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Gene vectors for the treatment of cystic fibrosis lung disease inofanira kunanga kuitisa mhepo nekuti peripheral lung transduction haipei rubatsiro rwekurapa.Viral transduction performance inoenderana zvakananga nevector residence time.Zvisinei, zvinwiwa zvekuendesa zvakadai semagene carriers zvinopararira mualveoli panguva yekufemerwa, uye kurapa kweanorapirwa ari kukurumidza kujekesa chero nzira yekufambisa. nguva yevatakuri vemajini mumigwagwa yemhepo yakakosha asi yakaoma kuwana.Gene carrier-conjugated magnetic particles inogona kutungamirirwa pamusoro pemhepo inogona kuvandudza chinangwa chenharaunda.Nekuda kwematambudziko ekuona mu vivo, maitiro ezvidiki zvakadai zvemagnetic particles pamusoro pemhepo yemhepo muhupo hwemagineti yakashandiswa hainzwisisiki.Chinangwa chekudzidza uku kwechidzidzo chemagineti chaive chekushandisa magineti ekuona magineti ye synchrotrosion yekuona mutsara zvimedu zviri mutrachea yemakonzo anesthetized kuti aongorore simba uye mapatani emunhu uye akawanda particle maitiro mu vivo. kuzvuzvurudzwa pamwe chete nepamusoro pehupenyu hwemhepo ine magineti, asi panguva yekufambisa, madhipoziti akaiswa mumunda wekuona uko iyo magineti yakasimba.Transduction performance yakawedzerawo kakapetwa katanhatu apo lentiviral magineti particles akaunzwa pamberi pemagineti.
Cystic fibrosis (CF) inokonzerwa nekusiyana kwejini rimwe chete rinonzi CF transmembrane conductance regulator (CFTR) .Protein yeCFTR ndiyo ion channel inowanikwa mumasero akawanda epithelial mumuviri wose, kusanganisira iyo inofambisa mhepo, nzvimbo huru yeCF pathogenesis.CFTR defects inotungamirira kune abnormal abnormal trafficway of the airway of the airway of the airway of the airway of the airway of the airway of the airway of the airway of the airway of the airway of the airway of the airway of the airway. layer.Izvi zvakare zvinokanganisa kugona kwemucociliary transport (MCT) system yekubvisa inhaled particles uye utachiona kubva mumhepo.Chinangwa chedu ndechekugadzira lentiviral (LV) gene therapy kuendesa kopi chaiyo yeCFTR gene uye kuvandudza ASL, MCT, uye hutano hwemapapu, uye kuenderera mberi nekugadzira michina mitsva inokwanisa paramita1 memesuro.
LV Vectors ndeimwe yevatungamiri veCF Airway Gume kurapa Apa ndipo CF mapapu chirwere chinotanga.Kuendeswa kwevector yakadzika mumapapu kunogona kuita kuti alveolar transduction, asi izvi hazvina rubatsiro rwekurapa muCF.Zvisinei, zvinwiwa zvakadai semagene carriers zvinowanzoenda kune alveoli pakufemerwa mushure mekusununguka3,4 uye zvipembenene zvekurapa zvinokurumidza kucheneswa mumuromo weLV transduction kurefu kusvika kune yakanangana neMCT masero anotevera. bvumira kushandiswa kwemasero - "nguva yekugara" 5 - iyo inoderedzwa nyore nyore neyakajairwa mhepo yedunhu pamwe chete necoordinated particle mucus capture uye MCT.Kune CF, kukwanisa kuwedzera nguva yekugara kweLV mukati memugwagwa wemhepo kunokosha kuti uwane mazinga akakwirira ekufambisa munharaunda ino, asi kusvika ikozvino yave yakaoma.
Kuti tikunde chipingamupinyi ichi, tinokurudzira kuti LV magnetic particles (MPs) inogona kubatsira nenzira mbiri dzinopindirana.Kutanga, vanogona kutungamirirwa nemagnetic kumhepo yemhepo kuti vavandudze kutarisa uye kubatsira mashizha emagetsi anogara munzvimbo yaidiwa yemhepo; uye ASL) kutamira kune cell layer 6.MPs dzakashandiswa zvakanyanya sedzinonangwa mota dzekutakura zvinodhaka padzinosunga kune masoja ekudzivirira chirwere, chemotherapeutic zvinodhaka, kana mamwe mamorekuru madiki anonamira kune cell membranes kana kusunga kune akakodzera cell surface receptors uye kuungana panzvimbo dzebundu pamberi pemagetsi akamira. Magnetic Fields for Cancer Treatment 7. Dzimwe nzira dze "hyperthermal" dzinovavarira kupisa MPs pavanenge vachisangana ne oscillating magnetic fields, nokudaro vachiparadza tumor cell.Nheyo yemagnetic transfection, iyo magnetic field inoshandiswa sechinhu chinoshandura kuwedzera kuendeswa kweDNA kumasero, inowanzoshandiswa mu vitro kushandisa mutsara we-non-viral uye viral cell kuoma kweLV-transfection. magnetotransfection yakagadzwa, ine in vitro kuendeswa kweLV-MPs kune yemunhu bronchial epithelial cell line pamberi peiyo static magnetic field, ichiwedzera transduction performance by 186-fold zvichienzaniswa neLV vector chete. sputum10.Zvisinei, in vivo magnetotransfection yenhengo yakagamuchira kutariswa kuduku uye yakangoongororwa mune zvidzidzo zvishoma zvemhuka11,12,13,14,15, kunyanya mumapapu16,17.Zvisinei, mikana yemagnetic transfection muCF lung therapy yakajeka.Tan et20 proof of magnetic et al. nanoparticle pulmonary delivery ichavhura nzira yeramangwana CFTR inhalation mazano ekuvandudza mhedzisiro yekiriniki muvarwere veCF”6.
