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Ama-nanoparticles e-insulin (ama-NP) anokuqukethwe okuphezulu kokulayisha athole ukusetshenziswa okuhlukile ngezindlela ezahlukene zomthamo. Lo msebenzi uhlose ukuhlola umphumela wezinqubo zokomisa ngokuqandisa kanye nokomisa ngokufutha esakhiweni sama-nanoparticles e-chitosan agcwele i-insulin, ane-mannitol noma engenayo njenge-cryoprotectant. Siphinde sahlola ikhwalithi yala ma-nanoparticles ngokuwaxuba kabusha. Ngaphambi kokuphelelwa amanzi emzimbeni, usayizi wezinhlayiya ze-chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles walungiswa waba ngu-318 nm, i-PDI yayingu-0.18, ukusebenza kahle kwe-encapsulation kwakungu-99.4%, kanti umthwalo wawungu-25.01%. Ngemva kokuvuselelwa, wonke ama-nanoparticles, ngaphandle kwalawo akhiqizwe ngendlela yokomisa ngokuqandisa ngaphandle kokusebenzisa i-mannitol, agcina isakhiwo sawo sezinhlayiya eziyindilinga. Uma kuqhathaniswa nama-nanoparticles aqukethe i-mannitol ancishiswe ngamanzi yi-spray, ama-nanoparticles angenawo ama-mannitol nawo abonise usayizi wezinhlayiya ezincane kakhulu (376 nm) kanye nokuqukethwe okuphezulu kokulayisha (25.02%). ngesilinganiso esifanayo sokumbozwa (98.7%) kanye ne-PDI (0.20) ngobuchwepheshe bokomisa noma bokuqandisa. Ama-nanoparticles omisiwe ngokufutha ngaphandle kwe-mannitol nawo aphumele ekukhishweni okusheshayo kwe-insulin kanye nokusebenza kahle kakhulu kokuthathwa kwamaseli. Lo msebenzi ukhombisa ukuthi ukomisa ngokufutha kungakhipha amanzi kuma-nanoparticles e-insulin ngaphandle kwesidingo sama-cryoprotectants uma kuqhathaniswa nezindlela zokomisa ngokuqandisa ezivamile, okudala umthamo omkhulu wokulayisha, izidingo zokwengeza eziphansi kanye nezindleko zokusebenza inzuzo enkulu.
Kusukela ekutholakaleni kwayo ngo-1922, 2, 3, i-insulin kanye nemithi yayo kusindise izimpilo zeziguli ezinesifo sikashukela sohlobo 1 (T1DM) kanye nesifo sikashukela sohlobo 2 (T1DM). Kodwa-ke, ngenxa yezakhiwo zayo njengeprotheni enesisindo sama-molecule amaningi, i-insulin ihlanganiswa kalula, ihlukaniswe ngama-enzyme e-proteolytic, futhi isuswe ngomphumela wokuqala. Abantu abatholakale benesifo sikashukela sohlobo 1 badinga imijovo ye-insulin impilo yabo yonke. Iziguli eziningi ezitholakale zinesifo sikashukela sohlobo 2 ekuqaleni nazo zidinga imijovo ye-insulin yesikhathi eside. Imijovo ye-insulin yansuku zonke iwumthombo omkhulu wobuhlungu nokungakhululeki kwansuku zonke kulaba bantu, nemiphumela emibi empilweni yengqondo. Ngenxa yalokho, ezinye izinhlobo zokuphathwa kwe-insulin ezibangela ukungakhululeki okuncane, njengokuphathwa kwe-insulin ngomlomo, ziyafundwa kabanzi5 njengoba zinamandla okubuyisela ikhwalithi yokuphila yabantu ababalelwa ezigidini ezi-5 abanesifo sikashukela emhlabeni jikelele.
Ubuchwepheshe be-nanoparticle buhlinzeke ngentuthuko enkulu emizamweni yokuthatha i-insulin yomlomo4,6,7. Ehlanganisa ngempumelelo futhi ivikele i-insulin ekuwohlokeni ukuze ithunyelwe ezindaweni ezithile zomzimba.Kodwa-ke, ukusetshenziswa kwe-nanoparticle formulations kunemikhawulo eminingana, ikakhulukazi ngenxa yezinkinga zokuzinza kokumiswa kwezinhlayiya. Okunye ukuhlangana kungenzeka ngesikhathi sokugcina, okunciphisa ukutholakala kwe-nanoparticles efakwe i-insulin8.Ngaphezu kwalokho, ukuzinza kwamakhemikhali kwe-polymer matrix yama-nanoparticles kanye ne-insulin kumele kucatshangelwe ukuqinisekisa ukuzinza kwama-nanoparticles e-insulin (NPs).Njengamanje, ubuchwepheshe bokomisa ngokuqandisa buyindlela yegolide yokwakha ama-NP azinzile ngenkathi kuvimbela izinguquko ezingafuneki ngesikhathi sokugcina9.
Kodwa-ke, ukomisa ngeqhwa kudinga ukungezwa kwama-cryoprotectants ukuvimbela isakhiwo esiyindilinga sama-NP ukuthi singathinteki ukucindezeleka komshini kwamakristalu eqhwa. Lokhu kunciphisa kakhulu umthwalo wama-nanoparticles e-insulin ngemuva kwe-lyophilization, njengoba i-cryoprotectant ithatha ingxenye enkulu yesisindo. Ngakho-ke, ama-insulin NP akhiqizwayo avame ukutholakala engafaneleki ekwenziweni kwe-dry powder formulations, njengamaphilisi omlomo namafilimu omlomo, ngenxa yesidingo senani elikhulu lama-nanoparticles omile ukuze kufezwe iwindi lokwelapha le-insulin.
Ukomisa ngesifutho kuyinqubo eyaziwayo nengabizi kakhulu yezimboni yokukhiqiza impuphu eyomile evela ezigabeni zoketshezi embonini yezemithi10,11. Ukulawula inqubo yokwakheka kwezinhlayiya kuvumela ukufakwa kahle kwamakhemikhali amaningana asebenzayo 12, 13. Ngaphezu kwalokho, sekuyindlela ephumelelayo yokulungiselela amaprotheni afakwe ngefutho ukuze aphathwe ngomlomo. Ngesikhathi sokomisa ngefutho, amanzi ayahwamuka ngokushesha okukhulu, okusiza ukugcina izinga lokushisa le-particle core liphansi11,14, okuvumela ukusetshenziswa kwalo ukufaka izingxenye ezizwela ukushisa. Ngaphambi kokomisa ngefutho, izinto zokumboza kufanele zihlanganiswe kahle nesisombululo esiqukethe izithako ezifakwe ngefutho11,14. Ngokungafani nokomisa ngeqhwa, ukufakwa ngefutho ngaphambi kokufakwa ngefutho ekomiseni ngefutho kuthuthukisa ukusebenza kahle kwefutho ngesikhathi sokuphelelwa amanzi emzimbeni. Njengoba inqubo yokufaka ngefutho ngefutho ingadingi ama-cryoprotective, ukomisa ngefutho kungasetshenziswa ukukhiqiza ama-NP omisiwe anokuqukethwe okuphezulu kokulayisha.
