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Kua kitea ngā whakamahinga rerekē o ngā matūriki nano o te insulin (NP) he nui te ihirangi utaina i roto i ngā momo horopeta rerekē. Ko te whāinga o tēnei mahi he aromatawai i te pānga o ngā tukanga whakamaroke-tio me te whakamaroke-rehu ki te hanganga o ngā matūriki nano chitosan utaina-insulin, me te mannitol, kāore rānei hei ārai-kiri. I aromatawaihia hoki e mātou te kounga o ēnei matūriki nano mā te whakarewa anō i a rātou. I mua i te whakamaroketanga, i arotauhia te rahi o te matūriki o ngā nano chitosan/sodium tripolyphosphate/insulin cross-linked kia 318 nm, ko te PDI he 0.18, ko te whai huatanga o te encapsulation he 99.4%, ā, ko te utaina he 25.01%. I muri i te hanganga anō, ko ngā matūriki nano katoa, engari ko ērā i hangaia e te tikanga whakamaroke-tio me te kore e whakamahi i te mannitol, i mau tonu tō rātou hanganga matūriki porowhita. Ki te whakatauritea ki ngā matūriki kei roto te mannitol i whakamarokehia e tētahi o ngā rehu, ko ngā matūriki rehu-maroke kore-mannitol i whakaatu hoki i te rahi matūriki toharite iti rawa (376 nm) me te ihirangi utaina teitei rawa. (25.02%) me te tere whakakī rite (98.7%) me te PDI (0.20) mā te whakamaroke, mā te whakamaroke tio rānei. Ko ngā matūriki nano maroke mā te whakamaroke rehu me te kore mannitol i hua ake ai te tukunga tere o te insulin me te tino whai huatanga o te tango pūtau. E whakaatu ana tēnei mahi ka taea e te whakamaroke rehu te whakamaroke i ngā matūriki nano insulin me te kore e hiahiatia he cryoprotectants ki te whakataurite ki ngā tikanga whakamaroke tio tuku iho, ka nui ake te kaha uta, ka iti ake ngā whakaritenga tāpiri me ngā utu whakahaere he painga nui.
Mai i tōna kitenga i te tau 19221,2,3, kua ora ngā tūroro e pangia ana e te mate huka momo 1 (T1DM) me te mate huka momo 2 (T1DM) i te insulin me āna rongoā. Heoi, nā ōna āhuatanga hei pūmua taumaha ngota teitei, ka ngāwari te whakakotahi i te insulin, ka pakaruhia e ngā whākōkī proteolytic, ā, ka whakakorea mā te pānga tuatahi. Ko te hunga kua tātaitia he mate huka momo 1 me werohanga insulin mō te toenga o ō rātou oranga. He maha ngā tūroro i tātaitia tuatahitia he mate huka momo 2 me werohanga insulin mō te wā roa. Ko ngā werohanga insulin o ia rā he pūtake nui o te mamae me te kore whakamarie o ia rā mō ēnei tāngata, me ngā pānga kino ki te hauora hinengaro. Nō reira, kei te rangahauhia whānuitia ētahi atu momo whakahaere insulin e kore e nui ake te mamae, pērā i te whakahaere insulin ā-waha,5 nā te mea he kaha ki te whakahoki mai i te kounga o te oranga o te tata ki te 5 piriona tāngata e pangia ana e te mate huka puta noa i te ao.
Kua whakaratohia e te hangarau matūriki nano tētahi whanaketanga nui i roto i ngā ngana ki te tango i te insulin ā-waha4,6,7. He mea e whai hua ana ki te whakauru me te tiaki i te insulin mai i te pirau kia tukuna ki ngā wāhi tinana motuhake. Heoi, he maha ngā herenga o te whakamahinga o ngā hanganga matūriki nano, ko te nuinga nā ngā take pumau o ngā whakatārewatanga matūriki. Tērā pea ka puta ētahi whakahiato i te wā rokiroki, ka whakaiti i te waatea o ngā matūriki nano kua utaina ki te insulin8. Hei tāpiri, me whai whakaaro hoki ki te pumau matū o te matrix polymer o ngā matūriki nano me te insulin kia mau ai te pumau o ngā matūriki nano insulin (NP). I tēnei wā, ko te hangarau whakamaroke-tio te paerewa koura mō te hanga NP pumau me te ārai i ngā huringa kāore e hiahiatia i te wā rokiroki9.
Heoi, me tāpirihia ngā matū tiaki-whakatio hei ārai i te hanganga porowhita o ngā NP kei pāngia e te ahotea ā-pūkaha o ngā tioata hukapapa. Ka whakaitihia nuitia e tēnei te utaina o ngā matūriki nano insulin i muri i te lyophilization, nā te mea ko te nuinga o te ōwehenga taumaha kei roto i te matū tiaki-whakatio. Nō reira, he maha ngā wā ka kitea kāore e tika ana ngā NP insulin kua hangaia mō te hanga i ngā whakatakotoranga paura maroke, pērā i ngā papa ā-waha me ngā kiriata ā-waha, nā te mea e hiahiatia ana he nui ngā matūriki maroke hei whakatutuki i te matapihi rongoā o te insulin.
He tukanga ahumahi rongonui, he iti hoki te utu te whakamaroke rehu, mō te whakaputa paura maroke mai i ngā āhua wai i roto i te umanga rongoā10,11. Mā te whakahaere i te tukanga hanganga matūriki ka taea te whakakī tika i ētahi matū koiora 12, 13. Hei tāpiri, kua noho hei tikanga whai hua mō te whakarite i ngā pūmua kua whakakīia mō te whakahaere ā-waha. I te wā whakamaroke rehu, ka tere te mimiti o te wai, ka āwhina i te pupuri i te pāmahana o te matua matūriki kia iti11,14, ka taea ai te whakamahi hei whakakī i ngā wāhanga tairongo-wera. I mua i te whakamaroke rehu, me tino whakaranu te rauemi paninga ki te otinga kei roto ngā kai kua whakakīia11,14. Kāore i rite ki te whakamaroke tio, ko te whakaranu i mua i te whakakī i roto i te whakamaroke rehu ka whakapai ake i te whai huatanga o te whakakī i te wā maroke. Nā te mea kāore e hiahiatia he cryoprotectants mō te tukanga whakakī whakamaroke rehu, ka taea te whakamahi i te whakamaroke rehu hei whakaputa i ngā NP maroke me te ihirangi utaina teitei.
