Lysozyme crystallization in hydrogel media under ultrasound irradiation

Ul asonics Sonochemis y 88 (2022) 106096 A ailable online 18 July 2022 1350-4177/© 2022 The Au ho s. Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). Lysozyme c ys alliza ion in hyd ogel media unde ul asound i adia ion Ma iia Sa chenko a , c , d , Manuel Hu ado b , e , , g , Modes o T. Lopez-Lopez c , g , Guille mo Rus e , , g , Luis ´ Al a ez de Cien uegos a , g , * , Juan Melcho b , , g , * , Jos´ e A. Ga i a d , * a Uni e sidad de G anada (UGR), Depa amen o de Química O g´ anica, Unidad de Excelencia Química Aplicada a Biomedicina y Medioambien e (UEQ), C. U. Fuen enue a, A da. Se e o Ochoa s/n, E-18071 G anada, Spain b Uni e sidad de G anada (UGR), Depa amen o de Es adís ica e In es igaci´ on Ope a i a, Spain c Uni e sidad de G anada (UGR), Depa amen o de Física Aplicada, C. U. Fuen enue a, A da. Se e o Ochoa s/n, E-18071 G anada, Spain d Labo a o io de Es udios C is alog ´ a icos, Ins i u o Andaluz de Ciencias de la Tie a (Consejo Supe io de In es igaciones Cien í icas-UGR), UEQ, A enida de las Palme as 4, 18100 A milla, G anada, Spain e Depa amen o de Mec´ anica de Es uc u as e Ingenie ía Hid ´ aulica, Ul asonics Lab TEP-959, Uni e sidad de G anada, Spain Unidad de Excelencia Modeling Na u e MNAT, Uni e sidad de G anada, Spain g Ins i u o de In es igaci´ on Biosani a ia Ibs, GRANADA, G anada, Spain ARTICLE INFO Keywo ds: P o ein c ys alliza ion Nuclea ion Ul asound Hyd ogels Lysozyme ABSTRACT Sonoc ys alliza ion implies he applica ion o ul asound adia ion o con ol he nuclea ion and c ys al g ow h depending on he ac ua ion ime and in ensi y. I s applica ion allows o induce nuclea ion a lowe supe sa u- a ions han equi ed unde s anda d condi ions. Al hough ex ended in ino ganic and o ganic c ys alliza ion, i has been sca cely explo ed in p o ein c ys alliza ion. Now, ha indus ial p o ein c ys alliza ion is gaining momen um, he in e es on new ways o con ol p o ein nuclea ion and c ys al g ow h is ad ancing. In his wo k we p esen he de elopmen o a no el ul asound bio eac o o s udy i s in luence on p o ein c ys alliza ion in aga ose gel. Gel media minimize con en ion cu en s and sedimen a ion, a o ing a mo e homogeneous and s able condi ions o s udy he e ec o an ex e nally gene a ed low ene gy ul asonic i adia ion on p o ein c ys alliza ion a oiding o he undesi ed e ec s such as empe a u e inc ease, in oduc ion o su aces which induce nuclea ion, des uc i e ca i a ion phenomena, e c. In-dep h s a is ical analysis o he esul s has shown ha he impac o ul asound in gel media on c ys al size popula ions a e s a is ically signi ican and ep oducible. 1. In oduc ion P o ein c ys alliza ion is an essen ial ool o deciphe he h ee- dimensional s uc u e o a p o ein in i s c ys alline s a e by means o X- ay di ac ion, as well as he ca aly ic cen e o an enzyme, allowing us o unde s and i s mechanism o ac ion [1]. The e o e, p o ein c ys- alliza ion is needed, among o he s, in ields o s uc u al biology and d ug de elopmen and disco e y. Mo eo e , p o ein c ys alliza ion can also be use ul in p o ein pu i ica ion and isola ion being used in bio- pha maceu ical indus ies ou inely [2,3]. Ne e heless, due o he high molecula weigh s and dynamic na u e o p o eins in solu ion; p o ein c ys alliza ion is a complex p ocess ha equi es high supe sa u a ion a ios and usually p oceeds by slow nuclea ion and c ys al g ow h ki- ne ics [4,5]. In his ega d, i is undamen al o de elop no el echniques o p o ocols ha allow a mo e e icien and eliable c ys alliza ion p ocess. P o ein c ys alliza ion in gel media has u ned ou o be an excellen s a egy o ob ain p o ein c ys als o high quali y and size, ideal o X- ay di ac ion, mainly due o he educ ion o con ec ion cu en s, a oiding c ys als sedimen a ion and empe a u e o concen- a ion g adien s [6]. A such, c ys alliza ion in gels is simila o c ys- alliza ion unde educed g a i y condi ion p oducing c ys als o excellen quali y [7]. Mo eo e , gel ibe s ge inco po a ed wi hin he c ys als ans o ming hem in o new composi e ma e ials [8,9]. P o ein c ys alliza ion is also highly a ec ed by ex e nal s imuli, such as elec ic [10–14] and magne ic ields [15–18], ligh [19,20], audible sounds [21], mic owa e [22] and ul asound (US) i adia ion [23–26], ha ing a di ec in luence on nuclea ion [14]. Among hese s a egies, in he las decade, sonoc ys alliza ion, ha is, he applica ion o US in c ys alliza ion p ocess, is ecei ing a g owing in e es since i has shown o: inc ease nuclea ion a e, educe he induc ion ime and * Co esponding au ho s a : Ins i u o de In es igaci´ on Biosani a ia Ibs, GRANADA, G anada, Spain (L.