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Insulin nanoparticles (NPs) loading content sang tak te hian dosage form hrang hrangah hmanna hrang hrang an hmu a.He hna hian freeze-drying leh spray-drying process te hian insulin-loaded chitosan nanoparticles te structure a nghawng dan tehna a tum a, mannitol chu cryoprotectant atan hman emaw hmang lo emaw pawh nise.Heng nanoparticles te quality hi redissolving hmangin kan zirchiang bawk.Before dehydration, chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles te particle size chu 318 nm-ah optimized a ni a, PDI chu 0.18, encapsulation efficiency 99.4%, leh loading chu 25.01% a ni.Reconstitution hnuah nanoparticle zawng zawng, freeze-drying method hmanga siam chhuah tih loh chu hman lohvin a awm vek a ni of mannitol, maintained their spherical particle structure.Spray emaw hmanga dehydrated mannitol-containing nanoparticles te nen khaikhin chuan mannitol-free spray-dried nanoparticles te pawhin mean particle size tenau ber (376 nm) leh loading content sang ber (25.02%) an nei a, encapsulation rate inang (98.7%) leh PDI (0.20) chu drying emaw freeze-drying techniques.Mannitol tel lova spray drying hmanga dried nanoparticles te hian insulin release rang ber leh cellular uptake efficiency sang ber a thlen bawk.He hnathawh hian spray drying hian freeze drying method pangngai nena khaikhin chuan cryoprotectants mamawh lovin insulin nanoparticles te chu a dehydrate thei tih a tarlang a, loading capacity nasa zawk a siam thei a, additive mamawhna a hniam zawk a, operating pawh a ti tlem thei bawk costs significant advantage a ni.
Kum 19221,2,3-a hmuhchhuah a nih atang khan insulin leh a damdawi siamna te hian type 1 diabetes (T1DM) leh type 2 diabetes (T1DM) vei te nunna a chhanhim ta a ni.Amaherawhchu, high molecular weight protein a nih avangin insulin hi awlsam takin a inzawm khawm a, proteolytic enzymes hmangin a tichhia a, first-pass hmangin a bo ta a ni effect.Type 1 diabetes vei nia hriatte chuan an dam chhung zawngin insulin injection an mamawh a ni.Damlo tam tak chu a tir lama type 2 diabetes vei nia hriatte pawhin hun rei tak chhung insulin injection an mamawh bawk.Nitin insulin injection hi heng mite tan hian nitin natna leh hrehawmna thlentu lian tak a ni a, rilru hriselnaah nghawng tha lo tak a nei a ni.Chumi avang chuan insulin pek dan dang, harsatna tlem zawk thlentu, chutiang as oral insulin administration, khawvel puma zunthlum vei mi tluklehdingawn 5 vel nunphung siam\hat theihna an neih avangin nasa takin zirchian mek an ni5.
Nanoparticle technology hian oral insulin lak tumnaah hmasawnna nasa tak a pe a4,6,7.Insulin chu taksa hmun biktea targeted delivery atan a tha taka encapsulate leh degradation atanga humhimtu a ni.Mahse, nanoparticle formulations hman hian tihkhawtlai engemaw zat a nei a, a chhan ber chu particle suspensions stability issues vang a ni.Some aggregation may occur during storage, which reduces the bioavailability of insulin-loaded nanoparticles8.Chu bakah, insulin nanoparticles (NPs) te stability a awm theih nan nanoparticles leh insulin te polymer matrix chemical stability pawh ngaihtuah a ngai a ni.Tun dinhmunah chuan freeze-drying technology hi NPs stable siamna atana gold standard a ni a, chutih rualin dahkhawmnaah duh loh zawng inthlak danglamna a awm lo
Mahse, freeze-drying-ah hian ice crystal-te mechanical stress-in NP-te spherical structure a nghawng loh nan cryoprotectants dah belh a ngai a ni.Hei hian lyophilization hnua insulin nanoparticles loading chu nasa takin a tihtlem a, cryoprotectant hian weight ratio tam zawk a luah avangin.Chuvangin, insulin NPs siam chhuah chu a tan a tling lo tih hmuhchhuah a ni fo thin insulin therapeutic window tihhlawhtlin nan dry nanoparticles tam tak mamawh a nih avangin dry powder formulation siam, oral tablet leh oral film te a ni.
Spray drying hi damdawi industry-a liquid phase atanga dry powder siamna atana hmanraw hriat lar leh man tlawm tak industrial-scale process a ni10,11.Particle formation process control hian bioactive compound engemaw zat encapsulation dik tak a siam thei 12, 13 .Chubakah, oral administration atana encapsulated proteins siamna atana hmanraw tangkai tak a lo ni ta a ni.Durring spray drying, tui chu a vawt rang hle a, chu chuan particle core temperature a hniam tir thei a ni11,14, a hmanna chuan heat-sensitive components te chu a encapsulate thei a ni.Spray drying hmain coating material chu encapsulated ingredients awmna solution nen uluk taka homogenized tur a ni11,14.Unlike freeze-drying, homogenization before spray-drying-a encapsulation hian dehydration laiin encapsulation efficiency a ti sang a ni.Spray-drying encapsulation process-ah hian cryoprotectants a ngai lo a, chuvangin spray-drying hmangin dried NPs loading content sang tak siam theih a ni.
