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Insulin nanoparticles (NPs) zili aamanguzu manji zyakajana nzila zisiyene-siyene zyakubelesya munzila ziindene-indene. Mulimo ooyu uyanda kulanga-langa nzila zyakuyumya-yumya akusansaila kubupange bwa insulin-loaded chitosan nanoparticles, kakuli naa kakwiina mannitol mbuli cryoprotectant. Alimwi twakalanga-langa bubotu bwama nanoparticles aaya kwiinda mukuzisanzya alimwi. kubula maanzi, bupati bwazibeela zya chitosan/sodium tripolyphosphate/insulin zyakabambwa kuti zibe 318 nm, PDI yakali 0.18, kubeleka kabotu kwakuvwumbilila kwakali 99.4%, mpawo kubikkwa kwakali 25.01%. Nikwakamana kubambululwa, zyoonse zyakali kuchitwa, kunze lyanzila yakugwisya kakwiina kubelesya mannitol, bakabamba bupange bwabo bwazibeela zya bbola. Kweezyanisya a mannitol-ijisi nanoparticles zyakagwisigwa maanzi kwiinda mukusansaila, mannitol-free spray-dried nanoparticles azyalo zyakatondezya bupati bwatunsiyansiya tuniini (376 nm) antoomwe alubono lupati (25.02%) kakuli kuvwumbililwa kukozyenye (DI987%) antoomwe a PDI. (0.20) kwiinda mukuyuminizya naakuti nzila zyakuyuminizya. Ma nanoparticles aayumide kwiinda mukusansaila kakunyina mannitol akapa kuti insulini ifwambaane kuzwa akubeleka kabotu kwamaseelo. Mulimo ooyu utondezya kuti kuyuminizya kulakonzya kugwisya maanzi muma nanoparticles aa insulini kakunyina kuyandika ma cryoprotectants kweelanisigwa anzila zyakale zyakuyuminizya, kupa kuti kube kuyuma kupati nguzu, ziyandikana zyakuyungizya ziche antoomwe amali aakubelesya zilagwasya kapati.
Kuzwa muchiindi chakajanwa mu 19221,2,3, insulini amisamu yayo yakafwutula maumi aabalwazi bachiswa bulwazi bwasyuga bwamusyobo wakusaanguna (T1DM) abulwazi bwasyuga bwamusyobo wabili (T1DM). Nokuba boobo, akaambo kabube bwayo mbuli ma protein aajisi ma molecule manji, insulini ilafwambaana kubungana, ilatyompokelwa ama enzymes aa proteolytic, mpawo ilagwisyigwa apenzi lyakusanguna. bachiswa bulwazi bwasyuga bwamusyobo wakusaanguna beelede kujaigwa insulini buumi bwabo boonse. Balwazi biingi bachiswa bulwazi bwasyuga bwamusyobo wabili bayandika kujaigwa kwa insulini kwachiindi chilamfu. Kujaigwa kwa insulini abuzuba nchintu chipa kuchisa akubula luumuno kubantu aaba, mpawo nchintu chibi kubuumi bwabo. Akaambo kaako, nzila zimwi zyakubelesya insulini zitakatazyi pe, zilimbuli kubelesya insulini muchiindi chilamfu bakavwuntauzya5 nkaambo balakonzya kubambulula buumi bwabantu babalilwa ku 5 billion bachiswa bulwazi bwasyuga munyika yoonse.
Busongo bwa nanoparticle bwakapa kuyaambele kupati mukusola kunywa insulini yakumulomo4,6,7. Imwi iibamba kabotu akukwabilila insulini kuti itanyonyooki kutegwa isike mumasena amwi aamubili. Nokuba boobo, kubelesya nzila zya nanoparticle kuli azintu zimwi zikonzya kulesyegwa, ikapati akaambo kakutazungaana kwama particle suspensions. Zimwi zilakonzya kucitika ciindi noziyobololwa, eezyo zicesya kuyobolwa kwabuumi insulin-loaded nanoparticles8. Mukuyungizya, kusima kwamakemikalu kwa polymer matrix ya nanoparticles a insulin akwalo kweelede kulanga-langwa kuchitila kuti kube kukkalwa kwa insulin nanoparticles (NPs). Kwalino, nzila yakuyuminizya mumulilo njenzila mbotu yakupanga NPs zitazungaani kumwi kakwiina kuchincha kutayandiki muchiindi chakuyobola9.
Nokuba boobo, kuyumya-yumya kuyandika kuyungizya ma cryoprotectants kuchitila kutabilila bupange bwa NPs kuti butanyonyooki akaambo kakukatazyigwa kwa ice crystals. Eezi zicesya kapati kubikkwa kwa insulini nanoparticles musule aakusanduka, nkaambo cryoprotectant ilatola bunji bwabulemu. Akaambo kaceeci, NPs zilajanika kuti zipangwa a NPs kanjikanji zilajanwa kuti zipangwa a NPs. tazyeelede pe mukupanga misamu iikola, mbuli mapilisi aakumulomo amafilimu aakumulomo, akaambo kakuyandika kwama nanoparticle manji aayumu kutegwa kube kusilika kwa insulini.
Kuyuminizya kwiinda mukusansaila nchintu chizyibidwe loko alimwi chitayandiki mali manji mukupanga mapowda aayumide kuzwa mumaanzi mumakwebo aamisamu10,11. Kweendelezya nzila yakupanga tuniini kupa kuti kube kuvwumbilila kabotu kwazintu zyiingi zipona 12, 13 .Kuyungizya waawo, kwaba nzila iigwasya yakubamba ma protein naakuti ma puloteni Kubelesya. Muchiindi chakuyuminizya, maanzi alafwambaana kuzwa, eecho chigwasya kuti kupya kwatuniini kakuli ansi11,14, kupa kuti kubelesya kwawo kukonzye kuvwumbilila zibeela zikonzya kupya. Kakutanaba kuyuminizya, zintu zyakuvwumba zyeelede kusanganyigwa kabotu-kabotu amusamu uulaa zintu zibikkidwe, mbuli U1114, akuvwungulula. kubamba zintu zyoonse antoomwe kakutanaba kuvwumbililwa mukusansaila kupa kuti kuvwumbilila kube kabotu muchiindi chakumana maanzi. Mbwaanga nzila yakusansaila taiyandiki ma cryoprotectants, kusansaila kulakonzya kubelesegwa kupanga ma NP aayumide ajisi zyintu zinji.
