Intermatrix synthesis of monometallic and magnetic metal/metal oxidenanoparticles with bactericidal activity on anionic exchange polymers†

In e ma ix syn hesis o monome allic and magne ic me al/me al oxide nanopa icles wi h bac e icidal ac i i y on anionic exchange polyme s{ Amanda Alonso,* a Xa ie Mun˜oz-Be bel, b Nu´ ia Vigue´s, c Rosalı´a Rod ı´guez-Rod ı´guez, d Jo ge Macana´s, e Jo di Mas, b Ma ı´a Mun˜oz a and Dmi i N. Mu a ie a Recei ed 7 h Feb ua y 2012, Accep ed 17 h Ma ch 2012 DOI: 10.1039/c2 a20216 In his communica ion, he syn hesis o nanopa icles on anionic exchange polyme s by he In e ma ix Syn hesis me hod is epo ed. Monome allic (Ag) and co e–shell me al/me al oxide (Ag@Fe 3 O 4 ) nanocomposi es we e syn hesized and cha ac e ized. Thei magne ic and bac e icidal ac i i ies we e e alua ed. In e ma ix Syn hesis (IMS) is a simple and as p o ocol o he syn hesis o nanopa icles (NPs) onpolyme icma icesbasedon he loading o ion me al p ecu so s and hei chemical educ ion gene ally unde so expe imen al condi ions. 1,2 Addi ionally, his me hod is also ad an ageous in e ms o NP dis ibu ion since he di icul y o he ionic p ecu so s o pene a e inside he ma ix a ou s NP- o ma ion on he polyme su ace 3,4 whe e hey a e highly accessible and unc ional. All hese easons make IMS one o he mos p omising ou es o p oduce polyme s abilized me al o me al oxide NPs (PS-MNPs o PS-MONPs), as demons a ed by he numbe o publica ion in ol ing NP syn hesis on polyme ic ma ices by IMS. 5,6 I is no ewo hy ha , e en i amines a e known o s abilize NPs agains agg ega ion wi hou dis u bing hei p ope ies, mos o he wo k epo ed up o now ha e been based on ca ionic exchange polyme s and li le a en ion has been paid o anion exchange polyme s (i.e. amine- ype, qua e na y ammonium- ype). This is basically due o he ac ha in he case o anion exchange s bo h he polyme ic ma ix and ion p ecu so s a e posi i ely cha ged. This limi a ion has been o e come using di e en s a egies. The i s consis ed o changing he sign o he cha ge o ei he he NPs o he polyme ic ma ix. Hence, Yonezawa and Kuni ake 7a and P aha aj e al. 7b modi ied Au-NPs wi h nega i ely cha ged molecules (3-me cap op opiona e o ci a e, espec i ely) o a ou hei immobiliza ion on posi i ely cha ged polyme ic ma ices. Al hough being a good al e na i e, hese app oaches equi ed s ong expe imen al condi ions (high empe a u e, e lux, e c.)and could no be di ec ly used o syn hesize o he MNPs o MONPs. Ano he s a egy was p oposed by Sa ka e al. 8 .In his case, he anionic exchange polyme was ini ially loaded wi h a nega i ely cha ged oxidizing agen (e.g.,NaOCl) ha in si u oxidized Fe 2+ ions o Fe 3+ which, in he p esence o hyd oxyl anions, p ecipi a ed gene a ing hyd a ed e ic oxide NPs. This app oach was limi ed o insoluble me al oxide NPs and i was no possible o ob ain me allic NPs. Con e sely, his communica ion epo s an IMS me hod o he syn hesis o me al and co e–shell me al/me al oxide NPs on posi- i ely cha ged ma ices. Pa icula ly, Ag- and supe pa amagne ic Ag@Fe 3 O 4 -NPs wi h bac e icidal ac i i y we e syn hesized on anionic exchange polyme s o he eagen - ee disin ec ion o wa e . The g anula ed esin A520E 9 (Pu oli e), consis ing o a poly( inyl- benzyl chlo ide) backbone c oss-linked wi h di inylbenzene and con aining qua e na y ammonium unc ional g oups (–NR 3+ ), was used as he polyme ic ma ix. The o al ion exchange capaci y (IEC) o he ma e ial was 1.4 