Lolu cwaningo lubika ukukhiqizwa kwama-NP agcwele i-insulin ngokuxhumanisa i-chitosan ne-sodium tripolyphosphate kusetshenziswa indlela ye-ion gel. I-Ion gelation iyindlela yokulungiselela evumela ukukhiqizwa kwama-nanoparticles ngokusebenzisana kwe-electrostatic phakathi kwezinhlobo ezimbili noma ngaphezulu ze-ionic ngaphansi kwezimo ezithile. Zombili izindlela zokomisa ngokuqandisa kanye nokomisa ngokufutha zasetshenziswa ukumunca amanzi kuma-nanoparticles e-chitosan/sodium tripolyphosphate/insulin ahlanganiswe kahle. Ngemuva kokuphelelwa amanzi, isimo sazo sahlaziywa yi-SEM. Amandla azo okuphinda ahlanganiswe ahlolwe ngokulinganisa ukusatshalaliswa kosayizi wazo, ukushaja kwendawo, i-PDI, ukusebenza kahle kwe-encapsulation, kanye nokuqukethwe kokulayisha. Ikhwalithi yama-nanoparticles aphinde ancibilikiswa akhiqizwe ngezindlela ezahlukene zokuphelelwa amanzi nawo ahlolwe ngokuqhathanisa ukuvikelwa kwawo kwe-insulin, ukuziphatha kokukhululwa, kanye nokusebenza kahle kokuthathwa kwamaseli.
I-pH yesisombululo esixubile kanye nesilinganiso se-chitosan ne-insulin yizici ezimbili ezibalulekile ezithinta usayizi wezinhlayiya kanye nokusebenza kahle kwe-encapsulation (EE) yama-NP okugcina, njengoba zithinta ngqo inqubo ye-ionotropic gelation. I-pH yesisombululo esixubile iboniswe ukuthi ihlobene kakhulu nosayizi wezinhlayiya kanye nokusebenza kahle kwe-encapsulation (Isithombe 1a). Njengoba kuboniswe ku-Fig. 1a, njengoba i-pH ikhuphuka kusuka ku-4.0 kuya ku-6.0, usayizi wezinhlayiya ojwayelekile (nm) wehla kanti i-EE ikhuphuke kakhulu, kuyilapho lapho i-pH ikhuphuka yaya ku-6.5, usayizi wezinhlayiya ojwayelekile waqala ukwanda kanti i-EE ayizange ishintshe. Njengoba isilinganiso se-chitosan ne-insulin sanda, usayizi wezinhlayiya ojwayelekile nawo uyakhula. Ngaphezu kwalokho, akukho shintsho ku-EE olubonwe lapho ama-nanoparticles elungiswa ngesilinganiso se-chitosan/insulin esiphezulu kune-2.5:1 (w/w) (Isithombe 1b). Ngakho-ke, izimo zokulungiselela ezifanele kulolu cwaningo (i-pH 6.0, isilinganiso se-chitosan/insulin mass esingu-2.5:1) zasetshenziswa ukuze lungiselela ama-nanoparticles agcwele i-insulin ukuze uthole olunye ucwaningo. Ngaphansi kwalesi simo sokulungiselela, usayizi ojwayelekile wezinhlayiya zama-nanoparticles e-insulin wenziwe waba ngu-318 nm (Isithombe 1c), i-PDI yayingu-0.18, ukusebenza kahle kokushumeka kwakungu-99.4%, amandla e-zeta kwakungu-9.8 mv, kanti umthwalo we-insulin wawungu-25.01% (m/m ). Ngokusekelwe emiphumeleni ye-transmission electron microscopy (TEM), ama-nanoparticles alungiselelwe ayecishe abe yindilinga futhi ahlukene ngosayizi ofanayo (Isithombe 1d).
Ukwenziwa ngcono kwepharamitha yama-nanoparticles e-insulin: (a) umphumela we-pH kububanzi obuphakathi kanye nokusebenza kahle kwe-encapsulation (EE) kwama-nanoparticles e-insulin (okulungiselelwe ngesilinganiso esingu-5:1 sobuningi be-chitosan kanye ne-insulin); (b) i-chitosan kanye nethonya lesilinganiso sobuningi be-insulin kububanzi obuphakathi kanye nokusebenza kahle kwe-encapsulation (EE) kwama-insulin NPs (okulungiselelwe ku-pH 6); (c) ukusatshalaliswa kosayizi wezinhlayiya zama-nanoparticles e-insulin alungiselelwe; (d) i-TEM micrograph yama-insulin NPs alungiselelwe.
Kwaziwa kahle ukuthi i-chitosan iyi-polyelectrolyte ebuthakathaka ene-pKa engu-6.5. Ishajwa kahle kwimidiya ye-acidic ngoba iqembu layo eliyinhloko lama-amino likhiqizwa yi-hydrogen ions15. Ngakho-ke, ivame ukusetshenziswa njengesithwali ukufaka ama-macromolecule ashajwe kabi. Kulolu cwaningo, i-chitosan yasetshenziswa ukufaka i-insulin ngephuzu le-isoelectric elingu-5.3. Njengoba i-chitosan isetshenziswa njengento yokumboza, ngokwanda kwesilinganiso sayo, ukujiya kwengqimba yangaphandle yama-nanoparticles kuyanda ngokufanayo, okuholela kusayizi omkhulu wezinhlayiya. Ngaphezu kwalokho, amazinga aphezulu e-chitosan angahlanganisa i-insulin eningi. Esimweni sethu, i-EE yayiphezulu kakhulu lapho isilinganiso se-chitosan ne-insulin sifinyelela ku-2.5:1, futhi akukho shintsho olubalulekile ku-EE lapho isilinganiso siqhubeka nokukhula.
Ngaphandle kwesilinganiso se-chitosan ne-insulin, i-pH nayo idlale indima ebalulekile ekulungiseleleni i-NPs. UGan et al. 17 bafunde ngomphumela we-pH kusayizi wezinhlayiya ze-chitosan nanoparticles. Bathole ukwehla okuqhubekayo kosayizi wezinhlayiya kwaze kwaba yilapho i-pH ifinyelela ku-6.0, futhi ukwanda okukhulu kosayizi wezinhlayiya kwabonwa ku-pH > 6.0, okuhambisana nokubona kwethu. Lesi simo sibangelwa iqiniso lokuthi ngokwanda kwe-pH, i-molecule ye-insulin ithola ishaja engemihle ebusweni, ngaleyo ndlela, ithanda ukusebenzisana kwe-electrostatic ne-chitosan/sodium tripolyphosphate (TPP) complex, okuholela kusayizi wezinhlayiya ezincane kanye ne-EE ephezulu. Kodwa-ke, lapho i-pH ilungiswa ibe ngu-6.5, amaqembu ama-amino ku-chitosan asuswa, okuholela ekugoqweni kwe-chitosan. Ngakho-ke, i-pH ephezulu iholela ekuvezweni okuncane kwama-amino ion ku-TPP kanye ne-insulin, okuholela ekuxhumekeni okuphansi, usayizi omkhulu wokugcina wezinhlayiya kanye ne-EE ephansi.