He zirchianna hian ion gel method hmanga chitosan leh sodium tripolyphosphate cross-linking hmanga insulin-loaded NPs siam chhuah dan a report a ni.Ion gelation hi preparation method a ni a, condition thenkhatah ionic species pahnih emaw a aia tam emaw inkara electrostatic interaction hmanga nanoparticles siam chhuah theihna a ni.Freeze-drying leh spray-drying technique pahnih hmangin optimized chitosan/sodium chu dehydrate a ni tripolyphosphate/insulin cross-linked nanoparticles.Dehydration hnuah an morphology chu SEM hmangin an zirchiang a.An recombination ability chu an size distribution, surface charge, PDI, encapsulation efficiency, leh loading content te tehna hmangin an zirchiang bawk.Dehydration method hrang hrang hmanga siam resolubilized nanoparticles quality pawh an insuline protection, release behavior, 1000 te khaikhin a ni bawk. leh cellular uptake efficacy te a ni.
Mixed solution pH leh chitosan leh insulin ratio te hi final NPs te particle size leh encapsulation efficiency (EE) tichhe thei thil pawimawh pahnih a ni a, ionotropic gelation process direct a nghawng avangin.Mixed solution pH hi particle size leh encapsulation efficiency nen a inzawm tlat tih hmuhchhuah a ni (Fig. 1a).Fig. 1a-a kan hmuh angin, pH a san chhoh zel avangin 4.0 atanga 6.0 a nih chuan average particle size (nm) a tlahniam a, EE pawh nasa takin a sang a, pH 6.5 a a san chuan average particle size a sang chho tan a, EE pawh a danglam lo.Chitosan leh insulin ratio a san chuan average particle size pawh a sang chho zel bawk.Chubakah, nanoparticles mass ratio 1000-a siam a nih chuan EE-ah danglamna hmuh tur a awm lo chitosan/insulin 2.5:1 (w/w) aia sang (Fig. 1b).Chuvangin, he zirchiannaa optimal preparation conditions (pH 6.0, chitosan/insulin mass ratio 2.5:1) hmangin insulin-loaded nanoparticles siam belh a ni.He preparation condition hnuaiah hian nanoparticles average particle size chu optimized a ni 318 nm (Fig. 1c), PDI chu 0.18 a ni a, embedding efficiency chu 99.4% a ni a, zeta potential chu 9.8 mv a ni a, insulin loading chu 25.01% (m/m) a ni.Transmission electron microscopy (TEM) result atanga chhut chuan optimized nanoparticles te chu roughly spherical leh discrete an ni a, an size pawh a inang tlang hle (Fig. 1d).
Insulin nanoparticles parameter optimization: (a) insulin nanoparticles (chitosan leh insulin mass ratio 5:1-a siam) mean diameter leh encapsulation efficiency (EE)-a pH-in nghawng a neih dan (b) chitosan leh Insulin NPs (pH 6-a siam) mean diameter leh encapsulation efficiency (EE)-a insulin mass ratio-in nghawng a neih dan (c) insulin nanoparticles optimized te particle size insem dan; (d) Insulin NPs optimized te TEM micrograph hmanga lak chhuah .
Chitosan hi polyelectrolyte chak lo tak, pKa 6.5 nei a ni tih hriat lar tak a ni.Acidic media-ah chuan positive charge a nei a, a chhan chu a amino group ber chu hydrogen ions-in a protonated a ni15.Chuvangin, negative charged macromolecules encapsulate turin carrier atan an hmang fo thin.He zirchiannaah hian chitosan hi insulin chu isoelectric point of 5.3.Since chitosan is used as a coating material, with the increase of its proportion, the thickness of the outer layer of the nanoparticles increases correspondingly, resulting in a larger average particle size.In addition, higher levels of chitosan can encapsulate more insulin.In our case, EE was highest when the ratio of chitosan and insulin reached 2.5:1, and there was no significant change in EE ratio a pung zel lai khan.
Chitosan leh insulin ratio bakah hian pH hian NPs siamnaah hmun pawimawh tak a chang bawk.Gan et al. 17 te chuan chitosan nanoparticles particle size-a pH-in nghawng a neih dan an zirchiang a.PhH 6.0 a thlen thlengin particle size a tlahniam chhunzawm zel tih an hmu a, pH > 6.0-ah particle size nasa takin a pung tih an hmu a, hei hi kan hmuh dan nen a inmil hle.He thil thleng hi pH tihpun rualin insulin molecule chuan negative surface charge a neih vang a ni a, chuvangin, a duh zawk a ni electrostatic interactions with the chitosan/sodium tripolyphosphate (TPP) complex, resulting small particle size and high EE .Amaherawhchu, pH chu 6.5-a siamrem a nih chuan chitosan-a amino group-te chu deprotonated an ni a, chu chuan chitosan folding a thlen a ni.Chutiang chuan pH sang chuan amino ion-te chu TPP leh insulin-ah an exposure tlem zawk a, chu chuan cross-linking a hniam zawk a, final average a lian zawk a ni particle size leh EE hniam zawk.