E pūrongo ana tēnei rangahau i te hanga o ngā NP kua utaina ki te insulin mā te whakawhiti i te chitosan me te sodium tripolyphosphate mā te whakamahi i te tikanga ion gel. Ko te ion gelation he tikanga whakarite e āhei ai te hanga o ngā nanoparticles mā te taunekeneke hiko i waenga i ngā momo ionic e rua, neke atu rānei, i raro i ētahi tikanga. I whakamahia ngā tikanga whakamaroke-tio me te whakamaroke-rehu hei whakamaroke i ngā nanoparticles kua whakawhitia ki te chitosan/sodium tripolyphosphate/insulin kua arotauhia. I muri i te whakamaroketanga, i tātarihia ō rātou āhua mā te SEM. I aromatawaihia ō rātou kaha whakakotahi mā te ine i tō rātou tohatoha rahi, te utu mata, te PDI, te pai o te whakakī, me te ihirangi uta. I aromatawaihia hoki te kounga o ngā nanoparticles kua whakarewahia i hangaia e ngā tikanga whakamaroke rerekē mā te whakataurite i ō rātou tiaki insulin, te whanonga tuku, me te whai huatanga o te tango pūtau.
Ko te pH o te otinga whakauru me te ōwehenga o te chitosan me te insulin he mea matua e rua e pā ana ki te rahi o te matūriki me te whai huatanga o te encapsulation (EE) o ngā NP whakamutunga, nā te mea ka pā tika ki te tukanga ionotropic gelation. I whakaatuhia te hononga nui o te pH o te otinga whakauru ki te rahi o te matūriki me te whai huatanga o te encapsulation (Pikitia 1a). E ai ki te Pikitia 1a, i te pikinga o te pH mai i te 4.0 ki te 6.0, ka heke te rahi toharite o te matūriki (nm) ā, ka piki nui te EE, i te pikinga o te pH ki te 6.5, ka tīmata te rahi toharite o te matūriki ki te piki ake, ā, kāore i rerekē te EE. I te pikinga o te ōwehenga o te chitosan ki te insulin, ka piki ake hoki te rahi toharite o te matūriki. I tua atu, kāore i kitea he huringa i roto i te EE i te wā i whakaritea ai ngā nanoparticles i te ōwehenga papatipu o te chitosan/insulin neke atu i te 2.5:1 (w/w) (Pikitia 1b). Nō reira, ko ngā tikanga whakarite pai rawa atu i roto i tēnei rangahau (pH 6.0, ōwehenga papatipu chitosan/insulin o te 2.5:1) i whakamahia hei te whakarite i ngā matūriki nano kua utaina ki te insulin mō ētahi atu rangahau. I raro i tēnei āhuatanga whakarite, i arotauhia te rahi toharite o ngā matūriki nano insulin kia 318 nm (Pikitia 1c), ko te PDI he 0.18, ko te whai huatanga whakauru he 99.4%, ko te pūmanawa zeta he 9.8 mv, ā, ko te uta insulin he 25.01% (m/m). I runga i ngā hua o te karu hiko tuku (TEM), he porowhita, he motumotu hoki ngā matūriki nano kua arotauhia, he rite tonu te rahi (Pikitia 1d).
Te arotautanga o ngā tawhā o ngā matūriki nano o te insulin: (a) te pānga o te pH ki te diameter toharite me te whai huatanga o te whakakī (EE) o ngā matūriki nano o te insulin (i whakaritea i te ōwehenga papatipu 5:1 o te chitosan me te insulin); (b) te chitosan me te awe o te ōwehenga papatipu o te insulin ki te diameter toharite me te whai huatanga o te whakakī (EE) o ngā NP insulin (i whakaritea i te pH 6); (c) te tohatoha rahi matūriki o ngā matūriki nano o te insulin kua arotauhia; (d) te whakaahua iti TEM o ngā NP insulin kua arotauhia.
E mōhiotia whānuitia ana ko te chitosan he polyelectrolyte ngoikore me te pKa o te 6.5. He pai te utu i roto i te pāpāho waikawa nā te mea ko tōna rōpū amino matua he ion hauwai15. Nō reira, he maha ngā wā ka whakamahia hei kawe hei whakakī i ngā macromolecules kua utu kino. I roto i tēnei rangahau, i whakamahia te chitosan hei whakakī i te insulin me te pūwāhi isoelectric o te 5.3. Mai i te mea e whakamahia ana te chitosan hei rauemi paninga, me te pikinga o tōna ōwehenga, ka piki ake te matotoru o te paparanga o waho o ngā nanoparticles, ka hua ake he rahi matūriki toharite nui ake. Hei tāpiri, ka taea e ngā taumata teitei ake o te chitosan te whakakī i te insulin. I roto i tā mātou take, ko te EE te mea teitei rawa atu i te wā i tae ai te ōwehenga o te chitosan me te insulin ki te 2.5:1, ā, kāore he huringa nui i roto i te EE i te wā i piki haere tonu ai te ōwehenga.
Haunga te ōwehenga o te chitosan me te insulin, he mea nui anō hoki te pH i roto i te whakarite o ngā NP. I rangahau a Gan et al. 17 i te pānga o te pH ki te rahi o te matūriki o ngā nanoparticles chitosan. I kitea he hekenga tonu o te rahi o te matūriki tae noa ki te pH ki te 6.0, ā, i kitea he pikinga nui o te rahi o te matūriki i te pH > 6.0, he rite ki ā mātou kitenga. Ko te take o tēnei āhuatanga ko te pikinga o te pH, ka whiwhi te ngota insulin i tētahi utu mata kino, nō reira, ka pai ake ngā taunekeneke hiko me te matatini chitosan/sodium tripolyphosphate (TPP), ka hua ake he rahi matūriki iti me te EE teitei. Heoi, i te whakatikatikaina o te pH ki te 6.5, ka tangohia te proton i ngā rōpū amino i runga i te chitosan, ka hua ake he takai chitosan. Nō reira, ko te pH teitei ka hua ake he iti ake te whakaaturanga o ngā katote amino ki te TPP me te insulin, ka hua ake he iti ake te hononga whakawhiti, he nui ake te rahi matūriki toharite whakamutunga me te EE iti iho.