Ukuhlaziywa kwezakhiwo ze-morphological ze-NP ezomisiwe ngokuqandisa kanye nezomisiwe ngokufutha kungaqondisa ukukhethwa kwezindlela ezingcono zokuqeda amanzi emzimbeni kanye nokwakheka kwempuphu. Indlela ekhethwayo kufanele inikeze ukuzinza kwemithi, ukuma kwezinhlayiya okufanayo, umthwalo ophezulu wemithi kanye nokuncibilika okuhle esixazululweni sokuqala. Kulolu cwaningo, ukuze kuqhathaniswe kangcono lawa masu amabili, ama-insulin NP ane-1% mannitol noma angenayo asetshenziswa ngesikhathi sokuphelelwa amanzi emzimbeni. I-Mannitol isetshenziswa njenge-bulking agent noma i-cryoprotectant ekwakhiweni kwempuphu eyomile ehlukahlukene yokomisa ngokuqandisa kanye nokomisa ngokufutha. Kuma-nanoparticles e-insulin angenawo i-mannitol, njengoba kuboniswe kuMfanekiso 2a, isakhiwo sempuphu esinezimbobo eziningi esinezindawo ezinkulu, ezingajwayelekile nezinolaka sabonwa ngaphansi kwe-scanning electron microscopy (SEM). Zimbalwa izinhlayiya ezihlukene ezitholakale kumpuphu ngemva kokuphelelwa amanzi emzimbeni (Umfanekiso 2e). Le miphumela ibonise ukuthi ama-NP amaningi abolile ngesikhathi sokumiswa ngokuqandisa ngaphandle kwanoma iyiphi i-cryoprotectant. Kuma-nanoparticles e-insulin afakwe ngokuqandisa kanye nomisiwe ngokufutha aqukethe i-1% mannitol, kwabonwa ama-nanoparticles ayindilinga anezindawo ezibushelelezi (Umfanekiso 2). 2b,d,f,h).Ama-insulin nanoparticles omiswe ngokufutha ngaphandle kwe-mannitol ahlala eyindilinga kodwa egobile ebusweni (Isithombe 2c). Izindawo eziyisiyingi nezigobile kuxoxwa ngazo kabanzi ekuziphatheni kokukhululwa kanye nokuhlolwa kokuthathwa kwamaseli ngezansi. Ngokusekelwe ekubukekeni okubonakalayo kwama-NP omisiwe, ama-NP omiswe ngokufutha ngaphandle kwe-mannitol kanye nama-NP omiswe ngokuqandisa kanye nowomiswe ngokufutha nge-mannitol akhiphe izimpushana ze-NPs ezincane (Isithombe 2f,g,h). Uma indawo enkulu phakathi kwezinhlayiya, kulapho ukuncibilika kukhuphuka khona futhi ngenxa yalokho izinga lokukhululwa liphakeme.
Ukwakheka kwama-NP e-insulin ahlukene angenamanzi: (a) Isithombe se-SEM sama-NP e-insulin afakwe i-lyophilized ngaphandle kwe-mannitol; (b) Isithombe se-SEM sama-NP e-insulin afakwe i-lyophilized ane-mannitol; (c) ama-NP e-insulin afakwe i-spray ngaphandle kwe-mannitol Isithombe se-SEM sika-; (d) Isithombe se-SEM sama-NP e-insulin afakwe i-spray nge-mannitol; (e) isithombe sempuphu ye-NPs ye-insulin efakwe i-lyophilized ngaphandle kwe-mannitol; (f) isithombe sama-NPs e-insulin afakwe i-lyophilized ane-mannitol; (g) Isithombe sempuphu ye-NPs ye-insulin efakwe i-spray ngaphandle kwe-mannitol; (h) isithombe sempuphu ye-NPs ye-insulin efakwe i-spray ene-mannitol.
Ngesikhathi sokumisa ngeqhwa, i-mannitol isebenza njenge-cryoprotectant, igcina ama-NP esesimweni esingaguquki futhi ivimbele umonakalo ngamakristalu eqhwa19. Ngokuphambene nalokho, akukho sinyathelo sokubanda ngesikhathi sokumisa ngeqhwa. Ngakho-ke i-mannitol ayidingeki kule ndlela. Eqinisweni, ama-NP omiswe ngeqhwa ngaphandle kwe-mannitol akhiqize ama-NP amahle njengoba kuchaziwe ngaphambili. Kodwa-ke, i-mannitol isasebenza njengesigcwalisi enkambisweni yokumisa ngeqhwa ukuze inikeze ama-NP isakhiwo esiyindilinga20 (Isithombe 2d), okusiza ukuthola ukuziphatha okufanayo kokukhululwa kwama-NP anjalo ahlanganisiwe. Ngaphezu kwalokho, kusobala ukuthi ezinye izinhlayiya ezinkulu zingatholakala kokubili kuma-NP e-insulin omiswe ngeqhwa kanye nawomiswe ngeqhwa aqukethe i-mannitol (Isithombe 2b, d), okungase kube ngenxa yokuqongelela kwe-mannitol enhlotsheni yezinhlayiya kanye ne-insulin ehlanganisiwe. Kuya ku-Chitosan layer. Kubalulekile ukuqaphela ukuthi kulolu cwaningo, ukuze kuqinisekiswe ukuthi isakhiwo esiyindilinga sihlala sinjalo ngemva kokuphelelwa amanzi emzimbeni, isilinganiso se-mannitol ne-chitosan sigcinwa ku-5:1, ukuze inani elikhulu lokugcwalisa likwazi nokwandisa usayizi wezinhlayiya ze-NPs ezomisiwe.
I-Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy ichaze ingxube ebonakalayo ye-insulin yamahhala, i-chitosan, i-chitosan, i-TPP kanye ne-insulin. Wonke ama-NP aphelelwe amanzi emzimbeni abonakaliswe ngokusebenzisa i-FTIR-ATR spectroscopy. Okuphawulekayo ukuthi, amandla e-band angu-1641, 1543 kanye no-1412 cm-1 abonwe kuma-NP afakwe i-capsulated aqandisiwe nge-mannitol kanye nama-NP afafazwayo nge-mannitol kanye nangaphandle kwayo (Isithombe 3). Njengoba kubikiwe ngaphambilini, lokhu kwanda kwamandla kwakuhlotshaniswa nokuxhumanisa phakathi kwe-chitosan, i-TPP kanye ne-insulin. Uphenyo lokuxhumana phakathi kwe-chitosan ne-insulin lubonise ukuthi kuma-spectra e-FTIR ama-nanoparticles e-chitosan agcwele i-insulin, i-chitosan band ihambisana ne-insulin, okwandisa amandla e-carbonyl (1641 cm-1) kanye ne-amine (1543 cm-1). Amaqembu e-tripolyphosphate e-TPP axhunywe namaqembu e-ammonium ku-chitosan, akha ibhande eliyi-1412 cm-1.