Ooku kuvwuntauzya kwaambilizya kupanga kwa insulin-loaded NPs kwiinda mukuswaanganya chitosan a sodium tripolyphosphate kubelesya nzila ya ion gel. Ion gelation ninzila yakulibambila iizumizya kupanga ma nanoparticles kwiinda mukubelesya magesi aakati ka ionic species zyobile naa kwiinda waawo mubukkale bumwi. Zyoonse zyobile nzila zyakuyuminizya akusansaila zyakabelesegwa kugwisya maanzi chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles. Musule akugwisigwa maanzi, bupange bwazyo bwakavwuntauzyigwa a SEM. Ikukonzya kwazyo kuswaangana kwakali kulinguwe kwiinda mukupima bupati bwazyo, nguzu zyaatala, PDI, kubeleka kabotu kwakuvwumbilila, antoomwe akubikkwa kwamaanzi. Bubotu bwa nanoparticles zyakabambwa kwiinda mukubelesya nzila ziindene-indene zyakugwisya maanzi mumaanzi zyakavwuntauzyigwa kwiinda mukweezyanisya insulin lukwabililo, kuchita kwakugwisya, antoomwe akubeleka kabotu kwamaseelo.
I pH ya musamu uusanganizidwe antoomwe a mweelwe wa chitosan a insulin nzintu zyobile zipati zijatikizya bupati bwatubeela antoomwe akubeleka kabotu kwakuvwumbilila (EE) kwa NPs zyakumamanino, mbuli mbozijatikizya nzila ya ionotropic gelation process. pH ya musamu uusanganizidwe yakatondezyegwa kuti ilaswaangene kapati abupati bwatubeela alimwi akubeleka kabotu kwakuvwumbilila (Fig. 1A, mbuli mbokutondezyedwe mu Fig. 1a). 4.0 kusika ku 6.0, mweelwe waakati aakati wazibeela (nm) wakaselemuka mpawo EE yakatanta loko, mpawo pH niyakatanta kusika ku 6.5, mweelwe waakati aakati wakatalika kuyaambele mpawo EE tiiyakachincha pe. Mbuli mweelwe wa chitosan a insulin mbouyaabukomena, mweelwe waakati aakati wazibeela wakatanta. Kunze lyaboobo, taakwe EE yakabambilwa aawo nokwakabambilwa pe mweelwe wa chitosan/insulin wiinda 2.5:1 (w/w) (Fig. 1b). Aboobo, bukkale bwakulibambila buli kabotu muchiiyo eechi (pH 6.0, chitosan/insulin mweelwe wa 2.5:1) zyakabelesegwa kubamba insulin-loaded nanoparticles kuti zibelesyegwe mukwiiya kunembo. Muchiimo eechi chakulibambila, mweelwe waakati aakati wa tuniini twa insulini twakabikkwa kabotu 318 nm (Mufanikisyo 1c), PDI yakali 0.18, kubeleka kabotu kwakunjizya kwakali 99.4%, nguzu zya zeta zyakali 9.8 mv, mpawo kubikkwa kwa insulini kwakali 25.01% (m/m ). Kweelana azyakazwa mu transmission electron microscopy (TEM), ma nanoparticles aakabambululwa akali mbuli bbola mpawo ajisi bupati bweelene (Mufanikisyo. 1d).
Kubamba kabotu ma nanoparticles aa insulini: (a) mpindu ya pH aamweelwe waakati-kati akujatika kabotu (EE) kwama nanoparticles aa insulini (yakabambilwa a 5:1 mass ratio ya chitosan a insulini); (b) chitosan a Nsungwaazyo yabupati bwa insulini abulemu bwa insulini akubeleka kabotu (EE) kwa insulini NPs (yakabambilwa a pH 6); (c) kwaabwa kwabupati bwazibeela zya insulin nanoparticles; (d) TEM micrograph ya insulin NPs iibambidwe kabotu.
Chilizyibidwe kuti chitosan ni polyelectrolyte iitajisi nguzu ilijisi pKa ya 6.5. Ili aamagesi mabotu mumasena aajisi acidi nkaambo kabunga ka amino kayo kapati ka protoned a hydrogen ions15. Aboobo, kanjikanji ilabelesyegwa mbuli chintu chinyamuna kuvwumbilila ma macromolecules aajisi nguzu zitali kabotu. Mukuvwuntauzya ooku, chitosan yakabelesegwa kuvwumbilila insulini 5.3.Mbwaanga chitosan ibelesyegwa mbuli chintu chakuvwumba, akuyungizya mweelwe wayo, bupati bwacibeela chaanze cha nanoparticles bulayungizya kweendelana, eezi zipa kuti kube bupati bwazibeela zipati. Mukuyungizya, mweelwe mupati wa chitosan ulakonzya kuvwumbilila insulini myiingi. Mubukkale bwesu, EE yakali mpati aawo mweelwe wa chitosan nowakasika ku 25:15, kwakanyina kucinca kupati. mu EE aawo mweelwe nowakazumanana kuyaambele.
