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Kodi ma nanoparticles ang'onoang'ono amakhala bwino nthawi zonse?Kumvetsetsa zachilengedwe zakuphatikizana kodalira kukula kwa nanoparticles zasiliva pansi pamikhalidwe yoyenera
Olemba: 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 of Environmental Chemistry, Hungary, Hungary, Hungary, Hungary , Yunivesite ya Szeged;2 Dipatimenti ya Biochemistry ndi Molecular Biology, Faculty of Science and Information, University of Szeged, Hungary;3 Dipatimenti ya Microbiology, Faculty of Science and Information, University of Szeged, Hungary;4MTA-SZTE Reaction Kinetics and Surface Chemistry Research Group, Szeged, Hungary* Olemba awa anathandizira mofananamo pantchitoyi.Kuyankhulana: Zoltán Kónya Dipatimenti ya Applied and Environment Chemistry, Faculty of Science and Informatics, University of Szeged, Rerrich Square 1, Szeged, H-6720, Hungary Phone +36 62 544620 Email [chitetezo cha imelo] Cholinga: Silver nanoparticles (AgNPs) ndi imodzi mwazinthu zomwe zimaphunziridwa kwambiri ndi nanomatadium, makamaka chifukwa cha ntchito zawo zamankhwala.Komabe, chifukwa cha kuphatikizika kwa nanoparticles, ntchito yawo yabwino kwambiri ya cytotoxicity ndi antibacterial nthawi zambiri imasokonekera muzachilengedwe.Mu ntchitoyi, machitidwe ophatikizana ndi zochitika zokhudzana ndi chilengedwe za zitsanzo zitatu za nanoparticle za siliva za citrate-kuthetsedwa ndi mainchesi a 10, 20, ndi 50 nm anaphunzira.Njira: Gwiritsani ntchito maikulosikopu ya ma elekitironi kuti mupange ndikuwonetsa ma nanoparticles, kuwunika momwe amalumikizirana pamitundu yosiyanasiyana ya pH, NaCl, kuchuluka kwa shuga ndi glutamine mwa kubalalika kwamphamvu komanso mawonekedwe owoneka bwino a ultraviolet.Kuphatikiza apo, mu cell culture sing'anga zigawo monga Dulbecco amawongolera machitidwe ophatikizana mu Eagle Medium ndi Fetal Calf Serum.Results: Zotsatira zikuwonetsa kuti acidic pH ndi physiological electrolyte content nthawi zambiri imapangitsa kuti micron-scale aggregation, yomwe imatha kukhala mkhalapakati ndi kupanga biomolecular corona.Ndikoyenera kudziwa kuti tinthu tating'onoting'ono timawonetsa kukana kwakukulu ku zikoka zakunja kuposa anzawo ang'onoang'ono.Kuyesa kwa in vitro cytotoxicity ndi antibacterial kunachitika pochiza maselo okhala ndi nanoparticle aggregates pamagawo osiyanasiyana ophatikiza.Kutsiliza: Zotsatira zathu zikuwonetsa kulumikizana kwakukulu pakati pa kukhazikika kwa colloidal ndi kawopsedwe ka ma AgNP, chifukwa kuphatikizika kwakukulu kumabweretsa kutaya kwathunthu kwachilengedwe.Kuchuluka kwa anti-aggregation komwe kumawonedwa pa tinthu tating'onoting'ono kumakhudza kwambiri kawopsedwe ka m'galasi, chifukwa zitsanzo zoterezi zimasunga ma cell antimicrobial ndi mammalian cell.Zomwe zapezazi zimapangitsa kuti, ngakhale malingaliro ambiri m'mabuku oyenerera, kutsata ma nanoparticles ochepa kwambiri sangakhale njira yabwino kwambiri.Mfundo zazikuluzikulu: Kukula kokhala ndi mbewu, kukhazikika kwa colloidal, kutengera kukula kwamagulu, kawopsedwe ka kuwonongeka kophatikizana
Pamene kufunikira ndi kutulutsa kwa nanomatadium kukukulirakulira, chidwi chochulukirapo chimaperekedwa ku biosafety yawo kapena zochitika zachilengedwe.Silver nanoparticles (AgNPs) ndi imodzi mwazinthu zomwe zimagwiritsidwa ntchito kwambiri, zofufuzidwa komanso zogwiritsidwa ntchito pagululi la zida chifukwa champhamvu kwambiri, zowoneka bwino komanso zachilengedwe.1 Nthawi zambiri amakhulupirira kuti mawonekedwe apadera a nanomatadium (kuphatikiza ma AgNPs) makamaka amachokera kudera lawo lalikulu lapadera.Choncho, vuto losapeŵeka ndilo ndondomeko iliyonse yomwe imakhudza mbali yofunikayi, monga kukula kwa tinthu, kuphimba pamwamba kapena kuphatikizika, kaya kudzawononga kwambiri katundu wa nanoparticles omwe ali ovuta kwambiri ku ntchito zenizeni.
Zotsatira za kukula kwa tinthu ndi stabilizers ndi nkhani zomwe zalembedwa bwino m'mabuku.Mwachitsanzo, malingaliro ovomerezeka ndi akuti nanoparticles ang'onoang'ono ndi oopsa kwambiri kuposa ma nanoparticles akuluakulu.2 Mogwirizana ndi zolemba zambiri, maphunziro athu am'mbuyomu awonetsa ntchito yodalira kukula kwa nanosilver pama cell a mammalian ndi tizilombo tating'onoting'ono.3- 5 Kupaka pamwamba ndi chikhalidwe china chomwe chimakhala ndi mphamvu zambiri pazinthu za nanomatadium.Pongowonjezera kapena kusintha ma stabilizer pamwamba pake, ma nanomaterial omwewo amatha kukhala ndi mawonekedwe osiyanasiyana akuthupi, mankhwala, komanso zachilengedwe.Kugwiritsa ntchito capping agents nthawi zambiri kumachitika ngati gawo la nanoparticle synthesis.Mwachitsanzo, citrate-kutha siliva nanoparticles ndi imodzi mwa AgNPs zofunika kwambiri mu kafukufuku, amene apanga ndi kuchepetsa mchere siliva mu osankhidwa stabilizer njira monga sing'anga anachita.6 Citrate imatha kupezerapo mwayi pamtengo wake wotsika mtengo, kupezeka, kuyanjana kwachilengedwe, komanso kuyanjana kwamphamvu kwa siliva, zomwe zitha kuwonetsedwa pazokambirana zosiyanasiyana, kuyambira pakubweza kumtunda mpaka kuyanjana kwa ionic.Mamolekyu ang'onoang'ono ndi ma ion a polyatomic pafupi ndi 7,8, monga citrate, ma polima, ma polyelectrolytes, ndi ma biological agents amagwiritsidwanso ntchito kukhazikika kwa nano-silver ndikupanga magwiridwe antchito apadera pamenepo.9-12
Ngakhale kuti kuthekera kosintha ntchito ya nanoparticles mwadala kukwera pamwamba ndi malo osangalatsa kwambiri, ntchito yaikulu ya chophimba ichi ndi yosasamala, kupereka kukhazikika kwa colloidal kwa dongosolo la nanoparticle.Dera lalikulu la ma nanomatadium limatulutsa mphamvu yayikulu pamwamba, zomwe zimalepheretsa mphamvu ya thermodynamic ya dongosolo kuti ifikire mphamvu zake zochepa.13 Popanda kukhazikika koyenera, izi zitha kuyambitsa kuphatikizika kwa nanomatadium.Aggregation ndi mapangidwe aggregates a particles zosiyanasiyana akalumikidzidwa ndi makulidwe kuti zimachitika pamene omwazika particles kukumana ndi panopa thermodynamic ankachita kulola particles kumamatira wina ndi mzake.Choncho, stabilizers ntchito kupewa aggregation mwa kuyambitsa mokwanira lalikulu mphamvu zonyansa pakati particles kulimbana ndi kukopa thermodynamic.14
