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Uduce duto twa insuline (NPs) dufite umutwaro mwinshi twasanze dukoreshwa mu buryo butandukanye mu buryo butandukanye bw'ingano. Uyu murimo ugamije gusuzuma ingaruka zo kumisha no gusya ku miterere ya chitosan nanoparticles za insuline, hamwe na mannitol cyangwa ntayo nk'umuti urinda ubushyuhe. Twanasuzumye ubwiza bw'utu duce duto dusya. Mbere yo kubura amazi, ingano y'uduce duto twa chitosan/sodium tripolyphosphate/insuline twashyizwe hamwe yatunganyijwe neza ikaba 318 nm, PDI yari 0.18, ubushobozi bwo gusya bwari 99.4%, naho umutwaro wari 25.01%. Nyuma yo kongera kuvugurura, uduce duto twa insuline twose, uretse utwakozwe hakoreshejwe uburyo bwo gusya butagira mannitol, twagumanye imiterere y'uduce duto. Ugereranyije n'uduce duto dufite mannitol twakuwemo amazi hakoreshejwe spray, uduce duto twa mannitol twagaragaje ingano ntoya y'uduce duto (376 nm) n'umutwaro munini cyane (25.02%). hamwe n'igipimo kimwe cyo gufunga (98.7%) na PDI (0.20) hakoreshejwe uburyo bwo kumisha cyangwa gukonjesha. Utwuma tw’uturemangingo twumye hakoreshejwe spray yumisha nta mannitol twanatumye insuline irekurwa vuba cyane kandi ikagira akamaro kanini mu gufata uturemangingo. Uyu murimo ugaragaza ko kumisha spray bishobora gukurura utwuma tw’uturemangingo twa insuline tudakeneye cryoprotectants ugereranije n'uburyo busanzwe bwo kumisha bukonjesha, bigatuma ubushobozi bwo gutwara ibintu burushaho kwiyongera, ibisabwa mu kongeramo ibintu bike n'ikiguzi cyo gukora kikaba cyiza cyane.
Kuva yavumburwa mu 1921,2,3, insuline n'imiti yayo byarokoye ubuzima bw'abarwayi ba diyabete yo mu bwoko bwa 1 (T1DM) na diyabete yo mu bwoko bwa 2 (T1DM). Ariko, kubera ko ifite imiterere myinshi ya poroteyine, insuline irahuzwa byoroshye, igasenywa na enzymes za proteolytic, kandi igakurwaho n'ingaruka za mbere. Abantu basanzwe barwaye diyabete yo mu bwoko bwa 1 bakeneye inshinge za insuline ubuzima bwabo bwose. Abarwayi benshi basanzwe barwaye diyabete yo mu bwoko bwa 2 nabo bakeneye inshinge za insuline z'igihe kirekire. Inshinge za insuline za buri munsi ni isoko ikomeye y'ububabare n'imibabaro bya buri munsi kuri aba bantu, bigira ingaruka mbi ku buzima bwo mu mutwe. Kubera iyo mpamvu, ubundi buryo bwo gutanga insuline butuma umuntu adakomeza kumererwa nabi, nko gutanga insuline mu kanwa, burimo kwigwaho cyane5 kuko bufite ubushobozi bwo gusubiza ubuzima bwiza bw'abantu bagera kuri miliyari 5 barwaye diyabete ku isi yose.
Ikoranabuhanga rya Nanoparticle ryatanze iterambere rikomeye mu kugerageza gufata insuline yo mu kanwa4,6,7. Ihuriro rikingira insuline neza kandi rikayirinda kwangirika kugira ngo igere ahantu runaka. Ariko, ikoreshwa rya nanoparticle rifite imbogamizi nyinshi, ahanini bitewe n'ibibazo byo kudahinduka kw'uduce duto. Guterana bishobora kubaho mu gihe cyo kubika, bigatuma nanoparticles zifite insuline zigabanuka8. Byongeye kandi, ubushobozi bwo guhagarara neza bwa polymer matrix ya nanoparticles na insuline nabwo bugomba kwitabwaho kugira ngo habeho kudahinduka kwa nanoparticles za insuline (NPs). Muri iki gihe, ikoranabuhanga ryo kumisha mu buryo bwa fringe ni ryo hame ry'agaciro ryo gukora NPs zihamye mu gihe hirindwa impinduka zidakenewe mu gihe cyo kubika9.
Ariko, gukonjesha bisaba kongeramo ibintu bikingira kugira ngo imiterere y’uruziga rwa NPs idakorwaho n’imbaraga z’urubura. Ibi bigabanya cyane umutwaro w’uduce duto twa insuline nyuma yo guhindurwamo lyophilization, kuko uduce duto twa insuline dufata igice kinini cy’uburemere. Kubwibyo, NP za insuline zikorwa akenshi ziboneka ko zitabereye mu gukora ifu yumye, nk'ibinini binyobwa n'udupira two kunywa, bitewe nuko hakenewe uduce duto twinshi tw’uduce duto tw’umunyu kugira ngo insuline igere ku rwego rwo kuvura.
Kumisha imiti ni inzira izwi kandi ihendutse mu nganda mu gukora ifu yumye iva mu byiciro by'amazi mu nganda zikora imiti10,11. Kugenzura uburyo bwo gukora uduce duto bituma habaho uburyo bwiza bwo gufunga neza ibintu byinshi bikora ku mubiri 12, 13. Byongeye kandi, byabaye uburyo bwiza bwo gutegura poroteyine zifunze kugira ngo zinyorwe. Mu gihe cyo kumisha imiti, amazi ahita ashira vuba cyane, bifasha kugumana ubushyuhe bw'igice cy'umubiri 11,14, bigatuma ikoreshwa ryayo rishyirwa mu gufunga ibice by'ibice bifata ubushyuhe. Mbere yo kumisha imiti, ibikoresho byo gusiga bigomba guhuzwa neza n'umuti urimo ibintu bifunze11,14. Bitandukanye no kumisha muri firigo, guhuza mbere yo gufunga mu gufunga imiti byongera ubushobozi bwo gufunga mu gihe cyo kubura amazi. Kubera ko uburyo bwo kumisha imiti budasaba imiti ikingira ubushyuhe, kumisha imiti bishobora gukoreshwa mu gukora NPs zumye zifite umutwaro mwinshi.
