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MaInsulin nanoparticles (NPs) ane huwandu hwakawanda hwemutoro akawana mashandisirwo akasiyana mumhando dzakasiyana dzemutero. Basa iri rine chinangwa chekuongorora mhedzisiro yekuomesa nekuisa mufiriji uye kuisa mufiriji pachimiro chechitosan nanoparticles ine insulin, ine kana isina mannitol se cryoprotectant. Takaongororawo mhando yeava nanoparticles nekudzinyungudutsa patsva. Tisati tapedza mvura, saizi yechitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles yakagadziriswa kuti ive 318 nm, PDI yaive 0.18, kushanda kwe encapsulation kwaive 99.4%, uye mutoro waiva 25.01%. Mushure mekugadzirisazve, ma nanoparticles ese, kunze kweaya akagadzirwa nenzira yekuomesa nefreeze pasina kushandisa mannitol, akachengetedza chimiro chawo chespheric particles. Zvichienzaniswa ne ma nanoparticles ane mannitol akabviswa mvura ne spray, ma nanoparticles asina spray-dried mannitol akaratidzawo saizi diki yepakati pe particles (376 nm) uye huwandu hwepamusoro hwemutoro (25.02%). nechiyero chakafanana chekuvharwa kwemasero (98.7%) uye PDI (0.20) kuburikidza nekuomesa kana kuomesa nechando. Zvidimbu zvakaomeswa nekuomesa nespray pasina mannitol zvakakonzerawo kuburitswa kweinsulin nekukurumidza uye kushanda kwakanyanya kwekushandiswa kwemasero. Basa iri rinoratidza kuti kuomesa nespray kunogona kunyungudutsa insulin nanoparticles pasina kudiwa kwe cryoprotectives zvichienzaniswa nenzira dzekare dzekuomesa nechando, zvichigadzira kugona kukuru kwekurodha, zvinodiwa zvekuwedzera zvishoma uye mari yekushanda inobatsira zvikuru.
Kubva pakuwanikwa kwayo muna 1922, 2, 3, insulin nemishonga yayo yakaponesa hupenyu hwevarwere vane chirwere cheshuga cherudzi rwekutanga (T1DM) uye chirwere cheshuga cherudzi rwechipiri (T1DM). Zvisinei, nekuda kwehunhu hwayo seprotein ine huremu hwakawanda, insulin inounganidzwa zviri nyore, inoputswa nema enzymes eproteolytic, uye inobviswa nemhedzisiro yekutanga. Vanhu vanoonekwa vane chirwere cheshuga cherudzi rwekutanga vanoda jekiseni re insulin kwehupenyu hwavo hwese. Varwere vazhinji vanoonekwa vaine chirwere cheshuga cherudzi rwechipiri pakutanga vanodawo jekiseni re insulin kwenguva refu. Majekiseni e insulin ezuva nezuva inokonzera kurwadziwa uye kusagadzikana kwezuva nezuva kune vanhu ava, nemhedzisiro yakaipa pahutano hwepfungwa. Nekuda kweizvozvo, mamwe marudzi e insulin anokonzera kusagadzikana kushoma, akadai se insulin yemuromo, ari kuongororwa zvakanyanya5 sezvo aine mukana wekudzorera hupenyu hwevanhu vanosvika mabhiriyoni mashanu vane chirwere cheshuga pasi rese.
Tekinoroji yeNanoparticle yakapa kufambira mberi kukuru mukuyedza kutora insulin yemuromo4,6,7. Imwe inonyatso vhara uye inodzivirira insulin kubva mukuora kuti iendeswe kunzvimbo dzakatarwa dzemuviri. Zvisinei, kushandiswa kwe nanoparticle formulations kune zvipingamupinyi zvakawanda, kunyanya nekuda kwekusagadzikana kwezvikamu zvesimbi. Kumwe kuungana kunogona kuitika panguva yekuchengetwa, izvo zvinoderedza bioavailability ye nanoparticles dzakazadzwa ne insulin8. Pamusoro pezvo, kugadzikana kwemakemikari kwepolymer matrix ye nanoparticles ne insulin kunofanirawo kufungwa nezvako kuti kuve nechokwadi chekugadzikana kwe insulin nanoparticles (NPs). Parizvino, tekinoroji yekuomesa nechando ndiyo chiyero chegoridhe chekugadzira NPs dzakagadzikana uku uchidzivirira shanduko dzisingadiwe panguva yekuchengetwa9.
Zvisinei, kuomesa nechando kunoda kuwedzerwa ma cryoprotectants kudzivirira chimiro chedenderedzwa cheNPs kuti chisakanganiswa nekumanikidzwa kwemakristaro echando. Izvi zvinoderedza zvakanyanya mutoro we insulin nanoparticles mushure me lyophilization, sezvo cryoprotectant inotora huwandu hwakawanda hwehuremu. Nokudaro, insulin NPs dzinogadzirwa dzinowanzoonekwa dzisina kukodzera kugadzirwa kwemafuta eupfu hwakaoma, akadai semapiritsi emuromo nemafirimu emuromo, nekuda kwekudiwa kwehuwandu hwakawanda hwema nanoparticles akaoma kuti insulin iwane mukana wekurapa.
Kuomesa nepfapfaidzo inzira inozivikanwa uye isingadhuri yeindasitiri yekugadzira hupfu hwakaoma kubva muzvikamu zvemvura muindasitiri yemishonga10,11. Kudzora maitiro ekuumbwa kwezvikamu kunobvumira kuvharwa kwakakodzera kwemakemikari akati wandei anoshanda mumuviri 12, 13. Uyezve, yave nzira inoshanda yekugadzira mapuroteni akavharirwa kuti apihwe nemuromo. Panguva yekuomesa nepfapfaidzo, mvura inopwa nekukurumidza, izvo zvinobatsira kuchengetedza tembiricha yepakati pezvikamu yakaderera11,14, zvichiita kuti ishandiswe kuvharwa kwezvinhu zvinonzwa kupisa. Usati waomesa nepfapfaidzo, zvinhu zvekufukidza zvinofanirwa kusanganiswa zvakakwana nemushonga une zvinhu zvakavharirwa11,14. Kusiyana nekuomesa nechando, kuvharwa nepfapfaidzo usati wavharwa nechando mukuomesa nepfapfaidzo kunovandudza kushanda kwekuvhara nepfapfaidzo panguva yekushaya mvura. Sezvo maitiro ekuvhara nepfapfaidzo asingade cryoprotectives, kuomesa nepfapfaidzo kunogona kushandiswa kugadzira maNP akaomeswa ane huwandu hwakawanda hwekuisa.