Mā te tātari i ngā āhuatanga āhua o ngā NP maroke-tio me ngā NP rehu-maroke ka taea te ārahi i te kōwhiringa o ngā tikanga whakamaroke me te hanga paura pai ake. Ko te tikanga pai me whakarato i te pumau o te rongoā, te āhua matūriki ōrite, te uta rongoā teitei me te wairewa pai i roto i te otinga taketake. I roto i tēnei rangahau, kia pai ake ai te whakataurite i ngā tikanga e rua, i whakamahia ngā NP insulin me te 1% mannitol, kāore rānei i te 1% mannitol i te wā whakamaroke. Ka whakamahia te Mannitol hei kaihoko whakapūmau, hei tiaki-whakamaroke rānei i roto i ngā momo whakatakotoranga paura maroke mō te whakamaroke-tio me te whakamaroke-rehu. Mō ngā matūriki insulin lyophilized kāore he mannitol, e whakaaturia ana i te Pikitia 2a, i kitea he hanganga paura tino porowhita me ngā mata nui, kāore i te rite, me te taratara i raro i te karu hiko matawai (SEM). He iti noa ngā matūriki motuhake i kitea i roto i te paura i muri i te whakamaroke (Pikitia 2e). I tohu ēnei hua ko te nuinga o ngā NP i pakaru i te wā whakamaroke-tio me te kore he tiaki-whakamaroke. Mō ngā matūriki insulin maroke-tio me te rehu-maroke kei roto he 1% mannitol, i kitea ngā matūriki porowhita me ngā mata maeneene (Pikitia 1). 2b,d,f,h). Ko ngā matūriki nano o te inihua i whakamaroketia ki te wai me te kore mannitol i noho porowhita engari he miro i runga i te mata (Pikitia 2c). Ka matapakihia anō ngā mata porowhita me ngā mata miro i roto i ngā whakamātautau whanonga tuku me te tango pūtau i raro nei. I runga i te āhua kitea o ngā NP maroke, ko ngā NP i whakamaroketia ki te wai me te kore mannitol me ngā NP i whakamaroketia ki te tio me te mannitol i puta ai he paura NP pai (Pikitia 2f,g,h). Ko te nui ake o te horahanga mata i waenga i ngā mata matūriki, ko te teitei ake o te wairewa, ā, ko te teitei ake o te tere tuku.
Ngā āhua o ngā momo NP insulin maroke: (a) Whakaahua SEM o ngā NP insulin kua whakamaroketia, kāore he mannitol; (b) Whakaahua SEM o ngā NP insulin kua whakamaroketia me te mannitol; (c) Ngā NP insulin kua rehu-maroketia, kāore he mannitol Whakaahua SEM o ; (d) Whakaahua SEM o ngā NP insulin kua rehu-maroketia ki te mannitol; (e) Whakaahua o te paura NP insulin kua rehu-maroketia, kāore he mannitol; (f) Whakaahua o ngā NP insulin kua rehu-maroketia me te mannitol; (g) Whakaahua o te paura NP insulin kua rehu-maroketia, kāore he mannitol; (h) Whakaahua o te paura NP insulin kua rehu-maroketia me te mannitol.
I te wā e whakamaroke ana i te tio, ka mahi te mannitol hei ārai i te wai, ka pupuri i ngā NP i roto i te āhua kore-āhua, ā, ka ārai i te kino o ngā tioata hukapapa19. He rerekē, kāore he taahiraa tio i te wā e whakamaroke ana i te rehu. Nō reira, kāore e hiahiatia te mannitol i roto i tēnei tikanga. Inaa, ko ngā NP i whakamarokehia ki te rehu kāore he mannitol i puta he NP pai ake e ai ki te whakaahuatanga o mua. Heoi, ka taea tonu e te mannitol te mahi hei whakakī i roto i te tukanga whakamaroke rehu hei hoatu i te hanganga porowhita ki ngā NP20 (Pikitia 2d), e āwhina ana ki te whiwhi i te whanonga tuku ōrite o aua NP kua kapihia. Hei tāpiri, he mārama ka taea te kite i ētahi matūriki nui i roto i ngā NP insulin kua whakamarokehia ki te tio me te rehu kua whakamarokehia kei roto te mannitol (Pikitia 2b,d), tera pea nā te kohikohinga o te mannitol i roto i te matua matūriki me te insulin kua kapihia. Ki. Paparanga chitosan. He mea tika kia mōhiotia i roto i tēnei rangahau, kia mau tonu ai te hanganga porowhita i muri i te whakamaroketanga, ka puritia te ōwehenga o te mannitol me te chitosan ki te 5:1, kia taea ai e te nui o te whakakī te whakanui ake i te rahi o te matūriki o ngā NP maroke.
Nā te tātaritanga whakaata katoa ā-iraira whatuwhatu (FTIR-ATR) i whakaahua te ranunga ā-tinana o te insulin kore utu, te chitosan, te chitosan, te TPP me te insulin. I whakaahuahia ngā NP maroke katoa mā te whakamahi i te tātaritanga FTIR-ATR. He mea nui, i kitea ngā kaha o te roopu o te 1641, 1543 me te 1412 cm-1 i roto i ngā NP kua whakakīia kua whakamaroketia ki te mannitol me ngā NP kua whakamaroketia ki te mannitol me te kore mannitol (Pikitia 3). E ai ki te pūrongo o mua, i honoa ēnei pikinga kaha ki te hononga whakawhiti i waenga i te chitosan, te TPP me te insulin. I whakaatuhia e te rangahau o te taunekeneke i waenga i te chitosan me te insulin i roto i ngā tātaritanga FTIR o ngā matūriki chitosan kua utaina ki te insulin, i taupatupatu te roopu chitosan ki te roopu o te insulin, ka whakanui ake i te kaha o te carbonyl (1641 cm-1) me te whitiki amine (1543 cm-1). Ko ngā rōpū tripolyphosphate o te TPP e honoa ana ki ngā rōpū ammonium i roto i te chitosan, ka hanga he roopu. i te 1412 cm-1.
