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Gin ma ki lwongo ni Insulin nanoparticles (NPs) ma tye ki rwom ma malo gunongo tic ma pat pat i kit ma pat pat. Tic man tye me temo adwogi pa yub me juku ki juku i kom jami ma ki lwongo ni insulin-loaded chitosan nanoparticles, ki nyo labongo mannitol ma calo cryoprotectant. Wa bene wa temo rwom pa nanoparticles magi kun wa dwoko gi cen. rwenyo pii, dit pa jami ma ki lwongo ni chitosan/sodium tripolyphosphate/insulin ma ki keto i kin jami ma ki lwongo ni chitosan/sodium tripolyphosphate/insulin onongo ki yubu me bedo 318 nm, PDI onongo tye 0.18, kit ma ki keto kwede jami onongo tye 99.4%, ki kit ma ki keto kwede onongo tye 25.01%. Iyonge dwoko ne, jami ma ki yubu ni weng, ma pe ki kwanyo ki i yoo me freeze labongo tic ki mannitol, ogwoko kit ma gi nen kwede. Ka kiporo ki mannitol-containing nanoparticles ma ki kwanyo ki i pii, mannitol-free spray-dried nanoparticles bene onyutu rwom me particle matidi loyo (376 nm) ki rwom me keto ma malo loyo (25.02%) ki encapsulation rate marom (DI987%) ki PDI. (0.20) ki yoo me juku onyo juku ma ngiic. Nanoparticles ma ki juku ki juku labongo mannitol bene okelo gonyo insulin oyot ki tic maber pa cell. Tic man nyutu ni juku twero balo insulin nanoparticles labongo miiti pa cryoprotectants ka kiporo ki yoo macon me juku, ma kelo looing madit kero, jami ma mite ma nok ki wel cente me tic ma kelo adwogi maber.
Cake i kare ma kinongo ne i mwaka 19221,2,3, insulin ki jami ma ki yubu me cango two ni gu laro kwo pa lutwo ma tye ki two cukari me acel (T1DM) ki two cukari me aryo (T1DM). Ento, pi kit ma en tye kwede ma calo gin ma pek pa molecule, insulin yot me ribbe, ki turo ki enzymes ma gwoko kom, ki kwanyo woko ki i kom two cukari. dano ma tye ki two cukari me acel mito yat insulin pi kare me kwo gi weny. Dano mapol ma kinongo ni gitye ki two cukari me aryo bene mito yat insulin pi kare malac. Yat insulin nino ducu kelo arem ki peko bot jo magi, ki adwogi marac i kom yotkom me tam. Macalo adwogi ne, yoo mukene me miyo insulin ma kelo peko manok, calo miyo insulin madwong, onyo miyo insulin kare ki kare ki kwano5 ma calo gi tye ki kero me dwoko kwo pa dano ma romo bilion 5 ma tye ki two cukari i wilobo weny.
Diro me Nanoparticle okelo dongo ma pire tek i temo tic ki insulin me dog4,6,7. Gin acel ma gwoko ki gwoko insulin ki i balo ne me cwalo ne i kom dano. Ento, tic ki nanoparticle formulations tye ki peko mapol, ma loyo pi peko me cung matek pa particle suspensions. Mukene twero time i kare me gwoko jami ma kwo, ma dwoko piny rwom me gwoko jami ma kwo insulin-loaded nanoparticles8.Medo ikom meno, kit ma jami ma ki lwongo ni polymer matrix pa nanoparticles ki insulin omyera ki nen kwede me neno ni insulin nanoparticles (NPs) tye maber. I kare ni, diro me juku jami ma ki lwongo ni freeze-drying aye obedo gin maber loyo me yubu NPs ma cung matek kun gengo alokaloka ma pi miite i kare me gwoko gi9.
Ento, freeze-drying mito medo cryoprotectants me gengo kit ma NPs nen kwede ki i kom peko me ice crystals. Man dwoko piny rwom me insulin nanoparticles inge lyophilization, ma calo cryoprotectant ni kwanyo wel pek pa gin ni. Pi meno, NPs ma ki yubu pol kare ki nongo ni NPs ni ki nongo ni gi tye ki insulin ma ki yubu. pe opore pi yubu jami ma ki lwongo ni dry powder formulations, ma calo yat ma ki tiyo kwede i dog ki oral films, pi miiti pi jami mapol ma ki tiyo kwede me nongo yat ma ki lwongo ni insulin.
Yub me juku jami ma ki lwongo ni Spray drying obedo gin ma ki ngeyo maber ki ma wel ne yot ma ki tiyo kwede me yubu jami ma otwo ki i pii i tic me yat10,11. Gwoko kit me yubu jami ma ki lwongo ni particle miyo kare me keto jami mapol ma tiyo i kom dano 12, 13 . Medo ikom meno, odoko yoo maber me yubu jami ma ki lwongo ni proteins onyo proteins tic kwede. I kare me juku, pii rweny oyotoyot, ma konyo me gwoko lyeto pa gin ma tye iye piny11,14, ma weko tic kwede me gwoko jami ma lyeto. Mapwod pi juku, gin ma ki tiyo kwede omyera ki yub maber ki gin ma tye ki jami ma ki keto iye, ma calo Un14, ma weko bedo agonya. keto gin acel acel ma peya ki keto i spray-drying weko encapsulation bedo maber i kare me dehydration. Kit ma spray-drying encapsulation process pe mito cryoprotectants, spray-drying twero bedo me yubu NPs ma otwo ma tye ki rwom ma malo.
