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Ii-nanoparticles ze-insulin (ii-NP) ezinomthamo ophezulu wokulayisha zifumene ukusetyenziswa okwahlukeneyo kwiindlela ezahlukeneyo zokulinganisa. Lo msebenzi ujolise ekuvavanyeni impembelelo yeenkqubo zokomisa ngokukhenkceza kunye nokomisa ngokutshiza kwisakhiwo se-nanoparticles ze-chitosan ezifakwe kwi-insulin, kunye ne-mannitol okanye ngaphandle kwayo njenge-cryoprotectant. Sikwavavanye umgangatho wezi nanoparticles ngokuzinyibilikisa kwakhona. Ngaphambi kokuphelelwa ngamanzi emzimbeni, ubungakanani be-particles ze-chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles buphuculwe baba yi-318 nm, i-PDI yayiyi-0.18, ukusebenza kakuhle kwe-encapsulation yayiyi-99.4%, kwaye umthwalo wawuyi-25.01%. Emva kokulungiswa kwakhona, zonke ii-nanoparticles, ngaphandle kwezo zenziwe ngendlela yokukhenkceza ngaphandle kokusebenzisa i-mannitol, zigcine isakhiwo sazo se-particle esingqukuva. Xa kuthelekiswa nee-nanoparticles ezine-mannitol eziye zaphelelwa ngamanzi yi-spray, ii-nanoparticles ezomileyo ezingena-mannitol nazo zibonise ubungakanani be-particles obuncinci (376 nm) kunye nomxholo ophezulu wokulayisha (25.02%). ngesantya esifanayo sokugquma (98.7%) kunye ne-PDI (0.20) ngeendlela zokomisa okanye zokuqandisa. Ii-nanoparticles ezomisiweyo ngokufutha ngaphandle kwe-mannitol nazo zikhokelele ekukhutshweni kwe-insulin ngokukhawuleza kunye nokusebenza kakuhle kokuthathwa kweeseli. Lo msebenzi ubonisa ukuba ukomisa ngokufutha kunokufutha ii-nanoparticles ze-insulin ngaphandle kwesidingo se-cryoprotectants xa kuthelekiswa neendlela eziqhelekileyo zokomisa ngokuqandisa, okudala amandla amakhulu okulayisha, iimfuno zokongeza eziphantsi kunye neendleko zokusebenza inzuzo enkulu.
Ukususela ekufumanekeni kwayo ngo-1922, 2, 3, i-insulin kunye nemveliso yayo yamayeza isindise ubomi bezigulane ezinesifo seswekile sohlobo loku-1 (T1DM) kunye nesifo seswekile sohlobo lwesibini (T1DM). Nangona kunjalo, ngenxa yeempawu zayo njengeprotheyini enobunzima obukhulu bemolekyuli, i-insulin ihlanganiswa ngokulula, yahlulwe zii-enzymes zeproteolytic, kwaye isuswe ngumphumo wokuqala. Abantu abafunyaniswe benesifo seswekile sohlobo loku-1 bafuna inaliti ye-insulin ubomi babo bonke. Izigulane ezininzi ezifunyaniswe zinesifo seswekile sohlobo lwesibini ekuqaleni zikwafuna inaliti ye-insulin yexesha elide. Inaliti ye-insulin yemihla ngemihla ngumthombo omkhulu wentlungu kunye nokungonwabi kwaba bantu, kunye nemiphumo emibi kwimpilo yengqondo. Ngenxa yoko, ezinye iindlela zolawulo lwe-insulin ezibangela ukungonwabi okuncinci, njengolawulo lwe-insulin yomlomo, zifundwa ngokubanzi5 njengoko zinamandla okubuyisela umgangatho wobomi babantu abamalunga ne-5 yeebhiliyoni abanesifo seswekile kwihlabathi liphela.
Itekhnoloji yeNanoparticle ibonelele ngenkqubela phambili ebalulekileyo kwimizamo yokuthatha i-insulin yomlomo4,6,7.Eyona igquma ngempumelelo kwaye ikhusele i-insulin ekuwohlokeni ukuze ihanjiswe kwiindawo ezithile zomzimba.Nangona kunjalo, ukusetyenziswa kwe-nanoparticle formulations kunemida eliqela, ikakhulu ngenxa yeengxaki zozinzo lwe-particle suspensions.Eminye i-coggregation inokwenzeka ngexesha lokugcina, nto leyo enciphisa ukufumaneka kwe-nanoparticles ezifakwe kwi-insulin8.Ukongeza, uzinzo lweekhemikhali ze-polymer matrix ye-nanoparticles kunye ne-insulin kufuneka ziqwalaselwe ukuqinisekisa uzinzo lwe-insulin nanoparticles (NPs).Okwangoku, itekhnoloji yokomisa ngokukhenkceza yeyona ndlela ilungileyo yokwenza i-NPs ezizinzileyo ngelixa ithintela utshintsho olungafunekiyo ngexesha lokugcina9.
Nangona kunjalo, ukomisa ngokukhenkceza kufuna ukongezwa kwee-cryoprotectants ukuthintela ulwakhiwo oluyindilinga lwee-NPs ekuchatshazelweni luxinzelelo loomatshini lweekristale zomkhenkce. Oku kunciphisa kakhulu umthwalo wee-nanoparticles ze-insulin emva kwe-lyophilization, njengoko i-cryoprotectant ithatha uninzi lomlinganiselo wobunzima. Ke ngoko, ii-insulin NPs eziveliswayo zihlala zifunyaniswa zingafanelekanga ekwenzeni iifomyula zomgubo owomileyo, ezifana neepilisi zomlomo kunye neefilimu zomlomo, ngenxa yesidingo senani elikhulu lee-nanoparticles ezomileyo ukuze kufezekiswe ifestile yonyango ye-insulin.
Ukomisa ngokutshiza yinkqubo eyaziwayo nengabizi kakhulu yemizi-mveliso yokuvelisa iipowders ezomileyo ezivela kwizigaba zolwelo kwishishini lamayeza10,11. Ulawulo lwenkqubo yokwakheka kwamasuntswana luvumela ukufakwa ngokufanelekileyo kweekhompawundi ezininzi ezisebenzayo 12, 13. Ngaphezu koko, iye yaba yindlela esebenzayo yokulungiselela iiproteni ezifakwe ngaphakathi ukuze zisetyenziswe ngomlomo. Ngexesha lokomisa ngokutshiza, amanzi ayaphela ngokukhawuleza, nto leyo enceda ukugcina ubushushu besiseko samasuntswana buphantsi11,14, nto leyo evumela ukusetyenziswa kwawo ukufaka izinto ezinobuzwelo kubushushu. Ngaphambi kokuba ukomise ngokutshiza, izinto zokugquma kufuneka zihlanganiswe ngokupheleleyo nesisombululo esinezithako ezifakwe ngaphakathi11,14. Ngokungafaniyo nokomisa ngokukhenkceza, ukufakwa ngokutshiza ngaphambi kokugquma ngokutshiza kuphucula ukusebenza ngokugquma ngexesha lokuphelelwa ngamanzi emzimbeni. Ekubeni inkqubo yokugquma ngokutshiza ngokutshiza ayifuni zithinteli zintsholongwane, ukomisa ngokutshiza kungasetyenziselwa ukuvelisa ii-NPs ezomileyo ezinomthamo ophezulu wokulayisha.