Kunze lya mweelwe wa chitosan a insulin, pH ayalo yakatola lubazu lupati mukubamba NPs.Gan et al. 17 bakaiya aatala a pH ku bupati bwa tuniini twa chitosan. Bakajana kuti bupati bwa tuniini bwakali kuyaansi kusikila pH yakasika ku 6.0, mpawo kuyungizigwa kupati kwa bupati bwa tuniini kwakabonwa a pH > 6.0, eezi zyeendelana azyeezyo nzyotwakabona. Eechi chintu chichitika akaambo kakuti aawo pH noiyaabukomena, molecule ya insulini ilajana nguzu zitali kabotu, aboobo ilagwasya kuswaangana kwa magesi a chitosan/sodium tripolyphosphate (TPP) complex, kwakapa kuti kube tuniini tuniini antoomwe a EE mpati. Nokuba boobo, aawo pH niyakacincwa kusika ku 6.5, tubunga twa amino twakali a chitosan twakagwisigwa maprotoni, eezi zyakapa kuti chitosan ivwunguluke. Aboobo, pH mpati yakapa kuti amino ions zitanjili mu TPP, mpawo kupa kuti insulini ibe mpati mweelwe waakati-kati wamamanino wamapati a EE nsyoonto.
Kuvwuntauzya bube bwa NPs zyakayuminizyigwa akusansaila kulakonzya kusololela kusala nzila zibotu zyakugwisya maanzi mumubili akupanga fulaulu. Nzila iiyandika yeelede kupa musamu uukkalilila, bube bwezibeela zyeelene, bunji bwamisamu alimwi akunyonyooka kubotu mumusamu wakusaanguna. Mukuvwuntauzya ooku, kweezyanisya nzila zyobile eezyi, NPs zya NPs zitakwe insulini naa 1% manolt zyakabelesyegwa muchiindi chakugwisya maanzi. chibelesegwa mbuli chintu chivwumbilila naa chikwabilizyo chakutontola mumabuwa aasiyene-siyene aakuyuminizya akusansaila. Kuli lyophilized insulin nanoparticles kakunyina mannitol, mbuli mbokutondezyedwe muchifanikisyo 2a, bupange bwabusi bujisi mapopwe mapati, butali kabotu alimwi buteteete bwakabonwa munsi lya microscopy microscopy (SEM microcles yakalangwa-langwa). zyakajanika mumusamu nikwakamana kumana maanzi (Mufanikisyo 2e). Eezyi mpindu zyakatondezya kuti bunji bwa NPs zyakabola muchiindi chakuyuminizya kakunyina chikwabilizyo. Kuma nanoparticles aa insulini aayuminizyigwa akusansaila aajisi 1% mannitol, ma nanoparticles aajisi zibeela ziteteete akabonwa (Fig. 2, 2e). nanoparticles zyakayuminizyigwa kakunyina mannitol zyakasyaala kazili mbuli bbola pesi kazinyonyooka atala (Mufanikisyo 2c). Zibeela zili mbuli bbola azivwumbilidwe zilabandikwa lubo mukusunkwa kwakugwisya akutola kwamaseelo ansi aawa. Kweelana akuboneka kwa NPs zyakayuminizyigwa, zyoonse zyobile NPs zyakasansailwa kakunyina mannitol alimwi a NPs zyakasansailwa kakunyina mannitol alimwi zyakayuminizyigwa a mannitol zyakapa busi bubotu bwa NPs (Mufanikisyo 2f,g,h). Busena bupati aakati kazibeela, mbobuli bupati bwakunyonyooka aboobo mbobuli bupati bwakuzwa.
Bupange bwa insulin NPs zitakwe maanzi: (a) Cifwanikiso cha SEM cha lyophilized insulin NPs kakunyina mannitol; (b) Cifwanikiso cha SEM cha lyophilized insulin NPs antoomwe a mannitol; (c) insulini yakayuminizyigwa akusansaila NPs kakunyina mannitol SEM mufanikisyo wa ; (d) Cifwanikiso cha SEM cha insulini NPs yakayuminizyigwa a mannitol; (e) mufanikisyo wa lyophilized insulin NPs powder kakunyina mannitol; (f) mufanikisyo wa lyophilized insulin NPs antoomwe a mannitol; ( g) Cifwanikiso cha insulin NPs yakayuminizyigwa kakunyina mannitol; (h) mufanikisyo wa musamu wa insulin NPs wakayuminizyigwa a mannitol.
Muchiindi chakuyuminizya, mannitol ibelekela mbuli chintu chikwabilila kupya, ikubamba ma NP muchimo chitalibonyi akukwabilila kunyonyoonwa amabwe aa aisi19. Mukwiimpana, taakwe ntaamu yakutontola muchiindi chakuyuminizya. Aboobo mannitol taiyandiki pe munzila eyi. Mubunike, NPs ziyuminidwe kakunyina mannit zyakali kupa ma NP mabotu mbuli kaindi chakapandululwa. Nokuba boobo, mannitol ilakonzya kubeleka mbuli chintu chizuzya munzila yakusansaila kuti ipe NPs bupange buli mbuli bbola20 (Fig. 2d), eecho chigwasya kujana nzila iikozyenye yakuzwa kwama NPs aaliboobo. Mukuyungizya, nchaantangalala kuti zimwi zintu zipati zilakonzya kujanwa muli NPs zyoonse zyobile zyakayuminizyigwa akusansaila zili a insulini (Fig. 2d). 2b,d), eecho chikonzya kuba akaambo kakuyoboloka kwa mannitol mukati kachintu antoomwe a insulini iijisilidwe. Ku. Chitosan layer. Nchintu chibotu kuzyiba kuti mukwiiya ooku, kuchitila kuti bupange bwabbola buzumanane kuba kabotu nikwakamana kumana maanzi, mweelwe wa mannitol a chitosan ulabikkwa a 5:1, kuchitila kuti mweelwe mupati wa filler ulakonzya kukomezya bupati bwazibeela zya NPs ziyumu.
Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy yakatondeezya kusanganya kwa insulin, chitosan, chitosan, TPP antoomwe a insulin. Ma NP woonse aatakwe maanzi akajanwa kwiinda mukubelesya FTIR-ATR spectroscopy. Chazibinkene, ma band intensities aa 1641, 1543 antoomwe a 14 cm-141-1200000 akajanwa mu NPs zyakayuminizyigwa a mannitol mpawo mu NPs zyakayuminizyigwa a mannitol azitakwe mannitol (Mufanikisyo 3). Mbuli mbukwakaambwa musule, ooku kuyungizigwa kwanguzu kwakaliswaangene akuswaangana aakati ka chitosan, TPP antoomwe a insulini. Kuvwuntauzya kwakujatana aakati ka chitosan a insulini kwakatondezya kuti mu FTIR spectra ya chitosan iizwide insulini, chitosan chitosan yakazwide insulini, bbanti lya chitosan lyakazwide insulini Kuyungizya nguzu zya carbonyl (1641 cm-1) antoomwe a amine (1543 cm-1) bbanti. Tubunga twa tripolyphosphate twa TPP tuliswaangene atubunga twa ammonium mu chitosan, akupanga bbandi a 1412 cm-1.
FTIR-ATR spectra ya insulin, chitosan, misanganisyo ya chitosan/TPP/insulin antoomwe a NPs zyakagwisigwa maanzi munzila zisiyene.
Kuyungizya waawo, eezyi mpindu zileendelana azeezyo zitondezyedwe mu SEM, eezyo zyakatondezya kuti ma NP aakabikkidwe akazumanana kuba kabotu aawo naakasansaila akuyuminizyigwa a mannitol, pesi mukubula mannitol, buyo kusansaila kwakapa kuti kube tuniini twavwumbidwe. Mukwiimpana, FTIR-ATR spectral results ya NPs zyakavwumbidwe zyakali kukozyanya kapati a mannitol iitakwe mannitol kusanganya kwa chitosan, TPP, antoomwe a insulin. Eechi chizwa chitondeezya kuti kujatana aakati ka chitosan, TPP antoomwe a insulin takuchiliko pe mu NPs zyakayuminizyigwa kakunyina mannitol. Bupange bwa NPs bwakanyonyoonwa muchiindi chakuyuminizyigwa kakunyina cryoprotectant, eezyo zinga zilabonwa mu SEM results (Fig. 2Aa morphology ya BaFT alimwi a BaIR zyakagwisigwa). insulini NPs, buyo lyophilized, spray-dried, antoomwe a mannitol-free NPs zyakabelesegwa mukusola kubamba alimwi a mannitol-free NPs akaambo kakubola kwa mannitol-free NPs muchiindi chakubula maanzi. zuwa.
Kugwisya maanzi mumubili kulabelesyegwa kubamba kwachiindi chilamfu akuchita zimwi zintu. Ikukonzya kwa NPs ziyumu kuti zibambululwe musule akuyobolwa nchintu chipati mukubelesyegwa muzintu zisiyene-siyene mbuli mapilisi amafilimu. Twakabona kuti mweelwe waakati-kati wazibeela zya insulini NPs kakunyina mannitol wakatanta mbichaani nikwakamana kubambululwa, mpawo bupati bwazibeela zyakayungizyigwa. Insulini yakayumizigwa akuyuminizyigwa a mannitol yakatanta kapati (Table 1).PDI a EE tiizyakali kuchincha pe (p > 0.05) nikwakamana kusanganyigwa ma NP woonse muchiiyo eechi (Table 1). Eechi chakazwa chitondeezya kuti bunji bwama particles akachili kabotu nikwakamana kunyonyooka. Nokuba boobo, kuyungizya kwa mannitol kwacesya kapati insulini lyophilized and spray-dried mannitol nanoparticles (Table 1). Mukwiimpana, insulin load content ya NPs spray-dried kakunyina mannitol yakachili mbubonya mbuli kaindi (Table 1).
Chilizyibidwe kuti kubikka ma nanoparticles nchintu chipati loko kuti kwabelesyegwa kutola misamu. Kuma NPs ajisi ma loads masyoonto, zintu zinji ziyandikana kuti zisike aawo mpozigolela. Nokuba boobo, bunji bwa viscosity bwa NPs bupati bupa kuti kutabi kukatazya akukatazyigwa mukubelesya misamu iikola alimwi akujaigwa, insulini 2 2 NP ilakonzya kuyungizya. alimwi zilakonzya kubelesegwa kupanga mapilisi ama biofilms aajisi busani23, 24, eezyo ziyandika kubelesya ma NPs miingi muchiindi chifwiifwi, eezi zipa kuti kube mapilisi mapati ama biofilms mapati aatakonzyi kubelesegwa mumulomo. Aboobo, ma NPs aatakwe maanzi aajisi insulini mpati ngaayandikana loko. Zyakajanwa zyesu zitondezya kuti bunji bwama NPs aajisi insulini iitakwe busani ilakonzya kupa bubotu bunji bwanzila zimbi zyakutola.
Ma NP woonse aatakwe maanzi akabikkwa mufridge kwamyezi yotatwe. Izyazwa mu SEM zyakatondezya kuti bupange bwama NP oonse aatakwe maanzi tiibwakachincha pe mumyezi yotatwe yakubamba (Mufanikisyo 4). Nikwakamana kubikkwa mumaanzi, ma NP oonse akatondezya kuseluka mbichani mu EE mpawo akagwisya insulini nsyoonto (~5%) muchiindi chakubamba kwamyezi yotatwe (T 2).Nokuba boobo, mweelwe waakati aakati wama nanoparticles wakatanta. Bupati bwazibeela zya NPs zyakayuminizyigwa kakunyina mannitol zyakatanta kusika ku 525 nm, mpawo zya NPs zyakayuminizyigwa a mannitol zyakayungizyigwa kusika ku 872 a 921 nm, kweendelana (Table 2).
Bupange bwa insulin NPs zitakwe maanzi zyakabambwa kwamyezi yotatwe: (a) Cifwanikiso cha SEM cha lyophilized insulin NPs antoomwe a mannitol; (b) Cifwanikiso cha SEM cha insulini yakayumizigwa kakunyina mannitol; (c) kakwiina zifwanikiso zya mannitol SEM zya insulini NPs.
