Whither magnetic hyperthermia? A tentative roadmap

ma e ials Opinion Whi he Magne ic Hype he mia? A Ten a i e Roadmap I ene Rubia-Rod íguez 1, An onio San ana-O e o 1, Simo Spasso 2, E elka Tombácz 3, Ch is e Johansson 4, Pa icia De La P esa 5,6 , F ancisco J. Te an 1,7 , Ma ía del Pue o Mo ales 8, Sabino Vein emillas-Ve dague 8, Nguyen T. K. Thanh 9,10 , Maximilian O. Besenha d 11 , Clai e Wilhelm 12, Flo ence Gazeau 12, Quen in Ha me 13, E ic Mayes 13, Bella B. Manshian 14 , S e aan J. Soenen 14 , Yuanyu Gu 15,Ángel Millán15 , Eleni K. E himiadou 16, Je Gaude 17, Pa ick Goodwill 17 , James Mans ield 17, Uwe S einho 18 , James Wells 18, F ank Wiekho s 18 and Daniel O ega 1,19,20,*   Ci a ion: Rubia-Rod íguez, I.; San ana-O e o, A.; Spasso , S.; Tombácz, E.; Johansson, C.; De La P esa, P.; Te an, F.J.; Mo ales, M.P. ; Vein emillas-Ve dague , S.; Thanh, N.T.K.; e al. Whi he Magne ic Hype he mia? A Ten a i e Roadmap. Ma e ials 2021,14, 706. h ps:// doi.o g/10.3390/ma14040706 Academic Edi o : Vadim Kessle Recei ed: 2 Decembe 2020 Accep ed: 25 Janua y 2021 Published: 3 Feb ua y 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). 1IMDEA Nanoscience, Fa aday 9, 28049 Mad id, Spain; i ene. [email p o ec ed] (I.R.-R.); [email p o ec ed] (A.S.-O.); [email p o ec ed]g (F.J.T.) 2 Geophysical Cen e o he Royal Me eo ological Ins i u e, 1 ue du Cen e Physique, 5670 Dou bes, Belgium; [email p o ec ed] 3Soós Wa e Technology Resea ch and De elopmen Cen e , Uni e si y o Pannonia, 8200 Nagykanizsa, Hunga y; [email p o ec ed] 4RISE Resea ch Ins i u es o Sweden, Senso s and Ma e ials, A id Hed alls Backe 4, 411 33 Gö ebo g, Sweden; ch is e [email p o ec ed] 5 Ins i u o de Magne ismo Aplicado UCM-ADIF-CSIC, A6 22,500 km, 29260 Las Rozas, Spain; [email p o ec ed] 6Depa amen o de Física de Ma e iales, Uni e sidad Complu ense de Mad id, A da. Complu ense s/n, 28048 Mad id, Spain 7Nano ech Solu ions, C a Mad id, 23, 40150 Villacas ín, Spain 8 Depa men o Ene gy, En i onmen and Heal h, Ins i u o de Ciencia de Ma e iales de Mad id (ICMM/CSIC), So Juana Inés de la C uz 3, 28049 Mad id, Spain; [email p o ec ed] (M.P.M.); [email p o ec ed] (S.V.-V.) 9UCL Heal hca e Biomagne ics and Nanoma e ials Labo a o ies, 21 Albema le S ee , London W1S 4BS, UK; [email p o ec ed] 10 Biophysics G oup, Depa men o Physics and As onomy, Gowe S ee , London WC1E 6BT, UK 11 Depa men o Chemical Enginee ing, Uni e si y College London, To ing on Place, London WC1E 7JE, UK; [email p o ec ed] 12 Labo a oi e Ma iè e e Sys èmes Complexes MSC, Uni e si éde Pa is/CNRS, 75013 Pa is, F ance; clai e.wilhelm@uni -pa is-dide o . (C.W.); lo ence.gazeau@uni -pa is-dide o . (F.G.) 13 Endomag, The Je eys Building, S John’s Inno a ion Pa k, Cowley Road, Camb idge CB4 0WS, UK; [email p o ec ed] (Q.H.); [email p o ec ed] (E.M.) 14 Biomedical Sciences G oup, T ansla ional Cell and Tissue Resea ch Uni , Depa men o Imaging and Pa hology, 3000 Leu en, Belgium; [email p o ec ed] (B.B.M.); [email p o ec ed] (S.J.S.) 15 INMA Ins i u o de Nanociencia de Ma e iales de A agón, Ped o Ce buna 12, 50009 Za agoza, Spain; [email p o ec ed] (Y.G.); amillan@uniza .es (Á.M.) 16 Chemis y Depa men , Ino ganic Chemis y Labo a o y, Na ional and Kapodis ian Uni e si y o A hens, Panepis imiopolis Zog a ou, 15771 A hens, G eece; [email p o ec ed] 17 Magne ic Insigh , Alameda, CA 94501, USA; [email p o ec ed] (J.G.); [email p o ec ed] (P.G.); [email p o ec ed] (J.M.) 18 Physikalisch-Technische Bundesans al , Abbes aße 2-12, 10587 Be lin, Ge many; [email p o ec ed] (U.S.); [email p o ec ed] (J.W.); [email p o ec ed] (F.W.) 19 Ins i u e o Resea ch and Inno a ion in Biomedical Sciences o he P o ince o Cádiz (INiBICA), 11002 Cádiz, Spain 20 Condensed Ma e Physics Depa men , Facul y o Sciences, Campus Uni e si a io de Pue o Real s/n, 11510 Pue o Real, Spain *Co espondence: [email p o ec ed] Abs ac : The scien i ic communi y has made g ea e o s in ad ancing magne ic hype he mia o he las wo decades a e going h ough a sizeable esea ch lapse om i s es ablishmen . All he p og ess made in a ious opics anging om nanopa icle syn hesis o biocompa ibiliza ion and in i o es ing ha e been seeking o push he o e on owa ds some new clinical ials. As many, hey did no go a he expec ed pace. Today, ui ul in e na ional coope a ion and he wisdom gain a e a ca e ul analysis o he lessons lea ned om seminal clinical ials allow us o ha e a u u e wi h be e gua an ees o a mo e de ini i e akeo o his genuine nano he apy agains cance . Delibe a ely gi ing p ominence o a numbe o c i ical aspec s, his opinion e iew o e s a blend o Ma e ials 2021,14, 706. h ps://doi.o g/10.3390/ma14040706 h ps://www.mdpi.com/jou nal/ma e ials Ma e ials 2021,14, 706 2 o 36 s a e-o - he-a hin s and glimpses in o he u u e o he he apy, conside ing he expec ed e olu ion o science and echnology behind magne ic hype he mia. Keywo ds: magne ic hype he mia; magne ic nanopa icles; hys e esis losses; cance ; magne ic pa icle imaging; he anos ics; nanopa icles syn hesis; he mome y; s anda diza ion; nano oxici y 1. In oduc ion The scien i ic communi y in ol ed wi h magne ic hype he mia may be on he e ge o ano he u ning poin a e some yea s wi hou ele an news on he ou comes o clinical esea ch: new clinical s udies on di e en indica ions a e cu en ly aking place. Fo example, MagFo ce AG ecen ly announced ha i s Ame ican subsidia y MagFo ce USA, Inc. ob ained app o al om he U. S. Food and D ug Adminis a ion (FDA) o a pi o al single-a m s udy o he ocal abla ion o in e media e- isk p os a e cance wi h hei NanoThe m ® he apy sys em [ 1 ]. In Eu ope, bo h he Vall d’Heb on Uni e si y Hospi al and he Fuenlab ada Uni e si y Hospi al a e home o a new easibili y s udy on ea ing locally ad anced panc ea ic duc al adenoca cinoma (PDAC) wi hin he emi o he NoCanThe p ojec [2]. Wi hou any doub , behind he p og ess so a on he clinical ansla ion o magne ic hype he mia, he e is an e e mo e in e wined scien i ic ne wo k wo ldwide ha is keeping a cons an in lux o basic esea ch, consolida ing he de elopmen s unde he ligh o consensual new p ocedu es, and expanding links wi h key ac o s in he ansla ional and clinical a ena. In e na ional ne wo king ini ia i es, such as he “RADIOMAG” COST ac ion [ 3 ], ha e helped in igh ing agains he geog aphical dispe sion o scien i ic and human esou ces ela ed o magne ic hype he mia, as well as elimina ing duplica ion o esea ch lines and con ibu ing o he ha moniza ion o key concep s and p ocedu es. In any case, he coope a ion be ween clinical and non-clinical wo lds has become much mo e luid, as i should be o achie e a sus ainable imp o emen in he coming decades [ 4 ]. The exis ence o unique in as uc u es o eliable, dedica ed and widesp ead cha ac e iza- ion echniques o nanomedicines is pa ing he way o a as e ansla ion o p omising nanop oduc s. A sup ana ional example is he Eu opean Nanomedicine Cha ac e isa ion Labo a o y [ 5 ], c ea ed back in 2015 unde he auspices o he H2020 amewo k p og am, and a mo e es ablished na ional example is he Nano echnology Cha ac e iza ion Labo- a o y in he USA, ounded by he Na ional Cance Ins i u e (NCI) in collabo a ion wi h he FDA and he Na ional Ins i u e o S anda ds and Technology (NIST) [ 6 ]. Howe e , as i could no be o he wise, he e a e some impo an issues s anding in he way o wide clinical adop ion o magne ic hype he mia, some o which a e common o many o he nanomedicines [ 7 ]. The economic bu den o aking he leap om basic nanomedicine esea ch o ansla ion [ 8 ] appea s o be insu moun able in he opinion o he scien i ic communi y, abo e all wi h he cu en unding schemes, which despi e being ega ded as insu icien and poo ly coo dina ed, a e also beginning o su e signi ican cu s. This is exempli ied by he ecen u moil a ound he decision o he Uni ed S a es Na ional Cance Ins i u e (NCI) in hal ing unding o he Cen e s o Cance Nano echnology Excellence (CCNEs) [ 9 ], he commen a y published by Kinam Pa k— he o me Edi o -in-Chie o he Jou nal o Con olled Release—in a o o he con o e sial decision [ 10 ], and he coun e - eac ion ha ollowed om he boa d o he Nanomedicine and Nanoscale Deli e y Focus G oup o he Con olled Release Socie y [ 11 ] and he o me p esiden o he Eu opean Resea ch Council [12]. Magne ic hype he mia he apy mainly comp ises wo key elemen s: injec able mag- ne ic nanopa icles (MNPs) and a magne ic ield applica o , bo h o which we e app o ed in mos cases as medical de ices. A leas in Eu ope, he e is s ill a deba e a ound whe he a mo e speci ic egula o y amewo k—beyond he mo e ecen egula ion (EU) 2017/745 on medical de ices epealing he 93/42/EEC and 90/385/EEC di ec i es—is needed o Ma e ials 2021,14, 706 3 o 36 nanomedical de ices. The ma e only wo sens when conside ing he p ocess in di e en pha maceu ical ju isdic ions [ 13 ]. This unce ain y a ound well-de ined p e-no ma i e and egula o y amewo ks is discou aging p i a e in es o s and pha maceu ical compa- nies om aking he ini ia i e in leading new indus ial p ojec s o sponso ing he mos p omising cu en de elopmen s. Added o his is he eluc ance o use MNPs in humans a e se e al cases o wi hd awals o nanop oduc s bo h om he ma ke and om he egula o y p ocess, in addi ion o he abandonmen o he p oduc ion o o he o mula ions based on MNPs (see Sec ion 5). All hese aspec s, along wi h many o he s shaping he p esen and he u u e o magne ic hype he mia, a e commen ed on he e by in e na ional expe s, aking he cu en s a e-o - he-a as a s a ing poin . 