Olu phononongo luxela ukuveliswa kwee-NPs ezine-insulin ngokudibanisa i-chitosan kunye ne-sodium tripolyphosphate kusetyenziswa indlela ye-ion gel. I-Ion gelation yindlela yokulungiselela evumela ukuveliswa kwee-nanoparticles ngokusebenzisana kwe-electrostatic phakathi kweentlobo ezimbini okanye ngaphezulu ze-ionic phantsi kweemeko ezithile. Zombini iindlela zokuqandisa ngokukhenkceza kunye nezokomisa ngokutshiza zisetyenzisiwe ukunyibilikisa i-chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles. Emva kokuphelelwa ngamanzi emzimbeni, imo yazo yahlalutywa yi-SEM. Amandla azo okuphinda ahlanganiswe avavanywe ngokulinganisa ukusasazwa kobungakanani bazo, itshaja yomphezulu, i-PDI, ukusebenza kakuhle kwe-encapsulation, kunye nomxholo wokulayisha. Umgangatho wee-nanoparticles ezinyibilikisiweyo eziveliswe ngeendlela ezahlukeneyo zokuphelelwa ngamanzi emzimbeni nawo wavavanywa ngokuthelekisa ukhuseleko lwazo lwe-insulin, indlela yokukhulula, kunye nokusebenza kakuhle kokuthathwa kweeseli.
I-pH yesisombululo esixutyiweyo kunye nomlinganiselo we-chitosan kunye ne-insulin zizinto ezimbini ezibalulekileyo ezichaphazela ubungakanani be-particle kunye nokusebenza kakuhle kwe-encapsulation (EE) ye-NPs yokugqibela, njengoko zichaphazela ngokuthe ngqo inkqubo ye-ionotropic gelation. I-pH yesisombululo esixutyiweyo ibonakaliswe ukuba inxulumene kakhulu nobukhulu be-particle kunye nokusebenza kakuhle kwe-encapsulation (Umzobo 1a). Njengoko kubonisiwe kumzobo 1a, njengoko i-pH inyuka ukusuka kwi-4.0 ukuya kwi-6.0, ubungakanani be-particle obuqhelekileyo (nm) behla kwaye i-EE inyuke kakhulu, ngelixa xa i-pH inyuka ukuya kwi-6.5, ubungakanani be-particle obuqhelekileyo buqale ukwanda kwaye i-EE ayizange itshintshe. Njengoko umlinganiselo we-chitosan kwi-insulin uyanda, ubungakanani be-particle obuqhelekileyo buyanda. Ngaphezu koko, akukho tshintsho kwi-EE olubonwe xa ii-nanoparticles zilungiswa kumlinganiselo wobunzima be-chitosan/insulin ongaphezulu kwe-2.5:1 (w/w) (Umzobo 1b). Ke ngoko, iimeko ezifanelekileyo zokulungiselela kolu phononongo (pH 6.0, umlinganiselo wobunzima be-chitosan/insulin we-2.5:1) zisetyenzisiwe lungiselela ii-nanoparticles ezifakwe kwi-insulin ukuze ufundisise ngakumbi. Phantsi kwale meko yokulungiselela, ubungakanani obuqhelekileyo bee-nanoparticles ze-insulin bulungiselelwe ukuba bube yi-318 nm (Umzobo 1c), i-PDI yayiyi-0.18, ukusebenza kakuhle kokubethela yayiyi-99.4%, amandla e-zeta yayiyi-9.8 mv, kwaye umthwalo we-insulin yayiyi-25.01% (m/m ). Ngokusekelwe kwiziphumo ze-transmission electron microscopy (TEM), ii-nanoparticles ezilungiselelweyo zaziphantse zibe yi-spherical kwaye zi-discrete ngobukhulu obufanayo (Umzobo 1d).
Ukulungiswa kweeparameter ze-nanoparticles ze-insulin: (a) impembelelo ye-pH kwi-avareji ye-diameter kunye nokusebenza kakuhle kwe-encapsulation (EE) yee-nanoparticles ze-insulin (ezilungiselelwe kwi-5:1 mass ratio ye-chitosan kunye ne-insulin); (b) i-chitosan kunye nempembelelo yomlinganiselo wobunzima be-insulin kwi-avareji ye-avareji kunye nokusebenza kakuhle kwe-encapsulation (EE) yee-insulin NPs (ezilungiselelwe kwi-pH 6); (c) ukusasazwa kobungakanani bee-particles zee-nanoparticles ze-insulin ezilungiselelweyo; (d) i-TEM micrograph yee-insulin NPs ezilungiselelweyo.
Kuyaziwa ukuba i-chitosan yi-polyelectrolyte ebuthathaka ene-pKa ye-6.5. Itshajwa kakuhle kwi-acidic media kuba iqela layo eliphambili le-amino liprotonwa yi-hydrogen ions15. Ke ngoko, isetyenziswa njengesithwali ukufaka ii-macromolecules ezitshajwe kakubi. Kolu phononongo, i-chitosan isetyenziselwe ukufaka i-insulin ngenqaku le-isoelectric elingu-5.3. Ekubeni i-chitosan isetyenziswa njengezinto zokugquma, ngokonyuka kwenani layo, ubukhulu bomaleko wangaphandle wee-nanoparticles buyanda ngokufanayo, nto leyo ebangela ubungakanani obukhulu be-particles. Ukongeza, amanqanaba aphezulu e-chitosan anokufaka i-insulin engaphezulu. Kwimeko yethu, i-EE yayiphezulu xa umlinganiselo we-chitosan kunye ne-insulin ufikelele kwi-2.5:1, kwaye akukho tshintsho lubalulekileyo kwi-EE xa umlinganiselo wawuqhubeka usanda.
Ngaphandle komlinganiselo we-chitosan kunye ne-insulin, i-pH ikwadlale indima ebalulekileyo ekulungiseleleni i-NPs. UGan et al. 17 bafunde ngempembelelo ye-pH kubungakanani be-particle ye-chitosan nanoparticles. Bafumene ukwehla okuqhubekayo kubukhulu be-particle de i-pH ifikelele kwi-6.0, kwaye ukwanda okukhulu kubukhulu be-particle kwabonwa kwi-pH > 6.0, nto leyo ehambelana nokubona kwethu. Le nto ibangelwa kukuba xa i-pH isanda, i-molecule ye-insulin ifumana i-negative surface charge, ngaloo ndlela, ikhetha ukusebenzisana kwe-electrostatic kunye ne-chitosan/sodium tripolyphosphate (TPP) complex, nto leyo ebangela ubungakanani be-particle obuncinci kunye ne-EE ephezulu. Nangona kunjalo, xa i-pH yalungiswa kwi-6.5, amaqela e-amino kwi-chitosan asuswa, nto leyo ebangela ukuba i-chitosan igobe. Ke ngoko, i-pH ephezulu ibangela ukuba ii-amino ions zingavezwa kakhulu kwi-TPP kunye ne-insulin, nto leyo ebangela ukuba i-cross-linking ibe sezantsi, ubungakanani be-avareji yokugqibela ibe nkulu kunye ne-EE ephantsi.