Ubu bushakashatsi bugaragaza ikorwa rya NPs zifite insuline binyuze mu guhuza chitosan na sodium tripolyphosphate hakoreshejwe uburyo bwa gel ya ion. Ion gelation ni uburyo bwo gutegura butuma habaho nanoparticles binyuze mu mikoranire ya electrostatic hagati y’ubwoko bubiri cyangwa burenze bwa ionic mu bihe bimwe na bimwe. Uburyo bwo gukonjesha no gusukura bukoresheje spray-kwumisha bwakoreshejwe mu gukamura nanoparticles za chitosan/sodium tripolyphosphate/insuline cross-linked nanoparticles. Nyuma yo kubura amazi, imiterere yazo yasesenguwe na SEM. Ubushobozi bwazo bwo kongera kuzura bwapimwe hapimwa ingano yazo, imbaraga zo hejuru, PDI, ubushobozi bwo gufunga, n'ingano y'umutwaro. Ubwiza bwa nanoparticles zasubiwemo zikorwa hakoreshejwe uburyo butandukanye bwo kubura amazi bwapimwe hagereranywa uburyo bwo kurinda insuline, imyitwarire yo kurekura, n'ubushobozi bwo gufata uturemangingo.
PH y'umuti uvanze n'igipimo cya chitosan na insuline ni ibintu bibiri by'ingenzi bigira ingaruka ku bunini bw'uduce duto n'imikorere myiza ya encapsulation (EE) ya NPs za nyuma, kuko bigira ingaruka ku buryo butaziguye ku mikorere ya ionotropic gelation. PH y'umuti uvanze byagaragaye ko ifitanye isano rikomeye n'ubunini bw'uduce duto n'imikorere myiza ya encapsulation (Ishusho ya 1a). Nkuko bigaragara ku Ishusho ya 1a, uko pH yiyongeraga kuva kuri 4.0 kugeza kuri 6.0, ingano mpuzandengo y'uduce duto (nm) yaragabanutse maze EE yiyongera cyane, mu gihe iyo pH yiyongeraga ikagera kuri 6.5, ingano mpuzandengo y'uduce duto yatangiye kwiyongera maze EE iguma idahinduka. Uko igipimo cya chitosan na insuline cyiyongera, ingano mpuzandengo y'uduce na yo iriyongera. Byongeye kandi, nta mpinduka muri EE yagaragaye ubwo uduce duto twategurwaga ku gipimo cy'uburemere bwa chitosan/insuline kiri hejuru ya 2.5:1 (w/w) (Ishusho ya 1b). Kubwibyo, uburyo bwiza bwo gutegura muri ubu bushakashatsi (pH 6.0, igipimo cy'uburemere bwa chitosan/insuline cya 2.5:1) byakoreshejwe kugira ngo Tegura uduce duto tw’inyongera dufite insuline kugira ngo urusheho kwiga. Muri ubu buryo bwo gutegura, ingano mpuzandengo y’uduce duto tw’inyongera twa insuline yatunganyijwe kugira ngo ibe 318 nm (Ishusho ya 1c), PDI yari 0.18, ubushobozi bwo gushyiramo ibintu bwari 99.4%, ubushobozi bwa zeta bwari 9.8 mv, naho umutwaro wa insuline wari 25.01% (m/m ). Hashingiwe ku byavuye muri microscopy ya transmission electron (TEM), uduce duto tw’inyongera twari hafi kuzunguruka kandi dufite ingano imwe (Ishusho ya 1d).
Uburyo bwo kunoza ibipimo bya insuline nanoparticles: (a) ingaruka za pH ku bunini bw'umurambararo n'ubushobozi bwo gufunga (EE) bw'uduce duto twa insuline (twateguwe ku gipimo cya 5:1 cy'ubunini bwa chitosan na insuline); (b) chitosan n'ingaruka z'ubunini bw'igipimo cya insuline ku bunini bw'umurambararo n'ubushobozi bwo gufunga (EE) bwa insuline NPs (twateguwe kuri pH 6); (c) ikwirakwizwa ry'uduce duto twa insuline nanoparticles nziza; (d) TEM mikorographe ya insuline NPs nziza.
Birazwi neza ko chitosan ari polyelectrolyte idakomeye ifite pKa ya 6.5. Ifite amashanyarazi meza mu buryo bw’aside kuko itsinda ryayo ry’ingenzi ry’amino riterwa na iyoni za hydrogen15. Kubwibyo, ikunze gukoreshwa nk'umutwaro wo gufunga macromolecules zifite amashanyarazi mabi. Muri ubu bushakashatsi, chitosan yakoreshejwe mu gufunga insuline ifite ingingo ya isoelectric ya 5.3. Kubera ko chitosan ikoreshwa nk'ibikoresho byo gutwikira, uko igipimo cyayo cyiyongera, ubunini bw'urwego rwo hanze rw'uduce duto tw’ingirabuzimafatizo buriyongera, bigatuma ingano y’uduce duto irushaho kuba nini. Byongeye kandi, urugero rwo hejuru rwa chitosan rushobora gufunga insuline nyinshi. Muri iki gihe, EE yari hejuru cyane igihe igipimo cya chitosan na insuline cyageraga kuri 2.5:1, kandi nta mpinduka nini yabayeho muri EE igihe igipimo cyakomeje kwiyongera.
Uretse igipimo cya chitosan na insuline, pH yagize uruhare runini mu gutegura NPs. Gan et al. 17 bigaga ku ngaruka za pH ku bunini bw'uduce twa chitosan nanoparticles. Basanze ubunini bw'uduce bukomeza kugabanuka kugeza pH igeze kuri 6.0, kandi ubwiyongere bugaragara bw'uduce bwagaragaye kuri pH > 6.0, ibi bikaba bihuye n'ibyo twabonye. Iki kintu giterwa n'uko iyo pH yiyongera, molekile ya insuline igira charge mbi ku buso, bityo, bigatuma habaho imikoranire ya electrostatic na chitosan/sodium tripolyphosphate (TPP), bigatuma ingano y'uduce duto na EE nyinshi. Ariko, ubwo pH yahindurwaga kuri 6.5, amatsinda ya amino kuri chitosan yarakuweho, bituma chitosan igwa. Bityo, pH nyinshi ituma amino ions zigaragara kuri TPP na insuline nke, bigatuma habaho cross-link nke, ingano nini y'uduce duto kandi EE nke.