Ngā irahiko FTIR-ATR o te insulin kore utu, te chitosan, ngā ranunga ā-tinana o te chitosan/TPP/insulin me ngā NP i whakamaroketia mā ngā tikanga rerekē.
I tua atu, he rite tonu ēnei hua ki ērā i whakaaturia i roto i te SEM, i whakaatu i te noho pūmau o ngā NP kua whakakīia i te wā i rehuhia ai, i whakamarokehia ai hoki ki te mannitol, engari i te korenga o te mannitol, ko te whakamaroke-rehu anake i puta ai ngā matūriki kua whakakīia. He rerekē, ko ngā hua tirohanga FTIR-ATR o ngā NP i whakamaroke-rehu me te kore mannitol he tino rite ki te ranunga ā-tinana o te chitosan, TPP, me te insulin. E tohu ana tēnei hua kāore ngā hononga whakawhiti i waenga i te chitosan, TPP me te insulin e kitea ana i roto i ngā NP i whakamaroke-rehu me te kore mannitol. I whakangaromia te hanganga NP i te wā i whakamaroke-rehu me te kore cryoprotectant, ka kitea i roto i ngā hua SEM (Pikitia 2a). I runga i te āhua me ngā hua FTIR o ngā NP insulin kua whakamarokehia, ko ngā NP lyophilized, i whakamaroke-rehu, me te kore mannitol anake i whakamahia mō ngā whakamātautau whakahou me ngā NP kore mannitol nā te pirau o ngā NP kore mannitol i te wā i whakamarokehia ai. matapakihia.
Ka whakamahia te whakamaroke mō te rokiroki mō te wā roa me te tukatuka anō ki ētahi atu whakatakotoranga. He mea nui te kaha o ngā NP maroke ki te hanga anō i muri i te rokiroki mō tā rātou whakamahinga i roto i ngā whakatakotoranga rerekē pēnei i ngā papa me ngā kiriata. I kite mātou ko te rahi matūriki toharite o ngā NP insulin i whakamaroketia i te korenga o te mannitol i piki ake i muri i te hanga anō. I tētahi atu taha, ko te rahi matūriki o ngā nanoparticles insulin i whakamaroketia me te tio-maroke me te mannitol i piki ake nui (Ripanga 1). Kāore i tino rerekē te PDI me te EE (p > 0.05) i muri i te whakakotahitanga o ngā NP katoa i roto i tēnei rangahau (Ripanga 1). E whakaatu ana tēnei hua i noho tonu te nuinga o ngā matūriki i muri i te rewa anō. Heoi, ko te tāpiri o te mannitol i hua ake ai te tino whakaiti i te utaina o te insulin o ngā nanoparticles mannitol lyophilized me te rehu-maroke (Ripanga 1). He rerekē, ko te ihirangi utaina o te insulin o ngā NP i whakamaroketia me te kore mannitol i noho tonu ki te rite ki mua (Ripanga 1).
E mōhiotia whānuitia ana he mea nui te utaina o ngā matūriki nano ina whakamahia mō ngā kaupapa tuku rongoā. Mō ngā NP he iti ngā uta, he nui rawa ngā rauemi e hiahiatia ana kia eke ki te paepae rongoā. Heoi, ko te matotoru teitei o aua kukū NP teitei ka arahi ki te whakararuraru me te uaua ki te whakahaere ā-waha me ngā whakatakotoranga werohanga, ia 22. Hei tāpiri, ka taea hoki te whakamahi i ngā NP insulin ki te hanga papa me ngā biofilm matotoru23, 24, e hiahia ana kia whakamahia he nui ngā NP i ngā taumata uta iti, ka hua ake he papa nui me ngā biofilm matotoru kāore e pai mō ngā tono ā-waha. Nō reira, he tino hiahiatia ngā NP maroke me te kawenga insulin teitei. E kī ana ā mātou hua he maha ngā painga ātaahua o te kawenga insulin teitei o ngā NP rehu-maroke kore-mannitol mō ēnei tikanga tuku rerekē.
I puritia ngā NP maroke katoa ki roto i te pouaka whakamātao mō ngā marama e toru. I whakaatuhia e ngā hua SEM kāore i tino rerekē te āhua o ngā NP maroke katoa i te wā e toru marama te rokiroki (Pikitia 4). Whai muri i te whakaranu ki te wai, i whakaatuhia e ngā NP katoa he iti te hekenga o te EE, ā, i tukuna he iti noa iho (~5%) o te insulin i te wā e toru marama te rokiroki (Ripanga 2). Heoi, i piki ake te rahi toharite o ngā matūriki nano katoa. I piki ake te rahi o ngā matūriki o ngā NP i whakamaroketia ki te rehu me te kore mannitol ki te 525 nm, ko te rahi o ngā NP i whakamaroketia ki te rehu me te tio me te mannitol i piki ake ki te 872 me te 921 nm, ia (Ripanga 2).
Te āhua o ngā momo NP insulin maroke kua rongoatia mō ngā marama e toru: (a) Whakaahua SEM o ngā NP insulin kua whakamaroketia me te mannitol; (b) Whakaahua SEM o ngā matūriki insulin kua rehu-maroketia, kāore he mannitol; (c) kāore he mannitol Ngā whakaahua SEM o ngā NP insulin kua rehu-maroketia.
I tua atu, i kitea ngā matūriki matūriki insulin kua hangaia anō, i whakamaroketia ki te mannitol, ā, i whakamaroketia ki te tio (Pikitia S2). Tērā pea nā ngā matūriki nunui kāore i te taurite tika i roto i te wai tēnei. E whakaatu ana ngā hua katoa o runga ake nei ka taea e te tikanga whakamaroke rehu te tiaki i ngā matūriki matūriki insulin mai i te maroke, ā, ka taea te whiwhi i ngā utaina nui o ngā matūriki matūriki insulin me te kore he whakakī, he ārai-wai rānei.