milliequi alen o unc ional g oup pe polyme g am (meq g 21 ). The syn hesis o NPs on his ma e ial was pe o med as ollows. Ini ially, he polyme was p e- ea ed wi h 1.0 M NaCl o 1 h o neu alize he coun e ions o he NR 3+ g oups in he polyme by Cl 2 . A e washing wi h deionized wa e (3 imes) and d ying o 24 h a 80 uC, he polyme pa icles we e sie ed o ob ain a homogeneous bead size close o 500 mm. F om ha poin , he syn hesis p o ocol depended on he NP ype. Fo Ag-NPs, he aw polyme was i s ly loaded wi h 20 mL o he educing agen solu ion (Na 2 S 2 O 4 o NaBH 4 ) o 1h.Fou Na 2 S 2 O 4 concen a ions anging om 0.025 o 0.5 M and a single concen a ion o NaBH 4 (0.5 M) we e used. The ma e ial was hen washed wi h deionized wa e (3 imes) o elimina e he excess o educing agen and loaded wi h 10 mL o AgNO 3 , heionic p ecu so o he Ag-NPs. The AgNO 3 concen a ion depended on he expe imen , a ying om 0.01 o 0.5 M. Du ing his s ep, he Ag + ions we e in si u educed, as illus a ed in eqn (1) (R9= o ganic chain). This esul ed in he o ma ion o he polyme –me al nanocomposi es (NCs) con aining Ag-NPs. (R9–NR 3+ ) 2 (S 2 O 422 ) + 4Ag + + 4OH 2 A (R9–NR 3+ ) 2 (S 2 O 622 ) + 4Ag 0 +2H 2 O(1) a Depa men o Chemis y, Uni e si a Au ono`ma de Ba celona (UAB), Bella e a, Ba celona, Spain. E-mail: [email p o ec ed]a ; Fax: +34-(0)5812379; Tel: +34 935811017 b Cen e Nacional de Mic oelec o`nica (IMB-CNM, CSIC), Bella e a, Ba celona, Spain c Depa men o Gene ics and Mic obiology, UAB, Ba celona, Spain d Depa men o Pha macology, Uni e sidad de Se illa, Se illa, Spain e Depa men o Chemical Enginee ing, Uni e si a Poli e`cnica de Ca alunya (UPC), Te assa, Spain {Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: 10.1039/ c2 a20216 RSC Ad ances Dynamic A icle Links Ci e his: RSC Ad ances, 2012, 2, 4596–4599 www. sc.o g/ad ances COMMUNICATION 4596 |RSC Ad ., 2012, 2, 4596–4599 This jou nal is ßThe Royal Socie y o Chemis y 2012 Published on 28 Ma ch 2012. Downloaded on 12/17/2021 12:28:37 PM. View A icle Online / Jou nal Homepage / Table o Con en s o his issue Finally, samples we e washed (3 imes) wi h deionized wa e and d ied o 24ha 80uC. I is wo hy o no e ha he syn he ic p o ocol o anionic exchange polyme s is ad an ageous when compa ed wi h con en ional IMS me hods o ca ionic exchange ma e ials. The main ad an age is ha , in his case, he loading and he educ ion o he ionic p ecu so akes place simul aneously, hus a oiding me al leaking du ing he syn he ic p ocess. This is especially ele an o expensi e me als. On he o he hand, he syn hesis o Ag@Fe 3 O 4 -NPs equi ed he combina ion o a co-p ecipi a ion me hod, commonly used o e i e NPs p epa a ion, 10,11 wi h he IMS me hod, as desc ibed below. Ini ially, he aw ma e ial was p e- ea ed wi h 1.0 M isodium ci a e a 70 uC o 1 h o exchange he Cl 2 ions (eqn (2), whe e ci = ci a e). 3(R9–NR 3+ )(Cl 2 ) + (Na + ) 3 (ci 32 )A (R9–NR 3+ ) 3 (ci 32 ) + 3NaCl (2) A e washing wi h deionized wa e (3 imes), 0.2 g o polyme we e incuba ed in 100 mL o a solu ion con aining 26 mM FeCl 2 and 41 mM FeCl 3 (Fe 2+ :Fe 3+ mola a io = 1 : 2) o 1 h a 80 uC, wi h con inuous s i ing and unde A a mosphe e (eqn (3)). 