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Ti nanoparticles ta insulin (NPs) letingana nhlayo yale henhla ya loading ti kumile switirhisiwa swohambana hambana eka swivumbeko swohambana hambana swa mpimo.Ntirho lowu wu kongomisa eka ku kambela mbuyelo wa maendlelo ya freeze-drying na spray-drying eka xivumbeko xa ti insulin-loaded chitosan nanoparticles, na kumbe handle ka mannitol tani hi cryoprotectant.Hi tlhele hi kambela khwalithi ya ti nanoparticles leti hiku ti dissolving nakambe.Emahlweni ka sweswo dehydration, vukulu bya ti particle ta chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles byi antswisiwile kuva 318 nm, PDI ayiri 0.18, encapsulation efficiency ayiri 99.4%, naswona ku layicha akuri 25.01%.Endzhaku ka ku vumbiwa nakambe, ti nanoparticles hinkwato, handle ka leti humesiweke hi ndlela ya freeze-drying handle ko tirhisiwa ya mannitol, yi hlayisile xivumbeko xa yona xa swiphemuphemu swa xirhendzevutani.Loko ku pimanisiwa na ti nanoparticles leti nga na mannitol leti nga riki na mati hi ku fafazela, ti nanoparticles leti omisiweke hi ku fafazela leti nga riki na mannitol ti tlhele ti kombisa mpimo wutsongo swinene wa swiphemuphemu (376 nm) na nhlayo ya le henhla ya ku layicha (25.02%) na mpimo wa encapsulation lowu fanaka (98.7%) na PDI (0.20) hi ku omisa kumbe freeze-drying techniques.Ti nanoparticles leti omisiweke hiku fafazela handle ka mannitol titlhele ti endla leswaku kuva na ku humesiwa hiku hatlisa swinene ka insulin xikan’we naku tirha kahle swinene ka cellular uptake.Ntirho lowu wukombisa leswaku ku omisa hiku fafazela swinga susa mati eka ti nanoparticles ta insulin handle ka xilaveko xa cryoprotectants loko ku pimanyisiwa na tindlela ta ntolovelo ta freeze drying, leswi endlaka leswaku kuva na vuswikoti lebyikulu byo layicha, swilaveko swale hansi swa additive xikan’we naku tirha swi durha ku vuyeriwa lokukulu.
Kusukela loko yi tshuburiwile hi 19221,2,3, insulin na malunghiselelo ya yona ya mirhi swi ponise vutomi bya vavabyi lava nga na vuvabyi bya chukele bya muxaka wo sungula (T1DM) na vuvabyi bya chukele bya muxaka wa vumbirhi (T1DM).Hambiswiritano, hikwalaho ka swivumbeko swa yona tanihi protein ya ntiko wa molekhuli ya le henhla, insulin yi hlengeletiwa hi ku olova, yi tshoveka hi ti-enzyme ta proteolytic, naswona yi herisiwa hi ku hundza ko sungula effect.Vanhu lava kumiweke va ri na vuvabyi bya chukele bya muxaka wo sungula va lava ku cheriwa ka insulin vutomi bya vona hinkwabyo.Vavabyi vo tala lava eku sunguleni va kumiweke va ri na vuvabyi bya chukele bya muxaka wa vumbirhi na vona va lava ku cheriwa ka insulin nkarhi wo leha.Ku cheriwa ka insulin siku na siku i xihlovo lexikulu xa ku vava ka siku na siku na ku nga tsaki eka vanhu lava, na vuyelo byo biha eka rihanyo ra miehleketo.Hikokwalaho ka sweswo, swivumbeko swin’wana swa ku nyikiwa ka insulin leswi vangaka ku nga tsaki kutsongo, swo tano tanihi vulawuri bya insulin ya nomu, ti kambisisiwa swinene5 tanihileswi ti nga na vuswikoti byo vuyisela khwalithi ya vutomi bya kwalomu ka 5 wa tibiliyoni ta vanhu lava nga na vuvabyi bya chukele emisaveni hinkwayo.
Thekinoloji ya ti nanoparticle yi nyikile nhluvuko lowukulu eka matshalatshala yo teka insulin ya nomu4,6,7.Yin’we leyi hi ndlela leyinene yi hlanganisaka no sirhelela insulin eka ku onhaka ku yisiwa loku kongomisiweke eka tindhawu to karhi ta miri.Hambiswiritano, ku tirhisiwa ka swivumbeko swa nanoparticle swi na swipimelo swo hlayanyana, ngopfungopfu hikwalaho ka timhaka ta ku tshamiseka ka ku yimisiwa ka swiphemuphemu.Ku hlengeletiwa kun’wana ku nga humelela hi nkarhi wa ku hlayisiwa, leswi hungutaka ku kumeka ka bioavailability ya insulin-loaded nanoparticles8.Ku engetela kwalaho, ku tshamiseka ka tikhemikhali ka matrix ya polymer ya ti nanoparticles na insulin na swona swi fanele ku tekeriwa enhlokweni ku tiyisisa ku tshamiseka ka ti nanoparticles ta insulin (NPs).Sweswi, thekinoloji ya freeze-drying i mpimanyeto wa nsuku wo tumbuluxa ti NP leti tiyeke loko ku ri karhi ku sivela ku cinca loku nga lavekiki hi nkarhi wa vuhlayiselo9.