I whakamātauria te pupuri i te insulin i roto i te pH = 2.5 me te pepsin, te trypsin, me te α-chymotrypsin hei whakaatu i te kaha tiaki o ngā NP ki te nakunaku whākōkī i muri i te maroke. I whakaritea te pupuri insulin o ngā NP maroke ki tērā o ngā NP hou i whakaritea, ā, i whakamahia te insulin kore utu hei mana whakahaere kino. I roto i tēnei rangahau, i whakaatuhia e te insulin kore utu te tere o te whakakorenga insulin i roto i te 4 hāora i roto i ngā maimoatanga enzymatic e toru (Pikitia 5a–c). He rerekē, ko te whakamātautau whakakorenga insulin o ngā NP i whakamaroketia ki te mannitol me ngā NP i rehuhia ki te mannitol, kāore rānei, i whakaatu i te tiaki nui ake o ēnei NP ki te nakunaku whākōkī, he rite ki tērā o ngā NP insulin hou i whakaritea (pikitia 1).5a-c). Mā te āwhina o ngā nanoparticles i roto i te pepsin, te trypsin, me te α-chymotrypsin, neke atu i te 50%, 60%, me te 75% o te insulin i taea te tiaki i roto i te 4 hāora, ia (Pikitia 5a–c). Tērā pea ka taea e tēnei kaha tiaki-insulin ka whakanui ake i te tūponotanga o te mimiti nui ake o te insulin ki roto i te toto25. E whakaatu ana ēnei hua ka taea e te whakamaroke rehu me te mannitol, me te whakamaroke-tio me te mannitol te tiaki i te kaha tiaki-insulin o ngā NP i muri i te maroke.
Te tiaki me te whanonga tuku o ngā NP insulin maroke: (a) te tiaki i te insulin i roto i te otinga pepsin; (b) te tiaki i te insulin i roto i te otinga trypsin; (c) te tiaki i te insulin e te otinga α-chymotrypsin; (d) Te whanonga tuku o ngā NP maroke i roto i te otinga pH = 2.5; (e) te whanonga tuku o ngā NP maroke i roto i te otinga pH = 6.6; (f) te whanonga tuku o ngā NP maroke i roto i te otinga pH = 7.0.
I whaowhia ngā NP insulin maroke kua oti te whakarite me te hanga anō ki roto i ngā momo arai (pH = 2.5, 6.6, 7.0) i te 37 °C, me te whakatauira i te taiao pH o te puku, te duodenum, me te whekau iti o runga, hei tirotiro i te pānga o te insulin ki te ātete ki te insulin. Te whanonga tuku i roto i ngā taiao rerekē. Te wāhanga o te ara kai. I te pH = 2.5, ko ngā NP kua utaina ki te insulin me ngā NP insulin maroke kua whakarewahia i whakaatu i te tukunga pakaru tuatahi i roto i te haora tuatahi, ā, i muri mai ko te tukunga puhoi i roto i ngā haora 5 e whai ake nei (Pikitia 5d). Ko tēnei tukunga tere i te tīmatanga he hua pea nō te mimiti tere o ngā ngota pūmua kāore i te tino mau i roto i te hanganga o roto o te matūriki. I te pH = 6.5, ko ngā NP kua utaina ki te insulin me ngā NP insulin maroke kua hangaia anō i whakaatu i te tukunga maeneene me te puhoi i roto i te 6 haora, i te mea he rite te pH o te otinga whakamātautau ki te otinga kua whakaritea e te NP (Pikitia 5e). I te pH = 7, kāore i te pumau ngā NP, ā, tata kore e pirau i roto i ngā haora tuatahi e rua (Pikitia 5f). Nā te mea ka puta te deprotonation o te chitosan i te pH teitei ake, ka hua ake he whatunga polymer iti ake te kiato me te tukunga o te insulin kua utaina.
I tua atu, ko ngā NP insulin i whakamaroketia me te kore mannitol i whakaatu i te tere o te tukunga atu i ngā NP kua whakamaroketia kē atu (Pikitia 5d–f). E ai ki te whakaahuatanga o mua, ko ngā NP insulin i hangaia anō i whakamaroketia me te kore mannitol i whakaatu i te rahi matūriki iti rawa. He nui ake te horahanga mata o ngā matūriki iti, nō reira ko te nuinga o te rongoā e tika ana ka noho ki te mata matūriki, ka tata rānei ki te mata, ka hua ake he tukunga tere o te rongoā26.
I tirotirohia te paitini pūtau o ngā NP mā te whakamātautau MTT. E ai ki te whakaaturanga i te Pikitia S4, i kitea kāore he pānga nui o ngā NP maroke katoa ki te oranga o te pūtau i ngā kukū o te 50–500 μg/ml, e kī ana ka taea te whakamahi haumaru i ngā NP maroke katoa kia tae atu ki te matapihi rongoā.
Ko te ate te okana matua e whakamahia ai e te insulin ana mahi ā-tinana. Ko ngā pūtau HepG2 he raina pūtau hepatoma tangata e whakamahia whānuitia ana hei tauira tangohanga hepatocyte in vitro. I konei, i whakamahia ngā pūtau HepG2 hei aromatawai i te tangohanga pūtau o ngā NP kua whakamaroketia mā te whakamahi i ngā tikanga whakamaroke-tio me te whakamaroke-rehu. Ko te tangohanga pūtau mā te matawai laser confocal mā te whakamahi i te cytometry rere me te tirohanga i muri i ētahi haora o te whaowhia me te insulin FITC kore utu i te kukū o te 25 μg/mL, ngā NP FITC kua utaina ki te insulin me ngā NP FITC kua whakamaroketia ki te insulin i ngā kukū insulin ōrite. I mahia ngā tirohanga karuārai tauanga (CLSM). Ko ngā NP Lyophilized kāore he mannitol i whakangaromia i te wā whakamaroke, ā, kāore i aromatawaitia i roto i tēnei whakamātautau. Ko ngā kaha fluorescence intracellular o ngā NP kua utaina ki te insulin, ngā NP lyophilized me te mannitol, me ngā NP kua whakamaroketia ki te rehu me te kore mannitol (Pikitia 6a) he 4.3, 2.6, 2.4, me te 4.1-ngā wā teitei ake i te ngā mea kore utu. Rōpū FITC-insulin, ia (Pikitia 6b). E whakaatu ana ēnei hua he kaha ake te tangohanga pūtau o te insulin kua whakakīia ki roto i te capsule i te insulin kore utu, nā te iti ake o ngā matūriki nano kua utaina ki te insulin i hangaia i roto i te rangahau.