Freeze-dried leh spray-dried NPs te morphological property thlirletna chuan dehydration leh powder siam dan tha zawk thlan dan tur a kaihruai thei a ni.A duh zawk chuan damdawi stability, uniform particle shape, damdawi loading sang tak leh original solution-a solubility tha tak a pe tur a ni.He zirchiannaah hian technique pahnih hi khaikhin tha zawk tur chuan insulin NPs 1% mannitol nei emaw nei lo emaw chu dehydration laiin hman a ni.Mannitol hi as freeze drying leh spray drying atana dry powder formulations hrang hranga bulking agent emaw cryoprotectant emaw.Mannitol tel lo lyophilized insulin nanoparticles tan chuan Figure 2a-a kan hmuh ang hian scanning electron microscopy (SEM) hmangin powder structure lian tak, mumal lo leh rough tak tak nei chu hmuh a ni.Few discrete particles chu powder-ah hian dehydration hnuah hmuhchhuah a ni (Fig. 2e).Hetiang result hian NP tam zawk chu cryoprotectant engmah awm lovin freeze-drying laiin a chhe vek tih a tilang.Ffreeze-dried leh spray-dried insulin nanoparticles 1% mannitol awmna tan chuan spherical nanoparticles, a chunglam hrual (smooth surfaces) te chu hmuh a ni (Fig. 2b,d,f,h).Insulin nanoparticles spray-dried te chu mannitol tel lovin spherical angin an awm reng a ni mahse a chung lamah a wrinkled (Fig. 2c).Spherical leh wrinkled surface te hi a hnuaia release behavior leh cellular uptake test-ah hian sawi belh a ni.Dryed NPs hmuh theiha a lan dan a zirin, spray-dried NPs mannitol tel lo leh NPs te pahnih freeze-dried leh spray-dried with mannitol te hian NPs powder tha tak tak an siam chhuak (Fig. 2c). 2f,g,h).Particle surface inkar surface area a lian chuan solubility a sang zawk a, chuvangin release rate a sang zawk thin.
Dehydrated insulin NP hrang hrangte morphology: (a) Mannitol tel lo lyophilized insulin NPs SEM image; (b) Mannitol hmanga lyophilized insulin NPs SEM image; (c) mannitol tel lo insulin NPs spray-dried SEM image of ; (d) Insulin NPs mannitol hmanga spray-dried te SEM image; (e) mannitol tel lo lyophilized insulin NPs powder thlalak; (f) mannitol nena lyophilized insulin NPs thlalak; ( g) Mannitol tel lo insulin NPs powder spray-a vawt thlalak; (h) mannitol hmanga insulin NPs powder spray-a vawt thlalak.
Freeze-drying lai hian mannitol hian cryoprotectant angin hna a thawk a, NPs chu amorphous form-ah a dah a, ice crystals-in a tihchhiat loh nan a veng bawk19.Chu danglamna chu spray drying-ah hian freezing step a awm lo.Chuvangin he method-ah hian mannitol hi a ngai lo.A nihna takah chuan spray-dried NPs mannitol tel lo chuan a hmaa kan sawi tawh angin NPs finer zawk a pe chhuak a ni.Amaherawhchu, mannitol hian spray-drying process-ah filler angin a la thawk thei a, NPs chu spherical structure20 (Fig. 2d) zawk a pe thei a, hei hian chutiang encapsulated NPs te chu uniform release behavior neih theihna turin a pui a ni.Chu bakah, mannitol awmna freeze-dried leh spray-dried insulin NPs-ah te pawh particle lian thenkhat hmuh theih a ni tih a chiang hle (Fig. 2b,d), chu chu a accumulation vang pawh a ni thei mannitol chu particle core-ah a awm a, chu chu insulin encapsulated nen a inzawm khawm a ni. To.Chitosan layer.Hriat tur chu, he zirchiannaah hian, dehydration hnuah pawh spherical structure chu a awm reng theih nan, mannitol leh chitosan ratio chu 5:1-ah dah a ni a, chutiang chuan filler tam tak chuan NPs vawt te particle size pawh a ti lian thei a ni.
Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy hmangin free insulin, chitosan, chitosan, TPP leh insulin te physical mixture chu a characterized a ni.Dehydrated NP zawng zawng chu FTIR-ATR spectroscopy hmangin characterized vek a ni.Hriat tur pawimawh tak chu encapsulated NPs-ah hian band intensity 1641, 1543 leh 1412 cm-1 hmuh a ni freeze-dried with mannitol and in NPs spray-dried with and without mannitol (Fig. 3).Tun hmaa kan sawi tawh ang khan heng chakna tihpun te hi chitosan, TPP leh insulin inkara cross-linking nen a inzawm a ni.Chitosan leh insulin inkara inzawmna chhui chiannaah chuan insulin-loaded chitosan nanoparticles FTIR spectra-ah chuan chitosan band chu insulin nen a inzawm tlat a, chu chuan a tipung a ni carbonyl intensity (1641 cm-1) leh amine (1543 cm-1) belt.TPP-a tripolyphosphate group-te hi chitosan-a ammonium group-te nen an inzawm a, 1412 cm-1-ah band an siam a ni.