Mafambiro ezvimedu zviduku zvemagineti panzvimbo yemhepo muhupo hwemagineti akaiswa zvakaoma kuona uye kudzidza, uye nokudaro kusanzwisiswa.Mune zvimwe zvidzidzo, takagadzira synchrotron-propagation-based phase-contrast X-ray imaging (PB-PCXI) nzira yekusaona uye kuenzanisa miniti mu vivo kuchinja muCanCTal kudzika kwepamusoro-192 uye MAC20 kudzika kweASL uye M20. inoshandiswa sechiratidzo chekutanga chekubudirira kwekurapa.Pakuwedzera, nzira yedu yekuongorora yeMCT inoshandisa 10-35 µm dhayamita zvidimbu zvakaumbwa nealumina kana high refractive index glass seMCT markers inooneka uchishandisa PB-PCXI21.Maitiro ose ari maviri akakodzera kuonekwa kwemhando dzakasiyana-siyana dzezvikamu, kusanganisira MP.
Nekuda kwekukwirira kwayo kwenzvimbo uye kugadzirisa kwechinguva, yedu PB-PCXI-based ASL uye MCT yekuongorora matekiniki akanyatso kuenderana nekuongorora madhiraivha uye mapatani eimwe uye yakawanda particle maitiro muvivo kutibatsira kunzwisisa uye kugonesa MP gene rekutumira maitiro.Maitiro atinoshandisa pano anobva muzvidzidzo zvedu tichishandisa SPring-8 BL20B mukufamba kwatinoita dhamu rekuona. nasal uye pulmonary airways yemakonzo kuti ibatsire kutsanangura yedu Non-uniform gene kutaura maitiro akaonekwa mune yedu gene carrier dose zvidzidzo zvemhuka 3,4.
Chinangwa chechidzidzo ichi chaive chekushandisa synchrotron PB-PCXI kufungidzira mafambiro emu vivo enhengo dzeMP mutrachea yemakonzo mhenyu.Izvi zvidzidzo zvePB-PCXI zvekufungidzira zvakagadzirirwa kuyedza huwandu hwemaMP, simba remagineti, uye nzvimbo dzekuona mhedzisiro yavo pane MP kufamba. tsvaga magineti magadzirirwo anowedzera huwandu hwezvimedu zvakachengetwa mutrachea mushure mekuiswa.Muchikamu chechipiri chezvidzidzo, takatsvaga kushandisa iyi yakakwana gadziriso kuratidza maitiro ekutapurirana anokonzerwa nekupa vivo kuendeswa kweLV-MPs kunzira yemhepo yemakonzo, zvichibva pafungidziro yekuti kuendesa kweLV-MPs mune inokonzeresa kufambiswa kwemhepo.
Zvidzidzo zvose zvemhuka zvakaitwa maererano nemitemo yakagamuchirwa neYunivhesiti yeAdelaide (M-2019-060 uye M-2020-022) uye SPring-8 Synchrotron Animal Ethics Committee.Kuedza kwakaitwa maererano neARRIVE mirayiridzo.
Yese X-ray imaging yakaitwa paBl20XU beamline paSPring-8 synchrotron muJapan, ichishandisa setup yakafanana neyakatsanangurwa kare21,22.Muchidimbu, bhokisi rekuyedza rakange riri 245 m kubva kune synchrotron yekuchengetedza mhete.Sample-to-detector kureba kwe0.6 m inoshandiswa pakuita particles imaging effects mA zvidzidzo zve particle imaging mA. monochromatic beam energy ye25 keV yakashandiswa.Mifananidzo yakatorwa pachishandiswa high-resolution X-ray converter (SPring-8 BM3) yakabatana ne sCMOS detector.Shanduro inoshandura X-rays kuita chiedza chinooneka uchishandisa 10 µm thick scintillator (Gd3Al2Ga3C inotungamirirwa neMOS 12), inova microscope × 12 sensor. (NA 0.3) .SCMOS detector yaive Orca-Flash4.0 (Hamamatsu Photonics, Japan) ine array size ye 2048 × 2048 pixels uye mbishi pixel saizi ye6.5 × 6.5 µm.Iyi setup inopa inoshanda isotropic pixel saizi ye0.51 × 1 × . mm.An exposure length ye 100 ms yakasarudzwa kuti iwedzere chiyero chechiratidzo-ku-ruzha chezvikamu zvemagineti mukati nekunze kwemhepo apo ichideredza kufema-induced motion artifacts.Kune zvidzidzo zve vivo, kukurumidza X-ray shutter yakaiswa munzira ye-X-ray kuderedza dhigirii yemwaranzi nekuvharira X-ray danda pakati pekuratidzwa.
Iyo LV carrier haina kushandiswa mune chero SPring-8 PB-PCXI imaging zvidzidzo nekuti iyo BL20XU imaging chamber haisi Biosafety Level 2 certified. Pane kudaro, takasarudza ruzhinji rwevamiriri veMP kubva kune vaviri vatengesi vekutengesa-inovhara huwandu hwehukuru, zvinhu, iron concentrations, uye maapplication mukati megineti yekugara, magineti magirasi uye magirasi ekufambisa zvinokanganisa maMP. nzira dzemhepo. pamusoro pepamusoro.MPs inokura muhukuru kubva ku0.25 kusvika ku18 μm uye inogadzirwa kubva kune zvakasiyana-siyana zvezvinhu (ona Tafura 1), asi kuumbwa kwemuenzaniso wega wega, kusanganisira ukuru hwemagineti mukati meMP, hazvizivikanwi.Kubva pane zvidzidzo zvedu zvakakura zveMCT 19, 20, 21, 23, 24, tinotarisira kuti MPs 5 μ panzira yemhepo inogona kuonekwa sediki, semuenzaniso. kubvisa mafuremu akatevedzana kuti aone kuoneka kwakawedzerwa kwekufamba kweMP. Imwe 0.25 μm-yakakura MP idiki pane kugadziriswa kwechigadzirwa chekufungidzira, asi PB-PCXI inotarisirwa kuona kusiyana kwevhoriyamu yavo uye kufamba kwemvura yepamusoro painoiswa mushure mekuiswa.
