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Iivektha zeGene zonyango lwesifo semiphunga se-cystic fibrosis kufuneka zijolise kwiindlela zomoya eziqhubayo kuba ukudluliselwa kwemiphunga engaphandle akuboneleli ngenzuzo yonyango. Ukusebenza kakuhle kokudluliselwa kwentsholongwane kunxulumene ngokuthe ngqo nexesha lokuhlala kwevektha. Nangona kunjalo, ulwelo lokuhambisa olufana nabathwali be-gene lusasazeka ngokwendalo kwi-alveoli ngexesha lokuphefumlelwa, kwaye amasuntswana onyango alo naluphi na uhlobo asuswa ngokukhawuleza kuthutho lwe-mucociliary. Ukwandisa ixesha lokuhlala kwabathwali be-gene kwiindlela zomoya kubalulekile kodwa kunzima ukulifezekisa. Amasuntswana emagnethi ahlanganiswe yi-gene carrier anokujoliswa kumphezulu weendlela zomoya anokuphucula ukujoliswa kwengingqi. Ngenxa yemingeni yokubona kwi-vivo, ukuziphatha kwamasuntswana amancinci emagnethi kumphezulu wendlela yomoya xa kukho intsimi yemagnethi esetyenzisiweyo akuqondwa kakuhle. Injongo yolu phononongo yayikukusebenzisa i-synchrotron imaging ukubona intshukumo ye-in vivo yothotho lwamasuntswana emagnethi kwi-trachea yeempuku ezinesifo sokuphefumla ukuhlola amandla kunye neepatheni zokuziphatha komntu ngamnye kunye nobuninzi be-particle kwi-vivo. Emva koko savavanya nokuba ukuhanjiswa kwamasuntswana emagnethi e-lentiviral phambi kwentsimi yemagnethi kuya kwandisa ukusebenza kakuhle kokudluliselwa kwi-rat. I-trachea.I-Synchrotron X-ray imaging ityhila indlela amasuntswana emagnethi asebenza ngayo kwiindawo ezimileyo nezihambayo zemagnethi kwi-vitro nakwi-vivo. Amasuntswana awanakutsalwa lula kumphezulu womoya ophilayo ngeemagnethi, kodwa ngexesha lokuthuthwa, iidipozithi zixinene kwindawo yokujonga apho intsimi yemagnethi inamandla khona. Ukusebenza kakuhle kwetransduction nako kwandiswe ngokuphindwe kathandathu xa amasuntswana emagnethi e-lentiviral aziswa phambi kwentsimi yemagnethi. Zizonke, ezi ziphumo zibonisa ukuba amasuntswana emagnethi e-lentiviral kunye namasuntswana emagnethi anokuba ziindlela ezixabisekileyo zokuphucula ukujoliswa kwe-gene vector kunye nokwandisa amanqanaba etransduction ekuqhubeni iindlela zemimoya kwi-vivo.
I-Cystic fibrosis (CF) ibangelwa kukwahluka kwijini enye ebizwa ngokuba yi-CF transmembrane conductance regulator (CFTR). Iprotheyini ye-CFTR ngumjelo we-ion okhoyo kwiiseli ezininzi ze-epithelial emzimbeni wonke, kubandakanya neendlela zomoya eziqhubayo, indawo ephambili ye-CF pathogenesis. Iziphene ze-CFTR zikhokelela ekuthuthweni kwamanzi okungaqhelekanga, zicoca umphezulu womoya kwaye zinciphise ubunzulu bomaleko wolwelo lomphezulu womoya (ASL). Oku kukwaphazamisa amandla enkqubo yokuthutha i-mucociliary (MCT) ukususa amasuntswana aphefumliweyo kunye neentsholongwane kwiindlela zomoya. Injongo yethu kukuphuhlisa unyango lwejini le-lentiviral (LV) ukuhambisa ikopi echanekileyo yejini ye-CFTR kunye nokuphucula impilo ye-ASL, i-MCT, kunye nemiphunga, kunye nokuqhubeka nokuphuhlisa ubuchwepheshe obutsha obukwaziyo ukulinganisa ezi parameters kwi-vivo1.
Iivektha ze-LV zezinye zezona zibalaseleyo kunyango lwe-CF airway gene, kuba zinokudibanisa i-gene yonyango ngokusisigxina kwiiseli ze-basal zomoya (iiseli ze-airway stem). Oku kubalulekile kuba zinokubuyisela umswakama oqhelekileyo kunye nokucocwa kwe-mucus ngokwahlulahlula kwiiseli zomoya ezihambelana ne-CF ezisebenzayo ezilungisiweyo yi-gene, nto leyo ekhokelela kwiingenelo zobomi bonke. Iivektha ze-LV kufuneka zijoliswe kwi-conducting airway, njengoko kulapho isifo se-CF lung siqala khona. Ukuhanjiswa kwe-vektha nzulu emaphaphu kunokubangela i-alveolar transduction, kodwa oku akunazo izibonelelo zonyango kwi-CF. Nangona kunjalo, ulwelo olufana nabathwali be-gene lufudukela kwi-alveoli emva kokuphefumlelwa emva kokuzalwa3,4 kwaye amasuntswana onyango asuswa ngokukhawuleza emlonyeni yi-MCT. Ukusebenza kakuhle kwe-LV transduction kunxulumene ngokuthe ngqo nobude bexesha i-vektha ehlala ecaleni kweeseli ekujoliswe kuzo ukuvumela ukufunxwa kweseli - "ixesha lokuhlala"5 - elincitshiswa lula kukuhamba komoya okuqhelekileyo kwengingqi kunye nokubanjwa kwe-particle mucus kunye ne-MCT. Kwi-CF, ukukwazi ukwandisa ixesha lokuhlala kwe-LV ngaphakathi kwendlela yomoya kubalulekile ukufezekisa amanqanaba aphezulu e-transduction kule ndawo, kodwa kuye kwaba njalo. kube nzima kakhulu.
Ukuze soyise lo mqobo, sicebisa ukuba ii-LV magnetic particles (MPs) zinokunceda ngeendlela ezimbini ezihambelanayo. Okokuqala, zinokukhokelwa ngemagnethi kumphezulu womoya ukuphucula ukujolisa kunye nokunceda ii-gene carrier particles zihlale kwindawo yomoya oyifunayo; kunye ne-ASL) ukuba zifudukele kumaleko weseli 6. Ii-MPs zisetyenziswa kakhulu njengezithuthi zokuhambisa amayeza ekujoliswe kuzo xa zibopha kwi-antibodies, amayeza e-chemotherapeutic, okanye ezinye iimolekyuli ezincinci ezinamathela kwi-membranes yeseli okanye ezibopha kwi-receptors ze-cell surface ezifanelekileyo kwaye ziqokelelane kwiindawo ze-tumor xa kukho umbane ongashukumiyo. IiMagnetic Fields zoNyango loMhlaza 7. Ezinye iindlela "ezishushu kakhulu" zijolise ekufudumaleni ii-MPs xa zichatshazelwe zii-magnetic fields ezijikelezayo, ngaloo ndlela zitshabalalisa iiseli zethumba. Umgaqo wokudluliselwa kwe-magnetic, apho i-magnetic field isetyenziswa njenge-arhente yokudluliselwa kwe-DNA kwiiseli, usetyenziswa rhoqo kwi-vitro kusetyenziswa uluhlu lwee-vectors ze-gene ezingezizo iintsholongwane kunye neentsholongwane kwiiseli ezinzima ukudluliselwa. Ukusebenza kwe-LV magnetotransfection kuye kwasekwa, ngokuhanjiswa kwe-LV-MPs kwi-epithelial cell line yomntu phambi kwe-static magnetic field, okwandisa ukusebenza kwe-transduction ngokuphindwe ka-186 xa kuthelekiswa ne-LV vector yodwa. I-LV-MP ikwasetyenziswe kwimodeli ye-CF ye-in vitro, apho ukudluliselwa kwe-magnetic kwandisa ukudluliselwa kwe-LV kwiinkcubeko zonxibelelwano lomoya-ulwelo ngokuphindwe ka-20 phambi kwe-CF sputum10. Nangona kunjalo, ukudluliselwa kwe-magnetotransfection kwi-vivo yezitho kuye kwafumana ingqalelo encinci kwaye kuye kwavavanywa kuphela kwizilwanyana ezimbalwa. izifundo11,12,13,14,15, ingakumbi emiphungeni16,17. Nangona kunjalo, amathuba okufakelwa kwe-magnetic kwi-CF lung therapy acacile. UTan et al. (2020) bathi "uphononongo olungqinayo lokuhanjiswa kwe-magnetic nanoparticle pulmonary olusebenzayo luya kuvula indlela yezicwangciso zokuphefumla ze-CFTR ezizayo ukuphucula iziphumo zeklinikhi kwizigulane ze-CF"6.