Uhlalutyo lweempawu zemo ye-NPs ezomisiweyo ngokuqandisa kunye nezomisiweyo ngokufuthwa lunokukhokela ukukhethwa kweendlela ezingcono zokuphelelwa ngamanzi kunye nokwakheka komgubo. Indlela ekhethwayo kufuneka ibonelele ngozinzo lweziyobisi, imo efanayo yamasuntswana, umthwalo ophezulu wamayeza kunye nokunyibilika okuhle kwisisombululo sokuqala. Kolu phononongo, ukuze kuthelekiswe ngcono ezi ndlela zimbini, ii-insulin NPs ezine-1% ye-mannitol okanye ezingenayo zasetyenziswa ngexesha lokuphelelwa ngamanzi. I-Mannitol isetyenziswa njenge-bulking agent okanye i-cryoprotectant kwiifomyula ezahlukeneyo zomgubo owomileyo wokomisa ngokuqandisa kunye nokomisa ngokufuthwa. Kwi-lyophilized insulin nanoparticles ngaphandle kwe-mannitol, njengoko kubonisiwe kuMfanekiso 2a, isakhiwo somgubo esineembobo ezininzi esineendawo ezinkulu, ezingalinganiyo nezirhabaxa sabonwa phantsi kwe-scanning electron microscopy (SEM). Zimbalwa ii-particles ezingabonakaliyo ezifunyenwe kumgubo emva kokuphelelwa ngamanzi (Umzobo 2e). Ezi ziphumo zibonise ukuba uninzi lwee-NPs zabola ngexesha lokomisa ngokuqandisa ngaphandle kwe-cryoprotectant. Kwi-freeze-dryed kunye ne-spray-dryed insulin nanoparticles ezine-1% ye-mannitol, kwabonwa ii-spherical nanoparticles ezineendawo ezigudileyo (Umzobo 1). 2b,d,f,h).Ii-insulin nanoparticles ezitshiziweyo ngaphandle kwe-mannitol zahlala zingqukuva kodwa zingqukuva phezu komhlaba (Umzobo 2c).Iindawo ezingqukuva nezingqukuva zixutyushwa ngakumbi kwindlela yokukhululwa kunye novavanyo lokufunxwa kweeseli ngezantsi. Ngokusekelwe kwimbonakalo ebonakalayo yee-NP ezomileyo, zombini ii-NP ezomileyo ezitshiziweyo ngaphandle kwe-mannitol kunye nee-NP zomiswe ngokuqandisa kunye nezomiswe ngokutshiza nge-mannitol zivelise ii-NPs ezicolekileyo (Umzobo 2f,g,h).Okukhona indawo enkulu phakathi kweendawo zeesuntswana, kokukhona ukunyibilika kuphezulu kwaye ke ngoko izinga lokukhululwa liphezulu.
Imo yee-NPs ze-insulin eziphelelwe ngamanzi emzimbeni ezahlukeneyo: (a) Umfanekiso we-SEM wee-NPs ze-insulin ezifakwe i-lyophilized ngaphandle kwe-mannitol; (b) Umfanekiso we-SEM wee-NPs ze-insulin ezifakwe i-lyophilized ezine-mannitol; (c) ii-NPs ze-insulin ezomisiweyo ngaphandle kwe-mannitol Umfanekiso we-SEM we-; (d) Umfanekiso we-SEM wee-NPs ze-insulin ezifakwe i-spray-dryed nge-mannitol; (e) umfanekiso wee-NPs ze-insulin ezifakwe i-lyophilized ngaphandle kwe-mannitol; (f) umfanekiso wee-NPs ze-insulin ezifakwe i-lyophilized ezine-mannitol; (g) Umfanekiso wee-NPs ze-insulin ezomisiweyo ngaphandle kwe-mannitol; (h) umfanekiso wee-NPs ze-insulin ezomisiweyo ngaphandle kwe-mannitol.
Ngexesha lokomisa ngokukhenkceza, i-mannitol isebenza njenge-cryoprotectant, igcina ii-NP zikwimo engafaniyo kwaye ithintela umonakalo obangelwa ziikristale zomkhenkce19. Ngokwahlukileyo koko, akukho nyathelo lokukhenkceza ngexesha lokomisa ngokucheba. Ke ngoko i-mannitol ayifuneki kule ndlela. Enyanisweni, ii-NP ezomiswe ngokucheba ngaphandle kwe-mannitol zivelise ii-NP ezintle njengoko kuchaziwe ngaphambili. Nangona kunjalo, i-mannitol isenokusebenza njengesizalisi kwinkqubo yokomisa ngokucheba ukunika ii-NP isakhiwo esingqukuva20 (Umzobo 2d), esinceda ekufumaneni ukuziphatha okufanayo kokukhululwa kwee-NP ezifakwe ngaphakathi. Ukongeza, kuyacaca ukuba ezinye iisuntswana ezinkulu zinokufunyanwa kwi-NPs ze-insulin ezomiswe ngokukhenkceza kunye nezomiswe ngokucheba eziqulethe i-mannitol (Umzobo 2b, d), ezinokubangelwa kukuqokelelwa kwe-mannitol kwisiseko sesuntswana kunye ne-insulin efakwe ngaphakathi. Kumaleko weChitosan. Kubalulekile ukuqaphela ukuba kolu phononongo, ukuqinisekisa ukuba isakhiwo esingqukuva sihlala singaguquki emva kokuphelelwa ngamanzi emzimbeni, umlinganiselo wemannitol kunye ne-chitosan ugcinwa kwi-5:1, ukuze inani elikhulu le-filler likwazi ukwandisa ubungakanani be-particle ye-NPs ezomisiweyo.
I-Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy ibonakalise umxube we-insulin ekhululekileyo, i-chitosan, i-chitosan, i-TPP kunye ne-insulin. Zonke ii-NP eziphelelwe ngamanzi emzimbeni zibonakaliswe ngokusebenzisa i-FTIR-ATR spectroscopy. Okuphawulekayo kukuba, amandla e-band ye-1641, 1543 kunye ne-1412 cm-1 abonwe kwi-encapsulated NPs ezomiswe nge-mannitol kwaye kwi-NPs ezifakwe i-spray-dryed with and without mannitol (Umzobo 3). Njengoko bekuxeliwe ngaphambili, oku kunyuka kwamandla kunxulunyaniswa nokudibanisa phakathi kwe-chitosan, i-TPP kunye ne-insulin. Uphando lokunxibelelana phakathi kwe-chitosan kunye ne-insulin lubonise ukuba kwi-FTIR spectra ye-chitosan nanoparticles efakwe i-insulin, i-chitosan band idibene ne-insulin, inyusa amandla e-carbonyl (1641 cm-1) kunye ne-amine (1543 cm-1). Amaqela e-tripolyphosphate e-TPP adibene namaqela e-ammonium kwi-chitosan, enza ibhendi kwi-1412 cm-1.
