed sequ entially in 95% EtOH and 100% EtOH . The sections w er e then cle ared and mounted in s yn th etic r esin . Iron Staining- Perls Prussian Blue The P erls Prussian Blue kit (ab150674, A bc am, Cambridg e , UK) w as used t o st ain i r on. Tissue secti ons w er e d epar af finiz ed and h y drat ed in disti lled w at er . Equal v olumes of po t assium f err ocy anid e solutio n and h y dr ochloric acid solu tion w e r e mi x e d t o est a blish a w orking ir on st ain solu tio n. Slides w e r e incubat ed in ir o n s tain soluti on f or 3 minut es and rinsed t hor oughly in dis t illed wat er . The slides w e r e then st ain ed in Nucle ar F a s t R ed S olution (Abc am) f or 5 minut es and rinsed in 4 ch ang es of dis till ed w at er . Fin ally , they w ere deh y drat ed seq uentially in 95% EtOH and 100% EtOH , cleared, and mou n ted in s yn thetic resin. Analysis The sections w e r e an aly z ed and ph otographed unde r a Leic a DMRX micr osc o pe. An e xp erie nced assessor estimat e d the level of ir on st aini ng , e xpressed as th e per c ent age of lymph node area and lymph node cir cumf erence th at w as occupied b y ir on-positiv e cells. H emat oxylin/ eosin-s t ain ed sections were used as a re f e r enc e f or the PPB ir on s tain ing. ICP-OES bioanaly sis Brie fly , aft e r being w eighed , th e tissues w er e stor e d at -80 °C. The tissue w as thaw ed and digest ed in T e flon vials (Pr ot otyp v erks taden, M edicon Villag e, Lund) with 38.08% nitric acid, HNO 3 (Normatom, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 13 VWR Chemic als), 13.2% h y dr og en pe r o xi de, H 2 O 2 (Sigma Aldrich, Missouri, USA), and Milli-Q w at er (Milli- Q Plus 185, r esis tivity: 18.2 MΩ x cm). The samples w ere he at ed f or 90 minutes at 100 °C with shaking a t 240 rpm and c ooled f or a t le as t 30 min. T w o millilit e r s of each sample w as th en dil ut ed t o 5 ml 15.2% HNO 3 and 5.3% H 2 O 2 be f ore analy sis . The ICP-OES analy sis w as perf ormed on a n ICP-OES 710 tha t w as eq uipped with a quartz t o r ch with a 2.4-mm inject or , a single-pass glass cy clonic spr a y chamber , and a One Neb in ert concentric or glass nebuliz er (all fr om Agile n t, Sa nt a Clar a, U SA). The ins trume n t se t tings w e r e as f oll ow s: number of r eplicat es, 3 ; sample uptak e dela y time , 40 s; s t abiliza ti on dela y , 20 s; r epl ica te r e ad time, 5 s; plasma g as flow , 15 L /min; auxiliary g as flow , 1.5 L/m in; RF pow er , 1.2 k W ; ne buliz er flow , 0.75 L/min; and flow r a t e of peris taltic pump, 15 rpm. A w eight e d linear calibr ation cur v e w as generat ed, wi th a ma ximum 5% err o r allow ed . St atistics R esults are prese n ted as representa tive i mag es th a t show the r el a tive amou n ts a nd loc ations of the nanopar ticles. V alues fr om semiqua n ti ta t iv e analy ses a r e p r ese nt ed using descrip t iv e s ta ti s tics—i e, th e mean and st anda r d deviation— and gr ap hed as bar plo ts. No signific anc e tes ti ng w as applied due t o the qualita tive natu r e of this s tu dy . Re s u l t s T o xic ology (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 14 The t o xici ty of the injec t ed nan opar ticles t o th e g en er al he alth of all ra ts w as f ollow ed until termin a tion (see T able 1), f or a ma ximum of 24 hour s . No clinic al o r beha vioral abnormali ties w er e n oted in an y r at during this time, and all or g ans ap pea r ed normal. Spr ead of nan opar ticles fr om site of injection Spr ague Dawley r a ts w ere injected with Ferr o tr an® or M ag tr ac e® solution (10.3 –1 0.9 mg F e) or saline dor sally a t the root of th e tail. The injecti on sit e turne d br own-black aft er t he ad minis tr ation of F err o tr an® an d Mag trace® (Figur