Indlela ezisebenza ngayo ii-small magnetic particles kwi-airway surfaces xa kukho i-magnetic field esetyenzisiweyo kunzima ukuyicinga nokuyifunda, kwaye ngaloo ndlela ayiqondwa kakuhle. Kwezinye izifundo, siphuhlise indlela ye-synchrotron-propagation-based phase-contrast X-ray imaging (PB-PCXI) yokubona ngeliso elingangenisi gazi kwaye silinganise utshintsho lwemizuzu kwi-vivo kubunzulu be-ASL18 kunye nokuziphatha kwe-MCT19,20 ukulinganisa ngokuthe ngqo igesi Ukufuma komphezulu weCanal kwaye kusetyenziswe njengesalathisi sokuqala sokusebenza kakuhle konyango. Ukongeza, indlela yethu yovavanyo lwe-MCT isebenzisa ii-small particles ezingama-10–35 µm eziqulathwe yi-alumina okanye iglasi ye-refractive index ephezulu njengeempawu ze-MCT ezibonakalayo kusetyenziswa i-PB-PCXI21. Zombini ezi ndlela zifanelekile ekuboniseni uluhlu lweentlobo zee-small particles, kuquka ne-MP.
Ngenxa yesisombululo sayo esiphezulu sendawo kunye nexesha, iindlela zethu zohlalutyo lwe-ASL kunye ne-MCT ezisekwe kwi-PB-PCXI zifanelekile kakhulu ekuhloleni amandla kunye neepatheni zokuziphatha kwesuntswana elinye nelininzi kwi-vivo ukusinceda siqonde kwaye siphucule iindlela zokuhambisa i-MP gene. Indlela esiyisebenzisayo apha ivela kwizifundo zethu sisebenzisa i-SPring-8 BL20B2 beamline, apho sibone khona intshukumo yolwelo emva kokuhanjiswa kwedosi ye-sham vector kwi-nasal kunye ne-pulmonary airways yeempuku ukunceda ukuchaza iipatheni zethu zokubonakalisa i-gene ezingafaniyo ezibonwe kwizifundo zethu zezilwanyana ze-gene carrier dose 3,4.
Injongo yolu phononongo yayikukusebenzisa i-synchrotron PB-PCXI ukuze sibone iintshukumo ze-in vivo zoluhlu lwee-MP kwi-trachea yeempuku eziphilayo. Ezi zifundo zomfanekiso ze-PB-PCXI zenzelwe ukuvavanya uluhlu lwee-MP, amandla e-magnetic field, kunye neendawo ukuze kuqinisekiswe impembelelo yazo kwintshukumo ye-MP. Sicinge ukuba i-magnetic field esetyenziswa ngaphandle iya kunceda i-MP ehanjisiweyo ukuba ihlale okanye ihambe iye kwindawo ekujoliswe kuyo. Ezi zifundo zikwasivumele ukuba sichonge ulungelelwaniso lwemagnethi oluya kwandisa inani lamasuntswana agcinwe kwi-trachea emva kokufakwa. Kuluhlu lwesibini lwezifundo, sifuna ukusebenzisa olu lungelelwaniso lufanelekileyo ukubonisa ipateni yokudluliselwa ebangelwa kukuhanjiswa kwe-LV-MPs kwi-airway yempuku, ngokusekelwe kwingcinga yokuba ukuhanjiswa kwe-LV-MPs kumxholo wokujoliswa kwendlela yomoya kuya kubangela ukuphucula ukusebenza kwe-LV transduction.
Zonke izifundo zezilwanyana zenziwe ngokwemigaqo evunyiweyo yiYunivesithi yaseAdelaide (M-2019-060 kunye neM-2020-022) kunye neKomiti ye-SPring-8 Synchrotron Animal Ethics. Iimvavanyo zenziwe ngokwezikhokelo ze-ARRIVE.
Yonke imifanekiso ye-X-ray yenziwe kwi-BL20XU beamline kwi-SPring-8 synchrotron eJapan, kusetyenziswa iseti efana naleyo ichazwe ngaphambili21,22. Ngamafutshane, ibhokisi yovavanyo yayikwi-245 m ukusuka kwiringi yokugcina i-synchrotron. Umgama wesampulu ukuya kwi-detector we-0.6 m usetyenziselwa izifundo ze-particle imaging kunye ne-0.3 m kwizifundo ze-in vivo imaging ukuvelisa iziphumo ze-phase contrast. Kusetyenziswe amandla e-monochromatic beam angama-25 keV. Imifanekiso ithathwe kusetyenziswa i-high-resolution X-ray converter (SPring-8 BM3) edityaniswe ne-sCMOS detector. I-converter iguqula ii-X-rays zibe kukukhanya okubonakalayo kusetyenziswa i-10 µm thick scintillator (Gd3Al2Ga3O12), ethi emva koko iqondiswe kwi-sCMOS sensor kusetyenziswa i-× 10 microscope objective (NA 0.3). I-sCMOS detector yayiyi-Orca-Flash4.0 (Hamamatsu Photonics, eJapan) enobukhulu be-array Iiphikseli ezingama-2048 × 2048 kunye nobukhulu bephikseli eluhlaza obuyi-6.5 × 6.5 µm. Olu seto luvelisa ubungakanani bephikseli ye-isotropic esebenzayo eyi-0.51 µm kunye nebala lokujonga elimalunga ne-1.1 mm × 1.1 mm. Kukhethwe ubude bokuvezwa obuyi-100 ms ukuze kuphuculwe umlinganiselo wesignali-ingxolo yamasuntswana emagnethi ngaphakathi nangaphandle kwendlela yomoya ngelixa kuncitshiswa izinto ezishukumayo ezibangelwa kukuphefumla. Kwiizifundo ze-in vivo, i-shutter ye-X-ray ekhawulezayo ibekwe kwindlela ye-X-ray ukuze kuncitshiswe idosi yemitha ngokuthintela umqadi we-X-ray phakathi kokuvezwa.
