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Ngaba ii-nanoparticles ezincinci zihlala zingcono?Qonda imiphumo yebhayoloji yokuhlanganiswa kobungakanani obuxhomekeke kwi-nanoparticles yesilivere phantsi kweemeko ezifanelekileyo zebhayoloji
Ababhali: Bélteky P, Rónavári A, Zakupszky D, Boka E, Igaz N, Szerencsés B, Pfeiffer I, Vágvölgyi C, Kiricsi M, Kónya Z
Péter Bélteky,1,* Andrea Rónavári,1,* Dalma Zakupszky,1 Eszter Boka,1 Nóra Igaz,2 Bettina Szerencsés,3 Ilona Pfeiffer,3 Csaba Vágvölgyi,3 Mónika Kiricsi we-Environmental Chemistry, Hungary, Hungary , IYunivesithi yaseSzeged;I-2 iSebe le-Biochemistry kunye ne-Molecular Biology, i-Faculty of Science and Information, iYunivesithi yaseSzeged, eHungary;Isebe le-3 leMicrobiology, iFaculty of Science and Information, iYunivesithi yaseSzeged, eHungary;I-4MTA-SZTE Reaction Kinetics kunye ne-Surface Chemistry Research Group, Szeged, Hungary* Aba babhali banikele ngokulinganayo kulo msebenzi.Unxibelelwano: Zoltán Kónya iSebe le-Applied kunye ne-Environmental Chemistry, iFaculty of Science and Informatics, University of Szeged, Rerrich Square 1, Szeged, H-6720, Hungary Phone +36 62 544620 I-imeyile [ukhuseleko lwe-imeyile] Injongo: Iinanoparticles zeSilivere (AgNPs) zikho enye yezona zinto ziqhele ukufundwa ngama-nanomaterials, ngakumbi ngenxa yezicelo zabo zebhayoloji.Nangona kunjalo, ngenxa yokudityaniswa kwe-nanoparticles, i-cytotoxicity egqwesileyo kunye nomsebenzi we-antibacterial uhlala uphazamiseka kwimidiya yebhayoloji.Kulo msebenzi, ukuziphatha kokuhlanganiswa kunye nemisebenzi enxulumene nebhayoloji yeesampuli ezintathu ze-citrate-terinated zesilivere ze-nanoparticle ezinomlinganiselo ophakathi kwe-10, 20, kunye ne-50 nm zafundwa.Indlela: Sebenzisa i-electron microscope yokudlulisa ukudibanisa kunye nokubonakalisa iimpawu ze-nanoparticles, ukuvavanya ukuziphatha kwazo kokuhlanganiswa kumaxabiso ahlukeneyo e-pH, i-NaCl, i-glucose kunye nogxininiso lwe-glutamine ngokusasazwa kokukhanya okuguquguqukayo kunye ne-ultraviolet-kubonakala kwe-spectroscopy.Ukongeza, kumacandelo aphakathi kwenkcubeko yeseli efana ne-Dulbecco iphucula indlela yokuziphatha yokudibanisa kwi-Eagle Medium kunye ne-Fetal Calf Serum.Iziphumo: Iziphumo zibonisa ukuba i-pH ene-asidi kunye nomxholo we-electrolyte we-physiological ngokubanzi ibangela ukuhlanganiswa kwe-micron-scale, enokuthi ifakwe ngokusekwa kwe-biomolecular corona.Kuyafaneleka ukuba uqaphele ukuba amaqhekeza amakhulu abonisa ukuchasana okuphezulu kwiimpembelelo zangaphandle kunezincinci zabo ezincinci.I-In vitro cytotoxicity kunye novavanyo lwe-antibacterial lwenziwa ngokuphatha iiseli kunye ne-nanoparticle aggregates kwizigaba ezahlukeneyo zokuhlanganisa.Isiphelo: Iziphumo zethu zibonakalisa ulungelelwaniso olunzulu phakathi kozinzo lwe-colloidal kunye nobutyhefu be-AgNP, njengoko ukuhlanganiswa okugqithisileyo kukhokelela ekulahlekelweni ngokupheleleyo komsebenzi webhayoloji.Iqondo eliphezulu lokuchasana nokudityaniswa okujongwe kumasuntswana amakhulu kunempembelelo enkulu kwityhefu ye-vitro, kuba iisampulu ezinjalo zigcina umsebenzi weseli we-antimicrobial kunye ne-mammalian.Ezi ziphumo zikhokelela kwisigqibo sokuba, nangona uluvo oluqhelekileyo kwiincwadi ezifanelekileyo, ukujolisa kwi-nanoparticles encinci kunokwenzeka ukuba ingabi yinkqubo engcono kakhulu.Amagama angundoqo: Ukukhula okulungiselelwe yimbewu, uzinzo lwe-colloidal, ukuziphatha okudityanisiweyo okuxhomekeke kubungakanani, ukudityaniswa komonakalo wetyhefu
Njengoko imfuno kunye nemveliso ye-nanomaterials iqhubeka ikhula, ingqwalasela engakumbi ihlawulwa kwi-biosafety yabo okanye umsebenzi we-biological.ISilver nanoparticles (AgNPs) yenye yezona zinto ziqhele ukwenziwa, ziphandwe kwaye zisetyenziswe abameli bolu didi lwemathiriyeli ngenxa yeempawu zabo ezibalaseleyo ze-catalytic, optical and biological properties.1 Ngokuqhelekileyo kukholelwa ukuba iimpawu ezizodwa ze-nanomaterials (kubandakanywa ne-AgNPs) zibangelwa ubukhulu becala kwindawo yazo enkulu.Ngoko ke, ingxaki engenakuphepheka nayiphi na inkqubo echaphazela le nkalo ephambili, njengobungakanani be-particle size, i-surface coating okanye i-aggregation, nokuba iya kulimaza kakhulu iipropati ze-nanoparticles ezibaluleke kakhulu kwizicelo ezithile.