Hambiswiritano, freeze-drying swilava ku engeteriwa ka ti cryoprotectants ku sivela xivumbeko xa spherical xa NPs kuva xi khumbeka hi ntshikelelo wa michini wa ti ice crystals.Leswi swihunguta swinene ku layicha ka ti nanoparticles ta insulin endzhaku ka lyophilization, tani hileswi cryoprotectant yi tekaka vunyingi bya ratio ya ntiko.Hikokwalaho, ti insulin NPs leti humelerisiwaka titala ku kumeka tinga ringanelanga eka ku endliwa ka swivumbeko swa powder yo oma, kufana na ti tablets ta nomu na ti films ta nomu, hikokwalaho ka xilaveko xa nhlayo leyikulu ya ti nanoparticles to oma kuva ku fikeleriwa fasitere ra vutshunguri ra insulin.
Ku omisiwa hi xifafazeri i endlelo ra xikalo xa indasitiri leri tivekaka no ka ri nga durhi ro humelerisa ti powder to oma kusuka eka swiphemu swa mati eka indasitiri ya mirhi10,11.Vulawuri eka endlelo ro tumbuluxa swiphemuphemu swi pfumelela ku encapsulation leyi faneleke ya ti bioactive compounds to hlaya 12, 13 .Ku tlula kwalaho, yi hundzuke thekiniki leyi tirhaka ya ku lulamisela ti protein leti pfaleriweke ku tirhisiwa hi nomu.During ku omisiwa hi ku fafazela, mati ya khuluka hiku hatlisa swinene, leswi pfunetaka ku hlayisa mahiselo ya particle core yale hansi11,14, leswi endlaka leswaku ku tirhisiwa ka wona kuva ku encapsulate swiphemu leswi twaka ku hisa.Loko kungase omisiwa hiku fafazela, swilo swo peta swifanele kuva swifana kahle na solution leyingana swiaki leswi pfaleriweke11,14.Kuhambana na freeze-drying, homogenization before encapsulation eka spray-drying yi antswisa encapsulation efficiency hinkarhi wa dehydration.Tanihi leswi endlelo ra spray-drying encapsulation ringa laviki ti cryoprotectants, spray-drying yinga tirhisiwa ku humelerisa ti NPs leti omisiweke letingana nhlayo yale henhla ya loading.
Dyondzo leyi yi vika vuhumelerisi bya ti insulin-loaded NPs hiku cross-linking ya chitosan na sodium tripolyphosphate hiku tirhisa ndlela ya ion gel.Ion gelation i ndlela yo lulamisela leyi pfumelelaka vuhumelerisi bya ti nanoparticles hiku tirhisa electrostatic interactions exikarhi ka tinxaka timbirhi kumbe kutlula ta ionic ehansi ka swiyimo swokarhi.Havumbirhi bya ti freeze-drying na spray-drying techniques titirhisiwile ku susa mati eka chitosan/sodium leyi antswisiweke tripolyphosphate/insulin cross-linked nanoparticles.Endzhaku ka dehydration, morphology ya tona yi kamberiwile hi SEM.Vuswikoti bya tona byo hlanganisa nakambe byi kamberiwile hi ku pima ku hangalasiwa ka vukulu bya tona, surface charge, PDI, encapsulation efficiency, na loading content.Khwalithi ya resolubilized nanoparticles leyi humesiweke hi tindlela to hambana ta dehydration yi tlhele yi kamberiwa hi ku pimanisa insulin ya tona ya nsirhelelo, ku humesa, mahanyelo ya ku humesa, na ku tirha kahle ka ku amukeriwa ka tisele.
pH ya solution leyi pfanganisiweke xikan’we na ratio ya chitosan na insulin i swilo swimbirhi swa nkoka leswi khumbhaka vukulu bya ti particle na encapsulation efficiency (EE) ya ti NPs to hetelela, tani hileswi swi khumbaka hiku kongoma endlelo ra ionotropic gelation.PH ya solution leyi pfanganisiweke yi kombisiwile kuva yi fambelanisiwa swinene na vukulu bya ti particle xikan’we na encapsulation efficiency (Fig. 1a).Hilaha swi kombisiweke hakona eka Fig. 1a, tani hileswi pH yi tlakukeke kusuka eka 4.0 kuya eka 6.0, vukulu bya ti particle bya xikarhi (nm) byi hungutekile naswona EE yi tlakukile swinene, kasi loko pH yi tlakuka kuya eka 6.5, vukulu bya ti particle bya xikarhi byi sungurile ku tlakuka naswona EE yitshame yinga cinci.Loko ratio ya chitosan kuya eka insulin yikula, vukulu bya ti particle bya xikarhi na byona bya tlakuka.Ku tlula kwalaho, akuri hava ku cinca eka EE loku voniweke loko ti nanoparticles ti lulamisiwile eka mass ratio ya chitosan/insulin yale henhla kutlula 2.5:1 (w/w) (Fig. 1b).Hikokwalaho, swiyimo swa kahle swaku lulamisela eka ndzavisiso lowu (pH 6.0, chitosan/insulin mass ratio ya 2.5:1) switirhisiwile ku lulamisa ti nanoparticles leti layichiweke hi insulin eka dyondzo yin’wana.Ehansi ka xiyimo lexi xo lulamisela, vukulu bya swiphemuphemu swa xikarhi swa ti nanoparticles swi antswisiwile kuva 318 nm (Fig. 1c), PDI a yi ri 0.18, embedding efficiency a yi ri 99.4%, zeta potential a yi ri 9.8 mv, naswona insulin loading a yi ri 25.01% (m/m ).Hi ku ya hi mbuyelo wa transmission electron microscopy (TEM), ti nanoparticles leti antswisiweke a ti ri roughly spherical na discrete na vukulu lebyi fanaka (Fig. 1d).
