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Inoshanda photosensitizers inonyanya kukosha kune yakapararira kiriniki yekushandiswa kwephototherapy.Nekudaro, akajairwa mafotosensitizers anowanzo tambura nekupfupika kwewavelength absorption, kusakwana photostability, yakaderera quantum goho reiyo reactive oxygen marudzi (ROS), uye aggregation-induced quenching yeROS.Pano tinoshuma pedyo-infrared (NIR) supramolecular photosensitizer (RuDA) inopindirana nekuzvibatanidza kweRu (II) -arene organometallic complexes mune aqueous solution.RuDA inogona kungogadzira singlet okisijeni (1O2) munharaunda yakaunganidzwa, uye inoratidza pachena kuunganidzwa-induced 1O2 chizvarwa maitiro nekuda kwekuwedzera kukuru kweiyo crossover process pakati peiyo singlet-triplet system.Pasi pechiito che808 nm laser light, RuDA inoratidza 1O2 quantum goho re16.4% (FDA-yakatenderwa indocyanine girini: ΦΔ=0.2%) uye yakakwirira photothermal kutendeuka kushanda kwe24.2% (yekutengesa goridhe nanorods) ine yakanakisa photostability.: 21.0%, nanoshells dzegoridhe: 13.0%).Pamusoro pezvo, RuDA-NPs ine biocompatibility yakanaka inogona kuunganidza munzvimbo dzebundu, zvichikonzera kuderera kwebundu panguva yekurapa kwephotodynamic ne95.2% kuderedzwa kwebundu vhoriyamu mu vivo.Iyi aggregation-inosimudzira photodynamic therapy inopa zano rekugadzira photosensitizers ine yakanaka photophysical uye photochemical zvimiro.
Kana ichienzaniswa neyakajairwa kurapa, photodynamic therapy (PDT) mushonga unoyevedza wegomarara nekuda kwezvakanaka zvawo zvakakosha senge kwakaringana spatiotemporal control, kusapindira, kushomeka kwezvinodhaka, uye kuderedza mhedzisiro 1,2,3.Pasi pechiedza chekuvhenekera, mafotosensitizers anoshandiswa anogona kugadziridzwa kuti aumbe zvakanyanya reactive oxygen marudzi (ROS), zvichiita kuti apoptosis/necrosis kana immune immune4,5. Nekudaro, mazhinji akajairika mafotosensitizers, akadai seklorini, porphyrins, uye anthraquinones, ane pfupi-wavelength absorption (frequency <680 nm), zvichikonzera kusapinda kwechiedza nekuda kwekunyanya kunyura kwemamorekuru ehupenyu (semuenzaniso, hemoglobin uye melanin) mu nharaunda inooneka6,7. Nekudaro, mazhinji akajairika mafotosensitizers, akadai seklorini, porphyrins, uye anthraquinones, ane pfupi-wavelength absorption (frequency <680 nm), zvichikonzera kusapinda kwechiedza nekuda kwekunyanya kunyura kwemamorekuru ehupenyu (semuenzaniso, hemoglobin uye melanin) mu nharaunda inooneka6,7. Однако большинство обычных фотосенсибилизаторов, таких как хлорины, порфирины и антрахиноны, обладают относительно коротковолновым поглощением (частота < 680 нм), что приводит к плохому проникновению света из-за интенсивного поглощения биологических молекул (например, гемоглобина и меланина) в видимая область6,7. Zvakadaro, mafotosensitizers akajairika akadai seklorini, porphyrins uye anthraquinones ane kupfupika kwewavelength (<680 nm) zvichikonzera kusapinda kwechiedza nekuda kwekunyudzwa kwakanyanya kwemamorekuru ehupenyu (eg hemoglobin nemelanin) munzvimbo inooneka6,7.然而, 大多数 传统 的 光敏剂, 二 二 卟酚, 和 和 蒽醌, 相对 <680 NM), 因此 由于 对 生物 分子 (如 血红 蛋白 和 黑色素) 的 强烈 吸收,导致光穿透性差.然而, 大多数 传统 的 光敏剂, 氢 氢, 卟啉, 具有, 频率 相对 较 的 波长 (频率 频率 <680 nm) 因此 由于 对 的 (吸收 吸收 吸收 吸收 吸收 HI导致光穿透性差. Однако большинство традиционных фотосенсибилизаторов, таких как хлорины, порфирины и антрахиноны, имеют относительно коротковолновое поглощение (частота < 680 нм) из-за сильного поглощения биомолекул, таких как гемоглобин и меланин, что приводит к плохому проникновению света. Nekudaro, mazhinji echinyakare photosensitizers akadai seklorini, porphyrins uye anthraquinones ane ipfupi wavelength absorption (frequency <680 nm) nekuda kwekutorwa kwakasimba kwemabiomolecules akadai sehemoglobin uye melanin zvichikonzera kusapinda kwechiedza.Inoonekwa nzvimbo 6.7.Naizvozvo, pedyo-infrared (NIR) inotora photosensitizers iyo inogadziriswa mu700-900 nm "hwindo rekurapa" rakanyatsokodzera phototherapy.Sezvo pedyo nechiedza che infrared chiri chishoma chinotorwa ne biological tissues, inogona kutungamirira kune yakadzika kupinda uye kushoma photodamage8,9.
Nehurombo, iripo NIR-inotora photosensitizers kazhinji haina kusimba kwemifananidzo, yakaderera singlet okisijeni (1O2) generating kugona, uye aggregation-induced 1O2 quenching, iyo inoganhura yavo yekiriniki application10,11.Kunyangwe hazvo kuedza kukuru kwakaitwa kuvandudza iyo photophysical uye photochemical zvimiro zveyakajairwa photosensitizers, kusvika parizvino mishumo yakati wandei yakataura kuti NIR-inotora photosensitizers inogona kugadzirisa matambudziko ese aya.Pamusoro pezvo, akati wandei mafotosensitizers aratidza vimbiso yekugadzirwa kwakanaka kwe1O212,13,14 kana yakavhenekerwa nechiedza pamusoro pe800 nm, sezvo simba rephoton richidzikira nekukurumidza munzvimbo iri pedyo-IR.Triphenylamine (TFA) seanopa maerekitironi uye [1,2,5] thiadiazole-[3,4-i] dipyrido[a,c] phenazine (TDP) seboka rinogamuchira maerekitironi Donor-acceptor (DA) mhando inodhayi kirasi. yedhayi, inonwisa pedyo-infrared, iyo yakave yakanyatsodzidza kune pedyo-infrared bioimaging II uye photothermal therapy (PTT) nekuda kwebhendi ravo rakatetepa.Nekudaro, madhayi emhando yeDA anogona kushandiswa kuPDT aine ari pedyo-IR excitation, kunyangwe asina kumbodzidzwa sephotosensitizer yePDT.
Zvinonyatsozivikanwa kuti kushanda kwepamusoro kwe intersystem crossing (ISC) ye photosensitizers inosimudzira kuumbwa kwe1O2.Yakajairika zano rekusimudzira iyo ISC maitiro ndeyekusimudzira spin-orbit coupling (SOC) yephotosensitizers nekuunza inorema maatomu kana yakakosha organic moieties.Nekudaro, nzira iyi ichine zvimwe zvisingabatsiri uye zvinogumira19,20.Munguva ichangopfuura, supramolecular self-assembly yakapa pasi-up intelligent approach yekugadzirwa kwezvinhu zvinoshanda pa molecular level, 21,22 ine zvakawanda zvakanakira mu phototherapy: (1) self-assembled photosensitizers inogona kuva nekwanisi yekugadzira ribhoni zvimiro.Zvakafanana nezvivakwa zvemagetsi zvine denser kugovera kwemazinga esimba nekuda kwekupindirana orbits pakati pezvivharo zvekuvaka.Nokudaro, simba rekuenzanisa pakati pepasi singlet inofadza nyika (S1) uye yakavakidzana triplet excited state (Tn) ichavandudzwa, iyo inobatsira kune ISC process 23, 24.(2) Supramolecular assembly ichaderedza kusina-radiative relaxation zvichienderana ne intramolecular motion limitation mechanism (RIM), iyo inokurudzirawo ISC process 25, 26.(3) Iyo supramolecular gungano inogona kuchengetedza mamorekuru emukati memonomeri kubva pakusvibiswa uye kuparara, nekudaro ichivandudza zvakanyanya kutsvuka kwefotosensitizer.Tichifunga zvakanakira zviri pamusoro, isu tinotenda kuti supramolecular photosensitizer masisitimu inogona kuve inovimbisa imwe nzira yekukunda kukanganisa kwePDT.