Mienzaniso yeMP yega yega muTable 1 yakagadzirwa mu20 μl girazi capillaries (Drummond Microcaps, PA, USA) ine dhayamita yemukati ye 0.63 mm.Corpuscular particles inowanikwa mumvura, nepo CombiMag particles inowanikwa mumugadziri weproprietary fluid.Pubhu imwe neimwe ine hafu izere nemvura inogadzika (inenge 1 μ inoiswa pachiyero che μ (inenge 1 μ). 1) .Magirazi capillaries akaiswa horizontally pamusoro muenzaniso pachikuva mu imaging box, zvichiteerana, uye nzvimbo micheto fluid.A 19 mm dhayamita (28 mm kureba) nickel goko isingawanzowanikwi pasi neodymium iron boron (NdFeB) magnet (N35, katsi. no. LM1652, Australia1 magnetization of Electronics. yakanga yakanamirwa kune imwe nhanho yekushandura kuti ibudirire Shandura nzvimbo yayo iri kure panguva yekufungidzira.Kutora mufananidzo weX-ray kunotanga apo magineti yakamira inenge 30 mm pamusoro pemuenzaniso, uye mifananidzo inowanikwa pamwero we 4 frames pasekondi.Panguva yekufungidzira, magineti yakaunzwa pedyo negirazi capillary tube (inenge 1 mm kure) uye yakabva yashandurirwa pamwe chete nechubhu kune simba uye mashizha.
In vitro imaging setup ine MP samples mugirazi capillaries pamuenzaniso xy dudziro nhanho.Nzira ye X-ray danda inoratidzwa ne red dashed line.
Pane imwe nguva iyo in vitro kuonekwa kweMPs yakasimbiswa, chikamu chavo chakaedzwa mu vivo mumhuka dzomusango dzechikadzi albino Wistar rats (~ mavhiki e12, ~ 200 g) .0.24 mg / kg medetomidine (Domitor®, Zenoaq, Japan), 3.2 mg / kg midazolam (Dormicum®, mg4 Phaorpha / Japan) buts (Vetorphale®, Meiji Seika) Makonzo akaitwa anesthetized nemusanganiswa wePharma), Japan) ne intraperitoneal injection.After anesthesia, ivo vakagadzirirwa kufungidzira nekubvisa fur kumativi e trachea, kuisa endotracheal tube (ET; 16 Ga iv cannula, Terumo BCT) uye immobilizing plate ine tsika-inogadzirisa iyo inomira-inopisa kune muviri wekushisa. 22 .Iplate yekufungidzira yakabva yabatanidzwa kumuenzaniso wekushandura nhanho mubhokisi rekufungidzira pane imwe kona kuti ienzanise trachea yakatarisa mumufananidzo we-X-ray, sezvinoratidzwa mumufananidzo 2a.
(a) In vivo imaging setup mu SPring-8 imaging box, nzira ye X-ray danda inotaridzirwa nemutsara wakatsvuka. (b, c) Magnet localization pa trachea yakaitwa kure nekushandisa maviri orthogonally akaiswa IP makamera.Kuruboshwe kwechifananidzo chekrini, tambo yetambo inobata musoro inogona kuonekwa, uye nzvimbo yekutumira inogona kuonekwa mukati meET tube.
A remote controlled syringe pump system (UMP2, World Precision Instruments, Sarasota, FL) achishandisa 100 μl girasi sirinji yakabatanidzwa nePE10 tubing (OD 0.61 mm, ID 0.28 mm) kuburikidza ne 30 Ga tsono. Maka iyo tube kuti uone kuti chidimbu chiri munzvimbo yakarurama. syringe plunger yakabviswa apo muromo wechubhu yakanyudzwa muMP sampu kuti iendeswe.Iyo yakatakurwa yekutakura chubhu yakabva yaiswa mukati me endotracheal tube, ichiisa chidimbu mukati mechikamu chakasimba chetarisiro yedu inoshandiswa magnetic field.Image acquisition yakadzorwa nekushandisa detector yekufema yakabatanidzwa kune yedu Arduino based timing box, bhokisi rekuvhara / rekuvhara, uye mifananidzo yose yekuvhura, uye zviratidzo zvose zvekuvhura kutora) zvakanyorwa uchishandisa Powerlab uye LabChart (AD Instruments, Sydney, Australia) 22. Paunenge uchifungidzira Apo iyo yakavharwa yakanga isingasviki, makhamera maviri eIP (Panasonic BB-SC382) akaiswa pane anenge 90 ° kune mumwe nemumwe uye akashandiswa kutarisa nzvimbo yemagineti maererano netrachea, minib . zvigadzirwa, mufananidzo mumwe wakawanikwa pakufema panguva yekupedzisira-tidal flow plateau.
Magineti inonamirwa kune yechipiri nhanho inogona kuwanikwa iri kure kubva kunze kweiyo imaging housing.Various magineti nzvimbo uye configurations vakaedzwa, kusanganisira: Akaiswa pakona anenge 30 ° pamusoro trachea (magadzirirwo anoratidzwa Figures 2a uye 3a); imwe magineti pamusoro pemhuka uye imwe iri pasi, ine mapango akaiswa kuti akwezve (Mufananidzo 3b); imwe magineti pamusoro pemhuka uye imwe pazasi, ine mapango akaiswa kuti adzinge (Mufananidzo 3c); uye imwe magineti kumusoro uye perpendicular kune trachea (Figure 3d) .Kana mhuka nemagineti zvagadziriswa uye MP inofanirwa kuongororwa inoiswa mupombi yesirinji, endesa 50 μl dose pamwero we 4 μl / sec uchiwana mifananidzo.Magineti inobva yafambiswa mberi nekudzoka pamwe chete kana kuti ichienderera mberi kuwana trachea.