Ngakhale kuti nkhani ya kukula kwa tinthu tating'onoting'ono komanso kufalikira kwapadziko lapansi yafufuzidwa bwino potsata malamulo ake achilengedwe omwe amayamba chifukwa cha nanoparticles, kuphatikizika kwa tinthu ndi gawo losanyalanyazidwa.Palibe pafupifupi kafukufuku wokwanira kuti athetse kukhazikika kwa colloidal kwa nanoparticles pansi pazikhalidwe zogwirizana ndi biologically.10,15-17 Kuphatikiza apo, chopereka ichi ndi chosowa kwambiri, pomwe poizoni wokhudzana ndi kuphatikizika adaphunziridwanso, ngakhale angayambitse zovuta, monga vascular thrombosis, kapena kutayika kwa mawonekedwe omwe amafunidwa, monga kawopsedwe ake, monga kuwonetsedwa mu Chithunzi 1.18, 19 chowonetsedwa.Ndipotu, imodzi mwa njira zochepa zomwe zimadziwika kuti siliva nanoparticle resistance imakhudzana ndi kugwirizanitsa, chifukwa mitundu ina ya E. coli ndi Pseudomonas aeruginosa imanenedwa kuti imachepetsa mphamvu zawo za nano-silver pofotokozera puloteni flagellin, flagellin.Ili ndi kuyanjana kwakukulu kwa siliva, motero kumapangitsa kuphatikizika.20
Pali njira zingapo zosiyana zokhudzana ndi kawopsedwe ka nanoparticles zasiliva, ndipo kuphatikiza kumakhudza njira zonsezi.Njira yomwe AgNP amakambitsirana kwambiri, yomwe nthawi zina imatchedwa "Trojan Horse", imawona ma AgNP ngati onyamula Ag+.1,21 The Trojan horse mechanism ikhoza kuonetsetsa kuti kuwonjezeka kwakukulu kwa ndende ya Ag +, yomwe imayambitsa kubadwa kwa ROS ndi membrane depolarization.22-24 Kuphatikizika kungakhudze kutulutsidwa kwa Ag +, potero kumakhudza kawopsedwe, chifukwa kumachepetsa magwiridwe antchito pomwe ma ayoni asiliva amatha kukhala oxidized ndi kusungunuka.Komabe, ma AgNP sangowonetsa kawopsedwe kudzera mu kutulutsidwa kwa ion.Zambiri zokhudzana ndi kukula ndi morphology ziyenera kuganiziridwa.Pakati pawo, kukula ndi mawonekedwe a nanoparticle pamwamba ndizofotokozera makhalidwe.4,25 Kutolere kwa njirazi kutha kugawidwa ngati "njira zopangira poizoni."Pali zochitika zambiri za mitochondrial ndi pamwamba pa membrane zomwe zimatha kuwononga organelles ndikupangitsa kufa kwa cell.25-27 Popeza mapangidwe amagulu amakhudza mwachibadwa kukula ndi mawonekedwe a zinthu zomwe zili ndi siliva zomwe zimadziwika ndi machitidwe amoyo, kuyanjana kumeneku kungakhudzidwenso.
Mu pepala lathu lapitalo lokhudza kuphatikizika kwa ma nanoparticles a siliva, tidawonetsa njira yowunikira yomwe ili ndi kuyesa kwamankhwala ndi in vitro kuti tiphunzire vutoli.19 Dynamic Light Scattering (DLS) ndiyo njira yabwino yowunikira mitundu iyi chifukwa zinthuzo zimatha kumwaza ma photon pamlingo wofanana ndi kukula kwa tinthu tating'onoting'ono.Popeza Brownian kuyenda liwiro la particles mu sing'anga madzi okhudzana ndi kukula, kusintha mphamvu ya kuwala anamwazikana angagwiritsidwe ntchito kudziwa pafupifupi hydrodynamic m'mimba mwake (Z-kutanthauza) wa chitsanzo madzi.28 Kuphatikiza apo, pogwiritsa ntchito voliyumu pachitsanzo, kuthekera kwa zeta (ζ kuthekera) kwa nanoparticle kumatha kuyesedwa mofanana ndi mtengo wapakati wa Z.13,28 Ngati mtengo wokwanira wa zeta ukhoza kukhala wokwanira (malinga ndi malangizo ambiri> ± 30 mV), idzapanga mphamvu yamphamvu ya electrostatic pakati pa tinthu tating'onoting'ono tolimbana ndi kuphatikizika.Characteristic surface plasmon resonance (SPR) ndi chinthu chapadera chowoneka bwino, chomwe chimapangidwa ndi nanoparticles zamtengo wapatali (makamaka Au ndi Ag).29 ​ Kutengera ma oscillation amagetsi (ma plasmons) a zida izi pa nanoscale, zimadziwika kuti ma AgNP ozungulira ali ndi mawonekedwe a UV-Vis mayamwidwe pachimake pafupi ndi 400 nm.30 The mphamvu ndi wavelength kusintha kwa particles ntchito kuwonjezera zotsatira DLS, monga njira angagwiritsidwe ntchito kudziwa nanoparticle aggregation ndi pamwamba adsorption wa biomolecules.
Kutengera zomwe zapezedwa, cell viability (MTT) ndi antibacterial assays amachitidwa m'njira yomwe kawopsedwe ka AgNP imafotokozedwa ngati ntchito yamagulu ophatikizana, m'malo mwa (chomwe chimagwiritsidwa ntchito kwambiri) ndende ya nanoparticle.Njira yapaderayi imatithandiza kusonyeza kufunikira kwakukulu kwa msinkhu wophatikizana muzochitika zamoyo, chifukwa, mwachitsanzo, ma AgNP omwe amathetsedwa ndi citrate amataya ntchito yawo yamoyo mkati mwa maola angapo chifukwa cha kuphatikizika.19
Mu ntchito yamakono, tikufuna kukulitsa kwambiri zopereka zathu zam'mbuyomu pakukhazikika kwa ma colloid okhudzana ndi bio ndi momwe zimakhudzira zochitika zamoyo mwa kuphunzira momwe kukula kwa nanoparticle kumakhudzira kuphatikizika kwa nanoparticle.Izi mosakayikira ndi imodzi mwa maphunziro a nanoparticles.Malingaliro apamwamba kwambiri ndi 31 Kuti afufuze nkhaniyi, njira ya kukula kwa mbeu inagwiritsidwa ntchito kupanga ma AgNP a citrate-termized mumitundu itatu yosiyana (10, 20, ndi 50 nm).6,32 ngati imodzi mwa njira zodziwika bwino.Kwa ma nanomatadium omwe amagwiritsidwa ntchito mochulukira komanso nthawi zonse pazachipatala, ma AgNP amitundu yosiyanasiyana amasankhidwa kuti aphunzire kuchuluka kwa kudalira kwazinthu zokhudzana ndi kuphatikizika kwachilengedwe kwa nanosilver.Titapanga ma AgNP amitundu yosiyanasiyana, tidawonetsa zitsanzo zomwe zidapangidwa ndi ma electron microscopy (TEM), kenako ndikuwunika tinthu tating'onoting'ono pogwiritsa ntchito njira yomwe tatchulayi.Kuphatikiza apo, pamaso pa mu vitro cell zikhalidwe za Dulbecco's Modified Eagle's Medium (DMEM) ndi Fetal Bovine Serum (FBS), machitidwe odalira kukula ndi machitidwe ake adawunikidwa pamitundu yosiyanasiyana ya pH, NaCl, glucose, ndi glutamine.Makhalidwe a cytotoxicity amatsimikiziridwa pansi pamikhalidwe yonse.Kugwirizana kwa sayansi kumasonyeza kuti nthawi zambiri, tinthu tating'onoting'ono timakonda;kufufuza kwathu kumapereka nsanja yamankhwala ndi zachilengedwe kuti tidziwe ngati ndi choncho.