Chidzidzo ichi chinoshuma kugadzirwa kweinsulin-loaded NPs kuburikidza nekubatanidza chitosan nesodium tripolyphosphate uchishandisa nzira yeion gel. Ion gelation inzira yekugadzirira inobvumira kugadzirwa kwenanoparticles kuburikidza nekubatana kwemagetsi pakati pemarudzi maviri kana anopfuura eionic pasi pemamwe mamiriro. Maitiro ese ari maviri ekuomesa nechando uye espray-drying akashandiswa kubvisa mvura mu optimized chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles. Mushure mekushaya mvura, chimiro chavo chakaongororwa neSEM. Kugona kwavo kudzokorodza kwakaongororwa nekuyera kupararira kwavo kwehukuru, surface charge, PDI, encapsulation efficiency, uye loading content. Hunhu hwe resolubilized nanoparticles dzakagadzirwa nenzira dzakasiyana dzekushaya mvura hwakaongororwawo nekuenzanisa dziviriro yavo yeinsulin, maitiro ekuburitsa, uye kushanda kwemasero.
PH yemusanganiswa wemvura pamwe nechiyero che chitosan ne insulin zvinhu zviviri zvakakosha zvinokanganisa saizi ye particles uye encapsulation efficiency (EE) yeNPs yekupedzisira, sezvo zvichikanganisa zvakananga maitiro e ionotropic gelation. PH yemusanganiswa wemvura yakaratidzwa kuti ine hukama hwakanyanya nehukuru hwe particles uye encapsulation efficiency (Mufananidzo 1a). Sezvakaratidzwa muMufananidzo 1a, sezvo pH yakawedzera kubva pa4.0 kusvika 6.0, avhareji ye particles size (nm) yakaderera uye EE yakawedzera zvakanyanya, nepo pH payakawedzera kusvika pa6.5, avhareji ye particles size yakatanga kuwedzera uye EE yakaramba isina kuchinja. Sezvo chiyero che chitosan ne insulin chichiwedzera, avhareji ye particles size inowedzerawo. Uyezve, hapana shanduko muEE yakaonekwa apo nanoparticles dzakagadzirwa pahuwandu hwe chitosan/insulin hwakakwira kupfuura 2.5:1 (w/w) (Mufananidzo 1b). Nokudaro, mamiriro ekugadzirira akanakisa muchidzidzo ichi (pH 6.0, chitosan/insulin mass ratio ye 2.5:1) akashandiswa gadzirirai nanoparticles dzakazadzwa neinsulin kuti mudzidze zvakawanda. Pasi pemamiriro aya ekugadzirira, avhareji yehukuru hweparticles dze insulin nanoparticles yakagadziriswa kuti ive 318 nm (Mufananidzo 1c), PDI yaive 0.18, kushanda zvakanaka kwe embedding kwaive 99.4%, zeta potential yaive 9.8 mv, uye insulin loading yaive 25.01% (m/m ). Zvichibva pamhedzisiro ye transmission electron microscopy (TEM), nanoparticles dzakagadziriswa dzaive dzakaita sedenderedzwa uye dzakaparadzana dzine saizi yakafanana (Mufananidzo 1d).
Kugadziriswa kwezvikamu zve insulin nanoparticles: (a) mhedzisiro yepH paavhareji yedhayamita uye encapsulation efficiency (EE) ye insulin nanoparticles (yakagadzirwa pa 5:1 mass ratio ye chitosan ne insulin); (b) chitosan uye Kupesvedzera kwehuwandu hwemasendi ye insulin paavhareji yedhayamita uye encapsulation efficiency (EE) ye insulin NPs (yakagadzirwa pa pH 6); (c) kugoverwa kwehukuru hwezvikamu zve insulin nanoparticles yakavandudzwa; (d) TEM micrograph ye insulin NPs yakavandudzwa.
Zvinozivikanwa kuti chitosan ipolyelectrolyte isina simba ine pKa ye6.5. Inochajwa zvakanaka mu acidic media nekuti boka rayo guru remaamino rinogadzirwa nehydrogen ions15. Nokudaro, inowanzo shandiswa semutakuri wekubatanidza macromolecules ane negative charge. Muchidzidzo ichi, chitosan yakashandiswa kuvhara insulin ne isoelectric point ye5.3. Sezvo chitosan ichishandiswa sechinhu chekufukidza, nekuwedzera kwehuwandu hwayo, ukobvu hwekunze kwe nanoparticles hunowedzera zvakafanana, zvichikonzera avhareji yakakura yehukuru hwe particles. Pamusoro pezvo, huwandu hwakakwira hwe chitosan hunogona kuvhara insulin yakawanda. Muchiitiko chedu, EE yaive yakakwira apo chiyero che chitosan ne insulin chakasvika 2.5:1, uye pakanga pasina shanduko huru muEE apo chiyero chakaramba chichiwedzera.