Isesengura ry’imiterere y’imiterere ya NPs zumye mu buryo bwa konje n’izumutse zishobora kuyobora uburyo bwiza bwo guhitamo uburyo bwo gukora ifu. Uburyo bwiza bukwiye gutanga ubushobozi bwo kudatakaza amazi, imiterere imwe y’uduce, ubushobozi bwinshi bwo gukurura imiti no gushonga neza mu gisubizo cy’umwimerere. Muri ubu bushakashatsi, kugira ngo tugereranye neza ubwo buryo bwombi, NPs za insuline zifite cyangwa zitari zo 1% ya mannitol zakoreshejwe mu gihe cyo kubura amazi. Mannitol ikoreshwa nk’ikintu gikingira cyangwa gikingira amazi mu buryo butandukanye bw’ifu yumye kugira ngo yumishe mu buryo bwa konje no kumisha. Ku duce twa insuline twa lyophilized tudafite mannitol, nk’uko bigaragara ku Ishusho ya 2a, imiterere y’ifu ifite imyenge myinshi ifite ubuso bunini, butaringaniye kandi bugoye yagaragaye hifashishijwe microscopy ya electron (SEM). Uduce duke twagaragaye mu ifu nyuma yo kubura amazi (Ishusho ya 2e). Ibi byagaragaye ko NPs nyinshi zarangiritse mu gihe cyo kumisha mu buryo bwa konje nta kintu na kimwe gikingira amazi. Ku duce twa insuline twa 1% twa mannitol twa 1%, utwuma duto dufite ubuso bworoshye twagaragaye (Ishusho ya 1). 2b, d, f, h). Uduce duto twa insuline twumishijwe nta mannitol twagumye turi uruziga ariko duhinduka iminkanyari ku buso (Ishusho ya 2c). Ubuso bumeze nk'uruziga n'ubufite iminkanyari biraganirwaho cyane mu imyitwarire yo kurekura no mu bipimo byo gufata uturemangingo hepfo. Hashingiwe ku kuntu bigaragara NPs zumye, NPs zombi zumye zidafite mannitol na NPs zarakonjeshejwe kandi zigashyirwamo mannitol zatanze ifu nziza za NPs (Ishusho ya 2f, g, h). Uko ubuso buri hagati y'ubuso bw'uduce bunini, niko gushonga kw'uturemangingo kuba kwinshi bityo n'umuvuduko wo kurekura ukaba munini.
Imiterere ya NPs zitandukanye za insuline zaciwe amazi: (a) Ishusho ya SEM ya NPs za insuline za lyophilized zidafite mannitol; (b) Ishusho ya SEM ya NPs za insuline za lyophilized zirimo mannitol; (c) NPs za insuline za lyophilized zirimo mannitol Ishusho ya SEM ya ; (d) Ishusho ya SEM ya NPs za insuline za lyophilized zirimo mannitol; (e) Ishusho ya NPs za insuline za lyophilized zirimo mannitol; (f) Ishusho ya NPs za insuline za lyophilized zirimo mannitol; (g) Ishusho ya NPs za insuline za lyophilized zirimo mannitol; (h) Ishusho ya NPs za insuline za lyophilized zirimo mannitol.
Mu gihe cyo kumisha mu bukonje, mannitol ikora nk'ikintu kirinda ubukonje, ikarinda NPs kuba mu buryo budafite isura kandi ikarinda kwangirika kwa kristu z'urubura19. Ibinyuranye n'ibyo, nta ntambwe yo gukonjesha ibaho mu gihe cyo kumisha mu bukonje. Bityo, mannitol ntabwo ikenewe muri ubu buryo. Mu by'ukuri, NPs zumye mu bukonje zidafite mannitol zatanze NPs nziza nk'uko byasobanuwe mbere. Ariko, mannitol iracyashobora gukora nk'icyuzuzo mu gikorwa cyo kumisha mu bukonje kugira ngo ihe NPs imiterere y'uruziga20 (Ishusho ya 2d), bifasha kubona imyitwarire imwe yo kurekura NPs zifunze. Byongeye kandi, biragaragara ko hari uduce duto dushobora kugaragara muri NPs za insuline zumye mu bukonje n'izifunze zifunze zirimo mannitol (Ishusho ya 2b, d), bishobora guterwa no kwirundanya kwa mannitol mu gice cy'imbere cy'uduce hamwe na insuline ifunze. Kuri.Urutonde rwa Chitosan. Ni ngombwa kumenya ko muri ubu bushakashatsi, kugira ngo imiterere y’uruziga ikomeze kuba myiza nyuma yo kubura amazi, igipimo cya mannitol na chitosan gikomeza kuba kuri 5:1, kugira ngo ingano nini y’ibinure ishobora kongera ingano y’uduce twa NPs twumye.
Spektroskopiya ya Fourier transform infrared attenuated total reflection (FTIR-ATR) yagaragaje uruvange rw’umubiri rwa insuline yigenga, chitosan, chitosan, TPP na insuline. NP zose zabuze amazi zaranzwe hakoreshejwe spectroscopy ya FTIR-ATR. By’umwihariko, imbaraga za band za 1641, 1543 na 1412 cm-1 zagaragaye muri NPs zafunzwe zikonjeshwa na mannitol no muri NPs zasizwe na mannitol hamwe na mannitol (Ishusho ya 3). Nkuko byatangajwe mbere, uku kwiyongera kw’imbaraga kwari gufitanye isano no guhuza chitosan, TPP na insuline. Iperereza ku mikoranire iri hagati ya chitosan na insuline ryagaragaje ko muri spectra ya FTIR y’uduce duto twa chitosan twashyizwemo insuline, umugozi wa chitosan wahuriranye n’uwa insuline, byongera imbaraga za carbonyl (1641 cm-1) na amine (1543 cm-1). Amatsinda ya tripolyphosphate ya TPP afitanye isano n’amatsinda ya ammonium muri chitosan, arema umugozi uri kuri cm 1412-1.