Kwano man nyutu kit ma ki yubu kwede insulin-loaded NPs kun ki keto chitosan ki sodium tripolyphosphate kun kitiyo ki yoo me ion gel. Ion gelation obedo yoo me yubu ma weko ki yubu nanoparticles kun kitiyo ki electrostatic interactions i kin ionic species aryo onyo mapol i te cik mogo. Kitiyo ki yoo me freeze-drying ki spray-drying me kwanyo pii chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles.Iyonge pii, kit ma gi nen kwede ki kwano ki SEM. Kit ma gi twero rwate kwede ki pimo kit ma gi poko kwede, rwom me kom gi, PDI, kit ma ki keto kwede, ki kit ma ki keto kwede. Kit ma ki yubu kwede nanoparticles ma ki yubu ki yoo me kwanyo pii ki iye bene ki poro ki insulin gwok, kit me gonyo, ki tic pa cell me gamo jami.
pH pa gin ma ki rubu ki ratio pa chitosan ki insulin obedo jami aryo ma pire tek ma weko dit pa jami ma ki rubu ki kit ma ki keto kwede (EE) pa NPs me agiki, ma calo gi gudo kit ma ionotropic gelation process tye kwede. pH pa gin ma ki rubu ni ki nyutu ni tye ki rwate ki dit pa gin ma ki rubu ki kit ma ki keto kwede (Fig. 1A ma ki nyutu ki i cal 1a). 4.0 onyo 6.0, dit pa jami ma ki lwongo ni particle (nm) odok piny ki EE omede matek, ma ka pH omede nio wa i 6.5, dit pa jami ma ki lwongo ni particle ocako mede ki EE odong ma pe oloko. Kit ma ratio pa chitosan ki insulin omede kwede, wel jami ma ki lwongo ni particle bene omedde. rwom me pek pa chitosan/insulin ma malo kato 2.5:1 (w/w) (Fig. 1b). Pi meno, kit me yubu maber loyo i kwan man (pH 6.0, rwom me pek pa chitosan/insulin me 2.5:1) ki tiyo kwede me yubu insulin-loaded nanoparticles pi kwan mukene. I te kit me yubu man, rwom me insulin ma ki nongo maber loyo me nongo nanoparticles 318 nm (Fig. 1c), PDI onwongo tye 0.18, embedding efficiency onwongo tye 99.4%, zeta potential onwongo tye 9.8 mv, ki insulin loading onwongo tye 25.01% (m/m). Malube ki adwogi me transmission electron microscopy (TEM), nanoparticles ma kiyubu ni onwongo tye ma rwate ki dit pa gin ma ki lwongo ni spherical ki discrete (Fig. 1c. 1d).
Kit me yubu insulin nanoparticles: (a) adwogi pa pH i kom dit pa insulin ki kit ma ki keto kwede (EE) pa insulin nanoparticles (ki yubu i 5:1 pek pa chitosan ki insulin); (b) chitosan ki adwogi pa pek pa insulin i kom dit pa insulin ki kit ma ki keto kwede (EE) pa insulin NPs (ma ki yubu i pH 6); (c) poko pa dul kom ma ki lwongo ni insulin nanoparticles; (d) TEM micrograph pa insulin NPs ma ki yubu maber.
Ki ngeyo maber ni chitosan tye polyelectrolyte magoro ki pKa me 6.5. En tye ki kero maber i kin piny ma tye ki acid pien dul amino ne ma pire tek ki proton ki hydrogen ions15. Pi meno, pol kare ki tiyo kwede calo gin ma tingo macromolecules ma tye ki kero marac. I kwan man, ki tiyo ki chitosan me keto teko i insulin ki insulin 5.3. Kit ma ki tiyo kwede ki chitosan calo gin ma ki umu kwede, ki medo rwom ne, pek pa dul kom ma woko pa nanoparticles ni mede, ma kelo dit pa particles. Medo ikom meno, rwom ma malo pa chitosan twero gwoko insulin madwong. I lok wa, EE onwongo tye malo ka ratio pa chisan ki insulin oo 25, onwongo pe ki loko. i EE ma wel ne omede ki mede.
Medo i kom rwom pa chitosan ki insulin, pH bene otiyo tic ma pire tek i yubu NPs.Gan et al. 17 gu kwano adwogi pa pH i kom dit pa jami ma ki lwongo ni chitosan nanoparticles. Gin onongo ni dit pa jami ma ki lwongo ni particle tye ka medde ameda nio ka pH oo 6.0, ki medo pa dit pa jami ma tye i pH > 6.0, ma rwate ki gin ma waneno. Gin ma tye ka time ni tye pien ka pH mede, insulin nongo teko ma pe opore, ma miyo teko ma pe ber tic pa mac ki chitosan/sodium tripolyphosphate (TPP) complex, ma okelo dit pa jami ma nok ki EE ma malo. Ento, ka pH ni ki dwoko piny me 6.5, dul amino ma tye i kom chitosan ni ki kwanyo protonated, ma okelo chitosan folding. Pi meno, pH ma malo kelo rwom ma lapiny pa amino ions ki TPP, ma kelo cross-linr madit, rwom me agiki me dul kom ki EE ma piny.