Kuya kunembo, zintu zyakasweeka zyakabonwa muma nanoparticles aa insulini aakasanzyigwa a mannitol mpawo akayuminizigwa (Mufanikisyo S2). Eezi zilakonzya kuchitwa azintu zipati zitazungaani kabotu mumaanzi. Zyoonse zyaambwa atala aawa zitondezya kuti nzila yakusansaila ilakonzya kukwabilila ma nanoparticles aa insulini kuzwa kukugwisigwa kwamaanzi mumubili akuzula kwama nanoparticles aa insulini zyajanwa kakunyina zintu zizuzya naakuti zikwabilila kupya.
Kubamba insulini kwakasunkwa mu pH = 2.5 aakati-akati a pepsin, trypsin, antoomwe a α-chymotrypsin kutondezya nguzu zyakukwabilila zya NPs kuzwa kukugaya kwa enzymatic nikwakamana kubula maanzi. Kubamba insulini kwa NPs zyakamana maanzi kwakakozyanisyigwa a NPs zyakabambilwa bupya, mpawo insulini yaalubazu yakabelesegwa mbuli chintu chibi. zyoonse zyotatwe zyabusilisi bwa enzymatic (Fig. 5a–c). Mukwiimpana, kusunkwa kwakugwisya insulini mu NPs zyakayuminizyigwa a mannitol antoomwe a NPs zyakayuminizyigwa a mannitol naa kakwiina mannitol kwakatondezya kukwabililwa kupati kwa NPs eezyi kuzwa kukugaya kwa enzymatic, ooko kwakali kukozyanya a insulin NPs zyakabambwa bupya ( Cifwanikiso 15a- nac, lugwasyo lwa pepsinnoparticle). trypsin, a α-chymotrypsin, kwiinda 50%, 60%, antoomwe a 75% ya insulini ilakonzya kukwabililwa mukati ka 4 h, kweendelana (Mufanikisyo 5a-c). Ooku kukwabililwa kwa insulini kulakonzya kuyungizya mweenya wakunjilila kwa insulini mubulowa25. Eezyi mpindu zitondezya kuti kusansaila kuyumya naa kakunyina manniol a manniol takukonzyi kukwabililwa kubamba nguzu zyakukwabilila insulini zya NPs nikwakamana kumana maanzi mumubili.
Kukwabilila akugwisigwa kwa insulin NPs zitakwe maanzi: (a) kukwabilila insulin mumusamu wa pepsin; (b) kukwabilila insulin mumusamu wa trypsin; (c) kukwabilila insulini a α-chymotrypsin solution; (d) Kuzwa kwa NPs zitakwe maanzi mu pH = 2.5 solution; (e) nzila yakuzwa kwa NPs zitakwe maanzi mu pH = 6.6 solution; (f) nzila yakuzwa kwa NPs zitakwe maanzi mu pH = 7.0 solution.
Insulin NPs zyakabambilwa akubambululwa zyakabikkwa muma buffer aasiyene (pH = 2.5, 6.6, 7.0) a 37 °C, kweezyanisya pH yamumatumbu, duodenum, antoomwe amala masyoonto, kuchitila kulanga-langa mulimo wa insulin kukukazya insulini. Kuchita kwakuzwa mumasena aasiyene. Chibeela chamubili. A pH = 2.5, insulin-loaded NPs antoomwe a resolubilized dry insulin NPs zyakatondezya kuzwa kwakusaanguna mukati kawoola lyomwe lyakusaanguna, kutobelwa akuzwa asyoonto-syoonto mumahola aali 5 aatobela (Fig. 5d). Ooku kuzwa kwakufwambaana kumatalikilo kulibonya mbuli kuti nkuzwa kwakufwambaana kwa ma protein kakunyina kweenda mukati kacibeela. A pH = 6.5, insulin-loaded NPs antoomwe a constituted dry insulin NPs zyakatondezya kuzwa kabotu-kabotu kwamawoola aali 6, nkaambo pH ya musamu wakusunka yakalikozyene a NPs-prepared solution (Fig. 5e). A pH = 7, NPs tiizyakali kukonzya kugwisigwa pe muzibeela zyobile zyakusaanguna mawoola (Mufanikisyo 5f). Eezi ziliboobu nkaambo kugwisigwa kwa chitosan kulachitika a pH mpati, eecho chipa kuti kutabi kujatana kwa polymer mpawo akugwisya insulin iizwide.
Kuyungizya waawo, insulini NPs zyakayuminizyigwa kakunyina mannitol zyakatondezya kufwambaana kuzwa kwiinda zimwi NPs zyakagwisigwa maanzi (Fig. 5d–f). Mbuli mbokwaambidwe musyule, insulini NPs zyakayuminizyigwa kakunyina mannitol zyakatondezya bupati bwazibeela zisyoonto. Tubeela tusyoonto tupa busena bupati bwaatala, aboobo bunji bwamusamu uuyandika ulakonzya kuba afwaafwi amubili, akupa kuti kube kufwambaana kwaangulula26.
Kujaya maseelo aa NPs kwakavwuntauzyigwa a MTT assay. Mbuli mbokutondezyedwe muchifanikisyo S4, NPs zyoonse zyakagwisigwa maanzi zyakajanwa kuti tazikwe mpindu mpati kubuumi bwamaseelo aa 50-500 μg/ml, kutondeezya kuti NPs zyoonse zyakamana maanzi zilakonzya kubelesegwa kabotu kusika kuchiindi chakusilikwa.