Ru(II)-based complexes ipuratifomu inovimbisa yekurapa yezvingango shandiswa mukuongororwa uye kurapwa kwezvirwere nekuda kwezvakasiyana uye zvinoyevedza zvipenyu zvipenyu28,29,30,31,32,33,34.Pamusoro pezvo, kuwanda kwenzvimbo dzinofadza uye iyo inogadziriswa photophysicochemical properties yeRu(II)-based complexes inopa mabhenefiti makuru ekuvandudzwa kweRu(II)-based photosensitizers35,36,37,38,39,40.Muenzaniso unozivikanwa ndeye ruthenium(II) polypyridyl complex TLD-1433, iyo parizvino iri muchikamu chechipiri chekiriniki miedzo sephotosensitizer yekurapa isiri-muscle invasive cancer cancer (NMIBC)41.Pamusoro pezvo, ruthenium(II)arene organometallic complexes inoshandiswa zvakanyanya sechemotherapeutic agents pakurapwa kwegomarara nekuda kwekuderera kwehupfu uye nyore kwekugadzirisa42,43,44,45.Ionic properties yeRu (II) -arene organometallic complexes haigoni kungovandudza kusvibiswa kwakashata kweDA chromophores mune zvakajairika zvinonyungudutsa, asiwo inovandudza kuungana kweDA chromophores.Mukuwedzera, iyo pseudooctahedral half-sandwich structure ye organometallic complexes yeRu (II) -arenes inogona kudzivirira zvakasimba H-aggregation yeDA-mhando chromophores, zvichiita kuti kuumbwa kweJ-aggregation ine redshifted absorption bands.Zvisinei, kusakanganiswa kweRu(II) -arene complexes, zvakadai sekudzikama kwakaderera uye / kana kuti bioavailability yakaipa, inogona kukanganisa kushanda kwekurapa uye mu vivo basa re arene-Ru (II) complexes.Nekudaro, zvidzidzo zvakaratidza kuti izvi zvinokanganisa zvinogona kukundwa nekuputira ruthenium complexes ine biocompatible mapolymer nekuvharirwa kwemuviri kana covalent conjugation.
Mubasa iri, tinoshuma DA-conjugated complexes yeRu (II) -arene (RuDA) ine NIR trigger kuburikidza nekubatanidza chisungo pakati peDAD chromophore uye Ru (II) -arene moiety.Iwo anokonzeresa macomplexes anogona kuzviunganidzira kuita metalosupramolecular vesicles mumvura nekuda kwekusawirirana kusinga wirirane.Zvinonyanya kukosha, iyo supramolecular assembly yakapa RuDA ne polymerization-induced intersystem crossing-over properties, iyo yakawedzera zvakanyanya ISC kunyatsoshanda, iyo yaifarira zvikuru PDT (Fig. 1A).Kuwedzera bundu kuunganidzwa uye mu vivo biocompatibility, FDA-yakatenderwa Pluronic F127 (PEO-PPO-PEO) yakashandiswa encapsulate RuDA47,48,49 kugadzira RuDA-NP nanoparticles (Figure 1B) iyo yakashanda seyakanyanya kushanda PDT/ Dual- modhi PTT proxy .Mukenza phototherapy (Mufananidzo 1C), RuDA-NP yakashandiswa kurapa mbeva dzisina kupfeka neMDA-MB-231 tumors kudzidza kushanda kwePDT uye PTT muvivo.
Schematic illustration yephotophysical mechanism yeRuDA mune monomeric uye aggregated mafomu ekenza phototherapy, synthesis yeB RuDA-NPs uye C RuDA-NPs yeNIR-activated PDT uye PTT.
RuDA, inosanganisira TPA uye TDP kushanda, yakagadzirirwa maererano nemaitiro akaratidzwa muSupplementary Figure 1 (Mufananidzo 2A), uye RuDA yakaratidzwa ne1H uye 13C NMR spectra, electrospray ionization mass spectrometry, uye elemental analysis (Supplementary Figures 2-4 )Iyo RuDA electron density musiyano mepu yeyakaderera singlet shanduko yakaverengerwa nenguva-inotsamira density functional theory (TD-DFT) kuti idzidze nzira yekuchajisa.Sezvinoratidzwa muSupplementary Figure 5, iyo electron density inodonha zvakanyanya kubva kutriphenylamine kuenda kuTDP yekugamuchira unit mushure mephotoexcitation, iyo inogona kukonzerwa neyakajairwa intramolecular charge transfer (CT) shanduko.
Chimiro cheKemikari cheOre B Kutora chimiro cheOre mumisanganiswa yezvikamu zvakasiyana zveDMF nemvura.C Normalized absorption values ​​​​yeRuDA (800 nm) uye ICG (779 nm) maringe nenguva pa 0.5 W cm-2 ye808 nm laser light.D The photodegradation yeABDA inoratidzwa neRuDA-induced formation ye1O2 muDMF / H2O yakasanganiswa nemhando dzakasiyana dzemvura pasi pechiito chemalaser radiation ine wavelength ye808 nm uye simba re 0.5 W / cm2.
Abstract-UV-inooneka absorption spectroscopy yakashandiswa kudzidza kuzvibatanidza zvimisikidzo zveOre mumisanganiswa yeDMF nemvura mumareshiyo akasiyana.Sezvinoratidzwa mufig.2B, RuDA inoratidza mabhendi ekunyura kubva pa600 kusvika 900 nm muDMF ine yakanyanya kunyura bhendi pa729 nm.Kuwedzera huwandu hwemvura kwakatungamira kune zvishoma nezvishoma kutsvuka kweiyo Ore absorption yakanyanya kusvika 800 nm, iyo inoratidza J-kuunganidzwa kweOre mune yakaunganidzwa system.Iyo photoluminescence spectra yeRuDA mune zvakasiyana-siyana zvinonyungudutsa zvinoratidzwa muSupplementary Figure 6. RuDA inoratidzika kuratidza yakajairika NIR-II luminescence ine yakanyanya emission wavelength ye ca.1050 nm muCH2Cl2 uye CH3OH, zvichiteerana.Kuchinja kukuru kweStokes (inenge 300 nm) yeRuDA inoratidza kuchinja kukuru kwejometri yehurumende inofadza uye kuumbwa kwepasi-simba rinofadza nyika.Iyo luminescence quantum goho reOre muCH2Cl2 uye CH3OH yakatemwa kuve 3.3 uye 0.6%, zvichiteerana.Zvisinei, mumusanganiswa wemethanol nemvura (5/95, v/v), zvishoma zvitsvuku zvekubuda uye kuderera kwehuwandu hwezvibereko (0.22%) zvakaonekwa, izvo zvingave zvakakonzerwa nekuzvibatanidza kweOre. .
Kuona kuungana kweORE, takashandisa liquid atomic force microscopy (AFM) kuona shanduko ye morphological muORE mune methanol mhinduro mushure mekuwedzera mvura.Apo mvura yemvura yakanga iri pasi pe80%, hapana kusanganiswa kwakajeka kwakaonekwa (Supplementary Fig. 7).Zvisinei, nekuwedzera kuwedzera kwehuwandu hwemvura kusvika ku90-95%, nanoparticles duku dzakaonekwa, iyo yakaratidza kuzvibatanidza kweOre.Kuwedzera, laser irradiation ine wavelength ye808 nm haina kukanganisa kunwa kweRuDA mune aqueous. mhinduro (Fig. 2C uye Supplementary Fig. 8).Kusiyana neizvi, kunyura kweindocyanine green (ICG sekutonga) kwakadonha nekukurumidza pa779 nm, zvichiratidza kugona kwakanyanya kweRuDA.Mukuwedzera, kugadzikana kweRuDA-NPs muPBS (pH = 5.4, 7.4 uye 9.0), 10% FBS uye DMEM (yakakwirira glucose) yakaongororwa neUV-inooneka absorption spectroscopy panguva dzakasiyana-siyana.Sezvinoratidzwa muSupplementary Figure 9, kuchinja kuduku muRuDA-NP absorption bands kwakaonekwa muPBS papH 7.4 / 9.0, FBS uye DMEM, zvichiratidza kugadzikana kwakanakisisa kweRuDA-NP.Zvisinei, mune imwe acidic medium (рН = 5.4) hydrolysis yeOre yakawanikwa.Isu takaongorora zvakare kugadzikana kweRuDA neRuDA-NP tichishandisa yakakwira performance liquid chromatography (HPLC) nzira.Sezvinoratidzwa muSupplementary Figure 10, RuDA yakanga yakagadzikana mumusanganiswa we methanol nemvura (50/50, v / v) kweawa yekutanga, uye hydrolysis yakaonekwa mushure memaawa mana.Nekudaro, chete yakafara concave-convex peak yakaonekwa yeRuDA NPs.Nokudaro, gel permeation chromatography (GPC) yakashandiswa kuongorora kugadzikana kweRuDA NPs muPBS (pH = 7.4).Sezvinoratidzwa muSupplementary Figure 11, mushure memaawa masere ekuvharirwa pasi pemamiriro akaedzwa, kukwirira kwepamusoro, kukwirira kwepamusoro uye nzvimbo yepamusoro yeNP RuDA haina kuchinja zvakanyanya, zvichiratidza kugadzikana kwakanakisisa kweNP RuDA.Mukuwedzera, mifananidzo yeTEM yakaratidza kuti morphology yeRuDA-NP nanoparticles yakaramba isingashanduki mushure meawa 24 mu diluted PBS buffer (pH = 7.4, Supplementary Fig. 12).
Nemhaka yekuti kuzvibatanidza kunogona kupa maitiro akasiyana-siyana ekushanda uye kemikari paOre, takaona kusunungurwa kwe9,10-anthracenediylbis (methylene) dimalonic acid (ABDA, chiratidzo 1O2) mumisanganiswa yemethanol-mvura.Ore ine mvura yakasiyana50.Sezvinoratidzwa muMufananidzo 2D uye Supplementary Figure 13, hapana kuora kweABDA kwakaonekwa apo mvura inenge iri pasi pe20%.Nekuwedzera kwehunyoro kusvika ku40%, kuderedzwa kweABDA kwakaitika, sezvinoratidzwa nekuderera kwesimba reABDA fluorescence.Izvo zvakaonekwawo kuti mvura yepamusoro inoguma inokurumidza kuparara, zvichiratidza kuti RuDA self-assembly inokosha uye inobatsira ABDA degradation.Ichi chiitiko chakasiyana zvakanyanya kubva kumazuva ano ACQ (aggregation-induced quenching) chromophores.Kana yakavhenekerwa nelaser ine wavelength ye808 nm, iyo quantum goho re1O2 RuDA mumusanganiswa we 98% H2O/2% DMF i16.4%, iyo inosvika makumi masere nemaviri kupfuura iyo ICG (ΦΔ = 0.2%)51, kuratidzira chizvarwa chinoshamisa chekubudirira 1O2 RuDA mumamiriro ekuunganidza.
