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Nanoparticles na insulin (NPs) masu yawan lodawa sun sami aikace-aikace daban-daban a cikin nau'ikan allurai daban-daban. Wannan aikin yana da nufin kimanta tasirin busarwa da daskarewa da busarwa da feshi akan tsarin nanoparticles na chitosan da aka ɗora da insulin, tare da ko ba tare da mannitol a matsayin mai kariya ba. Mun kuma tantance ingancin waɗannan nanoparticles ta hanyar sake wargaza su. Kafin bushewa, an inganta girman barbashi na chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles zuwa 318 nm, PDI shine 0.18, ingancin encapsulation shine 99.4%, kuma nauyin shine 25.01%. Bayan sake haɗawa, duk nanoparticles, banda waɗanda aka samar ta hanyar busarwa da daskarewa ba tare da amfani da mannitol ba, sun kiyaye tsarin barbashi mai zagaye. Idan aka kwatanta da nanoparticles masu ɗauke da mannitol waɗanda aka fitar da ruwa ta hanyar feshi, nanoparticles busarwa marasa feshi na mannitol suma sun nuna ƙaramin matsakaicin girman barbashi (376 nm) da mafi girman abun ciki (25.02%) tare da makamancin haka. Yawan rufewa (98.7%) da PDI (0.20) ta hanyar busarwa ko busarwa da daskare. Busassun ƙwayoyin nano ta hanyar busarwa da feshi ba tare da mannitol ba suma sun haifar da sakin insulin cikin sauri da kuma mafi girman ingancin ɗaukar ƙwayoyin halitta. Wannan aikin ya nuna cewa busarwa da feshi na iya busar da ƙwayoyin nano na insulin ba tare da buƙatar cryoprotectants ba idan aka kwatanta da hanyoyin busarwa da daskare na gargajiya, wanda ke haifar da ƙarin ƙarfin lodi, ƙarancin buƙatun ƙari da kuma farashin aiki mai mahimmanci.
Tun bayan gano shi a shekarar 19221,2,3, insulin da shirye-shiryen magunguna sun ceci rayukan marasa lafiya da ke fama da ciwon suga na nau'in 1 (T1DM) da ciwon suga na nau'in 2 (T1DM). Duk da haka, saboda halayensa na furotin mai nauyin kwayoyin halitta, ana iya haɗa insulin cikin sauƙi, a karya shi ta hanyar enzymes na proteolytic, sannan a kawar da shi ta hanyar tasirin farko. Mutanen da aka gano suna da ciwon suga na nau'in 1 suna buƙatar allurar insulin har tsawon rayuwarsu. Yawancin marasa lafiya da aka gano suna da ciwon suga na nau'in 2 da farko suma suna buƙatar allurar insulin na dogon lokaci. Allurar insulin ta yau da kullun babbar hanyar ciwo da rashin jin daɗi ce ga waɗannan mutane, tare da mummunan tasiri ga lafiyar kwakwalwa. Sakamakon haka, ana yin nazari sosai kan wasu nau'ikan allurar insulin waɗanda ke haifar da ƙarancin rashin jin daɗi, kamar allurar insulin ta baki,5 saboda suna da damar dawo da ingancin rayuwar kusan mutane biliyan 5 da ke fama da ciwon suga a duk duniya.
Fasahar Nanoparticle ta samar da ci gaba mai mahimmanci a ƙoƙarin ɗaukar insulin na baki4,6,7. Wanda ke ƙunshe da kuma kare insulin daga lalacewa don isar da shi zuwa takamaiman wuraren jiki. Duk da haka, amfani da tsarin nanoparticle yana da iyakoki da yawa, galibi saboda matsalolin kwanciyar hankali na dakatarwar barbashi. Wasu tarawa na iya faruwa yayin ajiya, wanda ke rage samuwar nanoparticles masu ɗauke da insulin8. Bugu da ƙari, dole ne a yi la'akari da daidaiton sinadarai na matrix na polymer na nanoparticles da insulin don tabbatar da daidaiton nanoparticles na insulin (NPs). A halin yanzu, fasahar bushewa daskararre ita ce ma'aunin zinare don ƙirƙirar NPs masu karko yayin hana canje-canje da ba a so yayin ajiya9.
Duk da haka, busar da daskare yana buƙatar ƙara cryoprotectants don hana tsarin zagaye na NPs ya shafi matsin lamba na injina na lu'ulu'u na kankara. Wannan yana rage yawan nauyin insulin nanoparticles bayan lyophilization, saboda cryoprotectant ya mamaye mafi yawan rabon nauyi. Saboda haka, ana ganin insulin NPs da aka samar ba su dace da ƙera busassun foda ba, kamar allunan baki da fina-finan baki, saboda buƙatar adadi mai yawa na busassun nanoparticles don cimma tagar maganin insulin.
Busar da feshi tsari ne na masana'antu da aka sani kuma mai araha don samar da busassun foda daga matakan ruwa a masana'antar magunguna10,11. Kulawa kan tsarin samar da barbashi yana ba da damar rufewa da kyau na mahaɗan bioactive da yawa 12, 13. Bugu da ƙari, ya zama dabara mai inganci don shirya furotin da aka lulluɓe don amfani da baki. A lokacin busar da feshi, ruwa yana ƙafewa da sauri, wanda ke taimakawa wajen kiyaye zafin jikin barbashi ƙasa da ƙasa11,14, yana ba da damar amfani da shi don lulluɓe abubuwan da ke damun zafi. Kafin busar da feshi, ya kamata a haɗa kayan shafa sosai tare da maganin da ke ɗauke da sinadaran da aka lulluɓe11,14. Ba kamar busar da daskare ba, haɗuwa kafin a lulluɓe a busar da feshi yana inganta ingancin rufewa yayin bushewa. Tunda tsarin busar da feshi ba ya buƙatar cryoprotectants, ana iya amfani da busar da feshi don samar da NPs busasshe tare da babban abun ciki.