Ama-spectra e-FTIR-ATR e-insulin yamahhala, i-chitosan, izingxube zomzimba ze-chitosan/TPP/insulin kanye nama-NP ancishiswe ngamanzi ngezindlela ezahlukene.
Ngaphezu kwalokho, le miphumela ihambisana naleyo eboniswe ku-SEM, ekhombisile ukuthi ama-NP ahlanganisiwe ahlala engashintshi lapho efuthwa futhi omiswa nge-mannitol, kodwa uma kungekho i-mannitol, ukomisa ngokufutha kuphela okwakhiqiza izinhlayiya ezihlanganisiwe. Ngokuphambene nalokho, imiphumela ye-spectral ye-FTIR-ATR yama-NP afriziwe ngaphandle kwe-mannitol yayifana kakhulu nengxube ebonakalayo ye-chitosan, i-TPP, ne-insulin. Lo mphumela ubonisa ukuthi izixhumanisi eziphakathi kwe-chitosan, i-TPP kanye ne-insulin azisekho kuma-NP afriziwe ngaphandle kwe-mannitol. Isakhiwo se-NPs sabhujiswa ngesikhathi sokomisa ngokufutha ngaphandle kwe-cryoprotectant, okungabonakala emiphumeleni ye-SEM (Isithombe 2a). Ngokusekelwe ku-morphology kanye nemiphumela ye-FTIR yama-NP e-insulin aphelelwe amanzi, ama-NP a-lyophilized, omiswe ngokufutha, kanye nama-mannitol angasetshenziswa kuphela ekuhlolweni kokuvuselela kanye nama-NP angenayo i-mannitol ngenxa yokubola kwama-NP angenayo i-mannitol ngesikhathi sokuphelelwa amanzi. xoxa.
Ukuphelelwa amanzi emzimbeni kusetshenziselwa ukugcina isikhathi eside kanye nokucubungula kabusha kwezinye izinhlobo. Amandla e-NP ezomile okuphinda akhiwe ngemva kokugcinwa abalulekile ekusetshenzisweni kwawo ezinhlotsheni ezahlukene ezifana namaphilisi namafilimu. Siqaphele ukuthi ubukhulu bezinhlayiya ezimaphakathi ze-insulin NPs ezomisiwe ngokufuthwa ngaphandle kwe-mannitol bukhuphuke kancane kuphela ngemva kokubuyiselwa. Ngakolunye uhlangothi, ubukhulu bezinhlayiya ze-nanoparticles ze-insulin ezomisiwe ngokufuthwa nangokuqandisa ezine-mannitol bukhuphuke kakhulu (Ithebula 1). I-PDI kanye ne-EE azishintshwanga kakhulu (p > 0.05) ngemva kokuhlanganiswa kabusha kwazo zonke i-NP kulolu cwaningo (Ithebula 1). Lo mphumela ubonisa ukuthi iningi lezinhlayiya zahlala zinjalo ngemva kokuphinda zincibilike. Kodwa-ke, ukwengezwa kwe-mannitol kwaholela ekunciphiseni kakhulu umthwalo we-insulin we-mannitol nanoparticles e-lyophilized ne-spray (Ithebula 1). Ngokuphambene nalokho, okuqukethwe komthwalo we-insulin we-NPs ezomisiwe ngokufuthwa ngaphandle kwe-mannitol kwahlala kufana nangaphambili (Ithebula 1).
Kwaziwa kahle ukuthi ukulayishwa kwama-nanoparticles kubalulekile uma kusetshenziselwa izinjongo zokulethwa kwemithi. Kuma-NP anemithwalo ephansi, kudingeka inani elikhulu kakhulu lezinto ukuze kufinyelelwe umkhawulo wokwelapha. Kodwa-ke, i-viscosity ephezulu yalezi zingxube eziphezulu ze-NP iholela ekuphazamisekeni kanye nobunzima ekuphathweni ngomlomo kanye namafomu okujova, ngokulandelana 22. Ngaphezu kwalokho, ama-insulin NP angasetshenziswa futhi ukwenza amaphilisi kanye nama-biofilms aqinile 23, 24, okudinga ukusetshenziswa kwamanani amakhulu ama-NP emazingeni aphansi okulayisha, okuholela kumaphilisi amakhulu kanye nama-biofilms aqinile angafaneleki ukusetshenziswa ngomlomo. Ngakho-ke, ama-NP aphelelwe amanzi emzimbeni anomthwalo ophezulu we-insulin ayafiseleka kakhulu. Imiphumela yethu iphakamisa ukuthi umthwalo ophezulu we-insulin wama-NPs omisiwe angenawo ama-mannitol unganikeza izinzuzo eziningi ezikhangayo zalezi zindlela zokulethwa ezihlukile.
Zonke i-NP eziphelelwe amanzi emzimbeni zagcinwa esiqandisini izinyanga ezintathu. Imiphumela ye-SEM ibonise ukuthi isimo sazo zonke i-NP eziphelelwe amanzi emzimbeni asishintshanga kakhulu ngesikhathi sokugcina izinyanga ezintathu (Isithombe 4). Ngemva kokubuyiselwa emanzini, zonke i-NP zabonisa ukwehla okuncane ku-EE futhi zakhipha cishe inani elincane (~5%) le-insulin phakathi nesikhathi sokugcina izinyanga ezintathu (Ithebula 2). Kodwa-ke, usayizi ojwayelekile wezinhlayiya zawo wonke ama-nanoparticles wanda. Usayizi wezinhlayiya ze-NP ezifakwe i-spray ngaphandle kwe-mannitol wanda wafika ku-525 nm, kuyilapho lowo we-NP ezifakwe i-spray kanye nezomiswe nge-freeze nge-mannitol wanda wafika ku-872 kanye no-921 nm, ngokulandelana (Ithebula 2).
Ukwakheka kwama-NP e-insulin ahlukene aphelelwe amanzi agcinwe izinyanga ezintathu: (a) Isithombe se-SEM sama-NP e-insulin afakwe i-lyophilized ane-mannitol; (b) Isithombe se-SEM sama-nanoparticles e-insulin eyomisiwe ngokufuthwa ngaphandle kwe-mannitol; (c) ngaphandle kwe-mannitol izithombe ze-SEM zama-NP e-insulin eyomisiwe ngokufuthwa.
Ngaphezu kwalokho, kwabonakala ukusha kwe-precipitate kuma-nanoparticles e-insulin aphinde afakwa i-spray-dryed nge-mannitol bese omiswa nge-freeze (Isithombe S2). Lokhu kungabangelwa yizinhlayiya ezinkulu ezingangeni kahle emanzini. Yonke imiphumela engenhla ikhombisa ukuthi inqubo yokomisa i-spray ingavikela ama-nanoparticles e-insulin ekuphelelweni amanzi emzimbeni nokuthi imithwalo ephezulu yama-nanoparticles e-insulin ingatholakala ngaphandle kwanoma yiziphi izigcwalisi noma ama-cryoprotectants.
