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Ingabe ama-nanoparticles amancane ahlala engcono?Qonda imiphumela yebhayoloji yokuhlanganisa okuncike kusayizi wama-nanoparticles esiliva ngaphansi kwezimo ezifanele 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, Isayensi Yezesayensi , Inyuvesi yaseSzeged;2 Umnyango we-Biochemistry kanye ne-Molecular Biology, i-Faculty of Science and Information, University of Szeged, Hungary;3 UMnyango Wezesayensi Yezinto Ezincane, I-Faculty of Science and Information, University of Szeged, Hungary;4MTA-SZTE Reaction Kinetics and Surface Chemistry Research Group, Szeged, Hungary* Laba babhali banikele ngokulinganayo kulo msebenzi.Ukuxhumana: Zoltán Kónya Department of Applied and Environment Chemistry, Faculty of Science and Informatics, University of Szeged, Rerrich Square 1, Szeged, H-6720, Hungary Ucingo +36 62 544620 I-imeyili [Ukuvikelwa kwe-imeyili] Injongo: Isiliva nanoparticles (AgNPs) kukhona enye yama-nanomatadium evame ukufundwa, ikakhulukazi ngenxa yokusetshenziswa kwayo kwe-biomedical.Kodwa-ke, ngenxa yokuhlanganiswa kwama-nanoparticles, umsebenzi wawo omuhle kakhulu we-cytotoxicity kanye nomsebenzi we-antibacterial kuvame ukuba sengozini emithonjeni yebhayoloji.Kulo msebenzi, ukuziphatha kokuhlanganisa kanye nemisebenzi ehlobene yebhayoloji yamasampuli amathathu ahlukene esiliva e-citrate-term nanoparticle anobubanzi obuyisilinganiso buka-10, 20, kanye no-50 nm.Indlela: Sebenzisa isibonakhulu se-electron yokudlulisa ukuze uhlanganise futhi ubonise ama-nanoparticles, uhlole ukuziphatha kwawo kokuhlanganisa ngamavelu ahlukahlukene we-pH, i-NaCl, i-glucose ne-glutamine ukugxila ngokusakazwa kokukhanya okuguquguqukayo kanye ne-ultraviolet-spectroscopy ebonakalayo.Ngaphezu kwalokho, ku-cell culture medium components ezifana ne-Dulbecco ithuthukisa ukuziphatha kokuhlanganisa ku-Eagle Medium kanye ne-Fetal Calf Serum.Imiphumela: Imiphumela ibonisa ukuthi i-pH ene-acidic kanye nokuqukethwe kwe-electrolyte ye-physiological ngokuvamile kudala ukuhlanganisa kwe-micron-scale, okungalamula ngokwakhiwa kwe-biomolecular corona.Kuyaphawuleka ukuthi izinhlayiya ezinkulu zibonisa ukumelana okuphezulu namathonya angaphandle kunozakwabo abancane.I-In vitro cytotoxicity nokuhlolwa kwe-antibacterial kwenziwa ngokwelapha amaseli ngama-nanoparticle aggregates ezigabeni ezihlukene zokuhlanganisa.Isiphetho: Imiphumela yethu yembula ukuhlobana okujulile phakathi kokuzinza kwe-colloidal kanye nobuthi be-AgNP, njengoba ukuhlanganisa ngokweqile kuholela ekulahlekelweni okuphelele komsebenzi webhayoloji.Izinga eliphakeme lokulwa nokuhlanganisa eliqashelwa izinhlayiya ezinkulu linomthelela omkhulu kubuthi be-in vitro, ngoba amasampula anjalo agcina umsebenzi weseli we-antimicrobial kanye nesilwane esincelisayo.Lokhu okutholakele kuholela esiphethweni sokuthi, naphezu kombono ojwayelekile ezincwadini ezifanele, ukukhomba ama-nanoparticles amancane kakhulu kungase kungabi inkambo engcono kakhulu yokwenza.Amagama angukhiye: ukukhula okuphakathi kwembewu, ukuzinza kwe-colloidal, ukuziphatha kokuhlanganisa okuncike kusayizi, ubuthi bokulimala kokuhlanganiswa
Njengoba isidingo nokukhishwa kwama-nanomaterials kuqhubeka ukwanda, kubhekwe lukhulu ku-biosafety yawo noma umsebenzi webhayoloji.Ama-nanoparticles esiliva (AgNPs) angomunye wabameleli abavame ukuhlanganiswa, abacwaningwa futhi abasetshenziswe kuleli klasi lezinto zokwakha ngenxa yezakhiwo zabo ezinhle kakhulu ze-catalytic, optical and biological.1 Kukholelwa ukuthi izici ezihlukile zama-nanomaterials (kuhlanganise nama-AgNP) zibangelwa kakhulu indawo yazo enkulu ethile.Ngakho-ke, inkinga engenakugwenywa yinoma iyiphi inqubo ethinta lesi sici esiyinhloko, njengosayizi wezinhlayiya, ukumbozwa kwendawo noma ukuhlanganisa, kungakhathaliseki ukuthi izolimaza kakhulu izakhiwo ze-nanoparticles ezibalulekile kuzinhlelo zokusebenza ezithile.
Imiphumela yobukhulu bezinhlayiyana neziqinisi yizihloko eziye zabhalwa kahle ezincwadini.Isibonelo, umbono owamukelwa ngokuvamile wukuthi ama-nanoparticles amancane anobuthi kakhulu kunama-nanoparticles amakhulu.2 Ngokuvumelana nezincwadi ezijwayelekile, izifundo zethu zangaphambilini zibonise umsebenzi oncike kusayizi we-nanosilver kumaseli ezincelisayo nama-microorganisms.3– 5 Ukufakwa phezu komhlaba ngesinye isici esinomthelela obanzi kuzakhiwo zama-nanomaterials.Ngokungeza nje noma ngokushintsha izinza endaweni yayo, i-nanomaterial efanayo ingaba nezakhiwo ezihluke ngokuphelele ezingokomzimba, zamakhemikhali, nezebhayoloji.Ukusetshenziswa kwama-ejenti we-capping ngokuvamile kwenziwa njengengxenye ye-nanoparticle synthesis.Isibonelo, ama-nanoparticles esiliva anqanyuliwe i-citrate angenye ye-AgNP ebaluleke kakhulu ocwaningweni, ahlanganiswa ngokunciphisa usawoti wesiliva esixazululweni se-stabilizer esikhethiwe njengendlela yokusabela.I-Citrate ye-6 ingakwazi ukusizakala kalula ngezindleko zayo eziphansi, ukutholakala, i-biocompatibility, kanye nokuhlobana okuqinile kwesiliva, okungabonakala ekusebenzelaneni okuhlukahlukene okuhlongozwayo, kusukela ekukhangiseni kwendawo okubuyisela emuva kuya ekusebenzisaneni kwe-ionic.Ama-molecule amancane nama-polyatomic ion aseduze no-7,8, njenge-citrate, ama-polymers, ama-polyelectrolytes, nama-ejenti ebhayoloji asetshenziswa ngokuvamile ukuze kunzinziswe i-nano-silver nokwenza imisebenzi eyingqayizivele kuyo.9-12