Magnet configuration ye in vivo imaging (a) magineti imwe chete pamusoro petrachea pakona inoita 30°, (b) magineti maviri akaiswa kuti akwezve, (c) magineti maviri akaiswa kuti adzore, (d) magineti imwe chete pamusoro uye perpendicular mutrachea.Mucherechedzi akatarisa pasi kubva pamuromo kuenda kumapapu achipfuura neparutivi rwetrachea nekuruboshwe, uye kuruboshwe rworuboshwe rworuboshwe rworuboshwe rworuboshwe rworuboshwe rwakapfuura nepakati petrachea. side.Magineti inofambiswa kureba kwenzira yemhepo kana kuruboshwe uye kurudyi pamusoro petrachea yakananga kuX-ray danda.
Takatsvagawo kuona kuonekwa uye mufambiro wezvikamu mumhepo mumhepo pasina kuvhiringidza kufema uye kufamba kwemwoyo.Saka, pakupera kwenguva yekufungidzira, mhuka dzakaurayiwa nenzira yevanhu nokuda kwepentobarbital overdose (Somnopenil, Pitman-Moore, Washington Crossing, USA; ~ 65 mg / kg / kg yakasiiwa pane imwe nguva, kufungidzira kwemhuka kwakange kwasara pane imwe nguva uye kufema papuratifomu). maitiro ekufungidzira akadzokororwa, achiwedzera imwe dose yeMP kana pasina MP yaionekwa pamhepo.
Iyo mifananidzo yakawanikwa yaive yakati sandara-munda uye yakasviba-munda yakagadziridzwa ichibva yaunganidzwa kuita bhaisikopo (makumi maviri mafuremu pasekondi; 15-25 × yakajairika kumhanya zvichienderana nekufema) uchishandisa tsika script yakanyorwa muMATLAB (R2020a, Iyo Mathworks).
Yese LV gene vector yekuendesa zvidzidzo yakaitwa paLaboratory Animal Research Facility paYunivhesiti yeAdelaide uye yakanangana nekushandisa zvakabuda muyedzo yeSPring-8 kuona kana LV-MP kuendesa pamberi pemagineti inogona kusimudzira kutamiswa kwemajini mu vivo. magineti.
LV gene vectors yakagadzirwa nekushandisa nzira dzakambotsanangurwa 25, 26. LacZ vector inoratidzira nyukliya-localized beta-galactosidase gene inotungamirirwa neanomiririra MPSV musimudziri (LV-LacZ), iyo inogadzira chigadzirwa cheblue reaction mumasero akachinjwa, anoonekwa mumapapu emapapu uye zvikamu zvetishu. hemocytometer kuverenga titer muTU / ml. Vatakuri vari cryopreserved pa -80 ° C, yakanyungudutswa isati yashandiswa, uye inosungirirwa kuCombiMag nekusanganisa pachiyero che1: 1 uye incubating pachando kwemaminitsi makumi matatu asati asvika.
Normal Sprague Dawley rats (n = 3 / boka, ~ 2-3 vakanga anesthetized intraperitoneally nemusanganiswa we 0.4 mg / kg medetomidine (Domitor, Ilium, Australia) uye 60 mg / kg ketamine (Ilium, Australia) mwedzi-yekare) ip) jekiseni uye isiri-yekuvhiya yemuromo cannulation 16 Kuti ive nechokwadi chemhepo yemuromo cannulation. inogamuchira LV transduction, yakagadziriswa kushandisa yedu yakambotsanangurwa mechanical perturbation protocol, umo tracheal airway surface yakakwizwa axially newaya tswanda (N-Circle, Nitinol Tipless Stone Extractor NTSE-022115) -UDH, Cook Medical, USA) 30 s28.Tracheal administration yakaitwa mushure mekuchengetedza maminitsi eLV-1MPological yeLV-1MPological.
Iyo magnetic field inoshandiswa mukuedza uku yakagadziridzwa nenzira yakafanana kune in vivo X-ray imaging study, ine magineti akafanana akabatwa pamusoro pe trachea achishandisa distillation stent clips (Mufananidzo 4) . A 50 μl volume (2 × 25 μl aliquots) yeLV-MP yakaunzwa mu trachea (n gel-tiptte yemhuka yakatsanangurwa kare). = Mhuka 3) dzakagamuchira maLV-MPs akafanana pasina kushandiswa kwemagineti.Mushure mokunge infusion yapera, cannula inobviswa kubva kuET tube uye mhuka inoputika.Magineti inoramba iripo kwemaminitsi gumi, yobva yabviswa.Makonzo akagamuchira subcutaneous dose ye meloxicam (1 ml/kg) (Ilium, Australian injection yeAtimaversp yeAtipazosthesia) hydrochloride (Antisedan, Zoetis, Australia) .Makonzo aichengetwa achidziya uye akatariswa kusvikira kupora kwakakwana kubva kune anesthesia.
LV-MP yekuendesa mudziyo mubhayoloji yekuchengetedza kabhineti.Iyo grey grey Luer hub yeET chubhu inogona kuoneka ichibuda mumuromo uye iyo gel tip yepipette inoratidzwa mumufananidzo inopinzwa kuburikidza neET chubhu kusvika pakadzika inodiwa mutrachea.