Ukugcinwa kwe-insulin kuhlolwe ku-pH = 2.5 medium nge-pepsin, trypsin, kanye ne-α-chymotrypsin ukuze kuboniswe ikhono lokuvikela lama-NP ekugayweni kwe-enzyme ngemva kokuphelelwa amanzi emzimbeni. Ukugcinwa kwe-insulin kwama-NP aphelelwe amanzi emzimbeni kwaqhathaniswa nokwe-NPs ezilungiselelwe, futhi i-insulin yamahhala yasetshenziswa njengokulawula okungekuhle. Kulolu cwaningo, i-insulin yamahhala ibonise ukususwa kwe-insulin okusheshayo zingakapheli amahora ama-4 kuzo zonke izindlela ezintathu zokwelapha nge-enzyme (Isithombe 5a–c). Ngokuphambene nalokho, ukuhlolwa kokususwa kwe-insulin kwama-NPs aqandisiwe nge-mannitol kanye nama-NPs afakwe i-spray-omiswe nge-mannitol noma ngaphandle kwayo kubonise ukuvikelwa okuphezulu kakhulu kwala ma-NP ekugayweni kwe-enzyme, okwakufana nokwe-insulin NPs ezilungiselelwe (isithombe 1).5a-c). Ngosizo lwezinhlayiya ezincane ku-pepsin, trypsin, kanye ne-α-chymotrypsin, okungaphezu kuka-50%, 60%, kanye no-75% we-insulin kungavikelwa zingakapheli amahora ama-4, ngokulandelana (Isithombe 5a–c). Leli khono lokuvikela i-insulin lingandisa amathuba okuba ne-insulin ephezulu. ukumuncwa egazini25. Le miphumela iphakamisa ukuthi ukomisa nge-spray nge-mannitol noma ngaphandle kwayo kanye nokomisa nge-freeze nge-mannitol kungalondoloza ikhono lokuvikela i-insulin lama-NP ngemva kokuphelelwa amanzi emzimbeni.
Ukuvikelwa kanye nokuziphatha kokukhululwa kwama-NP e-insulin aphelelwe amanzi emzimbeni: (a) ukuvikelwa kwe-insulin esixazululweni se-pepsin; (b) ukuvikelwa kwe-insulin esixazululweni se-trypsin; (c) ukuvikelwa kwe-insulin ngesisombululo se-α-chymotrypsin; (d) Ukuziphatha kokukhululwa kwama-NP aphelelwe amanzi emzimbeni kusisombululo se-pH = 2.5; (e) ukuziphatha kokukhululwa kwama-NP aphelelwe amanzi emzimbeni kusisombululo se-pH = 6.6; (f) ukuziphatha kokukhululwa kwama-NP aphelelwe amanzi emzimbeni kusisombululo se-pH = 7.0.
Ama-NP e-insulin eyomile asanda kulungiswa futhi akhiwe kabusha afakwe kuma-buffer ahlukahlukene (pH = 2.5, 6.6, 7.0) ku-37 °C, okulingisa indawo ye-pH yesisu, i-duodenum, kanye namathumbu amancane aphezulu, ukuze kuhlolwe umthelela we-insulin ekuphikisweni kwe-insulin. Ukuziphatha kokukhipha ezindaweni ezahlukene. Ingcezu yendlela yokugaya ukudla. Ku-pH = 2.5, ama-NP agcwele i-insulin kanye nama-NP e-insulin eyomile aphinde ancibilikiswa abonise ukukhishwa kokuqala kokuqhuma ngaphakathi kwehora lokuqala, kulandelwe ukukhishwa kancane emahoreni ama-5 alandelayo (Isithombe 5d). Lokhu kukhishwa okusheshayo ekuqaleni kungenzeka kakhulu ukuthi kuwumphumela wokususwa okusheshayo kwama-molecule amaprotheni ebusweni angavinjelwanga ngokuphelele esakhiweni sangaphakathi senhlayiya. Ku-pH = 6.5, ama-NP agcwele i-insulin kanye nama-NP e-insulin eyomile aphinde akhiwe abonise ukukhishwa okubushelelezi nokuhamba kancane emahoreni ayi-6, njengoba i-pH yesisombululo sokuhlola yayifana neyesisombululo esilungiselelwe ama-NP (Isithombe 5e). Ku-pH = 7, ama-NP ayengazinzile futhi cishe aqhekeka ngokuphelele emahoreni amabili okuqala (Isithombe 5f). Lokhu kungenxa yokuthi ukususwa kwe-chitosan kwenzeka ku-pH ephezulu, okuholela kunethiwekhi ye-polymer engaqinile kangako kanye nokukhululwa kwe-insulin elayishiwe.
Ngaphezu kwalokho, ama-insulin NPs omiswe ngokufutha ngaphandle kwe-mannitol abonise iphrofayili yokukhululwa okusheshayo kunezinye ama-NPs aphelelwe amanzi emzimbeni (Isithombe 5d–f). Njengoba kuchaziwe ngaphambili, ama-insulin NPs omiswe kabusha ngaphandle kwe-mannitol abonise usayizi wezinhlayiya ezincane kakhulu. Izinhlayiya ezincane zinikeza indawo enkulu, ngakho-ke iningi lomuthi ofanele lizoba seduze noma eduze kobuso bezinhlayiya, okuholela ekukhishweni komuthi ngokushesha26.
Ubuthi be-cytotoxicity be-NPs buhlolwe ngokuhlolwa kwe-MTT. Njengoba kuboniswe kuMfanekiso S4, wonke ama-NP aphelelwe amanzi emzimbeni atholakale engenawo umthelela omkhulu ekusebenzeni kahle kweseli ekugxilweni okungu-50–500 μg/ml, okuphakamisa ukuthi wonke ama-NP aphelelwe amanzi emzimbeni angasetshenziswa ngokuphephile ukufinyelela efasiteleni lokwelapha.
