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Majene vector ekurapa chirwere chemapapu chinonzi cystic fibrosis anofanira kunanga nzira dzekufema dzinofambisa mweya nekuti kupinza mapapu muropa hakupi rubatsiro rwekurapa. Kushanda zvakanaka kwehutachiona hwehutachiona kwakabatana zvakananga nenguva yekugara kwevector. Zvisinei, mvura dzinotakura dzakadai sedzinotakura majini dzinopararira mualveoli panguva yekufemerwa, uye zvidimbu zvekurapa zvechero rudzi zvinokurumidza kubviswa nekutakurwa kwemucociliary. Kuwedzera nguva yekugara kwevanotakura majini mumhepo kwakakosha asi kwakaoma kuwana. Zvikamu zvemagineti zvinobatwa negeneti zvinogona kutungamirirwa pamusoro pemhepo zvinogona kuvandudza kutarisa kwenzvimbo. Nekuda kwezvinetso zvekuona mu vivo, maitiro ezvimedu zvidiki zvemagineti pamusoro pemhepo pamberi pesimba remagineti rakashandiswa haanzwisiswi zvakanaka. Chinangwa chechidzidzo ichi chaive chekushandisa synchrotron imaging kufungidzira kufamba kwezvimedu zvemagineti mu trachea yemakonzo ane anesthesia kuti aongorore mafambiro uye maitiro emunhu neakawanda ezvimedu mu vivo. Takazoongororawo kana kuendeswa kwezvimedu zvemagineti zve lentiviral pamberi pesimba remagineti kwaizowedzera kushanda kwe transduction mugonzo. trachea. Kuongororwa kweX-ray neSynchrotron kunoratidza maitiro ezvimedu zvemagineti muminda yemagineti isingamire uye inofamba mu vitro uye mu vivo. Zvimedu hazvigone kudhonzwa zviri nyore pamusoro penzira yekufema iri mhenyu nemagineti, asi panguva yekufambisa, zvakachengetwa zvinowanikwa munzvimbo yekuona uko simba remagineti rakasimba. Kubudirira kwe transduction kwakawedzerwawo katanhatu apo zvimedu zvemagineti zve lentiviral zvakaunzwa pamberi pesimba remagineti. Pamwe chete, mhedzisiro iyi inoratidza kuti zvimedu zvemagineti zve lentiviral neminda yemagineti zvingave nzira dzinokosha dzekuvandudza gene vector targeting uye kuwedzera mazinga e transduction mukuita airways mu vivo.
Cystic fibrosis (CF) inokonzerwa nekusiyana kwejini rimwe chete rinonzi CF transmembrane conductance regulator (CFTR). Protein yeCFTR inzira yeion inowanikwa mumasero mazhinji epithelial mumuviri wese, kusanganisira nzira dzemhepo dzinofambisa mweya, nzvimbo huru yeCF pathogenesis. Kukanganisika kweCFTR kunotungamira mukutakurwa kwemvura kusingawanzoitiki, kusvibisa mvura pamusoro pemhepo uye kuderedza kudzika kwemvura yepamusoro pemhepo (ASL). Izvi zvinokanganisawo kugona kwemucociliary transport (MCT) system kubvisa zvidimbu zvinofema nehutachiona kubva mumhepo. Chinangwa chedu ndechekugadzira kurapa kwemajini e lentiviral (LV) kuti tipe kopi chaiyo yejini reCFTR uye kuvandudza hutano hweASL, MCT, nemapapu, uye kuramba tichigadzira matekinoroji matsva anokwanisa kuyera aya ma parameter mu vivo1.
Mavector eLV ndeimwe yeanotungamira pakurapa majini eCF airway, kunyanya nekuti anogona kubatanidza gene rekurapa zvachose mumasero emumhepo (masero emhepo). Izvi zvakakosha nekuti anogona kudzorera mvura yakajairika uye kubviswa kwematehwe nekuchinja kuita masero emhepo akabatana neCF, zvichikonzera mabhenefiti ehupenyu hwese. Mavector eLV anofanirwa kunangidzirwa pamhepo inofamba, sezvo ndipo panotanga chirwere chemapapu cheCF. Kuendeswa kwevector mukati memapapu kunogona kukonzera kuendeswa kwealveolar, asi izvi hazvina zvazvinobatsira pakurapa muCF. Zvisinei, mvura dzakadai sedzinotakura majini dzinofamba dzichienda kualveoli mushure mekufemerwa mushure mekusununguka3,4 uye zvidimbu zvekurapa zvinokurumidza kuburitswa mumuromo neMCT. Kubudirira kwekufambisa kweLV kwakabatana zvakananga nehurefu hwenguva iyo vector inoramba iri padyo nemasero anotarisirwa kuti ibvumire kutorwa kwemasero - "nguva yekugara"5 - iyo inoderedzwa nyore nyore nekufamba kwemhepo kwenzvimbo pamwe nekubatwa kwemudumbu neMCT. Kune CF, kugona kuwedzera nguva yekugara kweLV mukati memhepo kwakakosha kuti uwane huwandu hwakawanda hwekutumira mudunhu iri, asi kwakadaro. Zvave zvakaoma zvikuru.
Kuti tikunde chipingamupinyi ichi, tinokurudzira kuti LV magnetic particles (MPs) dzinogona kubatsira nenzira mbiri dzinoenderana. Chekutanga, dzinogona kutungamirirwa nemagineti kunzvimbo yemhepo kuti dzivandudze kunanga uye dzibatsire gene carrier particles kugara munzvimbo yemhepo inodiwa; uye ASL) kuti dzifambe kuenda kune cell layer 6. MPs dzave dzichishandiswa zvakanyanya senzira dzekupa mishonga dzinonangwa padzinobatana ne antibodies, mishonga ye chemotherapy, kana mamwe mamorekuru madiki anonamira pa cell membranes kana kunamatira kune cell surface receptors dzakakodzera uye dzinoungana panzvimbo dzegomarara kana paine static electricity. Magnetic Fields yekurapa Cancer 7. Dzimwe nzira dze "hyperthermal" dzinovavarira kupisa maMP kana akasangana nemasimba emagineti anotenderera, nokudaro achiparadza masero egomarara. Nheyo ye magnetic transfection, umo magnetic field inoshandiswa se transfection agent yekuwedzera kutamiswa kweDNA kumasero, inowanzo shandiswa in vitro uchishandisa akasiyana-siyana asiri mavhairasi uye mavhairasi gene vectors kune mitsetse yemasero yakaoma ku-transduce. Kushanda kweLV magnetotransfection kwakasimbiswa, nekuendeswa kweLV-MPs mu in vitro kune munhu bronchial epithelial cell line pamberi pesimba remagineti rakamira, zvichiwedzera kushanda kwe transduction ne186-fold zvichienzaniswa neLV vector chete.LV-MP yakashandiswawo kune in vitro CF model, uko magnetic transfection yakawedzera LV transduction mu air-liquid interface cultures ne20-fold pamberi peCF sputum10.Zvisinei, in vivo magnetotransfection yenhengo haina kutariswa zvakanyanya uye yakangoongororwa mumhuka shoma chete. zvidzidzo11,12,13,14,15, kunyanya mumapapu16,17. Zvisinei, mikana yekushandiswa kwe magnetic transfection mu CF lung therapy yakajeka.Tan et al.(2020) vakataura kuti "chidzidzo cheuchapupu hwepfungwa ye magnetic nanoparticle pulmonary delivery inoshanda chichagadzira nzira yeramangwana reCFTR inhale strategies yekuvandudza mhedzisiro yekiriniki muvarwere veCF"6.