Kwedo kit ma jami ma ki lwongo ni freeze-dried ki spray-dried NPs nen kwede twero konyo me yero yoo maber me kwanyo pii ki pii ki yubu buru. Yoo ma ki yero omyera omii yat ma cung matek, kit ma dul kom ma rom, rwom me yat ma malo ki rwate maber i gin ma ki yubu mukwongo. I kwan man, me poro yoo aryo ni maber, insulin NPs ma pe ki 1% Mantol mantniol ki tiyo kwede i kare me kwanyo pii. ki tiyo kwede ma calo gin ma weko piny doko tek onyo cryoprotectant i kit ma ki yubu kwede buru ma otwo ma pat pat ma ki tiyo kwede me juku ki pii. Pi insulin ma ki lwongo ni lyophilized insulin nanoparticles labongo mannitol, ma ki nyutu i cal 2a, gin ma ki lwongo ni highly porous powder structure ki kome ma dit, ma pe rwate ki ma goro ki neno i te microscope (SEM). ma ki nongo i buru ma ki kwanyo ki i pii (Fig. 2e). Adwogi eni onyutu ni pol pa NPs obale i kare me freeze-drying labongo cryoprotectant mo keken. Pi freeze-dried ki spray-dried insulin nanoparticles ma tye ki 1% mannitol, spherical nanoparticles ma tye ki kom ma pwot ki neno (Fig. 2e, Infulin). nanoparticles ma ki juku labongo mannitol odong ma nen calo dul kom ento opong ki dul kom (Fig. 2c). Dul kom ki dul kom ma opong ki lok i kom kit ma ki gonyo kwede ki cellular uptake tests ma piny kany. Malube ki kit ma nen kwede pa NPs ma ki juku, NPs ma ki juku labongo mannitol ki NPs ma ki juku ki mannitol ma ki juku ki mannitol okelo buru ma ki lwongo ni NPs (Fig. 2f,g,h). Ka dit pa ngom ma tye i kin dul kom ma pat pat, ci rwom me gonyo ne bedo malo ki dong rwom me gonyo ne bene malo.
Kit ma insulin NPs ma ki kwanyo ki i kom gi nen kwede: (a) cal me SEM pa insulin NPs ma ki keto i pii labongo mannitol; (b) cal me SEM pa lyophilized insulin NPs ki mannitol; (c) gin ma ki lwongo ni spray-dried insulin NPs labongo mannitol SEM cal pa ; (d) cal me SEM pa insulin NPs ma ki juku ki mannitol; (e) cal pa lyophilized insulin NPs powder ma pe ki mannitol; (f) cal pa lyophilized insulin NPs ki mannitol; (g) cal pa gin ma ki lwongo ni spray-dried insulin NPs powder ma pe ki mannitol; (h) cal pa gin ma ki lwongo ni spray-dried insulin NPs powder ki mannitol.
I kare me juku, mannitol tiyo calo cryoprotectant, gwoko NPs i kit ma pe nen ki gengo balo pa ice crystals19. I apoka poka, pe tye rwom me juku i kare me juku. Pi meno mannitol pi miite i yoo man. Ki lok adaa, NPs ma ki juku labongo mannit miyo NPs maber calo macon ma kiloko iye. Ento, mannitol pud twero tic calo gin ma pongo i yub me juku ne me miyo NPs bedo ma rwate20 (Fig. 2d), ma konyo me nongo kit me gonyo NPs ma ki keto i iye. Medo ikom meno, tye ka nen ni jami mogo madongo twero nonge i manniol ma tye ki insulin ma ki juku ki ma ki juku ki spray (Fig. 2d). 2b,d), ma twero bedo pi dongo pa mannitol i kom gin ma ki lwongo ni particle core kacel ki insulin ma ki keto i iye. To.Chitosan layer. Ber me poyo ni i kwan man, me neno ni dul kom ma nen calo dul kom ni odong maber inge kwanyo pii, ratio pa mannitol ki chitosan ki gwoko i 5:1, wek wel ma dit me pongo twero medo dit pa jami ma ki lwongo ni NPs ma otwo..
Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy onyutu dul kom ma ki lwongo ni free insulin, chitosan, chitosan, TPP ki insulin. NPs ma ki kwanyo ki i pii ki nyutu gi kun kitiyo ki FTIR-ATR spectroscopy. Ki ngeyo, band intensities me 1641, 1543 ki 14 cm 1641 ki neno i NP 14 ki juku ki mannitol ki i NPs ki juku ki ki labongo mannitol (Fig. 3). Kit ma onongo ki waco kwede con, medo teko ni onongo tye ki rwate ki rwate i kin chitosan, TPP ki insulin. Kwedo lok i kin chitosan ki insulin onyutu ni i FTIR spectra pa insulin-loaded chitosan chitosan, dul ma ki keto iye insulin, i kom chitosan ma ki keto iye insulin, medo rwom me carbonyl (1641 cm-1) ki amine (1543 cm-1) belt. Tripolyphosphate dul pa TPP gikubbe ki dul ammonium i chitosan, ma gi yubu dul ma tye 1412 cm-1.