´ A. Cien uegos and J. Melcho ). E-mail add esses: [email p o ec ed] (L. ´ Al a ez de Cien uegos), [email p o ec ed] (J. Melcho ), [email p o ec ed] (J.A. Ga i a). Con en s lis s a ailable a ScienceDi ec Ul asonics Sonochemis y jou nal homepage: www.else ie .com/loca e/ul son h ps://doi.o g/10.1016/j.ul sonch.2022.106096 Recei ed 29 Ap il 2022; Recei ed in e ised o m 4 July 2022; Accep ed 14 July 2022 Ul asonics Sonochemis y 88 (2022) 106096 2 he me as able zone wid h, inc eased c ys al g ow h a e, educed agglome a ion, and imp o e c ys al quali y and size dis ibu ion [27–30]. The e ec o ul asounds on he kine ic o c ys alliza ion has been known om mo e han 80 yea s wi h an ea ly e iew al eady published in 1967 and in which he mul iple possible mechanisms i.e. ca i a ion, agi a ion, cooling e ec o mechanical ib a ion we e hy- po hesized [31]. Besides an ex ended numbe o i s e ec s on he nuclea ion and g ow h o ino ganic/o ganic c ys als and e en a [32], only ecen ly he e ec o e p o ein c ys allogenesis has been epo ed [25,26]. Ne e heless, he applica ion o US in p o ein c ys alliza ion has no been sys ema ically s udied and sonica ion ac o s such as, sou ce, powe , ime, di ec ion and ampli ude o he ul asound wa es, a e s ill o be in es iga ed. Mo eo e , any s udy ca ies ou unde s anda d c ys alliza ion se -ups will be a ec ed by sedimen a ion and con ec ion mass anspo hinde ing he in e p e a ion o he esul s. Only ecen ly, Fe ei a and co-wo ke s ha e explo ed he e ec o US pulse o e lysozyme mic od ople s [25]. In o de o ex end he po en ial use o sonoc ys alliza ion o bigge olumes and o explo e he easi- bili y o his echnique in combina ion wi h o he c ys alliza ion p o- ocols ele an o indus ial c ys alliza ion, we ha e chosen o include aga ose o minimize con ec ion and c ys al sedimen a ion. Aga ose is one o he mos used media o gel a c ys allizing solu ion o ino ganic and o ganic compounds [33], coo dina ion polyme s [34] o p o eins [35], o p oduce new polymo phs [36] o o emula e in i o media in biomine aliza ion s udies [37]. Aga ose gels a e composed o in e connec ed uncha ged linea polysaccha ide chains easily ob ained by cooling he sol below i s gelling empe a u e and classi ied as phys- ical gel since he in e ac ions be ween polysaccha ide chains a e non- co alen . I uses is widely sp ead in biochemis y labs as suppo ing ma e ial o ho izon al elec opho esis. The physical p ope ies o he gel ha e been well cha ac e ized and i is well known ha below he c i ical gel concen a ion o 0.12 % (w/ ) aga ose solu ions beha e like non-New onian luids, while abo e his concen a ion i beha es as a egula iscoelas ic gel [38]. Mo eo e , a a concen a ion as low as 0.04 % (w/ ) aga ose gels a e able o o e come buoyancy and c ys al sedimen a ion [39]. Also, aga ose gels ha e been p e iously used o s udy he in luence o a magne ic ield while a oiding sedimen a ion and he associa e e ec s o con ec i e mass anspo on nuclea ion [18,40]. In he p esen wo k, we ha e s udied he in luence o US in p o ein c ys alliza ion a di e en aga ose concen a ion. To ca y ou his s udy a no el US bio eac o has been speci ically designed o ha e a mo e p ecise con ol o e he wa e pa ame e s o i adia ion. In-dep h mul i a ia e s a is ical analysis ha e shown ha , as al eady published, US induced he nuclea ion o lysozyme o e he induc o e ec o aga ose [41,42]. Ou esul s also shown ha abo e a h eshold con- cen a ion o aga ose he US e ec is de lec ed p obably due o he damping e ec o aga ose ibe s. The esul s ob ained by his se -up a e s a is ically signi ican allowing us o s ablished a clea e ec o he ul asound on he nuclea ion o lysozyme unde s udied condi ions. 2. Ma e ials and me hods 2.1. Reagen s and ma e ials Lysozyme (62971, HEWL, h ee- imes