Maitiro ezvimedu zvidiki zvemagineti panzvimbo dzemhepo dziripo kana paine simba remagineti rakashandiswa akaoma kuona nekudzidza, uye nokudaro haana kunzwisiswa zvakanaka. Mune zvimwe zvidzidzo, takagadzira nzira ye synchrotron-propagation-based phase-contrast X-ray imaging (PB-PCXI) kuti tione nekutarisa shanduko dzeminiti mu vivo muASL depth18 uye MCT behavior19,20 kuti tiongorore zvakananga gasi Canal surface hydration uye yakashandiswa sechiratidzo chekutanga chekushanda kwekurapa. Pamusoro pezvo, nzira yedu yekuongorora MCT inoshandisa zvimedu zve 10–35 µm dhayamita zvakaumbwa nealumina kana high refractive index glass seMCT markers zvinoonekwa uchishandisa PB-PCXI21. Maitiro ese ari maviri akakodzera kuona mhando dzakasiyana dzezvimedu, kusanganisira MP.
Nekuda kwekusagadzikana kwayo munzvimbo nenguva, matekiniki edu ekuongorora ePB-PCXI-based ASL neMCT akakodzera kuongorora mashandiro uye mapatani ehunhu hwechimwe chete uye hwakawanda mu vivo kuti atibatsire kunzwisisa nekugadzirisa matekiniki ekutumira majini eMP. Maitiro atinoshandisa pano anobva muzvidzidzo zvedu tichishandisa SPring-8 BL20B2 beamline, umo takaona kufamba kwemvura mushure mekuendeswa kwesham vector dose mumhino nemapapu emhepo yemakonzo kuti tibatsire kutsanangura maitiro edu ekutaura kwemajini asina kufanana akaonekwa muzvidzidzo zvedu zvemhuka zve gene carrier dose 3,4.
Chinangwa chechidzidzo ichi chaive chekushandisa synchrotron PB-PCXI kuti tifunge nezvemafambiro eMPs mu trachea yemakonzo mapenyu. Zvidzidzo izvi zvePB-PCXI zvakagadzirwa kuti zviongorore maMPs akasiyana-siyana, simba remagnetic field, nenzvimbo kuti zvione kuti zvinokanganisa sei kufamba kweMP. Takafungidzira kuti simba remagnetic rinoshandiswa kunze raizobatsira MP yakapihwa kuti igare kana kufamba kunzvimbo yainoda. Zvidzidzo izvi zvakatibvumirawo kuona magadzirirwo emagineti anowedzera huwandu hwezvimedu zvakachengetwa mu trachea mushure mekuiswa. Muongororo yechipiri, takatsvaga kushandisa iyi configuration yakanaka kuratidza transduction pattern inobva mukutumira maLV-MPs mu vivo kuenda kumhepo yemakonzo, zvichibva pakufungidzira kuti kutumira maLV-MPs muchimiro chekunanga mhepo kwaizoguma nekuvandudzwa kwekushanda kweLV transduction.
Zvidzidzo zvese zvemhuka zvakaitwa zvichienderana nemitemo yakatenderwa neYunivhesiti yeAdelaide (M-2019-060 naM-2020-022) pamwe nekomiti yetsika dzemhuka yeSPring-8 Synchrotron. Kuedza kwakaitwa zvichienderana nemirairo yeARRIVE.
Mifananidzo yese yeX-ray yakaitwa paBL20XU beamline paSPring-8 synchrotron muJapan, uchishandisa setup yakafanana neyakatsanangurwa kare21,22. Muchidimbu, bhokisi rekuyedza raive 245 m kubva pa synchrotron storage ring. Sample-to-detector distance ye0.6 m inoshandiswa pakudzidza kwe particle imaging uye 0.3 m yezvidzidzo zve in vivo kuti ibudise phase contrast effects. Monochromatic beam energy ye25 keV yakashandiswa. Mifananidzo yakatorwa uchishandisa high-resolution X-ray converter (SPring-8 BM3) yakabatana ne sCMOS detector. Iyo converter inoshandura X-rays kuita chiedza chinoonekwa ichishandisa 10 µm thick scintillator (Gd3Al2Ga3O12), iyo inozoendeswa kune sCMOS sensor ichishandisa × 10 microscope objective (NA 0.3). Iyo sCMOS detector yaive Orca-Flash4.0 (Hamamatsu Photonics, Japan) ine array size ye Mapikiseli e2048 × 2048 uye saizi yepikiseli isina kurongeka ye6.5 × 6.5 µm. Iyi setup inopa saizi inoshanda yepikiseli ye0.51 µm uye nzvimbo yekuona inosvika 1.1 mm × 1.1 mm. Hurefu hwekuratidzwa hwe100 ms hwakasarudzwa kuti kuwedzere chiyero chesignal-to-noise chezvimedu zvemagnetic mukati nekunze kwemhepo ukuwo kuchideredza mafambiro anokonzerwa nekufema. Kune zvidzidzo zve in vivo, shutter yeX-ray inokasira yakaiswa munzira yeX-ray kuti ideredze huwandu hwemwaranzi nekuvhara mwenje weX-ray pakati pekuratidzwa.