Ma nanoparticles atatu asiliva okhala ndi makulidwe osiyanasiyana adakonzedwa ndi njira yakukula yolumikizira mbewu yomwe Wan et al., Ndikusintha pang'ono.6 Njirayi imachokera ku kuchepetsa mankhwala, pogwiritsa ntchito silver nitrate (AgNO3) monga gwero la siliva, sodium borohydride (NaBH4) monga kuchepetsa, ndi sodium citrate monga stabilizer.Choyamba, konzani 75 mL ya 9 mM citrate aqueous solution kuchokera ku sodium citrate dihydrate (Na3C6H5O7 x 2H2O) ndi kutentha kwa 70 ° C.Kenako, 2 mL wa 1% w/v AgNO3 yankho anawonjezedwa kwa sing'anga anachita, ndiyeno mwatsopano okonzeka sodium borohydride njira (2 mL 0.1% w/v) anatsanuliridwa osakaniza dropwise.Kuyimitsidwa kwachikasu kofiirira kunasungidwa pa 70 ° C ndi kusonkhezera mwamphamvu kwa ola la 1, ndiyeno kuzizira kutentha.Chitsanzo chotsatira (chotchedwa AgNP-I kuyambira pano) chimagwiritsidwa ntchito ngati maziko a kukula kwa mbeu mu sitepe yotsatira ya kaphatikizidwe.
Kupanga sing'anga-kakulidwe tinthu kuyimitsidwa (kutchedwa AgNP-II), kutentha 90 mL 7.6 mM citrate njira 80 ° C, kusakaniza ndi 10 mL AgNP-I, ndiyeno kusakaniza 2 mL 1% w/v The AgNO3 njira. anasungidwa pansi mwamphamvu makina oyambitsa kwa 1 ora, ndiyeno chitsanzo anali utakhazikika firiji.
Kwa tinthu tating'onoting'ono (AgNP-III), bwerezaninso kukula komweko, koma munkhaniyi, gwiritsani ntchito 10 ml ya AgNP-II ngati kuyimitsidwa kwa mbewu.Zitsanzo zikafika kutentha kwa chipinda, zimayika ndende yawo ya Ag potengera kuchuluka kwa AgNO3 kufika pa 150 ppm powonjezera kapena kutulutsa zosungunulira zina pa 40 ° C, ndipo pamapeto pake zimazisunga pa 4 ° C mpaka kugwiritsidwa ntchito kwina.
Gwiritsani ntchito FEI Tecnai G2 20 X-Twin Transmission Electron microscope (TEM) (FEI Corporate Headquarters, Hillsboro, Oregon, USA) ndi 200 kV mathamangitsidwe voteji kuti muone morphological makhalidwe a nanoparticles ndi kujambula ma elekitironi diffraction (ED) chitsanzo.Zithunzi zosachepera 15 zoyimira (~ 750 particles) zidawunikidwa pogwiritsa ntchito pulogalamu ya ImageJ, ndipo ma histogram (ndi ma graph onse mu phunziro lonse) adapangidwa mu OriginPro 2018 (OriginLab, Northampton, MA, USA) 33, 34.
Avereji ya hydrodynamic diameter (Z-average), zeta kuthekera (ζ-zotheka) ndi characteristic surface plasmon resonance (SPR) ya zitsanzo zinayesedwa kuti zisonyeze makhalidwe awo oyambirira a colloidal.Kuchuluka kwa hydrodynamic m'mimba mwake ndi kuthekera kwa zeta kwachitsanzo kudayezedwa ndi chida cha Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) pogwiritsa ntchito ma cell a capillary otayidwa pa 37 ± 0.1 ° C.Ocean Optics 355 DH-2000-BAL UV-Vis spectrophotometer (Halma PLC, Largo, FL, USA) idagwiritsidwa ntchito kupeza mawonekedwe a SPR kuchokera ku UV-Vis kuyamwa kwa zitsanzo mumitundu ya 250-800 nm.
Pakuyesa konseko, mitundu itatu yoyezera yokhudzana ndi kukhazikika kwa colloidal idachitika nthawi imodzi.Gwiritsani ntchito DLS kuti muyese kuchuluka kwapakati pa hydrodynamic diameter (Z avareji) ndi kuthekera kwa zeta (ζ kuthekera) kwa tinthu ting'onoting'ono, chifukwa pafupifupi Z imagwirizana ndi kukula kwapakati pamagulu a nanoparticle, ndipo kuthekera kwa zeta kumawonetsa ngati kuthamangitsidwa kwa electrostatic m'dongosolo. ndi wamphamvu mokwanira kuti athetse kukopa kwa Van der Waals pakati pa nanoparticles.Miyeso imapangidwa katatu, ndipo kupatuka kwa Z mean ndi kuthekera kwa zeta kumawerengedwa ndi pulogalamu ya Zetasizer.Mawonekedwe a SPR a tinthu tating'onoting'ono amawunikidwa ndi UV-Vis spectroscopy, chifukwa kusintha kwamphamvu kwambiri komanso kutalika kwa mawonekedwe kumatha kuwonetsa kuphatikizika ndi kuyanjana kwapamtunda.29,35 Ndipotu, pamwamba pa plasmon resonance muzitsulo zamtengo wapatali ndi zamphamvu kwambiri moti zapangitsa njira zatsopano zowunikira ma biomolecules.29,36,37 Kuphatikizika kwa AgNPs mu kusakaniza koyesera kuli pafupi ndi 10 ppm, ndipo cholinga chake ndi kukhazikitsa mphamvu ya kuyamwa kwakukulu koyambirira kwa SPR ku 1. Kuyesera kunkachitika nthawi yodalira pa 0;1.5;3;6;Maola 12 ndi 24 pamikhalidwe yosiyanasiyana yokhudzana ndi biologically.Zambiri zofotokozera kuyesera zitha kuwoneka mu ntchito yathu yam'mbuyomu.19 Mwachidule, mitundu yosiyanasiyana ya pH (3; 5; 7.2 ndi 9), sodium kolorayidi (10 mm; 50 mm; 150 mm), shuga (3.9 mm; 6.7 mm) ndi glutamine (4 mm) ndende, ndi adakonzekeranso Dulbecco's Modified Eagle Medium (DMEM) ndi Fetal Bovine Serum (FBS) (m'madzi ndi DMEM) monga machitidwe achitsanzo, ndipo adaphunzira zotsatira zake pamakhalidwe ophatikizana a nanoparticles opangidwa ndi siliva.pH Miyezo ya, NaCl, shuga, ndi glutamine imawunikidwa kutengera momwe thupi limakhalira, pomwe kuchuluka kwa DEM ndi FBS ndi kofanana ndi milingo yomwe imagwiritsidwa ntchito pakuyesa konse kwa in vitro.38-42 Miyezo yonse idachitidwa pa pH 7.2 ndi 37 ° C ndi mchere wokhazikika wamchere wa 10 mM NaCl kuti athetse kuyanjana kulikonse kwa tinthu takutali (kupatulapo zoyeserera zina za pH ndi NaCl, pomwe izi ndizosiyana. kuphunzira).28 Mndandanda wa zikhalidwe zosiyanasiyana zikufotokozedwa mwachidule mu Table 1. Kuyesera kotchulidwa ndi † kumagwiritsidwa ntchito ngati chidziwitso ndipo kumagwirizana ndi chitsanzo chokhala ndi 10 mM NaCl ndi pH 7.2.