c ys allized powde ) and so- dium ace a e (AcONa 99 %) we e pu chased om Sigma-Ald ich (Mad id, Spain). Lysozyme was dissol ed in 50 mM AcONa, dialyzed (24 h) agains 50 mM AcONa (pH 4.5) in a a io 1:1000 a 4 ◦C and concen a ed by cen i uga ion a 4 ◦C (g =*5000/25 min) using 10-kDa cu o Cen icon concen a o s (Amicon) o ≈150 mg mL −1 de e mined spec opho ome ically a 280 nm using a heo e ical alue o he ex inc ion coe icien o 2.56 mL mg −1 . Then he solu ion was il e ed h ough a 0.45 μ m po e-size il e memb ane sys em (Millipo e). Sodium chlo ide (Sigma-Ald ich, Mad id, Spain) was p epa ed a 20 % (w/ ) in 50 mM AcONa (pH 4.5) and used as s ock solu ion. Solu ions o NaCl a desi ed concen a ion we e p epa ed by dilu ing wi h 50 mM AcONa and il e ed h ough a 0.45 μ m po e-size il e memb ane sys em (Mil- lipo e) p io using i . Aga ose D5 wi h a mel ing poin o 92 ◦C and gelling poin o 37 ◦C was supplied by Hispanaga (Mad id, Spain). Aga ose sols wi h desi able concen a ion we e ob ained by dissol ing aga ose in 50 mM AcONa (pH 4.5) and hea ed a 90 ◦C o ge a homogeneous anspa en solu ion. Then he solu ion was cooled down o 50 ◦C and kep a his empe a u e un il inally mixed wi h he p o ein and p ecipi an solu ion. 2.2. C ys alliza ion expe imen s Ba ch me hod was selec ed o s udy he in luence o ul asonic wa es on lysozyme c ys alliza ion in solu ion (Fig. 1.A). Fo he expe imen s in solu ion (Fig. 1.A1), lysozyme, NaCl and AcONa we e mixed oge he in Fig. 1. The expe imen al scheme: A) P epa a ion o he samples A1: Expe imen s in solu ion, A2: Expe imen s in gels made wi h aga ose; B) US-Bio eac o design and soni ica ion p ocedu e and illus a ion o he se -up; C) Obse a ion o he samples and da a analysis. M. Sa chenko e al. Ul asonics Sonochemis y 88 (2022) 106096 3 one Eppendo ube, homogenized and di ided in h ee aliquo s o 100 µL using mic o spec opho ome e isible-cu e es (B and, GMBH, CO- KG, Ge many) and kep a 20 ◦C. To selec he app op ia e condi ions, we sc eened lysozyme (25–50 mg mL −1 ) and NaCl (3.5–5 % w/ ). We ha e selec ed he inal con- cen a ion o lysozyme 40 mg mL −1 and NaCl 4.3 % (w/ ) o he ex- pe imen s in un-gelled solu ion and 40 mg mL −1 and NaCl 4.0 % (w/ ) o he expe imen in aga ose gels. The e olu ion o he expe imen s was ollowed by s anda d op ical mic oscopy (Nikon AZ100 zoom 2x2x0.6) obse ing he o ma ion o iny c ys als in all he samples (un-gelled solu ion as well as in samples wi h aga ose gels) a e 1 h. The in luence o aga ose (Fig. 1A2) was s udied by a ying he inal amoun o aga ose in he sys em: 0.010 %, 0.025 %, 0.050 %, 0.100 % and 0.200 % (w/ ). We se he uppe limi a 0.200 % (w/ ) o c oss he c i ical concen a ion alue o 0.120 % w/ de ining he ansi ion o he iscoelas ic beha io [43]. Fo each expe imen h ee aliquo s we e p epa ed and di ided as “silen ” o he sample wi hou ul asonic in- luence, “P o ocol US1” o he aliquo immedia ely i adia ed o 30 min long and “P o ocol US2” o he aliquo exposed o he ul asonic in luence 30 min a e p epa a ion and i adia ed o 30 min (Fig. 1B). All he expe imen s we e pe o med h ee old o s a is ical signi icance. Numbe and size o c ys als we e e alua ed a e 24 h by op ical mic oscopy using he Image-Focus-Alpha so wa e o he Nikon AZ100 mic oscope (zoom 2x2x0.6). C ys als numbe we e coun ed manually and size measu ed along he c axes om c ys als clea ly iden i ied. Each image was di ided in 25 equal egions (5 columns ×5 ows) a oiding zones nea cu e es bo de s whe e i was no possible o see he c ys als clea ly, and all he c ys als we e coun ed in all he egions. C ys als size we e analyzed by measu ing a minimum o 100 c ys als o each epli- ca e hus, a minimum o 300 measu emen s we e analyzed o each condi ion. Taking in o accoun all he con ols and di e en expe i- men al condi ions a o al o 5400 measu emen s we e done. 2.3. US-Bio eac o design To a oid a di ec con ac o he US emi e wi h he gel/c ys alliza- ion media ha could cause gel dis up ion o se e as he e ogeneous nuclean , a US-bio eac o was designed, manu ac u ed and p o o yped in he Ul asonics lab a he Uni e si y o G anada. I consis s o a con aine o hold he cu e e, a polyme hylme hac yla e (PMMA) chambe ha was chosen due o i s mechanical and low-densi