2. Es ablishing S anda d Ope a ional P ocedu es o S uc u al and Magne ic Cha ac e iza ion o Magne ic Nanopa icles Nowadays, nanoma e ials manu ac u e s ace a cons an inc ease o equi emen s ega ding speed p ocess and p oduc quali y con ol ha need eal- ime cha ac e iza ion echniques adap ed o nanoscale me ology and s anda dized ope a ional p ocedu es (SOP). Indeed, SOP and au oma ed ins umen a ion o cha ac e izing magne ic nanoma e ials will de ini i ely bene i bo h he indus ial demands in quali y con ol and also basic esea ch. Recen ini ia i es, as he “RADIOMAG” COST ac ion [ 3 ], showed signi ican a iabili y o esul s when compa ing physical pa ame e s, such as he speci ic abso p ion a e (SAR) o speci ic loss powe (SLP), ob ained in magne ic colloids by di e en esea ch labs [ 14 ]. Mo eo e , many physical pa ame e s o MNPs can be de e mined by dis inc cha ac e iza ion echniques (see Table 1), inc easing he a iabili y o he esul s. Hence, he e is a need o s anda dizing me hodologies o cha ac e izing ex emely ele an pa ame e s such as magne ic losses o MNPs. Table 1. MNP pa ame e s and he co esponding cha ac e iza ion echniques. Adap ed om [ 15 , 16 ]. See lis o ac onyms a he end o he documen . S uc u al P ope ies Pa icle, co e and agg ega e size TEM, XRD, DLS, NTA, SAXS, HRTEM, SEM, AFM, EXAFS, FMR, DCS, MALDI, NMR, TRPS, EPLS, magne ic suscep ibili y Mo phology TEM, HRTEM, AFM, EPLS, FMR, 3D- omog aphy Elemen al-chemical composi ion XRD, XPS, ICP-MS, ICP-OES, SEM-EDX, NMR, MFM, LEIS C ys allini y XRD, EXAFS, HRTEM, elec on di ac ion, STEM S uc u al de ec s HRTEM, EBSD Chemical s a e–oxida ion s a e XAS, EELS, XPS, Mössbaue Ligand-binding, su ace composi ion XPS, FTIR, NMR, SIMS, FMR, TGA, SANS Colloidal P ope ies Hyd odynamic and agg ega e size NTA, DLS, DCS, UV- is, SEM, TEM, C yo-TEM 3D isualiza ion 3D- omog aphy, AFM, SEM MNP cha ge Ze a po en ial, EPM Elemen concen a ion ICP-MS, UV- is, RMM-MEMS, PTA, DCS, TRPS Magne ic P ope ies Quasi-s a ic magne iza ion p ope ies SQUID, VSM, Mössbaue , MFM, FMR, XMCD, Dynamical magne iza ion p ope ies AC suscep ome y and magne ome y, magne o elaxome y, magne ic pa icle spec oscopy Magne ic losses AC calo ime y, AC suscep ome y and magne ome y Since his sec ion ocuses only on essen ial cha ac e iza ion echniques o magne ic hype he mia (MH) applica ions, i is wo h no ing ha magne ic losses a e s ongly in lu- enced by MNPs pa ame e s such as size [ 17 ] and shape [ 18 , 19 ], agg ega ion deg ee [20,21] magne ic aniso opy [ 22 ], magne ic dipola in e ac ions [ 23 , 24 ], unc ionaliza ion [25,26] , iscosi y o he dispe sion medium [ 27 , 28 ], and al e na ing magne ic ield condi ions Ma e ials 2021,14, 706 4 o 36 ( ield equency and ampli ude) [ 29 – 31 ]. Se e al EU p ojec s ocused on s anda diza ion and ha moniza ion o analysis me hods o MNPs ha e been/a e being ca ied ou , e.g., NanoMag, MagNaS and and RADIOMAG, as well as app o ed ISO s anda ds (ISO/TS 19807-1:2019) [ 32 , 33 ]. These achie emen s bene i he p epa a ion o SOPs o cha ac e izing ele an pa ame e s such as magne ic losses o MNPs o he design o s anda d e e ence nanoma e ials o ha monize he compa ison o esul s ob ained by dis inc esea ch g oups. Hence, SOPs aim o homogenize p ocedu es o cha ac e izing physicochemical pa ame e s o magne ic suspensions as he i s s ep owa ds In e na ional S anda ds. So a , e o s wi h good esul s ha e been done o cha ac e ize and ha monize analysis me hods o bo h suspended and immobilized MNPs [ 15 , 16 , 33 , 34 ]. He e we spo ligh selec ed essen ial me hods o MH applica ion. A gene al desc ip ion o analysis me hods o magne ic nanopa icle sys ems can be ound in e . [16]. 2.1. S uc u al Cha ac e iza ion Today, nanoscience canno exis wi hou nea - ield and elec on mic oscopy echniques such as TEM, HRTEM, SEM, EDX, AFM, e c. Wi hin he la e , TEM is he mos widely used o he s uc u al cha ac e iza ion o nanopa icles, which mainly comp ises MNP co e size, co e size dis ibu ion, shape, agg ega ion, e c. Howe e , due o he inhe en sample p epa a ion echniques, i is o en di icul o p ese e he o iginal colloidal s a e. In his sense, he use o c yo-TEM is encou aged o be e cap u e he spa ial a angemen o MNPs, hus p o iding mo e accu a e in o ma ion abou hei agg ega ion s a e. 2.2. Colloidal P ope ies These a e gene ally cha ac e ized unde andom condi ions, namely pu e wa e , bu e s (o en phospha e solu ions), e c. A p io i well-quali ied samples, howe e , o en end up ailing in i o due o a signi ican loss o e icacy and/o he onse o oxici y [ 35 ]. The easons behind his obse a ion can be di e se: sample con amina ion, MNPs ag- g ega ion, and in e acial in e ac ions wi h cell memb anes o blood componen s, among o he s [ 36 ]. MNPs quali ica ion mus be pe o med unde condi ions ha mimic he in i o en i onmen , mainly pH and salini y, bu also including p o eins, ca bohyd a es and lipids. In gene al, MNPs’ in e ac ions a bio-nano in e aces a e mainly de e mined by size, cha ge and hyd ophilici y/hyd ophobici y [ 37 ]. In ac , hese p ope ies a e closely ela ed o he pa en colloid s abili y— ia elec ic, s e ic and elec os e ic s abiliza ion—and pa icle agg ega ion in poo ly s abilized magne ic luids. Dynamic ligh sca e ing (DLS) is one o he mos employed me hods o measu e hyd odynamic sizes and size dis ibu ions in dilu e colloids by analyzing he in ensi y luc ua ion o sca e ed ligh caused by he B ownian mo ion o he cons i uen nanopa icles. The main sou ce o unce ain y he e is polydispe si y, bu in he li e a u e, he “DLS size” is o en p o ided wi hou epo ing some ele an measu emen condi ions like pH o ionic s eng h, making i di icul o es ablish he sou ce o polydispe si y. The la e could eside in he p ima y pa icles and hei agg ega ion due o weak colloidal s abili y [ 35 ], and i has a majo impac bo h on he sample’s shel li e and i s subsequen use. Fo example, app op ia e and inapp op ia e MNP manu ac u ing has been illus a ed in he li e a u e by human blood smea es s [ 36 ]. MNP cha ge can be cha ac e ized ia ze a po en ial ( ζ ) measu emen s, which is no cha ac e is ic o su ace cha ge as ound in he li e a u e [ 15 ]. I highly depends on he pH and ionic s eng h o he medium and he quali y and quan i y o speci ic ions (phospha es in bu e s, ca boxyla es, su ac an ions, e c.). I MNPs a e s abilized only elec os a ically, ζ alues highe han |25–30| mV, measu ed a low ionic s eng h, indica e good colloidal s abili y. A high sal concen a ion, ζ becomes ze o. A null alue also occu s bo h a a pH coinciding wi h he isoelec ic poin and in he p esence o speci ic ions, causing ζ e e sal. Consequen ly, epo ing ζ alues wi hou p o iding in o ma ion on pH, ionic s eng h, speci ic ions, e c., o he dispe sion solu ions is meaningless. In he case o concen a ed magne ic suspensions and gels, DLS canno be used; in hese cases, mo e powe ul sca e ing me hods such as SAXS and SANS a e needed [ 38 ]. The co e-shell Ma e ials 2021,14, 706 5 o 36 s uc u e and he p obabili y o agg ega ion in samples can be measu ed in p is ine samples as used in bio- ele an media, e en highly concen a ed o embedded in a gel. The hi d ele an colloidal pa ame e is he hyd ophilici y/hyd ophobici y o he MNP coa ing. O pa icula no e in he case o MNPs in ended o biological media is he p o ein adso p ion, leading o he so-called “p o ein co ona” a ound nanopa icles since i masks he o iginal cha ac e o he MNP su ace [35]. 2.3. AC Suscep ome y In AC suscep ibili y (ACS) s. equency measu emen s, a sinusoidal magne ic ield o cons an ampli ude is applied o e he sample, and he exci a ion equency is swep a a cons an empe a u e [ 39 – 42 ]. A supe imposed DC magne ic ield can also be applied. The AC ield in ensi y is gene ally su icien ly small, ul illing he low- ield limi whe e he magne iza ion is linea o he ield. The in-phase componen ( eal pa ) and ou -o -phase componen (imagina y pa ) o he ACS a e measu ed e sus exci a ion equency. In o de o calib a e he signal ampli ude and phase, he sys em should be calib a ed, e.g., wi h a sample wi h a known dynamic magne ic equency esponse, o ins ance, he pa amagne ic ma e ial Dy 2 O 3 in powde o m [ 43 ]. This also allows o compensa e o any ampli ude and phase e o s and also o con e he measu ed ACS in o a calib a ed olume, mola o mass suscep ibili y. ACS s. equency measu emen s ha e been ou inely used by nume ous g oups o cha ac e ize MNPs [ 43 – 46 ]. F om he ACS esponse, i is possible o es ima e he SLP alue by s udying he magne ic losses ob ained om he ACS ou -o -phase componen [41,47]. In AC suscep ibili y s. empe a u e measu emen s a cons an exci a ion equency, a small ampli ude sinusoidal magne ic ield is also used, and i s equency can be a ied up o abou 10 kHz [ 48 ]. In a ecen pape , an induc ion-based ACS sys em ha can be used a lowe empe a u es was designed o equencies up o he MHz ange [ 49 ]. Calib a ion is done in almos he same way as he ACS s. equency me hod using a sample wi h known dynamic magne ic p ope ies. The in-phase and ou -o -phase componen s o he ACS a e measu ed e sus he empe a u e o he sample. In addi ion, in his case, a supe imposed DC magne ic ield can be applied. In a speci ic empe a u e ange, he esponse becomes equency-dependen , and he ACS esul s p o ide in o ma ion abou he magne ic elaxa ion p ope ies o he MNP ensemble [ 49 – 55 ]. Thus, measu ing he dynamic magne ic p ope ies gi es in o ma ion on he magne iza ion dynamics in he sample by a ying he AC d i e equency (di e en ime scales). Tempe a u e-dependen ACS is a s anda d echnique o cha ac e iza ion o MNPs, o ins ance, o de e mine blocking empe a u es, magne ic elaxa ion p ope ies o magne ic in e ac ions; indeed, i is impo an o quan i y magne ic in e ac ions as hey will a ec he ene gy abso p ion and, he e o e, he hype he mia hea ing p ope ies [17,56–58]. 2.4. DC Magne iza ion In DC magne ome y (DCM), he magne ic momen o a sample is measu ed as a unc ion o bo h applied magne ic ield and empe a u e. DCM measu emen s a e ypically pe o med in comme cially a ailable magne ome e s, based on SQUID echniques, ib a ing sample magne ome e s (VSM) o al e na ing g adien magne ome e s (AGM) [ 59 ]. The maximum magne ic ields in he DCM me hod should be la ge enough o sa u a e he sample magne iza ion in o de o de e mine he in insic sa u a ion magne iza ion. DCM magne ome e s a e calib a ed agains a magne ic sample wi h known sa u a ion magne iza ion o suscep ibili y. The basic pa ame e s om a magne iza ion e sus ield a e in insic sa u a ion magne iza ion, whe e he measu ed magne ic momen is no malized o he mass o olume o he magne ic ma e ial unde in es iga ion. In addi ion, he emanence and coe ci i y om he hys e esis loop can be de e mined. Likewise, he abso bed ene gy by he MNP sys em a equilib ium can be ob ained by calcula ing he a ea enclosed unde he hys e esis loop [ 60 ]. DC magne iza ion measu emen s cons i u e Ma e ials 2021,14, 706 6 o 36 a basic magne ic cha ac e iza ion echnique ha has been ou inely used by nume ous g oups o cha ac e ize MNPs sys ems [61–64]. 2.5. AC Calo ime y Calo ime y is he mos employed echnique o quan i y magne ic losses in MNP suspensions subjec ed o an AC ield. The p ocedu e is based on measu ing he ini ial empe a u e inc ease a e immedia ely a e applying he AC ield. This expe imen al me hod has been widely employed and has con ibu ed o unde s and he in luence o in insic—s uc u al, colloidal, magne ic—o ex insic pa ame e s—AC ield—on SLP and ILP alues [ 20 , 23 , 24 , 26 , 30 , 31 , 36 , 47 , 56 – 58 , 63 , 65 – 68 ]. Calo ime ic measu emen s a e usually pe o med unde non-adiaba ic condi ions since adiaba ic ones a e a ely a ained [69,70] . Such non-adiaba ic sys ems equi e pa icula da a analysis o emo e a i ac s om di e - en e o sou ces [71,72]. 