e 1), as e xpec t e d due t o th e ir on— a change th a t w as not obser v ed in the c o n t r ol gr oup . The disc oloration of th e injection si t e did no t incr e ase in siz e fr o m 1 t o 24 hour s pos t- dose with Mag trace®. How ever , with Ferrotr an®, the br own a r ea sp r ead visibly fr o m the injection si t e, incr easing in diame t er fr om 10 mm 1 hour aft er injec tion t o 25 mm 24 hour s pos t -dose. Spr ead of nan opar ticles t o lymph nodes L ymph nodes c an be gr ouped as pr oxima l and dis t al fr om the injecti on site. In ra t , w e de fine d the superficial inguinal, de ep inguinal, a nd ex t e rnal iliac lymph nodes as pr oximal and the mese nt eric, a xilla ry , and mandibul ar lymph nodes as dis t al (Figure 2). The pr o ximal lymph nodes cont ain ed nan oparticl es 1 hour aft e r injection , visible by ey e (Figur e 3A). The nanopar ticles also r esided in lymph a tic vessels, as evidenced by dark ening of th e lympha tic v essels betw e en the sup erficial inguinal and a xill ary nodes, indi c ating th a t these tr acer s migr a te between nod es thr ough th e lymphatic v essels (Figur e 3C). Furth er , th e lymph nodes dark ened afte r 24 hour s v er sus 1 hour post-dose, indi c ating th a t nan opar ti cles accumula ted in th e lymph nodes after injection . After 24 hour s pos t-dos e, th e nanop articl es r e ach ed the a xill ary lymph nodes. Ferrotr an® a lso ent e r ed th e (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 15 mesent e ric lymph nodes by gr oss phot og r aph y and his tology (Figur es 3B and 5B , respectively); en t ry int o the mandibul ar lymph nodes w as evident only by his t ology (Figur e 5B). A more ho mog eneo us dis tribu tion of nanop articl es w as seen in the lymph nodes ne ar th e injection si t e a ft er 24 hour s c ompared with 1 hour . Dis tributi on of nanopar ticles in lymph no des T o ev alua t e thei r dis t ributio n within lymph nodes and th e injection si t e , the na nop articles wer e st a ined with PPB. Ther e w as no nonsp ecific s t aini ng of ir on in the lymph nodes or injecti o n sit e in c o n t r ol r ats. How ev er , a t th e injection si t e in the anim als that w e r e injec t ed with nan opar ticles , the nan opar ticles spr ead t o cells and fibrous s truc tures in t he dermis aft e r injection , as shown in Figur e 4A-B . R epresenta tive PPB-s t ained ima g es of pro ximal and dist al lymph nodes a r e shown in Figur e 5A and 5B , r espec tiv ely , and th e estimat ed p er ce nt a g es of the lymph node a r ea a nd cir cumf e r ence that w e r e s t ain ed by PPB ar e shown in Figur e 6A an d 6B , r esp ectiv ely . B ased on the st ains, i ron accumula ti on incr ease d in pr o ximal lymph nodes fr om 1 t o 5 to 24 hour s. A t 24 hour s po s t-dos e , F err o tr an® filled 10% t o 55% of the lymph node a r ea (e x c ept f or the mandi bular an d mesenteric lymph nodes), v er sus 2% t o 30% with Mag tr ace® (Figur e 6A). The ma ndibular lymph nodes, which c an o nly be r eached by nanopar ticles th r ough th e blood, did n ot c ont ain pa rticles a t 1 hou r and 5 hour s p os t-dose bu t appe ar s t o ha ve harbo r ed Ferrotr a n® par ticles at 24 hour s pos t-dose ; how ev er , no accumu la tio n of Mag tr ace® w as obser v ed. Unlik e the weak hist ological and pho t ogr aphic evidence of th e e n t r y of F err otr an® i nt o the mese nt eric lymph nodes in Figures 3 B and 5B , no such dis t ributi on w as obser v ed by PPB s t aining (Figur e 6A and B); th e low c ov er ag e of PP B s t aining in t he mandibul ar lymph node s by F err otr an® is c onsist e n t with th e w eak st aini ng by his tology (Figur e 6B vs Figur e 5B). Ov er all, th ese dat a d emons trat e the po t e n ti al upt ak e and tr a nspor ta