y p op- e ies and ul asonic ansduce s (Fig. 2). In pa allel, he p opaga ion o he wa es was allowed and maximized wi hou loss o ampli ude because o impedance be ween mediums. The suppo o he ansduce designed in Fig. 2C has he unc ion o holding he ansduce s, o be s able and pe pendicula o he block wi h a speci ic geome y showed in Fig. 2A-B. The decision was ocused a allowing he whole wa e incidence in o he chambe o he samples whe e he cu e e is in eg a ed, o ensu e maximum i adia ion. A p essu e on he block is also equi ed o ha e a di ec ansmission a oiding possible ai bubbles. Mo eo e , he bo om o he cu e e mus coincide wi h he cen al axis o he ansduce o achie e op imum in ensi y due o he maximiza ion o he esolu ion o beam in he cen al axis. The p opaga ion on o he mechanical ul asonic wa es is ansmi ed pe pendicula ly o he su ace o he ansduce in con ac wi h he con aine . The ela ionship be ween wa e p opaga ion, acous ic impedance o he ma e ials, nea ield, he possible empe a u e ise o he ansduce s and mechanical na u e o he sample ha e been also conside ed o he design o he con aine and con igu a ion o he ul asonic ansduce s. The impedance is cha ac e ized as a p oduc o densi y and eloci y o sound o he ma e ial as ollows: Fig. 2. A and B) Shows he la e al and op 1–2 ans e sal sec ion iews, espec i ely. The chambe Type 1 was designed o hold he cu e e and i is loca ed a 3.5 mm in on o he block. The dimensions o his chambe had 15 mm wid h, 12 mm o e ec i e high and 91.4 mm o hickness (app oxima ed olume o 16.4 cm 3 ). The chambe s Type 2 and Type 3 we e in oduced o hold se e al cu e es in he u u e conside ing di e en p essu es changing he main con igu a ion. C) Exhibi s he 3D p in ed suppo o main ain he ul asonic ansduce (100 KHz) in a co ec posi ion o ensu e he alignmen o he p opaga ing wa es. D) 3D schema ic ull con igu a ion o he US-Bio eac o . E) Pic u e o he US-Bio eac o p epa ed o he expe imen al i adia ion o gels. Table 1 Cha ac e iza ion o PMMA, Wa e and PS in e ms o impedance o quan i y he e ec o he ul asonic p opaga ion in he sample. Ma e ial ( ρ ) Densi y Kg/ m 3 (c) Veloci y (m/ s) (Z) Impedance (Pa.s/ m) PMMA 1180 2765 3,263,183 Wa e (27◦) 993 1525 1,515,584 Polys y ene (PS) 612 2340 1,432,080 M. Sa chenko e al. Ul asonics Sonochemis y 88 (2022) 106096 4 Z= ρ •c(1) whe e (Z) is he impedance, ( ρ ) is he densi y and (c) is he eloci y o he wa e h ough ma e ial. The mechanical pa ame e s o he US-bio eac o ma e ials we e cha ac e ized o analyze he p opaga ion h ough he sample, he im- pedances o PMMA, wa e and he cu e e-polys y ene (PS) we e also conside ed as is shown in Table 1 acco ding o Equa ion (1) and (2). Table 1 desc ibes densi ies, eloci ies o sound and impedances o hese ma e ials. The ansmission coe icien a he di e en in e aces is also necessa y o de e mine he p essu e in he sample a e p opaga ion h ough he di e en laye s. I is explici ly o mula ed as: D=4Z1Z2 [Z1+Z2]2(2) whe e (D) is he ansmission coe icien , (Z 1 ) is he impedance o ma- e ial 1 and (Z 2 ) is he impedance o ma e ial 2. The calcula ed alues a e summa ized in Table 2. T ansmi ed p essu e has been also de i ed o he ansi ion be- ween wo media acco ding o equa ion (2) (Fig. 2B). While, he wa e is oscilla ing a 100 kHz o equency om he ul asonic ansduce , i a els h ough he PMMA, a e he wa e and inally he polys y ene un il ge in con ac wi h he gel o solu ion sample. Fo his calcula ion, we ha e app oxima ed he densi y and he eloci y o sound o he gel o solu ion equal o wa e , because hey ha e almos he same alue. Thus, acous ic p essu e in wa e has been quan i ied as 618 Pa, conside ed well below he ca i a ion limi a his equency, and heo e ical de i- a ion o acous ic p essu e inside he gel o solu ion has been ex ac ed acco ding i s ansmission coe icien D, F om wa e o polys y ene → P =618 Pa ⋅ 0,99 =611,82 Pa F om polys y ene o gel o solu ion → P =611,82 Pa ⋅ 0,99 =605,7 Pa Se e al epo s in he li e a u e show ha he esponse o US is igge ed by empe a u e ac ua ion [44,45]. The e o e, o e i y his poin , empe a u e is also measu ed in wo in e als, a 5 min and a 15 min. The empe a u e ac ua ion is conside ed as , so i is no necessa y o measu e i o e a longe pe iod o