Chibindi ncechizo chipati oomo insulini moichita milimu yayo. Maselu aaHepG2 ngamaseelo aabantu aabelesyegwa mbuli chitondezyo cha hepatocyte. Aawa, maselu aaHepG2 akabelesegwa kulanga-langa kutambula kwa NPs zyakagwisigwa maanzi kwiinda mukubelesya nzila zyakuyuminizya akusansaila kwiinda mukubelesya laserfocal uptakescanning. cytometry akubona kutobela mawoola manji aakubikkwa a FITC insulini yaalubazu a 25 μg/mL, FITC insulin-loaded NPs zyakabambilwa bupya antoomwe a FITC insulin-loaded NPs zyakagwisigwa maanzi a insulini yeelene. musunko ooyu. Ikumweka kwamukati kamaseelo kwa NPs zyakabambilwa bupya zijisi insulini, NPs zyakayumizigwa a mannitol, antoomwe a NPs zyakayuminizyigwa a mannitol azitakwe mannitol (Mufanikisyo 6a) zyakali 4.3, 2.6, 2.4, antoomwe a 4.1-fold kwiinda kabunga ka insulin-ins iitakwe mannitol (Mufanikisyo 6). zyazwa zitondezya kuti insulini iijisilidwe ilaanguzu kapati mukutambula maseelo kwiinda insulini yaalubazu, ikapati akaambo kakuti ma nanoparticles aajisi insulini aakapangwa muciiyo aaya masyoonto.
Kutambula kwamaseelo aa HepG2 musule aa 4 h yakubikkwa antoomwe a NPs zyakabambilwa bupya antoomwe a NPs zitakwe maanzi: (a) Kwaabila kwa FITC-insulin kutambula kwamaseelo aa HepG2.(b) Mweelwe waakati-kati wa fluorescence intensities wakavwuntauzyigwa a flow cytometry (n = 3), *P < 0.05 kweezyanisyigwa a insulini iitakwe maanzi.
Mbubwenya buyo, zifwanikiso zya CLSM zyakatondezya kuti FITC fluorescence intensities ya FITC-insulin-loaded NPs zyakabambilwa bupya antoomwe a FITC-insulin-loaded spray-dried NPs (kakwiina mannitol) zyakali anguzu kwiinda zyamasampu aambi (Fig. 6a). Kuyungizya waawo, akuyungizigwa kwa mannitol, kusima kwa solution kwakayungizya kukutola kwamaseelo, eezi zyakapa kuti kucheya kwakuvwula kwa insulini. Eezyi mpindu zitondezya kuti mannitol-free spray-dried NPs yakatondeezya kuzwidilila kupati kwamaseelo nkaambo bupati bwatubeela twawo bwakali buniini kwiinda bwa freeze-dried NPs nikwakamana kunyoneka alimwi.
Chitosan (mweelwe wa 100 KDa, 75-85% deacetylated) yakaulwa kuzwa ku Sigma-Aldrich. (Oakville, Ontario, Canada). Sodium tripolyphosphate (TPP) yakaulwa kuzwa ku VWR (Radnor, Pennsylvania, USA). Insulin yabantu yakabelesyegwa muchiiyo eechi kuzwa ku Scientific, MAWalt, MA. USA).Fluorescein isothiocyanate (FITC)-labeled human insulin antoomwe a 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) zyakaulwa kuzwa ku Sigma-Aldrich.(Oakville, Ontario, Canada).I HepG2 cell line yakajanwa kuzwa ku ATCC (Manassas, Virginiaall, USAAll re analytic anagraphents). giledi.
Bambila 1 mg/ml CS solution kwiinda mukuunyonyoona mumaanzi aasalala (DD maanzi) ajisi 0.1% acetic acid. Bambila 1 mg/ml solutions ya TPP a insulini kwiinda mukuzinyonyoona mumaanzi aa DD a 0.1% acetic acid, kweendelana. I pre-emulsion yakapangwa a polyzer PCU-10-21-monizer (Brinkmann Ind. Westbury, NY, USA). Nzila yakulibambila ili boobu: chakusanguna, 2ml ya musamu wa TPP ilabikkwa kuli 4 ml ya musamu wa insulini, mpawo musamu ooyu ulavwundaulwa kwamaminiti aali 30 mpawo wasanganyigwa. Mpawo, musamu ooyu wakasanganyigwa wakabikkwa madosi-dosi kumusamu wa CS kwiinda mukubelesya syringe kakuvwundauka kapati (The rpm, 1000). misanganisyo yakabikkwa munsi lyakuzungaanisigwa chakufwambaana (15,000 rpm) mumusambi wa aisi kwamaminiti aali 30, mpawo yakacincwa kusika ku pH imwi kuti ijane insulini NPs. Kuti iyungizye kukozyanya akucesya bupati bwa tuniini twa insulini NPs, yakasonikwa kwamaminiti aambi aali 30 mu ice bath kubelesya sonicator (STUP-type 2000, 2000 Hielscher Ultrasonics, Teltow, Germany).
Insulin NPS yakasunkwa ku Z-average diameter, polydispersity index (PDI) antoomwe a zeta potential kubelesya dynamic light scattering (DLS) nzila zyakupima kubelesya Litesizer 500 (Anton Paar, Graz, Austria) kwiinda mukuzisofwaazya mumaanzi aa DD a 25°C. Bupange abupati bwabo bwakajanwa kwiinda mukubelesya nzila ya Hitachi electron transmission electron 0706 (TEM) (Hitachi, Tokyo, Japan), mpawo zifwanikiso zyakavwuntauzyigwa kubelesya pulogilamu ya Hitachi imaging (Hitachi, Tokyo, Japan). Kulanga-langa bubotu bwakuvwumbilila (EE) akukonzya kwakunyamuna (LC) kwa insulini NPs, ma NPs akabikwa mumabbodela aa ultrafiltration abulemu bwama molecule aasika ku 10-0 centifuge a Dafa 500 xg kwamaminiti aali 30. Insulin iitakwe mubbokesi mumusefa yakapimwa kubelesya nzila ya Agilent 1100 Series HPLC (Agilent, Santa Clara, California, USA) iijisi pompi ya quaternary, autosampler, column heater, antoomwe a DAD detector. Insulin yakavwuntauzyigwa a C18Zbax, 35 μm. × 150 mm, Agilent, USA) mpawo yakajanwa a 214 nm. Chibeela chakunyamuka chakali acetonitrile amaanzi, chakajisi 0.1% TFA, mweelwe wakutanta kuzwa ku 10/90 kusika ku 100/0, mpawo chakachitwa kwamaminiti aali kkumi. Chibeela chakunyamuka chakapompwa amweelwe wakupya kwa 10 m/min. 20 °C. Bala mweelwe wa EE a LC kubelesya zibalo.(1) a Eq.(2).