Isithwali se-LV asizange sisetyenziswe nakweyiphi na izifundo zomfanekiso we-SPring-8 PB-PCXI kuba igumbi lomfanekiso we-BL20XU alinaso isiqinisekiso se-Biosafety Level 2. Endaweni yoko, sikhethe uluhlu lwee-MPs ezichazwe kakuhle kubathengisi ababini bezorhwebo—ezigubungela uluhlu lobukhulu, izixhobo, ubuninzi bentsimbi, kunye nokusetyenziswa—okokuqala ukuqonda indlela amasimi emagnethi achaphazela ngayo intshukumo ye-MP ngaphakathi kwee-capillaries zeglasi, kwaye emva koko kwiindlela zomoya eziphilayo. phezu komhlaba. IiMP zinobukhulu obuqala kwi-0.25 ukuya kwi-18 μm kwaye zenziwe ngezinto ezahlukeneyo (jonga iTheyibhile 1), kodwa ukwakheka kwesampulu nganye, kuquka nobukhulu bee-particles zemagnethi ngaphakathi kwi-MP, akwaziwa. Ngokusekelwe kwizifundo zethu ezibanzi ze-MCT 19, 20, 21, 23, 24, silindele ukuba iiMP ezincinci njenge-5 μm zibonwe kumphezulu womoya we-tracheal, umzekelo ngokuthabatha iifreyimu ezilandelelanayo ukuze kubonwe ukubonakala okuphuculweyo kwentshukumo ye-MP. I-MP enye enobukhulu be-0.25 μm incinci kunesisombululo sesixhobo sokufota, kodwa i-PB-PCXI kulindeleke ukuba ibone umahluko wevolumu yazo kunye nentshukumo yolwelo lomphezulu ezifakwe kuwo emva kokufakwa.
Iisampulu ze-MP nganye kwiTheyibhile 1 zilungiselelwe kwii-capillaries zeglasi ezingama-20 μl (iDrummond Microcaps, PA, e-USA) ezinobubanzi obungaphakathi obuyi-0.63 mm. Ii-corpuscular particles ziyafumaneka emanzini, ngelixa ii-CombiMag particles zifumaneka kulwelo oluyimpahla yomenzi. Ityhubhu nganye izaliswe ngesiqingatha lulwelo (malunga ne-11 μl) kwaye ibekwe kwisibambi sesampulu (jonga uMfanekiso 1). Ii-capillaries zeglasi zibekwe ngokuthe tye kwinqanaba lesampulu kwibhokisi yokufota, ngokulandelanayo, kwaye zabekwa imiphetho yolwelo. I-magnet ye-nickel shell engaqhelekanga yomhlaba we-neodymium iron boron (NdFeB) eyi-19 mm ububanzi (28 mm ubude) (N35, cat. no. LM1652, Jaycar Electronics, e-Australia) ene-magnetization eseleyo ye-1.17 Tesla iqhotyoshelwe kwinqanaba lokuguqulela elahlukileyo ukuze kufezekiswe Ukutshintsha indawo yayo kude ngexesha lokufota. Ukufunyanwa komfanekiso we-X-ray kuqala xa i-magnet ibekwe malunga ne-30 mm ngaphezulu kwesampulu, kwaye imifanekiso ifunyanwa ngesantya seefreyimu ezi-4 ngomzuzwana. Ngexesha lokufota, i-magnet yasondezwa kufutshane neglasi. ityhubhu ye-capillary (malunga ne-1 mm ukusuka apho) ize iguqulelwe ecaleni kwetyhubhu ukuze kuvavanywe imiphumo yamandla entsimi kunye nendawo ekuyo.
Ukuseta imifanekiso ye-in vitro equlethe iisampulu ze-MP kwii-capillaries zeglasi kwinqanaba lokuguqulwa kwesampulu ye-xy. Indlela yomqadi we-X-ray iphawulwe ngomgca obomvu onemigca.
Emva kokuba ukubonakala kwe-MPs kwi-in vitro kuqinisekisiwe, iqela labo lavavanywa kwi-vivo kwiigundane ze-albino zaseWistar zasendle (~iiveki ezili-12 ubudala, ~200 g). 0.24 mg/kg medetomidine (Domitor®, Zenoaq, Japan), 3.2 mg/kg midazolam (Dormicum®, Astellas Pharma, Japan) kunye ne-4 mg/kg butorphanol (Vetorphale®, Meiji Seika) Iimpuku zathotywa i-anesthesia ngomxube wePharma), Japan) ngenaliti yangaphakathi kwesisu. Emva kwe-anesthesia, zalungiselelwa ukufota ngokususa uboya obujikeleze i-trachea, zifake ityhubhu ye-endotracheal (ET; 16 Ga iv cannula, Terumo BCT) kwaye zizivale zilele phantsi kwipleyiti yokufota eyenzelwe wena equlethe ibhegi yobushushu ukugcina ubushushu bomzimba 22. Ipleyiti yokufota yaxhonywa kwinqanaba lokuguqulela isampuli kwibhokisi yokufota nge-engile encinci ukuze i-trachea ilungelelaniswe ngokuthe tye kumfanekiso we-X-ray, njengoko kubonisiwe kuMfanekiso 2a.
(a) Ukuseta umfanekiso we-in vivo kwibhokisi yomfanekiso we-SPring-8, indlela ye-X-ray beam iphawulwe ngomgca obomvu onemigca.(b,c) Ukubekwa kwemagnethi kwi-trachea kwenziwe kude kusetyenziswa iikhamera ezimbini ze-IP ezifakwe kwi-orthogonally. Kwicala lasekhohlo lomfanekiso wesikrini, iluphu yentambo ebambe intloko ibonakala, kwaye i-cannula yokuhambisa ikwindawo ngaphakathi kwityhubhu ye-ET.
Inkqubo yokumpompa i-syringe elawulwa kude (UMP2, World Precision Instruments, Sarasota, FL) esebenzisa i-syringe yeglasi eyi-100 μl iqhagamshelwe kwityhubhu ye-PE10 (OD 0.61 mm, ID 0.28 mm) ngenaliti ye-30 Ga. Phawula ityhubhu ukuqinisekisa ukuba incam ikwindawo echanekileyo kwi-trachea xa ufaka ityhubhu ye-ET. Sisebenzisa i-micropump, i-syringe plunger yarhoxiswa ngelixa incam yetyhubhu intywiliselwa kwisampulu ye-MP eza kuhanjiswa. Ityhubhu yokuhambisa elayishiweyo yafakwa kwityhubhu ye-endotracheal, ibeka incam kwindawo enamandla kakhulu yentsimi yethu yemagnethi esetyenzisiweyo. Ukufunyanwa komfanekiso kwalawulwa kusetyenziswa isixhobo sokuphefumla esiqhagamshelwe kwibhokisi yethu yexesha esekelwe kwi-Arduino, kwaye zonke iimpawu (umz. ubushushu, ukuphefumla, ukuvulwa/ukuvalwa kwe-shutter kunye nokufunyanwa komfanekiso) zarekhodwa kusetyenziswa iPowerlab kunye neLabChart (AD Instruments, eSydney, e-Australia) 22. Xa kufotwa Xa indawo ebiyelweyo yayingafikeleleki, iikhamera ezimbini ze-IP (Panasonic BB-SC382) zabekwa malunga ne-90° ukuya enye kwenye kwaye zasetyenziswa ukujonga indawo yemagnethi xa kuthelekiswa ne-trachea ngexesha lokufota (Umzobo 2b, c). Ukunciphisa izinto ezishukumayo, umfanekiso omnye ufunyenwe ngokuphefumla ngexesha lokuhamba kolwandle ekupheleni kolwandle.