Vhiki imwe mushure meLV-MP dosing process, mhuka dzakaurayiwa nemunhu ne 100% CO2 inhalation uye LacZ kutaura kwakaongororwa uchishandisa maitiro edu e X-gal treatment.Itatu caudal most cartilaginous rings yakabviswa kuti ione kuti chero kukanganisa kwemagetsi kana kuchengetwa kwemvura kubva endotracheal tube kuiswa kwakanga kusina kuiswa mukuongorora. dhishi rine silicone rabha (Sylgard, Dow Inc) vachishandisa tsono yeMinutien (Fine Science Tools) kuti ione chiedza chepamusoro.Kugovera uye pateni yemasero akachinjwa akasimbiswa nemifananidzo yepamberi pachishandiswa Nikon maikorosikopu (SMZ1500) ine DigiLite kamera uye TCapture software (Tucsen Photonics, China). hupamhi hwetrachea), nehurefu hwese hwetrachea hwakafananidzirwa nhanho-ne-nhanho, kuve nechokwadi chekupindirana kwakakwana pakati pemufananidzo wega wega kuti ubvumire mufananidzo "kusona".Mifananidzo kubva mugwara rega rega rakabva raunganidzwa kuita mufananidzo umwechete unoshandiswa uchishandisa Image Composite Editor v2.0.3 (Microsoft Research) utilizing planar motion algorithm. automated MATLAB script (R2020a, MathWorks) sezvakatsanangurwa kare, uchishandisa marongero e0.35 0.15, uye Value <0.7.Nekutsvaga ma contours ematishu, mask yakagadzirwa nemaoko muGIMP v2.10.24 kuti ione chero nzvimbo yenhema yekuona nzvimbo yekunze kwekombiki tracheal tissue.Nzvimbo dzakasvibiswa kubva kumifananidzo yose yakasanganiswa kubva kumhuka imwe neimwe yakapfupiswa kuti ibudise nzvimbo yakasvibiswa yemhuka iyoyo.Nzvimbo yakasvibiswa yakabva yakamurwa nenzvimbo yose yemasiki kuti ibudise nzvimbo yakagadzikana.
Trachea imwe neimwe yakaiswa muparafini uye zvikamu zve 5 μm zvakatemwa. Zvikamu zvakasvibiswa ne neutral fast red for 5 min uye mifananidzo yakawanikwa uchishandisa Nikon Eclipse E400 microscope, DS-Fi3 kamera uye NIS element capture software (version 5.20.00).
Zvose zvinongororwa zvenhamba zvakaitwa muGraphPad Prism v9 (GraphPad Software, Inc.) .Kukosha kwenhamba kwakaiswa pa p ≤ 0.05.Normality yakasimbiswa uchishandisa Shapiro-Wilk test, uye kusiyana kweLacZ staining kwakaongororwa uchishandisa unpaired t-test.
MaParamende matanhatu anotsanangurwa muTebhenekeri 1 akaongororwa achishandisa PCXI, uye kuonekwa kunotsanangurwa muTebhu 2.Mapiresi maviri epolystyrene (MP1 neMP2; 18 μm uye 0.25 μm, maererano) akanga asingaonekwi pasi pePCXI, asi mamwe masampuli aizivikanwa (mienzaniso inoratidzwa muFigure% 3; 0.25 μm uye 0.9 μm, zvichitevedzana) zviri kuoneka zvisingaite.Kunyangwe iine zvimwe zvidiki-diki zvakaedzwa, MP5 (98% Fe3O4; 0.25 μm) ndiyo yainyanya kududzirwa.Chigadzirwa cheCombiMag MP6 chakaoma kuona.Muzviitiko zvese, kugona kwedu kuona MP nekuisa magineti kumashure kwakakwidziridzwa zvakanyanya. capillary.Apo magineti akasimuka kubva kune capillary, zvimedu zvakatambanudzwa mumatambo akareba, asi sezvo magineti aiswedera pedyo uye simba remagineti richiwedzera, tambo dzechidimbu dzakapfupika apo zvidimbu zvakatamira pamusoro pepamusoro pe capillary (ona Supplementary Video S1: MP4), ichiwedzera The particle density of the surface, simba remagineti rinobviswa kubva pamunda. inodzikira uye nhengo dzeMP dzinorongazve kuita tambo refu dzinobva kumusoro kwecapillary (ona Supplementary Video S2:MP4) Mushure mekunge magineti amira kufamba, zvimedu zvinoramba zvichifamba kwenguva pfupi mushure mekunge zvasvika panzvimbo yakaenzana.Apo MP inofamba ichienda uye ichienda kubva kumusoro kwecapillary, iyo inowanzoita magineti debrig particle.
Kuonekwa kweMP pasi pePCXI kunosiyana zvakanyanya pakati pemasamples.(a) MP3, (b) MP4, (c) MP5 uye (d) MP6.Mifananidzo yose yakaratidzwa pano yakatorwa nemagneti inowanikwa inenge 10 mm zvakananga pamusoro pecapillary.Madenderedzwa makuru anooneka mabhuru emhepo akavharirwa mumacapillaries, achiratidza zvakajeka dema uye white imaging edge ine mutsara webhokisi dzvuku mutsauko webhokisi tsvuku. magnification.Cherechedza kuti madhayamita ezvirongwa zvemagineti munhamba dzese haafanire kuyera uye akakura zvakapetwa ka100 pane zvaratidzwa.
Sezvo magineti achishandurwa kuruboshwe uye kurudyi pamwe chete nechepamusoro pe capillary, kona yeMP tambo inoshanduka kuti ienderane nemagnetti (ona Mufananidzo 6), nokudaro delineating mitsara yemagineti.Kune MP3-5, mushure mokunge chord yasvika pachikumbaridzo kona, zvikamu zvinodhonzwa pamwe chete nechepamusoro pe capillary. Supplementary Vhidhiyo S3:MP5).Izvi zvinonyanya kuoneka kana kufungidzira pedyo necapillary end, izvo zvinoita kuti MPs vaunganidze uye vanyatsoisa pfungwa pane fluid-air interface.Particles muMP6, iyo yainyanya kuoma kuona kupfuura MP3-5, haina kudhonzwa sezvo magineti yaifamba necapillary, asi MP tambo inosiya chidimbu chechikamu chechikamu chechikamu chekuona Video. S4: MP6) .Pane dzimwe nguva, apo magineti akaiswa akaderedzwa nekufambisa magineti kure kure nenzvimbo yekufungidzira, chero maMP akasara akadzika zvishoma nezvishoma kusvika pasi pechubhu negiravhiti achisara mutambo (ona Supplementary Video S5: MP3).