ime. We ha e used a classical he mome e o con ol his e ec , whose accu acy is 0.01 ◦C. Fo each case, in 100 kHz o equency, he measu emen s on nea es chambe s o ul asonic ansduce a e lowe han <0.01 ◦C, i.e., he hea ing e ec is negligible wi h ou con igu a ion. O he wise, he samples we e placed in he cu e e (Fig. 2D) o enhance he wa e p opaga ion. So ha he chosen dis ance was app oxima ely 8 cm om he ansduce s o a oid he nea ield a ea (in his egion he sound p essu e le els a y conside ably in e ms andom posi ions o ene gy and i is di icul o con ol he sound p essu e ho- mogenei y), he h eshold dis ance is calcula ed acco ding o Equa ion (3) as ollows, N=D2 4c(3) whe e (N) is he nea ield, (D) is he diame e , ( ) is he equency and (c) is he eloci y o sound o he inciden he wa e ha cha ac e ize he media o p opaga ion (Table 3). In his esea ch, aseline was used as coupling gel due o he du a ion p ope ies o his se o expe imen s, he common coupling gels a e d ied in a ew hou s. Addi ionally, con en ional ubbe s bands ha e been added o he US-bio eac o suppo o ix he ansduce o ensu e an op imal p essu e o con ac and no displacemen du ing he i adia ion expe imen s. 2.4. Expe imen al US con igu a ion The ul asonic se -up o he expe imen was designed o analyze he di e ences be ween he e ec on c ys alliza ion o he solu ion and aga ose gels a di e en concen a ions (Fig. 1). In consequence o he limi a ion o he wa e gene a o o 10 V, an ampli ie has been needed o each 180 peak- o-peak V as op imum scale le el o gene a ed he desi ed e ec . The wa e o m used was con igu ed wi h a 5 % du y cycle and 50 ms o bu s pe iod simula ing a con inuous p opaga ion o he wa e. The ampli ied wa e signal was emi ed a 100 kHz o cen al equency acco ding o a compa ible wa eleng h wi h he sample di- mensions [23]. Fo his design equi emen s, con ac ansduce s ha e been selec ed due o hei p ope ies as non- uned de ices. They p o ide a damped b oadband ha minimizes he undesi ed noise. This ype o ansduce s a e usually adequa e in Non-Des uc i e Tes ing (NDT) applica ions and Table 2 T ansmission coe icien s be ween wa e , PMMA and PS. D PMMA Wa e /gel PS PMMA 1 0,86 – Wa e /gel 0,86 1 0,992 PS – 0,9992 1 Table 3 Nea ield calcula ion in e ms o equency, eloci y and diame e o he ansduce . ( ) F equency (kHz) (D) Diame e (cm) (c) Veloci y (m/ s) (N) Nea ield (cm) 100 4,5 1525 3,32 Fig. 3. 3D-CAD illus a ion o he p o o yped o US-Bio eac o wi h a ec ea ion o mul ilaye media p opaga ion h ough aseline, PMMA, wa e , polys y ene and gel sample. M. Sa chenko e al. Ul asonics Sonochemis y 88 (2022) 106096 5 biomedical enginee ing [46]. Then, he eason o choose piezo-elec ic ansduce s is ha hey a e made o a single PZT ce amic elemen . They ypically gene a e a longi udinal monoch oma ic wa e in con ac wi h he sample, he e o e, hey mee he speci ica ions o gene a e a monoch oma ic wa e o be p opaga ed in mul ilaye media (Fig. 3). 2.5. Rheological cha ac e iza ion o hyd ogels Fo he heological cha ac e iza ion we used a Bohlin CS10 con olled s ess heome e , p o ided wi h a measu ing geome y o concen ic cylinde s wi h g oo ed su aces. (a) Kine ics o gela ion We measu ed he gela ion kine ics o he aga ose solu ions used o he c ys alliza ion du ing 1 h ( ha includes he ime applied o bo h P o ocol US1 and P o ocol US2) by subjec ing hem o oscilla o y s ain o 1 Hz o equency and 1 Pa o he s ess. The measu emen s s a ed 10 s a e he mix u e o he componen s ( he delay esul ed om he ime equi ed o lowe he inne cylinde o he measu ing posi ion and o s a he measu emen ). Th ee di e en samples we e measu ed o ensu e s a is ical signi icance o he esul s. The mean alues and s an- da d de ia ions o each magni ude we e p o ided in his wo k. (b) Mechanical p ope ies We cha ac e ized he mechanical p ope ies o 0.100 % and 0.200 % (w/ ) aga ose gels be o e and a e he i adia ion o US (using bo h P o ocol US1 and US2). Fo his aim, we ob ained he s o age (G’) and loss (G’’) moduli o he gels as unc ions o equency ( om 0.1 o 10 Hz) a a cons an s ess o 1 Pa. Th ee di e en samples we e measu ed o ensu e s a is ical signi icance o he esul s. The mean alues and s an- da d de ia ions o each magni ude we e p o ided in his wo k. 2.6. S a is