Wannan binciken ya ba da rahoton samar da NPs masu ɗauke da insulin ta hanyar haɗa chitosan da sodium tripolyphosphate ta amfani da hanyar gel na ion. Gelation na ion hanya ce ta shiri wadda ke ba da damar samar da nanoparticles ta hanyar hulɗar lantarki tsakanin nau'ikan ionic guda biyu ko fiye a ƙarƙashin wasu yanayi. An yi amfani da dabarun busar da daskare-daskare da busar da feshi don busar da nanoparticles na chitosan/sodium tripolyphosphate/insulin cross-linked da aka inganta. Bayan busar da jiki, an yi nazarin yanayin jikinsu ta hanyar SEM. An kimanta ikon sake haɗa su ta hanyar auna girman rarrabawarsu, cajin saman su, PDI, ingancin encapsulation, da abun ciki na lodawa. An kuma kimanta ingancin nanoparticles masu narkewa da aka samar ta hanyoyin busar da jiki daban-daban ta hanyar kwatanta kariyar insulin, halayen saki, da ingancin ɗaukar ƙwayoyin halitta.
pH na maganin da aka haɗa da rabon chitosan da insulin sune manyan abubuwa guda biyu da ke shafar girman barbashi da ingancin rufewa (EE) na NPs na ƙarshe, domin suna shafar tsarin haɗin ionotropic kai tsaye. An nuna cewa pH na maganin da aka haɗa yana da alaƙa sosai da girman barbashi da ingancin rufewa (Hoto na 1a). Kamar yadda aka nuna a Hoto na 1a, yayin da pH ya ƙaru daga 4.0 zuwa 6.0, matsakaicin girman barbashi (nm) ya ragu kuma EE ya ƙaru sosai, yayin da lokacin da pH ya ƙaru zuwa 6.5, matsakaicin girman barbashi ya fara ƙaruwa kuma EE ya kasance ba canzawa. Yayin da rabon chitosan da insulin ke ƙaruwa, matsakaicin girman barbashi shima yana ƙaruwa. Bugu da ƙari, babu wani canji a EE da aka lura lokacin da aka shirya nanoparticles a rabon taro na chitosan/insulin sama da 2.5:1 (w/w) (Hoto na 1b). Saboda haka, an yi amfani da yanayin shiri mafi kyau a cikin wannan binciken (pH 6.0, rabon taro na chitosan/insulin na 2.5:1) don shiryawa Nanoparticles masu ɗauke da insulin don ƙarin bincike. A ƙarƙashin wannan yanayin shiri, an inganta matsakaicin girman barbashi na nanoparticles na insulin zuwa 318 nm (Hoto na 1c), PDI shine 0.18, ingancin haɗawa shine 99.4%, ƙarfin zeta shine 9.8 mv, kuma nauyin insulin shine 25.01% (m/m). Dangane da sakamakon watsawa na'urar microscopy (TEM), nanoparticles da aka inganta sun kasance masu siffar ƙwallo da kuma masu bambanci tare da girman iri ɗaya (Hoto na 1d).
Inganta ma'auni na ƙwayoyin insulin: (a) tasirin pH akan matsakaicin diamita da ingancin encapsulation (EE) na ƙwayoyin insulin nanoparticles (wanda aka shirya a rabon taro na 5:1 na chitosan da insulin); (b) chitosan da Tasirin rabon taro na insulin akan matsakaicin diamita da ingancin encapsulation (EE) na insulin NPs (wanda aka shirya a pH 6); (c) rarraba girman barbashi na ƙananan ƙwayoyin insulin da aka inganta; (d) Micrograph na TEM na ingantattun NPs na insulin.
Sanannen abu ne cewa chitosan wani nau'in polyelectrolyte ne mai rauni tare da pKa na 6.5. Ana cajinsa da kyau a cikin kafofin acidic saboda babban rukunin amino ɗinsa yana da sinadarin hydrogen ions15. Saboda haka, sau da yawa ana amfani da shi azaman mai ɗaukar kaya don lulluɓe macromolecules masu caji mara kyau. A cikin wannan binciken, an yi amfani da chitosan don lulluɓe insulin tare da ma'aunin isoelectric na 5.3. Tunda ana amfani da chitosan azaman kayan shafa, tare da ƙaruwar rabonsa, kauri na waje na nanoparticles yana ƙaruwa daidai gwargwado, wanda ke haifar da matsakaicin girman barbashi. Bugu da ƙari, manyan matakan chitosan na iya lulluɓe ƙarin insulin. A yanayinmu, EE ya fi girma lokacin da rabon chitosan da insulin ya kai 2.5: 1, kuma babu wani canji mai mahimmanci a cikin EE lokacin da rabon ya ci gaba da ƙaruwa.
Baya ga rabon chitosan da insulin, pH kuma ya taka muhimmiyar rawa wajen shirya NPs. Gan et al. 17 sun yi nazarin tasirin pH akan girman barbashi na ƙwayoyin chitosan. Sun sami raguwar ci gaba a girman barbashi har sai pH ya kai 6.0, kuma an lura da ƙaruwa mai yawa a girman barbashi a pH > 6.0, wanda ya yi daidai da abin da muka lura. Wannan lamari ya faru ne saboda gaskiyar cewa tare da ƙaruwar pH, ƙwayar insulin tana samun cajin saman mara kyau, don haka, yana fifita hulɗar electrostatic tare da hadaddun chitosan/sodium tripolyphosphate (TPP), wanda ya haifar da ƙaramin girman barbashi da babban EE. Duk da haka, lokacin da aka daidaita pH zuwa 6.5, an cire rukunin amino akan chitosan, wanda ya haifar da naɗewar chitosan. Don haka, babban pH yana haifar da ƙarancin fallasa amino ions ga TPP da insulin, wanda ke haifar da ƙarancin haɗin giciye, babban matsakaicin matsakaicin barbashi na ƙarshe da ƙananan EE.