Isibindi yisitho esiyinhloko lapho i-insulin isebenzisa khona imisebenzi yaso yomzimba. Amaseli e-HepG2 awumugqa weseli le-hepatoma lomuntu ovame ukusetshenziswa njengemodeli yokuthatha i-hepatocyte ngaphakathi kwe-vitro. Lapha, amaseli e-HepG2 asetshenziswa ukuhlola ukumuncwa kwamaseli ama-NP aphelelwe amanzi kusetshenziswa izindlela zokuqandisa ngokuqandisa kanye nokomisa ngokufutha. Ukumuncwa kwamaseli ngokuskena kwe-confocal laser kusetshenziswa i-flow cytometry kanye nombono ngemuva kwamahora amaningana okufukamela nge-insulin yamahhala ye-FITC ekugxilweni okungu-25 μg/mL, ama-NP asanda kulungiswa e-FITC afakwe i-insulin kanye nama-NP aphelelwe amanzi e-FITC afakwe i-insulin ekugxilweni okulinganayo kwe-insulin. Kwenziwe ukubonwa kwe-Quantitative microscopy (CLSM). Ama-NP afakwe i-Lyophilized ngaphandle kwe-mannitol abhujiswa ngesikhathi sokuphelelwa amanzi futhi awahlolwanga kulolu vivinyo. Amandla e-fluorescence angaphakathi kweseli ama-NP asanda kulungiswa efakwe i-insulin, ama-NP afakwe i-lyophilized ane-mannitol, kanye nama-NP afakwe i-spray ane-mannitol nangenayo (Isithombe 6a) ayengu-4.3, 2.6, 2.4, kanye Iphakeme ngokuphindwe ka-4.1 kuneyamahhala. Iqembu le-FITC-insulin, ngokulandelana (Isithombe 6b). Le miphumela iphakamisa ukuthi i-insulin ehlanganisiwe inamandla kakhulu ekuthathweni kwamaseli kune-insulin yamahhala, ikakhulukazi ngenxa yobukhulu obuncane bezinhlayiya ezincane ezifakwe i-insulin ezikhiqizwe ocwaningweni.
Ukuthathwa kwamaseli e-HepG2 ngemva kokufakwa kwama-NPs asanda kulungiswa kanye nama-NPs aphelelwe amanzi emzimbeni: (a) Ukusatshalaliswa kokuthathwa kwe-insulin ye-FITC ngamaseli e-HepG2.(b) Isilinganiso se-geometric sokuqina kwe-fluorescence esihlaziywe yi-flow cytometry (n = 3), *P < 0.05 uma kuqhathaniswa ne-insulin yamahhala.
Ngokufanayo, izithombe ze-CLSM zibonise ukuthi amandla e-FITC fluorescence ama-NP asanda kulungiswa e-FITC-insulin-loaded kanye nama-NP omisiwe nge-FITC-insulin-loaded (ngaphandle kwe-mannitol) ayenamandla kakhulu kunalawo amanye amasampula (Isithombe 6a). Ngaphezu kwalokho, ngokufakwa kwe-mannitol, i-viscosity ephezulu yesisombululo yandisa ukumelana nokumuncwa kwamaseli, okwaholela ekunciphiseni ukwanda kwe-insulin. Le miphumela iphakamisa ukuthi ama-NP omisiwe nge-mannitol angenawo ama-spray abonise ukusebenza kahle kakhulu kokumuncwa kwamaseli ngoba usayizi wawo wezinhlayiya wawumncane kunowama-NP omisiwe nge-freeze ngemva kokuncibilika kabusha.
I-Chitosan (isisindo sama-molecule esimaphakathi esingu-100 KDa, esingu-75–85% esingena-acetylated) ithengwe kwa-Sigma-Aldrich. (Oakville, Ontario, Canada). I-Sodium tripolyphosphate (TPP) ithengwe kwa-VWR (Radnor, Pennsylvania, USA). I-insulin yomuntu ehlanganisiwe esetshenziswe kulolu cwaningo yayivela kwa-Fisher Scientific (Waltham, MA, USA). I-insulin yomuntu ene-Fluorescein isothiocyanate (FITC) kanye ne-4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) ithengwe kwa-Sigma-Aldrich. (Oakville, Ontario, Canada). Umugqa weseli we-HepG2 utholakale kwa-ATCC (Manassas, Virginia, USA). Zonke ezinye izithasiselo zaziyizinga lokuhlaziya noma le-chromatographic.
Lungisa isixazululo se-1 mg/ml se-CS ngokusincibilikisa emanzini acwecwe kabili (amanzi e-DD) aqukethe i-0.1% acetic acid. Lungisa izixazululo ze-1 mg/ml ze-TPP kanye ne-insulin ngokuzincibilikisa emanzini e-DD kanye ne-0.1% acetic acid, ngokulandelana. I-pre-emulsion yalungiswa nge-polytron PCU-2-110 high speed homogenizer (Brinkmann Ind. Westbury, NY, USA). Inqubo yokulungiselela imi kanje: okokuqala, isixazululo se-2ml se-TPP singezwa ku-4ml wesisombululo se-insulin, bese ingxube ivuselelwa imizuzu engama-30 bese ixutshwa ngokuphelele. Ngemuva kwalokho, isixazululo esixubile sangezwa ngokuwa phansi kwisisombululo se-CS ngesirinji ngaphansi kokuvuselelwa okusheshayo (10,000 rpm). Izingxube zigcinwe ngaphansi kokuvuselelwa okusheshayo (15,000 rpm) ebhavini leqhwa imizuzu engama-30, futhi zalungiswa ku-pH ethile ukuze kutholakale ama-NP e-insulin axhumene. Ukuze kuqhutshekwe kuhlanganiswe futhi kuncishiswe usayizi wezinhlayiya ze-insulin NPs, zafakwa i-sonication ukuze kutholakale okunye. Imizuzu engama-30 ebhavini leqhwa usebenzisa i-sonicator yohlobo lwe-probe (UP 200ST, Hielscher Ultrasonics, Teltow, Germany).
I-Insulin NPS ihlolwe ububanzi obuphakathi be-Z, i-polydispersity index (PDI) kanye ne-zeta potential kusetshenziswa izilinganiso ze-dynamic light scattering (DLS) kusetshenziswa i-Litesizer 500 (Anton Paar, Graz, Austria) ngokuyixuba emanzini e-DD ku-25°C. Ukwakheka kanye nokusatshalaliswa kosayizi kwaphawulwa yi-Hitachi H7600 transmission electron microscope (TEM) (Hitachi, Tokyo, Japan), futhi izithombe zahlaziywa kamuva kusetshenziswa isofthiwe ye-Hitachi imaging (Hitachi, Tokyo, Japan). Ukuze kuhlolwe ukusebenza kahle kwe-encapsulation (EE) kanye nomthamo wokulayisha (LC) we-insulin NPs, ama-NP afakwa ngamapayipi kumashubhu e-ultrafiltration ane-molecule weight cut-off engu-100 kDa futhi afakwa ku-centrifuge ku-500 xg imizuzu engama-30. I-insulin engafakwanga ku-filtrate yalinganiswa kusetshenziswa uhlelo lwe-Agilent 1100 Series HPLC (Agilent, Santa Clara, California, USA) oluqukethe i-quaternary pump, i-autosampler, ikholomu. i-heater, kanye ne-DAD detector. I-insulin ihlaziywe ngekholamu ye-C18 (i-Zorbax, 3.5 μm, 4.6 mm × 150 mm, e-Agilent, e-USA) futhi yatholakala ku-214 nm. Isigaba esiphathwayo sasiyi-acetonitrile namanzi, aqukethe i-0.1% TFA, izilinganiso ze-gradient kusuka ku-10/90 kuya ku-100/0, futhi sasebenza imizuzu eyi-10. Isigaba esiphathwayo sapompa ngesivinini sokugeleza esingu-1.0 ml/min. Izinga lokushisa lekholamu labekwa ku-20 °C. Bala amaphesenti e-EE kanye ne-LC usebenzisa izilinganiso.(1) kanye ne-Eq.(2).