Electron spins uchishandisa 2,2,6,6-tetramethyl-4-piperidinone (TEMP) uye 5,5-dimethyl-1-pyrroline N-oxide (DMPO) se spin traps Resonance spectroscopy (ESR) yakashandiswa kuona mhedzisiro yemhando. AFK.naRuDA.Sezvinoratidzwa muSupplementary Figure 14, yakave yakasimbiswa kuti 1O2 inogadzirwa panguva dzeirradiation pakati pe0 ne4 maminitsi.Mukuwedzera, apo RuDA yakavharwa neDMPO pasi pe irradiation, chimiro chechina-line EPR chiratidzo che1: 2: 2: 1 DMPO-OH · adduct yakaonekwa, ichiratidza kuumbwa kwe hydroxyl radicals (OH ·).Pakazara, mhedzisiro iri pamusoro inoratidza kugona kweRuDA kukurudzira kugadzirwa kweROS kuburikidza nemhando mbiri yemhando I/II photosensitization process.
Kuti unzwisise zviri nani zvigadzirwa zvemagetsi zveRuDA mune monomeric uye aggregated mafomu, iyo frontier molecular orbitals yeRuDA mu monomeric uye dimeric mafomu akaverengwa achishandisa nzira yeDFT.Sezvinoratidzwa mufig.3A, iyo yepamusoro-soro yakagarwa molecular orbital (HOMO) ye monomeric RuDA inobviswa padivi pe ligand musana uye iyo yakaderera isingabatanidzwe molecular orbital (LUMO) iri pakati peTDP yekugamuchira unit.Kusiyana neizvi, electron density mu dimeric HOMO yakanangidzirwa pa ligand yeimwe RuDA molecule, nepo electron density muLUMO inonyanya kuiswa pane inogamuchirwa yeimwe molecule yeRuDA, iyo inoratidza kuti RuDA iri mu dimer.Zvimiro zveCT.
A Iyo HOMO uye LUMO yeOre inoverengerwa mune monomeric uye dimeric mafomu.B Singlet uye katatu simba mazinga eOre mune monomers uye dimers.C Inofungidzirwa mazinga eRuDA uye inokwanisika ISC migero se monomeric C uye dimeric D. Miseve inoratidza dzingangoita ISC migero.
Kugoverwa kwema electrons nemakomba ari pasi-energy singlet excited states of RuDA mu monomeric uye dimeric mafomu akaongororwa achishandisa Multiwfn 3.852.53 software, iyo yakaverengwa uchishandisa nzira yeTD-DFT.Sezvinoratidzwa pane imwe label.Sezvinoratidzwa muFigure 1-2, maburi emonomeric RDA anonyanya kubviswa padivi pe ligand musana mune idzi singlet inonakidza nyika, nepo maerekitironi achinyanya kuwanikwa muboka reTDP, achiratidza intramolecular maitiro eCT.Pamusoro pezvo, kune idzi singlet dzinonakidza nyika, pane zvakanyanya kana kushoma kupindirana pakati pemakomba nemaerekitironi, zvichiratidza kuti idzi singlet dzinonakidza nyika dzinoita chimwe chinhu kubva kune emunharaunda excitation (LE).Kune ma dimers, kuwedzera kune intramolecular CT uye LE maficha, chimwe chikamu cheintermolecular CT maficha akaonekwa munzvimbo dzakateerana, kunyanya S3, S4, S7, uye S8, zvichibva paintermolecular CT kuongororwa, neCT intermolecular transitions seyakanyanya. (Supplementary Table).3).
Kuti tinzwisise zviri nani mhedzisiro yekuyedza, isu takaenderera mberi nekuongorora zvimiro zveRuDA zvinofadza nyika kuti tiongorore mutsauko pakati pe monomers uye dimers (Supplementary Tables 4-5).Sezvinoratidzwa muMufananidzo 3B, mazinga esimba eiyo singlet uye triplet inonakidza nyika dze dimer yakanyanya kusimba kupfuura iyo yemonomer, iyo inobatsira kudzikisa gaka resimba pakati peS1 neTn. Izvo zvave zvichishumwa kuti ISC shanduko inogona kuitika mukati mediki simba gap (ΔES1-Tn <0.3 eV) pakati peS1 neTn54. Izvo zvave zvichishumwa kuti ISC shanduko inogona kuitika mukati mediki simba gap (ΔES1-Tn <0.3 eV) pakati peS1 neTn54. Сообщалось, что переходы ISC inokwanisa быть реализованы в пределах небольшой энергетической щели (ΔES1-Tn <0,3 эВ) между S1 и Tn5 and Tn. Izvo zvave zvichishumwa kuti ISC shanduko inogona kuitika mukati mediki simba gap (ΔES1-Tn <0.3 eV) pakati peS1 neTn54.据报道，ISC 跃迁可以在S1 和Tn54 之间的小能隙（ΔES1-Tn < 0.3 eV）内实现.据报道，ISC 跃迁可以在S1 和Tn54 之间的小能隙（ΔES1-Tn < 0.3 eV）内实现. Сообщалось, что переход ISC inokwanisa kuburitsa реализован в пределах небольшой энергетической щели (ΔES1-Tn < 0,3 эВ) между S1 и Tn54. Izvo zvave zvichishumwa kuti ISC shanduko inogona kuitika mukati mediki simba gap (ΔES1-Tn <0.3 eV) pakati peS1 neTn54.Pamusoro pezvo, imwe chete orbital, yakagarwa kana kusagarwa, inofanirwa kusiyana mune yakasungwa singlet uye triplet nyika kuti ipe isiri-zero SOC yakakosha.Nokudaro, zvichibva pakuongororwa kwesimba rekukurudzira uye kutenderera kwe orbital, nzira dzose dzinogona kuitika dzeISC shanduko dzinoratidzwa muFigs.3C,D.Zvikuru, imwe chete ISC chiteshi inowanikwa mune monomer, nepo dimeric fomu ine ina ISC nzira dzinogona kuwedzera ISC shanduko.Naizvozvo, zvine musoro kufunga kuti iyo yakawanda RuDA mamorekuru akaunganidzwa, iyo inonyanya kusvikika iyo ISC migero ichave.Naizvozvo, RuDA aggregates anogona kuumba maviri-bhendi emagetsi zvimiro mu singlet uye triplet nyika, kuderedza simba gaka pakati S1 uye iripo Tn, nokudaro kuwedzera kushanda zvakanaka ISC kufambisa 1O2 chizvarwa.
Kuti tiwedzere kujekesa nzira yepasi, takagadzira chidimbu chetauro ye arene-Ru (II) yakaoma (RuET) nekutsiva mapoka maviri ethyl nemapoka maviri e-triphenylamine phenyl muRuDA (Fig. 4A, kuti izere hunhu, ona ESI, Supplementary 15). -21 ) Kubva kune donor (diethylamine) kusvika kubvuma (TDF), RuET ine zvakafanana intramolecular CT maitiro seRuDA.Sezvingatarisirwa, kutorwa kweRuET muDMF kwakaratidza bhendi yakaderera yekubhadhara mari yekufambisa ine simba rakasimba munzvimbo yepedyo infrared munharaunda ye600-1100 nm (Fig. 4B).Mukuwedzera, RuET aggregation yakacherechedzwawo nekuwedzera kwehuwandu hwemvura, iyo yakaratidzirwa mukutsvuka kwepamusoro wekutora, iyo yakawedzera kusimbiswa nemvura yeAFM imaging (Supplementary Fig. 22).Mhedzisiro yacho inoratidza kuti RuET, seRuDA, inogona kuumba intramolecular states uye kuzviunganidza muzvikamu zvakaunganidzwa.
Kemikari chimiro cheRuET.B Absorption spectra yeRuET mumisanganiswa yezvikamu zvakasiyana zveDMF nemvura.Plots C EIS Nyquist yeRuDA uye RuET.Photocurrent mhinduro D yeRuDA uye RuET pasi pechiito chemalaser radiation ine wavelength ye808 nm.
Iyo photodegradation yeABDA pamberi peRuET yakaongororwa neradiation ine laser ine wavelength ye808 nm.Zvinoshamisa kuti, hapana kuora kweABDA kwakaonekwa muzvikamu zvakasiyana-siyana zvemvura (Supplementary Fig. 23).Chikonzero chinogona kunge chiri chekuti RuET haigone kuumba bhendi yemagetsi chimiro nekuti iyo ethyl cheni haikurudzire inoshanda intermolecular charge transfer.Nokudaro, electrochemical impedance spectroscopy (EIS) uye zvishoma zvishoma photocurrent zviyero zvakaitwa kuenzanisa photoelectrochemical properties yeRuDA neRuET.Maererano neNyquist plot (Figure 4C), RuDA inoratidza radius duku zvikuru kupfuura RuET, izvo zvinoreva kuti RuDA56 inokurumidza intermolecular electron transport uye zviri nani conductivity.Mukuwedzera, iyo photocurrent density yeRuDA yakakwirira zvikuru kupfuura yeRuET (Fig. 4D), ichisimbisa zviri nani kutengesa kutengesa kushandiswa kweRuDA57.Nokudaro, boka re phenyl re triphenylamine muOre rinoita basa rinokosha mukupa intermolecular charge transfer uye kuumbwa kwebhandi yemagetsi chimiro.