Imagnethi iqhotyoshelwe kwinqanaba lesibini elinokufunyanwa kude ngaphandle kwendlu yokufota. Kuvavanywe iindawo ezahlukeneyo zemagnethi kunye nolwakhiwo, kuquka: Ifakwe kwi-engile emalunga nama-30° ngaphezulu kwe-trachea (ulwakhiwo oluboniswe kwiMifanekiso 2a kunye no-3a); imagnethi enye ngaphezulu kwesilwanyana kunye nenye engezantsi, kunye neepali ezibekwe ukutsala (Umfanekiso 3b); imagnethi enye ngaphezulu kwesilwanyana kunye nenye engezantsi, kunye neepali ezibekwe ukurhoxa (Umfanekiso 3c); kunye nemagnethi enye ngaphezulu kwaye imile ngokuthe nkqo kwi-trachea (Umfanekiso 3d). Nje ukuba isilwanyana kunye nemagnethi zilungiselelwe kwaye i-MP eza kuvavanywa ilayishwe kwipompo yesirinji, hambisa idosi ye-50 μl ngesantya se-4 μl/sec ngelixa ufumana imifanekiso. Imagnethi emva koko ihanjiswa emva nangaphambili ecaleni okanye ecaleni ngaphesheya kwe-trachea ngelixa iqhubeka nokufumana imifanekiso.
Uqwalaselo lwemagnethi kwimifanekiso ye-in vivo (a) imagnethi enye ngaphezulu kwe-trachea kwi-engile emalunga nama-30°, (b) iimagnethi ezimbini ezimiselwe ukutsala, (c) iimagnethi ezimbini ezimiselwe ukugxotha, (d) imagnethi enye ngaphezulu kwaye imile nkqo kwi-trachea. Umjongi ujonge ezantsi ukusuka emlonyeni ukuya emaphashini nge-trachea, kwaye umqadi we-X-ray udlule kwicala lasekhohlo lempuku uze uphume kwicala lasekunene. Imagnethi ishukunyiswa kubude bomoya okanye ekhohlo nasekunene ngaphezulu kwe-trachea kwicala le-X-ray beam.
Sikwafuna ukufumanisa ukubonakala kunye nokuziphatha kwamasuntswana kwiindlela zomoya ngaphandle kokuphefumla okuphazamisayo kunye nokuhamba kwentliziyo. Ke ngoko, ekupheleni kwexesha lokufota, izilwanyana zabulawa ngobuntu ngenxa yokugqithiswa kwe-pentobarbital (Somnopentil, Pitman-Moore, Washington Crossing, USA; ~65 mg/kg ip). Ezinye izilwanyana zashiywa kwiqonga lokufota, kwaye xa ukuphefumla kunye nokubetha kwentliziyo kuyekile, inkqubo yokufota yaphindwa, yongeza idosi eyongezelelweyo ye-MP ukuba akukho MP ibonakala kumphezulu womoya.
Imifanekiso efunyenweyo yalungiswa i-flat-field kunye ne-dark-field yaza yahlanganiswa yaba yi-movie (iifreyimu ezingama-20 ngomzuzwana; isantya esiqhelekileyo se-15-25 × kuxhomekeke kwisantya sokuphefumla) kusetyenziswa iskripthi esenziwe ngokwezifiso esibhalwe kwi-MATLAB (R2020a, The Mathworks).
Zonke izifundo zokuhanjiswa kwe-LV gene vector zenziwe kwiLaboratory Animal Research Facility kwiYunivesithi yaseAdelaide kwaye zazijolise ekusebenziseni iziphumo zovavanyo lwe-SPring-8 ukuvavanya ukuba ukuhanjiswa kwe-LV-MP phambi kwentsimi yemagnethi kunokuphucula ukudluliselwa kwe-gene emzimbeni. Ukuvavanya iziphumo ze-MP kunye nentsimi yemagnethi, amaqela amabini ezilwanyana anyangwa: elinye iqela lanikwa i-LV-MP kunye nemagnethi ebekwe, kwaye elinye iqela lafumana iqela lolawulo eline-LV-MP ngaphandle kwemagnethi.
Iivektha ze-LV gene zenziwe kusetyenziswa iindlela ezichazwe ngaphambili 25, 26. Ivektha yeLacZ iveza i-nuclear-localized beta-galactosidase gene eqhutywa yi-constitutive MPSV promoter (LV-LacZ), evelisa imveliso ye-blue reaction kwiiseli ezitshintshiweyo, ezibonakala kwimiphetho yezicubu zemiphunga kunye namacandelo ezicubu. I-Titration yenziwe kwiinkcubeko zeeseli ngokubala ngesandla inani leeseli zeLacZ positive nge-hemocytometer ukubala i-titer kwi-TU/ml. Abathwali bayagcinwa kwi--80 °C, banyibilike ngaphambi kokusetyenziswa, kwaye babotshelelwe kwiCombiMag ngokuxuba kwi-1:1 ratio kwaye bafakwe kwi-ice ubuncinane imizuzu engama-30 ngaphambi kokuhanjiswa.
Iigundane eziqhelekileyo zeSprague Dawley (n = 3/iqela, ~2-3 zafakwa i-anesthesia ngaphakathi kwesisu ngomxube we-0.4 mg/kg medetomidine (Domitor, Ilium, Australia) kunye ne-60 mg/kg ketamine (Ilium, Australia) enenyanga ubudala) ip) kunye ne-cannulation yomlomo engeyotyando ene-16 Ga iv cannula. Ukuqinisekisa ukuba izicubu zomoya we-tracheal zifumana i-LV transduction, yalungiswa kusetyenziswa i-mechanical perturbation protocol yethu echazwe ngaphambili, apho umphezulu womoya we-tracheal wahlikihlwa nge-axial ngebhasikithi yentambo (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28. Ulawulo lwe-tracheal lwe-LV-MP emva koko lwenziwa kwikhabhathi yokhuseleko lwebhayoloji malunga nemizuzu eli-10 emva kokuphazamiseka.
Intsimi yemagnethi esetyenzisiweyo kolu vavanyo yenziwe ngendlela efanayo nesifundo se-X-ray se-in vivo, kunye neemagnethi ezifanayo ezibanjwe ngaphezulu kwe-trachea kusetyenziswa ii-distillation stent clips (Umfanekiso 4). Umthamo we-50 μl (2 × 25 μl aliquots) we-LV-MP ungeniswe kwi-trachea (n = 3 izilwanyana) kusetyenziswa i-pipette equlethe incam yejeli njengoko kuchaziwe ngaphambili. Iqela lolawulo (n = 3 izilwanyana) lifumene ii-LV-MP ezifanayo ngaphandle kokusebenzisa imagnethi. Emva kokuba ukufakelwa kugqityiwe, i-cannula isuswa kwityhubhu ye-ET kwaye isilwanyana siyakhutshwa. Imagnethi ihlala endaweni yayo imizuzu eli-10, emva koko iyasuswa. Iigundane zifumene idosi ye-meloxicam engaphantsi komhlaba (1 ml/kg) (Ilium, Australia) elandelwa kukuguqulwa kwe-anesthesia ngokujova i-1 mg/kg ye-atipamazole hydrochloride (Antisedan, Zoetis, Australia). Iigundane zigcinwe zifudumele kwaye zijongiwe de ziphinde ziphile ngokupheleleyo kwi-anesthesia.