Izilinganiso ezahlukene ze-CS/insulin ezisukela ku-2.0 kuya ku-4.0 zahlolwa ukuze kuthuthukiswe i-insulin NP. Amanani ahlukene esisombululo se-CS angeziwe ngesikhathi sokulungiselela, kuyilapho ingxube ye-insulin/TPP igcinwa ingaguquguquki. Ama-Insulin NP alungiswa kububanzi be-pH obungu-4.0 kuya ku-6.5 ngokulawula ngokucophelela i-pH yengxube ngemva kokufaka zonke izixazululo (i-insulin, i-TPP kanye ne-CS). I-EE kanye nosayizi wezinhlayiya ze-insulin nanoparticles kwahlolwa ngamanani ahlukene e-pH kanye nezilinganiso ze-CS/insulin mass ukuze kuthuthukiswe ukwakheka kwama-insulin NPs.
Ama-insulin NP alungiselelwe kahle abekwa esitsheni se-aluminium futhi ambozwa ngezicubu eziqinisiwe ngeteyipu ethile. Ngemva kwalokho, izitsha ezifakwe izikulufo zafakwa kumshini wokomisa iqhwa iLabconco FreeZone (eLabconco, eKansas City, MO, e-USA) ohlonyiswe ngomshini wokomisa ithreyi. Izinga lokushisa kanye nomfutho we-vacuum kwabekwa ku--10 °C, 0.350 Torr emahoreni amabili okuqala, kanye no-0 °C kanye no-0.120 Torr emahoreni angama-22 asele emahoreni angama-24 ukuthola ama-insulin NPs amile.
I-Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Switzerland) yasetshenziswa ukukhiqiza i-insulin ehlanganisiwe. Amapharamitha okomisa akhethiwe ayengu: izinga lokushisa elingu-100 °C, ukugeleza kokudla okungu-3 L/min, kanye nokugeleza kwegesi okungu-4 L/min.
Ama-Insulin NP ngaphambi nangemva kokuphelelwa ngamanzi emzimbeni ahlonzwe kusetshenziswa i-FTIR-ATR spectroscopy. Ama-nanoparticles aphelelwe amanzi emzimbeni kanye ne-insulin yamahhala kanye ne-chitosan kwahlaziywa kusetshenziswa i-Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA) ehlonyiswe nge-ATR sampling accessory (PerkinElmer, Waltham, Massachusetts, USA). Izilinganiso zesiginali zitholwe kuma-scan angu-16 ngesisombululo esingu-4 cm2 ebangeni lemvamisa elingu-4000-600 cm2.
Ukwakheka kwama-NP e-insulin eyomile kuhlolwe ngezithombe ze-SEM zama-NP e-insulin eyomisiwe ngokuqandisa kanye neyomisiwe ngokufuthwa ethathwe yi-Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, USA). Ipharamitha eyinhloko esetshenzisiwe kwakuyi-voltage engu-5 keV kanye ne-current engu-30 mA.
Zonke i-insulin NPs eziphelelwe amanzi zaphinde zancibilikiswa emanzini ahlanzekile. Usayizi wezinhlayiya, i-PDI, i-EE kanye ne-LC kwahlolwa futhi kusetshenziswa indlela efanayo okukhulunywe ngayo ngaphambili ukuhlola ikhwalithi yazo ngemva kokuphelelwa amanzi. Ukuqina kwama-anhydroinsulin NPs kwalinganiswa ngokuhlola izakhiwo zama-NPs ngemva kokugcinwa isikhathi eside. Kulolu cwaningo, wonke ama-NPs ngemva kokuphelelwa amanzi agcinwe esiqandisini izinyanga ezintathu. Ngemva kwezinyanga ezintathu zokugcina, ama-NPs ahlolwa usayizi wezinhlayiya ze-morphological, i-PDI, i-EE kanye ne-LC.
Ncibilikisa ama-5 mL ama-NP aphinde ahlanganiswa ku-45 mL aqukethe uketshezi lwesisu olulinganisiwe (pH 1.2, oluqukethe i-1% pepsin), uketshezi lwamathumbu (pH 6.8, oluqukethe i-1% trypsin) noma isixazululo se-chymotrypsin (100 g/mL, ku-phosphate buffer, pH 7.8) ukuze kuhlolwe ukusebenza kahle kwe-insulin ekuvikeleni ama-NP ngemva kokuphelelwa amanzi emzimbeni. Afakwe e-incubator ku-37°C ngesivinini sokushukuma esingu-100 rpm. I-500 μL yesisombululo yaqoqwa ngezikhathi ezahlukene futhi ukuhlushwa kwe-insulin kwanqunywa yi-HPLC.
Ukuziphatha kokukhishwa kwe-insulin NPs esanda kulungiswa futhi eyomile kuhlolwe ngendlela yesikhwama se-dialysis (ukusika isisindo sama-molecule 100 kDa, i-Spectra Por Inc.). Ama-NPs omile asanda kulungiswa futhi avuselelwa afakwe ku-dialysis ku-pH 2.5, pH 6.6, kanye ne-pH 7.0 (0.1 M phosphate-buffered saline, PBS) ukuze kulingiswe indawo ye-pH yesisu, i-duodenum, kanye ne-upper small intestine, ngokulandelana. Wonke amasampula afakwa ku-37 °C ngokunyakaziswa okuqhubekayo ku-200 rpm. Phefumula uketshezi ngaphandle kwesikhwama se-dialysis esingu-5 mL ngezikhathi ezilandelayo: 0.5, 1, 2, 3, 4, kanye namahora angu-6, bese ugcwalisa ngokushesha ivolumu nge-dialysate entsha. Ukungcola kwe-insulin ku-fluid kwahlaziywa yi-HPLC, futhi izinga lokukhishwa kwe-insulin kusuka kuma-nanoparticles labalwa kusukela kusilinganiso se-insulin yamahhala ekhishwe ku-insulin ephelele ehlanganiswe kuma-nanoparticles (Isibalo 3).
Amaseli e-HepG2 ohlobo lwe-hepatocellular carcinoma cell line yabantu akhuliswe ezitsheni ezingama-60 mm ububanzi kusetshenziswa i-Dulbecco's Modified Eagle's Medium (DMEM) equkethe i-10% ye-fetal bovine serum, i-penicillin engu-100 IU/mL, kanye ne-100 μg/mL streptomycin. Ama-cultures agcinwe ku-37°C, umswakama ongu-95%, kanye ne-5% CO2. Ukuhlolwa kokuthathwa, amaseli e-HepG2 ahlwanyelwe kumaseli angu-1 × 105/ml ohlelweni lwesilayidi se-Nunc Lab-Tek chamber olunezimbobo ezingu-8 (Thermo Fisher, NY, USA). Ukuhlolwa kwe-cytotoxicity, ahlwanyelwe kumapuleti e-96-well (Corning, NY, USA) ngobuningi bamaseli angu-5 × 104/ml.