Kuwedzera bundu kuunganidzwa uye mu vivo biocompatibility, isu takawedzera kuvhara RuDA neF127.Ivhareji ye hydrodynamic dhayamita yeRuDA-NPs yakatsunga kuva 123.1 nm ine kugoverwa kwakamanikana (PDI = 0.089) uchishandisa dynamic light scattering (DLS) nzira (Figure 5A), iyo yakakurudzira kuunganidza bundu nekuwedzera permeability nekuchengetedza.EPR) mhedzisiro.Mifananidzo yeTEM yakaratidza kuti Ore NPs ine yunifomu spherical shape ine avhareji yedhayamita ye86 nm.Zvinonyanya kukosha, kubatwa kwepamusoro kweRuDA-NPs kwakaonekwa pa800 nm (Supplementary Fig. 24), zvichiratidza kuti RuDA-NPs inogona kuchengetedza mabasa uye zvinhu zvekuzvibatanidza maRuDAs.Iyo yakaverengerwa ROS quantum goho yeNP Ore ndeye 15.9%, iyo inofananidzwa neOre.The photothermal properties yeRuDA NPs yakadzidzwa pasi pechiito chemalaser radiation ine wavelength ye808 nm uchishandisa infrared kamera.Sezvinoratidzwa mufig.5B, C, boka rekutonga (PBS chete) rakawana kuwedzera kuduku kwekushisa, nepo kutonhora kweRuDA-NPs mhinduro yakawedzera nokukurumidza nekuwedzera kwekushisa (ΔT) kusvika 15.5, 26.1, uye 43.0 ° C.High concentrations yaiva 25, 50, uye 100 µM, maererano, iyo inoratidza simba rakasimba rephotothermal reRuDA NPs.Mukuwedzera, zviyero zvekupisa / kutonhora zvakatorwa kuti zviongorore kugadzikana kwe photothermal yeRuDA-NP uye kuenzanisa neCGG.Kupisa kweOre NPs hakuna kuderera mushure memazuva mashanu ekupisa / kutonhora (Fig. 5D), iyo inoratidza yakanakisa photothermal kugadzikana kweOre NPs.Kusiyana neizvi, ICG inoratidza kudzikama kwephotothermal sekuonekwa kubva pakunyangarika kuri pachena kweiyo photothermal tembiricha yepasi pasi pemamiriro akafanana.Maererano nenzira yapfuura58, photothermal conversion effectiveness (PCE) yeRuDA-NP yakaverengwa se24.2%, iyo yakakwirira kudarika iripo photothermal zvinhu zvakadai segoridhe nanorods (21.0%) uye nanoshells yegoridhe (13.0%)59.Nekudaro, NP Ore inoratidza yakanakisa photothermal zvivakwa, izvo zvinoita kuti vavimbise PTT vamiririri.
Kuongororwa kweDLS uye TEM mifananidzo yeRuDA NPs (inset).B Thermal mifananidzo yezvikamu zvakasiyana-siyana zveRuDA NPs inoratidzirwa kune laser radiation pane wavelength ye808 nm (0.5 W cm-2).C Photothermal kutendeuka curves yezvakasiyana-siyana zveore NPs, iyo yakawanda data.B. D Kuwedzera kwekushisa kweORE NP uye ICG pamusoro pe5 kupisa-kutonhora kutenderera.
Photocytotoxicity yeRuDA NPs inopesana neMDA-MB-231 masero ekenza yemazamu evanhu akaongororwa mu vitro.Sezvinoratidzwa mufig.6A, B, RuDA-NPs uye RuDA vakaratidza kusakosha cytotoxicity mukushayikwa kweirradiation, zvichireva kuderera kwakasviba kwehutu hweRuDA-NPs uye RuDA.Nekudaro, mushure mekuratidzwa nelaser radiation pawavelength ye808 nm, RuDA neRuDA NPs yakaratidza yakasimba photocytotoxicity inopesana neMDA-MB-231 cancer maseru ane IC50 values ​​​​(half-maximum inhibitory concentration) ye5.4 uye 9.4 μM, zvichiteerana, zvichiratidza kuti RuDA-NP neRuDA vane mukana wekenza phototherapy.Mukuwedzera, iyo photocytotoxicity yeRuDA-NP neRuDA yakaramba ichiongororwa pamberi pevhitamini C (Vc), ROS scavenger, kujekesa basa reROS muchiedza-induced cytotoxicity.Zviripachena, kushanda kwesero kwakawedzera mushure mekuwedzerwa kweVc, uye IC50 tsika dzeRuDA neRuDA NPs dzaive 25.7 uye 40.0 μM, zvichiteerana, izvo zvinoratidza basa rakakosha reROS muphotocytotoxicity yeRuDA neRuDA NPs.Light-induced cytotoxicity yeRuDA-NPs uye RuDA muMDA-MB-231 masero ekenza nehupenyu / akafa sero staining achishandisa calcein AM (green fluorescence yemasero mhenyu) uye propidium iodide (PI, tsvuku fluorescence yemasero akafa).inosimbiswa nemasero) se fluorescent probes.Sezvinoratidzwa muMufananidzo 6C, masero akabatwa neRuDA-NP kana RuDA akaramba achishanda pasina irradiation, sezvinopupurirwa neyakaoma green fluorescence.Pane zvinopesana, pasi pelaser irradiation, chete fluorescence tsvuku yakaonekwa, iyo inosimbisa photocytotoxicity inoshanda yeRuDA kana RuDA NPs.Zvinokosha kuziva kuti green fluorescence yakaonekwa pakuwedzera kweVc, iyo inoratidza kutyorwa kwephotocytotoxicity yeRuDA neRuDA NPs.Mhedzisiro iyi inoenderana ne in vitro photocytotoxicity assays.
Dose-inotsamira kushanda kweA RuDA- uye B RuDA-NP masero muMDA-MB-231 masero muhupo kana kusavapo kweVc (0.5 mM), zvichiteerana.Mabhawa ekukanganisa, zvinoreva ± kutsauka kwakajairwa (n = 3). Zvisina kupetwa, mativi maviri t bvunzo *p <0.05, **p <0.01, uye ***p <0.001. Zvisina kupetwa, mativi maviri t bvunzo *p <0.05, **p <0.01, uye ***p <0.001. Непарные двусторонние t-критерии *p <0,05, **p <0,01 uye ***p <0,001. t-bvunzo dzisina kupetwa mbiri *p<0.05, **p<0.01, uye ***p<0.001.未配对的双边t 检验*p < 0.05,**p < 0.01 和***p < 0.001.未配对的双边t 检验*p < 0.05,**p < 0.01 和***p < 0.001. Непарные двусторонние t-тесты *p <0,05, **p <0,01 uye ***p <0,001. t-bvunzo dzisina kupetwa mbiri *p<0.05, **p<0.01, uye ***p<0.001.C Rarama/yakafa sero staining ongororo uchishandisa calcein AM uye propidium iodide sefluorescent probes.Scale bar: 30 µm.Mifananidzo inomiririra yezvitatu zvebhayoloji inodzokorora kubva kuboka rega rega inoratidzwa.D Confocal fluorescence mifananidzo yeROS kugadzirwa muMDA-MB-231 masero pasi pemamiriro akasiyana ekurapa.Green DCF fluorescence inoratidza kuvapo kweROS.Irradiate nelaser ine wavelength ye808 nm ine simba re 0.5 W/cm2 kwemaminitsi gumi (300 J/cm2).Scale bar: 30 µm.Mifananidzo inomiririra yezvitatu zvebhayoloji inodzokorora kubva kuboka rega rega inoratidzwa.E Flow cytometry RuDA-NPs (50 µM) kana RuDA (50 µM) kuongororwa kwekurapa kana pasina 808 nm laser (0.5 W cm-2) muhupo uye kusavapo kweVc (0.5 mM) ye10 min.Mifananidzo inomiririra yezvitatu zvebhayoloji inodzokorora kubva kuboka rega rega inoratidzwa.F Nrf-2, HSP70 uye HO-1 yeMDA-MB-231 masero anobatwa neRuDA-NPs (50 µM) kana pasina 808 nm laser irradiation (0.5 W cm-2, 10 min, 300 J cm-2) , masero anotaura 2).Mifananidzo inomiririra yezvinyorwa zviviri zvinodzokorora kubva kuboka rega rega zvinoratidzwa.
Intracellular ROS kugadzirwa muMDA-MB-231 masero akaongororwa achishandisa 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method.Sezvinoratidzwa mufig.6D, masero anobatwa neRuDA-NPs kana RuDA airatidza zvakasiyana-siyana girini fluorescence payakavhenekerwa ne808 nm laser, zvichiratidza kuti RuDA-NPs neRuDA vane kugona kunoshanda kugadzira ROS.Pane zvinopesana, pasina chiedza kana pamberi peVc, chete chiratidzo chisina simba chefluorescent chemasero chakaonekwa, icho chairatidza kuumbwa kuduku kweROS.Intracellular ROS mazinga muRuDA-NP masero uye RuDA-yakabatwa MDA-MB-231 masero akawedzera kutsanangurwa nekuyerera cytometry.Sezvinoratidzwa muSupplementary Figure 25, iyo inonzi fluorescence intensity (MFI) yakagadzirwa neRuDA-NPs uye RuDA pasi pe808 nm laser irradiation yakawedzera zvakanyanya neinenge 5.1 uye 4.8 nguva, zvichienderana, zvichienzaniswa neboka rekutonga, richisimbisa maitiro avo akanaka AFK.kukwanisa.Zvisinei, intracellular ROS mazinga muRuDA-NP kana MDA-MB-231 masero akabatwa neRuDA aingofananidzwa nekutonga pasina laser irradiation kana pamberi peVc, zvakafanana nemigumisiro ye confocal fluorescence analysis.