FTIR-ATR spectra me insulin ma pe ki culu, chitosan, jami ma ki rubu ki chitosan/TPP/insulin ki NPs ma ki kwanyo pii ki i yoo ma pat pat.
Medo ikom meno, adwogi eni rwate ki ma ki nyutu i SEM, ma onyutu ni NPs ma ki gwoko ni odong maber i kare ma ki oru ki mannitol, ento ka pe ki mannitol, ki juku keken aye okelo jami ma ki keto i mannitol. I apoka poka, adwogi me FTIR-ATR spectral pa NPs ma ki keto i freeze onongo rom ki mannitol ma pe ki juku dul ma ki rubu ki chitosan, TPP, ki insulin. Adwogi man nyutu ni dul ma ki lwongo ni cross-links i kin chitosan, TPP ki insulin dong pe tye i NPs ma ki juku labongo mannitol. Kit ma NPs ni obale kwede i kare ma ki juku labongo cryoprotectant, ma ki twero neno i adwogi me SEM (Fig. 2A i kom adwogi pa BaIR ma ki kwanyo ki i pii). insulin NPs, lyophilized, spray-dried, ki mannitol-free NPs ki otiiyo kwede me temo dwoko rwom me tic piny ki mannitol-free NPs pi balo pa mannitol-free NPs i kare me pii. leyo tam.
Dehydration ki tiyo kwede me gwoko pi kare malac ki dwoko cen i jami mukene. Ker pa NPs ma otwo me dwogo cen i nge gwoko pire tek pi tic kwede i jami ma pat pat calo tablet ki film. Waneno ni rwom me dul kom ma ki lwongo ni spray-dried insulin NPs i kare ma pe ki keto iye mannitol omede manok keken inge dwoko cen, ki tung cing mukene dit pa dul kom. gin ma ki lwongo ni spray-dried ki freeze-dried insulin nanoparticles ki mannitol omede matek (Table 1).PDI ki EE pi gu loko tutwal (p > 0.05) ingee dwoko NPs weny i kwan man (Table 1). Adwogi man nyutu ni pol pa particles ni odong maber ingee dwoko gi cen. Ento, adwogi pa manniol odwoko piny rwom me insulin odok piny tutwal lyophilized ki spray-dried mannitol nanoparticles (Table 1). I apoka poka, rwom me insulin ma tye i NPs spray-dried labongo mannitol odong kit macon (Table 1).
Ki ngeyo maber ni keto nanoparticles pire tek ka kitiyo kwede me miyo yat. Pi NPs ma tye ki keto manok, jami mapol ma mite me oo i rwom me cango. Ento, rwom ma malo pa NP ma malo kelo peko ki peko i miyo ki dog ki kit ma ki keto kwede, 22 NPs twero medo. bene ki tiyo kwede me yubu jami ma ki lwongo ni tablets ki viscous biofilms23, 24, ma mito tic ki wel NPs madwong i rwom ma lapiny, ma kelo tablets madongo ki biofilms ma pek ma pe rwate ki tic kwede i dog. Pi meno, NPs ma ki keto iye insulin ma dwong tye gin ma mite. Adwogi wa nyutu ni NPs ma ki keto iye insulin madwong ma pe ki tiyo kwede twero miiyo adwogi maber mapol pi yoo mukene me cwalo jami ni.
NPs weng ma pii otum ki gwoko gi i fridge pi dwe adek. Adwogi me SEM onyutu ni kit ma NPs ma odong pii pe olokke tutwal i kare me gwoko pi dwe adek (Fig. 4). Iyonge dwoko gi i pii, NPs weny onyutu dwoko piny manok i EE ki gugonyo insulin manok (~5%) i kare me gwoko pi dwe adek (T 2).Ento, dit pa jami ma ki lwongo ni nanoparticles weng omede. Dit pa jami ma ki lwongo ni NPs ma ki juku labongo mannitol omede oo 525 nm, kun pa spray-dried ki freeze-dried NPs ki mannitol omede oo 872 ki 921 nm, kit meno (Table 2).
Kit ma jami ma ki lwongo ni insulin NPs ma ki gwoko pi dwe adek: (a) cal me SEM pa insulin NPs ma ki keto i pii ki mannitol; (b) cal me SEM pa gin ma ki lwongo ni insulin ma ki juku ma pe ki mannitol; (c) labongo mannitol SEM cal pa insulin NPs ma ki juku ki pii.
Medo ikom meno, ki neno jami ma odonyo i insulin nanoparticles ma ki juku ki mannitol ki ki juku i ngiico (Fig. S2). Man twero bedo ni oa ki i kom jami madongo ma pe gucung maber i pii. Adwogi ma malo ni nyutu ni yoo me juku insulin ni twero gwoko insulin nanoparticles ki i kom pii ki i pii ma twero bedo nanoparticles mapol ki nongo labongo gin mo ma ki keto i iye onyo jami ma gwoko kom.