tion of F err o tr an® by the blood in ra t wit hin 24 hour s (Figur e 5B). (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 16 Furthe r , as shown by the PPB s t ain at 1 hour pos t-dos e, th e nanop articl es r e ached the subc apsula r sinus (ie, the pe riphe ry of the lymph node), in dic ating r apid tr a nspor t t o p r o ximal lymp h nodes. How ever , aft er 24 h, th e nanop articl es pene tr ated deepe r int o lymph nodes an d spr ea d mor e homogeneo usly within them c omp ar e d with 1 h; this p a ttern w as more evident f or F err o tr an® tha n f or Mag tr ace®. A summary of the lymph nodes th a t wer e reached by each nanop articl e at each tim e point is pr ese nt ed in Figur e 6C. The c once n tra tio n of ir on in the lymph n odes w as measured by ICP-OES. Unf ortunat ely , w e had t oo f ew lymph nodes t o an aly z e t o g en era te an y signific an t calculations. Howev er , generall y , the c once n tra tio ns of ir on in lymph nodes w ere higher ov e r time aft e r injection . After 5 hour s (the o nly time poin t th a t w as a v ailable f or both n anopa rticles), th e c on cen tra ti on of ir on w as higher in lymph no des in r a ts that w e r e inject ed with Ferrotr a n® v er sus Mag t r ace ®, indic ating th a t Ferr o tr an® sp r eads f aster and more br oa dly; how ev er , w e h ad too little d a ta to dr aw an y c onclusions. T r ansport mech anism In the lymph nodes , ir on w as f ound prim arily in macr ophage-lik e cells but also be t w een cells, possibly in the lymph node sinuses . These findings sug g es t th a t na nopar ticles a r e transpor te d primarily by macr ophages, as r e por t ed [20]. In ad diti on, the macrophage-lik e cells th a t cont ai ned ir on w e r e la r ger at 24 hour s v er sus 1 hour post-dose (Figur e 7), indic a ting continuous uptak e of the tracer by these cells . L ymph node siz e (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 17 The lymph nodes in r a t (mean 3.2 mm; r a ng e 1.2–6 .6 mm) appr o ximate the meso rect al lymph nodes in human in siz e (mean 2.4 mm; r ang e 0.9 – 12.2 mm) [16]. In our s tudy , the long axis of lymph nodes in the r a ts r an g ed fr om 1.4 t o 7.3 mm, and t he short a xis r anged fr om 1 t o 3 .3 mm. Influence of massag e on nan opar ticle spread Ther e w as no obvious dif f e r enc e in the s pr eading of nano par ticles between ra ts t ha t ha d been massa g ed or not— nanop articl es ent e r ed the lymph a tic s y st em and sp r ead t o dist al lymph no des ev en withou t massag e.

In this s tudy , w e h a v e f ound th a t on subc ut an eous injection , F er r otran® and Magt r ace® ir on o xid e nanopar ticles e nt er the lymphatic s y s te m and ar e transpor ted to pr o ximal a nd di s t al dr ai ning lymph nodes in r at. Al though bo th c ompoun ds disseminat ed well, F e rr o tr an® accumulated bett e r and more r apidly in lymph nodes than Magtr ace® . Neith er nan opar ticle w as associ at ed with an y gr oss clinic al or beha vior al t o xici ty . In r ec t al c a ncer , th e st atus of r egion al an d mesor ec t al lymph nodes is centr al f or st aging the c ancer and pr edicti ng loc al and dist a n t r ecur r enc e— this inf orma tion will inf orm tr e a tm ent de cisions with r eg ar d t o pr eop er ativ e di agnos tic imaging , sur gic al t echniqu es, pathologi c al assessme n t, an d the use of r adi a tio n ther apy . Al though se n tin el lymph nodes ha v e bee n analy z ed in r ec t al c anc er p a ti e n ts, t here is no c onsensus on wheth er they exist [21,22]. Despit e some studies dispu ting thei r pre sence, oth er s ha ve r epo rted sentine l lymph nodes t o li e 1–1 0 cm fr om the primary tumor in colorectal c ancer p a ti ents [23]. Thus, one mus t also b e able t o an aly z e lymph