Zvave zvichiratidzwa kuti mitochondria ndiyo inonyanya kunangwa yeRu(II) -arene complexes60.Nokudaro, iyo subcellular localization yeRuDA neRuDA-NPs yakaongororwa.Sezvinoratidzwa muSupplementary Figure 26, RuDA neRuDA-NP inoratidza maitiro akafanana ekuparadzira masero ane kuunganidza kwakanyanya mumitochondria (62.5 ± 4.3 uye 60.4 ± 3.6 ng / mg mapuroteni, zvichiteerana).Zvisinei, zvishoma zvishoma zveRu zvakawanikwa muzvikamu zvenyukireya zveOre uye NP Ore (3.5 uye 2.1%, maererano).Chikamu chesero chakasara chine ruthenium yakasara: 31.7% (30.6 ± 3.4 ng / mg protein) yeRuDA uye 42.9% (47.2 ± 4.5 ng / mg protein) yeRuDA-NPs.Kazhinji, Ore neNP Ore inonyanya kuunganidzwa mumitochondria.Kuongorora kusashanda kwemitochondrial, takashandisa JC-1 uye MitoSOX Tsvuku tsvuku kuongorora mitochondrial membrane inogona uye superoxide kugadzirwa kwesimba, zvichiteerana.Sezvinoratidzwa muSupplementary Fig. 27, yakanyanya girini (JC-1) uye tsvuku (MitoSOX Red) fluorescence yakaonekwa mumasero akabatwa neRuDA uye RuDA-NPs pasi pe808 nm laser irradiation, zvichiratidza kuti zvose RuDA uye RuDA-NPs zvikuru fluorescent. Iyo inogona kunyatso kurudzira mitochondrial membrane depolarization uye superoxide kugadzirwa.Mukuwedzera, iyo nzira yekufa kwesero yakatemwa kushandisa kuyerera kwecytometry yakavakirwa ongororo yeannexin V-FITC/propidium iodide (PI).Sezvinoratidzwa muMufananidzo 6E, kana yakasvibiswa ne808 nm laser, RuDA neRuDA-NP yakakonzera kuwedzera kwakanyanya kweapoptosis rate (yakaderera kurudyi quadrant) muMDA-MB-231 masero achienzaniswa nePBS kana PBS pamwe nelaser.masero akagadzirwa.Zvisinei, apo Vc yakawedzerwa, iyo apoptosis rate yeRuDA neRuDA-NP yakaderera zvikuru kubva ku50.9% uye 52.0% kusvika ku15.8% uye 17.8%, maererano, iyo inosimbisa basa rinokosha reROS mu photocytotoxicity yeRuDA uye RuDA-NP..Mukuwedzera, masero mashoma necrotic akaonekwa mumapoka ose akaedzwa (kumusoro kuruboshwe quadrant), zvichiratidza kuti apoptosis inogona kunge iri iyo inonyanya kufa kwesero inokonzerwa neRuDA neRuDA-NPs.
Sezvo oxidative kusagadzikana kukuvadzwa kuri chinhu chikuru chinogadzirisa apoptosis, iyo nyukireya inobatanidza ne erythroid 2, factor 2 (Nrf2) 62, yakakosha regulator ye antioxidant system, yakaongororwa muRuDA-NPs-yakabatwa MDA-MB-231.Mechanism yekuita kweRuDA NPs inokonzerwa neradiation.Panguva imwecheteyo, kutaura kweprotein yakadzika heme oxygenase 1 (HO-1) yakaonekwa zvakare.Sezvakaratidzwa muMufananidzo 6F uye Supplementary Figure 29, RuDA-NP-mediated phototherapy yakawedzera Nrf2 uye HO-1 mazinga ekutaura akaenzaniswa neboka rePBS, zvichiratidza kuti RuDA-NPs inogona kukurudzira oxidative stress signaling pathways.Mukuwedzera, kuti udzidze photothermal effect yeRuDA-NPs63, kutaura kweprotein inopisa inopisa Hsp70 yakaongororwawo.Zviri pachena kuti masero anobatwa neRuDA-NPs + 808 nm laser irradiation yakaratidza kuwedzera kwekutaura kweHsp70 kana ichienzaniswa nemamwe mapoka maviri, ichiratidza mhinduro yemasero kune hyperthermia.
Iyo inoshamisa in vitro mhedzisiro yakatikurudzira kuti tiongorore in vivo mashandiro eRuDA-NP mu nude mbeva ane MDA-MB-231 tumors.Kugoverwa kwenyama yeRuDA NPs kwakadzidzwa nekugadzirisa zviri mukati meruthenium muchiropa, mwoyo, spleen, itsvo, mapapu, uye mapundu.Sezvinoratidzwa mufig.7A, iyo yakawanda yeOre NPs mumitezo yakajairwa yakaonekwa panguva yekutanga yekutarisa (4 h), nepo iyo yakanyanya kunyorwa yakatemerwa mu tumor tissues maawa masere mushure mejekiseni, pamwe nekuda kweOre NPs.EPR mhedzisiro yeLF.Maererano nemigumisiro yekugovera, nguva yakakwana yekurapa neNP ore yakatorwa maawa masere mushure mekutonga.Kuenzanisira nzira yekuunganidza kweRuDA-NPs munzvimbo dzebundu, photoacoustic (PA) zvinhu zveRuDA-NPs zvakaongororwa nekunyora zviratidzo zvePA zveRuDA-NPs panguva dzakasiyana mushure mejojo.Kutanga, chiratidzo chePA cheRuDA-NP mu vivo chakaongororwa nekunyora PA mifananidzo yenzvimbo yebundu mushure mejojo ​​ye intratumoral yeRuDA-NP.Sezvinoratidzwa muSupplementary Figure 30, RuDA-NPs yakaratidza chiratidzo chakasimba chePA, uye pakanga paine kuwirirana kwakanaka pakati peRuDA-NP concentration uye PA chiratidzo chesimba (Supplementary Figure 30A).Zvadaro, mu vivo PA mifananidzo yebundu nzvimbo yakanyorwa mushure mejekiseni remutsinga weRuDA neRuDA-NP panguva dzakasiyana mushure mejekiseni.Sezvinoratidzwa muMufananidzo 7B, chiratidzo chePA cheRuDA-NPs kubva panzvimbo yebundu zvishoma nezvishoma chakawedzera nekufamba kwenguva uye chakasvika pakakwirira pamaawa masere mushure mejekiseni, zvinoenderana nemigumisiro yekugovera tishu yakatemwa neICP-MS kuongorora.Nezve RuDA (Supplementary Fig. 30B), iyo yakawanda PA chiratidzo chesimba yakaonekwa maawa 4 mushure mejojo, zvichiratidza kukurumidza kwekupinda kweRuDA mubundu.Mukuwedzera, maitiro ekubuda kunze kweRuDA neRuDA-NPs akaongororwa nekugadzirisa huwandu hweruthenium muweti uye tsvina uchishandisa ICP-MS.Nzira huru yekubvisa RuDA (Supplementary Fig. 31) uye RuDA-NPs (Fig. 7C) iri kuburikidza netsvina, uye kubviswa kunobudirira kweRuDA neRuDA-NPs kwakaonekwa munguva ye8-day yekudzidza, zvinoreva kuti RuDA. uye RuDA-NPs inogona kunyatsobviswa kubva mumuviri pasina chepfu kwenguva refu.
A. Ex vivo kugoverwa kweRuDA-NP mumasumbu embeva yakatemwa neRu content (muzana weiyo inotemerwa dose yeRu (ID) pagiramu yetishu) panguva dzakasiyana mushure mejekiseni.Dhata zvinoreva ± chiyero chakatsauka (n = 3). Zvisina kupetwa, mativi maviri t bvunzo *p <0.05, **p <0.01, uye ***p <0.001. Zvisina kupetwa, mativi maviri t bvunzo *p <0.05, **p <0.01, uye ***p <0.001. Непарные двусторонние t-критерии *p <0,05, **p <0,01 uye ***p <0,001. t-bvunzo dzisina kupetwa mbiri *p<0.05, **p<0.01, uye ***p<0.001.未配对的双边t 检验*p < 0.05,**p < 0.01 和***p < 0.001.未配对的双边t 检验*p < 0.05,**p < 0.01 和***p < 0.001. Непарные двусторонние t-тесты *p <0,05, **p <0,01 uye ***p <0,001. t-bvunzo dzisina kupetwa mbiri *p<0.05, **p<0.01, uye ***p<0.001.B PA mifananidzo ye in vivo tumor sites pa 808 nm excitation mushure me intravenous administration yeRuDA-NPs (10 µmol kg-1) panguva dzakasiyana-siyana.Mushure mekutonga kwemukati kweRuDA NPs (10 µmol kg-1), C Ru yakabviswa kubva kumakonzo ane weti uye tsvina panguva dzakasiyana.Dhata zvinoreva ± chiyero chakatsauka (n = 3).