Binciken halayen NPs da aka busar da su da kuma waɗanda aka fesa na iya jagorantar zaɓin dabarun busar da su da kuma samar da foda mafi kyau. Hanyar da aka fi so ya kamata ta samar da daidaiton magani, siffar barbashi iri ɗaya, yawan shan magani da kuma kyakkyawan narkewa a cikin maganin asali. A cikin wannan binciken, don kwatanta dabarun biyu mafi kyau, an yi amfani da NPs na insulin tare da ko ba tare da 1% na mannitol ba yayin busar da su. Ana amfani da Mannitol azaman wakili mai bulking ko cryoprotectant a cikin nau'ikan busassun foda don busar da su da kuma busar da su da feshi. Ga ƙananan ƙwayoyin insulin da aka lyophilized ba tare da mannitol ba, kamar yadda aka nuna a Hoto na 2a, an lura da tsarin foda mai ramuka mai yawa tare da manyan saman da ba su dace ba, marasa tsari da kuma marasa ƙarfi a ƙarƙashin na'urar duba electron microscopy (SEM). An gano ƙananan ƙwayoyin da ba su da tsari a cikin foda bayan busar da su (Hoto na 2e). Waɗannan sakamakon sun nuna cewa yawancin NPs sun lalace yayin busar da su da feshi ba tare da wani cryoprotectant ba. Ga ƙananan ƙwayoyin insulin da aka busar da su da feshi waɗanda ke ɗauke da 1% na mannitol, an lura da ƙananan ƙwayoyin halitta masu siffar ƙwallo tare da saman santsi (Hoto na 2b,d,f,h). An fesa ƙwayoyin insulin da aka busar ba tare da mannitol ba, amma sun kasance masu siffar ƙwallo amma sun yi laushi a saman (Hoto na 2c). An ƙara tattauna saman da suka yi laushi da kuma waɗanda suka yi laushi a cikin gwajin fitar da ƙwayoyin halitta da ke ƙasa. Dangane da bayyanar da ake gani na busassun NPs, duka NPs da aka fesa ba tare da mannitol ba da NPs da aka daskare kuma aka fesa da mannitol suna samar da foda mai kyau na NPs (Hoto na 2f,g,h). Girman yankin saman tsakanin saman ƙwayoyin cuta, mafi girman narkewar su kuma mafi girman ƙimar fitarwa.
Tsarin halittar insulin NPs daban-daban da aka busar: (a) Hoton SEM na insulin NPs da aka busar ba tare da mannitol ba; (b) Hoton SEM na insulin NPs da aka busar da ba tare da mannitol ba; (c) insulin NPs da aka fesa ba tare da mannitol Hoton SEM na; (d) Hoton SEM na insulin NPs da aka fesa ba tare da mannitol ba; (e) Hoton insulin NPs da aka busar da ba tare da mannitol ba; (f) Hoton insulin NPs da aka busar da ba tare da mannitol ba; (g) Hoton insulin NPs da aka fesa ba tare da mannitol ba; (h) Hoton insulin NPs da aka fesa ba tare da mannitol.
A lokacin busar da daskare, mannitol yana aiki azaman kariya daga cryoprotectant, yana kiyaye NPs a cikin siffa mara tsari kuma yana hana lalacewa daga lu'ulu'u na kankara19. Sabanin haka, babu matakin daskarewa yayin busar da feshi. Saboda haka ba a buƙatar mannitol a cikin wannan hanyar. A zahiri, NPs busar da aka feshi ba tare da mannitol ba suna samar da NPs mafi kyau kamar yadda aka bayyana a baya. Duk da haka, mannitol har yanzu yana iya aiki azaman cikawa a cikin tsarin busar da feshi don ba NPs tsarin zagaye20 (Hoto na 2d), wanda ke taimakawa wajen samun yanayin sakin iri ɗaya na irin waɗannan NPs da aka lulluɓe. Bugu da ƙari, a bayyane yake cewa ana iya gano wasu manyan barbashi a cikin NPs na insulin busar da daskare da busar da feshi waɗanda ke ɗauke da mannitol (Hoto na 2b,d), wanda wataƙila ya faru ne saboda tarin mannitol a cikin tsakiyar barbashi tare da insulin da aka lulluɓe. Zuwa. Layin Chitosan. Ya kamata a lura cewa a cikin wannan binciken, domin tabbatar da cewa tsarin siffar ƙwallo ya kasance ba tare da wata matsala ba bayan bushewar ruwa, rabon mannitol da chitosan an kiyaye shi a 5:1, ta yadda babban adadin cikawa zai iya faɗaɗa girman ƙwayoyin NPs ɗin da aka busar.
FTIR-ATR spectroscopy ya nuna cakudawar jiki ta insulin kyauta, chitosan, chitosan, TPP da insulin. Duk NPs da aka bushe an siffanta su ta amfani da FTIR-ATR spectroscopy. Abin lura, an lura da ƙarfin band na 1641, 1543 da 1412 cm-1 a cikin NPs da aka rufe da aka daskare da mannitol kuma a cikin NPs da aka fesa tare da mannitol da kuma ba tare da su ba (Hoto na 3). Kamar yadda aka ruwaito a baya, waɗannan ƙaruwar ƙarfi suna da alaƙa da haɗin gwiwa tsakanin chitosan, TPP da insulin. Binciken hulɗar da ke tsakanin chitosan da insulin ya nuna cewa a cikin FTIR spectra na nanoparticles na chitosan da aka ɗora da insulin, band ɗin chitosan ya haɗu da na insulin, yana ƙara ƙarfin carbonyl (1641 cm-1) da amine (1543 cm-1). Rukunin tripolyphosphate na TPP suna da alaƙa da ƙungiyoyin ammonium a cikin chitosan, suna samar da band a 1412 cm-1.