Ku antswisiwa ka parameter ya ti nanoparticles ta insulin: (a) mbuyelo wa pH eka diameter yale xikarhi na encapsulation efficiency (EE) ya ti nanoparticles ta insulin (leti lulamisiweke eka 5:1 mass ratio ya chitosan na insulin); (b) chitosan na Nkucetelo wa mass ratio ya insulin eka diameter ya le xikarhi na encapsulation efficiency (EE) ya insulin NPs (leyi lulamisiweke eka pH 6); (c) ku hangalasiwa ka vukulu bya swiphemuphemu swa ti-nanoparticle ta insulin leti antswisiweke; (d) TEM micrograph ya ti-NP ta insulin leti antswisiweke .
Switiveka kahle leswaku chitosan i polyelectrolyte yo tsana leyingana pKa ya 6.5.Yi na charge ya kahle eka acidic media hikuva ntlawa wa yona lowukulu wa amino wu protonated hi hydrogen ions15.Hikokwalaho, yitala kutirhisiwa tani hi carrier ku encapsulate negatively charged macromolecules.Eka ndzavisiso lowu, chitosan yitirhisiwile ku encapsulate insulin hi isoelectric point ya 5.3.Tanihi leswi chitosan yi tirhisiwaka tanihi nchumu wo peta, hi ku engeteleka ka mpimo wa yona, ku enta ka leyara ya le handle ya ti nanoparticles ku tlakuka hi ku fambisana, leswi endlaka leswaku ku va na sayizi leyikulu ya swiphemuphemu swa xikarhi.Ku engetela kwalaho, swiyimo swa le henhla swa chitosan swi nga pfalela insulin yo tala.Eka xiyimo xa hina, EE a yi ri ehenhla swinene loko mpimo wa chitosan na insulin wu fika eka 2.5:1, naswona a ku ri hava ku cinca lokukulu eka EE loko mpimo wu tshama wu ri karhi wu andza.
Handle ka ratio ya chitosan na insulin, pH yitlhele yi tlanga xiave xa nkoka eka ku lulamisiwa ka NPs.Gan et al. 17 va dyondze hi mbuyelo wa pH eka vukulu bya ti particle ta chitosan nanoparticles.Va kume ku hunguteka loku yaka emahlweni ka vukulu bya ti particle ku kondza pH yi fika eka 6.0, naswona ku tlakuka lokukulu ka vukulu bya ti particle ku voniwe eka pH > 6.0, leswi fambelanaka na leswi hi swi xiyeke.Xiendlakalo lexi xi vangiwa hileswi hi ku engeteleka ka pH, molecule ya insulin yi kuma charge ya le henhla yo biha, xisweswo, yi tsakela electrostatic interactions na chitosan/sodium tripolyphosphate (TPP) complex, leswi endlaka leswaku kuva na vukulu bya ti particle letintsongo xikan’we na EE yale henhla .Hambiswiritano, loko pH yi lulamisiwile kuya eka 6.5, mintlawa ya amino eka chitosan yi deprotonated, leswinga endla leswaku kuva na chitosan folding.Xisweswo, pH yale henhla yi endla leswaku kuva na ku hlangana kutsongo ka ti amino ions eka TPP na insulin, leswi endlaka leswaku kuva na cross-linking yale hansi, avhareji yo hetelela leyikulu vukulu bya swiphemuphemu na EE ya le hansi.