Te tangohanga pūtau HepG2 i muri i te whaowhia mō te 4 hāora me ngā NP hou kua whakaritea me ngā NP kua maroke: (a) Te tohatoha o te tangohanga FITC-insulin e ngā pūtau HepG2.(b) Te toharite āhuahanga o ngā kaha fluorescence i tātarihia mā te flow cytometry (n = 3), *P < 0.05 ki te whakataurite ki te insulin kore utu.
Waihoki, i whakaatuhia e ngā whakaahua CLSM he kaha ake te kaha o te hihi o te FITC o ngā NP kua utaina ki te insulin FITC me ngā NP kua whakamaroketia ki te insulin FITC (kāore he mannitol) i ērā o ngā tauira kē atu (Pikitia 6a). I tua atu, i te tāpiritanga o te mannitol, ko te matotoru teitei ake o te otinga i whakanui ake i te ātete ki te mimiti pūtau, ā, ka heke te tipu o te insulin. E whakaatu ana ēnei hua ko ngā NP kua whakamaroketia ki te insulin kāore he mannitol te mea i whakaatu i te tino pai o te mimiti pūtau nā te mea he iti ake te rahi o te matūriki i ērā o ngā NP kua whakamaroketia ki te hukapapa i muri i te whakarewatanga anō.
I hokona te Chitosan (te taumaha ngota toharite 100 KDa, 75–85% deacetylated) mai i Sigma-Aldrich. (Oakville, Ontario, Canada). I hokona te Sodium tripolyphosphate (TPP) mai i VWR (Radnor, Pennsylvania, USA). Ko te insulin tangata recombinant i whakamahia i tēnei rangahau i ahu mai i Fisher Scientific (Waltham, MA, USA). Ko te insulin tangata kua tohua ki te Fluorescein isothiocyanate (FITC) me te 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) i ahu mai i Sigma-Aldrich. (Oakville, Ontario, Canada). I tangohia te raina pūtau HepG2 mai i ATCC (Manassas, Virginia, USA). Ko ngā atu matū katoa he tohu tātari, he tohu chromatographic rānei.
Whakapaia he otinga CS 1 mg/ml mā te whakarewa i roto i te wai whakapūkara takirua (wai DD) kei roto ko te 0.1% waikawa acetic. Whakapaia he otinga 1 mg/ml o te TPP me te insulin mā te whakarewa i roto i te wai DD me te 0.1% waikawa acetic. I whakaritea te pre-emulsion me te polytron PCU-2-110 tere-teitei homogenizer (Brinkmann Ind. Westbury, NY, USA). Ko te tukanga whakarite penei: tuatahi, ka tāpirihia te 2ml o te otinga TPP ki te 4ml o te otinga insulin, ā, ka whakaohokia te ranunga mō te 30 meneti ka whakaranua katoatia. Kātahi, ka tāpirihia te otinga whakaranu ki te otinga CS mā te syringe i raro i te whakaoho tere-teitei (10,000 rpm). I puritia ngā ranunga i raro i te whakaoho tere-teitei (15,000 rpm) i roto i te kaukau hukapapa mō te 30 meneti, ā, i whakatikatikaina ki tētahi pH hei whiwhi i ngā NP insulin hono-whiti. Hei whakakotahi me te whakaiti i te rahi o te matūriki o ngā NP insulin, i... i whakamātaohia mō te 30 meneti anō i roto i te kaukau hukapapa mā te whakamahi i te sonicator momo-probe (UP 200ST, Hielscher Ultrasonics, Teltow, Tiamana).
I whakamātauria te NPS o te insulin mō te diameter Z-average, te polydispersity index (PDI) me te zeta potential mā te whakamahi i ngā inenga marara mārama hihiri (DLS) mā te whakamahi i te Litesizer 500 (Anton Paar, Graz, Austria) mā te whakawaiwai i a rātou ki te wai DD i te 25°C. I tautuhia te āhua me te tohatoha rahi e te karu hiko tuku Hitachi H7600 (TEM) (Hitachi, Tokyo, Japan), ā, i tātarihia ngā whakaahua i muri mai mā te whakamahi i te pūmanawa whakaahua Hitachi (Hitachi, Tokyo, Japan). Hei aromatawai i te whai huatanga o te encapsulation (EE) me te kaha uta (LC) o ngā NP insulin, i pipetted ngā NP ki roto i ngā ngongo ultrafiltration me te tapahi taumaha ngota o te 100 kDa, ā, i centrifuged i te 500 xg mō te 30 meneti. I inehia te insulin kāore i roto i te filtrate mā te whakamahi i te pūnaha Agilent 1100 Series HPLC (Agilent, Santa Clara, California, USA) kei roto ko te papu quaternary, te autosampler, te pou. whakamahana, me te pūoko DAD. I tātarihia te inihua e te pou C18 (Zorbax, 3.5 μm, 4.6 mm × 150 mm, Agilent, USA) ā, i kitea i te 214 nm. Ko te wāhanga neke he acetonitrile me te wai, kei roto ko te 0.1% TFA, ngā ōwehenga piki me te heke mai i te 10/90 ki te 100/0, ā, i whakahaerehia mō te 10 meneti. I papuhia te wāhanga neke i te tere rere o te 1.0 ml/min. I whakatakotoria te pāmahana pou ki te 20 °C. Tātaihia ngā ōrau o te EE me te LC mā te whakamahi i ngā whārite.(1) me te Whārite.(2).