Kupisa simba reRuDA-NP mu vivo rakadzidzwa mu nude mice ne MDA-MB-231 uye RuDA tumors kuenzanisa.Sezvinoratidzwa mufig.8A uye kuwedzera Fig. 32, boka rekutonga (saline) rakaratidza kuderera kwekushisa (ΔT ≈ 3 ° C) mushure memaminitsi e10 ekuenderera mberi.Zvisinei, kutonhora kweRuDA-NPs neRuDA kwakawedzera nokukurumidza nepamusoro pekushisa kwe55.2 uye 49.9 ° C, maererano, kupa hyperthermia yakakwana ye in vivo cancer therapy.Kuonekwa kwekuwedzera kwekushisa kwepamusoro kweRuDA NPs (ΔT ≈ 24 ° C) yakaenzaniswa neRuDA (ΔT ≈ 19 ° C) inogona kunge yakakonzerwa nekukwanisa kwayo kupindira uye kuunganidza mumasumbu emaronda nekuda kweEPR effect.
Infrared thermal mifananidzo yemakonzo ane MDA-MB-231 tumors yakavhenekerwa ne808 nm laser panguva dzakasiyana 8 maawa mushure mejekiseni.Mifananidzo inomiririra yezvina biological inodzokorora kubva kuboka rega rega inoratidzwa.B Relative bundu vhoriyamu uye C Avhareji yebundu hukuru hwemapoka akasiyana emakonzo panguva yekurapwa.D Macurve ehuremu hwemuviri wemapoka akasiyana emakonzo.Irradiate nelaser ine wavelength ye808 nm ine simba re 0.5 W/cm2 kwemaminitsi gumi (300 J/cm2).Mabhawa ekukanganisa, zvinoreva ± kutsauka kwakajairwa (n = 3). Zvisina kupetwa, mativi maviri t bvunzo *p <0.05, **p <0.01, uye ***p <0.001. Zvisina kupetwa, mativi maviri t bvunzo *p <0.05, **p <0.01, uye ***p <0.001. Непарные двусторонние t-критерии *p <0,05, **p <0,01 uye ***p <0,001. t-bvunzo dzisina kupetwa mbiri *p<0.05, **p<0.01, uye ***p<0.001.未配对的双边t 检验*p < 0.05,**p < 0.01 和***p < 0.001.未配对的双边t 检验*p < 0.05,**p < 0.01 和***p < 0.001. Непарные двусторонние t-тесты *p <0,05, **p <0,01 uye ***p <0,001. t-bvunzo dzisina kupetwa mbiri *p<0.05, **p<0.01, uye ***p<0.001. E H & E inosvibisa mifananidzo yenhengo huru uye mapundu kubva kumapoka ekurapa akasiyana, kusanganisira Saline, Saline + Laser, RuDA, RuDA + Laser, RuDA-NPs, uye RuDA-NPs + Laser mapoka. E H & E inosvibisa mifananidzo yenhengo huru uye mapundu kubva kumapoka ekurapa akasiyana, kusanganisira Saline, Saline + Laser, RuDA, RuDA + Laser, RuDA-NPs, uye RuDA-NPs + Laser mapoka. Изображения окрашивания E H&E основных органов и опухолей из разных групп лечения, включая группы физиологического раствора, физиологического раствора + лазера, RuDA, RuDA + Laser, RuDA-NPs и RuDA-NPs + Laser. E H & E inosvibisa mifananidzo yenhengo huru uye mapundu kubva kumapoka akasiyana ekurapa, kusanganisira saline, saline + laser, RuDA, RuDA + Laser, RuDA-NPs, uye RuDA-NPs + Laser mapoka.來自不同治疗组的主要器官和肿瘤的E H&E 染色图像，包括盐水、盐水+ 激光、RuDA、RuDA +和激光、RuDA + 和激光 NPs、RuDA + 激光、RuDA來自不同治疗组的主要器官和肿瘤的E H&E Окрашивание E H&E основных органов и опухолей из различных групп лечения, включая физиологический раствор, физиологический раствор + лазер, RuDA, RuDA + лазер, RuDA-NPs и RuDA-NPs + лазер. E H & E kusvibiswa kwenhengo huru uye mapundu kubva kumapoka akasiyana-siyana ekurapa anosanganisira saline, saline + laser, RuDA, RuDA + laser, RuDA-NPs, uye RuDA-NPs + laser.Scale bar: 60 µm.
Mhedzisiro yephototherapy mu vivo neRuDA neRuDA NPs yakaongororwa umo mbeva dzisina kupfeka dzine MDA-MB-231 tumors dzakaiswa intravenously neRuDA kana RuDA NPs pamutengo mumwe we10.0 µmol kg-1 kuburikidza nemuswe wemuswe, uye ipapo 8 maawa mushure mejekiseni.laser irradiation ine wavelength ye808 nm.Sezvinoratidzwa muMufananidzo 8B, mavhoriyamu ebundu akawedzera zvakanyanya mumapoka esaline uye laser, zvichiratidza kuti saline kana laser 808 irradiation haina mhedzisiro pakukura kwebundu.Sezviri muboka resaline, kukurumidza kukura kwebundu kwakaonekwawo mumakonzo akabatwa neRuDA-NPs kana RuDA pasina laser irradiation, zvichiratidza kuderera kwavo kwakasviba kwakasviba.Kusiyana neizvi, mushure mekuvhenekerwa nelaser, kurapwa kweRuDA-NP neRuDA kwakakonzera kuderera kwebundu nebundu kuderedzwa kwe95.2% uye 84.3%, zvichiteerana, zvichienzaniswa neboka rakarapwa saline, zvichiratidza yakanakisa synergistic PDT., inopindirana neRuDA/CHTV effect.Kuenzaniswa neRuDA, RuDA NPs yakaratidza zviri nani phototherapeutic effect, iyo yainyanya kukonzerwa neEPR effect yeRuDA NPs.Tumor kukura inhibition mhedzisiro yakawedzera kuongororwa nehuremu hwebundu hwakabviswa pazuva 15 rekurapa (Fig. 8C uye Supplementary Fig. 33).Izvo zvinoreva bundu guru muRuDA-NP yakabata mbeva uye RuDA yakabata mbeva yaiva 0.08 uye 0.27 g, maererano, iyo yakanga yakanyanya kureruka kupfuura muboka rekutonga (1.43 g).
Mukuwedzera, uremu hwemuviri wemakonzo hwakanyorwa mazuva matatu ega ega kuti udzidze kusvibiswa kwerima kweRuDA-NPs kana RuDA in vivo.Sezvakaratidzwa muMufananidzo 8D, hapana misiyano yakakura muhuremu hwemuviri yakaonekwa kumapoka ose ekurapa. Uyezve, kusvibiswa kwehematoxylin uye eosin (H & E) yenhengo huru (moyo, chiropa, spleen, mapapu, uye itsvo) kubva kumapoka akasiyana-siyana ekurapa akaitwa. Uyezve, kusvibiswa kwehematoxylin uye eosin (H & E) yenhengo huru (moyo, chiropa, spleen, mapapu, uye itsvo) kubva kumapoka akasiyana-siyana ekurapa akaitwa. Кроме того, было проведено окрашивание гематоксилином и эозином (H&E) основных органов (сердца, печени, селезенки, легких и поразкыпы). Mukuwedzera, hematoxylin uye eosin (H & E) tsvina yenhengo huru (moyo, chiropa, spleen, mapapu, uye itsvo) kubva kumapoka akasiyana-siyana ekurapa akaitwa.此外，对不同治疗组的主要器官（心脏、肝脏、脾脏、肺和肾脏）进苏木精和伊红(H&E) (H&E) Кроме того, проводили окрашивание гематоксилином и эозином (H&E) основных органов (сердца, печени, селезенки, легких и почерук) в разлихупенция в разлиханов. Mukuwedzera, hematoxylin uye eosin (H & E) tsvina yenhengo huru (moyo, chiropa, spleen, mapapu, uye itsvo) yakaitwa mumapoka akasiyana-siyana ekurapa.Sezvinoratidzwa muFig.8E, iyo H&E inosvibisa mifananidzo yenhengo shanu huru kubva kumapoka eRuDA-NPs neRuDA inoratidza kusakanganiswa kuri pachena kana kukuvadzwa kwenhengo. 8E, iyo H&E inosvibisa mifananidzo yenhengo shanu huru kubva kumapoka eRuDA-NPs neRuDA inoratidza kusakanganiswa kuri pachena kana kukuvadzwa kwenhengo.Sezvinoratidzwa mufig.8E, изображения окрашивания H&E пяти основных органов из групп RuDA-NPs uye RuDA не демонстрируют явных аномалий или повреждений органий. 8E, H & E inosvibisa mifananidzo yezvishanu nhengo huru kubva kuRuDA-NPs uye mapoka eRuDA anoratidza kusaoneka kwenhengo yenhengo kana maronda.如图8E 所示，來自RuDA-NPs 和RuDA 组的五个主要器官的H&E 染色图像沒有显示出明显的异常或器官.如图8E 所示，來自RuDA-NPs 和RuDA 组的五个主要器官的H&E Как показано на рисунке 8E, изображения окрашивания H&E ichangoburwa neruda-NPs uye RuDA haina kumbomira mira. Sezvakaratidzwa muMufananidzo 8E, H & E inosvibisa mifananidzo yezvikamu zvishanu zvikuru kubva kuRuDA-NPs nemapoka eRuDA yakaratidza kusakanganiswa kuri pachena kana kukanganisa kwenhengo.Izvi zvakaratidza kuti hapana RuDA-NP kana RuDA yakaratidza zviratidzo zvehuturu mu vivo. Uyezve, H & E staining mifananidzo yemamota yakaratidza kuti mapoka maviri eRuDA + Laser uye RuDA-NPs + Laser anogona kukonzera kuparadzwa kwakanyanya kwekenza, zvichiratidza zvakanakisa mu vivo phototherapeutic efficacy yeRuDA neRuDA-NPs. Uyezve, H & E staining mifananidzo yemamota yakaratidza kuti mapoka maviri eRuDA + Laser uye RuDA-NPs + Laser anogona kukonzera kuparadzwa kwakanyanya kwekenza, zvichiratidza zvakanakisa mu vivo phototherapeutic efficacy yeRuDA neRuDA-NPs.Mukuwedzera, hematoxylin-eosin yakasvibiswa bundu mifananidzo yakaratidza kuti mapoka maviri eRuDA + Laser uye RuDA-NPs + Laser anogona kukonzera kuparadzwa kwakanyanya kwekenza masero, zvichiratidza hukuru hwephototherapeutic efficacy yeRuDA neRuDA-NPs in vivo.此外，肿瘤的H&E 染色图像显示，RuDA + Laser 和RuDA-NPs + Laser 组均可导致严重的癌细胞破坏，说夺坏，话公经亚会此外 , 肿瘤 的 & e 染色 显示 , ruda + laser 和 ruda-nps + laser 组均 导致 的 癌细胞 破坏 , 证明 组均的。 . ...Mukuwedzera, hematoxylin uye eosin yakasvibiswa mifananidzo yebundu yakaratidza kuti mapoka ose eRuDA + Laser uye RuDA-NPs + Laser akaguma nekuparadzwa kwakanyanya kwemasero emukenza, zvichiratidza hukuru hwephototherapeutic efficacy yeRuDA neRuDA-NPs in vivo.