Chinotakurika cheLV hachina kushandiswa mune chero zvidzidzo zveSPring-8 PB-PCXI imaging nekuti BL20XU imaging chamber haina chitupa cheBiosafety Level 2. Pane kudaro, takasarudza mhando dzakasiyana dzeMP dzakanyatsozivikanwa kubva kune vatengesi vaviri vemabhizinesi—vanosanganisira saizi dzakasiyana-siyana, zvinhu, huwandu hwesimbi, uye mashandisirwo—kutanga kuti tinzwisise kuti masimba emagineti anokanganisa sei kufamba kweMP mukati megirazi capillaries, uye ipapo munzira dzemhepo dziri mhenyu. pamusoro. MaMP anokura kubva pa0.25 kusvika 18 μm uye akagadzirwa nezvinhu zvakasiyana-siyana (ona Tafura 1), asi kuumbwa kwemuenzaniso wega wega, kusanganisira saizi yemagineti ari mukati meMP, hakuzivikanwe. Zvichibva pazvidzidzo zvedu zvakawanda zveMCT 19, 20, 21, 23, 24, tinotarisira kuti maMP madiki se5 μm anogona kuonekwa pamusoro penzira yekufema yetracheal, semuenzaniso nekubvisa mafuremu akatevedzana kuti aone kuoneka kwakanyanya kwekufamba kweMP. MP imwe chete ye0.25 μm-size idiki pane resolution yemuchina wekufungidzira, asi PB-PCXI inotarisirwa kuona kusiyana kwevhoriyamu yavo uye kufamba kwemvura yepamusoro yavanoiswa pairi mushure mekuiswa.
Mienzaniso yeMP yega yega muTafura 1 yakagadzirwa mu20 μl glass capillaries (Drummond Microcaps, PA, USA) ine dhayamita yemukati ye0.63 mm. Corpuscular particles inowanikwa mumvura, nepo CombiMag particles inowanikwa mumvura yemugadziri. Chubhu yega yega yakazadzwa nehafu yemvura (inenge 11 μl) uye yakaiswa pamubati wemuenzaniso (ona Mufananidzo 1). Magirazi capillaries akaiswa zvakatwasuka padanho remuenzaniso mubhokisi remifananidzo, zvichiteerana, uye akaiswa pamucheto wemvura. Magnetti ye19 mm dhayamita (28 mm kureba) nickel shell rare earth neodymium iron boron (NdFeB) (N35, cat. no. LM1652, Jaycar Electronics, Australia) ine residual magnetization ye1.17 Tesla yakabatanidzwa padanho rekushandura rakasiyana kuti iite Shanduko yenzvimbo yayo iri kure panguva yemifananidzo. Kuwanikwa kwemifananidzo yeX-ray kunotanga kana magineti akaiswa anenge 30 mm pamusoro pemuenzaniso, uye mifananidzo inowanikwa nechiyero chemafuremu mana pasekondi. Munguva yemifananidzo, magineti yakaswededzwa pedyo negirazi. chubhu ye capillary (inenge 1 mm kure) uye yozoshandurwa ichitevedza chubhu kuti iongorore mhedzisiro yesimba remunda nenzvimbo.
Kugadzira mifananidzo ye in vitro ine sampuro dzeMP mu capillaries dzegirazi padanho rekushandura re xy. Nzira ye X-ray beam yakanyorwa nemutsetse mutsvuku une dashboard.
Pakangoonekwa kuti MPs dzinoonekwa sei mu vitro, boka radzo rakaongororwa mu vivo mumakonzo echikadzi eWistar ane albino (anenge mavhiki gumi nemaviri, ~200 g). 0.24 mg/kg medetomidine (Domitor®, Zenoaq, Japan), 3.2 mg/kg midazolam (Dormicum®, Astellas Pharma, Japan) uye 4 mg/kg butorphanol (Vetorphale®, Meiji Seika) makonzo akarapwa nemusanganiswa wePharma), Japan) nekubaiwa jekiseni remukati medumbu. Mushure mekunzwa kurwadza, vakagadzirira kuratidzwa nekubvisa mvere dzakatenderedza trachea, vachiisa chubhu ye endotracheal (ET; 16 Ga iv cannula, Terumo BCT) uye vachidzimisa kufamba vakarara paplate yemifananidzo yakagadzirwa nemaoko ine bhegi rinopisa kuti varambe vachidziya muviri 22. Plate yemifananidzo yakazobatanidzwa padanho rekushandura muenzaniso mubhokisi remifananidzo nekona diki kuti trachea ienderane neyakatarisana mumufananidzo weX-ray, sezvakaratidzwa paMufananidzo 2a.
(a) Kugadzwa kwemifananidzo yemukati memuviri mubhokisi remifananidzo reSPring-8, nzira yeX-ray beam yakanyorwa nemutsetse mutsvuku wakachekwa.(b,c) Kuwanikwa kwemagineti patrachea kwakaitwa kure uchishandisa makamera maviri eIP akaiswa padivi. Kuruboshwe rwemufananidzo wechiratidziro, waya yakabata musoro inogona kuonekwa, uye cannula yekuendesa iri panzvimbo yayo mukati mechubhu yeET.
Sisitimu yepombi yesirinji inodzorwa kure (UMP2, World Precision Instruments, Sarasota, FL) ichishandisa sirinji yegirazi ye100 μl yakabatanidzwa nepeji yePE10 (OD 0.61 mm, ID 0.28 mm) kuburikidza netsono ye30 Ga. Maka chubhu kuti uve nechokwadi chekuti muromo uri panzvimbo chaiyo mutrachea paunoisa chubhu yeET. Uchishandisa micropomp, syringe plunger yakabviswa uku muromo wechubhu uchinyudzwa muMP sample kuti upinzwe. Chubhu yekutumira yakatakura yakazoiswa muchubhu ye endotracheal, ichiisa muromo mukati mechikamu chakasimba chesimba redu remagineti raitarisirwa. Kutora mifananidzo kwakadzorwa uchishandisa respiration detector yakabatana neArduino based timing box yedu, uye masaini ese (semuenzaniso tembiricha, respiration, shutter opening/closing uye image acquisition) akarekodhwa uchishandisa Powerlab neLabChart (AD Instruments, Sydney, Australia) 22. Pakutora mifananidzo Apo enclosure yakanga isingasvikike, makamera maviri eIP (Panasonic BB-SC382) akaiswa paanenge 90° kusvika uye zvakashandiswa kutarisa nzvimbo yemagineti maererano ne trachea panguva yekutora mifananidzo (Mufananidzo 2b, c). Kuti kuderedze mafambiro ezvishandiso, mufananidzo mumwe chete wakawanikwa pakufema panguva yekuyerera kwemvura.