Kunze kwehuwandu hwechitosan neinsulin, pH yakaitawo basa rakakosha mukugadzirira kweNPs.Gan et al. 17 vakadzidza nezvemhedzisiro yepH pahukuru hwechitosan nanoparticles. Vakaona kudzikira kuri kuramba kuripo muhukuru hwechitosan kusvika pH yasvika 6.0, uye kuwedzera kukuru kwehukuru hwechiparticle kwakaonekwa papH > 6.0, izvo zvinoenderana nezvatakaona. Chiitiko ichi chinokonzerwa nekuti nekuwedzera kwepH, molekyuru yeinsulin inowana negative surface charge, nokudaro, ichikurudzira electrostatic interactions ne chitosan/sodium tripolyphosphate (TPP) complex, zvichikonzera saizi diki yechitosan uye EE yakakwira. Zvisinei, pH payakagadziriswa kusvika 6.5, mapoka eamino ari pachitosan akabviswa protonated, zvichikonzera chitosan folding. Saka, pH yakakwira inokonzera kusabatanidzwa kwemaamino ions kuTPP neinsulin zvishoma, zvichikonzera kudzikira kwe cross-linking, kukura kweavhareji yekupedzisira yechiparticle uye EE yakaderera.
Kuongororwa kwehunhu hweNPs dzakaomeswa nechando uye dzakaoma nespray kunogona kutungamira kusarudzwa kwenzira dziri nani dzekubvisa mvura mumuviri uye kugadzira upfu. Nzira inosarudzwa inofanira kupa kugadzikana kwemushonga, chimiro chezvimedu zvakafanana, mutoro wemushonga wakawanda uye kunyungudika kwakanaka mumushonga wekutanga. Muchidzidzo ichi, kuti tienzanise zviri nani nzira mbiri idzi, insulin NPs dzine kana dzisina 1% mannitol dzakashandiswa panguva yekubvisa mvura. Mannitol inoshandiswa semushonga wekuwedzera kana cryoprotectant mumhando dzakasiyana dzeupfu hwakaoma hwekuomeswa nechando uye kuomeswa nespray. Kune insulin nanoparticles dzisina lyophilized dzisina mannitol, sezvakaratidzwa muMufananidzo 2a, chimiro cheupfu chine maburi akawanda chine nzvimbo hombe, dzisina kurongeka uye dzakaoma chakaonekwa pasi pe scanning electron microscopy (SEM). Zvimedu zvishoma zvakaonekwa muupfu mushure mekupera mvura (Mufananidzo 2e). Izvi zvakaratidza kuti maNP mazhinji akaora panguva yekuomeswa nechando pasina cryoprotectant. Kune insulin nanoparticles dzakaomeswa nechando uye dzakaoma nespray dzine 1% mannitol, nanoparticles dzakatenderera dzine nzvimbo dzakatsetseka dzakaonekwa (Mufananidzo 2). 2b,d,f,h).Mananoparticles einsulin akaomeswa nespray asina mannitol akaramba akaita sedenderedzwa asi akakwenya pamusoro (Mufananidzo 2c). Nzvimbo dzakakombama nedzakakwenya dzinokurukurwa zvakanyanya mumaitiro ekuburitswa uye bvunzo dzekutorwa kwemasero pazasi. Zvichibva pakuonekwa kunoonekwa kweNPs dzakaomeswa, maNPs ese akaomeswa nespray asina mannitol neNPs akaomeswa nechando uye akaomeswa nespray nemannitol akaburitsa hupfu hwakanaka hweNPs (Mufananidzo 2f,g,h). Nzvimbo iri pakati penzvimbo dzeparticle, inonyunguduka zvakanyanya uye nekudaro mwero wekuburitswa wakakwira.
Maitiro emhando dzakasiyana dzeinsulini dzisina mvura: (a) Mufananidzo weSEM we lyophilized insulin NPs dzisina mannitol; (b) Mufananidzo weSEM we lyophilized insulin NPs dzine mannitol; (c) insulini yakaomeswa nespray NPs dzisina mannitol Mufananidzo weSEM we ; (d) Mufananidzo weSEM we insulini NPs dzakaomeswa nemannitol; (e) mufananidzo we lyophilized insulin NPs upfu husina mannitol; (f) mufananidzo we lyophilized insulin NPs dzine mannitol; (g) Mufananidzo we insulini yakaomeswa nespray NPs upfu husina mannitol; (h) mufananidzo we insulini yakaomeswa nespray NPs upfu husina mannitol.
Munguva yekuomesa nechando, mannitol inoshanda se cryoprotectant, ichichengetedza maNPs muchimiro chisina chimiro uye ichidzivirira kukuvara nemakristaro echando. Kusiyana neizvi, hapana danho rechando panguva yekuomesa nespray. Saka mannitol haidiwi nenzira iyi. Kutaura zvazviri, maNPs akaomeswa nespray asina mannitol akaburitsa maNPs akapfava sezvakatsanangurwa kare. Zvisinei, mannitol inogona kushanda sekuzadza mukuita kwekuomesa nespray kuti ipe maNPs chimiro chakatenderera20 (Mufananidzo 2d), izvo zvinobatsira kuwana maitiro akafanana ekuburitsa maNPs akadaro akavharirwa. Pamusoro pezvo, zviri pachena kuti zvimwe zvidimbu zvikuru zvinogona kuonekwa mu maNPs e insulin akavharirwa nespray ane mannitol (Mufananidzo 2b, d), izvo zvinogona kunge zvichikonzerwa nekuunganidzwa kwemannitol muchikamu chepakati pamwe chete ne insulin yakavharirwa. Ku.Chitosan layer. Zvakakosha kuziva kuti muchidzidzo chino, kuitira kuti chimiro chedenderedzwa chirambe chakasimba mushure mekushaya mvura mumuviri, chiyero chemannitol nechitosan chinochengetwa pa5:1, kuitira kuti huwandu hwakawanda hwekuzadza hugonewo kuwedzera saizi yezvikamu zveNPs dzakaomeswa.
Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy yakaratidza musanganiswa wemuviri we insulin yemahara, chitosan, chitosan, TPP uye insulin. NP dzese dzisina mvura dzakaratidzwa nekushandisa FTIR-ATR spectroscopy. Zvinonyanya kukosha, simba re band re 1641, 1543 uye 1412 cm-1 rakaonekwa mu encapsulated NPs dzakaomeswa ne freeze ne mannitol uye muNPs dzakapfapfaidzwa ne spray-dryed ne uye dzisina mannitol (Mufananidzo 3). Sezvakambotaurwa, kuwedzera uku kwesimba kwakabatana ne cross-linking pakati pe chitosan, TPP ne insulin. Kuferefeta kwekudyidzana kuripo pakati pe chitosan ne insulin kwakaratidza kuti mu FTIR spectra ye insulin-loaded chitosan nanoparticles, chitosan band yakabatana ne insulin, zvichiwedzera carbonyl intensity (1641 cm-1) uye amine (1543 cm-1) belt. Mapoka e tripolyphosphate e TPP akabatana ne ammonium groups mu chitosan, zvichiumba bhendi riri pa1412 cm-1.
Ma spectra eFTIR-ATR e insulin yemahara, chitosan, musanganiswa chaiwo we chitosan/TPP/insulin neNPs zvakabviswa mvura nenzira dzakasiyana.
Uyezve, mhedzisiro iyi inoenderana neyakaratidzwa muSEM, iyo yakaratidza kuti maNP akavharirwa akaramba asina kukuvadzwa kana akapfapfaidzwa nemannitol, asi pasina mannitol, kupfapfaidzwa chete ndiko kwaiburitsa zvidimbu zvakavharirwa. Kusiyana neizvi, mhedzisiro yeFTIR-ATR spectral yeNPs dzakavharirwa pasina mannitol yakafanana zvikuru nemusanganiswa wepanyama we chitosan, TPP, uye insulin. Mhedzisiro iyi inoratidza kuti cross-links pakati pechitosan, TPP ne insulin hadzisisipo muNPs dzakavharirwa pasina mannitol. Maumbirwo eNPs akaparadzwa panguva yekuomesa pasina cryoprotectant, izvo zvinogona kuonekwa mumhedzisiro yeSEM ​​(Mufananidzo 2a). Zvichibva pachimiro uye mhedzisiro yeFTIR yeinsulin NPs dzakapera mvura, maNPs chete akashandiswa pakuedzazve uye maNPs asina mannitol nekuda kwekuora kweMantonitol-freeze panguva yekushaya mvura. kurukura.
Kushaya mvura mumuviri kunoshandiswa pakuchengetwa kwenguva refu uye kudzokororwa kuita mamwe maformulations. Kugona kweNPs dzakaomeswa kudzokororwa mushure mekuchengetwa kwakakosha pakushandiswa kwadzo mumafomu akasiyana akadai semapiritsi nemafirimu. Takaona kuti avhareji yehukuru hwezvikamu zveinsulin NPs dzakaomeswa nekupfapfaidzwa pasina mannitol yakawedzera zvishoma mushure mekugadziriswazve. Kune rumwe rutivi, hukuru hwezvikamu zveinsulin nanoparticles dzakaomeswa nekupfapfaidzwa nemannitol hwakawedzera zvakanyanya (Tafura 1). PDI neEE hazvina kuchinjwa zvakanyanya (p > 0.05) mushure mekudzokororwa kweNPs dzese muchidzidzo chino (Tafura 1). Mhedzisiro iyi inoratidza kuti zvikamu zvakawanda zvakaramba zviripo mushure mekunyungudutswazve. Zvisinei, kuwedzerwa kwemannitol kwakaita kuti insulin ideredzwe zvakanyanya yemannitol nanoparticles dzakapfapfaidzwa nekupfapfaidzwa (Tafura 1). Kusiyana neizvi, huwandu hweinsulin load yeNPs dzakaomeswa nekupfapfaidzwa pasina mannitol hwakaramba hwakafanana nepakutanga (Tafura 1).
Zvinozivikanwa kuti kurodha kwe nanoparticles kwakakosha kana kuchishandiswa pakutumira mishonga. Kune maNP ane mutoro wakaderera, huwandu hwakawanda hwezvinhu hunodiwa kuti husvike padanho rekurapa. Zvisinei, kusimba kwakanyanya kwehuwandu hwakadaro hweNP kunotungamira kune kusagadzikana uye kuoma mukupihwa nemuromo uye majekiseni, zvichiteerana 22. Pamusoro pezvo, insulin NPs inogona zvakare kushandiswa kugadzira mapiritsi ne viscous biofilms23, 24, izvo zvinoda kushandiswa kwehuwandu hwakawanda hweNPs padanho rakaderera rekurodha, zvichikonzera mapiritsi makuru ne biofilms dzakakora dzisingakodzeri kushandiswa nemuromo. Naizvozvo, maNP asina mvura yakawanda ane mutoro wakawanda we insulin anodiwa zvikuru. Mhedzisiro yedu inoratidza kuti huwandu hwakawanda hwe insulin ye mannitol-free spray-dryed NPs hunogona kupa mabhenefiti akawanda anoyevedza kune idzi dzimwe nzira dzekupa.
MaNP ese asina mvura akachengetwa mufiriji kwemwedzi mitatu. Zvakabuda muSEM zvakaratidza kuti chimiro cheNP dzese dzisina mvura hachina kuchinja zvakanyanya panguva yekuchengetwa kwemwedzi mitatu (Mufananidzo 4). Mushure mekugadziriswazve mumvura, maNP ese akaratidza kudzikira kudiki muEE uye akaburitsa huwandu hushoma (~5%) hweinsulin mukati menguva yekuchengetwa kwemwedzi mitatu (Tafura 2). Zvisinei, avhareji yehukuru hwezvinhu zvese zve nanoparticles yakawedzera. Hukuru hwezvinhu zveNPs zvakapfapfaidzwa zvisina mannitol hwakawedzera kusvika 525 nm, nepo hweNPs dzakapfapfaidzwa uye dzakaomeswa nechando nemannitol hwakawedzera kusvika 872 ne921 nm, zvichiteerana (Tafura 2).
Maitiro emhando dzakasiyana dzeinsulin dzakaoma dzakachengetwa kwemwedzi mitatu: (a) Mufananidzo weSEM we lyophilized insulin NPs ne mannitol; (b) Mufananidzo weSEM we nanoparticles dze insulin dzakaoma dzisina mannitol; (c) mifananidzo yeSEM ​​isina mannitol ye insulin dzakaoma dzisina mannitol.