4(R9–NR 3+ ) 3 (ci 32 )+Fe 2+ + 2Fe 3+ A (R9–NR 3+ ) 4 (ci 32 ) 4 (Fe 2+ , 2Fe 3+ )+8R9–NR 3+ (3) Nex , 125 mL o 0.5 M NaOH we e slowly added wi h con inuous s i ing in o he suspension con aining i on sal s and he polyme ic ma e ial ( inal pH = 9). The suspension was incuba ed o 1 h a 80 uC. Du ing he incuba ion, he polyme became black/b own in colou due o he o ma ion o magne i e NPs by he ollowing eac ion: (R9–NR 3+ ) 4 (ci 32 ) 4 (Fe 2+ , 2Fe 3+ ) + 8NaOH A 4(R9–NR 3+ )(ci 32 )(Na + ) 2 +Fe 3 O 4 +4H 2 O(4) The polyme beads con aining magne ic NPs we e washed wi h deionized wa e (4–5 imes), collec ed wi h a magne and d ied o 24 h a 80 uC. The d ied polyme ic ma e ial con aining Fe 3 O 4 -NPs was hen loaded wi h Ag + ions by incuba ion in 10 mL o 0.1 M AgNO 3 o 1 h a oom empe a u e. The polyme ic ma e ial was washed wi h deionized wa e (3 imes) and he immobilized Ag + ions we e educed by incuba ion in 10 mL o 0.5 M NaBH 4 (1 h, oom empe a u e) o ob ain Ag@Fe 3 O 4 -NPs. The inal polyme ic ma e ial was collec ed wi h a magne and d ied o 24 h a 80 uC. The me al con en (Table 1) on Ag and Ag@Fe 3 O 4 -NC, espec i ely, was de e mined by Induc i ely Coupled A omic Emission Spec ome y (ICP-AES) o ICP Mass Spec oscopy (ICP-MS) as de ailed in he ESI{(S.I.1). In he case o he Ag-NC, he Ag con en ( om he NPs) was ound o be ex emely dependen on he educing agen . Samples p epa ed wi h NaBH 4 showed a lowe me al con en han hose p epa ed wi h he same concen a ion o Na 2 S 2 O 4 (Table 1). This ac was also con i med by Scanning Elec on Mic oscopy (SEM). Ag-NC c oss-sec ions we e p epa ed o SEM imaging as de ailed in S.I.2{. In he images, he Ag-NPs appea ed as a mo e o less in ense b igh a ea, depending on he concen a ion o NPs. Hence, samples p epa ed using NaBH 4 (Fig. 1A) showed lowe in ensi ies (and a lowe NPs concen a ion) han hose p epa ed wi h Na 2 S 2 O 4 (Fig. 1C), e en when using a lowe educing agen concen a ion (Fig. 1B). Al hough he easons o his a e s ill con o e sial, he Donnan Exclusion E ec 12 was iden i ied o play a ele an ole in he loading o he educing agen . In his sense, molecules wi h a highe cha ge (e.g.,S 2 O 422 ) would be easily loaded on he polyme ic s uc u e, hus a ou ing he o ma ion o NPs. Besides, he me al con en was also ound o be e y sensi i e o he educing agen concen a ion. The Ag con en inc eased when inc easing he educing agen concen a ion un il 0.25 M Na 2 S 2 O 4 , when i eached a pla eau (Table 1 and Fig. 2A). This ac was con i med by SEM imaging (Fig. 1B and 1C). On he con a y, he me al con en in he sample did no signi ican ly change wi h he ionic p ecu so concen a ion (Table 1 and Fig. 2B). This sugges ed ha he mos limi ing s ep in he syn he ic p o ocol was he ini ial loading o he polyme ic ma ix wi h he educing agen . Fo he Ag@Fe 3 O 4 -NC, he Ag con en was highe han ha ob ained in he Ag-NCs, indica ing ha he magne i e co e did no a ec ei he he loading o he deposi ion o Ag. Mo eo e , Ag and Fe we e ound o co-localize in he Ag@Fe 3 O 4 -NC ma ix (Fig. 1D). The pa icles dis ibu ion in he NC was analyzed by SEM and T ansmission Elec on Mic oscopy (TEM), as de ailed in S.I.2{. SEM images showed ha , in all cases, he spo s co esponding o NPs we e mainly ound concen a ed on he polyme su ace (Fig. 1D). In ac , TEM images demons a ed ha he densi y o NP dec eased when mo ing om he polyme su ace o he cen e (Fig. 3A and 3B). This non-homogeneous dis ibu ion o he NPs may be a ibu ed o he Donnan Exclusion E ec . Tha is, he posi i e cha ge o he Table 1 Me al con en in Ag- and Ag@Fe 3 O 4 -NC pe g am o NC and pe meq o unc ional g oup (–NR 3+ ) mg M /g NC mmol/meq NaBH 4 /M Na 2 S 2 O 4 /M AgNO 3 /M Ag Fe Ag Fe 0.50 — 0.10 16.1 — 0.11 — — 0.50 0.10 86.2 — 0.57 — — 0.25 0.10 78.0 — 0.52 — — 0.10 0.10 18.4 — 0.12 — — 0.025 0.10 10.00 — 0.07 — — 0.10 0.50 23.2 — 0.15 — — 0.10 0.25 21.3 — 0.14 — — 0.10 0.10 18.4 — 0.12 — — 0.10 0.01 18.2 0.12 0.50 — 0.10 260 83 1.7 1.1 Fig. 1 SEM images o c oss-sec ions o Ag-NC samples p epa ed using (A) 0.5 M NaBH 4 ,(B)0.1MNa 2 S 2 O 4 and (C) 0.5 M Na 2 S 2 O 4 .