nodes th a t a r e dist al t o t he tumo r . (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 18 MMUS is a nov el, po t e n ti ally suit abl e tec hnique th a t ent ails US und er th e applicat ion of an e x t e rnal magnetic field. An MM US s y s tem loca tes lymph nodes by high-r esolution US and t r ack s the distribu tion of a c on tras t a g e n t th r oughou t lymph nodes [10,24]. MMUS has th e po t e n tial t o p r ovide imag es with higher r es olution c ompared with MR I in clinic al set t ings and has the signific a n t b e ne fit of being fr e e of ionizing r adiation . In addi tion, lymph nod es in the rectal area, which are often dif ficult t o ev alu at e du e to anat omical cir cums tances, can be ex ami ned via the rectum. Thus, a small , eas y-to-use, por t abl e MMUS- based r ec t al US p r obe would po t e n tially ha v e a gr e a t impac t in diagnosing rect al cancer , ben e fiting pa ti ents. Ir on-o xid e nanop articl es ha v e be en used in v arious s tudi es f or det ec ting lymph nodes and lymph node met ast ases [14-17,19] but ha v e o nly r ece n tly bee n applied in M MUS. Jansson et al . r epo r t ed th e fir s t MMUS imag e of human t issue using ir on o xide nan opar ticles in 2023, in which a pa ti ent w as injected with nanopar ticles pri or t o sur gery , and a n e x cised tissue sp ecimen w as imag ed o n a pr ototype M MUS s y s t em (NanoEch o , Lund, S weden) [13]. F err o tr an® an d Mag trace® are tw o iron oxide nano par ticles th a t a r e b eing s tudi ed in v arious c ancer s . Their diagnostic v alue in c ance r s is being es tablished —Mag trace® f or the de tectio n of lymph nodes in br east c anc er and Ferrotr an® in conjuncti on with MRI. Howev er , the spread of th e se nanopar ticles must be in v estig at ed b e f or e they c an be d eem ed suit abl e f or MMUS . In this s tudy , w e a naly z ed the spread of Ferr o tr an® and M ag tr ac e® t o and within lymph nodes in r at aft er subcut an eous injection t o und er st and th eir tr ansp ort , dissemination, and mechan isms as a subcut an eously administ ered c o n tras t a gen t . These pa rticles , siz ed f or upt ak e i n to the lympha tic s y st em alongside normal draining lymph flow fr o m the injection si t e, were f ound in macr o phag e-lik e cells and the inter cellul ar space in lymph nodes . Mechanistic ally , th e nanop articl es filled t he subc apsular sinus of lymph nodes be f or e ent e ring the inn er si nuses. These d at a sug g es t that aft e r subcut an eous injection , (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 19 the nano par ticles tr ansi t th r ough th e lymph s y s t em via th e af f erent lympha t ic v essels int o dr aining lymph nodes. The par ticles w e r e also f ou nd t o dif fuse i n to the tissue adjace n t t o t he injection si t e. Our dat a c onfirm the findings of McCauley et al . [19] that nanop articl es spr ea d t o n ear by and dis t al lymph nodes aft e r subcut a neous admini s tra tio n , as has been obser ved aft e r in tra v enous adminis tra tio n, ag ain indic ating th a t t hese na nopar ticles e nt er the lymphatic s y s tem aft er subcu t an eous injection. With reg ar d t o th e dynamics of spr eadin g , c onsidering our findings ov er all, Ferrot r an® seems to ha v e disper sed f ast er than M ag tr ace® , as seen in the macr oan aly sis of the lymph nodes , which w er e p a tently dark er after injecti on of F errotr an® a t eq uiv alent time poi n ts . Furth er , by PPB s t ai ning , a higher per ce n tag e of cells c o nt aine d ir on after t he adminis tration of F er r otran®, par ticul arly aft er 24 hour s , c ompared with Mag trace®. Only F errotr a n® ent e r ed th e mandibula r lymph node a ft er 24 hour s , also sug g es ting