Siffar FTIR-ATR ta insulin kyauta, chitosan, gaurayen jiki na chitosan/TPP/insulin da NPs waɗanda aka fitar da ruwa ta hanyoyi daban-daban.
Bugu da ƙari, waɗannan sakamakon sun yi daidai da waɗanda aka nuna a cikin SEM, wanda ya nuna cewa NPs ɗin da aka lulluɓe sun kasance ba tare da an fesa su ba kuma an busar da su da mannitol, amma idan babu mannitol, busar da su kawai yana samar da ƙwayoyin da aka lulluɓe su. Sabanin haka, sakamakon FTIR-ATR na NPs da aka daskare ba tare da mannitol ba sun yi kama da cakuda na zahiri na chitosan, TPP, da insulin. Wannan sakamakon yana nuna cewa haɗin gwiwa tsakanin chitosan, TPP da insulin ba su wanzu a cikin NPs da aka daskare ba tare da mannitol ba. An lalata tsarin NPs yayin busar da su ba tare da cryoprotectant ba, wanda za a iya gani a cikin sakamakon SEM (Hoto na 2a). Dangane da yanayin jiki da sakamakon FTIR na insulin NPs da aka busar da su, an yi amfani da NPs marasa lyophilized, waɗanda aka fesa, da waɗanda ba mannitol ba kawai don gwaje-gwajen sake ginawa da NPs marasa mannitol saboda rugujewar NPs marasa mannitol yayin busar da su. tattauna.
Ana amfani da bushewar ruwa don adanawa na dogon lokaci da sake sarrafawa zuwa wasu hanyoyin. Ikon busassun NPs na sake haɗawa bayan ajiya yana da mahimmanci don amfani da su a cikin hanyoyin magani daban-daban kamar allunan da fina-finai. Mun lura cewa matsakaicin girman barbashi na insulin NPs da aka fesa idan babu mannitol ya ƙaru kaɗan bayan sake haɗawa. A gefe guda kuma, girman barbashi na insulin nanoparticles da aka fesa da kuma waɗanda aka daskare tare da mannitol ya ƙaru sosai (Tebur 1). PDI da EE ba a canza su sosai ba (p > 0.05) bayan sake haɗawa da duk NPs a cikin wannan binciken (Tebur 1). Wannan sakamakon yana nuna cewa yawancin barbashi sun kasance ba tare da matsala ba bayan sake narkewa. Duk da haka, ƙarin mannitol ya haifar da raguwar yawan insulin na mannitol nanoparticles da aka fesa da waɗanda aka fesa (Tebur 1). Sabanin haka, yawan insulin na NPs da aka fesa ba tare da mannitol ya kasance iri ɗaya kamar da (Tebur 1).
An san cewa ɗaukar ƙwayoyin nanoparticles yana da matuƙar muhimmanci idan aka yi amfani da shi don isar da magani. Ga NPs masu ƙarancin lodi, ana buƙatar adadi mai yawa na kayan aiki don isa ga maƙasudin magani. Duk da haka, yawan danko na irin wannan babban yawan NP yana haifar da rashin jin daɗi da wahala a cikin shan magani ta baki da kuma allurar rigakafi, bi da bi. Bugu da ƙari, ana iya amfani da insulin NPs don yin allunan da biofilms masu ƙauri23, 24, wanda ke buƙatar amfani da adadi mai yawa na NPs a ƙananan matakan lodi, wanda ke haifar da manyan allunan da biofilms masu kauri waɗanda ba su dace da amfani da baki ba. Saboda haka, NPs masu bushewa tare da babban nauyin insulin suna da matuƙar kyawawa. Sakamakonmu ya nuna cewa yawan insulin na NPs busassun mannitol ba tare da feshi ba na iya bayar da fa'idodi masu kyau da yawa ga waɗannan hanyoyin isarwa.
An ajiye duk NPs ɗin da aka busar a cikin firiji na tsawon watanni uku. Sakamakon SEM ya nuna cewa yanayin dukkan NPs ɗin da aka busar bai canza sosai ba a lokacin ajiya na watanni uku (Hoto na 4). Bayan sake haɗawa a cikin ruwa, duk NPs sun nuna ɗan raguwa a cikin EE kuma sun saki kusan ƙaramin adadin (~5%) na insulin a cikin lokacin ajiya na watanni uku (Tebur na 2). Duk da haka, matsakaicin girman ƙwayoyin cuta na duk ƙwayoyin cuta ya ƙaru. Girman ƙwayoyin cuta na NPs da aka busar ba tare da mannitol ba ya ƙaru zuwa 525 nm, yayin da na NPs ɗin da aka busar da kuma waɗanda aka daskare da mannitol ya ƙaru zuwa 872 da 921 nm, bi da bi (Tebur na 2).
Tsarin halittar nau'ikan insulin NPs daban-daban da aka bushe da aka adana na tsawon watanni uku: (a) Hoton SEM na insulin NPs da aka yayyanka da lyophilized tare da mannitol; (b) Hoton SEM na insulin nanoparticles da aka fesa ba tare da mannitol ba; (c) ba tare da mannitol ba hotunan SEM na insulin NPs da aka fesa.
Bugu da ƙari, an ga abubuwan da suka fashe a cikin ƙwayoyin insulin da aka sake haɗawa, an fesa su da mannitol sannan aka daskare su (Hoto na S2). Wannan na iya faruwa ne saboda manyan ƙwayoyin da ba sa tsayawa sosai a cikin ruwa. Duk sakamakon da ke sama sun nuna cewa hanyar busar da feshi na iya kare ƙwayoyin insulin daga bushewa kuma ana iya samun yawan ƙwayoyin insulin ba tare da wani abin cikawa ko abubuwan kariya ba.