(D)SEMimage o he c oss-sec ion o Ag@Fe 3 O 4 -NC. The line scans (by EDS) show he dis ibu ion o he me al ions ac oss he pa icles’ diame e . Blue = Ag, ed = Fe and g een = O. This jou nal is ßThe Royal Socie y o Chemis y 2012 RSC Ad ., 2012, 2, 4596–4599 |4597 Published on 28 Ma ch 2012. Downloaded on 12/17/2021 12:28:37 PM. View A icle Online ma e ial impeded a deep di usion o he posi i ely cha ged ionic me al p ecu so s in o he polyme ic ma ix. This ac was e en mo e accen ua ed in he case o Ag@Fe 3 O 4 -NPs (Fig. 1), whe e NPs we e concen a ed in a e y hin laye on he polyme bead su ace. Thus, e en in he p esence o ci a e o compensa e he cha ge o he polyme , he pene a ion o Fe 2+ and Fe 3+ ions was impeded. TEMimageswe ealsoused ode e mine heNP’ssize.A e he analysis o mo e han 100 isola ed Ag-NPs, an a e age size o 14.4 ¡ 0.3 nm was ob ained (Fig. 3D). The de e mina ion o he pa icles size was made easie han when ca ionic exchange ib es we e used 4 due o helowdeg eeo agg ega iono heNPs(Fig.3C).Thismay be due o he p esence o amino g oups in he polyme ic ma ix since hey a e well known o s abilize NPs agains agg ega ion, wi hou modi ying hei p ope ies. 14 The magne ic p ope ies o he Ag@Fe 3 O 4 -NCs we e de e mined wi h a Supe conduc ing Quan um In e e ence De ice (SQUID) and compa ed wi h hose ob ained by polyme ic s uc u es only con aining Fe 3 O 4 -NPs (see S.I.3{). Simila magne ic hys e esis cu es and sa u a ion alues we e ob ained when compa ing bo h NCs, sugges ing ha he p esence o Ag did no a ec he magne ic p ope ies o he ma e ial (Fig. 4). This was especially ele an when conside ing he inal applica ion o he NCs o eagen - ee wa e pu i ica ion. In pa icula , Ag-NPs ha e been ound much mo e oxic han bulk Ag me al, 13 limi ing hei applica ion o eal li e en i onmen s. Thus, he possibili y o collec ing Ag@Fe 3 O 4 -NPs acciden ally eleased om he polyme ic ma ix wi h a simple magne ic ap would be ex emely desi able o wa e pu i ica ion. Mo eo e , he low le el o oxici y (p obed in S.I.4{) o he supe pa amagne ic e ic oxides made hem e y con enien o biological applica ions. The capaci y o he NCs o inhibi bac e ial p oli e a ion was e alua ed by using he Minimum Inhibi o y Concen a ion (MIC) es (as de ailed in S.I.5.1{) and con inuous low analysis. The MIC o bo h Ag- and Ag@Fe 3 O 4 -NCs was de e mined and compa ed wi h ha ob ained by he aw ma e ial wi hou NPs o con aining Fe 3 O 4 -NPs. The esul s a e plo ed in Fig. 5A. Bo h Ag- and Ag@Fe 3 O 4 -NCs showed high inhibi o y ac i i ies wi h a deep dec ease in he abso bance magni ude a 550 nm (Abs 550 ) when inc easing he numbe o NC beads in he suspension. In con as , he aw ma e ial and he Fe 3 O 4 -NC did no p esen signi ican inhibi o y ac i i y a his concen a ion ange, wi h a cons an Abs 550 alue a ound 0.4 A.U. in all cases. This esul indica ed ha Ag-NPs we e esponsible o he inhibi ion o bac e ia p oli e a ion and i was no a ec ed by he p esence o i on oxides. In he con inuous low analysis, he Ag-NC pe o mance was e alua ed wi h ime o e 20 days o con inuous ope a ion, as desc ibed in