Magineti inosungirirwa padanho rechipiri rinogona kuwanikwa kure kubva kunze kweimba yemifananidzo. Nzvimbo dzakasiyana-siyana dzemagineti uye magadzirirwo akaedzwa, anosanganisira: Yakaiswa pakona inosvika 30° pamusoro petrachea (magadzirirwo anoratidzwa muMifananidzo 2a ne3a); magineti rimwe pamusoro pemhuka nerimwe pazasi, nematanda akaiswa kuti akwezve (Mufananidzo 3b); magineti rimwe pamusoro pemhuka nerimwe pazasi, nematanda akaiswa kuti adzore (Mufananidzo 3c); uye magineti rimwe pamusoro uye rakatarisa trachea (Mufananidzo 3d). Kana mhuka nemagnet zvagadziriswa uye MP inofanira kuyedzwa yaiswa mupombi yesirinji, tumira dose ye50 μl nechiyero che4 μl/sec uchitora mifananidzo. Magineti inobva yafambiswa mberi nekumashure kana kuti padivi netrachea uku ichiramba ichitora mifananidzo.
Magadzirirwo emagineti ekufungidzira kwemumuviri (a) magineti rimwe chete pamusoro petrachea pakona inosvika 30°, (b) magineti maviri akaiswa kuti akwezve, (c) magineti maviri akaiswa kuti adzore, (d) magineti rimwe chete riri pamusoro uye rakamira mutrachea. Mucherechedzi akatarisa pasi kubva pamuromo kuenda kumapapu kuburikidza netrachea, uye danda reX-ray rakapfuura neparutivi rweruboshwe rwegonzo ndokubuda kurutivi rwerudyi. Magineti anofambiswa pamwe chete nehurefu hwemhepo inofefetera kana kuruboshwe nekurudyi pamusoro petrachea munzira yeX-ray beam.
Takatsvagawo kuona kuti zvidimbu zvemuviri zviri mumhepo zvinoonekwa sei uye maitiro azvo pasina kufema kunovhiringidza uye kufamba kwemoyo. Saka, pakupera kwenguva yekutora mifananidzo, mhuka dzakaurayiwa zvine hunhu nekuda kwekunwa pentobarbital zvakanyanya (Somnopentil, Pitman-Moore, Washington Crossing, USA; ~65 mg/kg ip). Dzimwe mhuka dzakasiyiwa pachikuva chekutora mifananidzo, uye kana kufema nekurova kwemoyo zvamira, maitiro ekutora mifananidzo akadzokororwa, zvichiwedzera imwe MP kana pasina MP yaionekwa pamusoro pemhepo.
Mifananidzo yakawanikwa yakagadziriswa munzvimbo yakati sandara uye munzvimbo ine rima yozounganidzwa kuita firimu (mafuremu makumi maviri pasekondi; kumhanya kwakajairika 15-25 × zvichienderana nekufema) uchishandisa chinyorwa chakagadzirwa chakanyorwa muMATLAB (R2020a, The Mathworks).
Zvidzidzo zvese zveLV gene vector delivery zvakaitwa paLaboratory Animal Research Facility paYunivhesiti yeAdelaide uye zvaida kushandisa mhedzisiro yekuyedza kweSPring-8 kuti vaone kana kuendeswa kweLV-MP pamberi pesimba remagineti kunogona kuwedzera kutamiswa kwemajini mumuviri. Kuti vaongorore mhedzisiro yeMP nesimba remagineti, mapoka maviri emhuka akarapwa: boka rimwe rakapihwa LV-MP nemagnet akaiswa, uye rimwe boka rakagamuchira boka rekudzora rine LV-MP risina magineti.
Mavector eLV gene akagadzirwa achishandisa nzira dzakatsanangurwa kare 25, 26. LacZ vector inoratidza nuclear-localised beta-galactosidase gene inotungamirwa ne constitutive MPSV promoter (LV-LacZ), iyo inogadzira blue reaction product mumasero akachinjirwa, inoonekwa mumativi emapfupa emapapu uye muzvikamu zvematishu. Titration yakaitwa mumasero emhando dzakasiyana nekuverenga nhamba yemasero eLacZ positive nehemocytometer kuti iverenge titer muTU/ml. Vatakuri vanochengetwa pa -80 °C, vanonyungudika vasati vashandiswa, uye vanosungirirwa kuCombiMag nekusanganisa pa 1:1 ratio uye vanoiswa muchando kwemaminitsi makumi matatu vasati vasvika.
Makonzo eSprague Dawley akajairika (n = 3/boka, ~2-3 akapinzwa mushonga wekurwadziwa mukati medumbu nemusanganiswa we0.4 mg/kg medetomidine (Domitor, Ilium, Australia) uye 60 mg/kg ketamine (Ilium, Australia) yemwedzi) ip) uye mushonga wemuromo usingavhiyiwi ne16 Ga iv cannula. Kuti zvive nechokwadi chekuti tissue yemhepo yemudumbu inogamuchira LV transduction, yakagadziriswa uchishandisa nzira yedu yakatsanangurwa kare yemechanical perturbation, umo pamusoro pemhepo yemudumbu yakakweshwa netambo yewaya (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28. Kupiwa kweLV-MP mudumbu kwakazoitwa mukabati rekuchengetedza masero anenge maminetsi gumi mushure mekukanganiswa.
Simba remagineti rakashandiswa mukuyedza uku rakagadzirwa nenzira yakafanana nekudzidza kweX-ray mu vivo, nemagineti akafanana akabatirwa pamusoro petrachea achishandisa distillation stent clips (Mufananidzo 4). 50 μl volume (2 × 25 μl aliquots) yeLV-MP yakaunzwa mutrachea (n = 3 mhuka) uchishandisa pipette ine gel tip sezvakatsanangurwa kare. Boka rekudzora (n = 3 mhuka) rakagamuchira LV-MPs dzakafanana pasina kushandisa magnet. Mushure mekunge infusion yapera, cannula inobviswa kubva muET tube uye mhuka inobviswa. Magneti inoramba iripo kwemaminitsi gumi, yobva yabviswa. Makonzo akapihwa meloxicam pasi peganda (1 ml/kg) (Ilium, Australia) zvichiteverwa nekudzoserwa kweanesthesia ne ip injection ye 1 mg/kg atipamazole hydrochloride (Antisedan, Zoetis, Australia). Makonzo aichengetwa achidziya uye achitariswa kusvika apora zvizere kubva kuanesthesia.
Mudziyo wekutumira weLV-MP mukabati rekuchengetedza remuviri. Hubhu yeLuer ye ET tube inoonekwa ichibuda mumuromo uye muromo wegel wepipette unoratidzwa mumufananidzo unopinzwa nepa ET tube kusvika pakadzika paunoda mu trachea.