FTIR-ATR spectra ya insuline y'ubuntu, chitosan, uruvange rw'umubiri rwa chitosan/TPP/insuline na NPs byakuwemo amazi hakoreshejwe uburyo butandukanye.
Byongeye kandi, ibi bisubizo bihuye n’ibyagaragajwe muri SEM, byagaragaje ko NPs zashyizwemo ibinini zagumye uko zari zimeze haba iyo zasutsweho mannitol ndetse no mu gihe cy’ingufu, ariko iyo mannitol idafite, gusa spray-suspension yatangaga uduce twashyizwemo ibinini. Ibinyuranye n’ibyo, ibisubizo bya FTIR-ATR bya NPs zasutsweho ibinini nta mannitol byari bisa cyane n’uruvange rw’ibinyabutabire bya chitosan, TPP, na insuline. Ibi bisubizo bigaragaza ko isano iri hagati ya chitosan, TPP na insuline itakiriho muri NPs zasutsweho ibinini nta mannitol. Imiterere ya NPs yarangiritse mu gihe cyo gukonjesha nta cryoprotectant, ibi bigaragara mu bisubizo bya SEM (Ishusho ya 2a). Hashingiwe ku miterere n’ibisubizo bya FTIR bya NPs za insuline zabuze amazi, gusa NPs za lyophilized, spray-suspensed, na mannitol-free nizo zakoreshejwe mu igerageza ryo kongera gukora isuzuma kandi NPs za mannitol-free NPs zangiritse bitewe no kubora kwa NPs za mannitol mu gihe cyo kubura amazi. Muganire.
Kubura amazi mu mubiri bikoreshwa mu kubika igihe kirekire no kongera gukoreshwa mu zindi formulaire. Ubushobozi bwa NP zumutse bwo kongera gukora nyuma yo kubika ni ingenzi cyane mu ikoreshwa ryazo mu buryo butandukanye nk'ibinini n'amafirime. Twabonye ko ingano mpuzandengo y'uduce twa insuline NPs twumutse mu gihe nta mannitol yiyongereyeho gato nyuma yo kongera gukora. Ku rundi ruhande, ingano y'uduce twa insuline zumutse mu buryo bwa spray na condensate hamwe na mannitol yiyongereye cyane (Imbonerahamwe ya 1). PDI na EE ntibyahindutse cyane (p > 0.05) nyuma yo kongera gukora NPs zose muri ubu bushakashatsi (Imbonerahamwe ya 1). Ibi byagaragaye ko uduce twinshi twagumye uko twari tumeze nyuma yo kongera gushonga. Ariko, kongeramo mannitol byatumye insuline igabanuka cyane mu gutwika mannitol nanoparticles (Imbonerahamwe ya 1). Mu buryo bunyuranye, ingano ya insuline mu gutwika NPs idafite mannitol yagumye uko yari imeze mbere (Imbonerahamwe ya 1).
Birazwi neza ko gukurura uduce duto tw’umuti ari ingenzi cyane iyo ukoreshejwe mu gutanga imiti. Kuri NP zifite umutwaro muto, hakenewe ibikoresho byinshi cyane kugira ngo bigere ku rwego rwo kuvura. Ariko, ubukana bwinshi bw’ubwo bunini bwa NP butera ingorane no kugorwa no gutanga imiti mu kanwa no mu gutera inshinge, uko bikurikirana. Byongeye kandi, NP za insuline zishobora no gukoreshwa mu gukora ibinini na biofilms viscous23, 24, bisaba gukoresha NP nyinshi ku rugero rwo hasi rw’umutwaro, bigatuma habaho ibinini binini na biofilms nini zidakwiye gukoreshwa mu kanwa. Kubwibyo, NPs zaciwe amazi zifite umutwaro mwinshi wa insuline zirakenewe cyane. Ibisubizo byacu bigaragaza ko umutwaro mwinshi wa insuline wa NPs zumye zidafite mannitol ushobora gutanga inyungu nyinshi nziza kuri ubu buryo bwo gutanga ubundi buryo.
NP zose zabuze amazi zabitswe muri firigo mu gihe cy'amezi atatu. Ibisubizo bya SEM byagaragaje ko imiterere ya NP zose zabuze amazi itahindutse cyane mu gihe cy'amezi atatu yo kubika (Ishusho ya 4). Nyuma yo kongera kuvugurura mu mazi, NP zose zagaragaje kugabanuka gato kwa EE kandi zisohora hafi ingano nto (~5%) ya insuline mu gihe cy'amezi atatu yo kubika (Imbonerahamwe ya 2). Ariko, ingano mpuzandengo y'uduce duto tw'uduce duto twiyongereye. Ingano y'uduce duto twa NPs twasizwe ifuro nta mannitol yiyongereye igera kuri 525 nm, mu gihe NPs zasizwe ifuro n'izasizwe ifuro hamwe na mannitol yiyongereye igera kuri 872 na 921 nm, uko bikurikirana (Imbonerahamwe ya 2).
Imiterere ya NP zitandukanye za insuline zangiritse zibikwa mu gihe cy'amezi atatu: (a) Ishusho ya SEM ya NP za insuline za lyophilized hamwe na mannitol; (b) Ishusho ya SEM y'uduce duto twa insuline twumye tudafite mannitol; (c) Ishusho ya SEM idafite mannitol ya insuline yumye.
Byongeye kandi, amazi yagaragaye mu duce twa insuline nanoparticles twasubiwemo twumishijwe na mannitol hanyuma twumishwa muri frigo (Ishusho ya S2). Ibi bishobora guterwa n'uduce tunini tudahagaze neza mu mazi. Ibisubizo byose byavuzwe haruguru bigaragaza ko uburyo bwo kumisha spray bushobora kurinda uduce twa insuline nanoparticles kubura amazi kandi ko uduce twinshi twa insuline nanoparticles dushobora kuboneka nta byuzuza cyangwa cryoprotectants.