2.6. AC Magne ome y AC magne ome y quan i ies he enclosed a ea o AC magne ic hys e esis loops o de e mine SLP alues ( ≈ a ea unde loops × ield equency). The applica ion o his echnique o measu e magne ic colloids is ecen , and mos o he ob ained esul s ha e been pe o med using home-made equipmen [ 73 – 76 ] since comme cial equi alen s a e e y sca ce. AC magne ome y has he ad an age ha he calcula ion o SLP alues is no in luenced by he mal pa ame e s o condi ions, allowing o quan i y o magne ic losses when MNPs a e inside biological ma ices, like cells o issues [ 28 ]. The analysis o hys e esis loops unde AC ields can shed ligh on he e ec o pa icle size, shape, agg ega ion, aniso opy, iscosi y and ield ampli ude and equency on he magne ic losses [27,28,77–79]. Howe e , dedica ed SOP a e also needed o his echnique. In summa y, he exis ence o SOP and au oma ed ins umen a ion o quan i y ele an physicochemical pa ame e s o magne ic hype he mia will wa an he eliabili y and ep oducibili y o he ob ained alues, which is manda o y o ensu e a eliable ansla ion o MH o clinics. 3. Scalable Syn hesis P o ocols 3.1. Gene al Challenges Today’s li e a u e p o ides a a ie y o p o ocols o syn hesize uni o m e i e MNPs wi h di e en sizes and shapes, sui able o magne ic hype he mia [ 80 ]. In many cases, epo ed nanopa icle p ope ies a e supe io o hose o cu en ly app o ed p oduc s and, he e o e, ha e he po en ial o inc ease he e iciency o hype he mia ea men s by eaching highe empe a u es a lowe nanopa icle concen a ions unde milde magne ic ield condi ions. Howe e , la ge-scale p oduc ion o hese MNPs wi h imp o ed o op imal p ope ies is associa ed wi h obs acles such as low yield and, mos impo an ly, limi ed ep oducibili y due o poo con ol and documen a ion o syn hesis condi ions. These challenges need o be add essed o a syn he ic p oduc o each ma ke ma u i y. On he o he hand, a cu en esea ch challenge is unde s anding nanopa icle o ma- ion mechanisms and kine ics ha a e essen ial o guide he de elopmen o syn heses ha a e ep oducible in a sys ema ic, con ollable and scalable manne . Since con inuous p o- cesses can p o ide ad an ages o e ba ch p ocesses o ep oducible and scalable syn hesis p o ocols, hey ha e ecen ly gained inc eased in e es . The inal s ep ela ed o nanopa icle unc ionaliza ion is s ill a di icul ask ha needs special a en ion o achie e scalable p oduc ion. The coa ing and he numbe o ac i e su ace si es a e c ucial o nanopa icle dispe sion/s abili y, and he e o e pa icle–pa icle magne ic in e ac ions and he pa icle hea ing p ope ies independen ly on he media iscosi y and he concen a ion, i.e., in i o and in i o condi ions. I is impo an o es ablish a ep oducible yield o he coa ing s ep and pu i ica ion o any byp oduc . Ma e ials 2021,14, 706 7 o 36 3.2. P epa a ion o MNPs and Func ionaliza ion We ha e iden i ied some majo challenges in he p epa a ion o uni o m MNPs wi h di e en syn he ic me hods, classi ied by he media whe e nuclea ion and g ow h ake place, i.e., aqueous and pola o nonpola o ganic sol en s. We ocus on magne ic i on oxide nanopa icles (IONPs) as hey ha e al eady been app o ed o humans [ 81 ] and a e he e o e he mos p omising hea ing agen candida es. The main challenge o syn hesizing IONPs o hype he mia in aqueous media is he p oduc ion o la ge (~20 nm) pa icles wi h good con ol o size and shape dis ibu ion. Despi e p og ess and b oad u iliza ion o he wa e -based co-p ecipi a ion me hod due o he high yield, he e a e se e al d awbacks, such as subop imal size (<15 nm, wi h excep ions, e.g., o me hods wi h a slow pH inc ease [ 82 ]), high polydispe si y, poo c ys allini y con ol, and consequen ly poo sa u a ion magne iza ion. Rega ding la ge pa icles o hype he mia applica ion wi h sa u a ion magne iza ion alues nea he bulk, hey can be ob ained by oxida i e p ecipi a ion o Fe(II) sal s in aqueous media (>15 nm). Recen ly, oxida i e p ecipi a ion has been scaled up o 20 g pe ba ch [ 83 ] and was made a con inuous p ocess [ 84 , 85 ]. The combina ion o low-cos eagen s such as FeSO 4 , NaOH, NaNO3and e hanol/wa e mix u es was shown o yield uni o m nanopa icles. The main challenge o he mal decomposi ion syn hesis in high boiling poin o ganic media (commonly nonpola sol en s such as 1-oc decene o pola polyol sol en s) is he s anda diza ion o expe imen al p ocedu es and con ol o syn he ic condi ions. This is due o he complexi y o he o ma ion p ocess o magne i e and he consequence o a se o he mally ac i a ed chemical eac ions [ 83 ]. E en sub le changes o chemical aspec s such as p ecu so / eagen concen a ion can p o oke conside able modi ica ions o he decomposi ion and o nuclea ion empe a u e wi h d ama ic consequences on he inal nanoma e ial p oduc . One pa ame e dese ing special a en ion is he hea ing a e, as i is known o a - ec (o used o con ol) nanopa icle sizes [ 86 ]. Since he hea ing a e can a y wi h he expe imen al p ocedu e, o example, he size and geome y o he eac ion essel (wi h limi s o la ge olumes in e ms o hea inpu ), ep oducing hea ing p o iles om small o especially la ge-scales is no i ial. Tempe a u e p o iles o he eac ion media should he e o e be documen ed ca e ully; epo ing hea e se ings is no enough. Conside ing scalable p oduc ion, e icien hea ing sou ces a e equi ed o acili a e homogeneous em- pe a u e p o iles (he e, he mixing sys em plays an impo an ole) and su icien hea ing a es. Hea ing ia mic owa e adia ion is a p omising al e na i e o classical hea e s and was shown o enable scale up he p oduc ion up o 1 kg [ 87 ]. Mic owa e hea ing was also used o syn hesis o lowe -like mul i-co e IONPs wi h good con ol o e co e sizes and usion be ween hem depending on he eagen s, empe a u e and hea ing ime. O he hea ing sys ems such as hyd o he mal using au ocla es p esen s he d awback o needing a special pilo plan o scale up sa ely he lab-scale p ocedu e. In addi ion o hea ing a es, also eagen s o age, pu i y and supplie , s i ing con- di ions (s i ing speed, ype and dimension o p opelle /s i e used), ine /non-ine gas condi ions du ing syn hesis [ 88 ] and washing/cen i uga ion p o ocols need o be documen ed and/o eco ded accu a ely du ing syn hesis o ob ain good ep oducibili y. Only accu a e documen a ion, and epo ing, allows esea che s in o he labo a o ies o un a he mal decomposi ion syn hesis “in he same way”, i.e., a bes possible deg ee o simila i y. Al hough (classical) he mal decomposi ion syn heses in nonpola sol en s can yield magne ic pa icles wi h excellen size and shape uni o mi y, as well as c ys allini y and magne iza ion, hey a e e y high in p ice, especially due o expensi e o ganic p ecu so s, and gene a e many byp oduc s. Polyol me hods a e usually cheape , bu scalable p o ocols o syn hesize pa icles o magne ic hype he mia a e s ill a he esea ch s age. Func ionaliza ion is ano he signi ican aspec , which d aws in e es when aiming o use nanoma e ials o magne ic hype he mia. Much wo k has been done in his aspec , and he p o ocols o coa ing he nanopa icles wi h laye s o o ganic o ino ganic agen s and Ma e ials 2021,14, 706 8 o 36 hei unc ionaliza ion depending on he applica ion a e gene ally well-es ablished [ 89 ]. Thei limi a ions a e well-known and o en ela ed o he la ge polydispe si y ha is gene a ed by he coa ing o agg ega es, he limi ed colloidal s abili y and i s deg ada ion when s o ed o a long ime. Mos p o ocols use highly dilu ed suspensions and a e di icul o scale up. A special p oblem ha needs mo e a en ion is ela ed o he o e all in luence o he coa ings on he hype he mia pe o mance in i o and in i o. 3.3. Imp o ing Rep oducibili y In o de o imp o e he ep oducibili y o all MNP syn hesis, we need o de elop de ailed epo s o me hods and p ocedu es, including used chemical eagen s (e.g., pu i y, supplie , and p oduc code), as well as SOPs ha should be published as supplemen a y in o wi h he pape o be able o become bench-ma k syn heses o he communi y. In he same way, accu a e epo ing o cha ac e iza ion p o ocols is essen ial. The use o ISO s an- da ds ha a e in e na ionally ag eed by expe s and gua an eed quali y equi emen s (e.g., analysis o TEM images, usage o Sche e equa ion, DLS o size, ζ -po en ial, VSM-SQUID o magne ic p ope ies, TGA o he de e mina ion o he amoun o o ganic ma e ial a he pa icles su ace, ICP-OES elemen al analysis o pu e me al con en quan i ica ion) should be used [ 15 ]. A good p ac ice is he publica ion o aw da a (especially TEM images, whe e an image is no p esen a i e due o a limi ed numbe o pa icles ha can be obse ed). In ac , in many cases, he e is a need o use a combina ion o di e en cha ac e iza ion echniques in o de o ge a ull de ailed and eliable pic u e o a gi en sample. Al eady published ISO s anda ds can help in he compa ison o he esul s and he samples. In he same sense, he de elopmen o b oadly accep ed p o ocols o he magne ic cha ac e iza ion o hype he mia- a ge ed nanopa icles will help o p o ide a highe deg ee o eliabili y o hype he mia measu emen s a an in e na ionally accep ed le el. In his way, he measu emen o bo h SLP and ILP ( o compa e measu emen s pe o med a di e en equencies and ield s eng hs o using di e en ins umen s) will be mo e easily compa able among all academic and non-academic pa ne s. These cha ac e iza ions will conce n all possible o ms, such as colloidal dispe sions/ensembles and powde samples. De ailed cha ac e iza ion o he nanopa icles is o ou s anding impo ance o es ablishing a ep oducible syn hesis. 