mor e r a pid spr e ading of F erro tr an®, as w ell as the sp r ead of F e rr o tr an® thr ough th e bloods t r e am. It would ha v e b een i n teres t ing t o de t ermin e the time poi n t a t which Mag tr ace® r each es the mandibul ar lymph nodes; thus, a limi t ation of this study w as th a t w e c onclude d the experim ents at 24 hour s and did not me asur e the sp r ead of ir on o xide nano par ticles at later times . The dis t a nce fr om the injec tion si t e t o th e superficial inguinal nod es in r at is appro ximat ely 4 cm v er sus 12 cm t o the a xilla ry lymph node— thus similar t o the length of the r ectum in hum an (12–15 cm). Furthe r , th e number of lymph nodes th a t w er e de t ec ted by MRI aft er subcu t an eo us adminis tra tion of F err o tr an® w as id entic al 1 and 3 da y s after administr ation, a nd aft er 7 da y s , f ew er lymph nodes c ould be identified [19]. Collectiv ely , th ese dat a h av e implic ations f or imaging , necessita ti ng the optimal dos e and time window f or adminis t e ring F er r otran ®, which w e ha v e att empted t o de t ermin e in this s tudy . T o be able t o de tect metast ases (or th eir absence) in lymph nodes, it is desir abl e to fill mos t of the lymph node with nano par ticles, ra th er tha n onl y popula ting th e peri phe ry of the lymph node. B y PPB s t aining , a t 24 hour s po s t-dose , F er r otran® filled 1 0% t o 55% of the lymph node area (e x ce pt f or the mandibula r (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 20 lymph node), c ompared with 2% t o 30% with Mag tr ace® , indica ting th at at least 2 4 hour s is needed t o achiev e adeq uat e filling. W heth er this ti me is suf ficien t f or MMUS in a clinic al se tting will need to be t ested in a clinic al in ves tiga tion . Our in di c a t ion fr om this s t udy , how ev er , is th a t 2 4 hour s is the minimal r equi r ed tim e. Similarly , biodis trib ution studi es with in tra v enous administra tion of F e rr o tr an® in ra ts ha ve shown that ir on c once n trations peak a t 24 hour s a nd decr eas e sev er al da y s po s t-dose [25], du e t o b r eak down of the particles t o iron by the body ’ s metabolis m of ir on and subsequent dis t ributi on of ir on to the bl ood and e x cr eti on of the d e x tr os e c o a ting in th e u rine. The op timal time poi n t f or imaging with loc ally adminis tered F er r ot r an® is thus expec t ed t o be 24–72 ho ur s post-dose—i e, when mos t of the injec t ed ma terial h as ent e r ed the lymph nodes bu t be f ore an y signific an t amo unt has been met aboliz ed . W e f ound that subcutaneous Ferrotr an® and Mag tr ac e® at the a dminist ered dose c ause no acu t e t oxicity in r a ts, consist e n t with what has be en re ported f or thes e and o ther similar n anop articles [14-17,19], although most s tudi es on F er r otran® ha ve administ ered it by in tra v eno us injectio n. Mag tr ac e® is a v ailable on t he mark e t, and F e rr o tr an® has adv anced t o Phase II I tri als. Sjins et al . [16] f ound tha t a dose of F errotr an® a t 2 .5 mg F e/kg body w eigh t (175 mg F e/70 kg) i s saf e in human. Also , subcu t ane ous Mag tr ace® a t a d ose of 55 mg F e has no to xic e f f ects. McCauley et al . [19] inject ed 9 pa tie n ts subcut an eously in v arious areas of the p elvis a t a dose of 19.6 mg /70 kg in t otal a nd obser v ed no sev e r e t o xici ty . In a c ontinu a ti on of these t oxicity s tudi es , w e w ant e d t o analy z e t he saf ety of subcut an eous F e rr o tr an® and Mag tr ac e® in r at with r eg a r d t o th eir loc al and s y st emic e ff ects . In our study , subcut an eous adminis tra tio n of 11 mg F e—which is a high loc al dose f or r at (250 g), c orr espondi ng t o 44 mg /kg body w eigh t —had no lo c al or s y st emic adver s e e f f ects aft e r 24 hour s. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 21 This s tudy s y s t em a ti c ally analy z ed the ly mph nodes f ollowing subcut aneous injec tion of nanopa rticles in a r elev a n t in viv o s y s tem, c ompl emented by a det ailed char ac t e riz ation of th e lev e ls and loc aliza tio n of the tracer s in th e lymph nodes. This s tud y pr ovides import a n t insights th a t suppo r t the futu r e e xpa nsion of these nano par ticles to unt e s ted clinic a l applic ations . In humans with r ec t al cancer , the tracer w ould be injected und er th e submuc osa near th e tumor , but w e c an not b e cer t ain th at nanop articl e mov ement through the tissues in th e mesore ctum is the same as in subcut an eous tissues, which c o uld af f ect the time t o o r amount of upt ak e by the lympha tic s y s tem. How ev er , once the tracer ent e r s the lympha tic s y st em, we w ould expec t the ir tr a nsport via th e lympha tic v essels into lymph nodes t o b e similar; thus, ou r findings in r a ts c an still be g ene r aliz ed t o lympha tic tr ansp ort in the meso r ect um in humans. This s tudy has sev er al limita ti ons. On e drawback w as tha t our study w as perf orm e d in r a ts, no t humans—between which th ere are dif f e rences with r eg a r d t o lymph nodes and m ethod of adminis tra tio n (ie, subcut a neous injecti o n in the tail vs the actual clinical diagnost ic r oute) that w ere not e x amine d in this s tudy . Clinic al s tudi es sh ould be perf orme d t o de termine how F e r r otr an® a nd Mag tr ace® cir cul at e in the relev ant tissue s in humans. Nev er thel ess, s tu dying thes e tr acer s in ra ts has allow ed us to dissect lymph nodes a t v ari ous times aft e r injection, p r oviding v aluable insigh ts into thei r spr ead an d loc alization in lymph nodes a nd f orming the basis f or futur e clinical s t udies. W e did no t c onduct an a ppr op riate t oxic ol ogy s tudy using specific measur es of health a nd be ha vior , which c ould be addr ess ed in a subseque n t study . Othe r limita ti ons of this s tudy include th e small sample siz e. Fur ther , we did not a dminis t e r F er r ot r an® and Mag tr ac e® int o animals that bore tu mor s, which dif f er fr om normal tissu e in man y aspects, potentially influencing the pene tra tion . (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 22 Furthe r , w e did no t c ompa r e t he lymph node s taining and pr og r ession of F e rr o tr a n® and Mag tr ac e® ag ains t nano c olloids that are used in r adi onuclide scin tigr a ph y . Finally , our s t udy w as primarily qualita tive. Fu ture s tudi es th a t in c orporat e a qu antita t iv e analy sis of nano par ticle dif fusion and dis tribu tion, p articul arly v er sus ot her na noparticl es, will pr ovide addi tional inf or ma tion . Neverth eless, our findings ar e suf ficient f or selecti on of a c andid a te tr ac er f or furthe r dev elopm en t a nd will be c onfirmed in humans in future s tudi es. MMUS is a pr omising diagnostic method f or r ect al c ance r s taging , and our results c ontribu t e t o v alida ting its clini c al application. Compa red with MRI, MMUS has th e po t e n ti al t o be easie r to perf orm and less e xp ensiv e, us es a smaller device , and r equi r es l ess tr aining , but most no tably , it is pot e n ti ally mor e sensi tiv e, as will be shown in a clinic al in v estig a ti on. O nce evidence of th e v alue of MMUS f or r ectal c ance r is demonstr ated in clinic al studies, i t c ould be impl emented in oth er diseases and applica tions that are moni t ored using tr a cer s, such as pr ost ate c ancer , st em cell th er apy , and arterioscle r osis.