Kona yetambo yeMP inoshanduka sezvo magineti achiturikirwa kurudyi pamusoro pecapillary.(a) MP3, (b) MP4, (c) MP5 uye (d) MP6. Bhokisi dzvuku rine magnification anowedzera musiyano.Cherechedza kuti mavhidhiyo ekuwedzera anodzidzisa sezvo anoratidza akakosha maumbirwo ezvikamu uye ruzivo rwakasimba rusingagone kuratidzwa mumifananidzo yakamira iyi.
Miedzo yedu yakaratidza kuti kufambisa magineti zvishoma nezvishoma kumashure uye kumashure pamwe netrachea kunobatsira kutaridzika kweMP mumamiriro ekufamba kwakaoma mu vivo.In vivo yekuongorora haina kuitwa se polystyrene beads (MP1 neMP2) yaisaonekwa mu capillary. Imwe neimwe yeMPs ina dzakasara dzakaedzwa mu vivo nemagneti refu axis configured pamusoro pe 30 axis pamusoro pemakona e-trachever. Figure 2b ne3a), sezvo izvi zvakakonzera kuti pave nengetani dzeMP dzakareba uye dzakanyanya kushanda kupfuura magineti configuration yapera.MP3, MP4 uye MP6 hazvina kuonekwa mutrachea yemhuka chero ipi zvayo mhenyu.Apo nzira yemhepo yemakonzo yakafananidzirwa mushure mokunge mhuka dzaurayiwa nemunhu, zvimedu zvakaramba zvisingaoneki kunyange apo imwe vhoriyamu yakawedzerwa kushandisa simbi uye oxide yepikisi yaionekwa chete. saka yakashandiswa kuongorora uye kuratidza in vivo maitiro eMP.
Kuisa magineti pamusoro petrachea panguva yekuendesa kweMP kwakaguma nevakawanda, asi kwete vose, MPs vakaiswa munzvimbo yekuona.Particles inopinda mu trachea inonyatsoonekwa mumhuka dzakabayirwa nevanhu.Figure 7 uye Supplementary Video S6: MP5 inoratidza kukurumidza kubatwa kwemagineti uye kurongeka kwezvimedu pamusoro pepamusoro pe ventral inotangisa iyo trachea inotungamirirwa ne trachea, inogona kutungamirira kune trachea. vachitsvaga zvakanyanya padivi petrachea mushure mekutumira MP, mamwe maMP akawanikwa ari pedyo necarina, zvichiratidza kuti simba remagineti rakanga risingakwanisi kuunganidza nekuchengetedza maMPs ese, sezvo aiendeswa kuburikidza nenharaunda yesimba remagineti panguva yekuyerera kwemvura.
Mifananidzo kubva (a) pamberi uye (b) mushure mekutumirwa kweMP5 mutrachea yegonzo richangobva kuburitswa rine magineti yakamira zvakananga pamusoro penzvimbo yekufungidzira. Nzvimbo ine mufananidzo iri pakati pemarin'i maviri ecartilage. MP isati yatumirwa, pane imwe mvura munzira yemhepo. Bhokisi dzvuku rine kukwidziridzwa-kunakisisa. Iyi mifananidzo inobva muvhidhiyo ye5MP inoratidzwa.
Kushandura magineti pamwe chete ne trachea mu vivo kwakaita kuti MP chain ichinje angle mukati memhepo yemhepo nenzira yakafanana neinooneka mu capillaries (ona Figure 8 uye Supplementary Video S7: MP5) .Zvisinei, mukudzidza kwedu, MPs yaisakwanisa kudhonzwa pamusoro penzira yemhepo mhenyu sezvavaigona necapillaries.Mune dzimwe nguva inoenda kuruboshwe, iyo ketani yekurudyi seMP. zvakare akawana kuti tambo yechidimbu inoratidzika kushandura hudzamu hwemvura yepamusoro kana magineti ichifambiswa kureba parutivi rwetrachea, uye inowedzera apo magineti inofambiswa zvakananga kumusoro uye tambo yechidimbu ichitenderedzwa ichienda panzvimbo yakamira (ona Supplementary Video S7). : MP5 pa 0:09, pasi kurudyi).Chimiro chechimiro chekufamba chakashanduka apo magineti yakashandurudzwa kumusoro kwetrachea nechemberi (ndiko kuti, kuruboshwe kana kurudyi kwemhuka pane kureba kwetrachea).Zvimedu zvakanga zvichiri kuoneka zvakajeka sezvazvaifamba, asi magineti payakabviswa mutrachea, matipi echikamu cheVhidhiyo chinotanga kuoneka pa8 Spplement: 0:08).Izvi zvinofambirana nemaitiro eMP atakaona pasi pesimba remagineti rinoiswa mugomba regirazi.
Mienzaniso yemifananidzo inoratidza MP5 mutrachea yembeva mhenyu.(a) Magineti inoshandiswa kutora mifananidzo kumusoro nekuruboshwe kwetrachea, ipapo (b) mushure mekunge magineti yaendeswa kurudyi.Bhokisi dzvuku rine kukwidziridzwa kunonakisa.Mifananidzo iyi inobva muvhidhiyo inoratidzwa muSupplementary Video S7:MP5.