3.4. Scalabili y Possibili ies P oduc ion a la ge scales can easily be achie ed due o epe i i e and pa allel p oduc ion, bu syn heses using lab-scale eac o s (<100 mL) will ha dly be cos -e ec i e. Only in ew cases, he solubili y o eagen s allows he la ge p oduc ion by inc easing he eagen concen a ion while keeping he same sol en olume [ 83 ]. In mos cases, keeping he p ecu so s/su ac an s/sol en s a io while scaling-up a gi en eac ion is no enough o assu e i s ep oducibili y in hose la ge scales unless demons a ed. I should be aken in o accoun ha he gap be ween he o dina y labo a o y scale (100 mL) and he indus ial pilo plan scale (100 L) is h ee o de s o magni ude. Expe imen s o scaling up o 10 L ha s ill could be done in he lab a e needed be o e going o he indus ial scale. The key o a success ul scale-up is o unde s and he syn hesis’ c i ical p ocess pa am- e e s, i.e., pa ame e s wi h a high impac on he nanopa icles’ c i ical quali y a ibu es ( o example, in he case o hype he mia, pa icle size dis ibu ion, su ace unc ionaliza ion, and mos impo an ly, he SLP). Hence, unde s anding nanopa icle o ma ion mechanisms and kine ics a e impo an as i shows, o example, i mixing imes, hea ing a es, o eagen addi ion ime scales (o a combina ion) is c ucial, i.e., i needs o be main ained a la ge scales. An in e es ing op ion o p oduce MNPs a la ge scales is he con inuous syn heses a low [ 85 , 90 – 92 ] and high empe a u es [ 93 – 96 ] (Figu e 1). Bo h p ocesses can be au oma ized a a labo a o y scale, bu up o now, none o hem ha e been passed o an indus ial pilo plan scale. Ma e ials 2021,14, 706 9 o 36 Ma e ials 2021, 14, x FOR PEER REVIEW 9 o 37 The key o a success ul scale-up is o unde s and he syn hesis’ c i ical p ocess pa- ame e s, i.e., pa ame e s wi h a high impac on he nanopa icles’ c i ical quali y a ib- u es ( o example, in he case o hype he mia, pa icle size dis ibu ion, su ace unc ion- aliza ion, and mos impo an ly, he SLP). Hence, unde s anding nanopa icle o ma ion mechanisms and kine ics a e impo an as i shows, o example, i mixing imes, hea ing a es, o eagen addi ion ime scales (o a combina ion) is c ucial, i.e., i needs o be main- ained a la ge scales. An in e es ing op ion o p oduce MNPs a la ge scales is he con inuous syn heses a low [85,90–92] and high empe a u es [93–96] (Figu e 1). Bo h p ocesses can be au om- a ized a a labo a o y scale, bu up o now, none o hem ha e been passed o an indus ial pilo plan scale. Figu e 1. Schema ic ep esen a ion o a con inuous low se up o la ge-scale p oduc ion o magne ic nanopa icles. Changes in he manu ac u ing p ocess can signi ican ly change pa icle p ope ies such as size, shape, and pu i y. The e o e, obus nanopa icle p oduc ion ou es need o be chosen. I is impo an o selec hose sol en s o highe chemical s abili y o a oid hei deg ada ion. O u mos impo ance o scalable p ocesses (and ce ainly when ansla ing ba ch p ocesses o low) is he equi ed eac ion ime. Fo p ac ical easons, highly exo- he mic eac ions a e undesi able, al hough low eac o s p o ide a sa e al e na i e as hey acili a e apid hea exchange. Rega ding he scale-up o he unc ionaliza ion s ep, he common need o s i ing may complica e he a emp s o scale up such expe imen s when applying a con inuous low p ocess ins ead o “ba ch” ones. I needs u he s udies o ansla e he syn hesis and unc ionaliza ion o MNPs in con inuous o segmen ed low eac o s o p oduc ion a indus ial scales. Besides he equi emen o ha ing a obus and ep oducible p ocess, wha o con- side as la ge-scale p oduc ion should be pu in o pe spec i e o demand and alue o he p oduc . Assuming ha a conse a i e es ima e 0.05% o he wo ld’s popula ion (8 billion) would su e om cance ha can be ea ed ia magne ic hype he mia, and assuming u he , ha each pa ien will need six cycles o ea men a yea equi ing each ime 0.3 g o MNPs [97], he global demand would be 144 ons a yea . We need o imp o e he hea - ing e iciency o IONPs, le us say h ee imes mo e, o educe he amoun o MNPs needed o each ime o 0.1 g. Al hough his p oduc ion a e appea s challenging, i mus be con- side ed oge he wi h he p oduc ion cos s. Assuming ha cos s o $100 pe ea men wi h he nanopa icles a e accep able, a easonable cap o p oduc ion cos s migh be $50/g (5% o ea men cos s). Al hough his is an ex emely o e simpli ied es ima ion, i indi- ca es wha can be conside ed as an economic la ge-scale syn hesis o MNPs o hype he - mia, i.e., a p ocess capable o ep oducibly p oducing he desi ed nanoma e ial a cos s < $50/g. In biomedical applica ions, when a small amoun o nanopa icles is needed, an al e na i e o scaling he p oduc ion o MNPs could be he ansla ion o he syn he ic p ocess owa ds a dose-on-demand syn hesis in he clinic [98,99]. 4. Long-Te m S abili y and Biodis ibu ion o Nano-Hea e s in Humans One key issue in he use o magne ic nanopa icles o magne ic hype he mia he - apy is hei biodis ibu ion, bio ans o ma ion and long- e m a e in he body. A i s conce n is o de ine he ime window o magne ic hype he mia e icacy, i.e., how long he pa icles will keep hei magne ic p ope ies and will be able o hea . The Figu e 1. Schema ic ep esen a ion o a con inuous low se up o la ge-scale p oduc ion o magne ic nanopa icles. Changes in he manu ac u ing p ocess can signi ican ly change pa icle p ope ies such as size, shape, and pu i y. The e o e, obus nanopa icle p oduc ion ou es need o be chosen. I is impo an o selec hose sol en s o highe chemical s abili y o a oid hei deg ada ion. O u mos impo ance o scalable p ocesses (and ce ainly when ansla ing ba ch p ocesses o low) is he equi ed eac ion ime. Fo p ac ical easons, highly exo he - mic eac ions a e undesi able, al hough low eac o s p o ide a sa e al e na i e as hey acili a e apid hea exchange. Rega ding he scale-up o he unc ionaliza ion s ep, he common need o s i ing may complica e he a emp s o scale up such expe imen s when applying a con inuous low p ocess ins ead o “ba ch” ones. I needs u he s udies o ansla e he syn hesis and unc ionaliza ion o MNPs in con inuous o segmen ed low eac o s o p oduc ion a indus ial scales. Besides he equi emen o ha ing a obus and ep oducible p ocess, wha o conside as la ge-scale p oduc ion should be pu in o pe spec i e o demand and alue o he p oduc . Assuming ha a conse a i e es ima e 0.05% o he wo ld’s popula ion (8 billion) would su e om cance ha can be ea ed ia magne ic hype he mia, and assuming u he , ha each pa ien will need six cycles o ea men a yea equi ing each ime 0.3 g o MNPs [ 97 ], he global demand would be 144 ons a yea . We need o imp o e he hea ing e iciency o IONPs, le us say h ee imes mo e, o educe he amoun o MNPs needed o each ime o 0.1 g. Al hough his p oduc ion a e appea s challenging, i mus be conside ed oge he wi h he p oduc ion cos s. Assuming ha cos s o $100 pe ea men wi h he nanopa icles a e accep able, a easonable cap o p oduc ion cos s migh be $50/g (5% o ea men cos s). Al hough his is an ex emely o e simpli ied es ima ion, i indica es wha can be conside ed as an economic la ge-scale syn hesis o MNPs o hype he mia, i.e., a p ocess capable o ep oducibly p oducing he desi ed nanoma e ial a cos s < $50/g. In biomedical applica ions, when a small amoun o nanopa icles is needed, an al e na i e o scaling he p oduc ion o MNPs could be he ansla ion o he syn he ic p ocess owa ds a dose-on-demand syn hesis in he clinic [98,99]. 4. Long-Te m S abili y and Biodis ibu ion o Nano-Hea e s in Humans One key issue in he use o magne ic nanopa icles o magne ic hype he mia he apy is hei biodis ibu ion, bio ans o ma ion and long- e m a e in he body. A i s conce n is o de ine he ime window o magne ic hype he mia e icacy, i.e., how long he pa icles will keep hei magne ic p ope ies and will be able o hea . The subsequen clinical challenge is o de ine he numbe and he ime o magne ic ield applica ions ha will be use ul o a ec he umo . I has been shown ha in acellula con inemen o magne ic nanopa icles in lyso- somes has a d ama ic impac on hei dynamical magne ic p ope ies and SLP, mos ly due o dipola in e pa icle in e ac ions and loss o o a ional mobili y [ 100 , 101 ], e en i he pa icles keep hei c ys alline in eg i y. Ano he c i ical aspec is ha he in acellula magne ic pa icles a e exposed o he ha sh en i onmen o lysosomes ha combine acidic pH (abou 4.5), enzymes ha egula e p o ein deg ada ion and edox egula o s. Since he unc ion o lysosomes is o deg ade undesi able p o eins and xenobio ics, i is impo an o de e mine o wha ex end lysosomes may be able o deg ade magne ic pa icles and make hem lose hei s uc u al in eg i y and magne ic p ope ies? Ma e ials 2021,14, 706 16 o 36 a e i has achie ed i s PMOA o hea ing. The e o e, he p ima y mode o ac ion would emain as a de ice. 5.2.2. D ug-De ice Combina ion P oduc Whe e a p oduc uses bo h d ug and de ice unc ions o achie e i s p ima y in ended pu pose, i is classi ied as a d ug-de ice combina ion p oduc [ 128 ]. A hype he mia pa icle ha has a a ge ing unc ion using a ecep o (i.e., chemical/pha macological ac ion), as well as hea ing, would be classi ied as a combina ion p oduc . Fo combina ion p oduc s in he US, he p ima y mode o ac ion de e mines whe he he p ima y egula o y pa hway will be d ug o de ice. In Eu ope, combina ion p oduc s a e ei he egula ed as d ugs o de ices depending on he p ima y e sus ancilla y unc ion. Fo pa icles combining mo e han one ype o ac ion, he app op ia e egula o y pa hway will depend on which mode o ac ion is p ima y, and ypical elemen s o bo h he d ug and de ice pa hway a e ollowed, depending on he e i o y. 5.3. S eamlined De elopmen Any would-be hype he mia he apy will need o demons a e clinical sa e y and e icacy, and o his, he e is no sho -cu , bu he e a e ways o s eamline he o e all p ocess. One app oach is o use an exis ing pa icle in a new way. Such “ e-pu posing” is he basis o physician-led “o -label” use o he apies. Fo example, he e a e a numbe o clinical s udies in es iga ing he use o pa icles app o ed o i on- eplacemen he apy as MRI con as agen s [ 129 ]. The a ionale is ha i a p oduc is shown o be sa e in one clinical indica ion, i will be easie o es ablish sa e y in a new one. The p ocess can also be s eamlined by aking in o accoun he equi emen s o la e s ages, e en in he ea lies s ages o pa icle de elopmen . Ensu ing ha manu ac u ing ques ions such as oxici y, biocompa ibili y, s abili y and p ocess scalabili y a e consid- e ed ea ly on can di ec he de elopmen away om “dead-ends” and sa e signi ican cos and ime. Fo example, ano he nano echnology, quan um do s, show p omise o cance he apy. Howe e , a i s , he mos commonly used co es con ained cy o oxic cadmium [ 130 ]. While in heo y, hese co es could be coa ed o minimize cy o oxici y, in p ac ice, es ablishing long- e m sa e y has p o ed challenging. Ini ial conside a ion o biocompa ibili y could ha e accele a ed p og ess owa ds clinical use by di ec ing e o owa ds he cadmium- ee non- oxic al e na i es now being explo ed [131]. In summa y, he challenging la e s ages o magne ic pa icle de elopmen can be mo e easily nego ia ed i , a he ea ly-s ages, downs eam equi emen s such as GMP manu ac u e, clinical sa e y and he egula o y pa hway can be inco po a ed and used o guide he de elopmen . This kind o in eg a ed app oach can help has en a b igh u u e o clinical magne ic hype he mia. 6. Nano oxici y o Nanopa icles o Magne ic Hype he mia The inc easing use o magne ic hype he mia in (p e)clinical se ings wa an s a p ope unde s anding and ca e ul e alua ion o how enginee ed nanoma e ials would be mos op imally sui ed. This conce ns bo h he e icacy o he mal con e sion, wi h maximal hea gene a ion o a minimal numbe o nanopa icles, as well as a comple e lack o any po en ial oxici y on heal hy cells om he pa ien . To da e, a ious s udies ha e looked in o he oxici y o IONPs, mainly d i en by hei clinical accep ance as con as agen s o MRI [ 132 , 133 ]. The majo i y o hese s udies conce n in i o expe imen s, whe e cell ypes o in e es a e used o e alua e po en ial oxici y upon