An gwada riƙe insulin a pH = 2.5 matsakaici tare da pepsin, trypsin, da α-chymotrypsin don nuna ikon kariya na NPs daga narkewar enzymatic bayan bushewa. An kwatanta riƙe insulin na NPs da aka bushe da na NPs da aka shirya sabo, kuma an yi amfani da insulin kyauta azaman iko mara kyau. A cikin wannan binciken, insulin kyauta ya nuna kawar da insulin cikin sauri cikin awanni 4 a cikin dukkan jiyya uku na enzymatic (Hoto na 5a–c). Sabanin haka, gwajin kawar da insulin na NPs da aka daskare da mannitol da NPs da aka fesa tare da mannitol ko ba tare da mannitol ba ya nuna babban kariya na waɗannan NPs daga narkewar enzymatic, wanda yayi kama da na insulin NPs da aka shirya sabo (hoto na 1).5a-c). Tare da taimakon nanoparticles a cikin pepsin, trypsin, da α-chymotrypsin, fiye da 50%, 60%, da 75% na insulin za a iya kare su cikin awanni 4, bi da bi (Hoto na 5a–c). Wannan ikon kariya na insulin na iya ƙara damar samun ƙarin insulin. Shanyewa cikin jini25. Waɗannan sakamakon sun nuna cewa busar da feshi tare da mannitol ko ba tare da shi ba da kuma busar da mannitol tare da daskarewa na iya kiyaye ikon kariyar insulin na NPs bayan bushewa.
Kariya da kuma sakin NPs na insulin da aka fitar daga ruwa: (a) kariyar insulin a cikin maganin pepsin; (b) kariyar insulin a cikin maganin trypsin; (c) kariyar insulin ta hanyar maganin α-chymotrypsin; (d) Halayyar sakin NPs da aka fitar daga ruwa a cikin pH = maganin 2.5; (e) halayen sakin NPs da aka fitar daga ruwa a cikin pH = maganin 6.6; (f) halayen sakin NPs da aka fitar daga ruwa a cikin pH = maganin 7.0.
An saka busasshen insulin NPs da aka shirya sabo kuma aka sake haɗa su a cikin buffers daban-daban (pH = 2.5, 6.6, 7.0) a zafin jiki na 37 °C, suna kwaikwayon yanayin pH na ciki, duodenum, da ƙananan hanji na sama, don bincika tasirin insulin akan juriyar insulin. Halayyar sakin abubuwa a wurare daban-daban. Rarrabuwar hanyoyin narkewar abinci. A pH = 2.5, NPs da aka ɗora wa insulin da busassun insulin NPs da aka sake narkewa sun nuna fitowar fashewar farko a cikin awa ɗaya ta farko, sannan aka sake sakin su a hankali a cikin awanni 5 masu zuwa (Hoto na 5d). Wannan sakin da sauri a farkon wataƙila sakamakon cire ƙwayoyin furotin da sauri a saman wanda ba su da cikakken motsi a cikin tsarin ciki na barbashi. A pH = 6.5, NPs da aka ɗora wa insulin da busassun insulin NPs da aka sake haɗawa sun nuna sakin su mai santsi da jinkiri a cikin awanni 6, saboda pH na maganin gwajin yayi kama da na maganin da NPs suka shirya (Hoto na 5e). A pH = 7, NPs ba su da ƙarfi kuma kusan sun ruɓe gaba ɗaya a cikin awanni biyu na farko (Hoto na 5f). Wannan saboda cire chitosan yana faruwa a mafi girman pH, wanda ke haifar da hanyar sadarwa ta polymer mara ƙanƙanta da sakin insulin da aka ɗora.
Bugu da ƙari, allurar insulin NPs da aka fesa ba tare da mannitol ba ta nuna saurin fitarwa fiye da sauran allurar insulin NPs da aka busar ba tare da mannitol ba (Hoto na 5d-f). Kamar yadda aka bayyana a baya, allurar insulin NPs da aka sake busar ba tare da mannitol ba ta nuna ƙaramin girman barbashi. Ƙananan barbashi suna samar da babban yanki na saman, don haka yawancin maganin da ya dace zai kasance a saman barbashi ko kusa da shi, wanda ke haifar da sakin magani cikin sauri26.
An binciki gubar NPs ta hanyar gwajin MTT. Kamar yadda aka nuna a Hoto na S4, an gano cewa duk NPs da suka bushe ba su da wani tasiri mai mahimmanci akan wanzuwar ƙwayoyin halitta a yawan 50-500 μg/ml, wanda ke nuna cewa duk NPs da suka bushe za a iya amfani da su lafiya don isa ga taga ta magani.
Hanta ita ce babbar gaɓar da insulin ke amfani da ita wajen gudanar da ayyukanta na jiki. Kwayoyin HepG2 layin ƙwayar hepatoma ne na ɗan adam wanda aka saba amfani da shi azaman samfurin ɗaukar hepatocyte a cikin vitro. A nan, an yi amfani da ƙwayoyin HepG2 don tantance ɗaukar NPs da aka bushe ta hanyar amfani da hanyoyin busar da daskare da busar da feshi. Ɗauka ta hanyar amfani da na'urar daukar hoto ta confocal laser scanning ta amfani da kwararar cytometry da gani bayan sa'o'i da yawa na haɗuwa tare da insulin FITC kyauta a yawan 25 μg/mL, sabbin NPs da aka ɗora insulin a FITC da busar da NPs da aka ɗora insulin a daidai adadin insulin An gudanar da lura da ƙididdigar microscopy (CLSM). An lalata NPs da aka ƙona ba tare da mannitol ba yayin bushewa kuma ba a tantance su a cikin wannan gwajin ba. Ƙarfin hasken cikin ƙwayoyin halitta na NPs da aka ɗora insulin a sabo, NPs da aka ɗora lyophilized tare da mannitol, da NPs da aka fesa tare da mannitol da ba tare da mannitol ba (Hoto na 6a) sun ninka 4.3, 2.6, 2.4, da ninki 4.1 fiye da kyauta. ones. FITC-rukunin insulin, bi da bi (Hoto na 6b). Waɗannan sakamakon sun nuna cewa insulin da aka lulluɓe ya fi ƙarfi a cikin ɗaukar ƙwayoyin halitta fiye da insulin kyauta, galibi saboda ƙaramin girman ƙwayoyin nano da aka ɗora wa insulin da aka samar a cikin binciken.