Isixhobo sokuhambisa i-LV-MP kwikhabhathi yokhuseleko lwebhayoloji. I-Luer hub engwevu ekhanyayo yetyhubhu ye-ET inokubonwa iphuma emlonyeni kwaye incam yejeli yepayipi eboniswe kumfanekiso ifakwe ngetyhubhu ye-ET ukuya kubunzulu obufunekayo kwi-trachea.
Emva kweveki emva kwenkqubo yokulinganisa i-LV-MP, izilwanyana zabulawa ngobuntu ngokuphefumla i-CO2 eyi-100% kwaye ukubonakaliswa kweLacZ kwavavanywa kusetyenziswa unyango lwethu oluqhelekileyo lwe-X-gal. Iindandatho ezintathu ze-caudal ezininzi ze-cartilaginous zasuswa ukuqinisekisa ukuba nawuphi na umonakalo woomatshini okanye ukugcinwa kolwelo oluvela kwindawo ye-endotracheal tube alufakwanga kuhlalutyo. I-trachea nganye yanqunyulwa ubude ukuze kwenziwe ii-halves ezimbini zohlalutyo, kwaye zafakwa kwisitya esine-silicone rubber (Sylgard, Dow Inc) kusetyenziswa inaliti yeMinutien (Fine Science Tools) ukuze kubonwe umphezulu we-luminal. Ukusasazwa kunye nepatheni yeeseli eziguquliweyo kwaqinisekiswa ngokufota kwangaphambili kusetyenziswa i-Nikon microscope (SMZ1500) ngekhamera yeDigiLite kunye nesoftware ye-TCapture (Tucsen Photonics, China). Imifanekiso yafunyanwa ngokukhulisa i-20x (kubandakanya indawo ephezulu yobubanzi obupheleleyo be-trachea), kunye nobude bonke be-trachea obuthathwe inyathelo ngenyathelo, ukuqinisekisa ukufana okwaneleyo phakathi komfanekiso ngamnye ukuvumela "ukuthunga" komfanekiso. Imifanekiso evela kwi-trachea nganye Emva koko i-trachea yahlanganiswa yaba ngumfanekiso omnye odityanisiweyo kusetyenziswa i-Image Composite Editor v2.0.3 (Microsoft Research) kusetyenziswa i-algorithm yokuhamba kwe-planar. Iindawo zokubonakaliswa kwe-LacZ kwimifanekiso edityanisiweyo ye-trachea evela kwisilwanyana ngasinye zalinganiswa kusetyenziswa iskripthi se-MATLAB ezenzekelayo (R2020a, MathWorks) njengoko kuchaziwe ngaphambili, kusetyenziswa useto lwe-0.35 < Hue < 0.58, Saturation > 0.15, kunye neXabiso < 0.7. Ngokulandela imilo yezicubu, imaski yenziwa ngesandla kwi-GIMP v2.10.24 kumfanekiso ngamnye odityanisiweyo ukuze kuchongwe indawo yezicubu kwaye kuthintelwe naluphi na ukufunyanwa okungachanekanga ngaphandle kwezicubu ze-tracheal. Iindawo ezidityanisiweyo kuzo zonke imifanekiso edityanisiweyo kwisilwanyana ngasinye zahlanganiswa ukuze kuvele indawo edityanisiweyo yeso silwanyana. Indawo edityanisiweyo yahlulwahlulwa yindawo edityanisiweyo yemaski ukuze kuvele indawo eqhelekileyo.
I-trachea nganye yayifakwe kwiparafini kwaye amacandelo angama-5 μm anqunyulwa. Amacandelo abekwe ngombala obomvu okhawulezayo ongathathi hlangothi kangangemizuzu emi-5 kwaye imifanekiso yafunyanwa kusetyenziswa imakroskopu yeNikon Eclipse E400, ikhamera yeDS-Fi3 kunye nesoftware yokubamba izinto ze-NIS (uhlobo 5.20.00).
Lonke uhlalutyo lwezibalo lwenziwe kwiGraphPad Prism v9 (GraphPad Software, Inc.). Ukubaluleka kwezibalo kumiselwe kwi-p ≤ 0.05. Ubungakanani buqinisekisiwe kusetyenziswa uvavanyo lweShapiro-Wilk, kwaye umahluko kwi-LacZ staining uhlolwe kusetyenziswa uvavanyo lwe-t olungabhangqwanga.
IiMP ezintandathu ezichazwe kwiTheyibhile 1 zihlolwe kusetyenziswa i-PCXI, kwaye ukubonakala kuchazwe kwiTheyibhile 2. IiMP ezimbini ze-polystyrene (MP1 kunye ne-MP2; 18 μm kunye ne-0.25 μm, ngokwahlukeneyo) azizange zibonakale phantsi kwe-PCXI, kodwa ezinye iisampulu zazibonakala (imizekelo iboniswe kuMfanekiso 5). I-MP3 kunye ne-MP4 (10-15% Fe3O4; 0.25 μm kunye ne-0.9 μm, ngokwahlukeneyo) zibonakala kancinci. Nangona ziqulathe ezinye zeempuku ezincinci ezivavanyiweyo, i-MP5 (98% Fe3O4; 0.25 μm) yayiyeyona ibonakalayo kakhulu. Imveliso ye-CombiMag MP6 kunzima ukuyibona. Kuzo zonke iimeko, amandla ethu okufumana i-MP aphuculwe kakhulu ngokuguqulela imagnethi ngasemva nangaphambili ngokuhambelana ne-capillary. Xa iimagnethi zazisuka kwi-capillary, iimpuku zanda ngeentambo ezinde, kodwa njengoko iimagnethi zazisondela kwaye amandla e-magnetic field enyuka, iintambo zeempuku zazimfutshane njengoko iimpuku zazifudukela kumphezulu we-capillary. (jonga iVidiyo eyoNgezelelweyo S1: MP4), inyusa uxinano lwamasuntswana omphezulu. Ngokwahlukileyo koko, xa imagnethi isuswa kwi-capillary, amandla entsimi ayancipha kwaye ii-MPs zihlengahlengisa zibe yimicu emide ephuma kumphezulu ophezulu we-capillary (jonga iVidiyo eyoNgezelelweyo S2: MP4). Emva kokuba imagnethi iyekile ukuhamba, amasuntswana ayaqhubeka nokuhamba ixesha elifutshane emva kokufikelela kwindawo yokulingana. Njengoko i-MP isiya phambili kwaye isuka kumphezulu ophezulu we-capillary, amasuntswana emagnethi adla ngokurhuqa inkunkuma kulwelo.
Ukubonakala kwe-MP phantsi kwe-PCXI kwahluka kakhulu phakathi kweesampuli.(a) MP3, (b) MP4, (c) MP5 kunye (d) MP6.Zonke imifanekiso eziboniswe apha zithathwe ngemagnethi ebekwe malunga ne-10 mm ngqo ngaphezulu kwe-capillary.Izangqa ezinkulu ezibonakalayo ngamaqamza omoya abanjwe kwi-capillaries, ebonisa ngokucacileyo iimpawu zomphetho omnyama nomhlophe wemifanekiso yomahluko wesigaba.Ibhokisi ebomvu iqulethe ukukhulisa okwandisa umahluko.Qaphela ukuba ububanzi be-magnet schematics kuzo zonke iifigure abulingani kwaye bukhulu ngokuphindwe kalikhulu kunokuba kubonisiwe.