Nxopaxopo wa swivumbeko swa xivumbeko xa ti NP leti omisiweke hi freeze na leti omisiweke hi ku fafazela swi nga kongomisa ku hlawuriwa ka tithekiniki to antswa ta ku pfumaleka ka mati na ku endla powder.Ndlela leyi tsakeriwaka yi fanele ku nyika ku tshamiseka ka murhi, xivumbeko xa swiphemuphemu leswi fanaka, ku layicha ka mirhi lokukulu na ku n’oka kahle eka solution yo sungula.Eka dyondzo leyi, ku pimanisa tindlela letimbirhi ku antswa, ti insulin NPs leti nga na kumbe leti nga riki na 1% mannitol ti tirhisiwile hi nkarhi wa ku pfumaleka ka mati.Mannitol yi tirhisiwa tanihi a bulking agent kumbe cryoprotectant eka swivumbeko swohambana hambana swa dry powder for freeze drying na spray drying.Eka ti lyophilized insulin nanoparticles letinga hava mannitol, tani hileswi swikombisiweke eka Figure 2a, xivumbeko xa powder lexinga na ti porous swinene lexinga na swiphemu leswikulu, leswinga tshamisekangiki na rough swivoniwe ehansi ka scanning electron microscopy (SEM).Few discrete particles swikumiwile eka powder endzhaku ka dehydration (Fig. 2e).These results indicated that most NPs were decomposed during freeze-drying without any cryoprotectant.For freeze-dried and spray-dried insulin nanoparticles containing 1% mannitol, spherical nanoparticles with smooth surfaces were observed (Fig. 2b,d,f,h).Insulin nanoparticles spray-dried without mannitol remained spherical kambe swi rhurhumerile ehenhla (Fig. 2c).Spherical na wrinkled surfaces swi ya emahlweni swi xopaxopiwa eka mahanyelo yo humesa na swikambelo swa ku amukeriwa ka tisele laha hansi.Hi ku ya hi ku languteka loku vonakaka ka ti NP leti omisiweke, havumbirhi bya ti NP leti omisiweke hi ku fafazela leti nga riki na mannitol na ti NP leti omisiweke hi freeze na ku fafazeriwa hi mannitol ti humese ti powder ta kahle ta NPs (Fig. 2c). 2f,g,h).Loko ndzhawu yale henhla exikarhi ka swiphemu swa swiphemuphemu yiva yikulu, ku olova ka yona kuva e henhla naswona hikokwalaho ka sweswo nhlayo yale henhla ya ku humesiwa.
Xivumbeko xa ti-NP to hambana ta insulin leti nga riki na mati: (a) Xifaniso xa SEM xa ti-NP ta insulin leti lyophilized leti nga riki na mannitol; (b) Xifaniso xa SEM xa ti-insulin NP leti lyophilized na mannitol; (c) ti-insulin NP leti omisiweke hi ku fafazela handle ka xifaniso xa manitol SEM xa ; (d) Xifaniso xa SEM xa ti-insulin NP leti omisiweke hi ku fafazeriwa hi mannitol; (e) xifaniso xa lyophilized insulin NPs powder leyi nga riki na mannitol; (f) xifaniso xa ti-insulin NP leti lyophilized na mannitol; ( g) Xifaniso xa phoyizeni ya insulin NPs leyi omisiweke hi ku fafazela leyi nga riki na mannitol; (h) xifaniso xa phoyizeni ya insulin NPs leyi omisiweke hi ku fafazela na mannitol.
Hi nkarhi wa freeze-drying, mannitol yi tirha tani hi cryoprotectant, yi hlayisa ti NPs eka xivumbeko xa amorphous xikan’we naku sivela ku onhaka hi ti ice crystals19.Kuhambana na sweswo, akuri na goza ro freezing hi nkarhi wa freeze drying.Hikokwalaho mannitol ayi laveki eka ndlela leyi.Entiyisweni, ti NPs leti omisiweke hiku fafazela handle ka mannitol ti humese ti NPs letinene tani hileswi swinga hlamuseriwa khale.Hambiswiritano, akuri na goza ro titimela hi nkarhi wa ku omisiwa hi xifafazeri.Hikokwalaho, ti NPs leti omisiweke hiku fafazela letinga hava mannitol ti humese ti NPs to saseka tani hileswi swinga hlamuseriwa khale.Hambiswiritano, akuri na goza ro titimela hi nkarhi wa ku omisiwa hi xifafazeri. mannitol ya ha tirha tani hi filler eka endlelo ro fafazela hi ku fafazela ku nyika ti NPs xivumbeko xa xirhendzevutani20 (Fig. 2d), leswi pfunetaka ku kuma mahanyelo yo fana ya ku humesiwa ya ti NPs to tano leti pfaleriweke.Ku engetela kwalaho, swi le rivaleni leswaku swiphemuphemu swin’wana leswikulu swi nga voniwa eka ti insulin NP leti omisiweke hi freeze na leti omisiweke hi ku fafazela leti nga na mannitol (Fig. 2b,d), leswi nga ha vaka swi vangiwa hi ku hlengeletiwa ka mannitol eka xiphemu xa swiphemu swin’we na insulin leyi pfaleriweke. To.Chitosan layer.Swifanele ku xiya leswaku eka ndzavisiso lowu, kutiyisisa leswaku xivumbeko xa xirhendzevutani xitshama xiri tano endzhaku ka ku pfumaleka ka mati, ratio ya mannitol na chitosan yi hlayisiwa eka 5:1, leswaku nhlayo leyikulu ya filler yinga tlhela yi kurisa vukulu bya ti particle ta ti NPs leti omisiweke.
Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy yi hlawulekile eka nkatsakanyo wa miri wa insulin ya mahala, chitosan, chitosan, TPP na insulin.Ti NP hinkwato letinga na mati ti tiveka hiku tirhisa FTIR-ATR spectroscopy.Lexi xiyekaka, matimba ya band ya 1641, 1543 na 1412 cm-1 ya voniwe eka ti NPs letinga encapsulated freeze-dried na mannitol na le ka NPs spray-dried na na handle ka mannitol (Fig. 3).Hilaha swi vikiweke hakona khale, ku tlakuka loku ka matimba ku fambelanisiwile na cross-linking exikarhi ka chitosan, TPP na insulin.Vulavisisi bya ku tirhisana exikarhi ka chitosan na insulin byi kombisile leswaku eka ti FTIR spectra ta ti nanoparticles ta chitosan leti layichiweke hi insulin, band ya chitosan yi hlangana na ya insulin, eku engeteleni ka carbonyl intensity (1641 cm-1) na amine (1543 cm-1) belt.Mintlawa ya tripolyphosphate ya TPP yi hlanganisiwa na mintlawa ya ammonium eka chitosan, yi endla band eka 1412 cm-1.
FTIR-ATR spectra ya insulin ya mahala, chitosan, swihlanganisi swa nyama swa chitosan/TPP/insulin na NPs leyi susiweke mati hi tindlela to hambana.
Kuya emahlweni, mbuyelo lowu wu fambisana na lowu kombisiweke eka SEM, leswi kombiseke leswaku ti NPs letinga encapsulated titshame tirikarhi ti onhaka loko ti fafazeriwa xikan’we naku omisiwa hi freeze hi mannitol, kambe loko kungari na mannitol, i ntsena ku fafazeriwa hiku omisiwa loku humeleriseke ti encapsulated particles.Kuhambana na sweswo, mbuyelo wa FTIR-ATR spectral wa ti NPs letinga omisiwa hi freeze handle ka mannitol aswifana swinene na nkatsakanyo wa xiviri ya chitosan, TPP, na insulin.Mbuyelo lowu wu kombisa leswaku cross-links exikarhi ka chitosan, TPP na insulin a ya ha ri kona eka ti NPs leti omisiweke hi freeze handle ka mannitol.Xivumbeko xa NPs xi onhiwile hi nkarhi wa freeze-drying handle ka cryoprotectant, leswi nga voniwaka eka mbuyelo wa SEM (Fig. 2a).Hi ku ya hi morphology na mbuyelo wa FTIR wa ti NPs ta insulin leyi nga riki na mati, ntsena ti lyophilized, spray-dried, na ti mannitol-free NPs titirhisiwile eka swikambelo swa reconstitution na ti mannitol-free NPs hikokwalaho ka ku bola ka ti mannitol-free NPs hinkarhi wa ku pfumaleka ka mati. kanela.
Ku pfumaleka ka mati emirini switirhisiwa eka vuhlayiselo bya nkarhi woleha xikan’we naku lulamisiwa nakambe eka swivumbeko swin’wana.Vuswikoti bya ti NPs leti omeke kuva ti vumbiwa nakambe endzhaku ka nhlayiso i swa nkoka swinene eka kutirhisiwa ka tona eka swivumbeko swohambana hambana swofana na ti tablets nati films.Hi xiye leswaku vukulu bya xikarhi bya ti particle ta ti insulin NPs leti omisiweke hiku fafazela loko kungari na mannitol byi tlakuke nyana ntsena endzhaku ka ku vumbiwa nakambe.Hi tlhelo rin’wana, vukulu bya ti particles ta leswi fafazeriweke na freeze-dried insulin nanoparticles with mannitol yi tlakukile swinene (Table 1).PDI na EE a swi cincanga ngopfu (p > 0.05) endzhaku ka ku hlanganisiwa nakambe ka ti NP hinkwato eka ndzavisiso lowu (Table 1).Mbuyelo lowu wu kombisa leswaku vunyingi bya swiphemuphemu swi tshamile swi nga onhakanga endzhaku ko tlhela swi n’oka.Hambiswiritano, ku engeteriwa ka mannitol swi endle leswaku ku hunguteka swinene ku layicha ka insulin ka lyophilized na spray-dried mannitol nanoparticles (Table 1).Kuhambana na sweswo, nhlayo ya ndzhwalo wa insulin ya ti NPs leti omisiweke hiku fafazela handle ka mannitol yi tshamile yifana na khale (Table 1).