Imiphumo yesayizi ye-particle size kunye ne-stabilizers yizifundo eziye zabhalwa ngokufanelekileyo kwiincwadi.Ngokomzekelo, imbono eyamkelekileyo ngokubanzi kukuba i-nanoparticles encinci inetyhefu kune-nanoparticles enkulu.I-2 Ngokuhambelana noncwadi oluqhelekileyo, izifundo zethu zangaphambili zibonise umsebenzi oxhomekeke kubukhulu be-nanosilver kwiiseli ze-mammalian kunye ne-microorganisms.I-3– 5 yokugquma ubuso yenye impawu enempembelelo ebanzi kwiipropati ze-nanomaterials.Ngokungongeza okanye ukuguqula izinzisi kumphezulu wayo, i-nanomaterial efanayo inokuba neempawu ezahlukeneyo zomzimba, iikhemikhali kunye nebhayoloji.Ukusetyenziswa kwee-agent ze-capping ngokuqhelekileyo kwenziwa njengenxalenye ye-nanoparticle synthesis.Ngokomzekelo, i-citrate-terinated silver nanoparticles yenye yezona zinto zibalulekileyo ze-AgNP kuphando, ezidityaniswe ngokunciphisa iityuwa zesilivere kwisisombululo esikhethiweyo sokuzinzisa njengendlela yokuphendula.I-6 I-Citrate inokuthatha lula iindleko zayo eziphantsi, ukufumaneka, ukuhambelana kwe-biocompatibility, kunye nobudlelwane obuqinileyo besilivere, enokubonakaliswa kwiintsebenziswano ezahlukeneyo ezicetywayo, ukusuka kwi-adsorption ye-reversible surface ukuya kwi-ionic interactions.Iimolekyuli ezincinci kunye ne-polyatomic ion kufuphi ne-7,8, njenge-citrate, ii-polymers, i-polyelectrolytes, kunye nee-agent ze-biological nazo zisetyenziswa ngokuqhelekileyo ukuzinzisa i-nano-silver kunye nokwenza imisebenzi ekhethekileyo kuyo.9-12
Nangona kunokwenzeka ukuguqula umsebenzi we-nanoparticles nge-colloidal surface capping yindawo enomdla kakhulu, indima ephambili yale ngqungquthela yomhlaba ayinanto, inikezela ukuzinza kwe-colloidal kwinkqubo ye-nanoparticle.Indawo enkulu yendawo ethile ye-nanomatadium iya kuvelisa amandla amakhulu omphezulu, nto leyo ethintela amandla e-thermodynamic enkqubo ukufikelela kumandla aphantsi.13 Ngaphandle kozinzo olufanelekileyo, oku kunokukhokelela kwi-agglomeration ye-nanomaterials.Ukuhlanganiswa kukubunjwa kweengqungquthela zeemilo ezahlukeneyo kunye nobukhulu okwenzekayo xa amasuntswana ahlakaziweyo adibana kunye nokusebenzisana kwangoku kwe-thermodynamic kuvumela amaqhekeza ukuba abambelele omnye komnye.Ngoko ke, i-stabilizers isetyenziselwa ukuthintela ukuhlanganisana ngokuzisa amandla amakhulu okwaneleyo okuhlambalaza phakathi kwamaqhekeza ukuchasana nomtsalane wabo we-thermodynamic.14
Nangona umxholo we-particle size kunye ne-surface coverage ihlolwe ngokucokisekileyo kumxholo wokulawulwa kwayo kwemisebenzi yezinto eziphilayo ezibangelwa yi-nanoparticles, i-particle aggregation yindawo engahoywayo kakhulu.Kukho phantse akukho sifundo esipheleleyo sokusombulula ukuzinza kwe-colloidal ye-nanoparticles phantsi kweemeko ezifanelekileyo ze-biologically.I-10,15-17 Ukongeza, eli galelo linqabile ngokukodwa, apho ubuthi obunxulumene nokuhlanganiswa kuye kwafundwa kwakhona, nokuba kunokubangela ukusabela okubi, njenge-vascular thrombosis, okanye ukulahlekelwa kweempawu ezifunwayo, ezifana netyhefu, njengoko iboniswe kuMfanekiso 1.18, 19 ebonisiweyo.Enyanisweni, enye yeendlela ezimbalwa ezaziwayo zokuxhathisa i-nanoparticle yesilivere inxulumene nokuhlanganiswa, kuba i-E. coli ethile kunye ne-Pseudomonas aeruginosa strains zixelwe ukuba zinciphise uvakalelo lwabo lwe-nano-silver ngokuvakalisa i-protein flagellin, i-flagellin.Inobudlelwane obuphezulu besilivere, ngaloo ndlela ikhuthaza ukuhlanganiswa.20
Kukho iindlela ezininzi ezahlukeneyo ezinxulumene netyhefu ye-nanoparticles yesilivere, kwaye ukuhlanganiswa kuchaphazela zonke ezi ndlela.Eyona ndlela kuxoxwe ngayo ye-AgNP yebhayoloji, ngamanye amaxesha ibizwa ngokuba yi-“Trojan Horse” indlela, ithatha ii-AgNP njengabathwali be-Ag+.I-1,21 Indlela yehashe yeTrojan inokuqinisekisa ukwanda okukhulu kwi-concentration ye-Ag + yendawo, ekhokelela ekuveliseni i-ROS kunye ne-membrane depolarization.I-22-24 Ukuhlanganiswa kunokuchaphazela ukukhululwa kwe-Ag +, ngaloo ndlela kuchaphazela ubuthi, kuba kunciphisa indawo esebenzayo esebenzayo apho i-ion zesilivere zinokuthi zixutywe kwaye zinyibilike.Nangona kunjalo, ii-AgNP aziyi kubonakalisa ubutyhefu kuphela ngokukhululwa kwe-ion.Ubukhulu obuninzi kunye nobudlelwane obunxulumene ne-morphology kufuneka buthathelwe ingqalelo.Phakathi kwabo, ubukhulu kunye nokuma kwendawo ye-nanoparticle yimpawu ezichazayo.4,25 Ukuqokelelwa kwezi ndlela zingahlelwa "njengeendlela zetyhefu ezibangelwayo."Kukho izinto ezininzi ezinokuthi zenzeke kwimitochondrial kunye ne-surface membrane enokuthi yonakalise i-organelles kwaye ibangele ukufa kweeseli.I-25-27 Ekubeni ukubunjwa kwee-aggregates ngokwemvelo kuchaphazela ubungakanani kunye nokumila kwezinto ezinesilivere eziqatshelwe yinkqubo ephilayo, oku kusebenzisana kunokuchaphazeleka.
Kwiphepha lethu langaphambili malunga nokudityaniswa kwe-nanoparticles yesilivere, sibonise inkqubo yokuhlola esebenzayo ebandakanya iikhemikhali kunye novavanyo lwebhayoloji ye-vitro yokufunda le ngxaki.I-19 i-Dynamic Light Scattering (i-DLS) yindlela ekhethwayo yolu hlobo lokuhlola ngenxa yokuba izinto eziphathekayo zinokusasaza iifotoni kwi-wavelength ethelekiswa nobukhulu bamasuntswana ayo.Ekubeni isantya sentshukumo seBrownian samasuntswana kulwelo oluphakathi lunxulumene nobukhulu, utshintsho kumandla okukhanya okusasazekileyo lunokusetyenziswa ukumisela i-avareji ye-hydrodynamic diameter (Z-mean) yesampulu yolwelo.28 Ukongezelela, ngokusebenzisa i-voltage kwisampuli, amandla e-zeta (ζ amandla) ye-nanoparticle inokulinganiswa ngokufanayo nexabiso eliphakathi kwe-Z.I-13,28 Ukuba ixabiso elipheleleyo le-zeta liphezulu ngokwaneleyo (ngokwezikhokelo eziqhelekileyo> ± 30 mV), liya kuvelisa ukunyanzeliswa okunamandla kwe-electrostatic phakathi kwamaqhekeza ukuchasana nokuhlanganiswa.I-characteristic surface plasmon resonance (SPR) yinto ekhethekileyo ye-optical phenomenon, ebangelwa ngokukodwa kwi-nanoparticles yentsimbi exabisekileyo (ingakumbi i-Au kunye ne-Ag).29​ Ngokusekwe kwii-oscillations ze-elektroniki (iiplasmons ezingaphezulu) kwezi zixhobo kwi-nanoscale, kuyaziwa ukuba ii-AgNP ezingqukuva zineempawu zokufunxa kwe-UV-Vis kufutshane ne-400 nm.I-30 Ubunzima kunye nokutshintshwa kwe-wavelength yeengqungquthela zisetyenziselwa ukuncedisa iziphumo ze-DLS, njengoko le ndlela ingasetyenziselwa ukufumanisa i-nanoparticle aggregation kunye ne-adsorption surface ye-biomolecules.