exposu e o he enginee ed nanopa icles [ 134 , 135 ]. While mo e limi ed, some s udies ha e also been pe o med in p eclinical animal models, mainly mice and a s, o e alua e po en ial sys emic oxici y om exposu e o he nanopa icles [136]. Howe e , inal conclusions ega ding he sa e y o oxici y o hese nanopa icles emain di icul o answe , mainly d i en by he wide a ie y o model sys ems used, ex- pe imen al se ings and nanopa icle p ope ies [ 137 ]. O e all, i is belie ed ha IONPs a e Ma e ials 2021,14, 706 17 o 36 ai ly sa e, up o concen a ions o 5 mM o i on [ 138 ]. Ye , hese ea ly opinions ha e been e- isi ed by a ious s udies, showing ha nanopa icle-speci ic p ope ies such as size, shape and su ace chemis y can ha e a signi ican in luence on he oxici y o he pa icles [ 139 ]. O e all, oxici y has mainly been linked wi h cellula up ake le els, whe e IONPs end o cause high le els o eac i e oxygen species, which, depending on he na u e o he cells, can lead o oxida i e s ess [ 140 ]. This, in u n, can mani es i sel in di e en ways, po en ially esul ing in geno oxici y, diminished s em cell di e en ia ion, neu o oxici y o in lamma ion [ 141 – 144 ]. In iew o cance he apy, he seques a ion o dex an-coa ed IONPs by umo -associa ed mac ophages has been shown o al e mac ophage s a us and p omo e p oin lamma o y M1 pheno ype [ 145 ]. While his is po en ially in e es ing o umo immuno he apy, he e ec o he pa icles on o he mac ophages and he induc ion o in lamma o y esponses may cause se e e side-e ec s. As men ioned abo e, one main p oblem lies in he seemingly con adic ing da a a ailable in he li e a u e, which can be na owed down o expe imen al a ia ions. Mino modi ica ions o nanopa icle p ope ies can ha e majo implica ions in iew o hei biodis ibu ion and oxici y, and, he e o e, no gene al conclusion can e e be made. IONPs also in e e e wi h a ious classical biochemis y es s, such as he MTT assay [ 135 ], and ca e mus be aken o p ope ly design s udies wi h sui able con ols o in in e p e ing da a om o he s udies. Fo IONPs, one aspec o in e es is hei biome abolism, whe e cellula up ake o he nanopa icles esul s in hei lysosomal seques a ion. The e, he low lysosomal pH and p esence o small molecules (e.g., ci a e) esul in he dissolu ion o he nanopa icles and he elease o e ic ions ha a e hen shu led in o he cy oplasm and become pa o he cellula labile i on pool [ 138 ]. While his deg ada ion esul s in p ope biop ocessing o he nanopa icles, i will a ec he magne ic p ope ies o he nanopa icles in he longe e m [ 146 , 147 ]. In he sho - e m, he kine ics o his deg ada ion mus be ca e ully con olled, as apid dissolu ion is linked o excessi e e ic ion concen a ions p esen locally ha can su pass oxic h esholds [ 148 ]. Ca e ully con olling he deg ada ion kine ics by uning nanopa icle p ope ies, such as he su ace coa ing, can play a key ole in de e mining he ole ance o he body o such nanopa icles. In o de o u he exploi he clinical use o hese nanopa icles in hype he mia applica ions, i is impe a i e ha he scien i ic ques ion is p ope ly posed. The ques ion: “a e nanopa icles sa e” is oo gene ic and can simply no be answe ed. By eph asing he sen ence in o “does o mula ion x cause any ha m when i is used o hype he mia when adminis e ed by y a dose z?”, i de ines be e he esea ch ha needs o be pe - o med in o de o p omo e his ield: (1) he exac nanopa icle o mula ion mus be well cha ac e ized and desc ibed. (2) The ou e o adminis a ion o he pa ien mus be clea ly de ined. (3) The dose and need o epea ed adminis a ions o no mus be speci ied. (4) The abili y o pe o m hype he mia a i s op imal ou pu mus be e alua ed and compa ed o cu en gold s anda ds and s a e-o - he-a me hods. While la ge se s o li e a u e da a a e a ailable, he da a needed o answe he ques ion abo e emain sca ce. Typical examples include classical oxici y s udies, in which nanopa icles a e adminis e ed sys emically by in a enous adminis a ion, while o mos hype he mia applica ions, he nanopa icles a e adminis e ed locally. IONPs ha e been s udied, bu o he nanoma e ials ha a e e alua ed as hype he mia media o s wi h possibly highe he apeu ic e icacy a e o en less commonly s udied [ 149 , 150 ]. The biodis ibu ion o he nanopa icles eleased om he umo a e hype he mia applica ion would be mo e in e es ing o s udy han sys emically adminis e ed nanopa icles. The oxici y o he nanopa icles on hei own is impo an , bu hei e ec mus also be e alua ed a e hype he mia applica ion. I is likely ha he su ace coa ing o he nanopa icles has changed due o he gene a ed hea , and his may a ec nanopa icle beha io (biodis ibu ion and oxici y) qui e d as ically. Apa om sa e y, he he apeu ic e icacy mus also be demons a ed. While his commonly happens using classical ea men (e.g., doxo ubicin), his is o en a om eali y, and clinically ele an ea men s, as well as o he no el s a e-o - he-a he apies, mus be used as a compa ison (e.g., small molecules, immuno he apy, e c.). Ma e ials 2021,14, 706 18 o 36 The clinical accep ance o a pa icula nanopa icle o mula ion also equi es egu- la o y app o al. While guidelines o chemicals o pass clinical ials a e a he clea , o nanopa icles, his emains a he ague. P og ess has been made, howe e , and wi h u he op imiza ion and eamwo k be ween expe s in he ield, he h oughpu o clinical ansla ion o nanomedicines will only inc ease. Thus, u he ad ances in he clinical ans- la ion o nano echnologies can be expec ed in he nea u u e [ 151 ]. To da e, nanopa icles can ei he be labeled as a “medical de ice” o as a “d ug”, which has majo implica ions on how egula o y app o al o clinical use can be ob ained. The label o “medical de ice” was p e iously p e e ed as i in ol ed less leng hy clinical s udies and was based on he no ion ha he nanopa icles hemsel es did no change o we e an ac i e subs ance bu me ely a ool by which he apy could be pe o med. As desc ibed in he excellen manusc ip by Jones e al. [ 152 ], he slow p og ess in clinical ansla ion is no in i sel caused by a lack o egula ions bu a he a b oad gap be ween heo e ical knowledge and p ac ice. This is in pa due o he lack o academics in ol ed in se ing up he documen s ega ding sa e y and e icacy es ing o medical de ices, esul ing in a lack o s anda dized me hods o ga he ing and p esen ing da a. E o s o b idge his gap a e ongoing, wi h he se up o he Nano echnology Cha ac e iza ion Labo a o y (NCL) o de elop s anda dized p o ocols o oxicology, pha macology and e icacy e alua ion o nanoma e ials. Fo mos agen s, he use o specialized clinical esea ch o ganiza ions (CROs) is wa an ed, who will design and/o pe o m he p eclinical s udies equi ed o ile an applica ion o clinical s udies. While a ious CROs exis wi h b oad expe ise in a wide ange o echnologies, CROs wi h expe knowledge on nanopa icle use emains limi ed. P og ess in his domain is expec ed as he ield o nanomedicine keeps expanding, whe e nano oxici y and nanomedicine expe s can hen liaise wi h, o o m pa o , egula o y o ices and CROs dedica ed o enhancing clinical p og ess in his exci ing ield. 7. Tempe a u e Measu ing and Moni o ing 7.1. Backg ound Apa om hei gene al applica ion o dis ance con olled non-con ac hea ing, MNPs p o ide an excellen oppo uni y o nano-ac ua ion. Fo ins ance, hey can be used o selec i e hea ing o nano-objec s o induce poin eac ions a he nanoscale. This ope a ion implies he gene a ion o empe a u e g adien s in he nano-objec wi h espec o i s su oundings, which can only be de e mined by he mome e s wi h a spa ial eso- lu ion also a he nanome e scale. Mo eo e , he simul aneous use o nanohea e s and nano he mome e s could be employed o in es iga e phenomena o hea ans e a he nanoscale, which is ac ually an unexplo ed e i o y and he objec o in ense deba e, especially conce ning in acellula hea ans e [ 153 , 154 ]. The ecen de elopmen o a e-ea h-doped luminescen MNPs has in oduced a new ield in he mal biosensing, implying less in asi e expe imen s, no only in li ing cells bu also in mo e challenging small animal models [ 155 – 158 ]. Joining he mome y and hea ing a he nanoscale can also be pa icula ly in e es ing o he de elopmen o high-pe o mance non-in asi e hype he mia he apy based on he local hea ing o speci ic in acellula si es o p o oke cell apop osis, wi hou he need o o e all massi e hea ing o he whole cance umo . The hypo hesis o local in acellula hype he mia a ises om he d awbacks o he cu en s a egy o o e all umo hea ing and a massi e injec ion o MNPs di ec ly in o he umo [ 159 ]. E en be o e being es ed, his hypo hesis has al eady been he subjec o in ense deba e [ 160 ]. Objec ions come mos ly om s anda d he modynamic conside a ions, which es ima e an in acellula concen a ion o MNPs equi ed o e adica e umo s ha is i ually un easible [ 160 ]. A he cen e o his deba e is he ques ion o whe he i is possible o c ea e a su icien ly high- empe a u e g adien in he icini y o MNP dispe sed in a liquid o in acellula media [ 154 ]. Indeed, heo e ical he modynamic calcula ions p edic he o ma ion o negligible empe a u e g adien s in MNPs when subjec ed o al e na ing magne ic ields wi h espec o he bulk [ 161 , 162 ], and a ew expe imen al epo s a e in ag eemen wi h hese p edic ions [ 163 ]. Howe e , nume ous Ma e ials 2021,14, 706 19 o 36 expe imen al s udies ha e ound subs an ial local empe a u e g adien s a hese condi ions ha sugges he exis ence o nanoscale hea ans e phenomena di e en om mac oscopic sys em beha io [ 160 , 164 – 169 ]. I is hus pe emp o y o he ad ancemen in he ield o dispose o eliable me hods o he de e mina ion o local empe a u e in he icini y o MNPs, and he de i a ion o he modynamical models o in acellula hea ans e a he nanoscale. 