Nakuba kungenzeka ukuguqula umsebenzi we-nanoparticles ngokufaka i-surface capping ngamabomu kuyindawo ethakazelisayo kakhulu, indima eyinhloko yalesi simbozo esingaphezulu ayinalutho, ihlinzeka ngokuzinza kwe-colloidal ohlelweni lwe-nanoparticle.Indawo enkulu ethize ye-nanomatadium izokhiqiza amandla amakhulu angaphezulu, okuvimbela ikhono le-thermodynamic lohlelo ukufinyelela amandla alo amancane.13 Ngaphandle kokuzinzisa okufanele, lokhu kungaholela ekuhlanganisweni kwama-nanomaterials.Ukuhlanganisa ukwakheka kwamaqoqo ezinhlayiya zomumo nosayizi abahlukahlukene okwenzeka lapho izinhlayiya ezihlakazekile zihlangana futhi ukusebenzisana kwamanje kwe-thermodynamic kuvumela izinhlayiya ukuthi zinamathelane.Ngakho-ke, ama-stabilizers asetshenziselwa ukuvimbela ukuhlanganisa ngokwethula amandla amakhulu ngokwanele aphikisayo phakathi kwezinhlayiya ukuze abhekane nokukhanga kwazo kwe-thermodynamic.14
Nakuba isihloko sosayizi wezinhlayiya kanye nokumbozwa kwendawo kuye kwahlolisiswa kahle kumongo wokulawulwa kwayo kwemisebenzi yezinto eziphilayo ezibangelwa ama-nanoparticles, ukuhlanganisa izinhlayiyana kuyindawo enganakiwe kakhulu.Cishe alukho ucwaningo olunzulu lokuxazulula ukuzinza kwe-colloidal ye-nanoparticles ngaphansi kwezimo ezifanele ze-biologically.10,15-17 Ngaphezu kwalokho, lo mnikelo awuvamile ikakhulukazi, lapho ubuthi obuhlobene nokuhlanganiswa buye bacwaningwa, ngisho noma bungabangela ukusabela okubi, njenge-vascular thrombosis, noma ukulahlekelwa izici ezifiselekayo, njengobuthi bayo, njengoba kuboniswe kuMfanekiso 1.18, 19.Eqinisweni, enye yezindlela ezimbalwa ezaziwayo zokumelana ne-nanoparticle yesiliva ihlobene nokuhlanganisa, ngoba izinhlobo ezithile ze-E. coli ne-Pseudomonas aeruginosa kubikwa ukuthi zehlisa ukuzwela kwazo kwe-nano-silver ngokuveza iphrotheni flagellin, i-flagelin.Inokuhambisana okuphezulu kwesiliva, ngaleyo ndlela ikhuthaza ukuhlanganisa.20
Kunezindlela eziningana ezihlukene ezihlobene nobuthi be-nanoparticles esiliva, futhi ukuhlanganisa kuthinta zonke lezi zindlela.Indlela okuxoxwa ngayo kakhulu yomsebenzi webhayoloji ye-AgNP, kwesinye isikhathi ebizwa ngokuthi “i-Trojan Horse” indlela, ibheka ama-AgNP njengabathwali be-Ag+.1,21 Indlela yehhashi leTrojani ingaqinisekisa ukwanda okukhulu kokugxiliswa kwe-Ag+ yendawo, okuholela ekukhiqizeni i-ROS kanye ne-membrane depolarization.22-24 Ukuhlanganisa kungase kuthinte ukukhululwa kwe-Ag+, ngaleyo ndlela kuthinte ubuthi, ngoba kunciphisa indawo esebenzayo esebenzayo lapho ama-ion esiliva angase afakwe i-oxidized futhi ancibilike.Kodwa-ke, ama-AgNP ngeke abonise ubuthi kuphela ngokukhululwa kwe-ion.Kufanele kucatshangelwe ukuhlangana okuningi okuhlobene nosayizi kanye ne-morphology.Phakathi kwazo, ubukhulu nokuma kwendawo ye-nanoparticle yizici ezichazayo.4,25 Ukuqoqwa kwalezi zindlela kungahlukaniswa “njengezindlela zobuthi obubangelwayo.”Kunokusabela okuningi kwe-mitochondrial kanye ne-surface membrane engalimaza ama-organelle futhi kubangele ukufa kwamaseli.25-27 Njengoba ukwakheka kwama-aggregate kuthinta ngokwemvelo ubukhulu kanye nokuma kwezinto eziqukethe isiliva ezibonwa izinhlelo eziphilayo, lokhu kusebenzisana kungase futhi kuthinteke.
Ephepheni lethu langaphambilini lokuhlanganiswa kwama-nanoparticles esiliva, sibonise inqubo yokuhlola esebenzayo ehlanganisa ukuhlolwa kwamakhemikhali kanye ne-in vitro biological ukutadisha le nkinga.19 I-Dynamic Light Scattering (i-DLS) iyindlela ekhethwayo yalezi zinhlobo zokuhlola ngoba okokusebenza kungasakaza ama-photon ngobude begagasi obuqhathaniswa nosayizi wezinhlayiya zayo.Njengoba isivinini sokunyakaza kwe-Brownian sezinhlayiyana endaweni ewuketshezi ihlobene nosayizi, ukuguqulwa kokuqina kokukhanya okuhlakazekile kungasetshenziswa ukunquma isilinganiso se-hydrodynamic diameter (Z-mean) yesampula yoketshezi.28 Ngaphezu kwalokho, ngokusebenzisa i-voltage kusampula, amandla e-zeta (ζ amandla) we-nanoparticle angalinganiswa ngendlela efanayo nenani eliyisilinganiso esingu-Z.13,28 Uma inani eliphelele lamandla e-zeta liphezulu ngokwanele (ngokweziqondiso ezijwayelekile> ± 30 mV), lizokhiqiza ukuphindisela okuqinile kwe-electrostatic phakathi kwezinhlayiya ukuze kuliwe nokuhlanganisa.I-Characteristic surface plasmon resonance (SPR) yinto eyingqayizivele yokubona, ikakhulukazi ebangelwa ama-nanoparticles ensimbi ayigugu (ikakhulukazi i-Au ne-Ag).29​​ Ngokusekelwe ku-electronic oscillations (ama-plasmons angaphezulu) alezi zinto ku-nanoscale, kuyaziwa ukuthi ama-AgNP ayindilinga anesici sokumuncwa kwe-UV-Vis eduze kuka-400 nm.30 Ukuqina nokuguquka kwe-wavelength kwezinhlayiya kusetshenziselwa ukwengeza imiphumela ye-DLS, njengoba le ndlela ingasetshenziswa ukuthola ukuhlanganisa kwe-nanoparticle kanye nokukhangiswa kwendawo kwama-biomolecules.