Gufata insuline byageragejwe kuri pH = 2.5 hagati hifashishijwe pepsin, trypsin, na α-chymotrypsin kugira ngo hagaragazwe ubushobozi bwo kurinda NPs mu igogora rya enzymatic nyuma yo kubura amazi. Gufata insuline bya NPs zaciwe amazi byagereranyijwe n'ibya NPs zateguwe vuba, kandi insuline y'ubuntu yakoreshejwe nk'uburyo bwo kugenzura nabi. Muri ubu bushakashatsi, insuline y'ubuntu yagaragaje ko insuline yakuweho vuba mu masaha 4 mu buryo butatu bwo kuvura enzymatic (Ishusho ya 5a–c). Mu buryo bunyuranye, isuzuma rya insuline rya NPs zakuweho zikonjeshejwe na mannitol na NPs zakuweho spray-dryed hamwe na mannitol cyangwa zitayikuweho ryagaragaje ko izi NPs zirinda igogora rya enzymatic, nk'uko byari bimeze kuri NPs zakozwe vuba (Ishusho ya 1).5a-c). Hakoreshejwe na nanoparticles muri pepsin, trypsin, na α-chymotrypsin, insuline irenga 50%, 60%, na 75% yashoboraga kurindwa mu masaha 4, uko bikurikirana (Ishusho ya 5a–c). Ubu bushobozi bwo kurinda insuline bushobora kongera amahirwe yo kugira insuline nyinshi. kwinjiza mu maraso25. Ibi bisubizo bigaragaza ko kumisha hakoreshejwe cyangwa nta mannitol no kumisha hakoreshejwe mannitol mu buryo bwa frigo bishobora kubungabunga ubushobozi bwa NPs bwo kurinda insuline nyuma yo kubura amazi.
Kurinda no kurekura insuline NPs zangiritse: (a) kurinda insuline mu gisubizo cya pepsin; (b) kurinda insuline mu gisubizo cya trypsin; (c) kurinda insuline hakoreshejwe α-chymotrypsin; (d) Imyitwarire yo kurekura NPs zangiritse muri pH = 2.5; (e) imyitwarire yo kurekura NPs zangiritse muri pH = 6.6; (f) imyitwarire yo kurekura NPs zangiritse muri pH = 7.0.
Insulini yumye nshya yateguwe kandi yongeye gushyirwamo NPs yashyizwe mu buryo bushya mu buryo butandukanye (pH = 2.5, 6.6, 7.0) kuri 37 °C, biganisha ku miterere ya pH y'igifu, duodenum, n'amara mato yo hejuru, kugira ngo harebwe ingaruka za insuline ku kudashobora kwa insuline. Imyitwarire yo kurekura ibintu mu buryo butandukanye. Igice cy'inzira y'igifu. Kuri pH = 2.5, NPs zifite insuline na NPs zumutse zasubiwemo zagaragaje ko zatangiye kurekura mu isaha imwe ya mbere, hagakurikiraho kurekura buhoro mu masaha 5 yakurikiyeho (Ishusho ya 5d). Uku kurekura vuba mu ntangiriro birashoboka cyane ko biterwa no gukurura vuba kwa molekile za poroteyine zitari mu buryo bwuzuye mu miterere y'imbere y'agace. Kuri pH = 6.5, NPs zifite insuline na NPs zumutse zasubiwemo zagaragaje ko zirekura neza kandi buhoro mu masaha 6, kuko pH y'umuti w'ikizamini yari isa n'iya NPs zateguwe (Ishusho ya 5e). Kuri pH = 7, NPs zari zihagaze neza kandi hafi ya zose zarangiritse mu masaha abiri ya mbere (Ishusho ya 5f). Ibi biterwa nuko gukuraho kwa chitosan biba kuri pH iri hejuru, bigatuma habaho urusobe ruto rwa polymer no kurekura insuline yuzuye.
Byongeye kandi, NP za insuline zumye zidafite mannitol zagaragaje ko zirekurwa vuba kurusha izindi NP zabuze amazi (Ishusho ya 5d–f). Nkuko byasobanuwe mbere, NP za insuline zumye zidafite mannitol zagaragaje ingano ntoya y'uduce. Uduce duto dutanga ubuso bunini, bityo umuti mwinshi uzaba uri ku buso bw'uduce cyangwa hafi yatwo, bigatuma umuti urekurwa vuba26.
Uburozi bwa NPs bwakoreweho ubushakashatsi hakoreshejwe isuzuma rya MTT. Nkuko bigaragara ku Ishusho ya S4, NPs zose zabuze amazi zagaragaye ko nta ngaruka nini zigira ku mikorere y’uturemangingo ku gipimo cya 50–500 μg/ml, bigaragaza ko NPs zose zabuze amazi zishobora gukoreshwa mu buryo bwizewe kugira ngo zigere ku idirishya ryo kuvura.
Umwijima ni rwo rugingo rw'ingenzi insuline ikoresha mu gukora imirimo yayo ya fiziki. Uturemangingo twa HepG2 ni urugingo rw'uturemangingo tw'umwijima rw'umuntu rukunze gukoreshwa nk'icyitegererezo cyo gufata hepatocyte muri vitro. Aha, uturemangingo twa HepG2 twakoreshejwe mu gusuzuma uburyo bwo gufata NPs zaciwe amazi hakoreshejwe uburyo bwo gukonjesha no gupfundikira. Gufata selile hakoreshejwe uburyo bwa confocal laser scanning hakoreshejwe flow cytometry no kureba nyuma y'amasaha menshi yo gupfundikira hakoreshejwe free FITC insulin ku gipimo cya 25 μg/mL, NPs za FITC zateguwe vuba hamwe na NPs zaciwe amazi muri FITC insulin-loaded ku gipimo kingana cya insuline. Hakozwe isuzuma rya microscopy ya Quantitative (CLSM). NPs zaciwe neza zidafite mannitol zangijwe mu gihe cyo kubura amazi kandi ntizasuzumwe muri iki kizamini. Ingufu za fluorescence intracellular za NPs zateguwe vuba zikozwe muri insuline, NPs zaciwe neza zikozwe muri mannitol, na NPs zaciwe neza zikozwe muri spray hamwe na mannitol (Ishusho ya 6a) zari 4.3, 2.6, 2.4, na Ikubye inshuro 4.1 hejuru y’izisanzwe. Itsinda rya FITC-insuline, uko bikurikirana (Ishusho ya 6b). Ibi bisubizo bigaragaza ko insuline ifungishijwe ifite imbaraga nyinshi mu gufata ingirabuzimafatizo kurusha insuline y’ubuntu, ahanini bitewe n’ingano nto y’uduce duto twa insuline twakozwe mu bushakashatsi.