Swi tiveka kahle leswaku ku layicha ka ti nanoparticles i swa nkoka swinene loko switirhisiwa eka swikongomelo swo yisa mirhi.Eka ti NP leti nga na ku layicha ka le hansi, nhlayo leyikulu swinene ya swilo swa laveka ku fikelela threshold ya vutshunguri.Hambiswiritano, viscosity yale henhla ya nhlayo yale henhla ya NP yo tano yi yisa eka ku nga olovi na ku tika eka vulawuri bya nomu na ti formulations leti cheriwaka, hiku landzelelana 22 .Ku engetela, ti NPs tinga swi tlhela swi tirhisiwa ku endla tiphilisi na ti biofilms ta viscous23, 24, leswi lavaka ku tirhisiwa ka nhlayo leyikulu ya ti NP eka swiyimo swa le hansi swa ku layicha, leswi endlaka leswaku ku va na tiphilisi letikulu na ti biofilms to tiya leti nga ringanelangiki ku tirhisiwa hi nomu.Hikokwalaho, ti NP leti nga riki na mati leti nga na ndzhwalo wa le henhla wa insulin ta laveka swinene.Mimbuyelo ya hina yi ringanyeta leswaku ndzhwalo wa le henhla wa insulin wa ti NP leti omisiweke hi ku fafazela handle ka mannitol wu nga nyika swo tala swipfuno leswi kokaka rinoko eka tindlela leti tin’wana to rhumela.
Ti NP hinkwato letinga na mati ti hlayisiwile efrijini kuringana tinhweti tinharhu.Mbuyelo wa SEM wu kombisile leswaku mofoloji ya ti NP hinkwato letinga na mati ayi cincanga ngopfu eka nkarhi wa vuhlayiselo bya tinhweti tinharhu (Fig. 4).Endzhaku ko vumbiwa nakambe ematini, ti NP hinkwato ti kombisile kuhunguteka nyana eka EE naswona ti humesile kwalomu ka nhlayo yitsongo (~5%) ya insulin eka nkarhi wa vuhlayiselo bya tinhweti tinharhu (Table 2).Hambiswiritano, vukulu bya ti particle ta xikarhi bya ti nanoparticles hinkwato byi tlakukile.Vukulu bya ti particle ta ti NPs leti omisiweke hiku fafazela handle ka mannitol byi tlakuke kuya eka 525 nm, kasi bya ti NPs leti omisiweke hiku fafazela na leti omisiweke hi freeze letingana mannitol byi tlakuke kuya eka 872 na 921 nm, hiku landzelelana (Table 2).
Xivumbeko xa ti-NP to hambana ta insulin leti nga riki na mati leti hlayisiweke ku ringana tin’hweti tinharhu: (a) Xifaniso xa SEM xa ti-NP ta insulin leti lyophilized leti nga na mannitol; (b) Xifaniso xa SEM xa ti-nanoparticle ta insulin leti omisiweke hi ku fafazela handle ka mannitol; (c) handle ka swifaniso swa mannitol SEM swa ti-insulin NP leti omisiweke hi ku fafazela.
Kuya emahlweni, ti precipitates ti voniwe eka ti reconstituted insulin nanoparticles leti fafazeriweke hi mannitol naswona ti omisiwa hi freeze (Fig. S2).Leswi swinga vangiwa hi ti particles letikulu letinga yimiki kahle ematini.Mimbuyelo hinkwayo leyi nga laha henhla yi kombisa leswaku thekiniki yo fafazela ti nga sirhelela ti nanoparticles ta insulin eka ku pfumaleka ka mati na leswaku ku layicha lokukulu ka ti nanoparticles ta insulin kunga kumiwa handle ka ti fillers kumbe swisirhelelo swa cryoprotectant.
Ku khomiwa ka insulin ku kamberiwile eka pH = 2.5 medium hi pepsin, trypsin, na α-chymotrypsin ku kombisa vuswikoti byo sirhelela bya ti NP eka ku gayela ka enzymatic endzhaku ka ku pfumaleka ka mati.Ku khomiwa ka insulin ya ti NP leti nga riki na mati ku pimanisiwile na ya ti NP leti nga ha ku lulamisiwaka, naswona insulin ya mahala yi tirhisiwile tanihi vulawuri byo biha.Eka ndzavisiso lowu, insulin ya mahala yi kombisile ku herisiwa ka insulin hi xihatla endzeni ka 4 h eka vutshunguri hinkwabyo byinharhu bya enzymatic (Fig. 5a–c).Ku hambana na sweswo, ku kamberiwa ka ku herisiwa ka insulin ka ti NP leti omisiweke hi xirhami hi mannitol na ti NP leti omisiweke hi xifafazeri na kumbe handle ka mannitol swi kombisile nsirhelelo wa le henhla swinene wa ti NP leti eka ku gayeriwa ka enzymatic, leswi a swi fana na swa ti NP ta insulin leti ha ku lulamisiwaka ( xifaniso 1).5a-c).Hi ku pfuniwa hi ti nanoparticles eka pepsin,. trypsin, na α-chymotrypsin, kutlula 50%, 60%, na 75% wa insulin swinga sirheleriwa eka 4 h, hiku landzelelana (Fig. 5a–c).Vuswikoti lebyi byo sirhelela insulin byi nga engetela nkarhi wa ku amukeriwa ka insulin yale henhla engatini25.Mimbuyelo leyi yi ringanyeta leswaku ku omisiwa hi xifafazeri na kumbe handle ka mannitol na ku omisiwa ka freeze hi mannitol swinga hlayisa vuswikoti byo sirhelela insulin bya ti-NP endzhaku ka ku pfumaleka ka mati emirini.