Ngokusekelwe olwazini olutholiwe, ukusebenza kweseli (MTT) kanye nokuhlolwa kwe-antibacterial kwenziwa ngendlela ubuthi be-AgNP buchazwa ngayo njengomsebenzi wezinga lokuhlanganisa, kunokuba (isici esisetshenziswa kakhulu) ukugxiliswa kwe-nanoparticle.Le ndlela eyingqayizivele isivumela ukuthi sibonise ukubaluleka okujulile kwezinga lokuhlanganisa emsebenzini webhayoloji, ngoba, isibonelo, ama-AgNP anqanyulwe nge-citrate alahlekelwa ngokuphelele umsebenzi wawo webhayoloji phakathi namahora ambalwa ngenxa yokuhlanganisa.19
Emsebenzini wamanje, sihlose ukwandisa kakhulu iminikelo yethu yangaphambilini ekuzinzeni kwama-colloid ahlobene ne-bio kanye nomthelela wawo emsebenzini webhayoloji ngokutadisha umphumela wosayizi we-nanoparticle ekuhlanganisweni kwe-nanoparticle.Lokhu ngokungangabazeki kungenye yezifundo ze-nanoparticles.Umbono wephrofayili ephezulu kanye no-31 Ukuze kuphenywe le nkinga, indlela yokukhula eqondiswa yimbewu yasetshenziswa ukuze kukhiqizwe ama-AgNP anqanyuliwe nge-citrate kububanzi obuthathu obuhlukene bosayizi (10, 20, kanye no-50 nm).6,32 njengenye yezindlela ezivame kakhulu.Kuma-nanomatadium asetshenziswa kakhulu futhi asetshenziswa njalo ezinhlelweni zezokwelapha, ama-AgNP anqanyulwe nge-citrate anosayizi abahlukahlukene akhethwa ukuze kufundwe ukuthi kungenzeka yini ukuncika kosayizi wezakhiwo zebhayoloji ezihlobene nokuhlanganiswa ze-nanosilver.Ngemuva kokwenza ama-AgNP osayizi abahlukene, siveze amasampula akhiqiziwe nge-transmission electron microscopy (TEM), bese sihlola izinhlayiya sisebenzisa inqubo yokuhlola eshiwo ngenhla.Ngaphezu kwalokho, ebukhoneni be-in vitro cell cultures ye-Dulbecco's Modified Eagle's Medium (DMEM) kanye ne-Fetal Bovine Serum (FBS), ukuziphatha kokuhlanganisa okuncike kusayizi nokuziphatha kwayo kwahlaziywa ngamavelu e-pH ahlukahlukene, i-NaCl, glucose, kanye ne-glutamine.Izici ze-cytotoxicity zinqunywa ngaphansi kwezimo ezibanzi.Ukuvumelana kwesayensi kubonisa ukuthi ngokuvamile, izinhlayiya ezincane zikhethwa;uphenyo lwethu luhlinzeka ngenkundla yamakhemikhali neyebhayoloji ukuze kutholwe ukuthi ngabe lokhu kunjalo.
Ama-nanoparticles amathathu esiliva anobubanzi obuhlukene bosayizi alungiselelwa indlela yokukhula eqondiswa yimbewu ehlongozwe u-Wan et al., ngokulungiswa okuncane.6 Le ndlela isekelwe ekunciphiseni kwamakhemikhali, kusetshenziswa i-nitrate yesiliva (AgNO3) njengomthombo wesiliva, i-sodium borohydride (NaBH4) njenge-ejenti yokunciphisa, kanye ne-sodium citrate njenge-stabilizer.Okokuqala, lungiselela u-75 mL we-9 mM citrate aqueous solution kusuka ku-sodium citrate dihydrate (Na3C6H5O7 x 2H2O) nokushisa ku-70°C.Bese, u-2 mL we-1% w/v AgNO3 isixazululo sengezwe endaweni yokusabela, kwase-ke isixazululo esisanda kulungiswa se-sodium borohydride (2 mL 0.1% w/v) sithelwa engxubeni ye-dropwise.Ukumiswa okuphuzile okuphuzi kwagcinwa ku-70 ° C ngokugqugquzela ngamandla ihora elingu-1, bese kupholiswa ekamelweni lokushisa.Isampula eliwumphumela (okubhekiselwa kulo njenge-AgNP-I kusukela manje kuqhubeke) lisetshenziswa njengesisekelo sokukhula okugxiliswe yimbewu esinyathelweni esilandelayo sokuhlanganisa.
Ukuze uhlanganise ukumiswa kwezinhlayiyana ezinosayizi omaphakathi (okuthiwa i-AgNP-II), shisa isixazululo se-citrate esingu-90 mL 7.6 mM sibe ngu-80°C, sixube no-10 mL AgNP-I, bese uhlanganisa u-2 mL 1% w/v Isixazululo se-AgNO3 igcinwe ngaphansi kokunyakaziswa okunamandla kwemishini ihora elingu-1, bese isampula lipholiswe ekamelweni lokushisa.
Ngezinhlayiyana ezinkulu kunazo zonke (i-AgNP-III), phinda inqubo efanayo yokukhula, kodwa kulesi simo, sebenzisa u-10 mL we-AgNP-II njengokumiswa kwembewu.Ngemva kokuba amasampula efinyelele izinga lokushisa legumbi, asetha ukugxila kwawo kwe-Ag ngokusekelwe enanini eliphelele le-AgNO3 libe ngu-150 ppm ngokwengeza noma ahwamulise isinyibilikisi esengeziwe ku-40°C, futhi ekugcineni azigcine ku-4°C kuze kuphinde kusetshenziswe.
Sebenzisa i-FEI Tecnai G2 20 X-Twin Transmission Electron Microscope (TEM) (FEI Corporate Headquarters, Hillsboro, Oregon, USA) ne-voltage yokusheshisa engu-200 kV ukuze uhlole izici ze-morphological zama-nanoparticles futhi uthwebule iphethini yawo ye-electron diffraction (ED).Okungenani izithombe ezimele abantu abangu-15 (~750 izinhlayiya) zahlolwa kusetshenziswa iphakheji yesofthiwe ye-ImageJ, futhi ama-histograms angumphumela (nawo wonke amagrafu ocwaningweni lonke) adalwe ku-OriginPro 2018 (OriginLab, Northampton, MA, USA) 33, 34.
I-avareji yobubanzi be-hydrodynamic (i-Z-isilinganiso), amandla e-zeta (ζ-amandla) kanye nesici se-plasmon resonance (SPR) yamasampula kulinganiswe ukuze kuboniswe izici zawo zokuqala ze-colloidal.Isilinganiso sobubanzi be-hydrodynamic namandla e-zeta esampula kukalwe ithuluzi le-Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) kusetshenziswa amaseli e-capillary agoqiwe alahlwayo kokuthi 37±0.1°C.I-Ocean Optics 355 DH-2000-BAL UV-Vis spectrophotometer (Halma PLC, Largo, FL, USA) yasetshenziselwa ukuthola izici ze-SPR ezivela ku-UV-Vis yokumuncwa kwamasampula ebangeni elingu-250-800 nm.
Phakathi nalo lonke ucwaningo, izinhlobo ezintathu zokulinganisa ezihlukene ezihlobene nokuzinza kwe-colloidal zenziwa ngesikhathi esisodwa.Sebenzisa i-DLS ukuze ulinganise ububanzi obumaphakathi be-hydrodynamic (isilinganiso esingu-Z) namandla e-zeta (amandla ka-ζ) ezinhlayiya, ngoba isilinganiso sika-Z sihlobene nosayizi omaphakathi wama-nanoparticle aggregates, futhi amandla e-zeta abonisa ukuthi ukuphindiselwa kwe-electrostatic ohlelweni iqine ngokwanele ukuvala ukukhanga kukaVan der Waals phakathi kwama-nanoparticles.Izilinganiso zenziwa ngokuphindwe kathathu, futhi ukuchezuka okujwayelekile kokuthi Z mean namandla e-zeta kubalwa isofthiwe ye-Zetasizer.Isici se-SPR sezinhlayiyana sihlolwa i-UV-Vis spectroscopy, ngoba izinguquko kubukhulu obuphakeme nobude begagasi zingabonisa ukuhlanganisa kanye nokusebenzisana kwendawo.29,35 Eqinisweni, i-surface plasmon resonance ezinsimbi eziyigugu inomthelela kangangokuthi kuye kwaholela ezindleleni ezintsha zokuhlaziya ama-biomolecules.29,36,37 Ukugxiliswa kwama-AgNP kungxube yokuhlola cishe ku-10 ppm, futhi inhloso ukusetha ukushuba kokumuncwa okuphezulu kokuqala kwe-SPR ku-1. Ukuhlolwa kwenziwe ngendlela encike esikhathini kokuthi 0;1.5;3;6;Amahora angu-12 nama-24 ngaphansi kwezimo ezihlukahlukene zebhayoloji efanele.Imininingwane eyengeziwe echaza isilingo ingabonwa emsebenzini wethu wangaphambilini.19 Ngamafuphi, amanani e-pH ahlukahlukene (3; 5; 7.2 kanye no-9), i-sodium chloride ehlukene (10 mM; 50 mM; 150 mM), i-glucose (3.9 mM; 6.7 mM) ne-glutamine (4 mM) ukugxila, kanye iphinde yalungiselela i-Dulbecco's Modified Eagle Medium (DMEM) kanye ne-Fetal Bovine Serum (FBS) (emanzini ne-DMEM) njengamamodeli amasistimu, futhi yafunda imiphumela yazo ekuziphatheni kokuhlanganisa kwama-nanoparticles esiliva ahlanganisiwe.pH Amanani e-, NaCl, glucose, kanye ne-glutamine ayahlolwa ngokusekelwe ekugxiliseni komzimba, kuyilapho amanani e-DMEM ne-FBS afana namazinga asetshenziswe kulo lonke ucwaningo lwe-in vitro.38-42 Zonke izilinganiso zenziwa ku-pH 7.2 no-37°C ngokugxiliswa kosawoti okungemuva okungashintshi okungu-10 mM NaCl ukuze kuqedwe noma yikuphi ukusebenzisana kwezinhlayiyana zebanga elide (ngaphandle kokuhlolwa okuthile okuhlobene ne-pH kanye ne-NaCl, lapho lezi zibaluli ziyiziguquko ngaphansi isifundo).28 Uhlu lwezimo ezihlukahlukene lufinyezwa kuThebula 1. Ukuhlolwa okumakwe ngokuthi † kusetshenziswa njengereferensi futhi kuhambisana nesampula equkethe u-10 mM NaCl kanye ne-pH 7.2.