Njengoko imagnethi iguqulelwa ngasekhohlo nasekunene phezulu kwe-capillary, i-engile yentambo ye-MP iyatshintsha ukuze ihambelane nemagnethi (jonga uMfanekiso 6), ngaloo ndlela ichaza imigca yentsimi yemagnethi. Kwi-MP3-5, emva kokuba i-chord ifikelele kwi-engile yomda, amasuntswana atsalwa kumphezulu we-capillary. Oku kudla ngokubangela ukuba ama-MP aqokelelene abe ngamaqela amakhulu kufutshane nendawo apho intsimi yemagnethi inamandla khona (jonga iVidiyo eyoNgezelelweyo S3:MP5). Oku kubonakala ngakumbi xa kufotwa kufutshane nesiphelo se-capillary, okubangela ukuba ama-MP ahlangane kwaye agxile kwi-interface yolwelo-moya. Amasuntswana kwi-MP6, awayenzima ukuwabona kune-MP3-5, awazange atsalwe njengoko imagnethi yayihamba ecaleni kwe-capillary, kodwa amasuntswana e-MP ahlukana, eshiya amasuntswana kwindawo yokujonga (jonga iVidiyo eyoNgezelelweyo S4:MP6). Kwezinye iimeko, xa intsimi yemagnethi esetyenzisiweyo yancitshiswa ngokususa imagnethi umgama omkhulu ukusuka kwindawo yomfanekiso, naziphi na ii-MP eziseleyo zehla kancinci ziye kumphezulu osezantsi wetyhubhu ngamandla omxhuzulane ngelixa zihlala kumtya (jonga iVidiyo eyoNgezelelweyo S5: MP3).
I-engile yentambo ye-MP iyatshintsha njengoko imagnethi iguqulelwa ngasekunene ngaphezulu kwe-capillary.(a) MP3, (b) MP4, (c) MP5 kunye (d) MP6. Ibhokisi ebomvu iqulethe ukukhulisa okwandisa umahluko.Qaphela ukuba iividiyo ezongezelelweyo zinolwazi njengoko zityhila isakhiwo esibalulekileyo samasuntswana kunye nolwazi olutshintshayo olungenakubonwa kule mifanekiso imileyo.
Uvavanyo lwethu lubonise ukuba ukuhambisa imagnethi kancinci ukuya phambili nangasemva kwi-trachea kwenza kube lula ukubona i-MP kwimeko yentshukumo enzima kwi-vivo. Uvavanyo lwe-in vivo aluzange lwenziwe njengoko ii-polystyrene beads (MP1 kunye ne-MP2) zazingabonakali kwi-capillary. I-MP nganye kwezine ezisele zavavanywa kwi-vivo nge-magnethi ende axis ebekwe ngaphezulu kwe-trachea kwi-engile emalunga ne-30° ukuya phezulu (jonga iMifanekiso 2b kunye ne-3a), njengoko oku kubangele iintambo ezinde ze-MP kwaye kwasebenza ngakumbi kunokumiswa kwemagnethi. I-MP3, i-MP4 kunye ne-MP6 azizange zifunyanwe kwi-trachea yezilwanyana eziphilayo. Xa iindlela zomoya zeempuku zazithathwa umfanekiso emva kokuba izilwanyana zibulewe ngobuntu, amasuntswana ahlala engabonakali nangona kongezwa ivolumu eyongezelelweyo kusetyenziswa ipompo yesirinji. I-MP5 yayinomxholo ophezulu we-iron oxide kwaye yayiyeyona suntswana ibonakalayo kuphela, kwaye ke ngoko yasetyenziselwa ukuvavanya nokuchonga ukuziphatha kwe-MP kwi-vivo.
Ukubeka imagnethi phezu kwe-trachea ngexesha lokuhanjiswa kwe-MP kubangele ukuba ii-MP ezininzi, kodwa kungekhona zonke, zigxile kwindawo yokujonga. Ii-particles ezingena kwi-trachea zibonwa kakuhle kwizilwanyana ezibingelelwe ngabantu. Umfanekiso 7 kunye neVidiyo eyongezelelweyo S6: I-MP5 ibonisa ukubanjwa ngokukhawuleza kwe-magnetic kunye nokulungelelaniswa kwama-particles kumphezulu we-ventral trachea, okubonisa ukuba ii-MPs zinokuthunyelwa kwiindawo ezifunwayo ze-trachea. Xa bekhangela kude kakhulu kwi-trachea emva kokuhanjiswa kwe-MP, amanye ama-MPs afunyenwe kufutshane ne-carina, nto leyo ebonisa ukuba amandla e-magnetic field ayenganelanga ukuqokelela nokugcina onke ama-MP, njengoko ayethunyelwa kummandla wamandla aphezulu e-magnetic field ngexesha lenkqubo yolwelo. Nangona kunjalo, amazinga e-MP emva kokuzalwa ayephezulu kwindawo enemifanekiso, nto leyo ebonisa ukuba ama-MP amaninzi ahlala kwimimandla yomoya apho amandla e-magnetic field asetyenziswayo ayephezulu.
Imifanekiso evela ku-(a) ngaphambi nasemva kokuhanjiswa kwe-MP5 kwi-trachea yegundane elisandula ukubulawa kunye nemagnethi ebekwe ngqo ngaphezulu kwendawo yokufota. Indawo efotayo iphakathi kweeringi ezimbini ze-cartilage. Ngaphambi kokuhanjiswa kwe-MP, kukho ulwelo oluthile kwindlela yomoya. Ibhokisi ebomvu iqulethe ukukhulisa okwandisa umahluko. Le mifanekiso ivela kwividiyo eboniswe kwiVidiyo eyoNgezelelweyo S6:MP5.
Ukuguqulela imagnethi ecaleni kwe-trachea in vivo kubangele ukuba itshaneli ye-MP itshintshe i-engile ngaphakathi komphezulu womoya ngendlela efana naleyo ibonwa kwi-capillaries (jonga uMfanekiso 8 kunye neVidiyo eyoNgezelelweyo S7: MP5). Nangona kunjalo, kuphononongo lwethu, ii-MP azikwazanga ukutsalwa kumphezulu womoya ophilayo njengoko zinokutsalwa kwi-capillaries. Kwezinye iimeko, itshaneli ye-MP iya kuba nde njengoko imagnethi ihamba ngasekhohlo nasekunene. Okubangel 'umdla kukuba, sifumanise nokuba intambo ye-particle ibonakala itshintsha ubunzulu bomaleko wolwelo lomphezulu xa imagnethi ishukunyiswa ngobude ecaleni kwe-trachea, kwaye iyanda xa imagnethi ishukunyiswa ngqo phezulu kwaye intambo ye-particle ijikeleziswa kwindawo ethe nkqo (jonga iVidiyo eyoNgezelelweyo S7). : MP5 ngo-0:09, ezantsi ngasekunene). Ipatheni yentshukumo ephawulekayo yatshintsha xa imagnethi iguqulelwa phezulu kwe-trachea ecaleni (oko kukuthi, ngasekhohlo okanye ngasekunene kwesilwanyana kunokuba ihambe ngobude be-trachea). Amasuntswana ayesabonakala ngokucacileyo njengoko ehamba, kodwa xa imagnethi isuswa kwi-trachea, iincam zeentambo zamasuntswana zabonakala (jonga iVidiyo eyoNgezelelweyo S8:MP5, eqala ngo-0:08). Oku kuhambelana nokuziphatha kwe-MP esikubonileyo phantsi kwentsimi yemagnethi esetyenzisiweyo kwi-capillary yeglasi.