Shawarar ƙwayoyin HepG2 bayan an gama shan su na tsawon awanni 4 tare da sabbin NPs da aka shirya da kuma bushewar NPs: (a) Rarraba shan FITC-insulin ta ƙwayoyin HepG2.(b) Matsakaicin ƙarfin haske na geometric wanda aka bincika ta hanyar kwararar cytometry (n = 3), *P < 0.05 idan aka kwatanta da insulin kyauta.
Haka kuma, hotunan CLSM sun nuna cewa ƙarfin hasken FITC na NPs da aka shirya sabo da FITC-insulin-loaded da FITC-insulin-loaded NPs (ba tare da mannitol) sun fi ƙarfin sauran samfuran (Hoto na 6a). Bugu da ƙari, tare da ƙarin mannitol, mafi girman danko na maganin ya ƙara juriya ga ɗaukar ƙwayoyin halitta, wanda ya haifar da raguwar yaduwar insulin. Waɗannan sakamakon sun nuna cewa NPs da aka busar ba tare da feshi ba da mannitol sun nuna mafi girman ingancin ɗaukar ƙwayoyin halitta saboda girman barbashinsu ya fi ƙanƙanta fiye da na NPs da aka busar bayan sake narkewa.
An sayi Chitosan (matsakaicin nauyin kwayoyin halitta 100 KDa, 75–85% deacetylated) daga Sigma-Aldrich. (Oakville, Ontario, Canada). An sayi Sodium tripolyphosphate (TPP) daga VWR (Radnor, Pennsylvania, Amurka). An yi amfani da insulin ɗin ɗan adam mai hadewa a cikin wannan binciken daga Fisher Scientific (Waltham, MA, Amurka). An sayi insulin ɗan adam mai suna Fluorescein isothiocyanate (FITC) da 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) daga Sigma-Aldrich. (Oakville, Ontario, Kanada). An samo layin ƙwayoyin HepG2 daga ATCC (Manassas, Virginia, Amurka). Duk sauran reagents an yi su ne da nazarce-nazarce ko kuma chromatographic.
Shirya maganin CS na 1 mg/ml ta hanyar narke shi a cikin ruwa mai narkewa sau biyu (ruwan DD) wanda ke ɗauke da 0.1% acetic acid. Shirya maganin TPP da insulin 1 mg/ml ta hanyar narke su a cikin ruwan DD da 0.1% acetic acid, bi da bi. An shirya maganin kafin emulsion da polytron PCU-2-110 mai saurin homogenizer (Brinkmann Ind. Westbury, NY, Amurka). Tsarin shiri shine kamar haka: da farko, ana ƙara 2ml na maganin TPP zuwa 4ml na maganin insulin, sannan a juya cakuda na tsawon minti 30 sannan a gauraya gaba ɗaya. Sannan, an ƙara maganin gauraya zuwa ga maganin CS ta hanyar sirinji a ƙarƙashin juyawa mai sauri (10,000 rpm). An ajiye haɗin a ƙarƙashin juyawa mai sauri (15,000 rpm) a cikin wanka mai kankara na tsawon minti 30, kuma an daidaita su zuwa wani pH don samun insulin NPs masu haɗin gwiwa. Don ƙara haɗuwa da rage girman barbashi na insulin NPs, an sonicated su don ƙarin minti 30 a cikin wanka mai kankara ta amfani da sonicator na nau'in bincike (UP 200ST, Hielscher Ultrasonics, Teltow, Jamus).
An gwada Insulin NPS don diamita na matsakaicin Z, ma'aunin polydispersity (PDI) da ƙarfin zeta ta amfani da ma'aunin watsa haske mai ƙarfi (DLS) ta amfani da Litesizer 500 (Anton Paar, Graz, Austria) ta hanyar narkar da su a cikin ruwan DD a 25°C. An gano yanayin jiki da rarraba girman ta hanyar na'urar hangen nesa ta lantarki ta Hitachi H7600 (TEM) (Hitachi, Tokyo, Japan), sannan aka yi nazarin hotuna ta amfani da software na daukar hoto na Hitachi (Hitachi, Tokyo, Japan). Don tantance ingancin encapsulation (EE) da ƙarfin lodi (LC) na insulin NPs, an saka NPs a cikin bututun ultrafiltration tare da yanke nauyin kwayoyin halitta na 100 kDa kuma centrifuge a 500 xg na tsawon mintuna 30. An auna insulin mara kauri a cikin tacewa ta amfani da tsarin Agilent 1100 Series HPLC (Agilent, Santa Clara, California, Amurka) wanda ya ƙunshi famfon quaternary, samfurin atomatik, na'urar dumama ginshiƙi, da kuma na'urar gano DAD. An yi nazarin insulin ta hanyar ginshiƙin C18 (Zorbax, 3.5 μm, 4.6 mm × 150 mm, Agilent, Amurka) kuma an gano shi a 214 nm. Matakin wayar hannu shine acetonitrile da ruwa, wanda ke ɗauke da 0.1% TFA, rabon gradient daga 10/90 zuwa 100/0, kuma yana aiki na tsawon mintuna 10. An tura matakin wayar hannu a ƙimar kwararar 1.0 ml/min. An saita zafin ginshiƙin zuwa 20 °C. Lissafa kashi-kashi na EE da LC ta amfani da daidaito.(1) da Eq.(2).