I-Human Prostate Cancer cell line (DU145) kanye nama-keratinocyte abantu abangafi (HaCaT) atholwe kwa-ATCC (Manassas, VA, USA).Amaseli akhiqizwa ngokujwayelekile kuKhozi elimaphakathi elibalulekile lase-Dulbecco (DMEM) eliqukethe i-4.5 g/L glucose (Sigma-Aldrich, Saint Louis, MO, USA), engezwe ngo-10% FBS, 2 mM L-glutamine, 0.01 % Streptomycin no-0.005% I-Penicillin (Sigma-Aldrich, St. Louis, Missouri, USA).Amaseli akhuliswa ku-incubator engu-37°C ngaphansi kuka-5% CO2 kanye nomswakama ongu-95%.
Ukuze uhlole izinguquko ku-AgNP cytotoxicity ebangelwa ukuhlanganisa izinhlayiyana ngendlela encike esikhathini, ukulinganisa kwe-MTT okuzinyathelo ezimbili kwenziwa.Okokuqala, ukusebenza kwezinhlobo zamaseli amabili kukalwa ngemva kokwelashwa nge-AgNP-I, AgNP-II kanye ne-AgNP-III.Kuze kube manje, lezi zinhlobo ezimbili zamangqamuzana zifakwe ku-96-well plates ekuxubeni kwamaseli angu-10,000 / kahle futhi ziphathwe ngamasayizi amathathu ahlukene we-nanoparticles esiliva ekugxilweni okwandayo ngosuku lwesibili.Ngemva kwamahora angu-24 okwelashwa, amaseli ahlanjululwa nge-PBS futhi afakwa ku-0.5 mg/mL MTT reagent (SERVA, Heidelberg, Germany) ehlanjululwe endaweni yesiko ihora elingu-1 ku-37°C.Amakristalu e-Formazan ahlakazwa ku-DMSO (Sigma-Aldrich, Saint Louis, MO, USA), futhi ukumuncwa kukalwa ku-570 nm kusetshenziswa i-Synergy HTX plate reader (BioTek-Hungary, Budapest, Hungary).Inani lokumuncwa lesampula yokulawula engelashiwe libhekwa njengesilinganiso sokusinda esingu-100%.Yenza okungenani izivivinyo ezi-3 usebenzisa izifaniso ezine ezizimele zebhayoloji.I-IC50 ibalwa kusukela kwijika lempendulo yomthamo ngokusekelwe emiphumeleni yamandla.
Ngemva kwalokho, esinyathelweni sesibili, ngokufukamela izinhlayiya nge-150 mM NaCl izikhathi ezihlukene (0, 1.5, 3, 6, 12, kanye namahora angu-24) ngaphambi kokwelashwa kwamaseli, izimo ezihlukene zokuhlanganisa ama-nanoparticles esiliva akhiqizwa.Kamuva, ukuhlolwa okufanayo kwe-MTT kwenziwa njengoba kuchazwe ngaphambilini ukuze kuhlolwe izinguquko ekusebenzeni kweseli okuthonywe ukuhlanganisa izinhlayiyana.Sebenzisa i-GraphPad Prism 7 ukuze uhlole umphumela wokugcina, ubale ukubaluleka kwezibalo kokuhlolwa ngokuhlolwa kwe-t okungabhanqiwe, bese umaka ileveli njengokuthi * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001 ) Futhi **** (p ≤ 0.0001).
Osayizi abathathu abahlukene be-nanoparticles esiliva (i-AgNP-I, i-AgNP-II ne-AgNP-III) basetshenziselwa ukuba sengozini yokulwa namagciwane ku-Cryptococcus neoformans IFM 5844 (IFM; Isikhungo Sokucwaninga se-Pathogenic Fungi kanye ne-Microbial Toxicology, i-Chiba University) kanye ne-Bacillus Test megaterium SZMC 60 (SZMC: Szeged Microbiology Collection) kanye ne-E. coli SZMC 0582 ku-RPMI 1640 medium (Sigma-Aldrich Co.).Ukuze kuhlolwe izinguquko emsebenzini we-antibacterial obangelwa ukuhlanganiswa kwezinhlayiya, okokuqala, ukugxila kwabo okuncane kakhulu kwe-inhibitory (MIC) kunqunywe yi-microdilution epuleti le-microtiter engu-96-well.Ku-50 μL wokumiswa kweseli okujwayelekile (amaseli angu-5 × 104/mL ku-RPMI 1640 medium), engeza u-50 μL we-nanoparticle yesiliva ukumiswa futhi uhlambulule ngokuphindwe kabili ukugxila (endaweni eshiwo ngenhla, ububanzi bungu-0 no-75 ppm, okungukuthi, isampula yokulawula iqukethe u-50 μL wokumiswa kweseli kanye no-50 μL we-medium ngaphandle kwama-nanoparticles).Ngemva kwalokho, ipuleti lafakwa ku-30°C amahora angu-48, futhi ukuminyana kokubona kwesiko kwalinganiswa ku-620 nm kusetshenziswa i-SPECTROstar Nano plate reader (BMG LabTech, Offenburg, Germany).Ukuhlolwa kwenziwa kathathu ngokuphindwe kathathu.
Ngaphandle kokuthi u-50 μL wamasampula e-nanoparticle ahlanganisiwe asetshenzisiwe ngalesi sikhathi, inqubo efanayo njengoba ichazwe ngaphambilini yasetshenziswa ukuhlola umthelela wokuhlanganisa emsebenzini wokulwa namagciwane ezinhlotsheni ezishiwo ngenhla.Izimo ezihlukene zokuhlanganisa ama-nanoparticles esiliva akhiqizwa ngokufukamela izinhlayiya nge-150 mM NaCl izikhathi ezihlukene (0, 1.5, 3, 6, 12, kanye namahora angu-24) ngaphambi kokucubungula iseli.Ukumiswa okuhlanganiswe no-50 μL we-RPMI 1640 medium kwasetshenziswa njengokulawula ukukhula, kuyilapho ukuze kulawulwe ubuthi, ukumiswa okunama-nanoparticles angahlanganisiwe kwasetshenziswa.Ukuhlolwa kwenziwa kathathu ngokuphindwe kathathu.Sebenzisa i-GraphPad Prism 7 ukuze uhlole umphumela wokugcina futhi, usebenzisa ukuhlaziya kwezibalo okufanayo nokuhlaziywa kwe-MTT.