7.2. Luminescence Nano he mome y Luminescence he mome e s a e p obably he bes op ion o non-con ac high spa ial esolu ion he mome y in gene al [ 155 ] and in acellula he mome y in pa icula [ 156 ]. The e a e se e al ypes o luminescen he mome ic p obes acco ding o hei na u e: lumi- nescen molecules (o ganic molecules such as dyes, p o eins like g een luo escence p o ein, GFP), ino ganic compounds (quan um do s, Si-do s, nanodiamond, lan hanides) o hyb id molecula o pa icula e ma e ials. Se e al empe a u es sensing op ical p ope ies a e also used, such as he in ensi y o emission, li e ime o pola iza ion aniso opy [ 157 ]. Li e ime measu emen s o e high sensi i i y and minimize in e e ences om o he luminopho es p esen in he medium. This is especially ue when using lan hanides luminescence p obes as hey show long li e imes, elimina ing any in e e ence e ec s om o he luminopho es. Howe e , li e ime measu emen s equi e a sophis ica ed de ec ion sys em, and he e o e in ensi y measu emen s, which can be pe o med on widely a ailable luo escence mic o- scopes, a e usually p e e ed. The simples luo escence he mome ic sys em is based on single emission in ensi y measu emen s, bu his sys em does no yield absolu e em- pe a u es, and i is likely o be a ec ed by he concen a ion o emi e s, he in ensi y o exci a ion ligh sou ce and he en i onmen . A way o o e come hese p oblems is o use he a io o wo emissions as he he mome ic pa ame e ( a iome ic he mome y), which is a mo e eliable, especially when he double emission comes om a single sou ce. The e a e se e al ways o implemen luminescence nano he mome y in o a magne ic nanohea e : he dual-pa icle app oach and he single-pa icle app oach [ 157 ]. The e a e also se e al kinds o empe a u e measu ing [ 155 – 157 , 169 ]: indi ec single empe a u e alue, di ec ins an , con inuous measu emen s o empe a u e changes, and in si u a io- me ic absolu e empe a u e measu emen s. In he nex sec ion, we will conside he bes a ailable op ions so a o magne ic hype he mia applica ions. 7.3. De e mina ion o Local Tempe a u e in MNPs When con enien ly endo sed wi h a ge ing agen s, magne ic NPs ha e he capaci y o selec i ely pene a e he memb ane o cance cells. P o iding ha hey can be di ec ed o speci ic in acellula o ganelles, he hea gene a ed by hese NPs can be used o induce local damage in hese o ganelles o igina ing cell apop osis wi hou he need o inc easing he empe a u e o he whole cell and hus subs an ially educing he amoun o MNPs necessa y o kill he cells. To e i y his hypo hesis, i is necessa y o check whe he he hea gene a ed by he MNPs is su icien o main ain a empe a u e g adien be ween he a ge ed o ganelle agains he hea conduc ion o he cy oplasm and he ex acellula ma ix. Mo eo e , his concep o local hype he mia he apy wi h AC ields equi es he use o MNPs wi h a high SLP a condi ions compa ible wi h in i o ea men . Dual MNP- he mome e nanopa icle sys ems lack p ecision as he empe a u e is measu ed a a dis ance om he hea e [ 167 ]. Single-pa icle sys ems based on he de achmen o luo opho es om he MNP shell abo e ce ain empe a u e alues ha e been used o ob ain empe a u e g adien s o abou 45 ◦ C [ 166 ] and 8 ◦ C [ 167 ] in he icini y o MNPs unde AC magne ic ield induc ion. Howe e , hese me hods equi e pos -analysis o he medium and he e o e canno be used o empe a u e moni o ing. The ideal solu ion is o inco po a e in a single MNP a he mome ic p obe ha yields ins an in o ma ion o he local absolu e empe a u e o he MNP du ing he applica ion o he al e na ing magne ic ield. The e a e wo possible ways: deco a ing he MNP su ace wi h molecula he mome e s o coa ing i wi h a solid he mome ic shell. The second case would o e Ma e ials 2021,14, 706 20 o 36 be e chemical s abili y agains he complex in acellula en i onmen , bu i is s ill a he s age o inding an adequa e ino ganic he mome ic coa ing o MNPs. Molecula lumi- nescence p obes may su e om luminescence quenching and bleaching e ec s p oduced by chemicals in he en i onmen . The e o e, he eal challenge in he design o his ype o p obes is o shield hem agains he ac ion o he complex biological en i onmen o p ese e hei op ical he mome ic p ope ies. As in oduced in Sec ion 7.2, se e al ypes o ma e ials ha e been p oposed as em- pe a u e nanop obes [ 156 ], some o hem ha e been e en used o in acellula he mom- e y [ 170 ], i.e., ER he mo yellow [ 171 ], bu only a ew ha e been ac ually used in local magne ic hype he mia s udies [ 170 ]. Fo ins ance, epo s o a 15 ◦ C empe a u e g adien we e epo ed on Mn- e i e NPs a ached o he memb ane o HEK 293 cells 15 s a e he applica ion o AC ields using DyLigh 549 as a he mome ic p obe [ 164 ]. Ad ances in his case also include he in si u measu emen o local empe a u e g adien s using hodamine [ 172 ] and lan hanide luminescence complexes [ 169 ]. The emission li e ime o GFP has also been used o epo la ge local empe a u es o 85 ◦ C, al hough hey we e es ima ed by ex apola ion o calib a ion cu es ob ained a much lowe empe a u es. The p obes we e no a ached o he hea he s, bu independen ly sp ead inside he cells. Using a double pa icle app oach, consis ing o MNPs and upcon e sion nanopa icles embedded in a silica ma ix, and a iome ic empe a u e de e mina ion, g adien s o abou 20 ◦ C we e epo ed on nanopa icles suspended in a liquid a e a 5 min exposu e o AC ields [ 168 ]. Ongoing in acellula expe imen s in ou lab using simila nanop obes ha e yielded qui e p omising esul s on 2D- empe a u e imaging o cells con aining MNPs du ing hype he - mia ea men (Figu e 6). Mos o he he mome ic p obes desc ibed so a a e based on isible ligh , lacking he necessa y issue pene a ion o body empe a u e moni o ing, and he e o e can only be use ul in cell cul u e expe imen s. Howe e , he ield is also mo ing owa ds he de elopmen o in a ed deep issue he mome y sys ems [ 173 ]. This op ion is possible in he case o op ical hype he mia, whe e single nanopa icles wi h a hea ing and empe a u e measu emen p ope ies a e al eady a ailable [ 174 ]. In a ed luminescence empe a u e p obes can also be possibly coupled o magne ic hea ing nanopa icles. Ma e ials 2021, 14, x FOR PEER REVIEW 21 o 37 moni o ing, and he e o e can only be use ul in cell cul u e expe imen s. Howe e , he ield is also mo ing owa ds he de elopmen o in a ed deep issue he mome y sys- ems [173]. This op ion is possible in he case o op ical hype he mia, whe e single nano- pa icles wi h a hea ing and empe a u e measu emen p ope ies a e al eady a ailable [174]. In a ed luminescence empe a u e p obes can also be possibly coupled o magne ic hea ing nanopa icles. Figu e 6. The mal images o MNPs in e nalized in cells, (a), be o e, and (b), du ing i adia ion wi h an AC magne ic ield; (c), a e age empe a u e shi upon he applica ion o an AC magne ic ield. We can easonably expec ha in a ew yea s, we would dispose o se e al ypes o MNPs inco po a ing local empe a u e p obes, bo h molecula and solid na u e, ha can yield eliable da a on he local empe a u e g adien gene a ed by MNPs exposed o al e - na ing magne ic ields ha can be used o he de elopmen o less in asi e, mo e e icien and mo e selec i e ad anced magne ic hype he mia ea men s. 8. T ea men Planning and Dosime y T ea men planning—which ep oduces he dose needed o des oy he umo s p e- se ing as much heal hy issue as possible— o many hype he mia modali ies ha e ben- e i ed om he de elopmen s made in adio he apy [175–179]. In he pa icula case o magne ic hype he mia, so a , he e is only one comme cially a ailable sys em o he pu pose de eloped by MagFo ce, he NanoPlan ® ( o me ly known as Hype Plan ® ) [180]. The ypical wo k low o a hype he mia ea men planning (HTP) ool s a s wi h a 3D model o he egion o in e es ha is buil om compu e ized omog aphy (CT), o MRI scans om pa ien s; hen, elec omagne ic ields, SAR and empe a u e dis ibu ions a e compu ed by sol ing Maxwell’s and biohea equa ions, espec i ely, unde he app op i- a e bounda y condi ions. Rega ding he i ual models used, a mo e o less ex ensi e col- lec ion is a ailable h oughou he li e a u e—see, o ins ance, e iew [181]. The use o hese pa ien -based models—despi e adding complexi y o he HTP p ocess—has been demons a ed o imp o e he es ima ion o SAR and empe a u e pa e ns compa ed o homogeneous phan om-based models [182]. Many physical models used o simula ing he in e ac ion o he applied elec omagne ic ields wi h issues ha e been p oposed [183– 188], mainly by de i ing om undamen al equa ions, like Maxwell’s equa ions— o cal- cula ing elec omagne ic ields and SAR—and Penne’s biohea equa ion— o empe a u e dis ibu ions. Un o una ely, modeling magne ic hype he mia wi hin he con ex o his body o knowledge s ill p o es di icul mainly due o an impo an challenge: o couple he di e en size scales— om 3D down o 1D, whe e he ele an magne ic and hea ing phenomena s em om—in ol ed in he mul iple physical phenomena con e ging in his he apy, namely luid dynamics, hea exchange, and elec omagne ic in e ac ion. Due o i s inhe en complexi y, his is a longs anding p oblem ha needs o be add essed in he Figu e 6. The mal images o MNPs in e nalized in cells, (a), be o e, and (b), du ing i adia ion wi h an AC magne ic ield; (c), a e age empe a u e shi upon he applica ion o an AC magne ic ield. We can easonably expec ha in a ew yea s, we would dispose o se e al ypes o MNPs inco po a ing local empe a u e p obes, bo h molecula and solid na u e, ha can yield eliable da a on he local empe a u e g adien gene a ed by MNPs exposed o al e na ing magne ic ields ha can be used o he de elopmen o less in asi e, mo e e icien and mo e selec i e ad anced magne ic hype he mia ea men s. Ma e ials 2021,14, 706 21 o 36 8. T ea men Planning and Dosime y T ea men planning—which ep oduces he dose needed o des oy he umo s p e- se ing as much heal hy issue as possible— o many hype he mia modali ies ha e bene i ed om he de elopmen s made in adio he apy [ 175 – 179 ]. In he pa icula case o magne ic hype he mia, so a , he e is only one comme cially a ailable sys em o he pu pose de eloped by MagFo ce, he NanoPlan ® ( o me ly known as Hype Plan ® ) [ 180 ]. The ypical wo k low o a hype he mia ea men planning (HTP) ool s a s wi h a 3D model o he egion o in e es ha is buil om compu e ized omog aphy (CT), o MRI scans om pa ien s; hen, elec omagne ic ields, SAR and empe a u e dis ibu ions a e compu ed by sol ing Maxwell’s and biohea equa ions, espec i ely, unde he app op ia e bounda y condi ions. Rega ding he i ual models used, a mo e o less ex ensi e col- lec ion is a ailable h oughou he li e a u e—see, o ins ance, e iew [ 181 ]. The use o hese pa ien -based models—despi e adding complexi y o he HTP p ocess—has been demons a ed o imp o e he es ima ion o SAR and empe a u e pa e ns compa ed o homogeneous phan om-based models [ 182 ]. Many physical models used o simula ing he in e ac ion o he applied elec omagne ic ields wi h issues ha e been p oposed [ 183 – 188 ], mainly by de i ing om undamen al equa ions, like Maxwell’s equa ions— o calcu- la ing elec omagne ic ields and SAR—and Penne’s biohea equa ion— o empe a u e dis ibu ions. Un o una ely, modeling magne ic hype he mia wi hin he con ex o his body o knowledge s ill p o es di icul mainly due o an impo an challenge: o couple he di e en size scales— om 3D down o 1D, whe e he ele an magne ic and hea ing phenomena s em om—in ol ed in he mul iple physical phenomena con e ging in his he apy, namely luid dynamics, hea exchange, and elec omagne ic in e ac ion. Due o i s inhe en complexi y, his is a longs anding p oblem ha needs o be add essed in he sho - e m o mo e owa ds wide clinical adop ion o magne ic hype he mia. A uni ied magne ic hype he mia heo y