Ngokusekelwe kulwazi olufunyenweyo, ukusebenza kweeseli (MTT) kunye novavanyo lwe-antibacterial lwenziwa ngendlela apho ubuthi be-AgNP buchazwa njengomsebenzi wezinga lokudibanisa, kunokuba (eyona nto isetyenziswa ngokuqhelekileyo) i-nanoparticle concentration.Le ndlela ekhethekileyo ivumela ukuba sibonise ukubaluleka okunzulu kwenqanaba lokudibanisa kumsebenzi we-biological, kuba, umzekelo, i-AgNP ye-citrate-iphelile ilahlekelwa ngokupheleleyo umsebenzi wabo webhayoloji kwiiyure ezimbalwa ngenxa yokuhlanganiswa.19
Kulo msebenzi wangoku, sijonge ukwandisa kakhulu iminikelo yethu yangaphambili ekuzinzeni kwee-colloids ezinxulumene ne-bio kunye nefuthe labo kumsebenzi we-biological ngokufunda umphumo we-nanoparticle ubukhulu kwi-nanoparticle aggregation.Oku ngokungathandabuzekiyo sesinye sezifundo ze-nanoparticles.Umbono weprofayili ephezulu kunye ne-31 Ukuphanda lo mbandela, indlela yokukhula kwembewu yayisetyenziselwa ukuvelisa i-AgNP ye-citrate-terinated kwiindidi ezintathu zobukhulu obuhlukeneyo (10, 20, kunye ne-50 nm).6,32 njengenye yeendlela eziqhelekileyo.Kuma-nanomaterials asetyenziswa ngokubanzi kwaye asetyenziswa ngokuqhelekileyo kwizicelo zonyango, ii-AgNP ze-citrate-terinated zobukhulu obuhlukeneyo zikhethwa ukuba zifunde ukuxhomekeka kobungakanani obunokuthi buxhomekeke kwi-aggregation-related biological properties of nanosilver.Emva kokudibanisa ii-AgNP zobukhulu obahlukeneyo, sibonakalise iisampuli eziveliswe ngokusasazwa kwe-electron microscopy (TEM), emva koko sivavanye amasuntswana sisebenzisa inkqubo yokuhlola ekhankanywe ngasentla.Ukongeza, phambi kweenkcubeko ze-in vitro cell Dulbecco's Modified Eagle's Medium (DMEM) kunye ne-Fetal Bovine Serum (FBS), indlela yokuziphatha exhomekeke kubungakanani kunye nokuziphatha kwayo kwavavanywa kumaxabiso ahlukeneyo e-pH, i-NaCl, i-glucose, kunye ne-glutamine.Iimpawu ze-cytotoxicity zichongwa phantsi kweemeko ezibanzi.Imvumelwano yenzululwazi ibonisa ukuba ngokubanzi, iincinci ezincinci zikhethwa;uphando lwethu lubonelela ngeqonga lemichiza kunye nebhayoloji ukufumanisa ukuba ngaba oku kunjalo.
Iinanoparticles ezintathu zesilivere ezinobungakanani obuhlukeneyo bobukhulu zilungiswe yindlela yokukhula kwembewu ecetywayo nguWan et al., Ngohlengahlengiso oluncinci.I-6 Le ndlela isekelwe ekunciphiseni imichiza, isebenzisa i-nitrate yesilivere (i-AgNO3) njengomthombo wesilivere, i-sodium borohydride (NaBH4) njenge-agent yokunciphisa, kunye ne-sodium citrate njenge-stabilizer.Okokuqala, lungiselela i-75 mL ye-9 mM citrate isisombululo samanzi kwi-sodium citrate dihydrate (Na3C6H5O7 x 2H2O) kunye nokushisa kwi-70 ° C.Emva koko, i-2 mL ye-1% yesisombululo se-w / v AgNO3 yongezwa kwi-reaction medium, kwaye isisombululo esandula ukulungiswa se-sodium borohydride (2 mL 0.1% w / v) sagalelwa kumxube we-dropwise.Ukunqunyanyiswa okubangelwa yi-yellow-brown kugcinwe kwi-70 ° C kunye nokuvuselela ngamandla kwi-1 iyure, kwaye ipholile kwiqondo lokushisa.Isampula esisiphumo (esibizwa ngokuba yi-AgNP-I ukususela ngoku ukuya phambili) sisetyenziswa njengesiseko sokukhula kwembewu kwinyathelo elilandelayo lokudibanisa.
Ukudibanisa i-particle-standard suspension (echazwe njenge-AgNP-II), ukushisa kwe-90 mL 7.6 mM isisombululo se-citrate ukuya kwi-80 ° C, xuba ne-10 mL AgNP-I, uze udibanise i-2 mL 1% w / v Isisombululo se-AgNO3 yayigcinwe phantsi kokuvuselela oomatshini be-1 iyure, kwaye isampuli yapholiswa kwiqondo lokushisa.
Kwi-particle enkulu (AgNP-III), phinda inkqubo efanayo yokukhula, kodwa kule meko, sebenzisa i-10 mL ye-AgNP-II njengokumiswa kwembewu.Emva kokuba iisampulu zifikelele kwiqondo lokushisa kwegumbi, ziseta i-Ag yoxinzelelo lwe-Ag ngokusekelwe kumxholo we-AgNO3 iyonke ukuya kwi-150 ppm ngokudibanisa okanye ukukhupha i-solvent eyongezelelweyo kwi-40 ° C, kwaye ekugqibeleni igcinwe kwi-4 ° C de isetyenziswe ngakumbi.
Sebenzisa i-FEI Tecnai G2 20 X-Twin Transmission Electron Microscope (TEM) (FEI Corporate Headquarters, Hillsboro, Oregon, USA) kunye ne-200 kV i-acceleration voltage ukuhlola iimpawu ze-morphological ze-nanoparticles kwaye ubambe ipateni ye-electron diffraction (ED).Ubuncinci imifanekiso ye-15 emele (~ 750 particles) ihlolwe kusetyenziswa iphakheji yesofthiwe ye-ImageJ, kunye ne-histograms ephumela (kunye nazo zonke iigrafu kwisifundo sonke) zenziwa kwi-OriginPro 2018 (OriginLab, Northampton, MA, USA) 33, 34.
I-avareji ye-hydrodynamic diameter (i-Z-avareji), i-zeta enokubakho (ζ-inokwenzeka) kunye neempawu ze-plasmon resonance (SPR) zesampulu zilinganiswe ukubonisa iimpawu zabo zokuqala ze-colloidal.I-avareji ye-hydrodynamic diameter kunye ne-zeta enokubakho kwisampulu ilinganiswe ngesixhobo se-Malvern Zetasizer Nano ZS (i-Malvern Instruments, i-Malvern, e-UK) isebenzisa iiseli ezigoqiweyo ze-capillary kwi-37±0.1 ° C.I-Ocean Optics 355 DH-2000-BAL UV-Vis spectrophotometer (Halma PLC, Largo, FL, USA) yasetyenziselwa ukufumana iimpawu ze-SPR ezivela kwi-spectra ye-UV-Vis yokufunxa iisampuli kuluhlu lwe-250-800 nm.