Izinga lokuhlanganisa lezinhlayiya ezincane kunazo zonke (i-AgNP-I) libonakalisiwe, futhi imiphumela yashicilelwa kancane emsebenzini wethu wangaphambilini, kodwa ukuze kuqhathaniswe kangcono, zonke izinhlayiya zahlolwa ngokucophelela.Idatha yokuhlola iyaqoqwa futhi kuxoxwe ngayo ezigabeni ezilandelayo.Osayizi abathathu be-AgNP.19
Izilinganiso ezenziwa yi-TEM, i-UV-Vis ne-DLS ziqinisekise ukuhlanganiswa okuyimpumelelo kwawo wonke amasampula e-AgNP (Umfanekiso 2A-D).Ngokomugqa wokuqala woMfanekiso 2, izinhlayiyana ezincane kunazo zonke (i-AgNP-I) zibonisa i-morphology eyindilinga efanayo enobubanzi obuyisilinganiso obungaba ngu-10 nm.Indlela yokukhula eqondiswa yimbewu iphinde inikeze i-AgNP-II ne-AgNP-III ngobubanzi obuhlukene bosayizi obunobubanzi bezinhlayiya ezimaphakathi cishe obungama-20 nm no-50 nm, ngokulandelana.Ngokuchezuka okujwayelekile kokusatshalaliswa kwezinhlayiyana, osayizi bamasampuli amathathu abadluleli, okubalulekile ekuhlaziyeni kwabo okuqhathanisayo.Ngokuqhathanisa i-aspect ratio eyisilinganiso kanye ne-thiness ratio ye-TEM-based particle 2D projection, kucatshangwa ukuthi i-sphericity yezinhlayiya ihlolwa i-plug-in yesihlungi somumo we-ImageJ (Umfanekiso 2E).43 Ngokohlaziyo lokuma kwezinhlayiya, i-aspect ratio (uhlangothi olukhulu/uhlangothi olufushane lukanxande oluncane kakhulu olubophayo) alithintwa ukukhula kwezinhlayiya, kanye nesilinganiso somzimba wazo omncane (indawo elinganiselwe yendingilizi ehambisanayo/indawo yethiyori. ) iyancipha kancane kancane.Lokhu kubangela izinhlayiya eziningi ze-polyhedral, eziyindilinga ngokuphelele ngokombono, ezihambisana nesilinganiso sobuncane esingu-1.
Umfanekiso 2 Isithombe se-electron microscope (TEM) sokudlulisa (A), iphethini ye-electron diffraction (ED) (B), i-histogram yokusabalalisa usayizi (C), isici esibonakalayo se-ultraviolet-visible (UV-Vis) i-spectrum yokumunca ukukhanya (D), kanye ne-avareji ye-Citrate yoketshezi -ama-nanoparticles esiliva anqanyuliwe anobubanzi bomshini (i-Z-avarage), amandla e-zeta, i-aspect ratio kanye nesilinganiso sokuqina (E) anezigaba ezintathu zosayizi abahlukene: I-AgNP-I ingu-10 nm (umugqa ophezulu), i-AgNP -II ingu-20 nm (umugqa ophakathi ), i-AgNP-III (umugqa ongezansi) ingu-50 nm.
Nakuba imvelo yomjikelezo wendlela yokukhula ithinte ukwakheka kwezinhlayiyana ngezinga elithile, okubangele i-sphericity encane yama-AgNP amakhulu, wonke amasampuli amathathu ahlala e-quasi-spherical.Ngaphezu kwalokho, njengoba kuboniswe kuphethini ye-electron diffraction ku-Figure 2B, nano I-crystallinity yezinhlayiya ayithinteki.Iringi ye-diffraction evelele-engahlotshaniswa nezinhlamvu zesiliva (111), (220), (200), kanye (311) ze-Miller zesiliva-ihambisana kakhulu nemibhalo yesayensi kanye neminikelo yethu yangaphambilini.9, 19,44 Ukuhlukaniswa kweringi ye-Debye-Scherrer ye-AgNP-II ne-AgNP-III kungenxa yokuthi isithombe se-ED sithwetshulwa ngokukhuliswa okufanayo, ukuze njengoba usayizi wezinhlayiyana ukhula, inani lezinhlayiya ezihlukanisiwe indawo yeyunithi iyanda futhi iyancipha.
Usayizi nokuma kwe-nanoparticles kwaziwa ukuthi kuthinta umsebenzi webhayoloji.3,45 Umsebenzi we-catalytic kanye ne-biological oncike ekubunjweni ungachazwa ngokuthi izimo ezihlukene zivame ukwandisa ubuso obucwebezelayo obuthile (ukuba nama-Miller indices ahlukene), futhi lobu buso bekristalu bunemisebenzi ehlukene.I-45,46 Njengoba izinhlayiya ezilungisiwe zihlinzeka ngemiphumela ye-ED efanayo ehambisana nezici ze-crystal ezifanayo kakhulu, kungacatshangwa ukuthi ekuzinzeni kwethu okulandelayo kwe-colloidal kanye nokuhlolwa komsebenzi wezinto eziphilayo, noma yikuphi ukungafani okubonwayo kufanele kufakwe kusayizi we-Nanoparticle, hhayi izakhiwo ezihlobene nokuma.
Imiphumela ye-UV-Vis efingqiwe ku-Figure 2D iphinde igcizelele ubunjalo obumangalisayo obuyindilinga be-AgNP ehlanganisiwe, ngoba iziqongo ze-SPR zawo womathathu amasampula acishe abe ngu-400 nm, okuyinani eliyisici lama-nanoparticles esiliva ayindilinga.29,30 I-spectra eqoshiwe iphinde yaqinisekisa ukukhula okuphumelelayo kwembewu ye-nanosilver.Njengoba usayizi wezinhlayiyana ukhula, ubude begagasi obuhambisana nokumuncwa kokukhanya okuphezulu kwe-AgNP-II-ngokugqama kakhulu-Ngokusho kwezincwadi, i-AgNP-III Ithole i-redshift.6,29
Ngokuphathelene nokuzinza kwe-colloidal kokuqala kohlelo lwe-AgNP, i-DLS yasetshenziswa ukukala ububanzi obumaphakathi be-hydrodynamic namandla e-zeta ezinhlayiya ku-pH 7.2.Imiphumela evezwe ku-Figure 2E ibonisa ukuthi i-AgNP-III inokusimama okuphezulu kwe-colloidal kune-AgNP-I noma i-AgNP-II, ngoba imihlahlandlela evamile ibonisa ukuthi amandla e-zeta we-30 mV absolute ayadingeka ukuze kube nokuzinza kwe-colloidal yesikhathi eside Lokhu okutholakele kusekelwa nakakhulu lapho. inani elingu-Z (elitholwe njenge-avareji ye-hydrodynamic diameter yezinhlayiya ezikhululekile nezihlanganisiwe) liqhathaniswa nosayizi wezinhlayiyana oyinhloko otholwe yi-TEM, ngoba uma amanani amabili esondele, iba mnene idigri ethi Qoqa kusampula.Eqinisweni, isilinganiso se-Z se-AgNP-I ne-AgNP-II siphakeme ngokunengqondo kunosayizi wezinhlayiyana ezihlaziywe nge-TEM, ngakho-ke uma kuqhathaniswa ne-AgNP-III, lawa masampuli abikezelwa ukuthi angaba maningi amathuba okuthi ahlanganiswe, lapho amandla e-zeta angemuhle kakhulu. ihambisana nosayizi oseduze Inani elingu-Z eliyisilinganiso.