Maselo a khansa ya prostate (DU145) ndi ma keratinocyte osafa aumunthu (HaCaT) adapezedwa kuchokera ku ATCC (Manassas, VA, USA).Maselo amabzalidwa pafupipafupi mu Dulbecco's minimum essential medium Eagle (DMEM) yokhala ndi 4.5 g/L shuga (Sigma-Aldrich, Saint Louis, MO, USA), yowonjezeredwa ndi 10% FBS, 2 mM L-glutamine, 0.01 % Streptomycin ndi 0.005% Penicillin (Sigma-Aldrich, St. Louis, Missouri, USA).Maselo amapangidwa mu chofungatira cha 37 ° C pansi pa 5% CO2 ndi 95% chinyezi.
Pofuna kufufuza kusintha kwa AgNP cytotoxicity chifukwa cha tinthu tating'onoting'ono tating'onoting'ono timene timadalira nthawi, kuyesa kwa MTT kwa magawo awiri kunachitika.Choyamba, kuthekera kwa mitundu iwiri ya maselo kunayesedwa pambuyo pa chithandizo ndi AgNP-I, AgNP-II ndi AgNP-III.Kuti izi zitheke, mitundu iwiri ya maselo inayikidwa mu mbale za 96-chitsime pa kachulukidwe ka maselo a 10,000 / bwino ndikuchiritsidwa ndi mitundu itatu yosiyana ya nanoparticles ya siliva pakuwonjezeka kwa tsiku lachiwiri.Pambuyo pa chithandizo cha maola a 24, maselowo adatsukidwa ndi PBS ndikuyikidwa ndi 0.5 mg / mL MTT reagent (SERVA, Heidelberg, Germany) kuchepetsedwa mu chikhalidwe chapakati kwa ola la 1 pa 37 ° C.Makhiristo a Formazan adasungunuka mu DMSO (Sigma-Aldrich, Saint Louis, MO, USA), ndipo kuyamwa kwake kunayesedwa pa 570 nm pogwiritsa ntchito owerenga mbale ya Synergy HTX (BioTek-Hungary, Budapest, Hungary).Mtengo wamayamwidwe wachitsanzo chosasamalidwa bwino umawerengedwa kuti ndi 100% ya kupulumuka.Chitani zoyeserera zosachepera zitatu pogwiritsa ntchito zofananira zinayi zodziyimira pawokha.IC50 imawerengeredwa kuchokera pamapindi a kuyankha kwa mlingo kutengera zotsatira zamphamvu.
Pambuyo pake, mu sitepe yachiwiri, poyika tinthu tating'onoting'ono ta 150 mM NaCl kwa nthawi zosiyanasiyana (0, 1.5, 3, 6, 12, ndi 24 maola) musanayambe chithandizo cha selo, zigawo zosiyana za nanoparticles zasiliva zinapangidwa.Pambuyo pake, kuyesa komweko kwa MTT kunachitika monga momwe tafotokozera kale kuti kuwunika kusintha kwa ma cell kukhudzidwa ndi kuphatikizika kwa tinthu.Gwiritsani ntchito GraphPad Prism 7 kuti muyese chotsatira chomaliza, kuwerengera kufunikira kwa kuyesako pogwiritsa ntchito mayeso osagwirizana ndi t, ndikuyika mulingo wake ngati * (p ≤ 0.05), ** (p ≤ 0.01), *** (p ≤ 0.001) ) Ndipo **** (p ≤ 0.0001).
Miyezo itatu yosiyana ya nanoparticles yasiliva (AgNP-I, AgNP-II ndi AgNP-III) idagwiritsidwa ntchito ngati antibacterial susceptibility to Cryptococcus neoformans IFM 5844 (IFM; Research Center for Pathogenic Fungi and Microbial Toxicology, Chiba University) ndi Bacillus Test megaterium SZMC 60 (SZMC: Szeged Microbiology Collection) ndi E. coli SZMC 0582 mu RPMI 1640 medium (Sigma-Aldrich Co.).Pofuna kuyesa kusintha kwa ntchito ya antibacterial chifukwa cha kuphatikizika kwa tinthu tating'onoting'ono, choyamba, ndende yawo yocheperako (MIC) idatsimikiziridwa ndi microdilution mu mbale ya 96-well microtiter.Ku 50 μL ya kuyimitsidwa kwa cell yokhazikika (ma cell 5 × 104/mL mu RPMI 1640 sing'anga), onjezani 50 μL ya kuyimitsidwa kwa nanoparticle siliva ndikuchepetsa kawiri kuchuluka kwake (mu sing'anga yomwe tatchulayi, mitunduyo ndi 0 ndi 75 ppm, ndiye kuti, chitsanzo chowongolera chimakhala ndi 50 μL ya kuyimitsidwa kwa selo ndi 50 μL yapakati popanda nanoparticles).Pambuyo pake, mbaleyo incubated pa 30 ° C kwa maola 48, ndipo kachulukidwe kuwala kwa chikhalidwe anayesedwa pa 620 nm ntchito SPECTROstar Nano mbale wowerenga (BMG LabTech, Offenburg, Germany).Kuyesera kunachitika katatu katatu.
Pokhapokha kuti 50 μL ya zitsanzo zophatikizika za nanoparticle zidagwiritsidwa ntchito panthawiyi, njira yofananira yomwe tafotokozayi idagwiritsidwa ntchito powunika momwe kuphatikizika kumagwirira ntchito pa antibacterial pamitundu yomwe tatchulayi.Mayiko osiyanasiyana ophatikizana a siliva nanoparticles amapangidwa ndikuyika tinthu tating'onoting'ono ndi 150 mM NaCl kwakanthawi kosiyanasiyana (0, 1.5, 3, 6, 12, ndi 24 maola) isanachitike ma cell.Kuyimitsidwa komwe kumaphatikizidwa ndi 50 μL ya RPMI 1640 sing'anga kunagwiritsidwa ntchito ngati kuwongolera kukula, pomwe pofuna kuwongolera kawopsedwe, kuyimitsidwa ndi nanoparticles osaphatikizana kunagwiritsidwa ntchito.Kuyesera kunachitika katatu katatu.Gwiritsani ntchito GraphPad Prism 7 kuti muwunikenso chotsatira chomaliza, pogwiritsa ntchito kusanthula kofananako monga kusanthula kwa MTT.