ical analysis To explo e an in e ence abou he di e ences be ween he samples wi h and wi hou US a wo le els o exposi ion, a mul iple eg ession analysis ia ANOVA was conside ed. The i s s ep was o check he p oo o no mali y ia he Kolmogo o -Smi no and Shapi o-Wilk es s. They we e calcula ed o de e mine he no mali y o he size and numbe o obse ed c ys als, espec i ely. In he cases whe e he dis ibu ion o he a iable was non-no mal, K uskal-Wallis es was pe o med o ob ain he simul aneous mean di e ences by expe imen and g oup o ea men o le el (Con ol, US1 and US2). Fu he mo e, hey ha e been co obo a ed wi h Dunn’s es o assess he p- alue signi icance in a- g oup. Fo he pa ame ic dis ibu ed a iables, we ha e applied clas- sical one-way ANOVA s a is ical me hodology o desc ibe he di e - ences o means by g oup o ea men . The p- alues ha e been compa ed wi h he signi icance le el o e alua e he null hypo hesis whe e he e we e no di e ences be ween means o when he null hypo hesis indica es ha he popula ion means a e all equal. A signi icance le el ype I e o o 0,05 was conside ed o be he minimum accep ed le el ha deno es a di e ence be ween means. The no a ion ha we ha e included he ea e is * p <0,05, ** p <0,001 and *** p <0,0001 when he di e ences be ween means a e s a is ically signi ican . Fo he eco d he indi idual analysis o each expe imen in numbe and c ys als size a e included in he supplemen- a y ma e ial Figu es S1 and S2. 3. Resul s and discussion Nuclea ion is a s ochas ic phenomenon ha equi es o o e come an ene gy ba ie . This ene gy ba ie can be educed by inc easing he supe sa u a ion bu , on he o he hand, an excess o supe sa u a ion educes he abili y o con ol he nuclea ion ime and densi y [4]. The e a e di e en ways o o e come he longes induc ion ime imposed by he hickness o he me as able zone. To slim down he me as able zone, nuclea ion has been p omo ed by using di e en su aces ([47] and e e ences he e in), gels [41][42], and ex e nal ields such as ligh i adia ion, elec ic and magne ic ield o US ([14] and e e ences he e in), all o hem p omo ing he nuclea ion and he e o e educing he nuclea ion induc ion ime and inc easing he numbe o c ys als [14]. The ul asonic ac i a ion o me as able solu ions o induce Fig. 4. Row A) Shows lysozyme c ys als ob ained in solu ion unde silen condi ion (Con ol) and ul asonic i adia ion o 30 min immedia ely a e p epa a ion o he expe imen (US1) and 30 min a e p epa a ion (US2). The s a is ical analysis o he numbe (B) and size (C) o he c ys als a e shown o he h ee eplica es. The scale ba in he op ical mic oscopy images is 500 µm in all he pic u es. M. Sa chenko e al. Ul asonics Sonochemis y 88 (2022) 106096 6 nuclea ion is he s anda d applica ion o sononuclea ion echnique o con ol c ys al size dis ibu ion, mo phology o polymo ph selec ion [48,49]. In o de o s udy he in luence o US a a ixed supe sa u a ion wi h an unce ainly me as able zone, he applica ion o US a di e en imes, a e he se ing up o he c ys alliza ion expe imen s, seems o be he simple way. We p oposed a se -up in which US is gene a ed ex e nally o he bulk c ys alliza ion solu ion and which includes a hyd ogel media o a oid c ys als sedimen a ion and con ec ion so ha he US e ec could be un- couple om any mass anspo e ec s. To es he e iciency o ou se - up we ca ied ou a i s se o expe imen s in an un-gelled solu ion. We es ed a ange o supe sa u a ion alues by changing he lysozyme concen a ion, om 25 o 50 mg⋅mL −1 , NaCl concen a ion om 3.5 % o 5.0 % (w/ ) and he i adia ion ime se a 10 s, 10 min and 30 min. F om his ini ial sc eening we de e mined ha using 40 mg⋅mL −1 o lysozyme and 4.3 % (w/ ) o NaCl he nuclea ion induc ion ime mo ed in he ange o he ini ial 60 min and he e o e i ed ou expe imen al equi emen s. We also de e mined ha sho i adia ion pe iods, 10 s o 10 min, a e p epa a ion did no in luence he nuclea ion beha io and he e o e a minimum o 30 min o i adia ion was equi ed which could be loca ed wi hin he 60 min o induc ion ime, i.e. a he beginning (P o ocol US1) and 30 min a e he p epa a ion (P o ocol US2). Numbe and size o c ys als we e e alua ed a e 24 h by op ical mic oscopy using he Image-Focus-Alpha so wa e o he Nikon AZ100 mic oscope (zoom 