Ukwinjira kwa selile za HepG2 nyuma y'amasaha 4 yo kwinjizwa hamwe na NPs nshya zateguwe hamwe na NPs zabuze amazi: (a) Ikwirakwizwa rya FITC-insuline ifatwa n'selile za HepG2. (b) Igipimo cya geometrike cy'ubukana bwa fluorescence cyasesenguwe hakoreshejwe flow cytometry (n = 3), *P < 0.05 ugereranije na insuline yigenga.
Mu buryo nk'ubwo, amashusho ya CLSM yagaragaje ko imbaraga za FITC fluorescence za NPs nshya zateguwe na FITC-insulin-loaded na NPs za FITC-insulin-loaded spray-yumye (zidafite mannitol) zari zikomeye cyane kurusha iz'izindi ngero (Ishusho ya 6a). Byongeye kandi, hamwe no kongeramo mannitol, ubukana bwinshi bw'umuti bwongereye ubudahangarwa ku ifatwa ry'uturemangingo, bigatuma ikwirakwira rya insuline rigabanuka. Ibi bisubizo bigaragaza ko NPs za mannitol-free spray-yumye zagaragaje ubushobozi bwo gufata uturemangingo kuko ingano yazo yari ntoya ugereranyije n'iya NPs za fungishijwe nyuma yo kongera gushonga.
Chitosan (uburemere bw'ingirabuzimafatizo 100 KDa, 75–85% deacetylated) yaguzwe muri Sigma-Aldrich. (Oakville, Ontario, Kanada). Sodium tripolyphosphate (TPP) yaguzwe muri VWR (Radnor, Pennsylvania, Amerika). Insulini y'abantu yakoreshejwe muri ubu bushakashatsi yakuwe muri Fisher Scientific (Waltham, MA, Amerika). Insulini y'abantu ifite label ya Fluorescein isothiocyanate (FITC) na 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) yaguzwe muri Sigma-Aldrich. (Oakville, Ontario, Kanada). Uduce tw'uturemangingo twa HepG2 twakuwe muri ATCC (Manassas, Virginia, Amerika). Izindi reagents zose zari isesengura cyangwa icyerekezo cya chromatographic.
Tegura umuti wa 1 mg/ml wa CS uwushongesha mu mazi abiri yaciwe (amazi ya DD) arimo aside asetiki 0.1%. Tegura umuti wa 1 mg/ml wa TPP na insuline ubishongesha mu mazi ya DD na aside asetiki 0.1%, uko bikurikirana. Pre-emulsion yateguwe hakoreshejwe polytron PCU-2-110 homogenizer yihuta cyane (Brinkmann Ind. Westbury, NY, USA). Uburyo bwo gutegura ni ubu bukurikira: mbere na mbere, 2ml y'umuti wa TPP yongerwa kuri 4ml y'umuti wa insuline, hanyuma uruvange rukabyukwa iminota 30 hanyuma rukavanga neza. Hanyuma, umuti uvanze wongewemo umuti wa CS unyuze muri seringe munsi yo kuvanga cyane (10.000 rpm). Uruvange rwabitswe munsi yo kuvanga cyane (15.000 rpm) mu bwogero bw'urubura mu gihe cy'iminota 30, kandi byahinduwe kuri pH runaka kugira ngo haboneke NP za insuline zifatanye. Kugira ngo birusheho guhuza no kugabanya ingano y'uduce twa insuline NPs, byashyizwemo sonic kugira ngo bikongererweho. Iminota 30 mu bwogero bw'urubura ukoresheje sonicator yo mu bwoko bwa probe (UP 200ST, Hielscher Ultrasonics, Teltow, Ubudage).
Insulin NPS yapimwe kuri Z-ikigereranyo cya diameter, polydispersity index (PDI) na zeta potential hakoreshejwe ibipimo bya dynamic light scattering (DLS) hakoreshejwe Litesizer 500 (Anton Paar, Graz, Otirishiya) binyuze mu kuyigabanya mu mazi ya DD kuri 25°C. Imiterere n'ikwirakwizwa ry'ingano byagaragajwe na mikorosikopi ya Hitachi H7600 transmission electron (TEM) (Hitachi, Tokyo, Ubuyapani), hanyuma amashusho asesengurwa hakoreshejwe porogaramu ya Hitachi imaging (Hitachi, Tokyo, Ubuyapani). Kugira ngo harebwe imikorere ya encapsulation (EE) n'ubushobozi bwo gutwara (LC) bya insuline NPs, NPs zashyizwe mu miyoboro ya ultrafiltration hamwe n'uburemere bwa molekile bwa 100 kDa hanyuma zishyirwa kuri centrifuge kuri 500 xg mu gihe cy'iminota 30. Insulin idafungishijwe muri filtrate yapimwe hakoreshejwe sisitemu ya Agilent 1100 Series HPLC (Agilent, Santa Clara, Californiya, Amerika) igizwe na pompe ya quaternary, autosampler, column. Igishyushya, na DAD detector. Insulini yasesenguwe hakoreshejwe inkingi ya C18 (Zorbax, 3.5 μm, 4.6 mm × 150 mm, Agilent, USA) maze iboneka kuri 214 nm. Icyiciro cyo kugenda cyari acetonitrile n'amazi, birimo 0.1% TFA, igipimo cy'ubushyuhe kuva kuri 10/90 kugeza 100/0, kandi cyakoreshejwe mu gihe cy'iminota 10. Icyiciro cyo kugenda cyapompewe ku gipimo cy'amazi cya 1.0 ml/min. Ubushyuhe bw'inkingi bwashyizwe kuri 20 °C. Barura ijanisha rya EE na LC ukoresheje ibigereranyo. (1) na Eq. (2).