Kuphatikizika kwa tinthu tating'onoting'ono kwambiri (AgNP-I) kwadziwika, ndipo zotsatira zake zidasindikizidwa pang'ono m'ntchito yathu yapitayi, koma kuti tifananize bwino, tinthu tating'onoting'ono tating'ono tating'ono tating'onoting'ono.Deta yoyesera imasonkhanitsidwa ndikukambidwa m'magawo otsatirawa.Miyezo itatu ya AgNP.19
Miyezo yochitidwa ndi TEM, UV-Vis ndi DLS idatsimikizira kaphatikizidwe kopambana kwa zitsanzo zonse za AgNP (Chithunzi 2A-D).Malinga ndi mzere woyamba wa Chithunzi 2, tinthu tating'ono kwambiri (AgNP-I) tikuwonetsa mawonekedwe ozungulira ozungulira omwe ali ndi mainchesi pafupifupi 10 nm.Njira ya kukula kwa mbeu imaperekanso AgNP-II ndi AgNP-III yokhala ndi mitundu yosiyanasiyana ya kukula ndi pafupifupi ma diameter a tinthu pafupifupi 20 nm ndi 50 nm, motero.Malinga ndi kupatuka kwapang'onopang'ono kwa kugawa kwa tinthu, kukula kwa zitsanzo zitatuzi sikulumikizana, zomwe ndizofunikira pakuwunika kwawo kofananiza.Poyerekeza pafupifupi mbali chiŵerengero ndi woonda chiŵerengero cha TEM ofotokoza tinthu 2D zolosera, akuganiza kuti sphericity wa particles ndi kuunika ndi ImageJ a mawonekedwe fyuluta pulagi (Chithunzi 2E).43 Malinga ndi kusanthula kwa mawonekedwe a tinthu tating'onoting'ono, mawonekedwe awo (mbali yayikulu / yaifupi ya rectangle yaying'ono kwambiri) samakhudzidwa ndi kukula kwa tinthu, komanso kuonda kwawo (gawo loyezedwa la bwalo lolingana / gawo lamalingaliro). ) pang'onopang'ono amachepetsa.Izi zimapangitsa kuti tinthu tating'ono ting'onoting'ono ta polyhedral, zomwe zimakhala zozungulira bwino, zomwe zimagwirizana ndi kuonda kwa 1.
Chithunzi 2 Chithunzi cha 2 Transmission electron maikulosikopu (TEM) (A), electron diffraction (ED) pattern (B), size distribution histogram (C), characteristic ultraviolet-visible (UV-Vis) light mayamwidwe sipekitiramu (D), ndi avareji madzimadzi Citrate Ma nanoparticles asiliva omwe amathetsedwa ndi makina awiri (Z-average), kuthekera kwa zeta, chiŵerengero cha mawonekedwe ndi makulidwe (E) ali ndi mitundu itatu yosiyana ya kukula: AgNP-I ndi 10 nm (mzere wapamwamba), AgNP -II ndi 20 nm (mzere wapakati ), AgNP-III (mzere wapansi) ndi 50 nm.
Ngakhale cyclic chikhalidwe cha kukula njira zinakhudza tinthu mawonekedwe kumlingo, chifukwa mu ang'onoang'ono sphericity lalikulu AgNPs, onse atatu zitsanzo anakhala quasi-ozungulira.Komanso, monga momwe ma elekitironi diffraction chitsanzo mu Chithunzi 2B, nano The crystallinity wa particles si anakhudzidwa.Ma diffraction mphete-omwe angagwirizane ndi (111), (220), (200), ndi (311) ma indices a Miller a siliva-amagwirizana kwambiri ndi zolemba zasayansi ndi zopereka zathu zam'mbuyomu.9, 19,44 Kugawikana kwa mphete ya Debye-Scherrer ya AgNP-II ndi AgNP-III ndi chifukwa chakuti chithunzi cha ED chimatengedwa pakukula komweko, kotero kuti kukula kwa tinthu kukuwonjezeka, chiwerengero cha particles diffracted pa. gawo la unit likuwonjezeka ndi kuchepa.
Kukula ndi mawonekedwe a nanoparticles amadziwika kuti amakhudza zochitika zachilengedwe.3,45 Mawonekedwe otengera mawonekedwe othandizira komanso zochitika zamoyo zitha kufotokozedwa ndikuti mawonekedwe osiyanasiyana amakonda kuchulukitsa nkhope za kristalo (kukhala ndi ma indices osiyanasiyana a Miller), ndipo nkhope za kristalozi zimakhala ndi ntchito zosiyanasiyana.45,46 Popeza tinthu tating'onoting'ono tomwe timapanga timapereka zotsatira zofanana za ED zofanana ndi mawonekedwe a kristalo ofanana kwambiri, tingaganize kuti m'mayesero athu otsatila a colloidal ndi zochitika zamoyo, kusiyana kulikonse kuyenera kukhala chifukwa cha kukula kwa Nanoparticle, osati mawonekedwe okhudzana ndi mawonekedwe.
Zotsatira za UV-Vis zomwe zafotokozedwa mwachidule mu Chithunzi 2D zimagogomezeranso kwambiri mawonekedwe ozungulira a AgNP opangidwa, chifukwa nsonga za SPR za zitsanzo zonse zitatu ndizozungulira 400 nm, zomwe ndi mtengo wamtengo wapatali wa nanoparticles wasiliva wozungulira.29,30 Chiwonetsero chojambulidwa chinatsimikiziranso kukula kwabwino kwa mbewu za nanosilver.Pamene tinthu kukula kukula, wavelength lolingana ndi pazipita kuwala mayamwidwe AgNP-II-odziwika kwambiri-Malinga ndi mabuku, AgNP-III Anakumana ndi redshift.6, 29
Ponena za kukhazikika kwa colloidal koyambirira kwa dongosolo la AgNP, DLS idagwiritsidwa ntchito kuyeza kuchuluka kwa hydrodynamic m'mimba mwake ndi kuthekera kwa zeta kwa particles pa pH 7.2.Zotsatira zomwe zikuwonetsedwa mu Chithunzi 2E zikuwonetsa kuti AgNP-III ili ndi kukhazikika kwamtundu wapamwamba kuposa AgNP-I kapena AgNP-II, chifukwa malangizo omwe amapezeka akuwonetsa kuti kuthekera kwa zeta kwa 30 mV mtheradi ndikofunikira kuti pakhale kukhazikika kwa colloidal kwa nthawi yayitali. Mtengo wapakati wa Z (womwe umapezeka ngati wapakati wa hydrodynamic wa tinthu tating'ono taufulu ndi ophatikizana) umafananizidwa ndi kukula kwa tinthu tating'ono tomwe timapeza ndi TEM, chifukwa kuyandikira kwa milingo iwiriyi ndi, kucheperako kwa Digiri Yosonkhanitsa mu zitsanzo.M'malo mwake, kuchuluka kwa Z kwa AgNP-I ndi AgNP-II ndikwambiri kuposa kukula kwa tinthu tating'onoting'ono ta TEM, kotero poyerekeza ndi AgNP-III, zitsanzozi zimanenedweratu kuti zitha kuphatikizika, pomwe kuthekera koyipa kwambiri kwa zeta. imatsagana ndi kukula kwapafupi The Z average value.