Iispectra ze-FTIR-ATR ze-insulin yasimahla, i-chitosan, imixube ebonakalayo ye-chitosan/TPP/insulin kunye ne-NPs ezicocwe ngeendlela ezahlukeneyo.
Ngaphezu koko, ezi ziphumo zihambelana nezo ziboniswe kwi-SEM, ezibonise ukuba ii-NP ezifakwe kwiikhaphu zahlala zinjalo zombini xa zitshizwa kwaye zomiswa nge-mannitol, kodwa xa kungekho mannitol, kuphela ukomisa nge-spray okuvelisa amasuntswana afakwe kwiikhaphu. Ngokwahlukileyo koko, iziphumo ze-FTIR-ATR spectral ze-NPs ezifakwe kwiikhaphu ngaphandle kwe-mannitol zazifana kakhulu nomxube we-chitosan, TPP, kunye ne-insulin. Esi siphumo sibonisa ukuba amakhonkco aphakathi kwe-chitosan, TPP kunye ne-insulin awasekho kwi-NPs ezifakwe kwiikhaphu ngaphandle kwe-mannitol. Ulwakhiwo lwe-NPs lwatshatyalaliswa ngexesha lokomisa nge-freeze ngaphandle kwe-cryoprotectant, enokubonwa kwiziphumo ze-SEM (Umzobo 2a). Ngokusekelwe kwi-morphology kunye neziphumo ze-FTIR ze-insulin NPs eziphelelwe ngamanzi emzimbeni, kuphela ii-NPs ezifakwe ii-lyophilized, ezifakwe kwiikhaphu, kunye nee-mannitol-free ezisetyenzisiweyo kwiimvavanyo zokuhlaziya kunye nee-NPs ezingenayo i-mannitol ngenxa yokubola kwee-NPs ezingenayo i-mannitol ngexesha lokuphelelwa ngamanzi emzimbeni. xoxa.
Ukuphelelwa ngamanzi emzimbeni kusetyenziselwa ukugcina ixesha elide kunye nokuphinda kwenziwe ezinye iindlela. Amandla ee-NP ezomileyo okuphinda ziphinde zisebenze emva kokugcinwa abalulekile ekusetyenzisweni kwazo kwiindlela ezahlukeneyo ezifana neetafile kunye neefilimu. Siqaphele ukuba ubungakanani obuphakathi bee-NP ze-insulin ezomisiweyo ngokutshizwa xa kungekho mannitol bunyuke kancinci emva kokuphinda zisebenze. Kwelinye icala, ubungakanani bee-particle ze-nanoparticles ze-insulin ezomisiweyo ngokutshizwa nangokuqandisa kunye ne-mannitol bunyuke kakhulu (Itheyibhile 1). I-PDI kunye ne-EE azitshintshwanga kakhulu (p > 0.05) emva kokuphinda zihlanganiswe zonke ii-NP kolu phononongo (Itheyibhile 1). Esi siphumo sibonisa ukuba uninzi lwee-particles zahlala zinjalo emva kokuphinda zinyibilike. Nangona kunjalo, ukongezwa kwe-mannitol kubangele ukwehla okukhulu komthwalo we-insulin wee-nanoparticles ze-mannitol ezifakwe i-lyophilized kunye ne-spray-dryed (Itheyibhile 1). Ngokwahlukileyo koko, umxholo womthwalo we-insulin wee-NP ezifakwe i-spray ngaphandle kwe-mannitol wahlala ufana nangaphambili (Itheyibhile 1).
Kuyaziwa ukuba ukulayishwa kwee-nanoparticles kubaluleke kakhulu xa kusetyenziselwa iinjongo zokuhambisa amayeza. Kwi-NPs ezinemithwalo ephantsi, kufuneka izinto ezininzi kakhulu ukuze kufikelele kumda wonyango. Nangona kunjalo, i-viscosity ephezulu yaloo manqanaba aphezulu e-NP ikhokelela kwingxaki kunye nobunzima ekusetyenzisweni ngomlomo kunye neefomyula ezifakwa ngenaliti, ngokulandelelana 22. Ukongeza, ii-insulin NPs zingasetyenziselwa ukwenza iipilisi kunye ne-viscous biofilms23, 24, nto leyo efuna ukusetyenziswa kwee-NPs ezininzi kumanqanaba aphantsi okulayisha, nto leyo ebangela iipilisi ezinkulu kunye ne-biofilms ezityebileyo ezingafanelekanga ukusetyenziswa ngomlomo. Ke ngoko, ii-NPs eziphelelwe ngamanzi emzimbeni ezinomthwalo ophezulu we-insulin ziyathandeka kakhulu. Iziphumo zethu zibonisa ukuba umthwalo ophezulu we-insulin we-NPs ezifakwe nge-mannitol-free spray-dryed NPs unokubonelela ngeenzuzo ezininzi ezikhangayo kwezi ndlela zokuhambisa ezinye.
Zonke ii-NP eziphelelwe ngamanzi emzimbeni zigcinwe efrijini kangangeenyanga ezintathu. Iziphumo ze-SEM zibonise ukuba imo yazo zonke ii-NP eziphelelwe ngamanzi emzimbeni azitshintshanga kakhulu ngexesha lokugcina iinyanga ezintathu (Umzobo 4). Emva kokuphinda zifakwe emanzini, zonke ii-NP zibonise ukwehla okuncinci kwi-EE kwaye zakhupha malunga nesixa esincinci (~5%) se-insulin ngexesha lokugcina iinyanga ezintathu (Itheyibhile 2). Nangona kunjalo, ubungakanani obuqhelekileyo bee-nanoparticles zonke bunyukile. Ubungakanani bee-NPs ezifakwe i-spray-dryed ngaphandle kwe-mannitol bunyuke bafikelela kwi-525 nm, ngelixa ezo ze-NPs ezifakwe i-spray-dryed kunye ne-freeze-dryed kunye ne-mannitol zinyuke zaya kwi-872 kunye ne-921 nm, ngokulandelelana (Itheyibhile 2).
Imo yee-NP ezahlukeneyo ze-insulin eziphelelwe ngamanzi emzimbeni ezigcinwe iinyanga ezintathu: (a) Umfanekiso we-SEM wee-NP ze-insulin ezifakwe i-lyophilized ezine-mannitol; (b) Umfanekiso we-SEM wee-nanoparticles ze-insulin ezomisiweyo ngaphandle kwe-mannitol; (c) ngaphandle kwe-mannitol imifanekiso ye-SEM yee-NP ze-insulin ezomisiweyo ngokufuthwa.