Ibipimo bitandukanye bya CS/insuline kuva kuri 2.0 kugeza kuri 4.0 byapimwe kugira ngo hakorwe insuline NP nziza. Ingano zitandukanye z'umuti wa CS zongewemo mu gihe cyo gutegura, mu gihe uruvange rwa insuline/TPP rwagumanye. Insulin NPs zateguwe mu rugero rwa pH kuva kuri 4.0 kugeza kuri 6.5 hagenzurwa neza pH y'uruvange nyuma yo kongeramo ibisubizo byose (insuline, TPP na CS). Ingano ya EE n'ingano y'uduce duto twa insuline byapimwe ku gaciro gatandukanye ka pH n'ibipimo bya CS/insuline kugira ngo hongerwe ikorwa rya insuline NPs.
NP za insuline nziza zashyizwe ku gikoresho cya aluminiyumu maze zipfundikirwa n'udupira duto dukonjeshejwe na kaseti. Nyuma yaho, udusanduku twari dufite vis twashyizwe mu cyuma cyumisha icyuma cya Labconco FreeZone (Labconco, Kansas City, MO, USA) gifite icyuma cyumisha icyuma. Ubushyuhe n'umuvuduko wa vacuum byashyizwe kuri -10 °C, 0.350 Torr mu masaha 2 ya mbere, na 0 °C na 0.120 Torr mu masaha 22 asigaye y'amasaha 24 kugira ngo haboneke NP za insuline zumutse.
Imashini yo kumisha ya Buchi Mini Spray B-290 (BÜCHI, Flawil, Suwisi) yakoreshejwe mu gukora insuline ifunze. Ibipimo byo kumisha byatoranijwe ni: ubushyuhe bwa 100 °C, urujya n'uruza rw'ibiryo L 3/minota, n'uruza rw'umwuka L 4/minota.
NP za insuline mbere na nyuma yo kubura amazi zaranzwe hakoreshejwe spectroscopy ya FTIR-ATR. Uduce duto tw’amazi twakuwemo hamwe na insuline y’ubuntu na chitosan byasesenguwe hakoreshejwe spectrophotometer ya Spectrum 100 FTIR (PerkinElmer, Waltham, Massachusetts, USA) ifite igikoresho cy’isuzuma cya ATR (PerkinElmer, Waltham, Massachusetts, USA). Impuzandengo y’ibimenyetso yabonetse mu gupima inshuro 16 ku gipimo cya cm2 4 mu ntera ya cm2 4000-600.
Imiterere ya NP za insuline yumye yagenzuwe hakoreshejwe amashusho ya SEM ya NP za insuline yumye zikonjeshejwe n’izipfundikiye zafashwe na Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, muri Amerika). Igipimo cy’ingenzi cyakoreshejwe cyari voltage ya 5 keV n’umuvuduko wa 30 mA.
NP zose za insuline zaciwe amazi zongeye gushonga mu mazi ya dd. Ingano y'uduce, PDI, EE na LC byongeye gupimwa hakoreshejwe uburyo bumwe twavuze mbere kugira ngo harebwe ubwiza bwabyo nyuma yo kubura amazi. Uburambe bwa NP za anhydroinsuline nabwo bwapimwe hapimwa imiterere ya NPs nyuma yo kubura amazi igihe kirekire. Muri ubu bushakashatsi, NPs zose nyuma yo kubura amazi zabitswe muri firigo mu gihe cy'amezi atatu. Nyuma y'amezi atatu yo kubikwa, NPs zapimwe ingano y'uduce, PDI, EE na LC.
Shyira mililitiro 5 za NPs zasubiwemo muri mililitiro 45 zirimo amazi yo mu gifu yakozwe mu buryo bwa "pH 1.2", irimo 1% ya pepsin), amazi yo mu mara (pH 6.8, irimo 1% ya trypsin) cyangwa umuti wa chymotrypsin (100 g/mL, muri phosphate buffer, pH 7.8) kugira ngo harebwe akamaro ka insuline mu kurinda NPs nyuma yo kubura amazi. Zashyizwe mu cyuma kuri dogere selisiyusi 37 hamwe n'umuvuduko wa 100 rpm. 500 μL y'umuti yakusanyijwe mu bihe bitandukanye kandi ingano ya insuline yagenwe na HPLC.
Imyitwarire yo kurekura insuline NPs nshya yateguwe kandi yabuze amazi mu mubiri yageragejwe hakoreshejwe uburyo bwa dialysis bag (kugabanya uburemere bwa molekile 100 kDa, Spectra Por Inc.). NPs zumutse zateguwe vuba kandi zongeye gushyirwamo dialysis zashyizwe mu mazi kuri pH 2.5, pH 6.6, na pH 7.0 (0.1 M phosphate-buffered saline, PBS) kugira ngo bigane pH ibidukikije byo mu gifu, duodenum, n'amara mato yo hejuru, uko bikurikirana. Ingero zose zashyizwe muri incubation kuri 37 °C no gutigita buri gihe kuri 200 rpm. Shyira amazi hanze y'agasanduku ka dialysis ka 5 mL mu masaha akurikira: 0.5, 1, 2, 3, 4, na 6, hanyuma wongere ubwinshi bwa dialysate nshya. Ubuhumane bwa insuline mu mazi bwasesenguwe na HPLC, kandi igipimo cyo kurekura insuline mu turemangingo twa nanoparticles cyabazwe uhereye ku gipimo cya insuline yigenga yasohotse na insuline yose yashyizwe muri nanoparticles (Ikigereranyo cya 3).
Uturemangingo twa HepG2 twatewe mu masahani ya mm 60 y'umurambararo hakoreshejwe Dulbecco's Modified Eagle's Medium (DMEM) irimo 10% by'amaraso y'inka yo mu nda, penisiline ya 100 IU/mL, na 100 μg/mL streptomycin29. Ibihingwa byakomeje kurangwa kuri 37°C, ubushyuhe bwa 95%, na CO2 ya 5%. Mu gupima, uturemangingo twa HepG2 twatewe kuri 1 × 105 selile/ml kuri sisitemu ya Nunc Lab-Tek chamber slide system ifite 8-well (Thermo Fisher, NY, USA). Mu gupima uburozi bwa cytotoxicity, twatewe mu masahani 96-wells (Corning, NY, USA) ku bucucike bwa 5 × 104 selile/ml.