Mukupedzisa, iyo Ru (II) -arene (RuDA) organometallic complex ine DA-type ligands yakagadzirirwa kufambisa chirongwa cheISC uchishandisa nzira yekuunganidza.Synthesized RuDA inogona kuzviunganidza kuburikidza nekusabatana kwekubatana kuumba RuDA-derived supramolecular systems, zvichiita kuti 1O2 igadzire uye inobudirira photothermal kutendeuka kwechiedza-induced cancer therapy.Zvinokosha kuziva kuti monomeric RuDA haina kuburitsa 1O2 pasi pelaser irradiation pa808 nm, asi inogona kuburitsa huwandu hukuru hwe1O2 munzvimbo yakaunganidzwa, ichiratidza kurongeka uye kugona kwedhizaini yedu.Zvidzidzo zvakazotevera zvakaratidza kuti supramolecular assembly inopa RuDA nekuvandudzwa kwefotophysical uye photochemical properties, zvakadai seredshift absorption uye photobleaching resistance, izvo zvinonyanya kudiwa kuPDT uye PTT kugadzirisa.Zvese zviri zviviri mu vitro uye mu vivo kuyedza zvakaratidza kuti RuDA NPs ine yakanaka biocompatibility uye yakanaka kuunganidzwa mubundu inoratidza yakanakisa chiedza-induced anticancer chiitiko pane laser irradiation pawavelength ye808 nm.Saka, RuDA NPs seanoshanda bimodal supramolecular PDT/PTW reagents ichapfumisa seti ye photosensitizers inogadziriswa pawavelengths pamusoro pe800 nm.Iyo yekufungidzira dhizaini yeiyo supramolecular system inopa inoshanda nzira yeNIR-activated photosensitizers ine yakanakisa photosensitizing mhedzisiro.
Zvese makemikari uye zvinonyungudutsa zvakawanikwa kubva kune vatengesi vekutengesa uye zvakashandiswa pasina kumwe kucheneswa.RuCl3 yakatengwa kubva kuBoren Precious Metals Co., Ltd. (Kunming, China).[(η6-p-cym)Ru(fendio)Cl]Cl (fendio = 1,10-phenanthroline-5,6-dione) uye 4,7-bis[4-(N,N-diphenylamino)phenyl]-5 ,6-Diamino-2,1,3-benzothiadiazole yakagadzirwa maererano nezvidzidzo zvekare64,65.NMR spectra yakarekodhwa paBruker Avance III-HD 600 MHz spectrometer kuSoutheastern University Analytical Test Center vachishandisa d6-DMSO kana CDCl3 se solvent.Kuchinja kwemakemikari δ kunopihwa muppm.nekuremekedza tetramethylsilane, uye mabatiro ekudyidzana J anopiwa muhutsika hwakakwana muhertz.High resolution mass spectrometry (HRMS) yakaitwa paAgilent 6224 ESI/TOF MS chiridzwa.Elemental ongororo yeC, H, uye N yakaitwa paVario MICROCHNOS elemental analyzer (Elementar).UV-inooneka spectra yakayerwa paShimadzu UV3600 spectrophotometer.Fluorescence spectra yakanyorwa paShimadzu RF-6000 spectrofluorimeter.EPR spectra yakanyorwa pane Bruker EMXmicro-6/1 chiridzwa.Iyo morphology uye chimiro chemasampuli akagadzirirwa akadzidzwa paFEI Tecnai G20 (TEM) uye Bruker Icon (AFM) zviridzwa zvinoshanda pamhepo ye200 kV.Dynamic light scattering (DLS) yakaitwa paNanobrook Omni analyzer (Brookhaven).Photoelectrochemical properties yakayerwa pane electrochemical setup (CHI-660, China).Photoacoustic mifananidzo yakawanikwa uchishandisa iyo FUJIFILM VisualSonics Vevo® LAZR system.Mifananidzo yeConfocal yakawanikwa uchishandisa Olympus FV3000 confocal microscope.Kuongororwa kweFACS kwakaitwa paBD Calibur flow cytometer.High performance liquid chromatography (HPLC) miedzo yakaitwa pa Waters Alliance e2695 system uchishandisa 2489 UV/Vis detector.Gel Permeation Chromatography (GPC) bvunzo dzakanyorwa pane Thermo ULTIMATE 3000 chiridzwa uchishandisa ERC RefratoMax520 refractive index detector.
[(η6-p-cym)Ru(fendio)Cl]Cl (fendio = 1,10-phenanthroline-5,6-dione)64 (481.0 mg, 1.0 mmol), 4,7-bis[4 -(N, N-diphenylamino)phenyl]-5,6-diamino-2,1,3-benzothiadiazole 65 (652.0 mg, 1.0 mmol) uye glacial acetic acid (30 mL) yakamutswa pa reflux firiji kwemaawa gumi nemaviri.The solvent yakabva yabviswa muvacuo uchishandisa rotary evaporator.Izvo zvakasara zvakasara zvakanatswa ne flash column chromatography (silica gel, CH2Cl2: MeOH = 20: 1) kuwana RuDA sehupfu hwakasvibirira (chibereko: 877.5 mg, 80%).anus.Yakaverengerwa C64H48Cl2N8RuS: C 67.84, H 4.27, N 9.89.Yakawanikwa: C 67.92, H 4.26, N 9.82.1H NMR (600 MHz, d6-DMSO) δ 10.04 (s, 2H), 8.98 (s, 2H), 8.15 (s, 2H), 7.79 (s, 4H), 7.44 (s, 8H), 7.21 (d, J = 31.2 Hz, 16H), 6.47 (s, 2H), 6.24 (s, 2H), 2.69 (s, 1H), 2 .25 (s, 3H), 0.99 (s, 6H).13c NMR (150 MHZ, D6-DMS), δ (ppm) 158.03, 139.68, 128.01, 125, 123.01, 125, 123.01, 125, 123.01, 125.40. , 103. , 86.52, 84.75, 63.29, 30.90, 22.29, 18.83.ESI-MS: m/z [M-Cl]+ = 1097.25.
Synthesis ye4,7-bis [4-(N,N-diethylamino) phenyl-5,6-diamino-2,1,3-benzothiadiazole (L2): L2 yakagadzirwa mumatanho maviri.Pd(PPh3)4 (46 mg, 0.040 mmol) yakawedzerwa kuN,N-diethyl-4-(tributylstannyl) aniline (1.05 g, 2.4 mmol) uye 4,7-dibromo-5,6-dinitro solution - 2, 1,3-benzothiadiazole (0.38 g, 1.0 mmol) mune yakaoma toluene (100 ml).Musanganiswa wacho wakamutswa pa100 ° C kwemaawa makumi maviri nemana.Mushure mekubvisa toluene muvacuo, iyo yakasimba yakagadziriswa yakagezwa nepetroleum ether.Zvadaro musanganiswa wechikamu ichi (234.0 mg, 0.45 mmol) uye iron powder (0.30 g, 5.4 mmol) muacetic acid (20 ml) yakamutswa pa80 ° C. kwemaawa mana.Iyo reaction musanganiswa yakadururwa mumvura uye yakakonzeresa brown solid yakaunganidzwa nekusefa.Chigadzirwa chacho chakacheneswa kaviri ne vacuum sublimation kuti ipe girini yakasimba (126.2 mg, 57% goho).anus.Yakaverengerwa kuC26H32N6S: C 67.79, H 7.00, N 18.24.Yakawanikwa: C 67.84, H 6.95, H 18.16.1H NMR (600 MHz, CDCl3), δ (ppm) 7.42 (d, 4H), 6.84 (d, 4H), 4.09 (s, 4H), 3.42 (d, 8H), 1.22 (s, 12H).13С NMR (150 MHz, CDCl3), δ (ppm) 151.77, 147.39, 138.07, 131.20, 121.09, 113.84, 111.90, 44.34, 12.77.ESI-MS: m/z [M+H]+ = 461.24.