Our s tu dy show s that Mag trace® and F e r r otr an® r each the pr oximal lymph nodes a t 1 hour p os t- injection. Over tim e, th eir spread within l ymph nodes bec ame mo r e homo g ene ou s, and the nanopar ticles w e r e obser ved in dist al lymph nodes (a xilla ry), appr o aching optim al lev els at 24 hour s pos t-injectio n. How ev er , only F er r otran® r each ed the mes ent e ric and mandibul ar l ymph nodes. Thus, w e c onclude t hat one should wait a t le as t 24 hour s aft er subcu t an eous administr ation of nanoparticl es be f or e imaging th e lymph nodes with M MUS. In ad dition , F er r otran® spr e ads f as ter than Mag tr ac e® thr oughou t th e lympha tic s y st em, indi c at ing tha t Ferrotr a n® is a potentially mor e suit abl e part icle f or MMUS. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 23 Abbr evia tions ICP-OES, inductiv ely c oupl ed plasma-opti c al emission spect r omet ry; MMUS , magnet omo tiv e ult r asound ; MRI, magnetic r esona nce imaging; PPB , P erls Prussian Blue; SPIO , supe rpar amag netic ir on o xid e; USPIO , ultr asmall supe rpar amagne tic ir on o xid e; US, ultr asound E thics appr ov al and i nf orm ed consen t This s tudy w as c onducted in acc o r danc e with license number s 18164-21 and M14 0-16 per the Malmö/Lund Ethics Commit t e e on Anima l T es ting. Consen t f or publica tion N o t a p p l ic a b le . Dat a a v ailability Da t a will be pr ovide d upon reasonab le re ques t . Funding The s tudy w as funded by NanoEcho A B. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 24 Competing in t er es ts U A and LP ar e employ ees of Na noEcho , a nd AM and SB a r e f ormer employees of NanoEcho . AM and LP own shar es in NanoEch o. Author s’ con trib utions All auth or s ha v e r ea d and appr oved th e manuscript. U A an aly z ed th e dat a , int e rp r eted th e r esul ts, g enerat ed the c onclusi ons, and wr o t e th e manuscript. SB pl anned a nd perf ormed the s tudy and analy z ed th e dat a . AM pl anned t he s t udy and perf ormed th e experim ents. LP ov er saw the s tudy , planned th e s tu dy , and wr ote the man uscript. Acknowledgmen ts T echnic al assis tance w as pr ovided by Micr omorph and Timelin e Bio r esea r ch. ICP-OES w as perf orme d by Spago Nanomedi c al. M edic al wri ting assi s t anc e w as pr ovided by Adv ansci R esea r ch Solutions. Re fe re n c e s 1. 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McCauley TR, Rifkin MD , Ledet CA: P elvic lymph node visualiz a tion with MR im aging using loc al adminis tra tio n of ultr a-small superp ar am agnetic ir on o xid e c o n tras t . J Magn R es o n Imaging. 2002, 15:492-497. 10.1002/jmri.10089 20. Moore A , W eissled er R, B og danov A , Jr .: Up t ak e of d e x tr an-c o at ed mon ocry st a lline ir on o xid es in tumor cells and macr o phages. J M agn R e son Imaging. 1997, 7:1140-1145. 10.100 2/jmri.1880070629 21. v an Oost end orp SE, Smits L JH, V r oom Y , et al. : Loc al recurrence after local e x cis ion of early r ec t al c ancer : a met a-an aly sis of c ompletion T ME, adjuv an t (chemo)r adi a tion , or no ad ditional tr e atment. B r J Sur g. 2020, 107:1719-1730. 10.1002/bjs.12040 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 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Enochs WS, Har sh G, Hoch ber g F , W ei ssleder R: Impr ov ed d eline a ti on of human br ain tumor s on MR imag es using a long-cir cula ti ng , superparamagnetic ir on o xi de agent. J M agn R eso n Imaging. 1999, 9:228-232. 