Incazelo yalesi simo ingaba kabili.Ngakolunye uhlangothi, ukugxiliswa kwe-citrate kugcinwa ezingeni elifanayo kuzo zonke izinyathelo zokuhlanganiswa, okuhlinzeka ngenani eliphezulu lamaqembu angaphezulu akhokhiswayo ukuze kuvinjelwe indawo ethile yezinhlayiya ezikhulayo ukuthi zinciphe.Kodwa-ke, ngokusho kukaLevak et al., ama-molecule amancane afana ne-citrate angashintshaniswa kalula ngama-biomolecules ebusweni be-nanoparticles.Kulokhu, ukuzinza kwe-colloidal kuzonqunywa yi-corona yama-biomolecules akhiqizwayo.31 Ngenxa yokuthi lokhu kuziphatha kuphinde kwabonwa ezilinganisweni zethu zokuhlanganisa (okuxoxwe ngemininingwane eyengeziwe kamuva), i-citrate capping iyodwa ayikwazi ukuchaza lesi sigameko.
Ngakolunye uhlangothi, usayizi wezinhlayiyana uhambisana ngokuphambene nokuthambekela kokuhlanganisa ezingeni le-nanometer.Lokhu kusekelwa ikakhulukazi indlela yendabuko ye-Derjaguin-Landau-Verwey-Overbeek (DLVO), lapho ukukhanga kwezinhlayiyana kuchazwa njengesamba samandla ahehayo nanyanyekayo phakathi kwezinhlayiya.Ngokusho kuka-He et al., inani eliphakeme lejika le-DLVO lamandla liyancipha ngobukhulu be-nanoparticles ku-hematite nanoparticles, okwenza kube lula ukufinyelela amandla amancane ayisisekelo, ngaleyo ndlela kukhuthaze ukuhlanganisa okungenakuhlehliswa (i-condensation).47 Nokho, kucatshangelwa ukuthi kukhona ezinye izici ezingaphezu kwemikhawulo yethiyori ye-DLVO.Nakuba amandla adonsela phansi e-van der Waals kanye ne-electrostatic double-layer repulsion kufana nokwanda kosayizi wezinhlayiyana, isibuyekezo sika-Hotze et al.iphakamisa ukuthi inomthelela onamandla ekuhlanganiseni kunokuvumela i-DLVO.14 Bakholelwa ukuthi ukugoba okungaphezulu kwama-nanoparticles ngeke kusalinganiswa njengendawo eyisicaba, okwenza ukulinganisa kwezibalo kungasebenzi.Ukwengeza, njengoba usayizi wezinhlayiyana uncipha, iphesenti lama-athomu akhona endaweni liba phezulu, okuholela ekwakhiweni kwe-elekthronikhi nokuziphatha kokushaja okungaphezulu.Futhi izinguquko ze-surface reactivity, ezingase ziholele ekwehleni kweshajethi esendlalelo esiphindwe kabili kagesi futhi kukhuthaze ukuhlanganisa.
Lapho siqhathanisa imiphumela ye-DLS ye-AgNP-I, AgNP-II, ne-AgNP-III kumfanekiso 3, siqaphele ukuthi womathathu amasampuli abonise ukuhlanganisa kwe-pH okufanayo.Imvelo ene-acidic kakhulu (pH 3) ishintsha amandla e-zeta esampula kuya ku-0 mV, okwenza izinhlayiya zenze ama-aggregate osayizi we-micron, kuyilapho i-alkaline pH ishintsha amandla ayo e-zeta abe inani elibi elikhudlwana, lapho izinhlayiya zakha ama-aggregate amancane (pH 5). ).Futhi 7.2) ), noma uhlale ungahlanganisiwe ngokuphelele (pH 9).Umehluko obalulekile phakathi kwamasampuli ahlukene nawo wabonwa.Kuso sonke isilingo, i-AgNP-I ibonise ukuthi izwela kakhulu ku-pH-induced zeta izinguquko ezingase zibe khona, ngoba amandla e-zeta alezi zinhlayiya ancishisiwe ku-pH 7.2 uma kuqhathaniswa ne-pH 9, kuyilapho i-AgNP-II ne-AgNP-III ibonise u-A kuphela. ushintsho olukhulu ku-ζ cishe luyi-pH 3. Ngaphezu kwalokho, i-AgNP-II ibonise izinguquko ezihamba kancane namandla e-zeta amaphakathi, kuyilapho i-AgNP-III ibonise ukuziphatha okuthambile kokuthathu, ngoba uhlelo lubonise inani eliphakeme kakhulu le-zeta kanye nokunyakaza kokuhamba kancane, okubonisa I-AgNP-III Imelana Kakhulu ekuhlanganisweni kwe-pH.Le miphumela ihambisana nesilinganiso semiphumela yokulinganisa ububanzi be-hydrodynamic.Uma kucatshangelwa usayizi wezinhlayiyana zama-primers azo, i-AgNP-I ibonise ukuhlanganisa kancane kancane kuwo wonke amanani e-pH, okungenzeka kakhulu ngenxa yesizinda se-NaCl esingu-10 mM, kuyilapho i-AgNP-II ne-AgNP-III zibonise ukubaluleka kuphela ku-pH 3 Yokuqoqwa.Umehluko othakazelisa kakhulu ukuthi naphezu kobukhulu bayo obukhulu be-nanoparticle, i-AgNP-III yenza izilinganiso ezincane kakhulu ku-pH 3 emahoreni angu-24, igqamisa izici zayo zokulwa nokuhlanganisa.Ngokuhlukanisa isilinganiso se-Z sama-AgNP ku-pH 3 ngemva kwamahora angu-24 ngevelu yesampula elungisiwe, kungaqashelwa ukuthi osayizi abahlanganisiwe abahlobene be-AgNP-I ne-AgNP-II bakhuphuke izikhathi ezingu-50, izikhathi ezingu-42, nezikhathi ezingu-22. , ngokulandelana.III.
Umfanekiso 3 Imiphumela yokuhlakazeka kokukhanya okuguquguqukayo yesampula ye-nanoparticles yesiliva enqanyuliwe ye-citrate enosayizi okhulayo (10 nm: AgNP-I, 20 nm: AgNP-II kanye no-50 nm: AgNP-III) avezwa njengobubanzi obumaphakathi be-hydrodynamic (isilinganiso se-Z ) (kwesokudla) Ngaphansi kwezimo ze-pH ezihlukene, amandla e-zeta (kwesokunxele) ashintsha phakathi namahora angu-24.