Vhiki imwe mushure mekupihwa mushonga weLV-MP, mhuka dzakaurayiwa zvine hunhu nekufemerwa kwe100% CO2 uye LacZ yakaongororwa tichishandisa nzira yedu yekurapa yeX-gal. Mhete nhatu dzecartilaginous dzakabviswa kuitira kuti pave nechokwadi chekuti chero kukuvara kwemuchina kana kuchengetwa kwemvura kubva mu endotracheal tube kwaisabatanidzwa muongororo. Trachea yega yega yakachekwa kwenguva refu kuti pave nehafu mbiri dzeongororo, uye dzakaiswa mundiro ine silicone rabha (Sylgard, Dow Inc) uchishandisa Minutien nerendi (Fine Science Tools) kuti ione pamusoro pechiedza. Kupararira uye maitiro emasero akachinjirwa zvakasimbiswa nemifananidzo yepamberi uchishandisa Nikon microscope (SMZ1500) neDigiLite kamera neTCapture software (Tucsen Photonics, China). Mifananidzo yakawanikwa pa20x magnification (kusanganisira nzvimbo yepamusoro yehupamhi hwese hwetrachea), nehurefu hwese hwetrachea hwakatorwa nhanho nhanho, zvichiita kuti pave nekupindirana kwakakwana pakati pemufananidzo wega wega kuti mufananidzo "ugone kusona". Mifananidzo kubva kune yega yega Trachea dzakazounganidzwa kuita mufananidzo mumwe chete wakagadzirwa uchishandisa Image Composite Editor v2.0.3 (Microsoft Research) uchishandisa planar motion algorithm. Nzvimbo dzeLacZ expression mumifananidzo yakabatanidzwa yetrachea kubva kumhuka yega yega dzakayerwa uchishandisa automated MATLAB script (R2020a, MathWorks) sezvakatsanangurwa kare, uchishandisa marongero e0.35 < Hue < 0.58, Saturation > 0.15, uye Value < 0.7. Nekutevera ma contours etishu, mask yakagadzirwa nemaoko muGIMP v2.10.24 yemufananidzo wega wega wakagadzirwa kuitira kuziva nzvimbo yetishu uye kudzivirira chero kuwanikwa kwenhema kubva kunze kwetishu yetrachea. Nzvimbo dzakasvibiswa kubva kumifananidzo yese yakabatanidzwa kubva kumhuka yega yega dzakaunganidzwa kuti dzigadzire nzvimbo yese yakavezwa yemhuka iyoyo. Nzvimbo yakasvibiswa yakazokamurwa nenzvimbo yese yakavharwa kuti igadzire nzvimbo yakajairika.
Trachea yega yega yakaiswa muparafini uye zvikamu zve5 μm zvakachekwa. Zvikamu zvakaiswa ne neutral fast red kwemaminitsi mashanu uye mifananidzo yakawanikwa uchishandisa maikorosikopu yeNikon Eclipse E400, kamera yeDS-Fi3 uye software yekutora zvinhu yeNIS element (vhezheni 5.20.00).
Ongororo dzese dzezviverengero dzakaitwa muGraphPad Prism v9 (GraphPad Software, Inc.). Kukosha kwezviverengero kwakaiswa pa p ≤ 0.05. Kurongeka kwakasimbiswa uchishandisa bvunzo yeShapiro-Wilk, uye mutsauko muLacZ staining wakaongororwa uchishandisa bvunzo yet isina kupatsanurwa.
MaMP matanhatu akatsanangurwa muTafura 1 akaongororwa achishandisa PCXI, uye kuoneka kwacho kunotsanangurwa muTafura 2. MaMP maviri epolystyrene (MP1 neMP2; 18 μm ne0.25 μm, zvichiteerana) aisaonekwa pasi pePCXI, asi mamwe masampuli akasara aizivikanwa (mienzaniso inoratidzwa muMufananidzo 5). MP3 neMP4 (10-15% Fe3O4; 0.25 μm ne0.9 μm, zvichiteerana) anooneka zvishoma. Kunyangwe aine mamwe matinji madiki akaedzwa, MP5 (98% Fe3O4; 0.25 μm) ndiyo yakanyanya kuoneka. Chigadzirwa cheCombiMag MP6 chakaoma kuona. Muzviitiko zvese, kugona kwedu kuona MP kwakawedzerwa zvakanyanya nekushandura magineti kumashure nemberi zvakafanana necapillary. Magineti paakabva pacapillary, tinji dzakatambanuka netambo refu, asi sezvo magineti aiswedera pedyo uye simba remagnetic field richiwedzera, tinji dzetinji dzakapfupika sezvo tinji dzakatama dzichienda pamusoro pecapillary. (ona Vhidhiyo Yekuwedzera S1: MP4), kuwedzera huwandu hwezvimedu pamusoro. Kusiyana neizvi, kana magineti yabviswa kubva mu capillary, simba remunda rinoderera uye maMP anogadziriswazve kuita tambo refu dzinobva pamusoro pe capillary (ona Vhidhiyo Yekuwedzera S2:MP4). Kana magineti yamira kufamba, zvimedu zvinoramba zvichifamba kwenguva pfupi mushure mekusvika panzvimbo ye equilibrium. Sezvo MP ichifamba ichienda uye ichibva pamusoro pe capillary, zvimedu zvemagineti zvinowanzo dhonza marara kuburikidza nemvura.
Kuonekwa kweMP pasi pePCXI kunosiyana zvakanyanya pakati pemasampuli.(a) MP3, (b) MP4, (c) MP5 uye (d) MP6.Mifananidzo yese inoratidzwa pano yakatorwa nemagnet iri pamusoro pe10 mm zvakananga pamusoro pe capillary. Madenderedzwa makuru anooneka imhepo yakavharirwa mu capillaries, zvichiratidza zvakajeka mativi matema nemachena emucheto we phase contrast imaging.Bhokisi dzvuku rine contrast-enhancing magnification.Ziva kuti dhayamita ye magnet schematics mumifananidzo yese haisi yehukuru uye yakakura kanenge ka100 pane inoratidzwa.