Kufotokozera za chodabwitsa ichi kungakhale pawiri.Kumbali imodzi, ndende ya citrate imasungidwa pamlingo wofanana pamasitepe onse ophatikizika, kupereka kuchuluka kwamagulu okwera pamwamba kuti tipewe kuchepera kwa magawo omwe akukula.Komabe, malinga ndi Levak et al., mamolekyu ang'onoang'ono monga citrate amatha kusinthanitsa mosavuta ndi ma biomolecules pamwamba pa nanoparticles.Pankhaniyi, kukhazikika kwa colloidal kudzatsimikiziridwa ndi korona wa ma biomolecule opangidwa.31 Chifukwa khalidweli lidawonedwanso mumiyeso yathu yophatikizira (kukambidwa mwatsatanetsatane pambuyo pake), citrate capping yokha sikungafotokoze chodabwitsa ichi.
Komano, tinthu kukula ndi inversely proportional kuti aggregation chizolowezi pa nanometer msinkhu.Izi zimathandizidwa makamaka ndi njira yachikhalidwe ya Derjaguin-Landau-Verwey-Overbeek (DLVO), pomwe kukopa kwa tinthu kumafotokozedwa ngati kuchuluka kwa mphamvu zowoneka bwino komanso zonyansa pakati pa tinthu tating'onoting'ono.Malinga ndi Iye et al., Kuchuluka kwa mphamvu ya DLVO mphamvu yokhotakhota kumachepa ndi kukula kwa nanoparticles mu hematite nanoparticles, zomwe zimapangitsa kuti zikhale zosavuta kuti zifike ku mphamvu yochepa kwambiri, potero kulimbikitsa kugwirizanitsa kosasinthika (condensation).47 Komabe, zikuganiziridwa kuti pali mbali zina zopyola malire a chiphunzitso cha DLVO.Ngakhale van der Waals mphamvu yokoka ndi electrostatic iwiri wosanjikiza repulsion ndi ofanana ndi kukula tinthu, ndemanga ndi Hotze neri Al.ikuwonetsa kuti imakhala ndi mphamvu pakuphatikiza kuposa momwe DLVO imalola.14 Amakhulupirira kuti kupindika kwa pamwamba kwa nanoparticles sikungathenso kuyerekezedwa ngati malo athyathyathya, kupangitsa kuyerekezera masamu kukhala kosagwira ntchito.Kuphatikiza apo, kukula kwa tinthu kumachepetsa, kuchuluka kwa ma atomu omwe amapezeka pamtunda amakhala apamwamba, zomwe zimapangitsa kuti pakhale mawonekedwe amagetsi ndi khalidwe lapamwamba.Ndipo kusintha kwa reactivity pamwamba, zomwe zingayambitse kuchepa kwa ndalama mu magetsi awiri osanjikiza ndikulimbikitsa kugwirizanitsa.
Poyerekeza zotsatira za DLS za AgNP-I, AgNP-II, ndi AgNP-III mu Chithunzi 3, tidawona kuti zitsanzo zonse zitatu zidawonetsa pH yofanana yomwe imapangitsa kuphatikizika.Malo okhala ndi acidic kwambiri (pH 3) amasintha mphamvu ya zeta ya chitsanzo ku 0 mV, kuchititsa kuti tinthu tipange magulu ang'onoang'ono a micron, pamene pH ya alkaline imasintha mphamvu zake za zeta ku mtengo waukulu woipa, kumene tinthu timapanga magulu ang'onoang'ono (pH 5). ).Ndipo 7.2) ), kapena kukhala osagawanika (pH 9).Kusiyana kwakukulu pakati pa zitsanzo zosiyanasiyana kunawonedwanso.Panthawi yonse yoyesera, AgNP-I adawonetsa kuti ndizovuta kwambiri ku pH-induced zeta kusintha, chifukwa zeta zomwe zingatheke za particleszi zachepetsedwa pa pH 7.2 poyerekeza ndi pH 9, pamene AgNP-II ndi AgNP-III anangosonyeza AgNP-III. kusintha kwakukulu kwa ζ kuli pafupi ndi pH 3. Kuwonjezera apo, AgNP-II inasonyeza kusintha pang'onopang'ono komanso mphamvu ya zeta, pamene AgNP-III inasonyeza khalidwe lochepa kwambiri la atatuwa, chifukwa dongosololi linasonyeza mtengo wapamwamba kwambiri wa zeta ndi kuyenda pang'onopang'ono. AgNP-III Kwambiri kugonjetsedwa ndi pH-induced aggregation.Zotsatirazi zikugwirizana ndi zotsatira za kuyeza kwa hydrodynamic m'mimba mwake.Poganizira kukula kwa tinthu tawo, AgNP-I idawonetsa kuphatikizika kwapang'onopang'ono pamitundu yonse ya pH, mwina chifukwa cha 10 mM NaCl maziko, pomwe AgNP-II ndi AgNP-III amangowonetsa kwambiri pH 3 Pakusonkhanitsa.Kusiyana kochititsa chidwi kwambiri ndikuti ngakhale kukula kwake kwakukulu kwa nanoparticle, AgNP-III imapanga magulu ang'onoang'ono pa pH 3 mu maola 24, kuwonetsa katundu wake wotsutsa-aggregation.Pogawa pafupifupi Z ya AgNPs pa pH 3 pambuyo pa maola 24 ndi mtengo wa chitsanzo chokonzekera, zikhoza kuwonedwa kuti kukula kwake kwa AgNP-I ndi AgNP-II kwawonjezeka ka 50, nthawi 42, ndi nthawi 22. , motero.III.
Chithunzi 3 The zazikulu kuwala kubalalitsa zotsatira za citrate-zitha siliva nanoparticles chitsanzo ndi kukula kukula (10 nm: AgNP-I, 20 nm: AgNP-II ndi 50 nm: AgNP-III) anasonyeza monga pafupifupi hydrodynamic m'mimba mwake (Z pafupifupi ) (kumanja) Pansi pa pH zosiyana, kuthekera kwa zeta (kumanzere) kumasintha mkati mwa maola 24.