should be made possible in he nea u u e, aking ad an age o 3D-1D coupling s a egies based on opological model educ ion [189]. New HTP sys ems based on con olu ional neu al ne wo ks and deep-lea ning ech- niques a e being es ed in a new clinical ial ocused on he ea men o locally ad anced panc ea ic duc al adenoca cinomas [ 2 ], allowing o ob ain a 3D model o each pa ien ins ead o elying on he s anda d ones al eady a ailable o pe o m he simula ions. The main bene i o he HTP is ha all cases can be s udied be o ehand, e en hose ha ma ch wi h any o he exclusion c i e ia, hus e alua ing hei isk and inally uling ou o no hei sui abili y o he ea men . One o he exclusion c i e ia o p ospec i e pa icipan s is ha no implan -bea ing pa ien s a e allowed, which may ep esen a p opo ion as high as 60% o he ini ial coho . The eason o his is ha pa o o ally me allic implan s may unde go no iceable hea ing du ing he he apy upon being exposed o he magne ic ield needed o exci e he MNPs (Figu e 7), e en inducing issue damage i no p ope ly con- olled. Ve y ecen esul s show how he implan hea ing p ocess akes place in di e en indica ions, and di e en ways o eco e a sizeable pe cen age o he ini ially excluded pa ien s ha e been p oposed [190,191]. Fu u e big imp o emen s in ea men planning o magne ic hype he mia a e on hei way, coming om he hand o speci ic eme ging nano-enabled diagnos ic echniques, like he case o magne ic pa icle imaging (MPI). The la e speci ically adds wha ea men planning in magne ic hype he mia is cu en ly lacking, namely he p ecise quan i ica ion and spa ial loca ion o he nanopa icles inside he body depending on he adminis a ion means and he physical p ope ies o he issues in ol ed [ 192 ]. This ea u e allows o a p elimina y quali y check o nanopa icle ins alla ion inside umo s, basing he p edic ions on he numbe o nanopa icles ha ha e ac ually eached he a ge and no he ough es ima ions made om he expec ed sp ead o he injec ed olume. In addi ion, he possibili y o a eal- ime ollow-up o he pos -injec ion a e o nanopa icles is c ucial o co ec he ini ial condi ions o simula ing subsequen ea men sessions. Conside ing ha he de elopmen o MPI scanne s o humans is cu en ly mo ing on [ 193 – 195 ], he Ma e ials 2021,14, 706 22 o 36 i s esul s om inco po a ing i in o he cu en HTP me hodologies may be seen in abou wo yea s. Ma e ials 2021, 14, x FOR PEER REVIEW 22 o 37 sho - e m o mo e owa ds wide clinical adop ion o magne ic hype he mia. A uni ied magne ic hype he mia heo y should be made possible in he nea u u e, aking ad- an age o 3D-1D coupling s a egies based on opological model educ ion [189]. New HTP sys ems based on con olu ional neu al ne wo ks and deep-lea ning ech- niques a e being es ed in a new clinical ial ocused on he ea men o locally ad anced panc ea ic duc al adenoca cinomas [2], allowing o ob ain a 3D model o each pa ien ins ead o elying on he s anda d ones al eady a ailable o pe o m he simula ions. The main bene i o he HTP is ha all cases can be s udied be o ehand, e en hose ha ma ch wi h any o he exclusion c i e ia, hus e alua ing hei isk and inally uling ou o no hei sui abili y o he ea men . One o he exclusion c i e ia o p ospec i e pa icipan s is ha no implan -bea ing pa ien s a e allowed, which may ep esen a p opo ion as high as 60% o he ini ial coho . The eason o his is ha pa o o ally me allic implan s may unde go no iceable hea ing du ing he he apy upon being exposed o he magne ic ield needed o exci e he MNPs (Figu e 7), e en inducing issue damage i no p ope ly con- olled. Ve y ecen esul s show how he implan hea ing p ocess akes place in di e en indica ions, and di e en ways o eco e a sizeable pe cen age o he ini ially excluded pa ien s ha e been p oposed [190,191]. Figu e 7. Calcula ed ield and empe a u e pa e ns in and a ound he hip implan o a p ospec i e pa ien o magne ic hype he mia o ea a p os a e umo . Fu u e big imp o emen s in ea men planning o magne ic hype he mia a e on hei way, coming om he hand o speci ic eme ging nano-enabled diagnos ic ech- niques, like he case o magne ic pa icle imaging (MPI). The la e speci ically adds wha ea men planning in magne ic hype he mia is cu en ly lacking, namely he p ecise quan i ica ion and spa ial loca ion o he nanopa icles inside he body depending on he adminis a ion means and he physical p ope ies o he issues in ol ed [192]. This ea- u e allows o a p elimina y quali y check o nanopa icle ins alla ion inside umo s, bas- ing he p edic ions on he numbe o nanopa icles ha ha e ac ually eached he a ge and no he ough es ima ions made om he expec ed sp ead o he injec ed olume. In addi ion, he possibili y o a eal- ime ollow-up o he pos -injec ion a e o nanopa icles is c ucial o co ec he ini ial condi ions o simula ing subsequen ea men sessions. Conside ing ha he de elopmen o MPI scanne s o humans is cu en ly mo ing on [193–195], he i s esul s om inco po a ing i in o he cu en HTP me hodologies may be seen in abou wo yea s. Figu e 7. Calcula ed ield and empe a u e pa e ns in and a ound he hip implan o a p ospec i e pa ien o magne ic hype he mia o ea a p os a e umo . Ano he signi ican s ep-up in HTP o magne ic hype he mia could be achie ed in he nea u u e by combining he exis ing echnology wi h adiomics. The la e com- p ises compu e -assis ed medical image analysis wi h dedica ed algo i hms o ea u e ex ac ion—much bene i ed om he boom o a i icial in elligence—p o iding spa ial and empo al in o ma ion ha is no a ained by da a om -omics [ 196 , 197 ]. Radiomics mainly nu u es om compu e omog aphy and magne ic esonance imaging da a, bu applying i s p inciples o MPI may b ing an unp eceden ed deg ee o accu acy o HTP in magne ic hype he mia. 9. Fu he E olu ion in o The anos ics: Combining Magne ic Hype he mia and MPI Combining he apy and diagnos ic imaging, o he anos ics has been an ac i e a ea o esea ch ac oss nume ous biomedical ields o e he pas decade. Fo example, he anos ics is an es ablished app oach in nuclea medicine whe eby he apy and diagnos ic imaging a e pe o med using he same molecule o simila molecules. The anos ic agen s enable simul aneous assessmen o clinical s a us, ea men , and con i ma ion o ea men dose. In his sec ion, we discuss a new he anos ic app oach ha combines MPI and localized magne ic hype he mia. Toge he , hese wo echnologies enable mapping o a MNP dis ibu ion, p esc ibing a hea ing dose, and hen ca e ully applying hea pe he p esc ibed hea dose o a local a ea. MPI is an eme ging imaging echnology ha di ec ly quan i a es MNP concen a ion in issue. MPI is ace -based and p oduces posi i e con as images, analogous o nuclea medicine o op ical. The signal is di ec ly de ec ed om he nanopa icle ace s and has he ad an age o being linea ly quan i a i e wi hou issue a enua ion. The physics ha unde lie he signal gene a ion and image o ma ion can be unde s ood using classical physics. An MPI sys em p oduces a s ong magne ic ield g adien con aining a ield- ee egion (FFR)—a egion whe e he magne ic ield is app oxima ely ze o (Figu e 8a). MNPs in he FFR a e magne ically unsa u a ed and p oduce a signal in a ecei e coil, while sa u a ed supe pa amagne ic i on oxide nanopa icles (SPIOs) ou side he FFR p oduce no signal. Images a e p oduced by as e scanning he FFR ac oss he subjec . Fi s published in Ma e ials 2021,14, 706 23 o 36 2005 [ 198 ], he ield has g own apidly, and he e a e now comme cially a ailable p eclinical MPI sys ems [199,200], and clinical-scale MPI sys ems a e unde de elopmen [193,194]. To da e, mos MPI has been pe o med o non-in asi ely image he dis ibu ion and quan i y o MNPs in mu ine models. Tumo s can be de ec ed by he passi e accumula ion o ace s h ough he enhanced pe meabili y and e en ion e ec [ 201 ], by he up ake o phagocy ic umo -associa ed mac ophages [ 202 ], o h ough he a ge ed use o unc- ionalized and a ge ed ace s [ 203 , 204 ]. No el nanopa icles enable new capabili ies o MPI sys ems. Song e al. demons a ed he syn hesis o mul imodal FeCo nanopa icles o imaging wi h nea -in a ed, MPI, MRI, and pho oacous ic echniques and he apeu ic p ope ies wi h pho o he mal and magne o he mal sys ems [205]. As p e iously discussed, magne ic hype he mia o ac i a ion o MNPs o e s consid- e able po en ial o nume ous biomedical applica ions, especially in he clinical ea men o cance s. Magne ic hype he mia elies on he deli e y o MNPs o umo s ollowed by he applica ion o AC ields, causing local hea ing o issue. The killing o umo cells occu s ei he di ec ly o by enhancing he cy o oxic e ec s o adio, immune, o chemo he - apy [ 206 ]. Magne ic hype he mia can be pe o med anywhe e in he body since AC ields pene a e issue wi hou a enua ion. Human clinical ials ha e demons a ed he bene i s o magne ic hype he mia o p os a e cance [ 207 ]; and, o e all su i al bene i s wi h adio he apy in ecu en glioblas oma esul ed in Eu opean egula o y app o al in 2010 [ 208 ]. Despi e i s demons a ed e ec i eness, cu en magne ic hype he mia imple- men a ions a e limi ed by he accumula ion o MNPs away om he lesion o in e es , he inabili y o isualize MNP dis ibu ion du ing ea men , and limi ed abili y o moni o issue empe a u e [ 209 ]. These limi a ions esul in poo MNP hea ing con ol, educed he apeu ic e ec , and inc eased colla e al damage. Recen ad ances ha e demons a ed ha applying he s ong g adien magne ic ield used o imaging in MPI du ing magne ic hype he mia enables localized magne ic hype - he mia, which can help o e come many o he limi a ions aced by adi ional magne ic hype he mia echnologies. Localized magne ic hype he mia allows esea che s o exe spa ial con ol o e which MNPs a e hea ed wi h millime e -scale esolu ion [210–212] . Tay e al. demons a ed he use o a magne ically localized magne ic hype he mia sys em o spa ially localize hea deposi ion o a umo while a oiding hea deposi ion o he heal hy li e [212]. Fu he , he hea dose can be op imized in eal ime by mo ing, expanding, o con ac ing he size o he FFR, changing he hea ing egion acco dingly. Ma e ials 2021, 14, x FOR PEER REVIEW 24 o 37 Recen ad ances ha e demons a ed ha applying he s ong g adien magne ic ield used o imaging in MPI du ing magne ic hype he mia enables localized magne ic hy- pe he mia, which can help o e come many o he limi a ions aced by adi ional mag- ne ic hype he mia echnologies. Localized magne ic hype he mia allows esea che s o exe spa ial con ol o e which MNPs a e hea ed wi h millime e -scale esolu ion [210– 212]. Tay e al. demons a ed he use o a magne ically localized magne ic hype he mia sys em o spa ially localize hea deposi ion o a umo while a oiding hea deposi ion o he heal hy li e [212]. Fu he , he hea dose can be op imized in eal ime by mo