FTIR-ATR spectra hmanga free insulin, chitosan, chitosan/TPP/insulin physical mixture leh NPs te chu hmanraw hrang hrang hmanga tui paih chhuah a ni.
Chubakah, heng result te hi SEM-a kan hmuhte nen a inmil a, chu chuan encapsulated NPs te chu mannitol hmanga spray leh freeze-dried a nih pawhin a awm reng tih a tarlang a, mahse mannitol awm lohnaah chuan spray-drying chauh hian encapsulated particles a siam chhuak a ni.Chu danglamna chu, mannitol tel lova freeze-dried NPs FTIR-ATR spectral result chu physical mixture nen a inang hle a ni of chitosan, TPP, and insulin.He result hian chitosan, TPP leh insulin inkara cross-links chu mannitol tel lo freeze-dried NPs-ah a awm tawh lo tih a tilang a ni.Cryoprotectant tel lova freeze-drying-ah NPs structure chu a chhe vek a, hei hi SEM result-ah hmuh theih a ni (Fig. 2a).Dehydrated insulin NPs morphology leh FTIR result atanga chhut chuan, chauh lyophilized, spray-dried, leh mannitol-free NPs te chu reconstitution experiment atan hman a ni a, dehydration laiin mannitol-free NPs te a chhe vek avangin mannitol-free NPs te hman a ni bawk. sawiho.
Dehydration hi hun rei tak chhunga dahkhawmna atan hman a ni a, formulation dangah siam thar lehna atan hman a ni.Dry NPs te hi dahkhawm hnua an insiam thar theihna hi tablet leh film ang chi formulation hrang hranga hman atan a pawimawh hle.Mannitol awm lohnaah chuan spray-dried insulin NPs te chu reconstitution hnuah tlemte chauh a pung tih kan hmuchhuak.Kawng lehlamah chuan spray-dried leh freeze-dried insulin nanoparticles with mannitol chu a pung nasa hle (Table 1).He ​​zirchiannaa NP zawng zawng recombination hnuah PDI leh EE hi a danglam vak lo (p > 0.05) (Table 1).He ​​result hian a tilang a, particle tam zawk chu redissolving hnuah pawh a awm reng tih a tarlang.Amaherawhchu, mannitol dah belh hian lyophilized leh spray-dried mannitol nanoparticles (Table 1).Chu danglamna chu, mannitol tel lova spray-dried NPs-a insulin load awm zat chu a hma ang bawkin a awm reng a ni (Table 1).
Damdawi pekna atana hman a nih chuan nanoparticles loading hi a pawimawh hle tih chu hriat lar tak a ni.Loding hniam NPs tan chuan therapeutic threshold thleng tur chuan material tam tak a ngai a ni.Amaherawhchu, hetiang NP concentration sang tak viscosity sang hian oral administration leh injectable formulations-ah harsatna leh harsatna a thlen a, a hnuaia mi ang hian 22 .Chu bakah, NPs te hian an ti thei bawk tablet leh viscous biofilms siamna atan pawh hman theih a ni23, 24, chu chuan loading level hniam takah NPs tam tak hman a ngai a, chu chuan tablet lian tak tak leh biofilm thick tak tak, oral application atana hman tlak loh a siam a ni.Chuvangin, insulin load sang tak nei dehydrated NPs te hi duhthusam tak an ni.Kan results chuan mannitol-free spray-dried NPs insulin load sang tak hian tam tak a pe thei tih a tilang heng delivery method dangte tan hian advantage hmuhnawm tak tak a awm.
Dehydrated NP zawng zawng chu thla thum chhung refrigerator-ah dah vek a ni.SEM result atanga a lan dan chuan thla thum chhung dahkhawm chhungin dehydrated NP zawng zawng morphology chu a danglam vak lo (Fig. 4).Tuiah reconstitution hnuah NP zawng zawng hian EE a tlahniam deuh vek a, thla thum chhunga dah chhungin insulin tlemte (~5%) vel an chhuah chhuak vek a ni (Table. 4). 2).Amaherawhchu, nanoparticle zawng zawng zingah chuan a zatve (average particle size) chu a pung a ni.Mannitol tel lova spray-dried NP-te particle size chu 525 nm-ah a pung a, mannitol hmanga spray-dried leh freeze-dried NP-te erawh chu 872 leh 921 nm-ah a pung thung (Table 2).
Thla thum chhunga dah dehydrated insulin NP hrang hrangte morphology: (a) Mannitol hmanga lyophilized insulin NPs SEM image; (b) Mannitol tel lo, spray-a tihthianghlim insulin nanoparticles SEM image; (c) spray-dried insulin NPs mannitol SEM images tel lovin.