I whakamātauria ngā ōwehenga CS/insulin mai i te 2.0 ki te 4.0 hei whakapai ake i te NP o te insulin. I tāpirihia ngā rahinga rerekē o te otinga CS i te wā o te whakarite, i te wā i puritia ai te ranunga insulin/TPP kia rite tonu. I whakaritea ngā NP o te insulin i te awhe pH o te 4.0 ki te 6.5 mā te āta whakahaere i te pH o te ranunga i muri i te tāpiri i ngā otinga katoa (insulin, TPP me te CS). I aromatawaihia te EE me te rahi o te matūriki o ngā nanoparticles o te insulin i ngā uara pH rerekē me ngā ōwehenga papatipu CS/insulin hei whakapai ake i te hanganga o ngā NP o te insulin.
I whakatakotoria ngā NP insulin kua arotauhia ki runga i te ipu konumohe, ā, i hipokina ki te kiko kua whakakīia ki te rīpene. I muri mai, i whakatakotoria ngā ipu kua whiria ki roto i te whakamaroke tio Labconco FreeZone (Labconco, Kansas City, MO, USA) he whakamaroke paepae kei roto. I whakatakotoria te pāmahana me te pēhanga korehau ki -10 °C, 0.350 Torr mō ngā haora tuatahi e 2, me te 0 °C me te 0.120 Torr mō ngā haora 22 e toe ana o ngā haora 24 hei whiwhi i ngā NP insulin maroke.
I whakamahia te Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Switzerland) hei whakaputa i te insulin kua whakakīia ki roto i te capsules. Ko ngā tawhā whakamaroke i tīpakohia ko: te pāmahana 100 °C, te rere whāngai 3 L/min, me te rere hau 4 L/min.
I tautuhia ngā NP insulin i mua atu, ā, i muri mai i te maroketanga mā te whakamahi i te FTIR-ATR spectroscopy. I tātarihia ngā matūriki nano maroke me te insulin kore utu me te chitosan mā te whakamahi i te Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA) kua rite ki te taputapu tauira ATR whānui (PerkinElmer, Waltham, Massachusetts, USA). I ​​whiwhihia ngā toharite tohu mai i te 16 matawai i te taumira o te 4 cm2 i te awhe auau o te 4000-600 cm2.
I aromatawaitia te āhua o ngā NP insulin maroke mā te whakamahi i ngā whakaahua SEM o ngā NP insulin kua whakamaroke-tio me ngā NP insulin kua rehu-maroke i hopukina e te Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, USA). Ko te tawhā matua i whakamahia ko te ngaohiko 5 keV me te iahiko 30 mA.
I whakarewaina anō ngā NP insulin maroke katoa ki te wai dd. I whakamātauria anō te rahi o te matūriki, te PDI, te EE me te LC mā te whakamahi i te tikanga kotahi i whakahuatia i mua ake nei hei aromatawai i tō rātou kounga i muri i te maroketanga. I inehia hoki te pumau o ngā NP anhydroinsulin mā te whakamātautau i ngā āhuatanga o ngā NP i muri i te rokiroki roa. I roto i tēnei rangahau, i rongoatia ngā NP katoa i muri i te maroketanga ki roto i te pouaka whakamātao mō ngā marama e toru. I muri i te rokiroki e toru marama, i whakamātauria ngā NP mō te rahi o te matūriki āhua, te PDI, te EE me te LC.
Whakarewahia te 5 mL o ngā NP kua hangaia anō ki roto i te 45 mL kei roto te wai puku whakaata (pH 1.2, kei roto te 1% pepsin), te wai puku (pH 6.8, kei roto te 1% trypsin) te otinga chymotrypsin rānei (100 g/mL, ki roto i te phosphate buffer, pH 7.8) hei aromatawai i te whai huatanga o te insulin ki te tiaki i ngā NP i muri i te maroke. I whaowhia rātou ki te 37°C me te tere whakaoho o te 100 rpm. I kohia he 500 μL o te otinga i ngā wā rerekē, ā, i whakatauhia te kukū o te insulin mā te HPLC.
I whakamātauria te whanonga tukunga in vitro o ngā NP insulin hou kua oti te whakarite me te whakamaroke mā te tikanga putea dialysis (te tapahi taumaha ngota 100 kDa, Spectra Por Inc.). Ko ngā NP maroke hou kua oti te whakarite me te hanga anō i whakamātauria ki roto i ngā wai i te pH 2.5, pH 6.6, me te pH 7.0 (0.1 M phosphate-buffered saline, PBS) hei whakatauira i te taiao pH o te puku, te duodenum, me te whekau iti o runga. I whakatōkia ngā tauira katoa i te 37 °C me te rūrū tonu i te 200 rpm. Mimia te wai i waho o te putea dialysis 5 mL i ngā wā e whai ake nei: 0.5, 1, 2, 3, 4, me te 6 h, ā, whakakīia tonutia te rōrahi ki te dialysate hou. I tātarihia te poke o te insulin i roto i te wai mā te HPLC, ā, i tatauhia te tere o te tukunga insulin mai i ngā nanoparticles mai i te ōwehenga o te insulin kore utu i tukuna ki te insulin katoa i roto i ngā nanoparticles (Whārite 3).
I whakatipuria ngā pūtau HepG2 o te momo pūtau mate pukupuku hepato tangata i roto i ngā rihi 60 mm te whānui mā te whakamahi i te Dulbecco's Modified Eagle's Medium (DMEM) kei roto ko te 10% te serum kau kukune, 100 IU/mL penicillin, me te 100 μg/mL streptomycin29. I puritia ngā ahurea ki te 37°C, 95% te haumākū whanaunga, me te 5% CO2. Mō ngā whakamātautau mimiti, i whakatōngia ngā pūtau HepG2 ki te 1 × 105 pūtau/ml ki runga i te pūnaha kiriata rūma Nunc Lab-Tek 8-pū (Thermo Fisher, NY, USA). Mō ngā whakamātautau paitini pūtau, i whakatōngia ki roto i ngā pereti 96-pū (Corning, NY, USA) ki te kiato o te 5 × 104 pūtau/ml.