Apo matanda maviri acho akagadziridzwa kumativi ekuchamhembe-kumaodzanyemba kumusoro uye pasi petrachea (kureva kukwezva; Fig. 3b), maMP chords akaonekwa kwenguva refu uye akanga ari padivi pechitsiko chetrachea panzvimbo pepamusoro pemvura (ona Supplementary Video S9: MP5) .Zvisinei, kuwanda kwepamusoro kwezvikamu, kwete pane imwe nzvimbo yakabatwa mushure mekutsvaga kwemvura (trachea). kuendesa pakashandiswa maviri-magineti mudziyo, izvo zvinowanzoitika kana imwe-magineti mudziyo inoshandiswa.Zvino apo imwe magineti yakagadziridzwa kuti idzore matanda akadzoserwa (Fig. 3c), nhamba yezvikamu zvinoonekwa mumunda wekuona haina kuratidzika kuwedzera mushure mekusununguka.Kugadzika kwezvose zviviri-magineti configurations kunonetsa nekuda kweiyo yepamusoro yekudhonza magineti yakashandurwa kuti iite magineti inoremekedzwa. single magnet parallel to the airway but passing through the airway at 90 degrees kuitira kuti mitsara yemunda yakayambuka tracheal wall orthogonally (Fig. 3d), orientation yakagadzirirwa kuona kana particle aggregation pamadziro emadziro aigona kuonekwa .Zvisinei, mukugadzirisa uku, pakanga pasina identifiable movement of MP accumulation, 3 magineti accumulation ose aya magineti. orientation configuration (Mufananidzo 3a) yakasarudzwa mu vivo gene carrier zvidzidzo.
Mhuka payaidzokororwa kufananidzirwa pakarepo mushure mekuuraya kwemunhu, kusavapo kwekuvhiringidza kufamba kwetishu kwaireva kuti mitsetse yakapfava uye mipfupi inogona kunzwisiswa mumunda wakajeka weinterchondral, "kuzununguka" zvinoenderana neshanduko yekufamba kwemagineti. Zvakadaro, haugone kuona zvakajeka kuvepo uye kufamba kweMP6 zvimedu.
Iyo LV-LacZ titer yaive 1.8 × 108 TU / ml, uye mushure me1: 1 kusanganiswa neCombiMag MP (MP6), mhuka dzakagamuchira 50 μl tracheal dose ye9 × 107 TU/ml LV motokari (kureva 4.5 × 106 TU/rat). .Muzvidzidzo izvi, panzvimbo yekushandura magineti panguva yebasa, takagadzirisa magineti mune imwe nzvimbo kuti tione kana LV transduction (a) inogona kuvandudzwa kana ichienzaniswa nevector delivery pasina magnetic field, uye (b) inogona kutariswa Airway masero anoendeswa kune magnetic target regions yemugwagwa wepamusoro wemhepo.
Kuvapo kwemagineti uye kushandiswa kweCombiMag yakasanganiswa neLV vectors haina kuratidzika kuva nemigumisiro yakaipa pahutano hwemhuka, sezvakaita chiyero chedu cheLV vector delivery protocol. Mifananidzo yepamberi yenharaunda yetracheal yakakonzerwa nekukanganiswa kwemagetsi (Supplementary Fig. 1) yakaratidza kuti pane mazinga akakwirira zvikuru ekufambisa muboka remhuka paibatwa neglyn9-MP . chiyero chebhuruu LacZ tsvina yaivepo muboka rekutonga (Fig. 9b) .Quantification of normalized X-Gal stained areas kwakaratidza kuti kutungamirirwa kweLV-MP muhupo hwemagineti kwakagadzira inenge 6-fold kunatsiridza (Fig. 9c).
Muenzaniso mifananidzo inoumbwa inoratidza tracheal transduction neLV-MP (a) pamberi pesimba remagineti uye (b) pasina magineti.(c) Statistically yakakosha kunatsiridza munzvimbo yakajairwa yeLacZ transduction area mukati metrachea kana uchishandisa magineti (*p = 0.029, t-test, n = 3 paboka, zvinoreva ± SEM).
Neutral inokurumidza tsvuku-yakasvibiswa zvikamu (muenzaniso unoratidzwa muSupplementary Fig. 2) yakaratidza masero ane LacZ-akasvibiswa aripo nenzira yakafanana uye nzvimbo sezvakambotaurwa.
Dambudziko guru remhepo yejene therapy rinoramba riri iro rakarurama renzvimbo yezvimedu zvinotakura kumatunhu anofarira uye kuwana mazinga akakwirira ekufambisa zvakanaka mumapapu anofamba muhupo hwemhepo inoyerera uye inoshingaira mucus clearance.Kune LV vatakuri vakagadzirirwa kurapa CF airway chirwere, kuwedzera nguva yekugara kwezvimedu zvinotakura mukati mekufambisa nzira yemhepo yave iri nguva yekushandiswa kwemagineti kusvika panguva ino. kunatsiridza transduction kune zvakanakira kana zvichienzaniswa nedzimwe nzira dzekutumira magene dzakadai se electroporation, sezvo inogona kusanganisa kuve nyore, kudhura-kubudirira, kuburitsa nzvimbo, kuwedzera kushanda zvakanaka, uye kupfupika incubation nguva, uye pamwe diki mutakuri dose10.Zvisinei, iyo in vivo deposition uye hunhu hwemagineti mumigwagwa yemhepo pasi pesimba rekunze remagineti masimba haana kumbobvira atsanangurwa nzira ye feasi yekutaura kweiyo feasi. mazinga mumhepo isina kusimba yekurarama.
Yedu in vitro synchrotron PCXI miyedzo yakaratidza kuti zvidimbu zvese zvatakaedza, kunze kwepolystyrene MP, zvaionekwa mukumisikidzwa kwekufungidzira kwatakashandisa.Muhupo hwemagnetic field, MPs inogadzira tambo dzine urefu hune hukama nechikamu chemhando uye simba remagineti (kureva kuswedera uye kufamba kwemagineti) .Sezvinoratidzwa mumufananidzo wegineti inogadzirwa nemunhu mumwe nomumwe, tinocherechedza chidimbu chegineti. inducing its own local magnetic field.Idzi minda dzakaparadzana dzinokonzera zvimwe zvimedu zvakafanana kuti zviunganidze uye zvibatane, neboka retambo-sekufamba-famba nekuda kwemasimba emunharaunda kubva kune inoyevedza uye inosembura masimba ezvimwe zvikamu.