Kit me gwoko insulin ki temo i pH = 2.5 ki pepsin, trypsin, ki α-chymotrypsin me nyutu teko pa NPs i kom jami ma ki lwongo ni enzymatic digestion inge rwenyo pii. Kit ma insulin gwoko kwede NPs ma ki kwanyo pii ki i kom gi ki poro ki pa NPs ma ki yubu nyen, ki insulin ma pe ki culu ki tiyo kwede calo gin ma pe ber, I kwan man onyutu insulin ma pe ki culu ki kwanyo oyotoyot i 4 h. jami adek ni weng ma ki tiyo kwede (Fig. 5a-c). I apoka poka, temo kwanyo insulin i kom NPs ma ki juku ki mannitol ki NPs ma ki juku ki mannitol onyo labongo mannitol onyutu gwok ma malo pa NPs magi i kom enzymatic digestion, ma onwongo rom ki pa insulin NPs ma ki yubu nyen ( cal 15a- Wic, kony pa pepsinnoparticle). trypsin, ki α-chymotrypsin, ma kato 50%, 60%, ki 75% pa insulin ki twero gwoko ne i kin cawa 4, kit meno (Fig. 5a-c). Kit me gwoko insulin ni twero medo rwom me donyo pa insulin i remo25. Adwogi eni nyutu ni tic ki manniol ma ki juku onyo labongo manniol ki manniol pe twero gwoko ne gwoko teko me gwoko insulin pa NPs inge rwenyo pii i kom.
Gwoko ki gonyo kit pa insulin NPs ma ki kwanyo pii iye: (a) gwoko insulin i pii ma ki lwongo ni pepsin; (b) gwoko insulin i gin ma ki lwongo ni trypsin; (c) gwoko insulin ki α-chymotrypsin solution; (d) Kit ma ki gonyo kwede NPs ma ki kwanyo ki i pH = 2.5 ma ki keto iye; (e) kit ma ki gonyo kwede NPs ma ki kwanyo ki i pH = 6.6 ma ki keto iye; (f) kit ma ki gonyo kwede NPs ma ki kwanyo ki pii i pH = 7.0 ma ki keto iye.
Insulin ma ki yubu manyen ki ma ki dwoko cen ki gwoko gi i buffers ma pat pat (pH = 2.5, 6.6, 7.0) i 37 °C, ma nyutu pH me ic, duodenum, ki ic ​​matidi ma malo, me neno adwogi pa insulin i kom teko pa insulin. Kit me gonyo i kabedo ma pat pat. Dul me dul kom ma i ic. I pH = 2.5, insulin-loaded NPs ki resolubilized dry insulin NPs onyutu gonyo ma oyabe i cawa acel mukwongo, ma lubo ki gonyo mot mot i cawa 5 ma lubo (Fig. 5d). Gonyo oyoto ni i acaki nen calo adwogi me rweny pa mole ma pe obedo mole ma pe ocung i kom gin ma ki lwongo ni particle.I pH = 6.5, insulin-loaded NPs ki reconstituted dry insulin NPs onyutu ni ki gonyo mot mot pi cawa 6, ma calo pH pa gin ma ki temo ni rom ki pa NPs-prepared solution (Fig. 5e). I pH = 7, NPs onongo dong pe ki twero kwanyo woko ki i gin aryo mukwongo cawa (Fig. 5f). Man tye pien deprotonation pa chitosan time i pH ma malo, ma kelo polymer network ma nok ki gonyo insulin ma ki keto iye.
Medo ikom meno, insulin NPs ma ki juku labongo mannitol onyutu rwom me gonyo oyot loyo NPs mukene ma ki juku (Fig. 5d–f). Kit ma onongo kiloko kwede con, insulin NPs ma ki juku labongo mannitol onyutu dit pa jami matino tino. Jami ma tiino miyo kabedo ma lac, pi meno pol pa yat ni obi bedo cok ki yat ni, oyot oyot gonyo26.
Kit ma NPs twero neko kwede kom dano ki kwano ki MTT assay. Kit ma ki nyutu kwede i cal S4, NPs ma ki kwanyo pii iye ki nongo ni pe ki adwogi mo i kom kwo pa cell i rwom me 50-500 μg/ml, ma nyutu ni NPs weng ma ki kwanyo pii ki iye ki twero tic kwede maber me oo i kare me cango two.
Adunu obedo dul kom ma pire tek ma insulin tiyo kwede me tic. HepG2 cells obedo dul kom ma ki lwongo ni human hepatoma cells ma pol kare kitiyo kwede calo in vitro hepatocyte uptake model. Kany, HepG2 cells kitiyo kwede me temo cellular uptake pa NPs ma ki kwanyo ki i pii kun kitiyo ki yoo me freeze-drying ki spray-drying kun kitiyo ki confocal scanning laser. cytometry ki neno ma lubo cawa mapol me gwoko ki FITC insulin ma pe ki culu i rwom me 25 μg/mL, FITC insulin-loaded NPs ma ki yubu nyen ki FITC insulin-loaded NPs ma ki dehydrated i rwom me insulin marom Quantitative microscopy (CLSM) observations were performed.Lyophilized manolids labongo dehydration onongo pe ki pimo temo man. Kit ma ler ma ki lwongo ni intracellular fluorescence intensities pa insulin-loaded NPs ma ki yubu nyen, lyophilized NPs ki mannitol, ki spray-dried NPs ki ki ma pe ki mannitol (Fig. 6a) onwongo tye 4.3, 2.6, 2.4, ki 4.1-fold ma malo loyo ones ma pe ki culu. FI-TC-inins group (Fig. 6). adwogi ni nyutu ni insulin ma ki keto i cell tye ki teko madwong i kom cell ma loyo insulin ma pe ki culu, tutwal pi dit pa insulin-loaded nanoparticles ma ki yubu i kwan ni.