An gwada nau'ikan rabon CS/insulin daban-daban daga 2.0 zuwa 4.0 don inganta insulin NP. An ƙara adadin maganin CS daban-daban yayin shirye-shiryen, yayin da aka ci gaba da daidaita cakuda insulin/TPP. An shirya NPs na insulin a cikin kewayon pH na 4.0 zuwa 6.5 ta hanyar sarrafa pH na cakuda a hankali bayan ƙara dukkan mafita (insulin, TPP da CS). An kimanta girman EE da barbashi na insulin nanoparticles a ƙimar pH daban-daban da rabon taro na CS/insulin don inganta samuwar NPs na insulin.
An sanya insulin NPs ɗin da aka inganta a kan kwalin aluminum sannan aka rufe su da tef ɗin da aka matse da tef. Daga nan, an sanya kwantena da aka murƙushe a cikin na'urar busar da daskare ta Labconco FreeZone (Labconco, Kansas City, MO, Amurka) wacce aka sanye da na'urar busar da tire. An saita zafin jiki da matsin lamba a -10 °C, 0.350 Torr na tsawon awanni 2 na farko, da kuma 0 °C da 0.120 Torr na sauran awanni 22 na awanni 24 don samun busasshen insulin NPs.
An yi amfani da na'urar busar da busasshen ruwa ta Buchi Mini B-290 (BÜCHI, Flawil, Switzerland) don samar da insulin mai ƙunshe. Sigogin busarwa da aka zaɓa sune: zafin jiki 100 °C, kwararar abinci 3 L/min, da kwararar iskar gas 4 L/min.
An gano NPs na insulin kafin da bayan bushewa ta amfani da FTIR-ATR spectroscopy. An yi nazarin ƙwayoyin nanoparticles da suka bushe da kuma insulin kyauta da chitosan ta amfani da na'urar aunawa ta Spectrum 100 FTIR (PerkinElmer, Waltham, Massachusetts, Amurka) wacce aka sanye da kayan haɗin samfurin ATR na duniya (PerkinElmer, Waltham, Massachusetts, Amurka). An sami matsakaicin sigina daga na'urori 16 a ƙudurin 4 cm2 a cikin kewayon mita na 4000-600 cm2.
An tantance yanayin busasshen insulin NPs ta hanyar hotunan SEM na insulin NPs da aka daskare da kuma busasshen da aka fesa ta hanyar na'urar Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, Amurka). Babban ma'aunin da aka yi amfani da shi shine ƙarfin lantarki 5 keV da halin yanzu 30 mA.
An sake narkar da duk insulin NPs da aka busar a cikin ruwan dd. An sake gwada girman barbashi, PDI, EE da LC ta amfani da wannan hanyar da aka ambata a baya don tantance ingancin su bayan bushewa. An kuma auna kwanciyar hankali na anhydroinsulin NPs ta hanyar gwada halayen NPs bayan ajiya mai tsawo. A cikin wannan binciken, an adana duk NPs bayan bushewa a cikin firiji na tsawon watanni uku. Bayan watanni uku na ajiya, an gwada NPs don girman barbashi, PDI, EE da LC.
A narkar da 5 mL na NPs da aka sake haɗawa a cikin 45 mL wanda ke ɗauke da ruwan ciki da aka yi kwaikwaya (pH 1.2, wanda ke ɗauke da 1% pepsin), ruwan hanji (pH 6.8, wanda ke ɗauke da 1% trypsin) ko kuma maganin chymotrypsin (100 g/mL, a cikin phosphate buffer, pH 7.8) don kimanta ingancin insulin wajen kare NPs bayan bushewa. An saka su a cikin 37°C tare da saurin tashin hankali na 100 rpm.500 μL na maganin da aka tattara a lokutan daban-daban kuma an ƙayyade yawan insulin ta hanyar HPLC.
An gwada halayen sakin insulin NPs da aka shirya da kuma wanda aka busar ta hanyar amfani da hanyar jakar dialysis (yanke nauyin kwayoyin halitta 100 kDa, Spectra Por Inc.). An yi dialyzed busassun NPs da aka shirya da kuma wanda aka sake haɗawa a cikin ruwa a pH 2.5, pH 6.6, da pH 7.0 (0.1 M phosphate-buffered saline, PBS) don kwaikwayon yanayin pH na ciki, duodenum, da ƙananan hanji, bi da bi. An saka duk samfuran a zafin jiki na 37 °C tare da ci gaba da girgiza a 200 rpm. A shafa ruwan a wajen jakar dialysis na 5 mL a lokutan masu zuwa: 0.5, 1, 2, 3, 4, da 6, sannan a sake cika ƙarar nan da nan da sabon dialysate. An yi nazarin gurɓatar insulin a cikin ruwan ta hanyar HPLC, kuma an ƙididdige yawan sakin insulin daga nanoparticles daga rabon insulin kyauta da aka saki zuwa jimlar insulin da aka lulluɓe a cikin nanoparticles (Equation 3).
An shuka ƙwayoyin cutar kansar hanta na ɗan adam ta hanyar amfani da Dulbecco's Modified Eagle's Medium (DMEM) wanda ke ɗauke da kashi 10% na sinadarin shanu na tayi, penicillin 100 IU/mL, da 100 μg/mL streptomycin29. An kiyaye al'adu a 37°C, da ɗanɗanon da ya dace da kashi 95%, da kuma kashi 5% na CO2. Don gwajin shan ƙwayoyi, an shuka ƙwayoyin HepG2 a ƙwayoyin 1 × 105/ml a kan tsarin zamewar ɗakin Nunc Lab-Tek mai rijiya 8 (Thermo Fisher, NY, Amurka). Don gwaje-gwajen gubar ƙwayoyin halitta, an shuka su a cikin faranti 96 (Corning, NY, Amurka) a yawan ƙwayoyin 5 × 104/ml.