Imifanekiso engumzekelo ebonisa i-MP5 kwi-trachea yegundane eliphilayo elithomalaliswe yi-anesthesia.(a) I-magnet isetyenziselwa ukufumana imifanekiso engasentla nangasekhohlo kwe-trachea, emva koko (b) emva kokuba i-magnet ishukunyiselwe ngasekunene. Ibhokisi ebomvu iqulethe ukukhulisa okwandisa umahluko. Le mifanekiso ivela kwividiyo eboniswe kwiVidiyo eyoNgezelelweyo S7:MP5.
Xa iipali ezimbini zazenziwe ngendlela ehambelana nomntla-ntshona ngaphezulu nangaphantsi kwe-trachea (oko kukuthi, ukutsala; Umfanekiso 3b), ii-MP chords zabonakala zinde kwaye zazikwicala le-trachea kunokuba zibe kwi-dorsal tracheal surface (jonga iVidiyo eyoNgezelelweyo S9:MP5). Nangona kunjalo, ubuninzi bee-particles kwindawo enye (oko kukuthi, umphezulu we-dorsal we-trachea) azizange zifunyanwe emva kokuhanjiswa kolwelo xa kusetyenziswa isixhobo se-dual-magnet, esiqhele ukwenzeka xa kusetyenziswa isixhobo se-single-magnet. Emva koko xa i-magnet enye yazenziwe ngendlela yokuba ibuyisele ii-poles eziguqulweyo (Umzobo 3c), inani lee-particles ezibonakalayo kwintsimi yokujonga alizange libonakale linyuka emva kokuhanjiswa. Ukuseta zombini ii-dual-magnet configurations kunzima ngenxa yamandla aphezulu e-magnetic field atsala okanye atyhala ii-magnets, ngokulandelelana. Ukuseta emva koko kwatshintshwa kwaba yi-single magnet ehambelana nomoya kodwa idlula kwindlela yomoya kwi-90 degrees ukuze imigca yentsimi iwele udonga lwe-tracheal ngokwe-orthogonally (Umzobo 3d), indlela eyenzelwe ukumisela ukuba i-particle aggregation ecaleni. udonga lwalunokubonwa. Nangona kunjalo, kolu qwalaselo, bekungekho ntshukumo ibonakalayo yokuqokelelwa kwe-MP okanye intshukumo yemagnet. Ngokusekelwe kuzo zonke ezi ziphumo, uqwalaselo lwe-single-magnet, oluyi-30-degree orientation configuration (Umfanekiso 3a) lukhethwe kwizifundo zokuthwala i-gene kwi-vivo.
Xa isilwanyana sasithathwa umfanekiso ngokuphindaphindiweyo emva kokubulawa komntu, ukungabikho kwentshukumo yezicubu ezididayo kwakuthetha ukuba imigca emifutshane nemfutshane yeenxalenye zezicubu yayinokubonwa kwintsimi ecacileyo ye-interchondral, "eshukumayo" ngokuhambelana nentshukumo yoguqulelo lwemagnethi. Nangona kunjalo, asikakwazi ukubona ngokucacileyo ubukho kunye nentshukumo yeenxalenye zeMP6.
I-LV-LacZ titer yayiyi-1.8 × 108 TU/ml, kwaye emva kokuxubana kwe-1:1 neCombiMag MP (MP6), izilwanyana zafumana idosi ye-tracheal ye-50 μl yesithuthi se-LV esingu-9 × 107 TU/ml (oko kukuthi, i-4.5 × 106 TU/rat). ). Kwezi zifundo, endaweni yokuguqulela imagnethi ngexesha lokubeleka, silungise imagnethi kwindawo enye ukuze siqinisekise ukuba i-LV transduction (a) ingaphucuka xa kuthelekiswa nokuhanjiswa kwevector xa kungekho magnetic field, kwaye (b) ingagxila. Iiseli ze-Airway zidluliselwa kwiindawo ezijoliswe kwimagnethi zendlela yomoya ephezulu.
Ubukho beemagnethi kunye nokusetyenziswa kweCombiMag kunye neevector ze-LV akubonakali ngathi kunemiphumo emibi kwimpilo yezilwanyana, njengoko kunjalo nangendlela yethu eqhelekileyo yokuhambisa iivector ze-LV. Imifanekiso yangaphambili yendawo ye-tracheal ejongene nokuphazamiseka koomatshini (Umzobo oNcedisayo 1) ibonise ukuba kukho amanqanaba aphezulu kakhulu okudluliselwa kwe-transduction kwiqela lezilwanyana eziphathwe nge-LV-MP xa imagnethi yayikhona (Umzobo 9a). Kuphela inani elincinci le-blue LacZ staining elalikho kwiqela lolawulo (Umzobo 9b). Ukulinganiswa kweendawo ezinemibala ye-X-Gal eqhelekileyo kubonise ukuba ukulawulwa kwe-LV-MP phambi kwentsimi yemagnethi kuvelise uphuculo oluphindwe kathandathu (Umzobo 9c).
Umzekelo wemifanekiso edibeneyo ebonisa ukudluliselwa kwe-tracheal yi-LV-MP (a) xa kukho intsimi yemagnethi kunye (b) xa kungekho magnethi. (c) Uphuculo olubalulekileyo ngokwezibalo kwindawo yokudluliselwa kwe-LacZ eqhelekileyo ngaphakathi kwe-trachea xa kusetyenziswa imagnethi (*p = 0.029, uvavanyo lwe-t, n = 3 ngeqela, i-mean ± SEM).
Amacandelo abomvu angathathi cala (umzekelo oboniswe kwiFig. 2 eyongezelelweyo) abonise iiseli ezibomvu ezikwiLacZ zikho kwipateni efanayo nakwindawo njengoko bekuxeliwe ngaphambili.
Umceli mngeni ophambili kunyango lwezakhi zofuzo zomoya usekukuba zibekwe ngokuchanekileyo kwiindawo ezinomdla kuzo iinxalenye zezakhi zomoya kunye nokufikelela kumanqanaba aphezulu okusebenza kakuhle kokudluliselwa kwezakhi zomoya kwimiphunga ehambayo xa kukho ukuhamba komoya kunye nokususwa kwe-mucus esebenzayo. Kwizithwali ze-LV ezenzelwe ukunyanga isifo somoya se-CF, ukunyusa ixesha lokuhlala kweenxalenye zezakhi zomoya kwimizila yomoya eqhubayo kube yinjongo engaqondakaliyo okwangoku. Njengoko kubonisiwe nguCastellani et al., ukusetyenziswa kweendawo zemagnethi ukuphucula ukudluliselwa kwezakhi zomoya kuneengenelo xa kuthelekiswa nezinye iindlela zokuhambisa izakhi zofuzo ezifana ne-electroporation, njengoko inokudibanisa ukulula, ukusebenza kakuhle kweendleko, indawo yokuhambisa, ukusebenza ngcono, kunye namaxesha amafutshane okufakwa, kwaye mhlawumbi idosi encinci yokuthwala. Nangona kunjalo, ukufakwa kwezakhi zomoya ngaphakathi kunye nokuziphatha kweenxalenye zemagnethi kwimizila yomoya phantsi kwempembelelo yamandla emagnethi angaphandle akukaze kuchazwe, kwaye akunakwenzeka ukuba le ndlela ibonakaliswe ngaphakathi emzimbeni ukuphucula amanqanaba okubonakaliswa kwezakhi zofuzo kwimizila yomoya ephilayo ephilileyo.