Makomponi akagadzirirwa uye akanatswa achitevera maitiro akafanana neRuDA.anus.Yakaverengerwa C48H48Cl2N8RuS: C 61.27, H 5.14, N 11.91.Zvawanikwa: C, 61.32, H, 5.12, N, 11.81,1H NMR (600 MHz, d6-DMSO), δ (ppm) 10.19 (s, 2H), 9.28 (s, 2H), 8.09 (s, 2H), 7.95 (s, 4H), 6.93 (s, 4H), 6.48 (d, 2H), 6.34 (s, 2H) , 3.54 (t, 8H), 2.80 (m, 1H), 2.33 (s, 3H), 1.31 (t, 12H), 1.07 (s, 6H).13C NMR (151 MHZ, CDCL3), δ (PPM) 158.20, 148.8.40, 128.35, 121.84, 104.8.70, 87.07, 87.0., 38.06, 31.22, 29.69, 22.29, 19.19, 14.98, 12.93.ESI-MS: m/z [M-Cl]+ = 905.24.
RuDA yakanyungudutswa muMeOH/H2O (5/95, v/v) pachikamu che10 μM.Kutora kweRuDA kwakayerwa maminetsi ega ega e5 paShimadzu UV-3600 spectrophotometer pasi pekuvhenekesa nelaser light ine wavelength ye808 nm (0.5 W/cm2).ICG spectra yakanyorwa pasi pemamiriro akafanana seyero.
EPR spectra yakanyorwa paBruker EMXmicro-6/1 spectrometer ine microwave simba re20 mW, scanning range ye100 G, uye field modulation ye1 G. 2,2,6,6-tetramethyl-4-piperidone. (TEMP) uye 5,5-dimethyl-1-pyrroline N-oxide (DMPO) yakashandiswa semisungo inotenderera.Electron spin resonance spectra yakanyorerwa mhinduro dzakasanganiswa dzeRuDA (50 µM) uye TEMF (20 mM) kana DMPO (20 mM) pasi pekuita kwemwaranzi yelaser ine wavelength ye808 nm (0.5 W/cm2).
DFT uye TD-DFT maverengero eRuDA akaitwa paPBE1PBE/6–31 G*//LanL2DZ mazinga mune aqueous solution uchishandisa chirongwa cheGaussian 1666,67,68.Iyo HOMO-LUMO, gomba uye maerekitironi kugoverwa kwepasi-simba singlet yakafara nyika RuDA yakarongwa uchishandisa chirongwa cheGaussView (vhezheni 5.0).
Isu takatanga kuedza kuyera chizvarwa che1O2 RuDA tichishandisa yakajairika UV-inooneka spectroscopy ine ICG (ΦΔ = 0.002) seyero, asi iyo photodegradation yeICG yakakanganisa zvakanyanya mhedzisiro.Nokudaro, huwandu hwehuwandu hwe1O2 RuDA hwakayerwa nekuona kuchinja kwehutano hweABDA fluorescence inenge 428 nm kana yakasvibiswa ne laser ine wavelength ye808 nm (0.5 W / cm2).Miedzo yakaitwa paRuDA neRuDA NPs (20 μM) mumvura / DMF (98/2, v/v) ine ABDA (50 μM).Kuwanda kwezvibereko zve1O2 kwakaverengwa uchishandisa chirevo chinotevera: ΦΔ (PS) = ΦΔ (ICG) × (rFS/APS)/(rICG/AICG).rPS uye rICG ndiwo maitiro ekuita kweABDA ne1O2 yakawanikwa kubva kune photosensitizer uye ICG, zvichiteerana.APS uye AICG ndiko kutora kwephotosensitizer uye ICG pa808 nm, zvichiteerana.
AFM zviyero zvaiitwa mumamiriro emvura uchishandisa scan mode pane Bruker Dimension Icon AFM system.Kushandisa chimiro chakashama nemasero emvura, masero akashambidzwa kaviri ne ethanol uye akaomeswa nerukova rwe nitrogen.Isai masero akaomeswa mumusoro weoptical wemicroscope.Pakarepo isa donhwe resample mudziva remvura woiisa pacantilever uchishandisa sirinji yepurasitiki inoraswa inoraswa uye tsono isina utachiona.Imwe donho rinoiswa zvakananga pamuenzaniso, uye kana musoro wemaziso wakadzikiswa, madonhwe maviri anobatana, achigadzira meniscus pakati pemuenzaniso uye reservoir yemvura.Zviyero zveAFM zvakaitwa pachishandiswa SCANASYST-FLUID V-yakaita nitride cantilever (Bruker, kuomarara k = 0.7 N m-1, f0 = 120–180 kHz).
HPLC chromatograms yakawanikwa paMvura e2695 system ine phoenix C18 column (250×4.6 mm, 5 µm) vachishandisa 2489 UV/Vis detector.Iyo yavelength ye detector ndeye 650 nm.Nharembozha A uye B yaive mvura uye methanol, zvichiteerana, uye mobile phase flow rate yaive 1.0 ml · min-1.Iyo gradient (solvent B) yaive sezvizvi: 100% kubva pa0 kusvika ku4 maminetsi, 100% kusvika 50% kubva pa5 kusvika 30 maminetsi, uye dzokera ku100% kubva pa31 kusvika 40 maminetsi.Ore yakanyungudutswa mune yakasanganiswa mhinduro ye methanol nemvura (50/50, nehuwandu) pahuwandu hwe50 μM.Huwandu hwejekiseni hwaive 20 μl.
GPC miyedzo yakanyorwa pachiridzwa cheThermo ULTIMATE 3000 chine maviri PL aquagel-OH MIXED-H makoramu (2 × 300 × 7.5 mm, 8 µm) uye ERC RefratoMax520 refractive index detector.Iyo GPC column yakakwenenzverwa nemvura pamwero wekuyerera we1 ml/min pa30°C.Ore NPs yakanyungudutswa muPBS mhinduro (pH = 7.4, 50 μM), jekiseni vhoriyamu yaive 20 μL.
Photocurrents yakayerwa pane electrochemical setup (CHI-660B, China).Mhinduro dzeoptoelectronic apo laser yakabatidzwa nekudzimwa (808 nm, 0.5 W / cm2) yakayerwa pamhepo ye0.5 V mubhokisi dema, zvichiteerana.Sero rechitatu-electrode cell rakashandiswa neL-shaped glassy carbon electrode (GCE) se electrode inoshanda, standard calomel electrode (SCE) sereferensi electrode, uye platinum disk se counter electrode.A 0.1 M Na2SO4 mhinduro yakashandiswa se electrolyte.
Mutsara wekenza yemazamu yemunhu MDA-MB-231 yakatengwa kubva kuKeyGEN Biotec Co., LTD (Nanjing, China, nhamba yekatalogi: KG033).Masero akakura ari monolayers muDulbecco's Modified Eagle's Medium (DMEM, high glucose) yakawedzerwa nemhinduro ye10% fetal bovine serum (FBS), penicillin (100 μg/ml) uye streptomycin (100 μg/ml).Masero ese akagadzirwa pa37 ° C mumhepo ine hunyoro ine 5% CO2.
Chiyero cheMTT chakashandiswa kuona cytotoxicity yeRuDA neRuDA-NPs muhupo uye kusavapo kwechiedza chechiedza, chine kana pasina Vc (0.5 mM).MDA-MB-231 maseru egomarara akakura mu96-tsime mahwendefa pasero density yeanenge 1 x 105 masero/ml/tsime uye akaiswa kwemaawa gumi nemaviri pa37.0 ° C mumhepo ye5% CO2 uye 95% mhepo.RuDA uye RuDA NPs yakanyungudutswa mumvura yakawedzerwa kumasero.Mushure memaawa gumi nemaviri ekukuchidzira, maseru akaiswa pa 0.5 W cm -2 laser mwaranzi pawavelength ye808 nm kwemaminitsi gumi (300 J cm -2) ndokuzoiswa murima kwemaawa makumi maviri nemana.Masero acho akabva aiswa neMTT (5 mg/ml) kwemamwe maawa mashanu.Chekupedzisira, shandura yepakati kuita DMSO (200 µl) kunyungudutsa mhedzisiro yepepuru formazan makristasi.OD maitiro akayerwa uchishandisa microplate muverengi ane wavelength ye570/630 nm.Iko IC50 kukosha kwemuenzaniso wega wega wakaverengerwa uchishandisa iyo SPSS software kubva kudosi-mhinduro macurves akawanikwa kubva kune angangoita matatu akazvimirira kuyedza.
MDA-MB-231 masero akabatwa neRuDA uye RuDA-NP pamusangano we 50 μM.Mushure memaawa gumi nemaviri ekuvharidzirwa, masero akasvibiswa ne laser ine wavelength ye808 nm uye simba re 0.5 W / cm2 ye 10 min (300 J / cm2).Muboka revhitamini C (Vc), maseru akabatwa ne 0.5 mM Vc isati yaitwa laser irradiation.Masero akazoiswa murima kwemamwe maawa makumi maviri nemana, obva asvibiswa necalcein AM uye propidium iodide (20 μg/ml, 5 μl) kwemaminetsi makumi matatu, ndokuzogezwa nePBS (10 μl, pH 7.4).mifananidzo yemasero ane mavara.