Sezvo magineti ichishandurwa kuruboshwe nekurudyi pamusoro pe capillary, kona yetambo yeMP inoshanduka kuti ienderane nemagnet (ona Mufananidzo 6), nokudaro ichitsanangura mitsetse yesimba remagineti. Kune MP3-5, mushure mekunge chord yasvika pakona yepakati, zvimedu zvinodhonzwa pamusoro pe capillary. Izvi zvinowanzoita kuti maMP aungane kuita mapoka makuru pedyo nepakasimba simba remagineti (ona Supplementary Video S3:MP5). Izvi zvinonyanya kuoneka kana uchitora mifananidzo iri pedyo nemucheto we capillary, izvo zvinoita kuti maMP aungane uye anyatsonangana pa fluid-air interface. Zvimedu zviri muMP6, zvaive zvakaoma kuona kupfuura MP3-5, hazvina kudhonzwa sezvo magineti yaifamba pamwe chete ne capillary, asi tambo dzeMP dzakaparadzaniswa, zvichisiya zvimedu zviri mumunda wekuona (ona Supplementary Video S4:MP6). Mune zvimwe zviitiko, apo simba remagineti rakashandiswa rakaderedzwa nekufambisa magineti kure kure nenzvimbo yekutora mifananidzo, chero maMP akasara aidzika zvishoma nezvishoma pasi pechubhu negravity achiramba ari mutambo (ona Supplementary Video S5: MP3).
Kona yetambo yeMP inoshanduka sezvo magineti ichishandurwa kurudyi pamusoro pe capillary.(a) MP3, (b) MP4, (c) MP5 uye (d) MP6. Bhokisi dzvuku rine kukura kunowedzera kusiyanisa. Cherechedza kuti mavhidhiyo ekuwedzera ane ruzivo sezvo achiratidza chimiro chakakosha chezvikamu uye ruzivo runoshanduka rusingagone kuonekwa mumifananidzo iyi isingachinji.
Miedzo yedu yakaratidza kuti kufambisa magineti zvishoma nezvishoma uchidzokera shure uchitevedza trachea kunoita kuti MP ionekwe zvakanaka kana paine kufamba kwakaoma mumuviri. Kuedza kwemuviri mu vivo hakuna kuitwa sezvo polystyrene beads (MP1 naMP2) dzaisaonekwa mu capillary. Imwe neimwe yeMP ina dzakasara dzakaedzwa mu vivo nemagnet long axis yakagadzirirwa pamusoro pe trachea pakona yeinenge 30° kusvika yakamira (ona Mifananidzo 2b na3a), sezvo izvi zvakakonzera cheni refu dzeMP uye zvaive zvichishanda kupfuura magnet configuration yakaguma.MP3, MP4 naMP6 hazvina kuonekwa mu trachea yemhuka mhenyu. Apo nzira dzemhepo dzemakonzo dzakatorwa mifananidzo mushure mekunge mhuka dzaurayiwa nenzira yemunhu, zvidimbu zvacho zvakaramba zvisingaonekwe kunyangwe pakawedzerwa huwandu uchishandisa syringe pombi.MP5 yaive nehuwandu hwepamusoro hwe iron oxide uye ndiyo yega chidimbu chinoonekwa, uye saka yakashandiswa kuongorora nekuratidza maitiro eMP mu vivo.
Kuisa magineti pamusoro petrachea panguva yekupa MP kwakaita kuti maMP akawanda, asi kwete ese, agare munzvimbo yekuona. Zvidimbu zvinopinda mutrachea zvinonyanya kuonekwa mumhuka dzakabayirwa nevanhu. Mufananidzo 7 neVhidhiyo Yekuwedzera S6: MP5 inoratidza kukurumidza kubatwa kwemagineti uye kurongeka kwezvidimbu pamusoro petrachea yemukati, zvichiratidza kuti maMP anogona kutungamirirwa kunzvimbo dzinodiwa dzetrachea. Pakutsvaga kure netrachea mushure mekupa MP, mamwe maMP akawanikwa ari pedyo ne carina, zvichiratidza kuti simba remagineti rakanga risina kukwana kuunganidza nekuchengeta maMP ese, sezvo aiendeswa kuburikidza nenzvimbo ine simba guru remagineti panguva yekushanda kwemvura. Zvisinei, huwandu hweMP mushure mekusununguka hwakanga hwakakwira munzvimbo yakatorwa mifananidzo, zvichiratidza kuti maMP mazhinji akasara munzvimbo dzemhepo uko simba remagineti rakashandiswa raive rakakwirira.
Mifananidzo kubva (a) pamberi uye (b) mushure mekuiswa kweMP5 mu trachea yegonzo richangobva kuurayiwa ne magnet iri pamusoro penzvimbo yemifananidzo. Nzvimbo yemifananidzo iri pakati pemaronda maviri e cartilage. MP isati yasvika, pane mvura mumhepo. Bhokisi dzvuku rine kukwidziridza kusiyanisa. Mifananidzo iyi inobva muvhidhiyo inoratidzwa muSupplementary Video S6:MP5.
Kushandura magineti ari patrachea mu vivo kwakaita kuti cheni yeMP ichinje kona iri pamusoro penzira yekufema nenzira yakafanana neinoonekwa mu capillaries (ona Mufananidzo 8 ne Supplementary Video S7:MP5). Zvisinei, muchidzidzo chedu, maMP haakwanise kudhonzwa pamusoro penzira yekufema iri mupenyu sezvavangaita nema capillaries. Mune zvimwe zviitiko, cheni yeMP icharamba yakareba sezvo magineti ichifamba kuruboshwe nekurudyi. Zvinofadza kuti, takaonawo kuti tambo ye particle inoita seyakachinja kudzika kwechikamu chemvura yepamusoro apo magineti inofambiswa kwenguva refu pa trachea, uye inokura kana magineti ikafambiswa zvakananga pamusoro uye tambo ye particle inotenderedzwa panzvimbo yakatwasuka (ona Supplementary Video S7). : MP5 pa0:09, pasi kurudyi). Maitiro ekufamba akachinja apo magineti yaipfuudzwa pamusoro petrachea (ndiko kuti, kuruboshwe kana kurudyi kwemhuka pane kutenderera nehurefu hwetrachea). Zvidimbu zvairamba zvichionekwa zvakajeka pazvaifamba, asi magineti payakabviswa kubva mutrachea, magumo etambo dzezvidimbu akatanga kuonekwa (ona Vhidhiyo Yekuwedzera S8:MP5, kutanga pa0:08). Izvi zvinoenderana nemaitiro eMP atakaona pasi pesimba remagineti rakaiswa mugirazi capillary.
Mienzaniso yemifananidzo inoratidza MP5 mu trachea yegonzo riri mhenyu rakararamiswa.(a) Magineti inoshandiswa kuwana mifananidzo iri pamusoro nekuruboshwe rwe trachea, wozobva (b) mushure mekunge magineti yaendeswa kurudyi. Bhokisi dzvuku rine kukwidziridzwa kunowedzera kusiyanisa. Mifananidzo iyi inobva muvhidhiyo inoratidzwa muSupplementary Video S7:MP5.