Ngaphezu koko, kubonakale ukuhla kwee-precipitates kwi-insulin nanoparticles ezifakwe i-spray-dryed nge-mannitol kwaye zomiswe ngokukhenkcezisiweyo (Umzobo S2). Oku kunokubangelwa zii-particles ezinkulu ezingaxhomeki kakuhle emanzini. Zonke iziphumo ezingentla zibonisa ukuba indlela yokomisa i-spray inokukhusela ii-nanoparticles ze-insulin ekuphelelweni ngamanzi emzimbeni kwaye imithwalo ephezulu yee-nanoparticles ze-insulin inokufunyanwa ngaphandle kwezizalisi okanye ii-cryoprotectants.
Ukugcinwa kwe-insulin kuvavanyiwe kwi-pH = 2.5 medium nge-pepsin, trypsin, kunye ne-α-chymotrypsin ukubonisa amandla okukhusela e-NPs ngokuchasene nokugaya i-enzyme emva kokuphelelwa ngamanzi emzimbeni. Ukugcinwa kwe-insulin ye-NPs eziphelelwe ngamanzi emzimbeni kuthelekiswa nokwe-NPs ezisandula ukwenziwa, kwaye i-insulin ekhululekileyo yasetyenziswa njengolawulo olubi. Kolu phononongo, i-insulin ekhululekileyo ibonise ukususwa kwe-insulin ngokukhawuleza kwiiyure ezi-4 kuzo zonke iindlela ezintathu zonyango lwe-enzyme (Umzobo 5a–c). Ngokwahlukileyo koko, uvavanyo lokususwa kwe-insulin ye-NPs eziqandisiweyo nge-mannitol kunye ne-NPs ezitshiziweyo nge-spray-dryed kunye okanye ngaphandle kwe-mannitol lubonise ukhuseleko oluphezulu kakhulu lwezi NPs ngokuchasene nokugaya i-enzyme, olufana nolwe-insulin NPs ezisandula ukwenziwa (umfanekiso 1).5a-c). Ngoncedo lwee-nanoparticles kwi-pepsin, trypsin, kunye ne-α-chymotrypsin, ngaphezulu kwe-50%, 60%, kunye ne-75% ye-insulin inokukhuselwa kwiiyure ezi-4, ngokulandelanayo (Umzobo 5a–c). Olu buchule bokukhusela i-insulin bunokunyusa amathuba e-insulin ephezulu. ukufunxwa egazini25. Ezi ziphumo zibonisa ukuba ukomisa nge-spray kunye ne-mannitol okanye ngaphandle kwayo kunye nokomisa nge-freeze nge-mannitol kunokugcina amandla okukhusela i-insulin ee-NPs emva kokuphelelwa ngamanzi emzimbeni.
Ukukhusela nokukhulula i-insulin NPs eyomileyo: (a) ukukhuselwa kwe-insulin kwisisombululo se-pepsin; (b) ukukhuselwa kwe-insulin kwisisombululo se-trypsin; (c) ukukhuselwa kwe-insulin yisisombululo se-α-chymotrypsin; (d) Ukukhululwa kwe-NPs eyomileyo kwisisombululo se-pH = 2.5; (e) Ukukhululwa kwe-NPs eyomileyo kwisisombululo se-pH = 6.6; (f) Ukukhululwa kwe-NPs eyomileyo kwisisombululo se-pH = 7.0.
Ii-NPs ze-insulin eyomileyo ezisandula ukulungiswa nezakhiwe ngokutsha zafakwa kwii-buffers ezahlukeneyo (pH = 2.5, 6.6, 7.0) kwi-37 °C, zilinganisa imeko ye-pH yesisu, i-duodenum, kunye namathumbu amancinci aphezulu, ukuze kuhlolwe impembelelo ye-insulin ekuchaseni i-insulin. Ukuziphatha kokukhululwa kwiindawo ezahlukeneyo. Iqhekeza lendlela yokugaya ukutya. Kwi-pH = 2.5, ii-NPs ezifakwe i-insulin kunye nee-NPs ze-insulin eyomileyo ezinyibilikisiweyo zibonise ukukhululwa kokuqala kokuqhuma kwiyure yokuqala, kulandele ukukhululwa kancinci kwiiyure ezi-5 ezilandelayo (Umzobo 5d). Oku kukhululwa ngokukhawuleza ekuqaleni kusenokwenzeka ukuba kungenxa yokutsalwa ngokukhawuleza komphezulu kweemolekyuli zeprotheni ezingashukumi ngokupheleleyo kwisakhiwo sangaphakathi sesuntswana. Kwi-pH = 6.5, ii-NPs ezifakwe i-insulin kunye nee-NPs ze-insulin eyomileyo ezilungisiweyo zibonise ukukhululwa okugudileyo nokucothayo kwiiyure ezi-6, njengoko i-pH yesisombululo sovavanyo yayifana neyesisombululo esilungiselelwe ii-NPs (Umzobo 5e). Kwi-pH = 7, ii-NPs zazingazinzanga kwaye phantse zabola ngokupheleleyo kwiiyure ezimbini zokuqala (Umzobo 5f). Oku kungenxa yokuba ukususwa kwe-chitosan kwenzeka kwi-pH ephezulu, nto leyo ekhokelela kwinethiwekhi ye-polymer encinci kunye nokukhululwa kwe-insulin efakwe.
Ngaphezu koko, ii-insulin NPs ezomiswe ngespray ngaphandle kwe-mannitol zibonise iprofayili yokukhululwa ngokukhawuleza kunezinye ii-NPs eziphelelwe ngamanzi emzimbeni (Umzobo 5d–f). Njengoko kuchaziwe ngaphambili, ii-insulin NPs ezomiswe kwakhona ngaphandle kwe-mannitol zibonise ubungakanani obuncinci be-particle. Ii-particles ezincinci zibonelela ngendawo enkulu yomphezulu, ngoko ke uninzi lweyeza elifanelekileyo luya kuba kufutshane okanye lukufutshane nomphezulu we-particle, nto leyo ekhokelela ekukhutshweni kweyeza ngokukhawuleza26.
Uphando malunga nokuba ii-NPs zinobuthi kangakanani na luphandwe ngovavanyo lwe-MTT. Njengoko kubonisiwe kuMfanekiso S4, zonke ii-NPs eziphelelwe ngamanzi emzimbeni zifunyenwe zingenampembelelo ibalulekileyo ekusebenzeni kweeseli kumanqanaba angama-50–500 μg/ml, nto leyo ebonisa ukuba zonke ii-NPs eziphelelwe ngamanzi emzimbeni zingasetyenziswa ngokukhuselekileyo ukufikelela kwifestile yonyango.