Uyezve, mvura yakabuda mu insulin nanoparticles dzakagadziriswazve dzakapfapfaidzwa ne mannitol ndokuzoomeswa nechando (Mufananidzo S2). Izvi zvinogona kukonzerwa nezvidimbu zvikuru zvisina kumira zvakanaka mumvura. Zvese zvabuda pamusoro apa zvinoratidza kuti nzira yekuomesa ne spray inogona kudzivirira insulin nanoparticles kubva pakushaya mvura mumuviri uye kuti huwandu hwakawanda hwe insulin nanoparticles hunogona kuwanikwa pasina ma fillers kana cryoprotectants.
Kuchengetwa kweinsulin kwakaedzwa mu pH = 2.5 medium ne pepsin, trypsin, uye α-chymotrypsin kuratidza kugona kwekudzivirira kweNPs kubva mukugaya kwe enzymatic mushure mekushaya mvura. Kuchengetwa kwe insulin kweNPs dzisina mvura kwakaenzaniswa nekweNPs dzichangobva kugadzirwa, uye insulin yemahara yakashandiswa senzira yekudzora negative. Muchidzidzo ichi, insulin yemahara yakaratidza kubviswa kwe insulin nekukurumidza mukati memaawa mana mukurapa kwese kwe enzymatic (Mufananidzo 5a–c). Kusiyana neizvi, kuyedzwa kwe insulin kubvisa NPs dzakaomeswa ne mannitol ne NPs dzakapfapfaidzwa ne kana dzisina mannitol kwakaratidza dziviriro yakakura yeNPs idzi kubva mukugaya kwe enzymatic, iyo yakafanana neye insulin NPs dzichangobva kugadzirwa (Mufananidzo 1).5a-c). Nerubatsiro rwe nanoparticles mu pepsin, trypsin, uye α-chymotrypsin, anopfuura 50%, 60%, uye 75% ye insulin inogona kudzivirirwa mukati memaawa mana, zvichiteerana (Mufananidzo 5a–c). Kugona uku kudzivirira insulin kunogona kuwedzera mukana we insulin yakakwira. kunyudzwa muropa25. Izvi zvinoratidza kuti kuomesa nespray kana pasina mannitol uye kuomesa nechando nemannitol zvinogona kuchengetedza kugona kwekudzivirira insulin kweNPs mushure mekupera mvura mumuviri.
Kudzivirira uye kuburitsa insulin NPs dzisina mvura mumuviri: (a) kudzivirirwa kwe insulin mumushonga we pepsin; (b) kudzivirirwa kwe insulin mumushonga we trypsin; (c) kudzivirirwa kwe insulin nemushonga we α-chymotrypsin; (d) Maitiro ekuburitsa insulin dzisina mvura mumuviri mu pH = 2.5 solution; (e) maitiro ekuburitsa insulin dzisina mvura mumuviri mu pH = 6.6 solution; (f) maitiro ekuburitsa insulin dzisina mvura mumuviri mu pH = 7.0 solution.
Ma NP e insulin yakaoma akagadzirwa uye akagadzirwazve akaiswa muzvikamu zvakasiyana-siyana (pH = 2.5, 6.6, 7.0) pa37 °C, zvichitevedzera pH yemudumbu, duodenum, uye ura hwepamusoro, kuti vaongorore simba re insulin pakuramba insulin. Maitiro ekuburitsa zvinhu munzvimbo dzakasiyana. Chidimbu chemudumbu. Pa pH = 2.5, ma NP ane insulin uye ma NP e insulin akaoma akagadziriswazve akaratidza kuburitswa kwekutanga mukati meawa imwe yekutanga, zvichiteverwa nekuburitswa zvishoma nezvishoma mumaawa mashanu anotevera (Mufananidzo 5d). Kuburitswa uku nekukurumidza pakutanga kungangove kuri mugumisiro wekubviswa nekukurumidza kwemamorekuru eprotein pamusoro asina kuvharwa zvizere mukati mechimiro chemukati mechikamu. Pa pH = 6.5, ma NP ane insulin uye ma NP e insulin akaoma akagadziriswazve akaratidza kuburitswa kwakanaka uye zvishoma nezvishoma kwemaawa matanhatu, sezvo pH yemhinduro yekuyedza yaive yakafanana neyemhinduro yakagadzirwa ne NPs (Mufananidzo 5e). Pa pH = 7, ma NP akanga asina kugadzikana uye akapotsa aora zvachose mukati memaawa maviri ekutanga (Mufananidzo 5f). Izvi zvinodaro nekuti kubviswa kwe chitosan kunoitika pa pH yakakwira, izvo zvinoguma ne network isina polymer yakawanda uye kuburitswa kwe insulin yakatakura.
Uyezve, insulin NPs dzakaomeswa pasina mannitol dzakaratidza kuburitswa nekukurumidza kupfuura dzimwe NPs dzisina mvura (Mufananidzo 5d–f). Sezvakatsanangurwa kare, insulin NPs dzakaomeswa dzisina mannitol dzakaratidza saizi diki diki yezvikamu. Zvidimbu zvidiki zvinopa nzvimbo yakakura, saka mushonga wakawanda unoenderana nawo uchava pamusoro pezvikamu kana kuti pedyo nazvo, zvichikonzera kuburitswa kwemishonga nekukurumidza26.
Kuonekwa kwechepfu mumasero eNP kwakaongororwa nekuyedzwa kweMTT. Sezvakaratidzwa muMufananidzo S4, maNP ese asina mvura mumuviri akawanikwa asina mhedzisiro yakakura pakushanda kwemasero pahuwandu hwe50–500 μg/ml, zvichiratidza kuti maNP ese asina mvura mumuviri anogona kushandiswa zvakachengeteka kusvika pahwindo rekurapa.