Mahanyelo ya nsirhelelo na ku humesiwa ka ti-NP ta insulin leti nga riki na mati: (a) nsirhelelo wa insulin eka xitofu xa pepsin; (b) nsirhelelo wa insulin eka xitofu xa trypsin; (c) nsirhelelo wa insulin hi α-chymotrypsin solution; ( d) Mahanyelo ya ku humesiwa ka ti NP leti nga riki na mati eka pH = 2.5 solution; (e) mahanyelo ya ku humesiwa ka ti NP leti nga riki na mati eka pH = 6.6 solution; (f) mahanyelo ya ku humesiwa ka ti NP leti nga riki na mati eka pH = 7.0 solution.
Ti NP ta insulin yo oma letinga lulamisiwa naku vumbiwa nakambe ti pfaleriwile eka ti buffers to hambana hambana (pH = 2.5, 6.6, 7.0) eka 37 °C, ku tekelela ndzhawu ya pH ya khwiri, duodenum, na marhumbu yale henhla yale henhla, ku kambela mbuyelo wa insulin eka ku lwisana na insulin. Mahanyelo yo humesa eka tindzhawu to hambana.Xiphemu xa xirho xa le ndzeni ka khwiri.Eka pH = 2.5, ti NP leti rhwaleke insulin na ti NP ta insulin yo oma leti resolubilized ti kombisile ku humesiwa ko sungula ka ku buluka eka awara yin’we yo sungula, ku landzela ku humesiwa hi ku nonoka eka tiawara ta 5 leti landzelaka (Fig. 5d).Ku humesiwa loku hi xihatla eku sunguleni swi nga endleka ngopfu ku va mbuyelo wa ku susiwa hi xihatla ka timolekhuli ta le henhla ka tiphrotheyini leti nga riki hi ku helela immobilized in the internal structure of the particle.Eka pH = 6.5, ti NPs leti layichiweke hi insulin na ti insulin NPs leti omeke leti vumbiweke nakambe ti kombisile ku humesiwa loku rhetaka no nonoka eka 6 h, tani hileswi pH ya solution ya xikambelo a yi fana na ya solution leyi lunghiseleriweke hi NPs (Fig. 5e).Eka pH = 7, ti NPs a ti nga tshamisekanga naswona ti lava ku bola hi ku helela eka tiawara timbirhi to sungula (Fig. 5f).Leswi swivangiwa hikuva deprotonation ya chitosan yihumelela eka pH yale henhla, leswi endlaka leswaku kuva na network ya polymer leyinga compact ngopfu xikan’we naku humesiwa ka insulin leyi layichiweke.
Ku tlula kwalaho, ti insulin NPs leti omisiweke hi ku fafazeriwa handle ka mannitol ti kombisile xivumbeko xo hatlisa xo humesiwa ku tlula ti NP tin’wana leti nga riki na mati (Fig. 5d–f).Hilaha swi hlamuseriweke hakona khale, ti insulin NPs leti vumbiweke nakambe leti omisiweke handle ka mannitol ti kombisile vukulu bya swiphemuphemu leswintsongo swinene.Swiphemu leswintsongo swi nyika ndhawu leyikulu ya le henhla, hikwalaho vunyingi bya murhi lowu faneleke wu ta va eka kumbe ekusuhi na vuandlalo bya murhi26.
Cytotoxicity ya NPs yi lavisisiwile hi MTT assay.Hilaha swi kombisiweke hakona eka Xifaniso xa S4, ti NP hinkwato leti nga riki na mati ti kumiwile ti nga ri na mbuyelo lowukulu eka ku hanya ka tisele eka nhlayo ya 50–500 μg/ml, leswi ringanyetaka leswaku ti NP hinkwato leti nga riki na mati ti nga tirhisiwa hi ku hlayiseka ku fikelela fasitere ra vutshunguri.
Xivindzi i xirho lexikulu lexi insulin yi tirhisaka mintirho ya yona ya physiological hi xona.Tisele ta HepG2 i layini ya tisele ta hepatoma ta munhu leti talaka ku tirhisiwa tanihi modele wa ku amukeriwa ka hepatocyte in vitro.Laha, tisele ta HepG2 ti tirhisiwile ku kambela ku amukeriwa ka tisele ka ti NP leti nga riki na mati hi ku tirhisa tindlela ta freeze-drying na spray-drying.Ku amukeriwa ka tisele hi ku tirhisa confocal laser scanning hi ku tirhisa ku khuluka cytometry na ku vona endzhaku ka tiawara to hlaya ta ku pfaleriwa na insulin ya mahala ya FITC eka nhlayo ya 25 μg/mL, ti NPs leti ha ku lulamisiwaka leti nga na FITC leti nga na insulin na ti NPs leti nga na mati hi FITC leti nga na insulin eka nhlayo yo ringana ya insulin Ku langutisisa ka quantitative microscopy (CLSM) ku endliwile.Ti NPs leti lyophilized leti nga riki na mannitol ti herisiwile hi nkarhi wa ku pfumaleka ka mati naswona a ti kamberiwanga eka leswi test.Matimba ya fluorescence ya le ndzeni ka sele ya ti-NP leti ha ku lunghiseleriweke leti layichiweke hi insulin, ti-NP leti lyophilized leti nga na mannitol, na ti-NP leti omisiweke hi ku fafazela leti nga na mannitol na leti nga riki na mannitol (Fig. 6a) a ti ri 4.3, 2.6, 2.4, na 4.1-fold higher than the free ones.FITC-insulin group, respectively (Fig. 6b).Mimbuyelo leyi yi ringanyeta leswaku insulin leyi encapsulated yina matimba swinene eka ku amukeriwa ka ti cellular kutlula insulin ya mahala, ngopfu ngopfu hikokwalaho ka vukulu lebyintsongo bya ti nanoparticles letinga tala insulin leti humesiweke eka nkambisiso.