Isibindi sisitho esiphambili apho i-insulin isebenzisa khona imisebenzi yayo yomzimba. Iiseli zeHepG2 ziluluhlu lweeseli zehepatoma zomntu olusetyenziswa rhoqo njengemodeli yokufunxa i-hepatocyte kwi-in vitro. Apha, iiseli zeHepG2 zisetyenziselwe ukuvavanya ukufunxa kweseli kwee-NPs ezimanzi kusetyenziswa iindlela zokuziqandisa ngokukhenkceza kunye nezomisa ngokufutha. Ukufunxa kweseli nge-confocal laser scanning kusetyenziswa i-flow cytometry kunye nombono emva kweeyure ezininzi zokufunxa nge-free FITC insulin kuxinzelelo lwe-25 μg/mL, ii-NPs ze-FITC ezisandula ukulungiswa kunye nee-NPs ze-FITC ezimanzi kumgangatho olinganayo we-insulin. Kwenziwe ukujongwa kwe-Quantitative microscopy (CLSM). Ii-NPs ze-Lyophilized ngaphandle kwe-mannitol zatshatyalaliswa ngexesha lokuphelelwa ngamanzi emzimbeni kwaye azizange zihlolwe kolu vavanyo. Ubunzulu be-intracellular fluorescence yee-NPs ezisandula ukulungiswa ezifakwe i-insulin, ii-NPs ze-lyophilized ezine-mannitol, kunye nee-NPs ezomileyo ezifakwe i-spray kunye ne-mannitol engenazo (Umzobo 6a) zaziyi-4.3, 2.6, 2.4, kunye Ingaphezulu ngokuphindwe ka-4.1 kunezo zikhululekileyo. Iqela le-FITC-insulin, ngokulandelelanayo (Umzobo 6b). Ezi ziphumo zibonisa ukuba i-insulin efakwe kwiikhaphu inamandla ngakumbi ekuthathweni kweeseli kune-insulin ekhululekileyo, ikakhulu ngenxa yobukhulu obuncinci bee-nanoparticles ezifakwe kwi-insulin eziveliswe kolu phononongo.
Ukuthathwa kweeseli zeHepG2 emva kweeyure ezi-4 zokufunxwa ngeeNP ezisandula ukulungiswa kunye neeNP eziphelelwe ngamanzi emzimbeni: (a) Ukusasazwa kokuthathwa kwe-FITC-insulin ziiseli zeHepG2. (b) I-geometric mean of fluorescence intensities ehlalutywe yi-flow cytometry (n = 3), *P < 0.05 xa kuthelekiswa ne-insulin yasimahla.
Ngokufanayo, imifanekiso ye-CLSM ibonise ukuba amandla e-FITC fluorescence e-NPs ezisandula ukulungiswa ze-FITC-insulin-loaded kunye ne-FITC-insulin-loaded spray-dried NPs (ngaphandle kwe-mannitol) zazinamandla kakhulu kunezinye iisampuli (Umzobo 6a). Ngaphezu koko, ngokongezwa kwe-mannitol, i-viscosity ephezulu yesisombululo yonyusa ukumelana nokufunxwa kweseli, nto leyo ebangele ukwehla kokwanda kwe-insulin. Ezi ziphumo zibonisa ukuba ii-NPs ezifunxwayo ezingena-mannitol zibonise ukusebenza okuphezulu kokufunxwa kweseli kuba ubungakanani bazo bezinto ezincinci babuncinci kunee-NPs ezifunxwayo emva kokunyibilika kwakhona.
I-Chitosan (ubunzima obuqhelekileyo be-molecular yi-100 KDa, i-75–85% deacetylated) ithengwe kwiSigma-Aldrich. (Oakville, Ontario, Canada). I-Sodium tripolyphosphate (TPP) ithengwe kwi-VWR (Radnor, Pennsylvania, USA). I-insulin yomntu ephinda-phinda esetyenzisiweyo kolu phononongo yayivela kwiFisher Scientific (Waltham, MA, USA). I-insulin yomntu ene-Fluorescein isothiocyanate (FITC) kunye ne-4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) ithengwe kwiSigma-Aldrich. (Oakville, Ontario, Canada). Umgca weseli ye-HepG2 ufunyenwe kwi-ATCC (Manassas, Virginia, USA). Zonke ezinye ii-reagents zazikumgangatho wohlalutyo okanye we-chromatographic.
Lungisa isisombululo se-1 mg/ml se-CS ngokusinyibilikisa emanzini acwecwe kabini (amanzi e-DD) aqulethe i-0.1% ye-acetic acid. Lungisa isisombululo se-1 mg/ml se-TPP kunye ne-insulin ngokusinyibilikisa emanzini e-DD kunye ne-0.1% ye-acetic acid, ngokulandelanayo. I-pre-emulsion yalungiswa nge-polytron PCU-2-110 high speed homogenizer (Brinkmann Ind. Westbury, NY, USA). Inkqubo yokulungiselela yile ilandelayo: okokuqala, i-2ml yesisombululo se-TPP yongezwa kwi-4ml yesisombululo se-insulin, kwaye umxube uvuselelwa imizuzu engama-30 kwaye uxutywe ngokupheleleyo. Emva koko, isisombululo esixutyiweyo songezwa kwisisombululo se-CS ngesirinji phantsi kokuvuselelwa ngesantya esiphezulu (10,000 rpm). Le mixtures igcinwe phantsi kokuvuselelwa ngesantya esiphezulu (15,000 rpm) kwindawo yokuhlambela iqhwa imizuzu engama-30, kwaye yalungiswa kwi-pH ethile ukuze kufunyanwe i-insulin NPs ezidityanisiweyo. Ukuze i-homogenize ngakumbi kwaye inciphise ubungakanani be-insulin NPs, zafakwa kwi-sonic ukuze zifumane i-insulin NPs eyongezelelweyo. Imizuzu engama-30 kwibhafu yomkhenkce usebenzisa i-sonicator yohlobo lwe-probe (UP 200ST, Hielscher Ultrasonics, Teltow, eJamani).
I-Insulin NPS ivavanywe kwi-Z-avareji yedayamitha, i-polydispersity index (PDI) kunye ne-zeta potential kusetyenziswa umlinganiselo we-dynamic light scattering (DLS) kusetyenziswa i-Litesizer 500 (Anton Paar, Graz, Austria) ngokuyixuba emanzini e-DD kwi-25°C. I-Morelogy kunye nokusasazwa kobungakanani kwabonakaliswa yi-Hitachi H7600 transmission electron microscope (TEM) (Hitachi, Tokyo, Japan), kwaye imifanekiso yahlaziywa kamva kusetyenziswa isoftware ye-Hitachi imaging (Hitachi, Tokyo, Japan). Ukuvavanya ukusebenza kakuhle kwe-encapsulation (EE) kunye nomthamo wokulayisha (LC) we-insulin NPs, ii-NP zafakwa kwiityhubhu ze-ultrafiltration ezine-molecular weight cut-off ye-100 kDa kwaye zafakwa kwi-centrifuge kwi-500 xg kangangemizuzu engama-30. I-insulin engafakwanga kwi-filtrate yalinganiswa kusetyenziswa inkqubo ye-Agilent 1100 Series HPLC (Agilent, Santa Clara, California, USA) equlathe i-quaternary pump, i-autosampler, ikholamu. iheater, kunye ne-DAD detector. I-Insulin ihlalutywe ngekholamu ye-C18 (iZorbax, 3.5 μm, 4.6 mm × 150 mm, e-Agilent, e-USA) yaza yafunyanwa kwi-214 nm. Isigaba esiphathwayo yayiyi-acetonitrile kunye namanzi, equlethe i-0.1% TFA, ii-gradient ratios ukusuka kwi-10/90 ukuya kwi-100/0, yaza yasebenza imizuzu eli-10. Isigaba esiphathwayo sipompelwe kwisantya sokuhamba se-1.0 ml/min. Ubushushu bekholamu bubekwe kwi-20 °C. Bala iipesenti ze-EE kunye ne-LC usebenzisa ii-equation.(1) kunye ne-Eq.(2).