Chubakah, reconstituted insulin nanoparticles-ah pawh mannitol hmanga spray-dried leh freeze-dried-ah precipitates hmuh a ni bawk (Fig. S2).Hei hi tuiah particle lian tak tak a innghat tha lo vang a ni thei bawk.A chunga result zawng zawng hian spray drying technique hian insulin nanoparticles te chu dehydration lakah a humhim thei tih leh insulin nanoparticles loading sang tak chu filler emaw, engmah awm lovin hmuh theih a nih thu a tarlang bawk cryoprotectants a ni.
Insulin retention chu pH = 2.5 medium-ah pepsin, trypsin, leh α-chymotrypsin hmangin test a ni a, chu chuan NPs te hian dehydration hnua enzymatic digestion laka an venghim theihna an lantir a ni h in all three enzymatic treatments (Fig. 5a–c).Chu danglamna chu, mannitol hmanga freeze-dried NPs leh mannitol hmanga spray-dried NPs leh mannitol tel lo emaw spray-dried NPs te insulin elimination testing-ah chuan heng NPs te hi enzymatic digestion laka venhimna sang zawk a ni tih hmuhchhuah a ni a, hei hi insulin NPs siam tharte nen a inang hle ( figure 1).5a-c).With the help of nanoparticles in pepsin, trypsin, leh α-chymotrypsin, insulin 50%, 60%, leh 75% aia tam chu darkar 4 chhungin a humhim thei a ni (Fig. 5a–c).He insulin-protective ability hian thisenah insulin tam zawk a luh theihna a tipung thei a ni25.Hetiang result hian spray drying with or without mannitol leh freeze-drying with mannitol can preserve the Dehydration hnua NPs te insulin-protective theihna.
Dehydrated insulin NPs venhimna leh tihchhuah dan: (a) pepsin solution-a insulin venhimna; (b) trypsin solution-a insulin venhimna; (c) α-chymotrypsin solution hmanga insulin venhimna; ( d) pH = 2.5 solution-a dehydrated NPs release dan; (e) pH = 6.6 solution-a dehydrated NPs release dan; (f) pH = 7.0 solution-a dehydrated NPs release dan.
Dry insulin NPs siam thar leh siam tharte chu buffer hrang hrang (pH = 2.5, 6.6, 7.0)-ah 37 °C-ah an incubate a, chu chu pum, duodenum leh upper small intestine-a pH environment simulating-in insulin resistance-a insulin-in nghawng a neih dan enfiah a ni. Environment hrang hranga release behavior.Fragment of the gastrointestinal tract.PH = 2.5-ah chuan insulin-loaded NPs leh resolubilized dry insulin NPs te chuan darkar khat hmasa chhungin initial burst release an nei a, chu chu darkar 5 hnuah slow release an nei leh a (Fig. 5d).A tir lama he rapid release hi protein surface molecules fully desorption rang tak avanga lo awm a nih a rinawm ber immobilized in the internal structure of the particle.PH = 6.5-ah chuan insulin-loaded NPs leh reconstituted dry insulin NPs te chuan 6 h chhungin a chhuak tha leh zawi lo hle a, test solution pH chu NPs-a siam solution nen a inang avangin (Fig. 5e).PH = 7-ah chuan NPs te chu a nghet lo va, darkar hnih chhungin a chhe vek tawh mai mai (Fig. 5e). 5f).Hei hi a chhan chu chitosan deprotonation hi pH sang zawkah a awm a, chu chuan polymer network compact lo zawk a siam a, loaded insulin a chhuah bawk.
Chubakah, mannitol tel lova spray-dried insulin NPs te hian dehydrated NP dangte aiin release profile rang zawk an nei a (Fig. 5d–f).A hmaa kan sawi tawh ang khan reconstituted insulin NPs mannitol tel lova dried te hian particle size tenau ber an lantir a ni.Small particles hian surface area lian zawk a pe a, chuvangin damdawi kaihhnawih tam zawk chu particle surface ah emaw a bulah emaw a awm ang a, chu chuan damdawi release rang zawk a thlen ang
NPs cytotoxicity chu MTT assay hmangin an zirchiang a.Figure S4-a kan hmuh angin dehydrated NPs zawng zawng hian cell viability-ah nghawng lian tham an nei lo tih hmuhchhuah a ni a, concentration 50–500 μg/ml-ah chuan dehydrated NPs zawng zawng hi therapeutic window thleng turin him takin hman theih a ni tih a tilang.