Ukuhlanganisa okuqashelwe okuncike ku-pH kuphinde kwathinta isici se-plasmon resonance (SPR) yamasampuli e-AgNP, njengoba kufakazelwa isibuko sabo se-UV-Vis.Ngokuvumelana ne-Supplementary Figure S1, ukuhlanganiswa kwakho konke ukumiswa kwe-nanoparticle esiliva kulandelwa ukuncipha kokuqina kweziqongo zazo ze-SPR kanye noshintsho olubomvu olumaphakathi.Izinga lalezi zinguquko njengomsebenzi we-pH lihambisana nezinga lokuhlanganisa elibikezelwe imiphumela ye-DLS, nokho, amathrendi athile athakazelisayo abonwe.Ngokuphambene nokucabanga, kuvela ukuthi i-AgNP-II yosayizi omaphakathi izwela kakhulu izinguquko ze-SPR, kuyilapho amanye amasampula amabili ezwela kancane.Ocwaningweni lwe-SPR, i-50 nm ingumkhawulo kasayizi wezinhlayiyana, osetshenziselwa ukuhlukanisa izinhlayiya ngokusekelwe ezintweni zazo ze-dielectric.Izinhlayiya ezincane kune-50 nm (i-AgNP-I ne-AgNP-II) zingachazwa njenge-dielectric dipoles elula, kuyilapho izinhlayiya ezifinyelela noma ezidlula lo mkhawulo (AgNP-III) zinezici ze-dielectric eziyinkimbinkimbi, futhi i-resonance yazo Ibhendi ihlukana ibe izinguquko ze-multimodal. .Endabeni yamasampula ezinhlayiyana ezimbili ezincane, ama-AgNP angathathwa njengama-dipoles alula, futhi i-plasma ingadlulana kalula.Njengoba usayizi wezinhlayiyana ukhula, lokhu kuhlangana kukhiqiza i-plasma enkulu, engachaza ukuzwela okuphezulu okubonwayo.29 Nokho, ezinhlayiyeni ezinkulu kakhulu, ukulinganisa okulula kwe-dipole akuvumelekile lapho ezinye izimo zokuhlanganisa zingase zenzeke, ezingachaza ukuthambekela okwehlisiwe kwe-AgNP-III ukukhombisa izinguquko ze-spectral.29
Ngaphansi kwezimo zethu zokuhlola, kufakazelwa ukuthi inani le-pH linomthelela omkhulu ekuzinzeni kwe-colloidal yama-nanoparticles esiliva ane-citrate anosayizi abahlukahlukene.Kulezi zinhlelo, ukuzinza kunikezwa amaqembu -COO- anecala elibi endaweni yama-AgNP.Iqembu elisebenzayo le-carboxylate le-ion citrate line-protoned enanini elikhulu lama-H+ ion, ngakho-ke iqembu le-carboxyl elakhiwe alisakwazi ukunikeza ukunyanyiswa kwe-electrostatic phakathi kwezinhlayiya, njengoba kuboniswe kumugqa ophezulu woMfanekiso 4. Ngokomgomo we-Le Chatelier, i-AgNP amasampula ngokushesha ahlanganisa ku-pH 3, kodwa kancane kancane aya ngokuya azinza njengoba i-pH ikhula.
Umfanekiso 4 Indlela eyisikimu yokusebenzelana kwendawo echazwe ngokuhlanganisa ngaphansi kwe-pH ehlukene (umugqa ophezulu), ukugxilisana kwe-NaCl (umugqa omaphakathi), nama-biomolecules (umugqa ongezansi).
Ngokomfanekiso wesi-5, ukuzinza kwe-colloidal ekumisweni kwe-AgNP kosayizi abahlukene nakho kwahlolwa ngaphansi kokwanda kokugxila kukasawoti.Ngokusekelwe emandleni e-zeta, usayizi we-nanoparticle okhulisiwe kulezi zinhlelo ze-AgNP ezinqanyulwe yi-citrate futhi unikeza ukumelana okuthuthukisiwe kumathonya angaphandle avela ku-NaCl.Ku-AgNP-I, i-10 mM NaCl yanele ukuhlanganisa ukuhlanganisa okuncane, futhi ukugxiliswa kukasawoti okungu-50 mM kunikeza imiphumela efanayo kakhulu.Ku-AgNP-II ne-AgNP-III, i-10 mM NaCl ayithinti kakhulu amandla e-zeta ngoba amanani ayo ahlala kokuthi (AgNP-II) noma ngaphansi (AgNP-III) -30 mV.Ukwandisa ukugxilisa kwe-NaCl ku-50 mM futhi ekugcineni ku-150 mM NaCl kwanele ukunciphisa ngokuphawulekayo inani eliphelele lamandla e-zeta kuwo wonke amasampuli, nakuba izinhlayiya ezinkulu zigcina ishaji elibi kakhulu.Le miphumela ihambisana ne-avareji ye-hydrodynamic diameter elindelwe yama-AgNP;imigqa yethrendi engu-Z elinganiselwe ku-10, 50, kanye ne-150 mM NaCl ibonisa amanani ahlukile, akhuphuka kancane kancane.Ekugcineni, izilinganiso zosayizi we-micron zitholwe kukho konke ukuhlolwa okuthathu kwe-150 mM.
Umfanekiso 5 Imiphumela yokuhlakazeka kokukhanya okuguquguqukayo yesampula ye-nanoparticles yesiliva enqanyuliwe ye-citrate enosayizi okhulayo (10 nm: AgNP-I, 20 nm: AgNP-II kanye no-50 nm: AgNP-III) avezwa njengobubanzi obumaphakathi be-hydrodynamic (isilinganiso se-Z ) (kwesokudla) kanye namandla e-zeta (kwesokunxele) ashintsha phakathi kwamahora angu-24 ngaphansi kokugxilisa okuhlukile kwe-NaCl.
Imiphumela ye-UV-Vis ku-Supplementary Figure S2 ibonisa ukuthi i-SPR engu-50 kanye ne-150 mM NaCl kuwo womathathu amasampuli inokwehla okuphazima kweso nokubalulekile.Lokhu kungachazwa yi-DLS, ngoba ukuhlanganisa okusekelwe ku-NaCl kwenzeka ngokushesha kunokuhlola okuncike ku-pH, okuchazwa umehluko omkhulu phakathi kwezilinganiso zangaphambi kwesikhathi (0, 1.5, kanye namahora angu-3).Ukwengeza, ukwandisa ukugxila kukasawoti kuzophinde kwandise ukuvunyelwa okuhlobene kwendawo yokuhlola, okuzoba nomthelela omkhulu ku-surface plasmon resonance.29
Umphumela we-NaCl ufingqiwe emgqeni ophakathi woMfanekiso 4. Ngokuvamile, kungaphethwa ngokuthi ukwandisa ukuhlushwa kwe-sodium chloride kunomphumela ofanayo nokwandisa i-asidi, ngoba ama-Na + ion anomkhuba wokuxhumanisa amaqembu e-carboxylate, ukucindezela ama-AgNP anamandla e-zeta.Ngaphezu kwalokho, i-150 mM NaCl ikhiqize ama-aggregate anosayizi we-micron kuwo wonke amasampuli amathathu, okubonisa ukuthi ukugxiliswa kwe-electrolyte ye-physiological kuyingozi ekuzinzeni kwe-colloidal kwama-AgNP anqanyuliwe yi-citrate.Ngokucabangela i-critical condensing concentration (CCC) ye-NaCl kumasistimu afanayo e-AgNP, le miphumela ingafakwa ngobuhlakani ezincwadini ezifanele.Huynh et al.ibale ukuthi i-CCC ye-NaCl yama-nanoparticles esiliva anqanyuliwe i-citrate enobubanzi obuyisilinganiso esingu-71 nm yayingu-47.6 mM, kuyilapho u-El Badawy et al.ibone ukuthi i-CCC ye-10 nm AgNPs ene-citrate coating yayingu-70 mM.10,16 Ngaphezu kwalokho, i-CCC ephakeme kakhulu engaba ngu-300 mM ikalwe ngu-He et al., okubangele indlela yabo yokuhlanganisa yehluke kulokho okushicilelwe ngaphambilini.48 Nakuba umnikelo wamanje ungahlosiwe ekuhlaziyweni okuphelele kwalawa manani, ngenxa yokuthi izimo zethu zokuhlola ziyanda enkingeni yalo lonke ucwaningo, ukugxiliswa kwe-NaCl okuhlobene nebhayoloji okungu-50 mM, ikakhulukazi u-150 mM NaCl, kubonakala kuphezulu kakhulu.I-coagulation eyenziwe, echaza izinguquko ezinamandla ezitholiwe.