HepG2 cell uptake ingee 4 h me gwoko ki NPs ma ki yubu nyen ki NPs ma ki kwanyo pii iye: (a) Poko pa FITC-insulin uptake ki HepG2 cells.(b) Geometric mean of fluorescence intensities analyzed by flow cytometry (n = 3), *P < 0.05 redred with free insulin.
I yo acel-lu, cal pa CLSM onyutu ni FITC ma ki lwongo ni fluorescence intensities pa FITC-insulin-loaded NPs ma ki yubu nyen ki FITC-insulin-loaded spray-dried NPs (labongo mannitol) onwongo tek loyo pa sampul mukene ni (Fig. 6a). Medo ikom meno, ka ki medo mannitol, rwom me tic pa gin ni omede me gamo cell, ma okelo dwoko piny rwom me insulin. Adwogi eni nyutu ni mannitol-free spray-dried NPs onyutu rwom ma malo me gamo cell pien dit pa jami gi onwongo nok loyo pa freeze-dried NPs ingee dwoko piny.
Chitosan (ma pek pa molecule 100 KDa, 75-85% deacetylated) ki wilo ki bot Sigma-Aldrich. (Oakville, Ontario, Canada). Sodium tripolyphosphate (TPP) ki wilo ki bot VWR (Radnor, Pennsylvania, USA). Recombinant insulin ma ki tiyo kwede i kwan man ki kwanyo ki bot Scientific Scientific Sherham, MA. USA).Fluorescein isothiocyanate (FITC)-labeled human insulin ki 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) ki wilo ki bot Sigma-Aldrich.(Oakville, Ontario, Canada).HepG2 cell line ki nongo ki bot ATCC (Manassas, VirginiaAll, ki nongo ki i reanatotic reagraphents onyo chromatic mukene). gured.
Yub 1 mg/ml CS ma ki rubu ne i pii ma ki lwongo ni double distilled water (DD water) ma tye ki 0.1% acetic acid. Yub 1 mg/ml me TPP ki insulin kun i juku gi i DD water ki 0.1% acetic acid, kit meno. Pre-emulsion ni ki yubu ki polly PCU-monizer ma tye ki specide madwong (Brinkmann Ind. Westbury, NY, USA). Kit me yubu ne tye kit man: mukwongo, 2ml me TPP solution ki medo i 4ml me insulin solution, ki gin ma ki rubu ni ki rubu pi dakika 30 ki dong ki rubu maber. I ngeye, ki medo gin ma ki rubu ni i CS solution kun kitiyo ki syringe ma ki rubu matek (The rpm, 1000). jami ma ki rubu ni ki gwoko gi i rwom ma malo (15,000 rpm) i ice bath pi dakika 30, ki ki dwoko gi i pH mo me nongo cross-linked insulin NPs. Me medo homogenize ki dwoko piny dit pa insulin NPs, ki keto gi i sonicated pi dakika 30 mukene i ice bath kun kitiyo ki sonicator (STUP, 2000) Hielscher Ultrasonics, Teltow, Germany).
Insulin NPS ki temo pi Z-average diameter, polydispersity index (PDI) ki zeta potential kun kitiyo ki dynamic light scattering (DLS) ma ki tiyo kwede ki Litesizer 500 (Anton Paar, Graz, Austria) kun ki keto gi i pii DD i 25°C. Kit ma gi nen kwede ki kit ma gi poko kwede ki nyutu ki microscope electron 607 (TEM) (Hitachi, Tokyo, Japan), ki cal ni ki kwano kun kitiyo ki jami ma ki lwongo ni Hitachi imaging software (Hitachi, Tokyo, Japan). Me neno kit ma encapsulation efficiency (EE) ki loading capacity (LC) pa insulin NPs, NPs ni ki keto gi i ultrafiltration tubes ki pek pa molecular cuffling ma romo 100 centifuge ki Dada 500 xg pi dakika 30. Insulin ma ki keto i gin ma ki lwongo ni filtrate ni ki kwano kun kitiyo ki Agilent 1100 Series HPLC system (Agilent, Santa Clara, California, USA) ma tye ki quaternary pump, autosampler, column heater, ki DAD detector. Insulin ki kwano ki C18Zbax, or column, 35 μm. × 150 mm, Agilent, USA) ki kinongo i 214 nm. Gin ma ki lwongo ni mobile phase onwongo tye acetonitrile ki pii, ma tye ki 0.1% TFA, gradient ratios cake ki 10/90 nio wa i 100/0, ki ki tiyo kwede pi dakika 10. Gin ma ki lwongo ni mobile phase onwongo ki pump i rwom me mol pa 10/min. 20 °C. Kwan wel pacenti pa EE ki LC kun itiyo ki equation.(1) ki Eq.(2).