Kuphatikizika komwe kumadalira pH kudakhudzanso mawonekedwe a plasmon resonance (SPR) a zitsanzo za AgNP, monga zikuwonekera ndi mawonekedwe awo a UV-Vis.Malinga ndi Supplementary Figure S1, kuphatikizika kwa kuyimitsidwa kwa nanoparticle siliva kumatsatiridwa ndikuchepetsa kuchuluka kwa nsonga zawo za SPR ndikusintha kofiira kocheperako.Kukula kwa zosinthazi monga ntchito ya pH kumagwirizana ndi kuchuluka kwa kuphatikizika komwe kumanenedweratu ndi zotsatira za DLS, komabe, zinthu zina zosangalatsa zawonedwa.Mosiyana ndi intuition, zimakhala kuti AgNP-II yapakatikati ndiyomwe imakhudzidwa kwambiri ndi kusintha kwa SPR, pamene zitsanzo zina ziwirizi ndizochepa.Pakafukufuku wa SPR, 50 nm ndiye malire a kukula kwa tinthu tating'ono, omwe amagwiritsidwa ntchito kusiyanitsa tinthu tating'onoting'ono potengera ma dielectric awo.Tinthu tating'onoting'ono kuposa 50 nm (AgNP-I ndi AgNP-II) zitha kufotokozedwa ngati ma dipoles osavuta a dielectric, pomwe tinthu tating'onoting'ono tomwe timafika kapena kupitilira malire awa (AgNP-III) amakhala ndi zinthu zovuta kwambiri za dielectric, ndipo resonance yawo Gulu limagawanika kukhala masinthidwe ambiri. .Pankhani ya tinthu tating'ono tating'ono tating'ono tating'ono, ma AgNP amatha kuwonedwa ngati ma dipoles osavuta, ndipo plasma imatha kupindika mosavuta.Pamene kukula kwa tinthu tating'onoting'ono kumawonjezeka, kuphatikizika kumeneku kumatulutsa plasma yokulirapo, yomwe imatha kufotokozera kukhudzika kwakukulu komwe kumawonedwa.29 Komabe, kwa tinthu tating'onoting'ono tating'onoting'ono, kuyerekeza kwa dipole kosavuta sikuli koyenera pamene mayiko ena ophatikizana atha kuchitika, zomwe zingafotokozere kuchepa kwa chizolowezi cha AgNP-III kuwonetsa kusintha kwa mawonekedwe.29
Pansi pa zoyesera zathu, zimatsimikiziridwa kuti pH mtengo umakhudza kwambiri kukhazikika kwa colloidal kwa nanoparticles ya siliva ya citrate yamitundu yosiyanasiyana.M'machitidwe awa, kukhazikika kumaperekedwa ndi magulu a COO-oyipa omwe ali pamwamba pa AgNPs.The carboxylate zinchito gulu la citrate ayoni ndi protonated ambiri H + ayoni, kotero kwaiye carboxyl gulu sangathenso kupereka electrostatic kunyansidwa pakati particles, monga tikuonera pamwamba mzere wa Chithunzi 4. Malinga ndi mfundo Le Chatelier, AgNP zitsanzo zimaphatikizana mwachangu pa pH 3, koma pang'onopang'ono zimakhala zokhazikika pamene pH ikuwonjezeka.
Chithunzi 4 Schematic mechanism of surface interaction imatanthauzidwa ndi kuphatikizira pansi pH yosiyana (mzere wapamwamba), ndondomeko ya NaCl (mzere wapakati), ndi ma biomolecules (mzere wapansi).
Malinga ndi Chithunzi 5, kukhazikika kwa colloidal mu kuyimitsidwa kwa AgNP kwamitundu yosiyanasiyana kudawunikidwanso pakuwonjezeka kwa mchere.Kutengera ndi kuthekera kwa zeta, kukula kwa nanoparticle kochulukira m'machitidwe a AgNP a citrate-kuthetsedwa kumaperekanso kukana kopitilira muyeso kuzinthu zakunja kuchokera ku NaCl.Mu AgNP-I, 10 mM NaCl ndi yokwanira kuyambitsa kuphatikizika pang'ono, ndipo kuchuluka kwa mchere wa 50 mm kumapereka zotsatira zofanana kwambiri.Mu AgNP-II ndi AgNP-III, 10 mM NaCl sichimakhudza kwambiri kuthekera kwa zeta chifukwa makhalidwe awo amakhalabe (AgNP-II) kapena pansi (AgNP-III) -30 mV.Kuonjezera chiwerengero cha NaCl ku 50 mM ndipo potsirizira pake ku 150 mM NaCl ndikokwanira kuchepetsa kwambiri mtengo wa zeta zomwe zingatheke mu zitsanzo zonse, ngakhale kuti tinthu tating'onoting'ono timasunga ndalama zambiri zoipa.Zotsatirazi zikugwirizana ndi kuchuluka kwapakati pa hydrodynamic diameter ya AgNPs;Avereji ya Z mizere yoyezedwa pa 10, 50, ndi 150 mM NaCl imawonetsa zosiyana, zomwe zikuwonjezeka pang'onopang'ono.Pomaliza, zophatikizira zazikuluzikulu za micron zidapezeka muzoyeserera zonse zitatu za 150 mM.
Chithunzi 5 The zazikulu kuwala kubalalitsa zotsatira za citrate-zitha siliva nanoparticles chitsanzo ndi kukula kukula (10 nm: AgNP-I, 20 nm: AgNP-II ndi 50 nm: AgNP-III) anasonyeza monga pafupifupi hydrodynamic m'mimba mwake (Z pafupifupi ) (kumanja) ndi kuthekera kwa zeta (kumanzere) kusintha mkati mwa maola 24 pansi pamagulu osiyanasiyana a NaCl.
Zotsatira za UV-Vis mu Supplementary Figure S2 zikuwonetsa kuti SPR ya 50 ndi 150 mM NaCl mu zitsanzo zonse zitatu imakhala yocheperako komanso yocheperako.Izi zitha kufotokozedwa ndi DLS, chifukwa kuphatikizika kwa NaCl kumachitika mwachangu kuposa kuyesa kodalira pH, komwe kumafotokozedwa ndi kusiyana kwakukulu pakati pa miyeso yoyambirira (0, 1.5, ndi 3 hours).Kuonjezera apo, kuonjezera ndende ya mchere kudzawonjezera kuvomerezeka kwachibale kwa sing'anga yoyesera, zomwe zidzakhudza kwambiri plasmon resonance.29
Zotsatira za NaCl zikufotokozedwa mwachidule pamzere wapakati wa Chithunzi 4. Kawirikawiri, tinganene kuti kuwonjezeka kwa sodium chloride kumakhala ndi zotsatira zofanana ndi kuwonjezeka kwa acidity, chifukwa Na + ions ali ndi chizolowezi chogwirizanitsa magulu a carboxylate, kupondereza zeta zomwe zingachitike ma AgNP.Kuonjezera apo, 150 mM NaCl inapanga ma micron-size aggregates mu zitsanzo zonse zitatu, kusonyeza kuti physiological electrolyte concentration ndi yowononga colloidal bata wa citrate-term AgNPs.Poganizira za Critical condensing concentration (CCC) ya NaCl pamakina ofanana a AgNP, zotsatirazi zitha kuyikidwa mwanzeru m'mabuku oyenera.Huynh et al.anawerengera kuti CCC ya NaCl ya citrate-terminated silver nanoparticles ndi pafupifupi awiri a 71 nm anali 47.6 mM, pamene El Badawy et al.adawona kuti CCC ya 10 nm AgNPs yokhala ndi zokutira za citrate inali 70 mM.10,16 Kuwonjezera apo, CCC yapamwamba kwambiri ya pafupifupi 300 mM inayesedwa ndi Iye et al., Zomwe zinachititsa kuti njira yawo yophatikizira ikhale yosiyana ndi yofalitsidwa kale.48 Ngakhale kuti chithandizo chamakono sichinayang'ane pa kufufuza mozama za makhalidwe awa, chifukwa miyeso yathu yoyesera ikuwonjezeka mu zovuta za kafukufuku wonse, biologically NaCl concentration ya 50 mM, makamaka 150 mM NaCl, ikuwoneka kuti ndi yokwera kwambiri.Anachititsa coagulation, kufotokoza amphamvu kusintha wapezeka.