Chiropa ndicho chikamu chikuru chinoshandiswa ne insulin kuita mabasa ayo emuviri. Masero eHepG2 imhando yemasero ehepatoma emunhu anowanzo shandiswa senzira yekutora hepatocyte muropa. Pano, masero eHepG2 akashandiswa kuongorora kutora kwemasero eNPs dzisina mvura mumuviri uchishandisa nzira dzekuomesa nefreeze uye spray-drying. Kutora kwemasero kuburikidza ne confocal laser scanning uchishandisa flow cytometry uye vision mushure memaawa akati wandei e incubation ne free FITC insulin pa concentration ye 25 μg/mL, FITC insulin-loaded NPs itsva yakagadzirwa uye FITC insulin-loaded NPs dzisina mvura mumuviri pa equal insulin concentrations. Kuongororwa kweQuantitative microscopy (CLSM) kwakaitwa. LPs dzisina mannitol dzakaparadzwa panguva yekushaya mvura mumuviri uye hadzina kuongororwa mubvunzo uyu. Intensities ye intracellular fluorescence ye NPs dzakagadzirwa insulin-loaded, lyophilized NPs ne mannitol, uye spray-dryed NPs dzine uye dzisina mannitol (Mufananidzo 6a) dzaive 4.3, 2.6, 2.4, uye Yakakwira ne4.1-kaviri kupfuura yemahara. FITC-insulin group, zvichiteerana (Mufananidzo 6b). Izvi zvinoratidza kuti insulin yakavharirwa ine simba guru mukutorwa kwemasero kupfuura insulin yemahara, kunyanya nekuda kwehukuru hudiki hwenanoparticles dzakagadzirwa muchidzidzo ichi.
Kutorwa kwemasero eHepG2 mushure memaawa mana ekuiswa mumasero neNPs dzakagadzirwa dzichangobva kugadzirwa uye NPs dzisina mvura: (a) Kugoverwa kweFITC-insulin uptake nemasero eHepG2.(b) Geometric avhareji yefluorescence intensities yakaongororwa ne flow cytometry (n = 3), *P < 0.05 kana ichienzaniswa ne free insulin.
Saizvozvowo, mifananidzo yeCLSM yakaratidza kuti simba reFITC fluorescence reFITC-insulin-loaded NPs idzva dzakagadzirwa uye FITC-insulin-loaded spray-dried NPs (isina mannitol) dzaive dzakasimba kupfuura dzemamwe masampuli (Mufananidzo 6a). Uyezve, nekuwedzera kwemannitol, viscosity yakanyanya yemushonga yakawedzera kuramba kutorwa kwemasero, zvichikonzera kudzikira kwehuwandu hweinsulin. Izvi zvinoratidza kuti mannitol-free spray-dryed NPs dzakaratidza kushanda kwepamusoro kwemasero nekuti saizi yavo yezvikamu yaive diki pane yeNPs dzakaomeswa nechando mushure mekunyungudika zvakare.
Chitosan (avhareji yehuremu hwemamorekuru 100 KDa, 75–85% deacetylated) yakatengwa kubva kuSigma-Aldrich. (Oakville, Ontario, Canada). Sodium tripolyphosphate (TPP) yakatengwa kubva kuVWR (Radnor, Pennsylvania, USA). Recombinant human insulin yakashandiswa muchidzidzo ichi yakabva kuFisher Scientific (Waltham, MA, USA). Fluorescein isothiocyanate (FITC)-labeled human insulin uye 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) zvakatengwa kubva kuSigma-Aldrich. (Oakville, Ontario, Canada). HepG2 cell line yakawanikwa kubva kuATCC (Manassas, Virginia, USA). Mamwe ma reagents ese aive analytical kana chromatographic grade.
Gadzirira mhinduro ye1 mg/ml yeCS nekuinyungudutsa mumvura yakasanganiswa kaviri (DD water) ine 0.1% acetic acid. Gadzirira mhinduro ye1 mg/ml yeTPP neinsulin nekuinyungudutsa mumvura yeDD uye 0.1% acetic acid, zvichiteerana. Pre-emulsion yakagadzirwa ne polytron PCU-2-110 high speed homogenizer (Brinkmann Ind. Westbury, NY, USA). Maitiro ekugadzirira ndeekuti: chekutanga, 2ml yeTPP solution inowedzerwa ku4ml yeinsulin solution, uye musanganiswa unokurunga kwemaminitsi makumi matatu uye wakasanganiswa zvizere. Zvadaro, mhinduro yakasanganiswa yakawedzerwa ku CS solution kuburikidza nesirinji pasi pekukurunga nekukurumidza (10,000 rpm). Misanganiswa yacho yakachengetwa pasi pekukurunga nekukurumidza (15,000 rpm) mu ice bath kwemaminitsi makumi matatu, uye yakagadziriswa kune pH yakati kuti iwane cross-linked insulin NPs. Kuti iwedzere homogenize uye kuderedza saizi yezvikamu zve insulin NPs, dzakaiswa sonicated kuti dziwedzere. Kugeza nechando kwemaminitsi makumi matatu uchishandisa sonicator yemhando yeprobe (UP 200ST, Hielscher Ultrasonics, Teltow, Germany).
Insulin NPS yakaedzwa kuti ionekwe kuti ine dhayamita yeZ-average, polydispersity index (PDI) uye zeta potential uchishandisa zviyero zve dynamic light scattering (DLS) uchishandisa Litesizer 500 (Anton Paar, Graz, Austria) nekudzisanganisa mumvura yeDD pa25°C. Maitiro uye kugoverwa kwehukuru zvakaratidzwa neHitachi H7600 transmission electron microscope (TEM) (Hitachi, Tokyo, Japan), uye mifananidzo yakazoongororwa uchishandisa software yeHitachi imaging (Hitachi, Tokyo, Japan). Kuti vaongorore kushanda kwe encapsulation (EE) uye loading capacity (LC) ye insulin NPs, ma NP akaiswa mumachubhu e ultrafiltration ane molecular weight cut-off ye100 kDa uye centrifuged pa500 xg kwemaminitsi makumi matatu. Unencapsulated insulin mu filtrate yakayerwa uchishandisa Agilent 1100 Series HPLC system (Agilent, Santa Clara, California, USA) ine quaternary pump, autosampler, column. hita, uye DAD detector. Insulin yakaongororwa neC18 column (Zorbax, 3.5 μm, 4.6 mm × 150 mm, Agilent, USA) uye yakaonekwa pa214 nm. Mobile phase yaive acetonitrile nemvura, ine 0.1% TFA, gradient ratios kubva pa10/90 kusvika 100/0, uye yakamhanya kwemaminitsi gumi. Mobile phase yakapombwa neflow rate ye1.0 ml/min. Colour tembiricha yakaiswa pa20 °C. Verenga ma percentages eEE neLC uchishandisa ma equation.(1) uye Eq.(2).