Ki temo rwom me CS/insulin ma pat pat ma cake ki 2.0 nio wa i 4.0 me yubu insulin NP. Wel CS ma pat pat ki medo i kare me yubu ne, kun insulin/TPP ma ki rubu ni ki gwoko ma rom. Insulin NPs ki yubu i pH ma tye 4.0 nio wa i 6.5 kun ki gwoko pH pa gin ma ki rubu ni maber ingee medo jami ma ki rubu ni (Tulins, TPP ki TPP CS). EE ki dit pa jami ma ki lwongo ni insulin nanoparticles ki kwano i pH ma pat pat ki CS/insulin mass ratios me yubu insulin NPs.
Gin ma ki lwongo ni insulin NPs ni ki keto i kom gin ma ki yubu ki aluminum ki ki umo ki del kom ma ki keto ki tape. I ngeye, ki keto gin ma ki lwongo ni screwed i Labconco FreeZone freeze dryer (Labconco, Kansas City, MO, USA) ma tye ki gin ma ki lwongo ni tray dryer. Ki keto rwom me lyeto ki vacuum i °C, -100 Torr. pi cawa 2 mukwongo, ki 0 °C ki 0.120 Torr pi cawa 22 ma odong me cawa 24 me nongo insulin NPs ma otwo.
Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Switzerland) ki tiyo kwede me yubu insulin ma ki keto i iye. Gin ma ki yero me juku ne obedo: rwom me lyeto 100 °C, rwom me cam 3 L/min, ki rwom me yamo 4 L/min.
Insulin NPs ma peya ki inge kwanyo pii ki ikom gi ki kwano kun kitiyo ki FTIR-ATR spectroscopy. Nanoparticles ma ki kwanyo ki i pii kacel ki insulin ki chitosan ma pe ki culu ki kwano kun kitiyo ki Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA) ma tye ki universal ATR accessory (Perkinlingham, Waltham, Waltham, Massachusetts, USA). Wel ma ki nongo ki i 16 ma ki kwanyo ki i rwom me 4 cm2 i kin rwom me 4000-600 cm2.
Kit ma dry insulin NPs nen kwede ki kwano ki cal me SEM pa insulin NPs ma ki juku ki spray ma ki mako ki Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, USA). Gin ma pire tek ma ki tiyo kwede onwongo tye voltage 5 keV ki current 30 mA.
Insulin NPs ma ki kwanyo ki i pii ni ki dwoko cen i pii dd. Dit pa dul kom, PDI, EE ki LC ki temo odoco kun kitiyo ki yoo acel ma kiloko iye con me neno kit ma gi tye kwede i nge kwanyo pii. Kit ma anhydroinsulin NPs gi cung kwede bene ki pimo ki temo kit pa NPs inge gwoko gi pi kare malac. I kwan man, NPs ni weng ki gwoko gi i refrigerator pi kare adek dwe. Iyonge dwe adek me gwoko, NPs ki temo pi dit pa jami ma nen, PDI, EE ki LC.
Lar 5 mL me NPs ma ki yubu i 45 mL ma tye ki pii ma ki yubu me ic (pH 1.2, ma tye ki 1% pepsin), pii me ic (pH 6.8, ma tye ki 1% trypsin) onyo chymotrypsin solution (100 g/mL, i phosphate buffer, pH 788) me neno kit ma insulin tiyo kwede me gwoko insulin. NPs inge kwanyo pii. Ki gwoko gi i 37°C ki rwom me 100 rpm. 500 μL me pii ni ki kwanyo i kare ma pat pat ki rwom me insulin ki nongo ki HPLC.
Kit ma ki gonyo kwede insulin NPs ma ki yubu nyen ki ma ki kwanyo pii iye ki temo ki yoo me dialysis bag (molecular weight cut-off 100 kDa, Spectra Por Inc.). Gin ma ki yubu manyen ki ma ki dwoko cen ma otwo ki keto i pii ma tye ki pH 2.5, pH 6.6, ki pH 7.0 ( M. 1.0, M. pholine saffered, saffered M.1. PBS) me nyutu pH me ic, duodenum, ki upper small intestine, kit meno. Kamu kene ni ki gwoko i 37 °C ki yengo ne i 200 rpm. Kwany pii ni woko ki i 5 mL dialysis bag i kare ma lubo ni: 0.5, 1, 2, 3, 4 h, ki dwoko cen ki dwonge cut cut dialysate. Insulin ma tye i pii ni ki kwano ki HPLC, ki wel insulin ma ki gonyo ki i nanoparticles ni ki kwano ki i ratio pa insulin ma ki gonyo ki i kom insulin ma ki keto i nanoparticles ni (Equation 3).
Gin ma ki lwongo ni Human hepatocellular carcinoma cell line HepG2 ki dongo gi i 60 mm diameter dishes kun kitiyo ki Dulbecco's Modified Eagle's Medium (DMEM) ma tye ki 10% fetal bovine serum, 100 IU/mL penicillin, ki 100 μg/mL streptomycin ma ki gwoko i 297°C. 95% rwom me yamo, ki 5% CO2. Pi temo kwanyo, cell pa HepG2 ki keto gi i 1 × 105 cells/ml i kom 8-well Nunc Lab-Tek chamber slide system (Thermo Fisher, NY, USA). Pi temo cytotoxicity, ki keto gi i plates 6-C, NY, USA i pek pa 5 × 104 cells/ml.