Ngexesha lovavanyo lonke, iintlobo ezintathu zokulinganisa ezahlukeneyo ezinxulumene nokuzinza kwe-colloidal zenziwa ngexesha elinye.Sebenzisa i-DLS ukulinganisa i-avareji ye-hydrodynamic diameter (i-Z i-avareji) kunye ne-zeta enokubakho (ζ enokubakho) yamasuntswana, kuba umyinge we-Z unxulumene nobukhulu obuphakathi be-nanoparticle aggregates, kunye ne-zeta enokubakho ibonisa ukuba ukunyanzeliswa kwe-electrostatic kwinkqubo. yomelele ngokwaneleyo ukucima umtsalane weVan der Waals phakathi kwe-nanoparticles.Imilinganiselo yenziwe ngokuphindwe kathathu, kwaye ukutenxa okusemgangathweni kwe-Z kuthetha kunye ne-zeta enokubakho ibalwa yi-software ye-Zetasizer.I-spectra ye-SPR ye-spectra yamasuntswana ihlolwe yi-UV-Vis spectroscopy, kuba utshintsho kwi-peak intensitety kunye nobude be-wavelength bunokubonisa ukudibanisa kunye nokusebenzisana komhlaba.I-29,35 Enyanisweni, i-surface plasmon resonance kwiintsimbi ezixabisekileyo inamandla kangangokuba ibangele iindlela ezintsha zokuhlalutya i-biomolecules.I-29,36,37 I-concentration ye-AgNPs kumxube wokulinga malunga ne-10 ppm, kwaye injongo kukuseta ubungakanani beyona nto iphezulu yokuxutywa kwe-SPR kwi-1. Uvavanyo lwenziwa ngexesha elixhomekeke kwi-0;1.5;3;6;Iiyure ezili-12 kunye ne-24 phantsi kweemeko ezahlukeneyo zebhayoloji ezifanelekileyo.Iinkcukacha ezingakumbi ezichaza umfuniselo zinokubonwa kumsebenzi wethu wangaphambili.19 Ngamafutshane, amaxabiso e-pH ahlukeneyo (3; 5; 7.2 kunye ne-9), i-sodium chloride eyahlukileyo (i-10 mM; i-50 mm; i-150 mM), i-glucose (3.9 mM; 6.7 mM) kunye ne-glutamine (4 mM) yoxinaniso, kunye iphinde yalungisa iDulbecco's Modified Eagle Medium (DMEM) kunye neFetal Bovine Serum (FBS) (emanzini kunye neDMEM) njengeenkqubo zemodeli, kwaye yafunda iziphumo zabo ekuziphatheni kokuhlanganiswa kwee-nanoparticles zesilivere ezenziweyo.pH Amaxabiso e-, NaCl, glucose, kunye ne-glutamine ayavavanywa ngokusekwe kugxininiso lwe-physiological, ngelixa izixa ze-DMEM kunye ne-FBS zifana namanqanaba asetyenziswe kulo lonke uvavanyo lwe-vitro.I-38-42 Yonke imilinganiselo yenziwe kwi-pH 7.2 kunye ne-37 ° C kunye ne-concentration yetyuwa engasemva ye-10 mM NaCl ukuphelisa nayiphi na intsebenziswano yamasuntswana omde (ngaphandle kwe-pH ethile kunye neemvavanyo ezinxulumene ne-NaCl, apho ezi mpawu ziguquguqukayo funda).28 Uluhlu lweemeko ezahlukeneyo lushwankathelwe kwiThebhile 1. Uvavanyo oluphawulwe nge-† lusetyenziswa njengesalathiso kwaye luhambelana nesampuli equkethe i-10 mM NaCl kunye ne-pH 7.2.
Umgca weseli yomhlaza wesibeleko somntu (DU145) kunye neekeratinocyte zabantu ezingafiyo (HaCaT) zifunyenwe kwi-ATCC (Manassas, VA, USA).Iiseli zikhuliswa ngokwesiqhelo kwi-Dulbecco encinci ebalulekileyo ye-Eagle (DMEM) equlethe i-4.5 g/L glucose (Sigma-Aldrich, Saint Louis, MO, USA), incediswa nge-10% FBS, 2 mM L-glutamine, 0.01 % Streptomycin kunye ne-0.005% I-Penicillin (Sigma-Aldrich, St. Louis, Missouri, USA).Iiseli zikhuliswa kwi-incubator engama-37°C ngaphantsi kwe-5% CO2 kunye nokufuma okungama-95%.
Ukuze uhlolisise utshintsho kwi-AgNP ye-cytotoxicity ebangelwa ukuhlanganiswa kwee-particle ngendlela exhomekeke kwixesha, i-assay ye-MTT yamanyathelo amabini yenziwa.Okokuqala, ukusebenza kweentlobo ezimbini zeeseli zalinganiswa emva konyango nge-AgNP-I, i-AgNP-II kunye ne-AgNP-III.Ukuza kuthi ga ngoku, ezi ntlobo zimbini zeeseli zifakwe kwi-96-well-plates ekuxineni kweeseli ze-10,000 / kakuhle kwaye ziphathwe ngeesayizi ezintathu ezahlukeneyo ze-nanoparticles zesilivere ekunyuseni ukunyuka ngosuku lwesibini.Emva kweeyure ze-24 zonyango, iiseli zihlanjwe nge-PBS kwaye zifakwe kwi-0.5 mg / mL i-reagent ye-MTT (i-SERVA, i-Heidelberg, eJamani) ihlanjululwe kwi-culture medium kwi-1 iyure kwi-37 ° C.I-crystals ye-Formazan yachithwa kwi-DMSO (i-Sigma-Aldrich, i-Saint Louis, i-MO, i-USA), kwaye i-absorption yalinganiswa kwi-570 nm usebenzisa i-Synergy HTX plate reader (BioTek-Hungary, Budapest, Hungary).Ixabiso lokufunxa kwisampulu yolawulo olungaphendulwanga lithathwa njengesantya se-100% sokusinda.Yenza ubuncinane imifuniselo emi-3 usebenzisa iikopi ezine ezizimeleyo zebhayoloji.I-IC50 ibalwa ukusuka kwigophe lempendulo yethamo ngokusekwe kwiziphumo zamandla.
Emva koko, kwisinyathelo sesibini, ngokufaka iinqununu kunye ne-150 mM NaCl ngamaxesha ahlukeneyo (0, 1.5, 3, 6, 12, kunye neeyure ezingama-24) ngaphambi kokunyangwa kweeseli, i-aggregation states ezahlukeneyo ze-nanoparticles zesilivere zenziwe.Emva koko, uvavanyo olufanayo lwe-MTT lwenziwa njengoko luchazwe ngaphambili ukuvavanya utshintsho ekusebenzeni kweeseli ezichatshazelwa yi-particle aggregation.Sebenzisa i-GraphPad Prism 7 ukuvavanya umphumo wokugqibela, ubale ukubaluleka kwezibalo zovavanyo ngovavanyo lwe-t-t-t-test, kwaye umaka inqanaba layo njenge * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001 ) Kwaye **** (p ≤ 0.0001).
Ubukhulu obuthathu obuhlukeneyo besilivere ye-nanoparticles (i-AgNP-I, i-AgNP-II kunye ne-AgNP-III) yayisetyenziselwa ukuxhatshazwa kwe-antibacterial kwi-Cryptococcus neoformans IFM 5844 (IFM; iZiko loPhando lwe-Pathogenic Fungi kunye ne-Microbial Toxicology, iYunivesithi yaseChiba) kunye ne-Bacillus Test megaterium SZMC 60 (SZMC: I-Szeged Microbiology Collection) kunye ne-E. coli SZMC 0582 kwi-RPMI 1640 medium (Sigma-Aldrich Co.).Ukuze kuhlolwe utshintsho kumsebenzi we-antibacterial obangelwa ukuhlanganiswa kweengqungquthela, okokuqala, i-concentration yabo encinci ye-inhibitory (MIC) inqunywe yi-microdilution kwi-96-well microtiter plate.Ukuya kwi-50 μL yokumiswa kweseli esemgangathweni (i-5 × 104 iiseli / mL kwi-RPMI 1640 medium), yongeza i-50 μL yesilivere ye-nanoparticle ukumiswa kwaye uhlambulule ngokuphindwe kabini ukuxininisa (kwindlela ekhankanywe ngasentla, uluhlu luyi-0 kunye ne-75 ppm, oko kukuthi, isampuli yokulawula iqulethe i-50 μL yokumiswa kweeseli kunye ne-50 μL ephakathi ngaphandle kwe-nanoparticles).Emva koko, i-plate ifakwe kwi-30 ° C kwiiyure ze-48, kwaye ukuxinwa kwe-optical yenkcubeko kulinganiswa kwi-620 nm usebenzisa i-SPECTROstar Nano plate reader (BMG LabTech, Offenburg, Germany).Uvavanyo lwenziwa kathathu ngokuphindwe kathathu.