Zibeela zya CS/insulin ziindene-indene kuzwa ku 2.0 kusikila ku 4.0 zyakasunkwa kutegwa insulini NP ibeleke kabotu. Mweelwe wa CS solution wakayungizyigwa muchiindi chakulibambila, mpawo insulin/TPP mixture yakabikkwa mbubwenya. Insulin NPs yakabambilwa mu pH ya 4.0 kusika ku 6.5 kwiinda mukulanganya kabotu pH ya mixture yoonse nikwakamana kusanganyigwa (Tulins, TPP antoomwe a TPP CS).I EE abwaamba bwazibeela zya insulin nanoparticles zyakali kulinguwe a pH ziindene-indene antoomwe a CS/insulin mass ratios kuchitila kuti kube kupanga kwa insulin NPs.
Insulin NPs zyakabikkwa kabotu zyakabikkwa achibiya cha aluminiyamu mpawo zyakavwumbidwe achisani chakajatilidwe atepu. Kuzwa waawo, zilongo zyakabikkwa mu Labconco FreeZone freeze dryer (Labconco, Kansas City, MO, USA) yakajisi tray dryer. Ikupya akutontola kwakabikkwa a - Torr -1000 °C. kwa 2 h yakusaanguna, a 0 °C antoomwe a 0.120 Torr kwa 22 h zyakasyeede zya 24 h kuti tujane insulin NPs ziyumu.
Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Switzerland) yakabelesegwa kupanga insulini. Zibelesyo zyakuyumya zyakasalwa zyakali: kupya 100 °C, kweenda kwachakulya 3 L/min, antoomwe akuzuza kwamuwo 4 L/min.
Insulin NPs kakutanaba akumana kwamaanzi zyakajanwa kwiinda mukubelesya FTIR-ATR spectroscopy. Ma nanoparticles aatakwe maanzi antoomwe a insulin a chitosan zyakavwuntauzyigwa kubelesya Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA) iijisi universal ATR accessory (Elmer, Waltham, Waltham, Massachusetts, USA). Mweelwe waakati-kati wazitondezyo wakajanwa kuzwa ku 16 scans a resolution ya 4 cm2 mu frequency range ya 4000-600 cm2.
Bupange bwa insulin NPs bwakali kulinguwe kwiinda muzifwanikiso zya SEM zya insulin NPs zyakayuminizyigwa a spray zyakajatwa a Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, USA). Chiimo chipati chakabelesegwa chakali changuzu zya 5 keV antoomwe a 30 mA.
Insulin NPs zyoonse zyakagwisigwa maanzi zyakasandulwa alimwi mumaanzi aa dd. Bupati bwazibeela, PDI, EE a LC zyakasunkwa alimwi kubelesya nzila njiyonya yakaambwa kale kuti ziboneke bubotu bwazyo nikwakamana kugwisigwa maanzi. Kukkalilila kwa anhydroinsulin NPs akwalo kwakapimwa kwiinda mukulanga bube bwa NPs nikwakamana kuyobolwa kwachiindi chilamfu. Mukuvwuntauzya ooku, NPs zyoonse nikwakamana kugwisigwa maanzi zyakayobolwa mufiriji yotatwe myezi. Nikwakainda myezi yotatwe yakubamba, ma NP akasunkwa kuti abone bupati bwazibeela, PDI, EE a LC.
Kunyonyoona 5 mL ya NPs yakabambululwa mu 45 mL iijisi maanzi aamumatumbu (pH 1.2, ajisi 1% pepsin), maanzi aamumala (pH 6.8, ajisi 1% trypsin) naakuti musamu wa chymotrypsin (100 g/mL, mu phosphate buffer, pH 788) kuchitila kulanga-langa kukwabilila kwa insulini. NPs nikwakamana kumana maanzi. Zyakabikkwa a 37°C akufwambaana kwakuzungaanya kwa 100 rpm. 500 μL yamusamu yakabungikwa muziindi zisiyene mpawo bunji bwa insulini bwakajanwa a HPLC.
Ikuzwa kwa in vitro kwa insulini NPs zyakabambilwa bupya alimwi zyakagwisigwa maanzi zyakasunkwa anzila ya dialysis bag (molecular weight cut-off 100 kDa, Spectra Por Inc.). Ma NPs mapya aakabambilwa akubambululwa akasanzyigwa mumaanzi aa pH 2.5, pH 6.6, antoomwe a pH 7.0 ( M. 1.0.0.0.0, phofoline, saffered M.1. PBS) kweezyanisya pH yamukati, duodenum, antoomwe amatumbu masyoonto, kweendelana. Zyoonse zitondezyo zyakabikkwa a 37 °C kazizungaanisigwa a 200 rpm. Kosowela maanzi aanze aa 5 mL ya dialysis bag muchiindi eechi: 0.5, 1, 2, 3, 6, 6, mpawo akuzuzya a volyumu mpawo mpawo dialysate. Kusofwaazyigwa kwa insulini mumaanzi kwakavwuntauzyigwa a HPLC, mpawo mweelwe wa insulini yakazwa muma nanoparticles yakabalilwa kuzwa ku mweelwe wa insulini yakazwa ku insulini yoonse yakabikkidwe muma nanoparticles (Equation 3).
Maseelo aa HepG2 aajisi kkansa yamumubili wamuntu akakomezyegwa muzilongo zya 60 mm diameter kubelesya Dulbecco's Modified Eagle's Medium (DMEM) iijisi 10% fetal bovine serum, 100 IU/mL penicillin, antoomwe a 100 μg/mL streptomycin 293°Cs ​​zyakabambwa a 3°C. 95% busi, antoomwe a 5% CO2. Mukulanga-langa, maselu aa HepG2 akabikkwa a 1 × 105 maselu/ml aa 8-well Nunc Lab-Tek chamber slide system (Thermo Fisher, NY, USA). Mukulanga-langa kujaya maseelo, akabikkwa mumapuleti aa 6-Corwellning (NY, USA, USA) abulemu bwa 5 × 104 maselu/ml.