Ukuhlolwa kwe-MTT kusetshenziswe ukuhlola ubuthi be-insulin NPs30 esanda kulungiswa futhi eyomile. Amaseli e-HepG2 ahlwanyelwa emapuletini angu-96-well ku-density yamaseli angu-5 × 104/mL futhi akhuliswa izinsuku eziyi-7 ngaphambi kokuhlolwa. Ama-insulin NPs ancibilikiswa kumazinga ahlukahlukene (50 kuya ku-500 μg/mL) endaweni yokukhulisa bese enikezwa amaseli. Ngemva kwamahora angu-24 okufukamela, amaseli agezwa izikhathi ezi-3 nge-PBS futhi afukamela endaweni equkethe i-0.5 mg/ml MTT amahora ama-4 engeziwe. Ubuthi be-cytotoxicity buhlolwe ngokulinganisa ukwehla kwe-enzyme kwe-tetrazolium MTT ephuzi kuya ku-purple formazan ku-570 nm kusetshenziswa i-Tecan infinite M200 pro spectrophotometer plate reader (Tecan, Männedorf, Switzerland).
Ukusebenza kahle kokuthathwa kwe-NPs ngamaseli kuhlolwe nge-confocal laser scanning microscopy kanye nokuhlaziywa kwe-flow cytometry. Umthombo ngamunye wesistimu yesilayidi segumbi le-Nunc Lab-Tek waphathwa nge-free FITC-insulin, i-FITC-insulin-loaded NPs, futhi wavuselela ama-25 μg/mL ama-FITC-insulin NPs aphelelwe amanzi emzimbeni ngesilinganiso esifanayo futhi afakwa amahora ama-4. Amaseli agezwa izikhathi ezi-3 nge-PBS futhi aqiniswa nge-4% paraformaldehyde. I-Nuclei yangcoliswa nge-4′,6-diamidino-2-phenylindole (DAPI). Ukutholakala kwe-insulin kwabonwa kusetshenziswa i-Olympus FV1000 laser scanning/two-photon confocal microscope (Olympus, Shinjuku City, Tokyo, Japan). Ekuhlaziyweni kwe-flow cytometry, amazinga afanayo e-10 μg/mL free FITC-insulin, i-FITC-insulin-loaded NPs, kanye ne-resolubilized FITC-insulin-dehydrated FITC-insulin. Ama-NP afakwe kumapuleti emithombo engu-96 ahlwanyelwe ngamaseli e-HepG2 futhi afakwa esikhumbeni amahora angu-4. Ngemva kwamahora angu-4 okufuya, amaseli asuswa futhi agezwa izikhathi ezintathu nge-FBS. Amaseli angu-5 × 104 ngesampula ngayinye ahlaziywa yi-BD LSR II flow cytometer (BD, Franklin Lakes, New Jersey, United States).
Zonke izindinganiso zivezwa njengokuphambuka okujwayelekile okungu-± okujwayelekile. Ukuqhathaniswa phakathi kwawo wonke amaqembu kuhlolwe kusetshenziswa i-ANOVA noma i-t-test eyodwa yi-IBM SPSS Statistics 26 ye-Mac (IBM, Endicott, New York, USA) kanye ne-p < 0.05 kwabhekwa njengokubalulekile ngokwezibalo.
Lolu cwaningo lubonisa ukuguquguquka kanye nekhono lokomisa ngokufutha ukuze kukhishwe amanzi kuma-nanoparticles e-chitosan/TPP/insulin axhumene ne-cross-linked nge-reconstruction engcono uma kuqhathaniswa nezindlela ezijwayelekile zokomisa ngokufutha kusetshenziswa ama-bulking agents noma amandla e-cryoprotectants kanye nomthamo ophezulu womthwalo. Ama-nanoparticles e-insulin alungiselelwe kahle akhiqize usayizi wezinhlayiya omaphakathi ongu-318 nm kanye nokusebenza kahle kwe-encapsulation okungu-99.4%. Imiphumela ye-SEM kanye ne-FTIR ngemuva kokuphelelwa amanzi emzimbeni ibonise ukuthi isakhiwo esiyindilinga sigcinwe kuphela kuma-NP omiswe ngokufutha ane-mannitol futhi angenayo futhi afakwe i-lyophilized nge-mannitol, kodwa ama-NP afakwe i-lyophilized ngaphandle kwe-mannitol abolile ngesikhathi sokuphelelwa amanzi emzimbeni. Ekuhlolweni kwamandla okuvuselelwa, ama-nanoparticles e-insulin afakwe i-spray ngaphandle kwe-mannitol abonise usayizi wezinhlayiya omaphakathi omncane kakhulu kanye nomthwalo ophezulu kakhulu lapho kuvuselelwa. Ukuziphatha kokukhululwa kwawo wonke la ma-NP aphelelwe amanzi emzimbeni kubonise ukuthi akhululwa ngokushesha ezixazululweni ze-pH = 2.5 kanye ne-pH = 7, futhi azinzile kakhulu esixazululweni se-pH = 6.5. Uma kuqhathaniswa namanye ama-NP aphelelwe amanzi emzimbeni aphinde ancibilika, ama-NP afakwe i-spray ngaphandle I-mannitol ibonise ukukhishwa okusheshayo. Lo mphumela uhambisana nalokho okubonwe ekuhlolweni kokuthathwa kweseli, njengoba ama-NP omisiwe ngokufuthwa ngaphandle kwe-mannitol cishe agcine ukusebenza kahle kokuthathwa kweseli kwama-NP asanda kulungiselelwa. Le miphumela iphakamisa ukuthi ama-nanoparticles e-insulin eyomile alungiselelwe ukomiswa kwesifutho okungena-mannitol afaneleka kakhulu ukucutshungulwa okwengeziwe kwezinye izinhlobo zomthamo ongenamanzi, njengamaphilisi omlomo noma amafilimu okunamathela.
Ngenxa yezinkinga zempahla yobuhlakani, amasethi edatha akhiqizwe kanye/noma ahlaziywe ngesikhathi socwaningo lwamanje awatholakali emphakathini, kodwa ayatholakala kubabhali abafanele uma kucelwa ngokufanele.
Kagan, A. Isifo sikashukela sohlobo 2: imvelaphi yezenhlalo neyesayensi, izinkinga zezokwelapha, kanye nemiphumela ezigulini nakwabanye. (McFarlane, 2009).
Singh, AP, Guo, Y., Singh, A., Xie, W. & Jiang, P. Ukuthuthukiswa kwe-insulin encapsulation: ingabe ukuphathwa ngomlomo manje kungenzeka? J. Pharmacy.bio-pharmacy.reservoir.1, 74–92 (2019).
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