Isuzuma rya MTT ryakoreshejwe mu gusuzuma uburozi bwa NPs30 za insuline ziherutse gutegurwa kandi zangiritse. Uturemangingo twa HepG2 twatewe mu masahani 96 y’ibiziba ku bucucike bwa 5 × 104 selile/mL hanyuma duhingwa iminsi 7 mbere yo gupimwa. NPs za insuline zavanzwe ku gipimo gitandukanye (50 kugeza 500 μg/mL) mu buryo bwo guhingwa hanyuma zigashyirwa mu turemangingo. Nyuma y’amasaha 24 yo gutwikirwa, uturemangingo twogejwe inshuro 3 na PBS hanyuma dushyirwa mu buryo burimo 0.5 mg/ml MTT mu gihe cy’amasaha 4 y’inyongera. Uburozi bwa Cytotoxicity bwapimwe hapimwa ku kugabanuka kwa enzymes ya tetrazolium MTT y’umuhondo ikaba formazan y’umuhengeri kuri 570 nm hakoreshejwe Tecan infinite M200 pro spectrophotometer plate reader (Tecan, Männedorf, Suwisi).
Ubuhanga bwo gufata neza NPs mu turemangingo bwapimwe hakoreshejwe microscopy ya confocal laser scanning na flow cytometry analysis. Buri riba rya Nunc Lab-Tek chamber slide system ryavuwe na FITC-insulin yigenga, FITC-insulin-loaded NPs, hanyuma hongerwamo 25 μg/mL ya FITC-insulin NPs zaciwe amazi ku rugero rumwe kandi zishyirwa mu gihe cy'amasaha 4. Uturemangingo twogejwe inshuro 3 na PBS kandi dukosorwa na 4% paraformaldehyde. Nuclei yasizwe ibara rya 4′,6-diamidino-2-phenylindole (DAPI). Gupima aho insulin iherereye byagaragaye hakoreshejwe Olympus FV1000 laser scanning/two-photon confocal microscope (Olympus, Shinjuku City, Tokyo, Japan). Ku isesengura rya flow cytometry, igipimo kimwe cya 10 μg/mL free FITC-insulin, FITC-insulin-loaded NPs, na FITC-insulin yaciwe amazi NP zongewe ku masahani 96 y’ibigega byatewemo uturemangingo twa HepG2 hanyuma zishyirwa mu bubiko mu gihe cy’amasaha 4. Nyuma y’amasaha 4 yo guterwa, uturemangingo twakuwemo twogezwa inshuro 3 na FBS. Uturemangingo 5 × 104 kuri buri gipimo twasesenguwe hakoreshejwe icyuma gipima BD LSR II (BD, Franklin Lakes, New Jersey, Leta Zunze Ubumwe za Amerika).
Agaciro kose kagaragazwa nk'ikigereranyo cya ± ihinduka risanzwe. Igereranya hagati y'amatsinda yose ryapimwe hakoreshejwe uburyo bumwe bwa ANOVA cyangwa test ya t na IBM SPSS Statistics 26 kuri Mac (IBM, Endicott, New York, USA) kandi p < 0.05 yafashwe nk'ingenzi mu mibare.
Ubu bushakashatsi bwerekana ubushobozi bwo kumisha spray kugirango ikure amazi mu tunyangingo twa chitosan/TPP/insuline hamwe n’uburyo bwo kongera kumisha amazi ugereranije n’uburyo busanzwe bwo gukonjesha hakoreshejwe ibikoresho byo gukurura cyangwa ubushobozi bwo gukingira amazi hamwe n’ubushobozi bwo hejuru bwo gutwika. Utunyangingo twa insuline twakozwe neza twatanze ingano mpuzandengo ya 318 nm n’ubushobozi bwo gufunga 99.4%. Ibisubizo bya SEM na FTIR nyuma yo kubura amazi byagaragaje ko imiterere y’umubumbe yagumanye gusa muri NPs zumye zirimo na mannitol kandi zidafite mannitol, ariko NPs zumye zidafite mannitol zarangiritse mu gihe cyo kubura amazi. Mu isuzuma ry’ubushobozi bwo kongera kumisha, insuline nanoparticles zumye zidafite mannitol zagaragaje ingano nto y’utunyangingo n’umutwaro mwinshi mu gihe cyo kongera kumisha amazi. Imyitwarire yo kurekura izi NPs zose zabuze amazi yagaragaje ko zarekuwe vuba mu bisubizo bya pH = 2.5 na pH = 7, kandi zihamye cyane mu gisubizo cya pH = 6.5. Ugereranyije n’izindi NPs zabuze amazi zisubiwemo, NPs zarangiritse zidafite Mannitol yagaragaje ko yasohotse vuba cyane. Ibi bihuye n'ibyagaragaye mu isuzuma ry'uburyo NP zumye mu buryo bwa spray-dryer, kuko NP zumye mu gihe nta mannitol yari ifite byakomeje ubushobozi bwo gufata neza NP nshya zateguwe mu buryo bwa spray. Ibi bisubizo bigaragaza ko uduce duto twa insuline twumye twakozwe na spray-free mannitol twumye ari two dukwiriye cyane mu gutunganya izindi ngero z'ibipimo bya anhydrous, nk'ibinini byo kunywa cyangwa filime zifata umubiri.
Bitewe n’ibibazo by’umutungo bwite mu by’ubwenge, amakuru yakozwe cyangwa yasesenguwe mu bushakashatsi buriho ntabwo aboneka ku mugaragaro, ahubwo araboneka ku banditsi babishinzwe iyo babisabye mu buryo bwumvikana.
Kagan, A. Diyabete yo mu bwoko bwa 2: inkomoko mu mibereho no mu bya siyansi, ingorane z'ubuvuzi, n'ingaruka ku barwayi n'abandi. (McFarlane, 2009).
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Wong, CY, Al-Salami, H. & Dass, CR Iterambere riherutse mu buryo bwo gutanga insuline yo mu kanwa irimo liposome mu kuvura diyabete. Interpretation.J. Pharmacy.549, 201–217 (2018).