Uvavanyo lwethu lwe-in vitro synchrotron PCXI lubonise ukuba zonke ii-particles esizivavanyeyo, ngaphandle kwe-polystyrene MP, zazibonakala kwisetingi yemifanekiso esiyisebenzisileyo. Xa kukho i-magnetic field, ii-MPs zenza ii-strings ezinobude obuhambelana nohlobo lwee-particles kunye namandla e-magnetic field (oko kukuthi, ukusondela kunye nentshukumo ye-magnet). Njengoko kubonisiwe kuMfanekiso 10, ii-strings esizibonayo zenziwa ngenxa yokuba i-particle nganye i-magnetized kwaye ibangela i-magnetic field yayo yendawo. Ezi fields zahlukeneyo zibangela ukuba ezinye ii-particles ezifanayo zihlangane kwaye ziqhagamshelane, kunye neentshukumo ezifana nee-string zeqela ngenxa yamandla asekuhlaleni avela kumandla asekuhlaleni anomtsalane nawagxothayo amanye ama-particles.
Ukubonisa ngokwesikimu (a,b) ii-particle trains eziveliswa ngaphakathi kwi-capillaries ezizaliswe lulwelo kunye ne-(c,d) i-trachea ezaliswe ngumoya. Qaphela ukuba i-capillaries kunye ne-trachea azidweliswanga kwisikali. Iphaneli (a) ikwaqulathe inkcazo ye-MP, equlathe ii-particles ze-Fe3O4 ezicwangciswe ngemitya.
Xa imagnethi yayishukunyiswa ngaphezulu kwe-capillary, i-engile yentambo yesuntswana yafikelela kumda obalulekileyo we-MP3-5 equlethe i-Fe3O4, emva koko intambo yesuntswana ayisahlali kwindawo yokuqala, kodwa yahamba phezu komphezulu yaya kwindawo entsha. imagnethi. Esi siphumo sinokuvela kuba umphezulu we-capillary yeglasi uthambile ngokwaneleyo ukuvumela le ntshukumo ukuba yenzeke. Okubangel 'umdla kukuba, i-MP6 (i-CombiMag) ayizange iziphathe ngale ndlela, mhlawumbi ngenxa yokuba amasuntswana ayemancinci, ayeneengubo ezahlukeneyo okanye iintlawulo zomphezulu, okanye ulwelo oluthwalayo oluyimpahla luchaphazele amandla azo okuhamba. Umahluko womfanekiso wamasuntswana e-CombiMag nawo ubuthathaka, nto leyo ebonisa ukuba ulwelo kunye namasuntswana anokuba nobunzima obufanayo kwaye ngenxa yoko angahambi lula omnye komnye. Amasuntswana anokunamathela ukuba imagnethi ihamba ngokukhawuleza kakhulu, nto leyo ebonisa ukuba amandla e-magnetic field akanakoyisa ukungqubana phakathi kwamasuntswana kulwelo, nto leyo ebonisa ukuba mhlawumbi akumangalisi ukuba amandla e-magnetic field kunye nomgama phakathi kwemagnethi kunye nendawo ekujoliswe kuyo Kubaluleke kakhulu. Xa zidibene, ezi ziphumo zikwabonisa ukuba, nangona iimagnethi zinokubamba ii-MP ezininzi ezihamba kwindawo ekujoliswe kuyo, akunakwenzeka ukuba iimagnethi zinokuthenjwa. ukuze kushukunyiswe amasuntswana eCombiMag kumphezulu we-trachea. Ke ngoko, sigqiba kwelokuba izifundo ze-LV-MP ze-in vivo kufuneka zisebenzise amasimi emagnethi angashukumiyo ukujolisa ngokwasemzimbeni kwiindawo ezithile zomthi womoya.
Xa amasuntswana efika emzimbeni, kunzima ukuwabona xa kujongwa izicubu zomzimba ezishukumayo, kodwa amandla okuwabona aphuculwe ngokuguqulela imagnethi ngokuthe tye ngaphezulu kwe-trachea ukuze "ishukumise" imitya ye-MP. Nangona ukufota bukhoma kunokwenzeka, kulula ukubona intshukumo yamasuntswana xa isilwanyana sibulewe ngobuntu. Uxinzelelo lwe-MP ngokubanzi lwaluphezulu kule ndawo xa imagnethi yayibekwe ngaphezulu kwendawo yokufota, nangona amanye amasuntswana ayehlala efumaneka kude kwi-trachea. Ngokungafaniyo nezifundo ze-in vitro, amasuntswana awanakutsalwa kwi-trachea ngokuguqulela imagnethi. Olu fumaniso luhambelana nendlela i-mucus egquma umphezulu we-trachea edla ngokuyicoca ngayo amasuntswana aphefumliweyo, iwabambe kwi-mucus kwaye emva koko isuswe yindlela yokucoca i-mucociliary.
Sicinge ukuba ukusetyenziswa kweemagnethi ekutsaleni ngaphezulu nangaphantsi kwe-trachea (Umzobo 3b) kunokubangela intsimi yemagnethi efanayo, endaweni yentsimi yemagnethi egxile kakhulu kwindawo ethile, nto leyo enokubangela ukusasazwa okufanayo kwee-particles. Nangona kunjalo, uphando lwethu lokuqala alufumananga bungqina bucacileyo bokuxhasa le ngcamango. Ngokufanayo, ukumisela isibini seemagnethi zokugxotha (Umzobo 3c) akuzange kubangele ukubekwa kwee-particles ezininzi kwindawo enemifanekiso. Ezi ziphumo zimbini zibonisa ukuba ukuseta i-dual-magnet akuphuculi kakhulu ulawulo lwendawo lwe-MP targeting, kwaye amandla emagnethi anamandla avelayo kunzima ukuwamisela, okwenza le ndlela ingasebenzi. Ngokufanayo, ukujolisa imagnethi ngaphezulu nangaphakathi kwe-trachea (Umzobo 3d) nako akwandisanga inani lee-particles ezigcinwe kwindawo enemifanekiso. Ezinye zezi zicwangciso zisenokungaphumeleli kuba zibangela amandla e-magnetic field aphantsi kwindawo enemifanekiso. Ke ngoko, ulungiselelo lwemagnethi ye-angle engama-30 degrees (Umfanekiso 3a) luthathwa njengeyona ndlela ilula nesebenzayo yovavanyo lwe-in vivo.
Uphononongo lwe-LV-MP lubonise ukuba xa ii-LV vectors zidibene neCombiMag kwaye zithunyelwa emva kokuphazamiseka ngokomzimba phambi kwentsimi yemagnethi, amanqanaba okudluliselwa kwe-trachea anyuswa kakhulu kwi-trachea xa kuthelekiswa nolawulo. Ngokusekelwe kwizifundo ze-synchrotron imaging kunye neziphumo zeLacZ, intsimi yemagnethi yayibonakala ikwazile ukugcina i-LV ngaphakathi kwi-trachea kwaye inciphise inani lamasuntswana e-vector angene nzulu ngoko nangoko emphungeni. Ukuphuculwa okunjalo kokujolisa kunokukhokelela ekusebenzeni okuphezulu ngelixa kunciphisa ii-titers ezihanjisiweyo, ukudluliselwa okungafunekiyo, iziphumo ebezingalindelekanga zokudumba kunye nokuzikhusela, kunye neendleko zokuthwala i-gene. Okubalulekileyo, ngokutsho komenzi, iCombiMag ingasetyenziswa kunye nezinye iindlela zokudlulisela i-gene, kubandakanya nezinye ii-vectors ze-viral (ezifana ne-AAV) kunye ne-nucleic acids.