2×2×0.6). As expec ed om p e ious esul s unde simila condi ions [26], he i adia ed sample in solu ion showed an inc ease o he numbe o c ys als, o smalle sizes, independen ly o he used p o ocol (Fig. 4 and Figu e S3). Wi hou i adia ion, 50 % o measu ed c ys als we e comp ised be ween 150 and 200 µm while nea 70 % o he immedia ely i adia ed c ys als (US1) showed sizes anging 50–100 µm and when i adia ed 30 min a e sample p epa a ion (US2) almos 90 % o he c ys als showed sizes anging 50–100 µm. No ably his no el bio eac o seems o be ideal o exe a con ol o e he nuclea ion p ocess. Mo e- o e , he highe con ol o e he c ys al size obse ed in p o ocol US2 could be explained as ollowing: i) since US2 is applied la e , c ys alline ma e ial is al eady o med; ii) he applica ion o he US o e his al eady o med nuclei may o could disagg ega e he c ys alline ma e ial and iii) he disagg ega ed agmen s ha e now he oppo uni y o g ow gi ing ise o a na owe c ys al size dis ibu ion. To a oid c ys al sedimen a ion, aga ose, a di e en concen a ions, was used as a media. Aga ose is a well-known nuclea ion induc ion media ully cha ac e ized unde egula condi ion bu no unde he in luence o US. The e o e, i s ly, we ully cha ac e ized he in luence o ou selec ed p o ocol o e he gel/gela ion beha io o aga ose. In o de o in es iga e how he ul asound may a ec he kine ic o gel o ma ion and he mechanical p ope ies o he gel we s ick o he concen a ion ange in which aga ose ansi om non-New onian luid (0.100 % w/ ) o a egula gel (0.200 % w/ ) [38]. As expec ed, a aga ose concen a ion below 0.100 %, no gel beha io was ob ained i.e. s o age modulus (G) being always smalle han loss modulus (G′′), e en a e moni o ing o 1 h. Fo 0.100 % and 0.200 % (w/ ) aga ose concen a ion G′was highe han G′′ om he e y beginning o he measu emen s. G′>G′′ is ypical o gel-like samples and he e o e, i can be concluded ha gel poin was eached e y quickly a e he solu ions we e p epa ed. Ne e heless, as Fig. 5. Gela ion kine ic o 0.100 % (A) and 0.200 % (B) solu ions o aga ose. Da ke lines ep esen he mean alues, whe eas he ligh e bands a ound hem ep esen he s anda d de ia ions. The bo om pa showing he iscoelas ic moduli as a unc ion o equency a a cons an s ess o 1 Pa o 0.100 % (C) and 0.200 % (D) aga ose gels be o e and a e applying he p o ocol US1. Da ke lines ep esen he mean alues, whe eas he ligh e bands a ound hem ep esen he s anda d de ia ions. No e ha he nonsymme ic appea ance o s anda d de ia ions is due o he loga i hmic scale. M. Sa chenko e al. Ul asonics Sonochemis y 88 (2022) 106096 7 obse ed in Fig. 5A-B, G′inc eased o e ime, which indica es ha gela ion p oceeded a e he gel poin was eached, and no s eady s a e was eached a e 3500 s o 0.100 %. Fo 0.200 % he ini ial enhancemen o G′was e y ab up , bu a e 2500 s a end owa ds a s eady s a e was obse ed indica ing he gel o ma ion. Fig. 5C-D, shows he iscoelas ic moduli as a unc ion o equency o a cons an shea s ess o 1 Pa a e he gela ion was comple ed. These cu es seem o co obo a e ha bo h samples con aining 0.100 % and 0.200 % o aga ose demons a ed a gel-like beha io , howe e , alues o G′′/G′a e in he ange 0.1–1, mos ly o sample 0.100 %, ypical o weak gels [50]. Al hough a i s i looks like he e is some di e ences on he iscoelas ic moduli be ween i adia ed and non-i adia ed samples, he s anda d de ia ion clea ly o e laps indica ing ha he e a e no signi ican di e ences. Also no e ha in he double loga i hmic scale, G′and G′′ a e weakly dependen on equency, which is ypical o gels. We also in es iga ed i he applica ion o US2 could a ec he gel beha io . As i is illus a ed in he supplemen ma e ial (Figu e S4) we did no obse e any e ec . The e o e, om ou esul s i could be concluded ha bo h samples a e gels, wi h sample 0.100 % aga ose p esen ing weake mechanical p ope ies han 0.200 % aga ose, which a e no a ec ed by he US i adia ion. Sonoc ys allyza ion s udies we e e alua ed a di e en aga ose gel concen a ions om 0.010 %, o 0.200 % (w/ ) by quan i ying he numbe o c ys als and c ys al size 24 h a e he applica ion o he US i adia ion p o ocols (US1 & US2) (Figs. 6 and 7). A he lowes aga ose concen a ion (0.010 % w/ ) esul s showed a signi ican inc ease in he numbe