Liver hi insulin hian a physiological functions a thawhna bulpui ber a ni.HepG2 cells hi mihring hepatoma cell line in vitro hepatocyte uptake model atana hman tlanglawn tak a ni.Hetah hian HepG2 cells hmangin freeze-drying leh spray-drying method hmanga NPs dehydrated cellular uptake tehna atan hman a ni.Cellular uptake by confocal laser scanning using flow cytometry leh vision chu darkar engemaw zat free FITC insulin concentration 25 μg/mL-a incubation hnua FITC insulin-loaded NPs siam thar leh dehydrated FITC insulin-loaded NPs insulin concentration inang chiah Quantitative microscopy (CLSM) observation tih a ni.Lyophilized NPs mannitol nei lo chu dehydration laiin tihchhiat a ni a, hei hi evaluate a ni lo test.Insulin-loaded NPs siam thar, mannitol hmanga lyophilized NP, leh spray-dried NPs mannitol nei leh nei lo (Fig. 6a) te chu free NPs aiin a let 4.3, 2.6, 2.4, leh 4.1-in a sang zawk a ni.FITC-insulin group, a hnuaia mi ang hian (Fig. 6b).Hetiang result hian encapsulated insulin hian free insulin aiin cellular uptake-ah a chak zawk a, hei hi zirchiannaa insulin-loaded nanoparticles siam chhuah a lian lo zawk vang a ni ber.
NPs siam thar leh dehydrated NPs nena 4 h incubation hnua HepG2 cell lakluh dan: (a) HepG2 cells ten FITC-insulin an lakluh dan insem dan.(b) Flow cytometry hmanga fluorescence intensities thlirletna geometric mean (n = 3), *P < 0.05 free insulin nena khaikhin chuan.
Chutiang bawkin CLSM images-ah pawh FITC-insulin-loaded NPs siam thar leh FITC-insulin-loaded spray-dried NPs (mannitol tel lo) te FITC fluorescence intensity chu sample dangte aiin a chak zawk hle tih hmuhchhuah a ni bawk (Fig. 6a).Chubakah, mannitol dah belh a nih chuan solution viscosity sang zawk chuan resistance a tisang zawk a ni to cellular uptake, resulting in decreased insulin proliferation.Heng results te hian mannitol-free spray-dried NPs te hian cellular uptake efficiency sang ber an nei tih a tilang a, a chhan chu an particle size chu re-dissolution hnua freeze-dried NPs aiin a tlem zawk a ni.
Chitosan (average molecular weight 100 KDa, 75–85% deacetylated) chu Sigma-Aldrich.(Oakville, Ontario, Canada) atangin lei a ni.Sodium tripolyphosphate (TPP) hi VWR (Radnor, Pennsylvania, USA) atangin lei a ni.He zirchiannaa recombinant human insulin hman chu Fisher Scientific (Waltham, MA, USA) atanga lak a ni.Fluorescein isothiocyanate (FITC)-labeled human insulin leh 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) te chu Sigma-Aldrich.(Oakville, Ontario, Canada) atangin lei a ni.HepG2 cell line hi ATCC (Manassas, Virginia, USA) atanga lak chhuah a ni.Reagent dang zawng zawng hi analytical emaw chromatographic grade a ni.
CS solution 1 mg/ml chu double distilled water (DD water)-a 0.1% acetic acid awmnaah hminin siam rawh.TPP leh insulin solution 1 mg/ml chu DD tui leh 0.1% acetic acid-ah hminin siam rawh.Pre-emulsion chu polytron PCU-2-110 high speed homogenizer (Brinkmann) hmanga siam a ni Ind. Westbury, NY, USA).A buatsaih dan chu hetiang hi a ni: a hmasa berah chuan TPP solution 2ml chu insulin solution 4ml-ah dah a ni a, chu chu 30min chhung hrual a ni a, a inhmeh vek a ni.Chumi hnuah, a mixed solution chu CS solution-ah syringe hmangin high-speed stirring (10,000 rpm) hnuaiah dropwise in dah a ni.A mixture te chu high-speed hnuaiah dah a ni ice bath-ah min 30 chhung stirring (15,000 rpm) a, cross-linked insulin NPs hmuh theih nan pH engemaw takah an siamrem a, insulin NPs te particle size homogenize leh tihtlem nan, probe-type sonicator (UP 200ST, Hielscher Ultrasonics, Teltow, Germany) hmangin ice bath-ah min 30 dang sonicated an ni bawk.
Insulin NPS te chu Z-average diameter, polydispersity index (PDI) leh zeta potential te chu dynamic light scattering (DLS) measurement hmangin Litesizer 500 (Anton Paar, Graz, Austria) hmangin DD tuiah 25°C-a diluting hmangin an test a.Morphology leh size distribution te chu Hitachi H7600 transmission electron microscope (TEM) (TEM) (Hitachi, 1) hmangin an zirchiang a ni. Tokyo, Japan), leh a hnuah Hitachi imaging software (Hitachi, Tokyo, Japan) hmangin images te chu an zirchiang leh a ni.Insulin NPs encapsulation efficiency (EE) leh loading capacity (LC) tehna atan NPs te chu ultrafiltration tube-ah pipet-in molecular weight cut-off 100 kDa-ah dahin 500 xg-ah 30 chhung centrifuged a ni min.Filtrate-a insulin dah loh chu Agilent 1100 Series HPLC system (Agilent, Santa Clara, California, USA) hmangin quantified a ni a, chu chu quaternary pump, autosampler, column heater, leh DAD detector hmanga siam a ni.Insulin chu C18 column (Zorbax, 3.5 μm, 4.6 mm × 150 mm, Agilent, USA) hmangin an zirchiang a ni. and detected at 214 nm.Mobile phase chu acetonitrile leh tui, 0.1% TFA awmna, gradient ratio 10/90 atanga 100/0, minute 10 chhung tlan a ni.Mobile phase chu flow rate 1.0 ml/min-in pump a ni.Column temperature chu 20 °C-ah dah a ni.Equation hmangin EE leh LC percentage chu chhut rawh.(1) leh Eq.(2) a ni.