Isinyathelo esilandelayo esivivinyweni se-polymerization siwukusebenzisa ama-molecule alula kodwa ahambisana nebhayoloji ukuze alingise ukusebenzisana kwe-nanoparticle-biomolecule.Ngokusekelwe ku-DLS (Izibalo 6 no-7) kanye nemiphumela ye-UV-Vis (Izibalo Ezingeziwe S3 kanye ne-S4), ezinye iziphetho ezijwayelekile zingagonyelwa.Ngaphansi kwezimo zethu zokuhlola, ama-molecule eglucose ne-glutamine ngeke abangele ukuhlangana kunoma iyiphi isistimu ye-AgNP, ngoba ithrendi engu-Z-mean ihlobene eduze nevelu yokulinganisa yesithenjwa ehambisanayo.Nakuba ubukhona bawo bungathinti ukuhlanganisa, imiphumela yokuhlola ibonisa ukuthi la ma-molecule akhangiswe ngokwengxenye ebusweni bama-AgNP.Umphumela ogqame kakhulu osekela lo mbono uguquko oluphawuliwe lokumuncwa kokukhanya.Nakuba i-AgNP-I ingabonisi izinguquko ezinengqondo zamaza noma ukuqina, ingabonwa ngokucacile ngokulinganisa izinhlayiya ezinkulu, okungenzeka kakhulu kungenxa yokuzwela okukhulu kokubona okukhulunywe ngakho ngaphambili.Kungakhathalekile ukuthi ingakanani i-glucose, i-glucose ingabangela ushintsho olukhulu olubomvu ngemva kwamahora angu-1.5 uma kuqhathaniswa nesilinganiso sokulawula, esingaba ngu-40 nm ku-AgNP-II kanye ne-10 nm ku-AgNP-III, okufakazela ukwenzeka kokusebenzelana kwendawo.I-Glutamine ibonise ukuthambekela okufanayo, kodwa ushintsho lwalungabonakali kangako.Ngaphezu kwalokho, kufanelekile ukusho ukuthi i-glutamine inganciphisa amandla aphelele we-zeta wezinhlayiya eziphakathi nendawo ezinkulu.Kodwa-ke, njengoba lezi zinguquko ze-zeta zingabonakali zithinta izinga lokuhlanganisa, kungacatshangwa ukuthi ngisho nama-biomolecules amancane afana ne-glutamine anganikeza izinga elithile lokuxoshwa kwendawo phakathi kwezinhlayiya.
Umfanekiso 6 Imiphumela yokuhlakazeka kokukhanya okuguquguqukayo yamasampuli esiliva e-citrate-terinated nanoparticle anosayizi okhulayo (10 nm: AgNP-I, 20 nm: AgNP-II kanye no-50 nm: AgNP-III) avezwa njengobubanzi obuyisilinganiso se-hydrodynamic (isilinganiso se-Z) (kwesokudla) Ngaphansi kwezimo zangaphandle zokugxila okuhlukile kwe-glucose, amandla e-zeta (kwesokunxele) ashintsha phakathi namahora angu-24.
Umfanekiso 7 Imiphumela yokuhlakazeka kokukhanya okuguquguqukayo yesampula ye-nanoparticles yesiliva enqanyuliwe ye-citrate enosayizi okhulayo (10 nm: AgNP-I, 20 nm: AgNP-II kanye no-50 nm: AgNP-III) avezwa njengobubanzi obumaphakathi be-hydrodynamic (isilinganiso se-Z ) (kwesokudla) Uma kukhona i-glutamine, amandla e-zeta (kwesokunxele) ashintsha phakathi namahora angu-24.
Ngamafuphi, ama-biomolecules amancane afana ne-glucose ne-glutamine akuthinti ukuzinza kwe-colloidal ekugxiliseni okulinganiselwe: nakuba athinta amandla e-zeta kanye nemiphumela ye-UV-Vis kumazinga ahlukahlukene, imiphumela emaphakathi ye-Z ayihambisani.Lokhu kubonisa ukuthi i-adsorption yangaphezulu yama-molecule ivimbela ukudonswa kwe-electrostatic, kodwa ngesikhathi esifanayo inikeza ukuzinza kwe-dimensional.
Ukuze sixhumanise imiphumela yangaphambilini nemiphumela yangaphambilini futhi silingise izimo zebhayoloji ngobuchule kakhudlwana, sikhethe ezinye zezingxenye ezivame ukusetshenziswa kakhulu zeseli futhi sazisebenzisa njengezimo zokuhlola zokutadisha ukuzinza kwe-AgNP colloid.Kuwo wonke ucwaningo lwe-in vitro, omunye wemisebenzi ebaluleke kakhulu ye-DMEM njengento ephakathi ukusungula izimo ezidingekayo ze-osmotic, kodwa ngokombono wamakhemikhali, isisombululo esiyinkimbinkimbi sikasawoti esinamandla e-ionic ephelele afana ne-150 mM NaCl. .40 Ngokuqondene ne-FBS, iyingxube eyinkimbinkimbi yama-biomolecules-ikakhulukazi amaprotheni-kusukela endaweni yokubuka i-adsorption, inokufana okuthile nemiphumela yokuhlola ye-glucose ne-glutamine, naphezu kokwakheka kwamakhemikhali nokwehlukahlukana Ubulili buyinkimbinkimbi kakhulu.I-19 DLS ne-UV-Imiphumela ebonakalayo ekhonjiswe ku-Figure 8 kanye Nomfanekiso Owengeziwe S5, ngokulandelanayo, ingachazwa ngokuhlola ukwakheka kwamakhemikhali kwalezi zinto kanye nokuzihlobanisa nezilinganiso ezisesigabeni sangaphambilini.
Umfanekiso 8 Imiphumela yokuhlakazeka kokukhanya okuguquguqukayo yesampula ye-nanoparticles yesiliva enqanyuliwe ye-citrate enosayizi okhulayo (10 nm: AgNP-I, 20 nm: AgNP-II kanye no-50 nm: AgNP-III) ivezwa njengobubanzi obumaphakathi be-hydrodynamic (isilinganiso se-Z ) (kwesokudla) Lapho kukhona izingxenye ze-cell culture i-DMEM ne-FBS, amandla e-zeta (kwesokunxele) ashintsha phakathi namahora angu-24.
Ukuhlanjululwa kwama-AgNP osayizi abahlukene ku-DMEM kunomthelela ofanayo ekuzinzeni kwe-colloidal kulokho okubonwa lapho kukhona ukugxila okuphezulu kwe-NaCl.Ukuhlakazwa kwe-AgNP ku-50 v/v% ye-DMEM kubonise ukuthi ukuhlanganiswa kwesilinganiso esikhulu kutholwe ngokukhuphuka kwamandla e-zeta kanye nenani elimaphakathi le-Z kanye nokwehla okubukhali kokuqina kwe-SPR.Kuyaphawuleka ukuthi ubukhulu besayizi obuhlanganisiwe obubangelwa i-DMEM ngemva kwamahora angu-24 buhambisana ngokuphambene nosayizi we-primer nanoparticles.
Ukusebenzisana phakathi kwe-FBS ne-AgNP kufana nalokho okubonwa lapho kukhona ama-molecule amancane njenge-glucose ne-glutamine, kodwa umphumela unamandla.Isilinganiso esingu-Z sezinhlayiya sihlala singathinteki, kuyilapho kutholwa ukwanda kwamandla e-zeta.Inani eliphakeme le-SPR libonise ukushintsha okubomvu okuncane, kodwa mhlawumbe okuthakazelisa nakakhulu, ukuqina kwe-SPR akuzange kwehle kakhulu njengasesilinganisweni sokulawula.Le miphumela ingachazwa nge-adsorption yokuzalwa yama-macromolecules ebusweni be-nanoparticles (umugqa ongezansi ku-Figure 4), manje oqondwa njengokwakheka kwe-biomolecular corona emzimbeni.49