Iireyithingi ezahlukeneyo ze-CS/insulin eziqala kwi-2.0 ukuya kwi-4.0 zivavanyiwe ukuze kuphuculwe i-insulin NP. Izixa ezahlukeneyo zesisombululo se-CS zongezwa ngexesha lokulungiselela, ngelixa umxube we-insulin/TPP ugcinwe ungaguquki. Ii-Insulin NPs zilungiselelwe kuluhlu lwe-pH oluyi-4.0 ukuya kwi-6.5 ngokulawula ngononophelo i-pH yomxube emva kokongeza zonke izisombululo (i-insulin, i-TPP kunye ne-CS). Ubungakanani be-EE kunye ne-particles ye-insulin nanoparticles zivavanywe kwiixabiso ezahlukeneyo ze-pH kunye ne-CS/insulin mass ratios ukuze kuphuculwe ukwakheka kwe-insulin NPs.
Ii-insulin NPs ezilungisiweyo zabekwa kwisikhongozeli se-aluminiyam zaza zagqunywa ngethishu eqinisiweyo ngeteyipu ethile. Emva koko, izikhongozeli ezinezikrufu zafakwa kwi-Labconco FreeZone freezer dryer (eLabconco, eKansas City, MO, e-USA) exhotyiswe nge-tray dryer. Ubushushu kunye noxinzelelo lwe-vacuum zamiselwa kwi--10 °C, i-0.350 Torr kwiiyure ezimbini zokuqala, kunye ne-0 °C kunye ne-0.120 Torr kwiiyure ezingama-22 eziseleyo kwiiyure ezingama-24 ukuze kufunyanwe ii-insulin NPs ezomileyo.
I-Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Switzerland) yasetyenziswa ukuvelisa i-insulin efakwe kwiikhaphu. Iiparameter zokomisa ezikhethiweyo zezi: ubushushu obuyi-100 °C, ukuhamba kokutya okuzii-L/min ezi-3, kunye nokuhamba kwegesi okuzii-L/min ezi-4.
Ii-Insulin NPs ngaphambi nasemva kokuphelelwa ngamanzi emzimbeni zichazwe kusetyenziswa i-FTIR-ATR spectroscopy. Ii-nanoparticles eziphelelwe ngamanzi emzimbeni kunye ne-insulin yasimahla kunye ne-chitosan zihlalutywe kusetyenziswa i-Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA) exhotyiswe ngesixhobo sokuvavanya i-ATR (PerkinElmer, Waltham, Massachusetts, USA). I-avareji yesignali ifunyenwe kwiiskeni ezili-16 kwisisombululo se-4 cm2 kuluhlu lwamaza angama-4000-600 cm2.
Imbonakalo yee-NP ze-insulin eyomileyo ihlolwe ngemifanekiso ye-SEM yee-NP ze-insulin ezomileyo eziqandisiweyo nezifakwe isitshizi ezithathwe yiHelios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, USA). Ipharamitha ephambili esetyenzisiweyo yayiyi-voltage eyi-5 keV kunye ne-current eyi-30 mA.
Zonke ii-NP ze-insulin eziphelelwe ngamanzi emzimbeni zaphinda zanyibilika emanzini. Ubungakanani beenxalenye, i-PDI, i-EE kunye ne-LC zavavanywa kwakhona kusetyenziswa indlela efanayo ekhankanyiweyo ngaphambili ukuvavanya umgangatho wazo emva kokuphelelwa ngamanzi emzimbeni. Uzinzo lwee-NP ze-anhydroinsulin lwalinganiswa ngokuvavanya iipropati zee-NP emva kokugcinwa ixesha elide. Kolu phononongo, zonke ii-NP emva kokuphelelwa ngamanzi emzimbeni zagcinwa efrijini kangangeenyanga ezintathu. Emva kweenyanga ezintathu zokugcina, ii-NP zavavanywa ubungakanani beenxalenye zomzimba, i-PDI, i-EE kunye ne-LC.
Nyibilikisa i-5 mL yee-NP ezilungisiweyo kwi-45 mL equlethe ulwelo lwesisu olulinganisiweyo (pH 1.2, equlethe i-1% ye-pepsin), ulwelo lwamathumbu (pH 6.8, equlethe i-1% ye-trypsin) okanye isisombululo se-chymotrypsin (100 g/mL, kwi-phosphate buffer, pH 7.8) ukuvavanya ukusebenza kwe-insulin ekukhuseleni ii-NP emva kokuphelelwa ngamanzi emzimbeni. Zafakwa kwi-incubation kwi-37°C ngesantya sokushukuma se-100 rpm. I-500 μL yesisombululo yaqokelelwa ngamaxesha ahlukeneyo kwaye uxinzelelo lwe-insulin lwamiselwa yi-HPLC.
Ukuziphatha kokukhululwa kwe-insulin NPs ezisandula ukulungiswa neziphelelwe ngamanzi emzimbeni kuhlolwe ngendlela ye-dialysis bag (i-molecular weight cut-off 100 kDa, Spectra Por Inc.). Ii-NPs ezomileyo ezisandula ukulungiswa nezilungisiweyo zafakwa kwi-dialyzed kwi-pH 2.5, pH 6.6, kunye ne-pH 7.0 (0.1 M phosphate-buffered saline, PBS) ukuze kulinganiswe imeko ye-pH yesisu, i-duodenum, kunye ne-upper small intestine, ngokulandelelana. Zonke iisampulu zafakwa kwi-incubated kwi-37 °C ngokushukuma okuqhubekayo kwi-200 rpm. Fudumeza ulwelo ngaphandle kwe-5 mL dialysis bag ngala maxesha alandelayo: 0.5, 1, 2, 3, 4, kunye ne-6 hrs, kwaye ngoko nangoko uzalise umthamo nge-dialysate entsha. Ungcoliseko lwe-insulin kulwelo luhlalutywe yi-HPLC, kwaye izinga lokukhululwa kwe-insulin kwi-nanoparticles libalwe ukusuka kumlinganiselo we-insulin ekhululekileyo ekhutshwe kwi-insulin iyonke efakwe kwi-nanoparticles (Equation 3).