Insulin NP tihchangtlun nan CS/insulin ratio hrang hrang 2.0 atanga 4.0 inkar test a ni.A siam lai hian CS solution zat hrang hrang dah a ni a, chutih laiin insulin/TPP mixture chu a awm reng bawk.Insulin NPs chu solution zawng zawng (insulin, TPP leh CS).Insulin NPs siam that nan insulin nanoparticles EE leh particle size chu pH value hrang hrang leh CS/insulin mass ratio hrang hrangah an zirchiang a.
Optimized insulin NPs chu aluminum container-ah dahin tissue tightened tape engemaw zat hmanga khuh a ni.Chumi hnuah chuan screwed container-te chu Labconco FreeZone freeze dryer (Labconco, Kansas City, MO, USA)-ah tray dryer dahna hmunah dah a ni.Temperature leh vacuum pressure chu -10 °C, 0.350 Torr-ah a hmasa ber 2 h, leh 0 °C leh 0.120 Torr-ah 24 h chhunga 22 h la awm chu dry insulin NPs hmuh theih nan.
Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Switzerland) hmangin encapsulated insulin siam a ni.Drying parameter thlan chhuah te chu: temperature 100 °C, feed flow 3 L/min, leh gas flow 4 L/min te an ni.
Dehydration hma leh hnua insulin NPs te chu FTIR-ATR spectroscopy hmangin an characterize a.Dehydrated nanoparticles bakah free insulin leh chitosan te chu universal ATR sampling accessory (PerkinElmer, Waltham, Massachusetts, USA) hmanga thuam Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA) hmangin an zirchiang a.Signal averages chu scan 16 atanga 4000-600 cm2 inkar resolution 4 cm2 atanga lak a ni.
Dry insulin NPs te morphology chu Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, USA) hmanga freeze-dried leh spray-dried insulin NPs te SEM image hmanga teh a ni. Parameter hman ber chu voltage 5 keV leh current 30 mA a ni.
Dehydrated insulin NPs zawng zawng chu dd water-ah redissolve vek a ni.Particle size, PDI, EE leh LC te chu a hmaa kan sawi tawh ang bawk khan dehydration hnua an quality tehna hmangin test leh a ni.Anhydroinsulin NPs te stability pawh hi rei tak dah hnua NPs te property test hmangin an teh bawk.He zirchiannaah hian dehydration hnua NPs zawng zawng chu refrigerator-ah vawi thum dah vek a ni thla.Thla thum chhung an dah hnuah NPs te chu morphological particle size, PDI, EE leh LC te test a ni.
Reconstituted NPs 5 mL chu simulated gastric fluid (pH 1.2, 1% pepsin awmna), intestinal fluid (pH 6.8, 1% trypsin awmna) emaw chymotrypsin solution (100 g/mL, phosphate buffer-ah, pH 7.8) awmna 45 mL-ah hmin la, insulin venhimna atana hman tangkai dan tehna atan dehydration.37°C-ah agitation speed 100 rpm-in an incubate a, solution 500 μL chu hun hrang hrangah lakkhawm a ni a, insulin concentration chu HPLC hmangin an teh a ni.
In vitro release behavior chu insulin NPs siam thar leh dehydrated te chu dialysis bag method (molecular weight cut-off 100 kDa, Spectra Por Inc.) hmangin an enfiah a.Dry NPs siam thar leh siam thar te chu fluid pH 2.5, pH 6.6, leh pH 7.0 (0.1 M phosphate-buffered saline, 0.1 M phosphate-buffered saline) ah dialyzed an ni. PBS) hmangin pum, duodenum, leh upper small intestine pH environment simulate turin.Sample zawng zawng chu 37 °C-ah 200 rpm-a tihnghing chhunzawm zelin incubate a ni.5 mL dialysis bag pawnah fluid chu a hnuaia mi ang hian aspirate rawh: 0.5, 1, 2, 3, 4, leh 6 h, leh volume chu fresh-in replenish nghal rawh dialysate.Fluid chhunga insulin contamination chu HPLC hmangin an zirchiang a, nanoparticles atanga insulin release rate chu free insulin release leh total insulin encapsulated in nanoparticles ratio atanga chhut a ni (Equation 3).