MTT assay ni ki tiyo kwede me pimo cytotoxicity pa insulin NPs30 ma ki yubu nyen ki ma ki kwanyo ki i pii. Ki keto cell me HepG2 i 96-well plates ma tye ki pek pa 5 × 104 cells/mL ki ki gwoko pi nino 7 ma peya ki temo. Insulin NPs ki keto i rwom ma pat pat (50 to 50 μm/50/mL ki culture medium) ki dong ki miyo bot cell. Iyonge cawa 24 me gwoko, cell ni ki lwoko tyen 3 ki PBS ki ki gwoko ki gin ma tye ki 0.5 mg/ml MTT pi cawa 4 mukene. Cytotoxicity ki temo kun ki pimo enzymatic reduction pa yellow tetrazolium MTT me doko 70 n ma ki tiyo kwede i Tetrazolium purzanite kun kitiyo ki afinite M200 pro spectrophotometer plate reader (Tecan, Männedorf, Switzerland).
Kit ma cell me gamo kwede NPs ki temo ki confocal laser scanning microscopy ki flow cytometry analysis. Kabedo acel acel me Nunc Lab-Tek chamber slide system ki tiyo kwede ki FITC-insulin ma pe ki culu, FITC-insulin-loaded NPs, ki ki dwoko 25 μg/mL me dehydrated FITC-insulin ma ki keto i rwom acel pi 4 cawa. Ki lwoko cell tyen 3 ki PBS ki ki keto ki 4% paraformaldehyde. Ki keto rangi i kom nuclei ki 4′,6-diamidino-2-phenylindole (DAPI). Kit ma insulin tye kwede ki neno kun kitiyo ki Olympus FV1000 laser scanning/two-photon microscope (Onju, City, Tokyo, City Japan).Pi kwan ma ki lwongo ni flow cytometry, wel ma rom pa 10 μg/mL FITC-insulin ma pe ki culu, FITC-insulin-loaded NPs, ki resolubilized dehydrated FITC-insulin NPs ki medo gi i 96-well plates ma ki keto iye HepG2 cells ki ki gwoko pi cawa 4. Iyonge 4 h me gwoko cells, ki kwanyo cells 3 ki i incubation. ki FBS.5 × 104 cell pi lanyut acel acel ki kwano ki BD LSR II flow cytometer (BD, Franklin Lakes, New Jersey, United States).
Wel weng ki nyutu calo rwom me rwom ± apoka poka. Apoka poka i kin gurup weng ki kwano kun kitiyo ki one-way ANOVA onyo t-test ki IBM SPSS Statistics 26 pi Mac (IBM, Endicott, New York, USA) ki p < 0.05 ki ngeyo ni tye ma pire tek.
Kwano man nyutu kit ma ki tiyo kwede ki jami ma ki lwongo ni spray drying me juku pii ma ki lwongo ni cross-linked chitosan/TPP/insulin nanoparticles ki dwoko piny maber ka kiporo ki yoo me freeze-drying ma ki tiyo kwede ki jami ma ki lwongo ni bulking agents onyo cryoprotectants capacity ki higher load capacity. tic maber pa 99.4%. Adwogi me SEM ki FTIR inge kwanyo pii i kom dano onyutu ni kit ma kome nen kwede ni ki gwoko i NPs ma ki juku ki mannitol ki ma pe ki mannitol ki lyophilized ki mannitol, ento lyophilized NPs labongo mannitol onongo gi rweny woko i kare me kwanyo pii. onyutu dit pa jami ma nok loyo ki rwom ma malo loyo i kare me yubu ne. Kit ma ki gonyo kwede NPs ma ki kwanyo pii ni onyutu ni ki gonyo gi oyotoyot i pii ma tye ki pH = 2.5 ki pH = 7, ki gicung matek i pii ma tye ki pH = 6.5. Ka kiporo ki NPs mukene ma ki kwanyo pii iye, NPs ma ki gonyo labongo pii ni onyutu adwogi ma oyot. tye ma rwate ki ma ki neno i cellular uptake assay, ma calo spray-dried NPs ma pe ki mannitol cokcok-ki ogwoko cellular uptake efficiency pa NPs ma ki yubu manyen. Adwogi eni nyutu ni dry insulin nanoparticles ma ki yubu ki mannitol-free spray drying aye ber loyo pi tic ki jami mukene ma calo pii, onyo jami mukene yat ma ki lwongo ni tablets onyo bioadhesive films.
Pi lok ma mako jami ma ki ngeyo, jami ma ki nongo ki/onyo ma ki nyamo i kare me kwan man pe tye bot lwak, ento tye ki bot lucoc ka ki penyo.
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Singh, AP, Guo, Y., Singh, A., Xie, W. & Jiang, P. Dongo pa insulin ma ki lwongo ni encapsulation: tic ki dog dong twere?J. Pharmacy.bio-pharmacy.reservoir.1, 74–92 (2019).
Wong, CY, Al-Salami, H. & Dass, CR Dongo manyen i kom jami ma ki lwongo ni insulin-loaded liposome delivery systems me cango two cukari. Gonye.J. Pharmacy.549, 201–217 (2018).