Schematic display (a,b) particle trains inogadzirwa mukati memvura-yakazadzwa capillaries uye (c,d) air-filled trachea.Ziva kuti capillaries uye trachea hazvikweverwi kusvika pachiyero.Panel (a) inewo tsanangudzo yeMP, iyo ine Fe3O4 zvikamu zvakarongwa mumatambo.
Apo magineti akatamiswa pamusoro pe capillary, kona ye particle tambo yakasvika pachikumbaridzo chakakomba cheMP3-5 ine Fe3O4, mushure meiyo tambo yechidimbu haina kuramba yakagara panzvimbo yepakutanga, asi yakatama pamusoro pepamusoro kune imwe nzvimbo.magnet.Ichi chiitiko chinogona kuitika nokuti girazi capillary pamusoro inotsvedzerera zvakakwana kubvumira kufamba uku kuti kuitike. twumedu twaive tudiki, twaive nemabhachi akasiyana-siyana kana machaji epamusoro, kana kuti mvura inotakura zvinhu yakakanganisa kugona kwavo kufamba.Musiyano wemufananidzo wezvikamu zveCombiMag haunawo simba, zvichiratidza kuti mvura nezvidimbu zvinogona kunge zviine densities zvakafanana uye naizvozvo hazvifambidzane zviri nyore. Zvimedu zvinogonawo kunamira kana magineti ikafamba zvakanyanya, zvichiratidza kuti simba remagineti rinogara richikunda. Hazvishamisi kuti simba remagineti uye chinhambwe chiri pakati pemagineti nenzvimbo yainotarirwa yakakosha.Kutorwa pamwe chete, mhinduro idzi dzinoratidzawo kuti, nepo magineti achigona kubata maMP akawanda anoyerera nenharaunda yakatariswa, hazvigoneke kuti magineti anogona kuvimbwa nawo kuti afambise zvimedu zveCombiMag pamwe nepamusoro petrachea. Naizvozvo, tinogumisa kuti mu vivo magineti anofanirwa kushandisa magineti chaiwo nharaunda yeLV-MP. muti.
Kana zvimedu zvinounzwa mumuviri, zvinonetsa kuziva mumamiriro ezvinhu emuviri wakaoma unofamba, asi kukwanisa kuvaona kwakasimudzirwa nekushandura magineti yakananga pamusoro petrachea "kuzungunusa" tambo dzeMP.Kunyange zvazvo kufungidzira mhenyu kuchibvira, zviri nyore kuona kufamba kwechidimbu kana mhuka yave ichinge yave yakafa pamusoro pemagineti epamusoro-soro apo magineti aya akaurayiwa. mamwe mapeji aiwanzowanikwa achienderera mberi netrachea.Kusiyana nezvidzidzo zve in vitro, zvimedu hazvigoni kukweverwa pamwe chete netrachea nekushandura magineti.Izvi zvakawanikwa zvinopindirana nemabatiro anoita mucus inoputira pamusoro petrachea inowanzogadzirisa zvimedu zvinoputirwa, zvichivabata mumucheka uye pashure pacho kucheneswa nemucociliary clearance mechanism.
Isu takafungidzira kuti kushandiswa kwemagineti ekukwezva kumusoro uye pasi petrachea (Fig. 3b) inogona kuguma kune imwe yunifomu yemagineti, panzvimbo yemagnetic field yakanyanyisa pane imwe nguva, iyo inogona kutungamirira kune imwe yunifomu kugoverwa kwezvikamu.Zvisinei, chidzidzo chedu chepakutanga hachina kuwana uchapupu hwakajeka hunotsigira iyi hypothesis.Saizvozvowo, kugadzirisa magineti kunokonzera 3 repel . zvimwe particle deposition in the imaged area.Izvi zviviri zvakawanikwa zvinoratidza kuti maviri-magineti setup haina zvakanyanya kuvandudza kudzora kwenzvimbo MP inonangwa, uye kuti zvinoguma simba remagineti masimba akaoma kugadzirisa, zvichiita kuti nzira iyi isashande.Similarly, orienting magineti kumusoro uye kuburikidza trachea (Fig. 3d) uyewo haana kuwedzera nhamba particles particles anochengeterwa mhedzisiro yemifananidzo iyi inogona kuchengetwa mumemero yemifananidzo iyi inogona kuchengetwa. mune yakaderera magineti simba mukati medeposition area.Naizvozvo, iyo imwe chete 30-degree angle magineti configuration (Mufananidzo 3a) inoonekwa senzira iri nyore uye inoshanda kwazvo yekuedza mu vivo.
Chidzidzo cheLV-MP chakaratidza kuti kana maVector eLV akabatanidzwa neCombiMag uye akaunzwa mushure mekuvhiringidzika kwemuviri pamberi pemagineti, mazinga ekuchinjisa akawedzera zvakanyanya mu trachea kana ichienzaniswa nekudzora.Kubva pane synchrotron imaging zvidzidzo uye nemhedzisiro yeLacZ, iyo magineti yakange yakakwanisa kuchengetedza LV mukati metrachea uye kuderedza zvidimbu zvakadzika mukati meiyo yakadzika chinangwa chevector. inogona kutungamirira kuhutano hwepamusoro paunenge uchideredza kuendeswa kwemazita, kunze kwechinangwa chekushandura, kuvhiringidza uye kudzivirira zvirwere, uye gene carrier cost.Importantly, maererano nemugadziri, CombiMag inogona kushandiswa pamwe chete nedzimwe nzira dzekutumira majeni, kusanganisira nemamwe mavhairasi (akadai seAAV) uye nucleic acids.