Mihring hepatocellular carcinoma cell line HepG2 cells chu 60 mm diameter dish-ah Dulbecco’s Modified Eagle’s Medium (DMEM) hmangin 10% fetal bovine serum, 100 IU/mL penicillin, leh 100 μg/mL streptomycin29 awmna hmangin an tipung a.Cultures chu 37°C, 95% relative-ah dah a ni humidity, leh 5% CO2.Uptake assay atan HepG2 cells chu 1 × 105 cells/ml-in 8-well Nunc Lab-Tek chamber slide system (Thermo Fisher, NY, USA)-ah seeded a ni.Cytotoxicity assay atan chuan 96-well plate (Corning, NY, USA)-ah density 5 × 104 cells/ml-ah seeded a ni.
MTT assay hmang hian insulin NPs30 siam thar leh tui tlakchham tawhte cytotoxicity tehna atan hman a ni.HepG2 cells chu 96-well plate-ah density 5 × 104 cells/mL-ah seeded niin test hmain ni 7 chhung culture a ni.Insulin NPs chu culture medium-ah concentration hrang hrang (50 to 500 μg/mL)-ah diluted a ni a, chutah chuan culture medium-ah diluted a ni administered to cells.Darkar 24 chhung an incubation hnuah cell te chu PBS hmangin vawi 3 silfai a ni a, 0.5 mg/ml MTT awmna medium nen darkar 4 dang incubate leh a ni.Cytotoxicity chu Tecan infinite M200 pro spectrophotometer hmanga yellow tetrazolium MTT to purple formazan 570 nm-a enzymatic reduction tehna hmangin an teh a ni plate chhiartu (Tecan, Männedorf, Switzerland) a ni.
NPs cellular uptake efficiency chu confocal laser scanning microscopy leh flow cytometry analysis hmangin an test a ni.Nunc Lab-Tek chamber slide system well tin chu free FITC-insulin, FITC-insulin-loaded NPs hmangin treat a ni a, dehydrated FITC-insulin NPs 25 μg/mL chu concentration inangah reconstituted a ni a, incubate a ni Darkar 4.Cell te chu PBS hmangin vawi 3 silfai a ni a, 4% paraformaldehyde hmangin fix a ni.Nuclei te chu 4′,6-diamidino-2-phenylindole (DAPI) hmangin stain a ni.Insulin localization chu Olympus FV1000 laser scanning/two-photon confocal microscope (Olympus, Shinjuku City, Tokyo, Japan) hmangin enfiah a ni.Flow atan cytometry analysis hmangin 10 μg/mL free FITC-insulin, FITC-insulin-loaded NPs, leh resolubilized dehydrated FITC-insulin NPs concentration inang chu 96-well plate-ah HepG2 cells seeded-ah dahin darkar 4 chhung incubate a ni .4 h incubation hnuah cells te chu lakchhuah a ni a, vawi 3 silfai a ni Sample khatah FBS.5 × 104 cells chu BD LSR II flow cytometer (BD, Franklin Lakes, New Jersey, United States) hmangin an zirchiang a.
Value zawng zawng hi mean ± standard deviation anga tarlan a ni.Group zawng zawng tehkhin dan chu one-way ANOVA emaw t-test hmangin IBM SPSS Statistics 26 for Mac (IBM, Endicott, New York, USA) hmanga teh a ni a, p < 0.05 chu statistically significant anga ngaih a ni.
He zirchianna hian spray drying hian cross-linked chitosan/TPP/insulin nanoparticles te chu bulking agents emaw cryoprotectants capacity leh load capacity sang zawk hmanga standard freeze-drying methods nena khaikhin chuan reconstitution tha zawk neiin a flexibility leh a tihzia a tilang a ni.The optimized insulin nanoparticles yielded an average particle size of 318 nm and an encapsulation efficiency of 99.4%.SEM and FTIR results after dehydration showed that the spherical structure was maintained only in spray-dried NPs with and without mannitol and lyophilized with mannitol, but lyophilized NPs without mannitol were decomposed during dehydration.In the reconstitution ability test, insulin nanoparticles spray-dried without mannitol showed the smallest mean particle size and the highest loading upon reconstitution.Heng dehydrated NP zawng zawngte release behaviors hian pH = 2.5 leh pH = 7 solution-ah rang takin an chhuak tih a tarlang a, pH = 6.5 solution-ah pawh an stable hle tih a tarlang bawk.Redissolved dehydrated NPs dangte nena khaikhin chuan mannitol tel lova spray-dried NPs te hian release rang ber an nei a ni.He result hi cellular uptake assay-a hmuh tawh nen a inmil a, as spray-dried NPs in the absence of mannitols almost completely maintained the cellular uptake efficiency of freshly prepared NPs.Heng results te hian mannitol-free spray drying hmanga siam dry insulin nanoparticles te hi anhydrous dosage form dang, oral tablet emaw bioadhesive film emaw-a siam belh zelna atan a tha ber tih a tilang.
Intellectual property chungchangah hian tun zirchianna neih chhunga dataset siam leh/ emaw thlirlet te hi mipui hriat theih a ni lo va, mahse a ziaktu hrang hrangte hnen atangin dilna dik tak a awm chuan hmuh theih a ni.
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