MTT assay yakabelesegwa kulanga-langa ntenda ya insulini NPs30 yakapangwa bupya alimwi yakagwisigwa maanzi. Maseelo aaHepG2 akabikkwa mumapuleti aali 96-well a density ya 5 × 104 cells/mL mpawo akukomezyegwa kwamazuba aali 7 kakutanaba kusunkwa. Insulin NPs akabikkwa muzibeela ziindene-indene (500 to 50 μm) mpawo yakapegwa kumaseelo. Nikwakainda mawoola aali makumi obilwe atune (24) yakukkala, maselu akasanzyigwa ziindi zyotatwe aPBS mpawo akabikkwa mumubili uujisi 0.5 mg/ml MTT kwamahola aambi aali 4. Kujaya maseelo kwakavwuntauzyigwa kwiinda mukupima kucesya kwa enzymatic kwa tetrazolium MTT yamubala wamubala wa yellow kuya ku 70 nfiniti ya Tetrazolium kubelesya Tetrazolium M200 pro spectrophotometer plate reader (Tecan, Männedorf, Switzerland).
Ikubeleka kabotu kwa NPs kwakasunkwa a confocal laser scanning microscopy antoomwe a flow cytometry analysis. Mugoti umwi aumwi wa Nunc Lab-Tek chamber slide system wakasilikwa a FITC-insulin, FITC-insulin-loaded NPs, mpawo wakabambululwa 25 μg/mL ya dehydrated NPs-baulins mubunji bweelene 4 mahola. Maseelo akasanzyigwa ziindi zyotatwe a PBS mpawo akabikkwa a 4% paraformaldehyde. Maseelo akasanzyigwa a 4′,6-diamidino-2-phenylindole (DAPI). Kujanika kwa insulini kwakabonwa kubelesya Olympus FV1000 laser scanning/two-photon microscope (Ompuly, Tokyo, City, Shikus Japan). Mukuvwuntauzya kwa flow cytometry, bunji bweelene bwa 10 μg/mL free FITC-insulin, FITC-insulin-loaded NPs, antoomwe a resolubilized dehydrated FITC-insulin NPs zyakabikkwa mu 96-well plates zyakabikkwa maselu aa HepG2 mpawo zyakabikkwa mubusena bwamawoola aali 4. Nikwakainda mawoola aali 4, maselu akagwisigwa mpawo akagwisigwa ziindi zili 3. a FBS.5 × 104 maselu aamusampu omwe akavwuntauzyigwa a BD LSR II flow cytometer (BD, Franklin Lakes, New Jersey, United States).
Mizeezo yoonse itondezyedwe mbuli mweelwe ± kusiyana. Kweezyanisya aakati kankamu zyoonse kwakali kulinguwe kubelesya one-way ANOVA naa t-test a IBM SPSS Statistics 26 for Mac (IBM, Endicott, New York, USA) mpawo p < 0.05 yakabonwa kuti ilayandika.
Ooku kuvwuntauzya kutondeezya kuchincha akukonzya kwakusansaila kugwisya maanzi muma chitosan/TPP/insulin nanoparticles antoomwe akubambululwa kubotu kweelanisigwa anzila zizibilikide zyakuyuminizya kubelesya misamu iikola naa cryoprotectants capacity alimwi a load capacity mpati. Insulin nanoparticles zyakabambwa kabotu zyakapa mweelwe waakati-kati wa n 18 kubeleka kabotu kwa 99.4%. SEM antoomwe a FTIR zyakazwa nikwakamana kumana maanzi zyakatondezya kuti bupange bwabbola bwakali buyo mu NPs zyakayuminizyigwa a mannitol azitakwe mannitol mpawo zyakasanzyigwa a mannitol, pesi NPs zyakasanzyigwa kakunyina mannitol zyakabola muchiindi chakubula maanzi. Mukusunkwa kwakujana insulini kakunyina mannitol yakatondeezya bupati bwatuniini tuniini loko antoomwe akujatikizigwa kupati aawo noyakabambululwa. Ikuzwa kwama NP oonse aaya kwakatondezya kuti kwakafwambaana kuzwa mumasolusi aa pH = 2.5 a pH = 7, alimwi kwakakkalilila kapati mumusamu wa pH = 6.5. Kweezyanisya ama NP aambi aakanyonyoonwa, ma NP aakasansailwa kakunyina mafuta akatondeezya kuti akafwambaana kuzwa. zileendelana azyeezyo zyakabonwa mukulanga-langa kwakunywa kwamaseelo, mbuli NPs zyakayuminizyigwa akusansaila kakunyina mannitol zyakazumanana kubamba kabotu kwamaseelo aa NPs zyakabambilwa bupya. Eezyi mpindu zitondezya kuti ma nanoparticles aa insulini aayumide aakabambwa kwiinda mukusansaila kakunyina mannitol ngaayelede kubelesegwa munzila imwi mbuli misamu iikola, naakuti misamu mapilisi naakuti mafilimu aajisi zintu zipona.
Akaambo katwaambo twabusongo, data yakabambwa a/naa yakavwuntauzyigwa muchiiyo chiliwo lino taikonzyi kujanika kubuleya, pesi ilajanwa kuzwa kuli balembi aabo kuti kwaba kulomba.
Kagan, A. Bulwazi bwashuga bwamusyobo wabili: matalikilo aabukkale asayensi, mapenzi aabusilisi, anzyozikonzya kuchitika kubalwazi abamwi. (McFarlane, 2009).
Singh, AP, Guo, Y., Singh, A., Xie, W. & Jiang, P. Kusumpuka kwa insulini: sena kubelesya mumulomo lino kulakonzyeka?J. Pharmacy.bio-pharmacy.reservoir.1, 74–92 (2019).
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