Apo matanda maviri aya akagadzirwa nenzira yekuchamhembe-kumaodzanyemba pamusoro nepasi petrachea (kureva kukwezva; Mufananidzo 3b), maMP chords aionekwa kwenguva refu uye aive padivi petrachea pane pamusoro petracheal dorsal (ona Supplementary Video S9:MP5). Zvisinei, kuwanda kwezvikamu panzvimbo imwe chete (kureva, pamusoro petrachea dorsal) hakuna kuonekwa mushure mekuburitswa kwemvura apo mudziyo une magineti maviri wakashandiswa, izvo zvinowanzoitika kana mudziyo une magineti rimwe chete ukashandiswa. Zvadaro, kana magineti rimwe chete rakagadzirwa kuti ridzore matanda akadzoserwa kumashure (Mufananidzo 3c), huwandu hwezvikamu zvinoonekwa mumunda wekuona hauna kuita sehunowedzera mushure mekuburitswa. Kugadzwa kwemaitiro ese ari maviri egineti kunonetsa nekuda kwesimba guru remagineti rinodhonza kana kusunda magineti, zvichiteerana. Kugadzwa kwacho kwakachinjwa kuita magineti rimwe chete rakafanana nemhepo yekufema asi richipfuura nepamhepo yekufema pamadhigirii 90 kuitira kuti mitsetse yemunda ipfuure madziro etracheal nenzira yakatenderedza (Mufananidzo 3d), nzira yakagadzirirwa kuona kana zvikamu zvakaungana parutivi. madziro aionekwa. Zvisinei, muchimiro ichi, pakanga pasina kufamba kunoonekwa kweMP accumulation kana kufamba kwemagnet. Zvichibva pane zvese izvi, gadziriro yekutarisa magineti imwe chete, 30-degree (Mufananidzo 3a) yakasarudzwa kuti ishandiswe muzvidzidzo zvevatakuri vemajini mu vivo.
Mhuka iyi payaidzokororwa kuratidzwa mifananidzo mushure mekuurayiwa kwevanhu, kusavapo kwekufamba kwenyama kwakakanganisa kwaireva kuti mitsetse mipfupi uye mipfupi yaigona kuonekwa mumunda wakajeka wepakati pechondral, "inodedera" zvichienderana nekufamba kwemagineti. Kunyange zvakadaro, haisati yanyatsoona kuvapo nekufamba kweMP6 particles.
Nhamba yeLV-LacZ yaive 1.8 × 108 TU/ml, uye mushure mekusanganiswa neCombiMag MP (MP6) 1:1, mhuka dzakawana 50 μl tracheal dose ye9 × 107 TU/ml LV vehicle (kureva 4.5 × 106 TU/rat). ). Muzvidzidzo izvi, pachinzvimbo chekushandura magineti panguva yekusununguka, takaisa magineti panzvimbo imwe chete kuti tione kana LV transduction (a) inogona kuvandudzwa zvichienzaniswa nevector delivery pasina simba remagineti, uye (b) inogona kutariswa. Masero emhepo anoendeswa kunzvimbo dzinotarirwa magineti dzenzira yepamusoro yekufema.
Kuvapo kwemagineti uye kushandiswa kweCombiMag pamwe chete nemavector eLV hazvina kuita sezvine mhedzisiro yakaipa pahutano hwemhuka, sezvakaitawo protocol yedu yeLV vector delivery. Mifananidzo yepamberi yenzvimbo yetracheal yakakanganiswa ne mechanical perturbation (Supplementary Fig. 1) yakaratidza kuti paive nemazinga akakwira e transduction muboka remhuka dzakarapwa neLV-MP apo magineti yaivepo (Fig. 9a). Huwandu hushoma hwe blue LacZ staining hwaivepo muboka rekutonga (Fig. 9b). Kuongororwa kwenzvimbo dzakasvibiswa neX-Gal kwakaratidza kuti kupihwa kweLV-MP pamberi pesimba remagineti kwakaburitsa kuvandudzwa kanenge ka6 (Fig. 9c).
Muenzaniso wemifananidzo yakasanganiswa inoratidza tracheal transduction neLV-MP (a) pamberi pesimba remagineti uye (b) pasina magineti. (c) Kuvandudzika kwakakosha muhuwandu hweLacZ transduction area mukati metrachea pakushandisa magineti (*p = 0.029, t-test, n = 3 paboka rega rega, avhareji ± SEM).
Zvikamu zvine ruvara rutsvuku zvisina kusimba (muenzaniso unoratidzwa muMufananidzo Wekuwedzera 2) zvakaratidza masero ane ruvara rweLacZ aripo muchimiro chakafanana nenzvimbo sezvakambotaurwa.
Dambudziko guru rekurapa majini emumhepo rinoramba riri rekuti masero emumhepo anowanikwa munzvimbo dzaanofarira uye kuwana huwandu hwakanyanya hwema "transduction" anoshanda zvakanaka mumapapu ari kufamba kana paine mhepo inoyerera uye mucus inobviswa. Kune vatakuri veLV vakagadzirirwa kurapa chirwere cheCF airway, kuwedzera nguva yekugara kwemasero emumhepo ari mu "conducting airway" kwave kuri chinangwa chisingawanikwe kusvika parizvino. Sezvakataurwa naCastellani et al., kushandiswa kwemagineti kuvandudza transduction kune zvakanakira zvichienzaniswa nedzimwe nzira dzekuendesa majini dzakadai se electroporation, sezvo zvichigona kusanganisa nyore, kushanda zvakanaka, nzvimbo yekutumira, kushanda kwakawedzerwa, uye nguva pfupi yekudzikisa, uye pamwe diki yekutakura.10. Zvisinei, kuiswa kwe "in vivo" uye maitiro e "magnetic particles" mumhepo pasi pesimba re "external magnetic forces" hakuna kumbobvira kwatsanangurwa, uye kugona kwenzira iyi hakuna kuratidzwa mu "vivo" kusimudzira huwandu hwemajini mumhepo dziri mhenyu dzisina kugadzikana.