Iiseli zeHepG2 zeHepatocellular carcinoma cell line zabantu zikhuliswe kwizitya ezinobubanzi obuyi-60 mm kusetyenziswa iDulbecco's Modified Eagle's Medium (DMEM) equlethe i-10% ye-fetal bovine serum, i-100 IU/mL penicillin, kunye ne-100 μg/mL streptomycin29. Iinkcubeko zigcinwe kwi-37°C, i-95% yomswakama, kunye ne-5% CO2. Kwiimvavanyo zokungenisa, iiseli zeHepG2 zityalwe kwi-1 × 105 cells/ml kwinkqubo yesilayidi se-Nunc Lab-Tek chamber ene-8-well (Thermo Fisher, NY, USA). Kwiimvavanyo ze-cytotoxicity, zityalwe kwiiplate ze-96-well (Corning, NY, USA) kubuninzi be-5 × 104 cells/ml.
Uvavanyo lwe-MTT lusetyenziselwe ukuvavanya ubuthi be-insulin NPs30 esandul’ ukulungiswa neyomileyo. Iiseli zeHepG2 zityalwe kwiiplate ezingama-96-well kuxinano lweeseli ezi-5 × 104/mL zaza zakhuliswa iintsuku ezi-7 ngaphambi kovavanyo. Ii-Insulin NPs zaxutywa kumanqanaba ahlukeneyo (50 ukuya kwi-500 μg/mL) kwindawo yokukhulisa emva koko zanikwa iiseli. Emva kweeyure ezingama-24 zokukhuliswa, iiseli zahlanjwa izihlandlo ezi-3 nge-PBS zaza zakhuliswa nge-medium equlethe i-0.5 mg/ml MTT kangangeeyure ezi-4 ezongezelelweyo. Ubuthi be-cytotoxicity buvavanywe ngokulinganisa ukunciphisa kwe-enzyme ye-tetrazolium MTT etyheli ukuya kwi-purple formazan kwi-570 nm kusetyenziswa i-Tecan infinite M200 pro spectrophotometer plate reader (Tecan, Männedorf, Switzerland).
Ukusebenza kakuhle kokuthathwa kwe-NPs kwiseli kuhlolwe nge-confocal laser scanning microscopy kunye nohlalutyo lwe-flow cytometry. Umthombo ngamnye we-Nunc Lab-Tek chamber slide system uphathwe nge-free FITC-insulin, i-FITC-insulin-loaded NPs, kwaye waphinda walungisa i-25 μg/mL ye-FITC-insulin NPs eyomileyo kwi-concentration efanayo kwaye yafakwa kwi-incubation iiyure ezi-4. Iiseli zahlanjwa izihlandlo ezi-3 nge-PBS kwaye zalungiswa nge-4% paraformaldehyde. I-Nuclei yadaywa nge-4′,6-diamidino-2-phenylindole (DAPI). Indawo ye-insulin ibonwe kusetyenziswa i-Olympus FV1000 laser scanning/two-photon confocal microscope (Olympus, Shinjuku City, Tokyo, Japan). Uhlalutyo lwe-flow cytometry, amazinga afanayo e-10 μg/mL free FITC-insulin, i-FITC-insulin-loaded NPs, kunye ne-resolubilized FITC-insulin eyomileyo. Ii-NP zongezwa kwiiplate ze-96-well ezityalwe ngeeseli ze-HepG2 zaza zafakwa kwiiyure ezi-4. Emva kweeyure ezi-4 zokufunxwa, iiseli zasuswa zaza zahlanjwa izihlandlo ezi-3 nge-FBS. Iiseli ezi-5 × 104 kwisampulu nganye zahlalutywa yi-BD LSR II flow cytometer (BD, Franklin Lakes, New Jersey, United States).
Zonke ixabiso zibonakaliswa njenge-mean ± standard deviation. Uthelekiso phakathi kwawo onke amaqela luvavanywe kusetyenziswa i-one-way ANOVA okanye i-t-test yi-IBM SPSS Statistics 26 ye-Mac (IBM, Endicott, New York, USA) kwaye i-p < 0.05 ithathwe njengebalulekileyo ngokwezibalo.
Olu phononongo lubonisa ukuguquguquka kunye nokukwazi ukomisa ngespray ukufunxa amanzi kwi-cross-linked chitosan/TPP/insulin nanoparticles ngokulungiswa ngcono xa kuthelekiswa neendlela eziqhelekileyo zokuqandisa ngefreeze kusetyenziswa ii-bulking agents okanye amandla e-cryoprotectants kunye nomthamo ophezulu womthwalo. Ii-insulin nanoparticles ezilungiselelweyo zivelise ubungakanani obuphakathi be-particle obuyi-318 nm kunye nokusebenza kakuhle kwe-encapsulation ye-99.4%. Iziphumo ze-SEM kunye ne-FTIR emva kokuphelelwa ngamanzi zibonise ukuba isakhiwo esingqukuva sigcinwe kuphela kwi-NPs ezomisiweyo ngespray kunye ne-mannitol kwaye zifakwe i-lyophilized nge-mannitol, kodwa ii-NPs ezomisiweyo ngaphandle kwe-mannitol zabola ngexesha lokuphelelwa ngamanzi. Kuvavanyo lokukwazi ukuphinda zisetyenziswe, ii-insulin nanoparticles ezomisiweyo ngaphandle kwe-mannitol zibonise ubungakanani obuncinci be-particle kunye nomthwalo ophezulu xa ziphinda zisetyenziswa. Iindlela zokukhulula zonke ezi NPs zomisiweyo zibonise ukuba zikhutshwa ngokukhawuleza kwizisombululo ze-pH = 2.5 kunye ne-pH = 7, kwaye zizinzile kakhulu kwisisombululo se-pH = 6.5. Xa kuthelekiswa nezinye ii-NPs ezomisiweyo kwakhona, ii-NPs zomisiweyo ngaphandle I-mannitol ibonise ukukhululwa okukhawulezayo. Esi siphumo sihambelana noko kubonwe kuvavanyo lokufunxwa kweseli, njengoko ii-NP ezomisiweyo ngokutshizwa ngaphandle kwe-mannitol zigcine phantse ngokupheleleyo ukusebenza kakuhle kokufunxwa kweseli kwee-NP ezisandula ukulungiswa. Ezi ziphumo zibonisa ukuba ii-nanoparticles ze-insulin ezomileyo ezilungiselelwe ngokufunxwa kwe-mannitol-free zilungele kakhulu ukucutshungulwa kwezinye iindlela zokulinganisa ezingenamanzi, ezifana neepilisi zomlomo okanye iifilimu zokuncamathelisa i-bioadhesive.
Ngenxa yeengxaki zepropathi yengqondo, iiseti zedatha ezenziweyo kunye/okanye ezihlalutyiweyo ngexesha lophando lwangoku azifumaneki kuluntu, kodwa ziyafumaneka kubabhali abafanelekileyo xa beceliwe ngokufanelekileyo.
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