o c ys als and a consequen educ ion o c ys als size inde- penden ly o he applied p o ocol, al hough in US2 c ys als size dis ibu ion was na owe (Fig. 7 and Figu e S5). A aga ose concen- a ion o 0.025 % and 0.050 % (w/ ) he numbe o c ys als is simila bu hey show a s a is ically signi ican smalle sizes (Fig. 7) and na - owe size dis ibu ion (Figu e S5) han in solu ion. A 0.100 % and 0.200 % (w/ ) aga ose concen a ion he e ec in he numbe o c ys als and c ys al sizes a e no signi ican ly di e en om he silen condi ions (Con ol) meaning ha he induc ion e ec o aga ose o e come he po en ial e ec o US. This can be clea ly obse ed in he numbe o c ys als ha a e highe and o smalle size. (Figu e S5 and S6). Taking in o accoun size dis ibu ion (Figu e S5) i was clea ha bo h expe imen al se -ups (US1 and US2) e ol ed simila ly as he aga ose concen a ion inc eased and became almos iden ical o he si- len con ol expe imen s when aga ose concen a ion was 0.100 % (w/ ). 4. Conclusions Sonoc ys alliza ion al hough e y p omising in p o ein c ys alliza- ion has no been s udied in-dep h and con adic o y esul s ha e been epo ed [25]. To shed ligh o e his ield and alida ed p e ious wo ks, we ha e compa ed he e ec o US in lysozyme c ys alliza ion in solu- ion and in gel media, bo h expe imen s ca ied ou unde he same US condi ions. We ha e obse ed an induc ion e ec o an US i adia ion on he nuclea ion o lysozyme when c ys allized in he absence o he p esence o aga ose a a concen a ion below 0.100 % w/ . In bo h media he e ec o ul asound is simila , he induc ion o he nuclea ion gi ing ise o a highe numbe o c ys als o smalle size. Abo e 0.100 % w/ aga ose concen a ion he e ec o US is hinde ed maybe due o he Fig. 6. Lysozyme c ys als ob ained in solu ion unde silen condi ion (Con ol) and ul asonic i adia ion o 30 min immedia ely a e p epa ing he expe imen (US1) and 30 min a e p epa a ion, i adia ed o 30 min (US2). F om le o igh i is shown he esul in solu ion and inc easing concen a ion o aga ose. The scale ba in he op ical mic oscopy images is 500 µm in all he pic u es. M. Sa chenko e al. Ul asonics Sonochemis y 88 (2022) 106096 8 enhancemen o he mechanical p ope ies o he gel. The esul s ob ained in solu ion and in aga ose gels a e s a is ically signi ican and co obo a es he e ec o US in c ys alliza ion. We ha e also demons a ed ha he combina ion o bo h echniques, he use o hyd ogel and US, a e compa ible in p o ein c ys alliza ion. CRediT au ho ship con ibu ion s a emen Ma iia Sa chenko: Fo mal analysis, In es iga ion, Valida ion. Manuel Hu ado: Fo mal analysis, In es iga ion, Valida ion. Modes o T. Lopez-Lopez: Me hodology, Funding acquisi ion, Fo mal analysis, W i ing – o iginal d a . Guille mo Rus: . Luis ´ Al a ez de Cien uegos: Concep ualiza ion, Funding acquisi ion, Me hodology, P ojec admin- is a ion, Supe ision, W i ing – o iginal d a , W i ing – e iew & edi ing. Juan Melcho : Funding acquisi ion, Me hodology, P ojec adminis a ion, Supe ision, W i ing – o iginal d a , W i ing – e iew & edi ing. Jos´ e A. Ga i a: Concep ualiza ion, Funding acquisi ion, Me hodology, P ojec adminis a ion, Supe ision, W i ing – o iginal d a , W i ing – e iew & edi ing. Decla a ion o Compe ing In e es The au ho s decla e ha hey ha e no known compe ing inancial in e es s o pe sonal ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape . Acknowledgemen s This s udy was suppo ed by p ojec PID2020-118498GB-I00, PID2020-116261GB-I00 and PID2020-115372RB-I00 unded by MCIN/AEI/ 10.13039/501100011033/ and PID2019-106947RA-C22 unded by MCIN/AEI/ 10.13039/501100011033/FEDER “Una mane a de hace Eu opa”, Spain, and by FEDER/Jun a de Andalucía-Conseje ía de T ans o maci´ on Econ´ omica, Indus ia, Conocimien o y Uni e sidades (Spain) p ojec s A-FQM-340-UGR20, P18-FR-3533, P18-RT-1653, B- TEP-026-UGR18. We would like o acknowledge Ra a Ma qu´ es who illus a ed he expe imen al p ocedu e in Fig. 1. Appendix A. Supplemen a y da a Supplemen a y da a o his a icle can be ound online a h ps://doi. o g/10.1016/j.ul sonch.2022.106096. Re e ences [1] A. McPhe son, J.A. Ga i a, In oduc ion o p o ein c ys alliza ion, Ac a C ys allog . Sec . F S uc . Biol. 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