S.I.5.2{. The aw polyme was used as a con ol. Acco ding o Fig. 3B, he Ag-NC showed high bac e icidal ac i i y (be ween 80–90% killing e iciency) o a leas 15 days bu , a e ha , i quickly dec eased. Se e al mechanisms may be in ol ed in he dec ease o bac e icidal ac i i y wi h ime. The Ag elease om he NC ma ix and he o ma ion o bac e ial bio ilms we e iden i ied as he mos plausible causes. In his sense, he amoun o Ag eleased om he NC ma ix (de e mined by ICP-AES) eached 25% o he o al immobilized a e 2 weeks o con inuous ope a ion. Addi ionally, bac e ial o ma ions we e also ound a ached o he NCs (see Fig. S.I.5.3{). Thus, bo h me al leaking and bio ilm o ma ionwe e ound ocon ibu ein he bac e icidal ac i i y dec ease. Hence, he ma e ial demons a ed good pe o mance o qui e long ope a ion pe iods (i.e. simila o wa e pu i ica ion ja s). In his wo k, a p o ocol o he syn hesis o monome allic and me al/me al oxide NPs on anionic exchange polyme s was p esen ed. Ag and Ag@Fe 3 O 4 -NPs we e syn hesized on g anula ed esins by Fig. 2 Rep esen a ion o he a ia ion o he Ag con en wi h he concen a ion o (A) he educing agen (Na 2 S 2 O 4 ) and (B) he me al ionic p ecu so (AgNO 3 ) du ing he loading s ep. Fig. 3 (A) TEM image o he ex e nal pa o Ag-A520E-NC samples and (B) he co esponding mask (NP = whi e spo s) ob ained using Image J so wa e. (C) Magni ied TEM image o Ag-NPs in he anionic ma ix. (D) NPs size his og am o Ag-A520E-NC samples. Fig. 4 SQUID hys e ic cu e o (#)Fe 3 O 4 -NC and (&)Ag@Fe 3 O 4 -NC. 4598 |RSC Ad ., 2012, 2, 4596–4599 This jou nal is ßThe Royal Socie y o Chemis y 2012 Published on 28 Ma ch 2012. Downloaded on 12/17/2021 12:28:37 PM. View A icle Online ollowing a p o ocol based on he IMS me hod. In bo h cases, NPs we e mainly ound on he polyme su ace a ou ing hei con ac wi h bac e ia. In ac , bo h NCs p esen ed excellen bac e icidal ac i i y, which combined wi h he magne ic p ope ies o e i e, made hem excellen candida es o eagen - ee wa e pu i ica ion. Acknowledgemen s This wo k was suppo ed by Resea ch G an MAT2006-03745, CSD2006-00044 TRAGUA (CONSOLIDER-INGENIO2010) and CTQ2009-14390-C02-02 om he Minis y o Science and Technology o Spain and by ACC1O ´ o VALTEC 09-02-0057 G an wi hin FEDER P og am. A. Alonso and X. Mun˜oz- Be bel espec i ely acknowledge he FI g an (AGAUR) and he Spanish Minis y o Science and Educa ion o he awa d o a Ramo´n y Cajal con ac . Re e ences 1 P. Wal e , E. Welcomme, P. 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Rana, G een Chem., 2009, 11, 1781–1786. 11 S. Lau en , D. Fo ge, M. Po , A. Roch, C. Robic, L. Vande and R. N. Mulle , Chem. Re ., 2008, 108(6), 2064. 12 V. Va anpou , S. S. Madaeni, R. Mo adian, S. Zinadini and B. As inchap, J. Memb . Sci.,2011,375, 284–284. 13 N. R. Panyala, E. M. Pen˜a-Me´ndez and J. Ha el, J. Appl. Biomed., 2008, 6, 117. 14 K. S. Soppima ha, T. M. Aminabha ia, A. R. Kulka ni and W. E. Rudzinski, J. Con olled Release, 2001, 70(1–2), 1–2. Fig. 5 (A) Rep esen a ion o he a ia ion o he abso bance a 550 nm (indica i e o bac e ial p oli e a ion) wi h he numbe o polyme beads o (&) he aw ma e ial, (e)Fe 3 O 4 -NC, (#)Ag-NCand(m)Ag@Fe 3 O 4 -NC (n= 3). (B) Rep esen a ion o he numbe o iable cells wi h he ea men ime o (&) he aw ma e ial and (#)Ag-NC. This jou nal is ßThe Royal Socie y o Chemis y 2012 RSC Ad ., 2012, 2, 4596–4599 |4599 Published on 28 Ma ch 2012. Downloaded on 12/17/2021 12:28:37 PM. View A icle Online