I whakamahia te whakamātautau MTT hei aromatawai i te paitini pūtau o ngā NPs insulin kua oti te whakarite me te whakamaroke30. I whakatōngia ngā pūtau HepG2 ki roto i ngā pereti 96-pū i te kiato o te 5 × 104 pūtau/mL, ā, i whakatipuria mō ngā rā e 7 i mua i te whakamātautau. I whakaranua ngā NPs insulin ki ngā kukū rerekē (50 ki te 500 μg/mL) i roto i te reo ahurea, kātahi ka tukuna ki ngā pūtau. I muri i te 24 hāora o te whakatipu, i horoia ngā pūtau e 3 ngā wā ki te PBS, ā, i whakatipuria ki te reo kei roto ko te 0.5 mg/ml MTT mō ētahi atu hāora e 4. I aromatawaihia te paitini pūtau mā te ine i te whakaiti whākōkī o te MTT tetrazolium kōwhai ki te formazan papura i te 570 nm mā te whakamahi i te kaipānui pereti spectrophotometer Tecan infinite M200 pro (Tecan, Männedorf, Switzerland).
I whakamātauria te whai huatanga o te tango pūtau o ngā NP mā te karuārai matawai laser confocal me te tātaritanga cytometry rere. I rongoatia ia poka o te pūnaha kiriata rūma Nunc Lab-Tek ki te FITC-insulin kore utu, FITC-insulin-loaded NPs, ā, i hangaia anōtia te 25 μg/mL o ngā FITC-insulin NPs maroke i te kukū kotahi, ā, i whaowhia mō ngā hāora e 4. I horoia ngā pūtau e 3 ngā wā ki te PBS, ā, i whakapūmautia ki te 4% paraformaldehyde. I pania ngā karihi ki te 4′,6-diamidino-2-phenylindole (DAPI). I tirohia te rohenga insulin mā te whakamahi i te karuārai matawai laser/rua-photon confocal Olympus FV1000 (Olympus, Shinjuku City, Tokyo, Japan). Mō te tātaritanga cytometry rere, i tāpirihia ngā kukū ōrite o te 10 μg/mL FITC-insulin kore utu, FITC-insulin-loaded NPs, me ngā FITC-insulin NPs maroke kua whakapūmautia ki ngā pereti 96-poka i whakatōngia ki ngā pūtau HepG2, ā, i whaowhia mō ngā hāora. 4 hāora. I muri i te 4 hāora o te whakatipuranga, i tangohia ngā pūtau, ā, i horoia kia 3 ngā wā ki te FBS. I tātarihia ngā pūtau 5 × 104 mō ia tauira mā te whakamahi i te mita rere BD LSR II (BD, Franklin Lakes, New Jersey, Te Hononga o Amerika).
Ko ngā uara katoa e whakaatuhia ana hei toharite ± paerewa rerekētanga. I aromatawaihia ngā whakataurite i waenga i ngā rōpū katoa mā te whakamahi i te ANOVA kotahi-ara, i te whakamātautau-t rānei mā te IBM SPSS Statistics 26 mō Mac (IBM, Endicott, New York, USA) ā, i kiia he hiranga tatauranga te p < 0.05.
E whakaatu ana tēnei rangahau i te ngāwari me te kaha o te whakamaroke rehu ki te whakamaroke i ngā matūriki nano chitosan/TPP/insulin kua honoa-whiti me te pai ake o te hanganga ki te whakataurite ki ngā tikanga whakamaroke-tio paerewa e whakamahi ana i ngā kaihoko whakapūmau, i ngā kaha tiaki-kiri rānei me te kaha kawenga teitei ake. Ko ngā matūriki nano insulin kua arotauhia i hua ake he rahi matūriki toharite o te 318 nm me te pai o te whakakī o te 99.4%. Ko ngā hua SEM me te FTIR i muri i te whakamaroke i whakaatu ko te hanganga porowhita i puritia noa i roto i ngā NP whakamaroke-rehu me te kore mannitol me te lyophilized ki te mannitol, engari ko ngā NP lyophilized me te kore mannitol i pirau i te wā o te whakamaroke. I roto i te whakamātautau kaha hanganga, ko ngā matūriki nano insulin i whakamaroke-rehu me te kore mannitol i whakaatu i te rahi matūriki toharite iti rawa me te kawenga teitei rawa atu i te hanganga. Ko ngā whanonga tukunga o ēnei NP whakamaroke katoa i whakaatu i te tere o te tukunga i roto i ngā otinga pH = 2.5 me te pH = 7, ā, he tino pumau i roto i te otinga pH = 6.5. Ki te whakataurite ki ētahi atu NP whakamaroke kua rewa anō, ko ngā NP Ko te whakamaroke-rehu me te kore mannitol i whakaatu i te tukunga tere rawa atu. He rite tonu tēnei hua ki tērā i kitea i roto i te whakamātautau mimiti pūtau, i te mea ko ngā NP whakamaroke-rehu i te korenga o te mannitol i tata tonu te pupuri i te whai huatanga mimiti pūtau o ngā NP hou kua whakaritea. E whakaatu ana ēnei hua ko ngā matūriki nano insulin maroke i whakaritea mā te whakamaroke rehu kore mannitol he mea tino pai mō te tukatuka atu ki ētahi atu momo horopeta kore wai, pērā i ngā papa ā-waha, i ngā kiriata piri-koiora rānei.
Nā ngā take mō te mana pupuri hinengaro, kāore ngā huinga raraunga i hangaia, i tātarihia rānei i te wā o te rangahau o nāianei e wātea ana ki te marea, engari e wātea ana mai i ngā kaituhi e tika ana mēnā ka tonoa mai.
Kagan, A. Te mate huka momo 2: ngā takenga pāpori me te pūtaiao, ngā raruraru hauora, me ngā pānga ki ngā tūroro me ētahi atu. (McFarlane, 2009).
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