Ngaphandle kokuba i-50 μL yeesampulu ezidibeneyo ze-nanoparticle zisetyenzisiwe ngeli xesha, inkqubo efanayo njengoko ichazwe ngaphambili yayisetyenziselwa ukuhlola umphumo wokudibanisa kumsebenzi we-antibacterial kwiintlobo ezikhankanywe ngasentla.Iimeko ezahlukeneyo zokuhlanganisa i-nanoparticles zesilivere ziveliswa ngokufukamela amasuntswana nge-150 mM NaCl ngamaxesha ahlukeneyo (0, 1.5, 3, 6, 12, kunye neeyure ezingama-24) ngaphambi kokulungiswa kweeseli.Ukumiswa okongeziweyo kunye ne-50 μL ye-RPMI 1640 medium isetyenziswe njengolawulo lokukhula, ngelixa ukwenzela ukulawula ubuthi, ukumiswa kunye ne-nanoparticles engaxutywanga isetyenziswe.Uvavanyo lwenziwa kathathu ngokuphindwe kathathu.Sebenzisa i-GraphPad Prism 7 ukuvavanya umphumo wokugqibela kwakhona, usebenzisa uhlalutyo olufanayo lwezibalo njengohlalutyo lwe-MTT.
Inqanaba lokudibanisa lawona mancinci amancinci (AgNP-I) libonakaliswe, kwaye iziphumo zapapashwa ngokuyinxenye kumsebenzi wethu wangaphambili, kodwa ukuthelekisa okungcono, zonke iinqununu zihlolwe ngokucokisekileyo.Idatha yovavanyo iqokelelwa kwaye ixoxwe kumacandelo alandelayo.Iisayizi ezintathu ze-AgNP.19
Imilinganiselo eyenziwa yi-TEM, i-UV-Vis kunye ne-DLS iqinisekise ukuhlanganiswa okuyimpumelelo kwazo zonke iisampuli ze-AgNP (Umfanekiso 2A-D).Ngokomqolo wokuqala we-Figure 2, i-particle encinci (i-AgNP-I) ibonisa i-morphology ye-spherical efanayo kunye nobubanzi obuphakathi kwe-10 nm.Indlela yokukhula yembewu ikwabonelela nge-AgNP-II kunye ne-AgNP-III enoluhlu oluhlukeneyo lobukhulu obunomlinganiselo wamasuntswana amadayamitha amalunga ne-20 nm kunye ne-50 nm, ngokulandelelanayo.Ngokutsho kokuphambuka okusemgangathweni kokusasazwa kwamasuntswana, ubukhulu beesampuli ezintathu azihambelani, okubalulekileyo kuhlalutyo lwabo lokuthelekisa.Ngokuthelekisa i-avareji ye-aspect ratio kunye ne-thiness ratio ye-TEM-based based particle 2D ingqikelelo, kucingelwa ukuba i-sphericity yamasuntswana ihlolwe yi-ImageJ's shape filter plug-in (Figure 2E).43 Ngokohlalutyo lobume bamasuntswana, umyinge wawo (icala elikhulu / icala elifutshane loxande oluncinane olunomda) alichatshazelwa kukukhula kwamasuntswana, kunye nomlinganiselo wokubhitya kwawo (indawo elinganiselweyo yesangqa esihambelanayo / indawo yethiyori. ) iyancipha ngokuthe ngcembe.Oku kubangela amasuntswana e-polyhedral amaninzi, ajikeleze ngokugqibeleleyo kwithiyori, ehambelana nomlinganiselo wokubhitya we-1.
Umzobo 2 Ukuhanjiswa kwe-electron microscope (TEM) umfanekiso (A), ipateni ye-electron diffraction (ED) (B), i-histogram yokusabalalisa ubungakanani (C), uphawu lwe-ultraviolet-kubonakala (UV-Vis) i-spectrum yokufunxa ukukhanya (D), kunye ne-avareji ye-Citrate yolwelo. -iphelile i-nanoparticles yesilivere ene-diameter ye-mechanical (i-Z-avareji), i-zeta enokubakho, i-aspect ratio kunye ne-tight ratio (E) ineentlobo ezintathu zobukhulu obuhlukeneyo: i-AgNP-I yi-10 nm (umqolo ophezulu), i-AgNP -II yi-20 nm (umqolo ophakathi ), i-AgNP-III (umqolo ongezantsi) yi-50 nm.
Nangona ubume be-cyclic yendlela yokukhula buchaphazele ubume be-particle ukuya kwinqanaba elithile, kubangele kwi-sphericity encinci ye-AgNPs enkulu, zonke iisampuli ezintathu zahlala zi-quasi-spherical.Ukongezelela, njengoko kuboniswe kwipatheni ye-electron diffraction kwi-Figure 2B, i-nano I-crystallinity yamaqhekeza ayichaphazeleki.Iringi ye-diffraction ebalaseleyo-enokuthi idityaniswe ne (111), (220), (200), kunye (311) i-Miller indices yesilivere-ihambelana kakhulu noncwadi lwesayensi kunye neminikelo yethu yangaphambili.9, 19,44 Ukuqhekeka kwe-Debye-Scherrer ring ye-AgNP-II kunye ne-AgNP-III kubangelwa ukuba umfanekiso we-ED uthathwe ngokukhuliswa okufanayo, ngoko ke njengoko ubungakanani be-particle sanda, inani leengqungquthela ezidityanisiweyo. indawo yeyunithi iyanda kwaye iyancipha.
Ubungakanani kunye nokuma kwee-nanoparticles ziyaziwa ngokuchaphazela umsebenzi we-biological.I-3,45 Umsebenzi oxhomekeke kwi-Catalytic kunye ne-biological exhomekeke kwimilo inokuchazwa yinyaniso yokuba iimilo ezahlukeneyo zivame ukwandisa ubuso obuthile be-crystal (ukuba ne-indices ye-Miller eyahlukeneyo), kwaye obu buso be-crystal bunemisebenzi eyahlukeneyo.I-45,46 Ekubeni iinqununu ezilungiselelwe zibonelela ngeziphumo ezifanayo ze-ED ezihambelana neempawu ze-crystal ezifanayo kakhulu, kunokucingelwa ukuba kwi-colloidal yethu elandelayo yokuzinza kunye neemvavanyo ze-biological activity, nayiphi na iintlukwano ezibonisiweyo kufuneka zibhekiswe kubukhulu be-Nanoparticle, kungekhona iipropati ezinxulumene nemilo.
Iziphumo ze-UV-Vis zishwankathelwe kuMfanekiso we-2D ngakumbi ukugxininisa ubume obungqukuva be-AgNP ehlanganisiweyo, kuba iincopho ze-SPR zazo zontathu iisampulu zijikeleze i-400 nm, elixabiso lophawu lwe-nanoparticles yesilivere engqukuva.I-29,30 I-spectra ebanjiweyo nayo yaqinisekisa ukukhula okuyimpumelelo kwembewu ye-nanosilver.Njengoko ubungakanani be-particle bukhula, ubude be-wavelength buhambelana nokukhanya okuphezulu kokukhanya kwe-AgNP-II-ngakumbi ngokuphawulekayo-Ngokutsho kweencwadi, i-AgNP-III Yafumana i-redshift.6,29
Ngokumalunga nokuzinza kokuqala kwe-colloidal yenkqubo ye-AgNP, i-DLS yayisetyenziselwa ukulinganisa i-avareji ye-hydrodynamic diameter kunye ne-zeta ye-zeta yamasuntswana kwi-pH 7.2.Iziphumo eziboniswe kuMfanekiso we-2E zibonisa ukuba i-AgNP-III inozinzo oluphezulu lwe-colloidal kune-AgNP-I okanye i-AgNP-II, kuba izikhokelo eziqhelekileyo zibonisa ukuba amandla e-zeta ye-30 mV absolute iyimfuneko ekuzinzeni kwexesha elide le-colloidal Oku kufunyaniswayo kuxhaswa ngakumbi xa Ixabiso elingu-Z (elifunyenwe njenge-avareji ye-hydrodynamic diameter yamasuntswana asimahla kunye ne-aggregated) ithelekiswa nobungakanani bamasuntswana angundoqo afunyenwe yi-TEM, kuba amaxabiso amabini akufutshane, kokukhona buthathaka isidanga Qokelela kwisampulu.Ngapha koko, umyinge we-Z we-AgNP-I kunye ne-AgNP-II uphezulu ngokufanelekileyo kunobungakanani bamasuntswana avavanyiweyo be-TEM, ngoko ke xa kuthelekiswa ne-AgNP-III, ezi sampuli ziqikelelwa ukuba zinokwenzeka ukuba zihlangane, apho i-zeta inokuba namandla kakhulu. Ikhatshwa yisayizi esondeleyo I-avareji yexabiso le-Z.