Kuedza kwedu kwe in vitro synchrotron PCXI kwakaratidza kuti zvidimbu zvese zvatakaedza, kunze kwe polystyrene MP, zvaionekwa mumufananidzo watakashandisa. Kana paine simba remagineti, ma MP anoumba tambo dzine hurefu hwadzo hwakabatana nerudzi rwezvidimbu uye simba remagineti (kureva kuva pedyo nekufamba kwemagnet). Sezvinoratidzwa muMufananidzo 10, tambo dzatinoona dzinoumbwa nekuda kwekuti zvidimbu zvega zvega zvinova nemagineti uye zvichikonzera simba rayo remagineti. Minda iyi yakasiyana inoita kuti zvimwe zvidimbu zvakafanana zviungane uye zvibatane, nemafambiro akafanana netambo nekuda kwemasimba emunharaunda kubva kumasimba emunharaunda anokwezva uye anosemesa ezvimwe zvidimbu.
Kuratidza zviratidzo (a,b) zvimedu zvinogadzirwa mukati me capillaries dzakazadzwa nemvura uye (c,d) trachea yakazadzwa nemhepo. Cherechedza kuti capillaries ne trachea hazvina kukwezvwa kuti zvienderane nehukuru. Panel (a) inewo tsananguro yeMP, ine zvimedu zveFe3O4 zvakarongwa mu tambo.
Magineti payakafambiswa pamusoro pe capillary, kona yetambo ye particle yakasvika padanho rakakosha reMP3-5 ine Fe3O4, mushure mezvo tambo ye particle haina kuzoramba iri panzvimbo yayo yekutanga, asi yakafamba pamusoro ichienda panzvimbo itsva. Magnet. Izvi zvinogona kuitika nekuti pamusoro pe capillary yegirazi wakatsetseka zvakakwana kuti izvi zviitike. Zvinofadza kuti MP6 (CombiMag) haina kuita seizvi, pamwe nekuti zvidimbu zvacho zvaive zvidiki, zvaive ne coatings dzakasiyana kana surface charges, kana kuti proprietary carrier fluid yakakanganisa kugona kwavo kufamba. Musiyano wemufananidzo we CombiMag particles hauna kusimba, zvichiratidza kuti fluid nezvidimbu zvingave ne densities dzakafanana uye nekudaro hazvifambi zviri nyore kune mumwe nemumwe. Particles dzinogonawo kunamira kana magnet ichifamba nekukurumidza, zvichiratidza kuti magnetic field strength haigoni kukunda kukweshana pakati pezvikamu zviri mumvura, zvichiratidza kuti pamwe hazvishamisi kuti magnetic field strength uye daro riri pakati pemagnet nenzvimbo yakanangwa Zvakakosha. Zvikatorwa pamwe chete, mhedzisiro iyi inoratidzawo kuti, nepo magnets achigona kubata maMP akawanda anoyerera munzvimbo yakanangwa, hazvigoneke kuti magnets anogona kuvimbwa nawo. kuti tifambise zvidimbu zveCombiMag pamusoro pe trachea. Saka, tinogumisa kuti zvidzidzo zve LV-MP mu vivo zvinofanirwa kushandisa masimba emagineti asingachinji kuti zvinange nzvimbo dzakatarwa dzemuti wemhepo.
Kana zvidimbu zvikaunzwa mumuviri, zvakaoma kuzviona kana tichitarisa mafambiro akaomarara emuviri, asi kugona kuzviona kwakawedzerwa nekushandura magineti yakatwasuka pamusoro petrachea kuti "itenderedze" tambo dzeMP. Kunyangwe mifananidzo chaiyo ichikwanisika, zviri nyore kuona kufamba kwezvidimbu kana mhuka yaurayiwa nenzira yemunhu. Kuwanda kweMP kwaiwanzo kuve kwakanyanya panzvimbo iyi apo magineti yaive pamusoro penzvimbo yemifananidzo, kunyangwe zvimwe zvidimbu zvaiwanzo kuwanikwa kure netrachea. Kusiyana nezvidzidzo zve in vitro, zvidimbu hazvigone kudhonzwa pamwe chete netrachea nekushandura magineti. Kuwanikwa uku kunoenderana nekuti manyemwe anofukidza pamusoro petrachea anowanzo gadzirisa zvidimbu zvinofemerwa, zvichivabata mumanyemwe uye zvinozobviswa ne mucociliary clearance mechanism.
Takafungidzira kuti kushandiswa kwemagineti pakukwezva pamusoro nepasi petrachea (Mufananidzo 3b) kunogona kukonzera simba remagineti rakafanana, pane simba remagineti rakanyanya kuzara pane imwe nzvimbo, zvichigona kukonzera kupararira kwakafanana kwezvikamu. Zvisinei, chidzidzo chedu chekutanga hachina kuwana humbowo hwakajeka hunotsigira fungidziro iyi. Saizvozvowo, kugadzirisa magineti maviri ekudzora (Mufananidzo 3c) hakuna kukonzera kuiswa kwezvikamu zvakawanda munzvimbo yakatorwa mifananidzo. Izvi zviviri zvakawanikwa zvinoratidza kuti kugadzwa kwemagineti maviri hakugadzirisi zvakanyanya kutonga kweMP targeting, uye kuti simba remagineti rakasimba rinobuda rakaoma kugadzirisa, zvichiita kuti nzira iyi isashande. Saizvozvowo, kutarisisa magineti ari pamusoro uye kuburikidza netrachea (Mufananidzo 3d) hakuna kuwedzerawo huwandu hwezvikamu zvakachengetwa munzvimbo yakatorwa mifananidzo. Mamwe emagadzirirwo aya anogona kusabudirira nekuti anokonzera kuderera kwesimba remagineti munzvimbo yakatorwa mifananidzo. Nokudaro, kugadzika kwemagineti e30-degree angle (Mufananidzo 3a) kunoonekwa senzira iri nyore uye inoshanda kwazvo yekuedza mu vivo.
Chidzidzo cheLV-MP chakaratidza kuti apo maLV vectors akabatanidzwa neCombiMag uye akaunzwa mushure mekuvhiringidzika kwemuviri pamberi pesimba remagineti, huwandu hwe transduction hwakawedzera zvakanyanya mu trachea zvichienzaniswa ne controls. Zvichibva pazvidzidzo zve synchrotron imaging uye LacZ results, simba remagineti raikwanisa kuchengetedza LV mukati me trachea uye kuderedza huwandu hwe vector particles dzakapinda mukati memapapu nekukurumidza. Kuvandudzwa kwakadaro kunogona kutungamira mukushanda kwakanyanya ukuwo kuchideredza ma titers akaunzwa, transduction isiri-target, migumisiro yekuzvimba uye yekudzivirira muviri, uye mitengo yekutakura majini. Zvakakosha, maererano nemugadziri, CombiMag inogona kushandiswa pamwe chete nedzimwe nzira dzekutumira majini, kusanganisira nedzimwe viral vectors (senge AAV) uye nucleic acids.