10.1002/(sici)1522 -2586(199 902)9:23.0 .c o ;2-k Figur e leg ends Figur e 1. R ep r es enta tive pho t ogr aphs of i njection sites at 1, 5, and 24 hour s after adminis tra tio n of Mag tr ace® a nd F er r otran®. The r ed ar r o w s denote th e injection si t e . Figur e 2. Loca ti on of pr o ximal and di s tal lymph nodes in r a t . Cr e a ted with BioRend er .com. Figur e 3. R ep r es enta tive pho t ogr aphs of A. pr oximal and B . dist al lymph nodes in r a ts 1, 5, and 24 hou r s aft er administra tion of Ferr o tr an® an d M ag tr ace® v e r sus saline c o n t r ol. Som e lymph nodes c ould no t be c ollec t ed f or technical r e asons and a r e th ere f or e missing. C. R ep r ese nt ativ e image s of lympha tic v essels in r a ts 24 hour s after injecti on with F er r o tr an® v er sus salin e c o n trol. As indi c at e d by the arr ow s, t he lymph v essels ar e st ain ed f or ir on by F errotr an® nan opar ticles. Figur e 4. Hist ol ogic al analy sis of skin and dermis at the injec tion site 1 and 24 hours aft er administr ation of F err o tr an® and M ag tr ace® by P erls Prussian Blue st aining. A. 2.5 x and B . 10x ma gnific a tion . The blue cells indic at e the p r esenc e of ir on. S c ale bar in A . and B. equals 100 μm and 400 μ m, r espectively . Figur e 5. Hist ol ogic al imag es of P erls Prus sian Blue-st ained A. p r o ximal and B. dist a l lymph nodes aft er adminis tra tio n of F err o tr an® a nd Mag tra ce®. Blue signals indicat e the presenc e of nanopar ticles. Som e (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 27 lymph nodes c ould not b e c oll ected f or technic al reasons and ar e the re f or e missi ng. Sc ale ba r s (black lines in t op l e ft pan els) equal 400 μm. Figur e 6. Es tim at ed p er ce n tag e of A. lymph node area and B . lymph node cir cumf er enc e s tained by P erls Prussian Blue. Ba r s are the me an, and w hisk er s ar e the st and ar d devi a tion . F or rats that had 2 of the same type of lymph node c ollec ted, th e nodes w e r e c onsid ered technical r epli c ates, and th eir mean w as plott ed . Some lymph nodes c o uld not b e c ollec t ed f or technical r e asons and a r e th ere f or e missing. C. Summary of the presence o r absence of each tr ace r in the v a rious lymph nodes at each time p oint. Figur e 7. Inc r eas e in siz e of macr ophage-lik e cells ov er time after administra tion of F err o tr an® by PPB s t aining . A t 24 h, dif fuse s taining is also o bser v ed betw e en cells. S c ale ba r equa ls 20 μm. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 5 h Magtrace 24 h1 h Ferrotran Figure 1 Saline (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint Deep inguinal Magtrace External illiac Superficial inguinal Saline 1 h 1 h 5 h 5 h 24 h 24 h Ferrotran 24 h A B 1 h 1 h 5 h 5 h 24 h 24 h 24 h Axillary MandibularMesenteric FerrotranSaline C Figure 3 Magtrace SalineFerrotran (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint Magtrace Saline Ferrotran B 1 h 24 h 10x A 1 h 24 h Magtrace Saline Ferrotran 2.5x Figure 4 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint Deep inguinal External illiac Superficial inguinal A Deep inguinal External illiac Superficial inguinal Ferrotran Magtrace Ferrotran Magtrace Axillary MandibularMesenteric Axillary MandibularMesenteric B Figure 5 1 h 5 h 24 h 1 h 5 h 24 h 1 h 5 h 24 h 1 h 5 h 24 h (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint 1 h 5 h 24 h Deep inguinal Both Both Both External iliac Both Both Both Superficial inguinal Both Both Both Axillary Magtrace1 Both Both Mandibular Neither Neither Ferrotran Mesenteric Neither Ferrotran2 Ferrotran3 1 Data for Ferrotran missing 2 Weak local staining observed 3 Based on lymph node image (Figure 3B) only; histology data missing C Figure 6 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint Magtrace 1 h 24 h5 h Ferrotran Figure 7 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 28, 2024. ; https://doi.org/10.1101/2024.08.28.610049doi: bioRxiv preprint