Ingcaciso yale nto inokuba mbini.Kwelinye icala, ugxininiso lwe-citrate lugcinwa kwinqanaba elifanayo kuwo onke amanyathelo okudibanisa, ukubonelela ngenani eliphezulu lamaqela ahlawuliswayo ukuthintela indawo ethile yamasuntswana akhulayo ukuba ehle.Nangona kunjalo, ngokutsho kweLevak et al., iimolekyuli ezincinci ezifana ne-citrate zinokutshintshwa ngokulula ngama-biomolecules kumphezulu we-nanoparticles.Kule meko, ukuzinza kwe-colloidal kuya kugqitywa yi-corona yee-biomolecules eziveliswayo.31 Ngenxa yokuba le ndlela yokuziphatha iphinde yabonwa kwimilinganiselo yethu yokuhlanganiswa (kuxoxwe ngokubanzi kamva), i-citrate capping iyodwa ayikwazi ukuchaza le nto.
Ngakolunye uhlangothi, ubungakanani be-particle ubukhulu buhambelana ngokungafaniyo ne-aggregation tendency kwinqanaba le-nanometer.Oku kuxhaswa kakhulu yindlela yeDerjaguin-Landau-Verwey-Overbeek (DLVO) yendabuko, apho umtsalane wamasuntswana uchazwa njengenani lezinto ezikhangayo kunye nezichasayo phakathi kwamaqhekeza.Ngokutsho kwe-He et al., Ixabiso eliphezulu le-curve ye-DLVO yamandla liyancipha kunye nobukhulu be-nanoparticles kwi-hematite nanoparticles, okwenza kube lula ukufikelela kumandla amancinci amancinci, ngaloo ndlela kukhuthazwa ukuhlanganiswa okungenakuguqulwa (i-condensation).47 Nangona kunjalo, kuqikelelwa ukuba kukho eminye imiba engaphaya kwemida yethiyori ye-DLVO.Nangona i-van der Waals ubunzima kunye ne-electrostatic double-layer repulsion ziyafana ngokunyuka kwamasuntswana, uphononongo olwenziwe nguHotze et al.icebisa ukuba inefuthe elinamandla kuhlanganiso kunokuba i-DLVO ivumela.14 Bakholelwa ukuba ukugoba komphezulu weenanoparticles akunakuphinda kuqikelelwe njengomphezulu osicaba, nto leyo eyenza uqikelelo lwemathematika lungasebenzi.Ukongeza, njengoko ubungakanani bamasuntswana buncipha, ipesenti yee-athomu ezikhoyo kumphezulu ziba phezulu, nto leyo ekhokelela kulwakhiwo lwe-elektroniki kunye nokuziphatha kwentlawulo yomphezulu.Kwaye utshintsho lwe-reactivity yomphezulu, olunokuthi lukhokhelele ekwehleni kwentlawulo kumbane ophindwe kabini kunye nokukhuthaza ukuhlanganisana.
Xa sithelekisa iziphumo ze-DLS ze-AgNP-I, i-AgNP-II, kunye ne-AgNP-III kwi-Figure 3, siye saqaphela ukuba zonke iisampuli ezintathu zibonisa i-pH efanayo ekhuthaza ukuhlanganiswa.Imekobume ene-acidic kakhulu (pH 3) iguqula amandla e-zeta yesampuli kwi-0 mV, ebangela ukuba iinqununu zenze i-micron-size aggregates, ngelixa i-alkaline pH iguqula amandla ayo e-zeta kwixabiso elikhulu elibi, apho iinqununu zenza i-aggregates encinci (pH 5). ).Kwaye i-7.2) ), okanye ihlale ingalingani ngokupheleleyo (pH 9).Ezinye iiyantlukwano ezibalulekileyo phakathi kweesampuli ezahlukeneyo nazo zabonwa.Kulo lonke uvavanyo, i-AgNP-I ibonakalise ukuba yi-pH-induced zeta ezinokuthi zitshintshe, kuba amandla e-zeta ala maqhekeza ancitshiswe kwi-pH 7.2 xa kuthelekiswa ne-pH 9, ngelixa i-AgNP-II kunye ne-AgNP-III ibonise kuphela i-A. Utshintsho olukhulu kwi-ζ lujikeleze i-pH 3. Ukongeza, i-AgNP-II ibonise utshintsho olucothayo kunye ne-zeta ephakathi, ngelixa i-AgNP-III ibonise ukuziphatha okuthobekileyo kwabathathu, kuba inkqubo ibonise ixabiso eliphezulu le-zeta kunye nentshukumo ehamba kade, ebonisa indlela yokuziphatha ephantsi. I-AgNP-III Uninzi oluxhathisayo kwi-pH-induced aggregation.Ezi ziphumo zihambelana neziphumo zomlinganiselo we-hydrodynamic diameter.Ukuqwalasela ubungakanani bamasuntswana eeprimers zazo, i-AgNP-I ibonise ukudityaniswa ngokuthe ngcembe kuwo onke amaxabiso e-pH, mhlawumbi ngenxa ye-10 mM ye-NaCl yangasemva, ngelixa i-AgNP-II kunye ne-AgNP-III zibonise kuphela ukubaluleka kwi-pH 3 yokuqokelela.Umahluko onomdla kakhulu kukuba nangona ubukhulu bayo be-nanoparticle enkulu, i-AgNP-III yenza i-aggregates encinci kwi-pH 3 kwiiyure ze-24, igxininisa iipropati zayo ezichasayo.Ngokwahlula umyinge we-Z we-AgNP kwi-pH 3 emva kweeyure ezingama-24 ngexabiso lesampulu elungisiweyo, kunokubonwa ukuba ubungakanani obudibeneyo be-AgNP-I kunye ne-AgNP-II bunyuke ngamaxesha angama-50, amaxesha angama-42, kunye namaxesha angama-22. , ngokulandelelanayo.III.
Umzobo 3 Iziphumo zokusasazwa kokukhanya okuguquguqukayo kwesampulu ye-citrate-terinated yesilivere ye-nanoparticles isampuli kunye nobukhulu obukhulayo (10 nm: AgNP-I, 20 nm: AgNP-II kunye ne-50 nm: AgNP-III) zibonakaliswa njenge-avareji ye-hydrodynamic diameter (Z umyinge ) (ngasekunene) Ngaphantsi kweemeko ezahlukeneyo ze-pH, amandla e-zeta (ngasekhohlo) atshintsha kwiiyure ezingama-24.