An yi amfani da gwajin MTT don tantance gubar da ke tattare da sinadarin insulin NPs30 da aka shirya kuma aka busar da shi. An shuka ƙwayoyin HepG2 a cikin faranti 96 a yawan ƙwayoyin 5 × 104/mL kuma an noma su na tsawon kwanaki 7 kafin a yi gwaji. An narkar da ƙwayoyin insulin NPs zuwa yawan da ya bambanta (50 zuwa 500 μg/mL) a cikin maganin gargajiya sannan a ba su ga ƙwayoyin. Bayan awanni 24 na haɗuwa, an wanke ƙwayoyin sau 3 da PBS kuma an saka su da matsakaici mai ɗauke da 0.5 mg/ml MTT na ƙarin awanni 4. An tantance gubar da ke tattare da ƙwayoyin ta hanyar auna raguwar enzymatic na tetrazolium MTT mai launin rawaya zuwa formazan mai launin shuɗi a 570 nm ta amfani da na'urar karanta farantin Tecan infinite M200 pro spectrophotometer (Tecan, Männedorf, Switzerland).
An gwada ingancin ɗaukar ƙwayoyin halitta na NPs ta hanyar amfani da na'urar daukar hoton laser confocal da kuma nazarin kwararar cytometry. Kowace rijiya ta tsarin zamewar ɗakin Nunc Lab-Tek an yi mata magani da FITC-insulin kyauta, FITC-insulin-loaded NPs, sannan an sake haɗa 25 μg/mL na FITC-insulin NPs da aka busar a daidai wannan taro sannan aka sake haɗa su na tsawon awanni 4. An wanke ƙwayoyin halitta sau 3 da PBS kuma an gyara su da 4% paraformaldehyde. An yi wa ƙwayoyin nuclei fenti da 4′, 6-diamidino-2-phenylindole (DAPI). An lura da wurin da ake amfani da insulin ta amfani da na'urar daukar hoton laser ta Olympus FV1000/maƙallin hoto mai ɗaukar hoto biyu (Olympus, Shinjuku City, Tokyo, Japan). Don nazarin kwararar cytometry, an ƙara yawan FITC-insulin guda 10 μg/mL kyauta, FITC-insulin-loaded NPs, da FITC-insulin-insulin-resolbilized resolbilized resolbilized NPs zuwa faranti 96 da aka shuka tare da ƙwayoyin HepG2 da an saka shi a cikin injin na tsawon awanni 4. Bayan awanni 4 na shigarsa, an cire ƙwayoyin halitta kuma an wanke su sau 3 da FBS. An yi nazarin ƙwayoyin 5 × 104 a kowane samfurin ta amfani da na'urar auna kwararar BD LSR II (BD, Franklin Lakes, New Jersey, Amurka).
Ana bayyana dukkan dabi'u a matsayin matsakaicin ± karkacewar daidaito. An tantance kwatancen da ke tsakanin dukkan ƙungiyoyi ta amfani da ANOVA ta hanya ɗaya ko gwajin t ta IBM SPSS Statistics 26 don Mac (IBM, Endicott, New York, Amurka) kuma an ɗauki p <0.05 a matsayin mahimmanci a kididdiga.
Wannan binciken ya nuna sassauci da ikon busar da feshi don busar da ƙwayoyin chitosan/TPP/insulin masu alaƙa da ruwa tare da ingantaccen sake haɗawa idan aka kwatanta da hanyoyin busar da daskare na yau da kullun ta amfani da wakilai masu ƙarfi ko ƙarfin cryoprotectants da ƙarfin kaya mafi girma. Insulin nanoparticles da aka inganta sun samar da matsakaicin girman barbashi na 318 nm da ingancin encapsulation na 99.4%. Sakamakon SEM da FTIR bayan busar da ruwa ya nuna cewa an kiyaye tsarin zagaye ne kawai a cikin NPs da aka fesa tare da kuma ba tare da mannitol ba kuma an lyophilized tare da mannitol, amma NPs da aka lyophilized ba tare da mannitol ba sun ruɓe yayin busar da ruwa. A cikin gwajin ikon sake haɗawa, insulin nanoparticles da aka fesa ba tare da mannitol ba sun nuna ƙaramin matsakaicin girman barbashi da mafi girman lodi lokacin sake haɗawa. Halayen sakin duk waɗannan NPs da aka yayyanka sun nuna cewa an saki su cikin sauri a cikin maganin pH = 2.5 da pH = 7, kuma suna da kwanciyar hankali sosai a cikin maganin pH = 6.5. Idan aka kwatanta da sauran NPs da aka yayyanka, NPs da aka yayyanka ba tare da mannitol ya nuna fitowar da ta fi sauri. Wannan sakamakon ya yi daidai da wanda aka gani a gwajin ɗaukar ƙwayoyin halitta, kamar yadda NPs busassun da aka fesa ba tare da mannitol ba kusan sun kiyaye ingancin ɗaukar ƙwayoyin halitta na NPs da aka shirya sabo. Waɗannan sakamakon sun nuna cewa busassun ƙwayoyin insulin da aka shirya ta hanyar busar da feshi ba tare da mannitol ba sun fi dacewa don ci gaba da sarrafawa zuwa wasu nau'ikan allurai marasa ruwa, kamar allunan baki ko fina-finan bioadhesive.
Saboda matsalolin mallakar fasaha, bayanan da aka samar da/ko aka yi nazari a kansu a lokacin binciken na yanzu ba a samun su a bainar jama'a, amma ana samun su daga marubutan idan aka buƙata.
Kagan, A. Ciwon suga na nau'in 2: asalin zamantakewa da kimiyya, matsalolin lafiya, da kuma tasirinsa ga marasa lafiya da sauransu. (McFarlane, 2009).
Singh, AP, Guo, Y., Singh, A., Xie, W. & Jiang, P. Ci gaban ƙunsar insulin: shin shan maganin baki yanzu zai yiwu? J. Pharmacy.bio-pharmacy.reservoir.1, 74–92 (2019).
Wong, CY, Al-Salami, H. & Dass, CR Ci gaban da aka samu kwanan nan a tsarin isar da liposome mai ɗauke da insulin ta baki don maganin ciwon suga.Interpretation.J. Pharmacy.549, 201–217 (2018).