ing, expanding, o con ac ing he size o he FFR, changing he hea ing egion acco dingly. Fu he imp o emen s in he accu acy o ea men can be made by combining bo h MPI and localized magne ic hype he mia o a comple e he anos ic wo k low ha com- bines imaging, ea men p esc ip ion, and applica ion o he apy (Figu e 8b). The im- po an linking ac o ac oss he wo echnologies is he a ailabili y o MNPs ha a e bo h isible in MPI and capable o being hea ed ia magne ic hype he mia [209,213]. In addi- ion o nanopa icle biodis ibu ion, he MPI signal con ains in o ma ion on he MNP’s mic oen i onmen and a combined MPI, and magne ic hype he mia can enable non-in- asi ely measu ing empe a u e du ing hea ing [212,214,215]. Accu a e empe a u e measu emen is pa icula ly impo an o p e en o e - and unde - ea men o umo s whe he he issue is being hea ed o apop osis o as an immune-s imula ing adju an he apy. (a) (b) Figu e 8. (a) MPI uses a selec ion ield o localize he nanopa icle signal. The di e ging magne ic ield lines p oduce a unique FFR in he cen e . By a ying he applied magne ic ield, he FFR can be ansla ed ac oss he sample; (b) wo k low o an MPI-di ec ed localized magne ic hype he mia he apy o solid umo s. Following ace adminis a ion, MPI was pe o med o iden i y umo loca ion and size and o iden i y ace up ake in heal hy issue such as he li e . The meas- u ed a ge dose and o - a ge a eas o isk a e hen used o op imize magne ic hype he mia ea men planning. Follow- up scans can be pe o med o assess esponse o he apy o e ime. Rep in ed (adap ed) wi h pe mission om [212]. The combina ion o MPI, magne ic hype he mia and localized magne ic hype he - mia opens new di ec ions o bo h esea ch and clinical ea men . In esea ch, new appli- ca ions a e possible such as a no el, magne ic hype he mia ac ua ed nano he apeu ics capable o he localized elease o d ugs o exp ession o hea -sensi i e gene ic p omo o s. Ul ima ely, as MPI and localized magne ic hype he mia a e clinically ansla ed, we can expec wo k low simila o X- ay/CT guided Radia ion The apy. 10. S anda diza ion o Magne ic Colloids o Magne ic Hype he mia 10.1. Gene al Aspec s The need o s anda diza ion o MNPs o magne ic hype he mia o ms pa o a much b oade demand o eliable, ep oducible, s able and well-cha ac e ized nanos uc- u ed magne ic ma e ials o use in eme ging applica ions in medicine and o he highly demanding sec o s. Howe e , s anda diza ion also includes he p o ision o well-de ined Figu e 8. ( a ) MPI uses a selec ion ield o localize he nanopa icle signal. The di e ging magne ic ield lines p oduce a unique FFR in he cen e . By a ying he applied magne ic ield, he FFR can be ansla ed ac oss he sample; ( b ) wo k low o an MPI-di ec ed localized magne ic hype he mia he apy o solid umo s. Following ace adminis a ion, MPI was pe o med o iden i y umo loca ion and size and o iden i y ace up ake in heal hy issue such as he li e . The measu ed a ge dose and o - a ge a eas o isk a e hen used o op imize magne ic hype he mia ea men planning. Follow-up scans can be pe o med o assess esponse o he apy o e ime. Rep in ed (adap ed) wi h pe mission om [212]. Ma e ials 2021,14, 706 24 o 36 Fu he imp o emen s in he accu acy o ea men can be made by combining bo h MPI and localized magne ic hype he mia o a comple e he anos ic wo k low ha com- bines imaging, ea men p esc ip ion, and applica ion o he apy (Figu e 8b). The impo - an linking ac o ac oss he wo echnologies is he a ailabili y o MNPs ha a e bo h isible in MPI and capable o being hea ed ia magne ic hype he mia [ 209 , 213 ]. In addi ion o nanopa icle biodis ibu ion, he MPI signal con ains in o ma ion on he MNP’s mic oen- i onmen and a combined MPI, and magne ic hype he mia can enable non-in asi ely measu ing empe a u e du ing hea ing [ 212 , 214 , 215 ]. Accu a e empe a u e measu emen is pa icula ly impo an o p e en o e - and unde - ea men o umo s whe he he issue is being hea ed o apop osis o as an immune-s imula ing adju an he apy. The combina ion o MPI, magne ic hype he mia and localized magne ic hype he mia opens new di ec ions o bo h esea ch and clinical ea men . In esea ch, new applica ions a e possible such as a no el, magne ic hype he mia ac ua ed nano he apeu ics capable o he localized elease o d ugs o exp ession o hea -sensi i e gene ic p omo o s. Ul ima ely, as MPI and localized magne ic hype he mia a e clinically ansla ed, we can expec wo k low simila o X- ay/CT guided Radia ion The apy. 10. S anda diza ion o Magne ic Colloids o Magne ic Hype he mia 10.1. Gene al Aspec s The need o s anda diza ion o MNPs o magne ic hype he mia o ms pa o a much b oade demand o eliable, ep oducible, s able and well-cha ac e ized nanos uc- u ed magne ic ma e ials o use in eme ging applica ions in medicine and o he highly demanding sec o s. Howe e , s anda diza ion also includes he p o ision o well-de ined and ep oducible measu emen me hods o he cha ac e iza ion o he ma e ials. These me hods a e equi ed o eliably de e mine magne ic (and s uc u al) pa ame e s o e e - ence ma e ials and o he MNPs employed in magne ic hype he mia. This s anda diza ion need, he ela ed science o MNPs and hei cha ac e iza ion ha e been p e iously explo ed in some de ail [ 216 , 217 ]. In he ollowing subsec ions, he key ools a ailable o aid in eal- izing he s anda diza ion o MNPs o hype he mia he apy will be desc ibed. Combined, he s eps desc ibed he e o m a oadmap o he u u e ealiza ion o a measu emen and s anda diza ion in as uc u e o MNPs o hype he mia he apy. 10.2. Valida ing Me ological T aceabili y a Key Labo a o ies While no a ealis ic goal o be achie ed a e e y measu emen labo a o y in he cha ac e iza ion communi y, i is o g ea impo ance ha he me ological aceabili y o SI uni s o a leas some measu emen labo a o ies is demons a ed o each in ol ed cha ac e iza ion echnique. Na ional me ology ins i u es a e ypical loca ions o his wo k o be unde aken, as hey combine he expe ise, equipmen and esou ces necessa y o unde ake his c i ically impo an wo k. This is he only possible way o uly e i y ha measu emen s a e accu a e, quan i a i e and cohe en , wi h e i ied unce ain ies. Eu opean me ology ins i u es al eady ha e a gene alized amewo k in place o unde - aking his kind o wo k, which has p e iously been applied o o he a eas o indus y wi h g ea success. Hence, a , none o he magne ic measu emen s o MNPs ha e been me ologically alida ed on calib a ed ins umen s, while some p elimina y wo k on o he non-magne ic cha ac e iza ion echniques has al eady been unde aken [ 218 ]. Es ablishing me ological aceabili y o SI uni s o key cha ac e iza ion echniques is necessa y o alida e MNPs o hype he mia he apy. This wo k is o u mos impo ance o achie ing s anda diza ion and dese es he highes p io i y. The ea ly in ol emen o he ele an end-use indus y igu es will ensu e he long- e m success o he alida ion wo k. 10.3. In e labo a o y Ring Compa isons o Ha monize Measu emen s In addi ion o es ablishing me ological aceabili y a indi idual key labo a o ies, de eloping an unde s anding o he o e all le el o measu emen ag eemen , accu acy and epea abili y ac oss he en i e measu emen communi y mus also be achie ed. Ring Ma e ials 2021,14, 706 25 o 36 compa isons (also called in e labo a o y compa isons) a e he p o en ool o unde aking his ype o wo k. By dis ibu ing iden ical sample se s o mul iple labo a o ies and moni o ing he indings, a huge amoun o in o ma ion can be ob ained. By changing a iables such as he use o in-house o cen ally de eloped ope a ing p ocedu es, he ex en o unce ain ies in oduced by measu emen appa a us, measu emen p ocedu es and analysis me hods can be isola ed, unde s ood and mi iga ed. To da e, ing compa isons ha e been pe o med o a ious nanopa icle pa ame- e s ele an o hype he mia cha ac e iza ion, including DLS [ 219 ], s a ic magne iza ion, SLP/ILP and AC suscep ibili y measu emen s. Ala mingly, hese s udies ha e e ealed a s ong need o u he ha moniza ion o achie e in e labo a o y ag eemen . Regula ing compa isons a e he only ealis ic way o moni o p og ess in he ha moniza ion o nanopa icle cha ac e iza ion echniques and e eal he ex en o in e labo a o y a ia ions. 10.4. De elopmen o Re e ence Ma e ials To da e, no e i ied o accep ed e e ence ma e ials exis o any o he p ope ies o MNPs ele an o magne ic hype he mia he apy. Re e ence ma e ial is cha ac e ized by being homogeneous and s able wi h espec o a ce ain ma e ial cha ac e is ic: his can be a physical quan i y like he ini ial magne ic suscep ibili y, bu i can also be a pe o mance cha ac e is ic like colloidal s abili y. Fo e e ence ma e ials wi h de ined physical p ope - ies, i is desi able o ha e a ce i ied measu emen o his p ope y (made wi h calib a ed ins umen s in a manne ha is me ologically aceable o SI uni s), oge he wi h an accu a ely ende ed unce ain y. Ma e ials o which his ype o alida ed measu emen has been conduc ed, and which a e alida ed as being s able, a e called ce i ied e e ence ma e ials (CRM). CRMs a e indispensable o e i ying he accu acy and empo al s abili y o indi idual measu emen equipmen , o achie ing success ul ing compa isons and accu a ely assessing in-house quali y assu ance sys ems. A p esen , la ge manu ac u e s o MNPs use hei own in-house e e ence ma e ials o e i y hei magne ic p ope ies measu emen s. Smalle companies and academic ins i u es ypically ely on o he comme cial MNP p oduc s as quasi- e e ence ma e ials while lacking de ailed knowledge abou he ac ual ba ch- o-ba ch o empo al s abili y o he ma e ial. The de elopmen o alida ed CRMs o magne ic nanopa icle p ope ies will bene i all le els o indus y and esea ch and is i al o he la ge-scale manu ac u ing o medical- g ade MNP ma e ials sui able o magne ic hype he mia he apy. The de elopmen o CRMs o his pu pose, he e o e, dese es o be he ocus o signi ican e o in he coming yea s. 10.5. Calib a ion and Ce i ica ion o Measu emen De ices and Se ices Indus ially accep ed es ing labo a o ies ypically ope a e unde s ic sys ems o quali y con ol and managemen . Guidance o in o m bes p ac ice in de eloping hese sys ems o biomedical p oduc s is a ailable om ISO [ 220 ]. Labo a o ies ha adhe e o hese quali y s anda ds can gain acc edi a ion om na ional bodies ha ope a e unde mu ual ecogni ion ag eemen s p o ided by he In e na ional Labo a o y Acc edi a ion Coope a ion (ILAC) o ensu e ha labo a o ies p oduce esul s ha a e o a known quali y ha is egula ly moni o ed. The le el o s anda diza ion in he MNP manu ac u ing and cha ac e iza ion communi ies is no ye su icien ly ad anced o his s ep o be possible. The equi emen s laid ou in poin s 10.2 o 10.4 mus be ealized i s . 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