Udityaniso olujongweyo oluxhomekeke kwi-pH luphinde lwachaphazela uphawu lomphezulu lwe-plasmon resonance (SPR) yeesampulu ze-AgNP, njengoko kungqinwa yi-UV-Vis spectra.Ngokutsho koMzobo we-Supplementary S1, ukudityaniswa kwazo zonke ezintathu zesilivere ze-nanoparticle suspensions zilandelwa kukunciphisa ubukhulu beencopho zabo ze-SPR kunye nokutshintsha okubomvu okuphakathi.Ubungakanani bolu tshintsho njengomsebenzi we-pH buhambelana neqondo lokudityaniswa okuxelwe kwangaphambili ziziphumo ze-DLS, nangona kunjalo, ezinye iindlela ezinomdla ziye zabonwa.Ngokuchaseneyo ne-intuition, kuvela ukuba i-AgNP-II yobungakanani obuphakathi yeyona nto ivakalelwa kakhulu kwiinguqu ze-SPR, ngelixa ezinye iisampuli ezimbini zincinci.Kuphando lwe-SPR, i-50 nm ngumda we-particle size limit, esetyenziselwa ukwahlula iinqununu ngokusekelwe kwiipropati zabo ze-dielectric.Iinqununu ezincinci kune-50 nm (i-AgNP-I kunye ne-AgNP-II) inokuchazwa njenge-dielectric dipoles elula, ngelixa iinqununu ezifikelela okanye ezidlulayo kulo mda (AgNP-III) zineepropati eziyinkimbinkimbi ze-dielectric, kunye ne-resonance yazo Ibhendi iyahlukana kwiinguqu ezininzi. .Kwimeko yeesampulu ezimbini ezincinci, ii-AgNP zinokuthathwa njenge-dipoles elula, kwaye i-plasma inokugqithisa ngokulula.Njengoko ubungakanani bamasuntswana bukhula, oku kudityaniswa kuvelisa iplasma enkulu, enokuthi ichaze ubuntununtunu obuphezulu obubonwayo.29 Nangona kunjalo, kumasuntswana amakhulu, uqikelelo olulula lwe-dipole alusebenzi xa ezinye iindawo zokudityaniswa zinokuthi zenzeke, ezinokuchaza ukuhla kwe-AgNP-III ukubonisa utshintsho lwe-spectral.29
Ngaphantsi kweemeko zethu zokulinga, kubonakaliswe ukuba ixabiso le-pH linempembelelo enzulu kwi-colloidal stability ye-citrate-coated silver nanoparticles zobukhulu obuhlukeneyo.Kwezi nkqubo, uzinzo lubonelelwa ngamaqela -COO- ahlawuliswa kakubi kumphezulu wee-AgNP.Iqela elisebenzayo le-carboxylate ye-ion ye-citrate i-protonated kwinani elikhulu le-H + ion, ngoko ke iqela le-carboxyl eliveliswayo alikwazi ukubonelela nge-electrostatic repulsion phakathi kwamaqhekeza, njengoko kubonisiwe kumqolo ophezulu we-Figure 4. Ngokomgaqo kaLe Chatelier, i-AgNP iisampulu zikhawuleza zidityaniswe kwi-pH 3, kodwa ngokuthe ngcembe ziye zizinza ngakumbi njengoko i-pH isanda.
Umzobo 4 Umzobo we-Schematic wokusebenzisana komphezulu ochazwe ngokudibanisa phantsi kwe-pH eyahlukileyo (umqolo ophezulu), ugxininiso lwe-NaCl (umqolo ophakathi), kunye ne-biomolecules (umqolo osezantsi).
NgokoMfanekiso 5, ukuzinza kwe-colloidal kwi-AgNP yokumiswa kobukhulu obuhlukeneyo kuye kwavavanywa phantsi kokunyuka kwetyuwa.Ngokusekelwe kwikhono le-zeta, ubungakanani be-nanoparticle eyongeziweyo kwezi nkqubo ze-AgNP ze-citrate ziphinda zinikeze ukuchasana okuphuculweyo kwiimpembelelo zangaphandle ezivela kwi-NaCl.Kwi-AgNP-I, i-10 mM i-NaCl yanele ukubangela ukuhlanganiswa okuthambileyo, kwaye ityuwa ye-50 mM inika iziphumo ezifanayo kakhulu.Kwi-AgNP-II kunye ne-AgNP-III, i-10 mM NaCl ayichaphazeli kakhulu i-zeta enokubakho kuba ixabiso labo lihlala (AgNP-II) okanye ngaphantsi (AgNP-III) -30 mV.Ukwandisa i-concentration ye-NaCl kwi-50 mM kwaye ekugqibeleni kwi-150 mM i-NaCl yanele ukunciphisa kakhulu ixabiso elipheleleyo le-zeta enokubakho kuzo zonke iisampuli, nangona iinqununu ezinkulu zigcina intlawulo embi kakhulu.Ezi ziphumo ziyangqinelana nomndilili olindelekileyo wedayamitha ye-hydrodynamic yee-AgNP;umyinge wemizila ye-Z elinganiselwa kwi-10, 50, kunye ne-150 mM NaCl ibonisa ngokwahlukileyo, inyusa ngokuthe ngcembe amaxabiso.Ekugqibeleni, ii-aggregates ezinobungakanani bemicron zichongiwe kuzo zontathu iimvavanyo ze-150 mM.
Umzobo 5 Iziphumo zokusasazwa kokukhanya okuguquguqukayo kwesampulu ye-citrate-terinated yesilivere ye-nanoparticles isampuli kunye nobukhulu obukhulayo (10 nm: AgNP-I, 20 nm: AgNP-II kunye ne-50 nm: AgNP-III) zibonakaliswa njenge-avareji ye-hydrodynamic diameter (i-Z avareji ) (ekunene) kunye ne-zeta enokubakho (ekhohlo) itshintshe kwiiyure ze-24 phantsi kweemeko ezahlukeneyo ze-NaCl.
Iziphumo ze-UV-Vis kwi-Supplementary Figure S2 zibonisa ukuba i-SPR ye-50 kunye ne-150 mM ye-NaCl kuzo zonke iisampuli ezintathu inokwehla ngokukhawuleza kunye nokubonakalayo.Oku kunokuchazwa yi-DLS, kuba ukuhlanganiswa kwe-NaCl-based aggregation kwenzeka ngokukhawuleza kuneemvavanyo ezixhomekeke kwi-pH, echazwa ngumahluko omkhulu phakathi kwemilinganiselo yokuqala (0, 1.5, kunye ne-3 iiyure).Ukongeza, ukunyusa ityuwa kuya kwandisa ukuvunyelwa okuhambelanayo kwendlela yokuvavanya, eya kuba nefuthe elinzulu kwi-surface plasmon resonance.29
Umphumo we-NaCl ushwankathelwa kumqolo ophakathi woMzobo 4. Ngokuqhelekileyo, kunokugqitywa ukuba ukwandisa ukuxinwa kwe-sodium chloride kunempembelelo efanayo nokwandisa i-acidity, kuba i-Na + ion inomdla wokulungelelanisa malunga namaqela e-carboxylate, ukucinezela i-negative zeta ezinokubakho ii-AgNP.Ukongeza, i-150 mM NaCl ivelise i-aggregates ye-micron-size aggregates kuzo zontathu iisampuli, ebonisa ukuba i-physiological electrolyte concentration iyonakalisa ukuzinza kwe-colloidal ye-AgNPs ye-citrate-terinated.Ngokuqwalasela i-critical condensing concentration (CCC) ye-NaCl kwiinkqubo ezifanayo ze-AgNP, ezi ziphumo zinokubekwa ngobuchule kuncwadi olufanelekileyo.UHuynh et al.ibalwa ukuba i-CCC ye-NaCl ye-citrate-terinated silver nanoparticles kunye nobubanzi obuphakathi kwe-71 nm yayingu-47.6 mM, ngelixa u-El Badawy et al.yaqaphela ukuba i-CCC ye-10 nm AgNPs ene-citrate coating yayingu-70 mM.I-10,16 Ukongezelela, i-CCC ephezulu kakhulu malunga ne-300 mM yalinganiswa nguYe et al., Okubangela ukuba indlela yabo yokudibanisa ihluke kupapasho olukhankanywe ngaphambili.48 Nangona igalelo langoku lingajoliswanga kuhlalutyo olubanzi lwala maxabiso, ngenxa yokuba iimeko zethu zokulinga zisanda kubunzima bophononongo lonke, i-biologically relevant NaCl concentration ye-50 mM, ngakumbi i-150 mM NaCl, ibonakala iphezulu kakhulu.I-coagulation eyenziwe, ichaza utshintsho olunamandla olufunyenweyo.