Zviyero zvakasiyana-siyana zveCS/insulin zvinotangira pa2.0 kusvika 4.0 zvakaedzwa kuti insulin NP ishande zvakanaka. Huwandu hwakasiyana hweCS solution hwakawedzerwa panguva yekugadzirira, ukuwo musanganiswa weinsulin/TPP wakaramba wakafanana. Insulin NPs dzakagadzirwa muhuwandu hwepH hwe4.0 kusvika 6.5 nekunyatso tarisa pH yemusanganiswa mushure mekuwedzera mhinduro dzese (insulin, TPP neCS). EE nehukuru hweparticles yeinsulin nanoparticles zvakaongororwa pamhando dzakasiyana dzepH uye huwandu hweCS/insulin mass kuti zvivandudze kuumbwa kweinsulin NPs.
Ma NP e insulin akagadziridzwa akaiswa pachirongo chearuminiyamu ndokufukidzwa netishu yakasimbiswa netepi. Zvadaro, ma screwed containers akaiswa muLabconco FreeZone freezer dryer (Labconco, Kansas City, MO, USA) ine tray dryer. Tembiricha uye vacuum pressure zvakaiswa pa -10 °C, 0.350 Torr kwemaawa maviri ekutanga, uye 0 °C uye 0.120 Torr kwemaawa makumi maviri nemaviri asara emaawa makumi maviri nemana kuti insulin NPs dziome.
Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Switzerland) yakashandiswa kugadzira insulin yakavharirwa. Zvinhu zvakasarudzwa zvekuomesa zvaive: tembiricha 100 °C, kuyerera kwemafuta 3 L/min, uye kuyerera kwegesi 4 L/min.
Insulin NPs isati yapera mvura mumuviri uye mushure mekupedza muviri yakaonekwa uchishandisa FTIR-ATR spectroscopy. Zvidimbu zvenano zvakapera mvura pamwe nefree insulin ne chitosan zvakaongororwa uchishandisa Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA) yakashongedzerwa ne universal ATR sampling accessory (PerkinElmer, Waltham, Massachusetts, USA). Avhareji yezviratidzo yakawanikwa kubva kuma scans gumi nematanhatu pa resolution ye 4 cm2 mu frequency range ye 4000-600 cm2.
Maitiro e insulin NPs dzakaoma akaongororwa nemifananidzo yeSEM ​​ye insulin NPs dzakaomeswa nefreeze uye dzakapfapfaidzwa dzakatorwa ne Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, USA). Chinonyanya kushandiswa chaive voltage 5 keV uye current 30 mA.
Ma NP ese e insulin asina mvura akanyungudutswa mumvura ye dd. Saizi yezvikamu, PDI, EE ne LC zvakaedzwa zvakare uchishandisa nzira imwecheteyo yambotaurwa kuti zvionekwe kuti zvakanaka sei mushure mekushaya mvura. Kugadzikana kwe anhydroinsulin NPs kwakayerwawo nekuyedza hunhu hweNPs mushure mekuchengetedza kwenguva refu. Muchidzidzo ichi, ma NP ese mushure mekushaya mvura akachengetwa mufiriji kwemwedzi mitatu. Mushure memwedzi mitatu yekuchengetera, ma NP akaedzwa kuti aone kana ane morphological particle size, PDI, EE ne LC.
Nyungudutsa 5 mL yeNPs dzakagadziriswazve mu45 mL ine mvura yemudumbu yakaenzaniswa (pH 1.2, ine 1% pepsin), mvura yemudumbu (pH 6.8, ine 1% trypsin) kana chymotrypsin solution (100 g/mL, mu phosphate buffer, pH 7.8) kuti uongorore kushanda kwe insulin mukudzivirira NPs mushure mekushaya mvura mumuviri. Dzakaiswa mu incubation pa37°C nekumhanya kwe100 rpm. 500 μL yemhinduro yakaunganidzwa panguva dzakasiyana uye huwandu hwe insulin hwakatarwa ne HPLC.
Maitiro ekuburitswa kwe insulin NPs itsva yakagadzirwa uye isina mvura mumuviri akaedzwa nenzira ye dialysis bag (molecular weight cut-off 100 kDa, Spectra Por Inc.). Ma NPs matsva akagadzirwa uye akagadzirwazve akaiswa dialyzed mumvura pa pH 2.5, pH 6.6, uye pH 7.0 (0.1 M phosphate-buffered saline, PBS) kuti vatevedzere pH yemudumbu, duodenum, uye upper small insulate, zvichiteerana. Samples dzese dzakaiswa pa37 °C nekuzungunuswa kunoramba kuripo pa200 rpm. Dururira mvura kunze kwe5 mL dialysis bag panguva dzinotevera: 0.5, 1, 2, 3, 4, uye 6 awa, uye wedzera huwandu hwayo ne dialysate itsva. Kusvibiswa kwe insulin mumvura kwakaongororwa neHPLC, uye mwero wekuburitswa kwe insulin kubva kuma nanoparticles wakaverengerwa kubva pachiyero che insulin yakasununguka yakaburitswa kune total insulin yakaiswa muma nanoparticles (Equation 3).