Chotsatira pakuyesa kwa polymerization ndikugwiritsira ntchito mamolekyu osavuta koma okhudzana ndi biologically kuti ayese kuyanjana kwa nanoparticle-biomolecule.Kutengera DLS (Zithunzi 6 ndi 7) ndi UV-Vis zotsatira (Supplementary Figures S3 ndi S4), mfundo zina wamba akhoza kunena.Pansi pa zoyeserera zathu, mamolekyu omwe taphunzira a glucose ndi glutamine sangapangitse kuphatikizika munjira iliyonse ya AgNP, chifukwa zomwe Z-kutanthauza zimagwirizana kwambiri ndi muyeso wofananira.Ngakhale kupezeka kwawo sikumakhudza kuphatikizika, zotsatira zoyesera zikuwonetsa kuti mamolekyuwa ndi adsorbed pang'ono pamwamba pa AgNPs.Chotsatira chodziwika kwambiri chochirikiza lingaliro ili ndikusintha kowoneka kwa kuyamwa kwa kuwala.Ngakhale AgNP-I sichiwonetsa kutalika kwa kutalika kwa mawonekedwe kapena kusintha kwamphamvu, imatha kuwonedwa bwino poyesa tinthu tating'onoting'ono, zomwe zimachitika chifukwa cha kutengeka kwakukulu kwa mawonekedwe omwe tawatchula kale.Mosasamala kanthu za kuchuluka kwa shuga, shuga imatha kupangitsa kusintha kwakukulu kofiira pambuyo pa maola 1.5 poyerekeza ndi muyeso wowongolera, womwe ndi pafupifupi 40 nm mu AgNP-II ndi pafupifupi 10 nm mu AgNP-III, zomwe zimatsimikizira kuchitika kwa kuyanjana kwapamtunda.Glutamine adawonetsa zofanana, koma kusintha sikunali koonekeratu.Kuonjezera apo, ndiyeneranso kutchula kuti glutamine ikhoza kuchepetsa mphamvu ya zeta yapakati ndi zazikulu.Komabe, popeza kusintha kwa zeta sikukuwoneka kuti kumakhudza mlingo wa aggregation, zikhoza kuganiziridwa kuti ngakhale ma biomolecules ang'onoang'ono monga glutamine angapereke mlingo wina wa kukana kwapakati pakati pa tinthu tating'onoting'ono.
Chithunzi 6 The zazikulu kuwala kubalalitsa zotsatira za citrate-zitha siliva nanoparticle zitsanzo ndi kukula kukula (10 nm: AgNP-I, 20 nm: AgNP-II ndi 50 nm: AgNP-III) anasonyeza monga pafupifupi hydrodynamic m'mimba mwake (Z avareji) (kumanja) Pansi pazikhalidwe zakunja zamitundu yosiyanasiyana ya shuga, kuthekera kwa zeta (kumanzere) kumasintha mkati mwa maola 24.
Chithunzi 7 The zazikulu kuwala kubalalitsa zotsatira za citrate-zitha siliva nanoparticles chitsanzo ndi kukula kukula (10 nm: AgNP-I, 20 nm: AgNP-II ndi 50 nm: AgNP-III) anasonyeza monga pafupifupi hydrodynamic m'mimba mwake (Z pafupifupi ) (kumanja) Pamaso pa glutamine, kuthekera kwa zeta (kumanzere) kumasintha mkati mwa maola 24.
Mwachidule, ma biomolecules ang'onoang'ono monga shuga ndi glutamine samakhudza kukhazikika kwa colloidal pamlingo woyezedwa: ngakhale amakhudza kuthekera kwa zeta ndi zotsatira za UV-Vis mosiyanasiyana, zotsatira zapakati za Z sizikugwirizana.Izi zikusonyeza kuti pamwamba adsorption wa mamolekyu linalake ndipo tikulephera repulsion electrostatic, koma pa nthawi yomweyo amapereka dimensional bata.
Pofuna kugwirizanitsa zotsatira zam'mbuyo ndi zotsatira zam'mbuyo ndikufanizira zamoyo mwaluso kwambiri, tinasankha zigawo zina zomwe zimagwiritsidwa ntchito kwambiri pamagulu amtundu wa maselo ndikuwagwiritsa ntchito ngati miyeso yoyesera pophunzira kukhazikika kwa AgNP colloids.Mu kuyesa konse kwa in vitro, imodzi mwa ntchito zofunika kwambiri za DMEM monga sing'anga ndikukhazikitsa zofunikira za osmotic, koma kuchokera kumaganizo a mankhwala, ndi njira yothetsera mchere wambiri ndi mphamvu ya ionic yofanana ndi 150 mM NaCl. .40 Ponena za FBS, ndizovuta zosakaniza za biomolecules-makamaka mapuloteni-kuchokera kumalo owonetsera pamwamba, zimakhala ndi zofanana ndi zotsatira zoyesera za shuga ndi glutamine, ngakhale kuti mankhwala amapangidwa ndi zosiyana Kugonana kumakhala kovuta kwambiri.19 DLS ndi UV-Zotsatira zowoneka bwino zomwe zikuwonetsedwa mu Chithunzi 8 ndi Supplementary Figure S5, motero, zikhoza kufotokozedwa poyang'ana mankhwala opangidwa ndi zipangizozi ndikuzigwirizanitsa ndi miyeso mu gawo lapitalo.
Chithunzi 8 The zazikulu kuwala kubalalitsa zotsatira za citrate-zitha siliva nanoparticles chitsanzo ndi kukula kukula (10 nm: AgNP-I, 20 nm: AgNP-II ndi 50 nm: AgNP-III) akufotokozedwa monga pafupifupi hydrodynamic m'mimba mwake (Z pafupifupi ) (kumanja) Pamaso pa zigawo za chikhalidwe cha maselo DMEM ndi FBS, zeta zomwe zingathe (kumanzere) zimasintha mkati mwa maola 24.
Kuchepetsedwa kwa ma AgNP a kukula kosiyana mu DEMM kumakhala ndi zotsatira zofanana pa kukhazikika kwa colloidal ndi zomwe zimawonedwa pamaso pa kuchuluka kwa NaCl.Kubalalika kwa AgNP mu 50 v / v% DMEM kunawonetsa kuti kuphatikizika kwakukulu kunadziwika ndi kuwonjezeka kwa zeta zomwe zingatheke ndi Z-avereji yamtengo wapatali komanso kuchepa kwakukulu kwa mphamvu ya SPR.Ndikoyenera kudziwa kuti kukula kwakukulu komwe kunayambika ndi DEMM pambuyo pa maola 24 kumayenderana ndi kukula kwa nanoparticles oyambira.
Kuyanjana pakati pa FBS ndi AgNP ndi kofanana ndi komwe kumawonedwa pamaso pa mamolekyu ang'onoang'ono monga shuga ndi glutamine, koma zotsatira zake zimakhala zamphamvu.Z avareji ya tinthu tating'onoting'ono timakhalabe osakhudzidwa, pomwe kuwonjezeka kwa kuthekera kwa zeta kumadziwika.Chiwonetsero cha SPR chinawonetsa kusintha pang'ono kofiira, koma mwinamwake chochititsa chidwi kwambiri, mphamvu ya SPR siinachepe kwambiri monga muyeso yolamulira.Zotsatirazi zitha kufotokozedwa ndi kutengera kwachilengedwe kwa ma macromolecules pamtunda wa nanoparticles (mzere wapansi pa Chithunzi 4), chomwe tsopano chimamveka ngati mapangidwe a biomolecular corona m'thupi.49