Isinyathelo esilandelayo kuvavanyo lwe-polymerization kukusebenzisa iimolekyuli ezilula kodwa ezihambelana ne-biological ukulinganisa ukusebenzisana kwe-nanoparticle-biomolecule.Ngokusekelwe kwi-DLS (Amanani 6 kunye ne-7) kunye neziphumo ze-UV-Vis (iMizobo eyoNgezelelweyo ye-S3 kunye ne-S4), ezinye izigqibo ngokubanzi zinokuthi zenziwe.Phantsi kweemeko zethu zovavanyo, iimolekyuli ezifundiweyo zeglucose kunye neglutamine aziyi kukhuthaza ukudityaniswa kuyo nayiphi na inkqubo ye-AgNP, kuba indlela ye-Z-mean inxulumene ngokusondeleyo nexabiso lereferensi ehambelana nayo.Nangona ubukho bazo bungachaphazeli ukudityaniswa, iziphumo zovavanyo zibonisa ukuba ezi molekyuli zithengiswa ngokuyinxenye kumphezulu wee-AgNP.Esona siphumo sibalaseleyo esixhasa lo mbono lutshintsho olujongiweyo lokufunxa ukukhanya.Nangona i-AgNP-I ingabonisi nguquko yobude obunentsingiselo okanye ubunzulu, inokujongwa ngokucacileyo ngokulinganisa amasuntswana amakhulu, okunokwenzeka ukuba kungenxa yovakalelo olukhulu olukhankanywe ngaphambili.Kungakhathaliseki ukuba i-concentration, i-glucose ingabangela utshintsho olukhulu olubomvu emva kweeyure ze-1.5 xa kuthelekiswa nomlinganiselo wokulawula, malunga ne-40 nm kwi-AgNP-II kunye ne-10 nm kwi-AgNP-III, ebonisa ukwenzeka kobudlelwane bomphezulu.I-Glutamine ibonise indlela efanayo, kodwa utshintsho lwalungabonakali kangako.Ukongezelela, kuyafaneleka ukukhankanya ukuba i-glutamine inokunciphisa amandla e-zeta aphakathi kunye namasuntswana amakhulu.Nangona kunjalo, ekubeni olu tshintsho lwe-zeta alubonakali luchaphazela inqanaba lokudibanisa, kunokucingelwa ukuba i-biomolecules encinci efana ne-glutamine inokubonelela ngeqondo elithile lokunyanzeliswa kwendawo phakathi kwamaqhekeza.
Umzobo 6 Iziphumo zokusasazwa kokukhanya okuguquguqukayo kweesampuli ze-citrate-terinated zesilivere ze-nanoparticle ezinobungakanani obukhulayo (10 nm: AgNP-I, 20 nm: AgNP-II kunye ne-50 nm: AgNP-III) zibonakaliswa njenge-avareji ye-hydrodynamic diameter (i-Z avareji) (ngasekunene) Ngaphantsi kweemeko zangaphandle ze-glucose ezihlukeneyo, amandla e-zeta (ngasekhohlo) atshintsha kwiiyure ze-24.
Umzobo 7 Iziphumo zokusasazwa kokukhanya okuguquguqukayo kwesampulu ye-citrate-terinated yesilivere ye-nanoparticles isampuli kunye nobukhulu obukhulayo (i-10 nm: AgNP-I, i-20 nm: i-AgNP-II kunye ne-50 nm: i-AgNP-III) ibonakaliswe njenge-avareji ye-hydrodynamic diameter (i-Z avareji ) (ngasekunene) Xa kukho i-glutamine, amandla e-zeta (ngasekhohlo) atshintsha kwiiyure ezingama-24.
Ngamafutshane, i-biomolecules ezincinci ezifana ne-glucose kunye ne-glutamine azichaphazeli ukuzinza kwe-colloidal kwi-concentration elinganisiweyo: nangona zichaphazela amandla e-zeta kunye neziphumo ze-UV-Vis kwiidigri ezihlukeneyo, iziphumo ze-Z azihambelani.Oku kubonisa ukuba i-adsorption ye-surface ye-molekyuli inqanda ukugxothwa kwe-electrostatic, kodwa kwangaxeshanye ibonelela ngokuzinza kwe-dimensional.
Ukuze sinxulumanise iziphumo zangaphambili kunye neziphumo zangaphambili kunye nokulinganisa iimeko zebhayoloji ngobuchule ngakumbi, sikhethe ezinye zezona nxalenye zisetyenziswa ngokuqhelekileyo kwiiseli zenkcubeko kwaye sizisebenzise njengeemeko zovavanyo lokufunda uzinzo lwe-AgNP colloids.Kulo lonke uvavanyo lwe-in vitro, enye yeyona misebenzi ibalulekileyo ye-DMEM njengento ephakathi kukuseka iimeko eziyimfuneko ze-osmotic, kodwa ukusuka kwindawo yokujonga iikhemikhali, isisombululo setyuwa esiyinkimbinkimbi esinamandla onke e-ionic afana ne-150 mM NaCl. .40 Ngokubhekiselele kwi-FBS, ngumxube onzima we-biomolecules-ikakhulukazi iiprotheyini-ukusuka kwindawo yokujonga i-adsorption ye-surface adsorption, inokufana okuthile kunye neziphumo zovavanyo lwe-glucose kunye ne-glutamine, nangona ukubunjwa kweekhemikhali kunye nokuhlukahluka kwe-Sex inzima kakhulu.I-19 DLS kunye ne-UV-Iziphumo ezibonakalayo eziboniswe kwi-Figure 8 kunye ne-Supplementary Figure S5, ngokulandelanayo, inokuchazwa ngokuhlola ukubunjwa kweekhemikhali zezi zinto kunye nokulungelelanisa kunye nemilinganiselo kwicandelo langaphambili.
Umzobo 8 Iziphumo zokusasazwa kokukhanya okuguquguqukayo kwesampulu ye-citrate-terinated yesilivere ye-nanoparticles isampuli kunye nobukhulu obukhulayo (10 nm: AgNP-I, 20 nm: AgNP-II kunye ne-50 nm: AgNP-III) zibonakaliswa njenge-avareji ye-hydrodynamic diameter (Z umyinge ) (ekunene) Phambi kwamacandelo enkcubeko yeseli iDMEM kunye neFBS, amandla e-zeta (ngasekhohlo) atshintsha kwiiyure ezingama-24.
Ukuhlanjululwa kwee-AgNP zobukhulu obuhlukeneyo kwi-DMEM kunempembelelo efanayo ekuzinzeni kwe-colloidal kwinto ebonwayo kubukho be-NaCl ephezulu.Ukusasazwa kwe-AgNP kwi-50 v / v% i-DMEM ibonise ukuba ukudibanisa okukhulu kufunyenwe ngokunyuka kwe-zeta enokubakho kunye ne-Z-avareji yexabiso kunye nokuncipha okubukhali kwe-SPR.Kuyafaneleka ukuba kuqatshelwe ukuba ubungakanani obukhulu be-aggregate obubangelwa yi-DMEM emva kweeyure ze-24 buhambelana ngokungafaniyo nobukhulu be-primer nanoparticles.
Ukusebenzisana phakathi kwe-FBS kunye ne-AgNP kufana noko kubonwa phambi kweeamolekyu ezincinci ezifana ne-glucose kunye ne-glutamine, kodwa umphumo unamandla.Umyinge we-Z wamasuntswana uhlala ungachaphazeleki, ngelixa ukwanda kwe-zeta kubonwa.Incopho ye-SPR ibonise utshintsho olubomvu oluncinci, kodwa mhlawumbi ngakumbi umdla, ubunzulu be-SPR abuzange bunciphe kakhulu njengakwimilinganiselo yolawulo.Ezi ziphumo zingachazwa nge-adsorption yangaphakathi ye-macromolecules ebusweni be-nanoparticles (umqolo ongezantsi kwi-Figure 4), ngoku iqondwa njengokubunjwa kwe-corona ye-biomolecular emzimbeni.49