Masero eHepG2 eHepatocellular carcinoma emunhu akakurira mundiro dzine dhayamita ye60 mm achishandisa Dulbecco's Modified Eagle's Medium (DMEM) ine 10% fetal bovine serum, 100 IU/mL penicillin, uye 100 μg/mL streptomycin29. Kukura kwacho kwakachengetwa pa37°C, 95% hunyoro, uye 5% CO2. Kuti zviongororwe, masero eHepG2 akadyarwa pa1 × 105 cells/ml pane 8-well Nunc Lab-Tek chamber slide system (Thermo Fisher, NY, USA). Kuti zviongororwe cytotoxicity, zvakadyarwa mumaplates e96-well (Corning, NY, USA) pahuwandu hwe5 × 104 cells/ml.
Kuongororwa kweMTT kwakashandiswa kuongorora kukuvadza kwemasero einsulin NPs30 achangobva kugadzirwa uye asina mvura mumuviri. Masero eHepG2 akadyarwa mumaplates ane matsime makumi mapfumbamwe nematanhatu pahuwandu hwemasero mashanu negumi nemana/mL uye akarimwa kwemazuva manomwe bvunzo isati yaitwa. MaInsulin NPs akasanganiswa kusvika pakuwanda kwakasiyana (50 kusvika 500 μg/mL) muchimiro chekurima uye akazopihwa kumasero. Mushure memaawa makumi maviri nemana ekuisirwa, masero akagezwa katatu nePBS uye akaisirwa mukati mechimiro chine 0.5 mg/ml MTT kwemaawa mana ekuwedzera. Kukuvadza kwemasero kwakaongororwa nekuyera kudzikira kweyellow tetrazolium MTT kuita purple formazan pa570 nm uchishandisa Tecan infinite M200 pro spectrophotometer plate reader (Tecan, Männedorf, Switzerland).
Kushanda zvakanaka kweNPs mumasero kwakaedzwa ne confocal laser scanning microscopy uye flow cytometry analysis. tsime rega rega reNunc Lab-Tek chamber slide system rakarapwa ne free FITC-insulin, FITC-insulin-loaded NPs, uye rakagadziriswazve 25 μg/mL ye FITC-insulin NPs dzakapera mvura mumuviri panguva imwe chete uye dzakaiswa mumaawa mana. Masero akagezwa katatu nePBS ndokugadziriswa ne 4% paraformaldehyde. Nuclei yakasanganiswa ne 4′,6-diamidino-2-phenylindole (DAPI). Kuwanikwa kweinsulin kwakaonekwa uchishandisa Olympus FV1000 laser scanning/two-photon confocal microscope (Olympus, Shinjuku City, Tokyo, Japan). Pakuongorora flow cytometry, huwandu hwakafanana hwe 10 μg/mL free FITC-insulin, FITC-insulin-loaded NPs, uye resolubilized dehydrated FITC-insulin. NPs dzakaiswa mumaplate ane matsime makumi mapfumbamwe nematanhatu akadyarwa nemasero eHepG2 uye dzakaiswa mumaawa mana. Mushure memaawa mana ekuiswa mumaseru, masero akabviswa ndokugezwa katatu neFBS. 5 × 104 masero pasampuli imwe neimwe akaongororwa neBD LSR II flow cytometer (BD, Franklin Lakes, New Jersey, United States).
Maitiro ese anoratidzwa seavhareji ± standard deviation. Kuenzanisa pakati pemapoka ese kwakaongororwa uchishandisa one-way ANOVA kana t-test neIBM SPSS Statistics 26 yeMac (IBM, Endicott, New York, USA) uye p < 0.05 yakaonekwa seyakakosha muhuwandu.
Chidzidzo ichi chinoratidza kuchinjika uye kugona kwekuomesa spray kuti ibvise mvura kubva ku cross-linked chitosan/TPP/insulin nanoparticles ine reconstruction iri nani zvichienzaniswa nenzira dzakajairwa dzekuomesa nefreeze uchishandisa zvinhu zvinogadzira kana cryoprotectants kugona uye mutoro wakakwira. Ma insulin nanoparticles akagadziriswa akaburitsa avhareji yehukuru hwe318 nm uye kushanda zvakanaka kwe encapsulation kwe99.4%. Mhedzisiro yeSEM ​​neFTIR mushure mekushaya mvura yakaratidza kuti chimiro chespherical chakachengetedzwa chete mu NPs dzakaomeswa nespray dzine uye dzisina mannitol uye dzakaomeswa nemannitol, asi NPs dzakaomeswa ne lyophilized dzisina mannitol dzakaora panguva yekushaya mvura. Muyedzo we reconstitution ability, insulin nanoparticles dzakaomeswa nespray dzisina mannitol dzakaratidza saizi diki diki yepakati uye mutoro wepamusoro pakudzokororwa. Maitiro ekuburitsa ma NPs ese aya asina mvura akaratidza kuti akaburitswa nekukurumidza mumhinduro dze pH = 2.5 uye pH = 7, uye akasimba kwazvo mumhinduro ye pH = 6.5. Zvichienzaniswa nedzimwe NPs dzakaomeswa ne dehydrated, ma NPs akaomeswa nespray asina mannitol yakaratidza kuburitswa nekukurumidza. Mhedzisiro iyi inoenderana neyakaonekwa mukuyedzwa kwemasero, sezvo maNPs akaomeswa nespray pasina mannitol akachengetedza kushanda kwemasero kweNPs dzichangobva kugadzirwa. Izvi zvinoratidza kuti ma nanoparticles e insulin akaoma akagadzirwa ne mannitol-free spray drying anonyanya kukodzera kugadziriswa mune mamwe mafomu e anhydrous dosage, akadai semapiritsi emuromo kana mafirimu anonamira.
Nekuda kwematambudziko epfuma yepfungwa, data rakagadzirwa uye/kana rakaongororwa panguva yekudzidza kuri kuitwa harisi kuwanikwa pachena, asi rinowanikwa kubva kuvanyori varo kana zvikakumbirwa zvine musoro.
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