Ku tekiwa ka tisele ta HepG2 endzhaku ka 4 h ya ku pfaleriwa na ti NP leti ha ku lulamisiwaka na ti NP leti nga riki na mati: (a) Ku hangalasiwa ka ku tekiwa ka FITC-insulin hi tisele ta HepG2.(b) Xiringaniso xa geometri xa matimba ya fluorescence lexi xopaxopiweke hi flow cytometry (n = 3), *P < 0.05 loko ku pimanisiwa na insulin ya mahala.
Hilaha ku fanaka, swifaniso swa CLSM swi kombisile leswaku matimba ya FITC fluorescence ya ti NPs letinga lulamisiwa hi FITC-insulin-loaded na FITC-insulin-loaded spray-dried NPs (handle ka mannitol) atiri na matimba swinene kutlula ya tisampulu tin’wana (Fig. 6a).Ku tlula kwalaho, hiku engeteriwa ka mannitol, viscosity yale henhla ya solution yi engetele ku lwisana ku ya eka ku amukeriwa ka tisele, leswi endlaka leswaku ku va na ku hunguteka ka ku andza ka insulin.Mimbuyelo leyi yi ringanyeta leswaku ti-NP leti omisiweke hi ku fafazela leti nga riki na mannitol ti kombisile vuswikoti bya le henhla bya ku tekiwa ka tisele hikuva vukulu bya ti particle ta tona a byi ri byintsongo ku tlula bya ti-NP leti omisiweke hi ku titimela endzhaku ko tlhela ti n’oka.
Chitosan (xiringaniso xa molecular weight 100 KDa, 75–85% deacetylated) yi xaviwile kusuka eka Sigma-Aldrich.(Oakville, Ontario, Canada).Sodium tripolyphosphate (TPP) yi xaviwile kusuka eka VWR (Radnor, Pennsylvania, USA).Insulin ya munhu leyi hlanganisiweke nakambe leyi tirhisiweke eka ndzavisiso lowu ayi huma eka Fisher Scientific (Waltham, MA, USA).Fluorescein isothiocyanate (FITC)-labeled human insulin na 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) swi xaviwile eka Sigma-Aldrich.(Oakville, Ontario, Canada).Layini ya tisele ta HepG2 yi kumiwile eka ATCC (Manassas, Virginia, USA).Ti reagents tin’wana hinkwato a ti ri ta grade ya analytical kumbe chromatographic.
Lulamisa 1 mg/ml ya CS solution hiku yi n’okisela eka mati ya double distilled (DD water) lawa yanga na 0.1% wa acetic acid.Lunghiselela 1 mg/ml solutions ya TPP na insulin hiku swi n’okisela eka mati ya DD na 0.1% acetic acid, hiku landzelelana.Pre-emulsion yi lulamisiwile hi polytron PCU-2-110 high speed homogenizer (Brinkmann Ind. Westbury, NY, USA).Maendlelo yo lulamisela hi ndlela leyi landzelaka: xosungula, 2ml ya TPP solution yi cheriwa eka 4ml ya insulin solution, naswona nkatsakanyo wu hlanganisiwa kuringana 30min naswona wu hlanganisiwa hiku hetiseka.Kutani, nkatsakanyo lowu pfanganisiweke wu cheriwa hi mathonsi eka CS solution hiku tirhisa syringe ehansi ka high-speed stirring (10,000 rpm).Mikatsakanyo yi hlayisiwile ehansi ka high-speed ku hlanganisiwa (15,000 rpm) eka bath ya ayisi kuringana 30 min, naswona ti lulamisiwile kuya eka pH yokarhi kuva ti kuma cross-linked insulin NPs.Ku ya emahlweni ti homogenize naku hunguta vukulu bya ti particle ta insulin NPs, ti sonicated kuringana 30 min yo engetelela eka ice bath hiku tirhisa probe-type sonicator (UP 200ST, Hielscher Ultrasonics, Teltow, Germany).
Insulin NPS yi kamberiwile eka Z-average diameter, polydispersity index (PDI) na zeta potential hiku tirhisa mipimo ya dynamic light scattering (DLS) hiku tirhisa Litesizer 500 (Anton Paar, Graz, Austria) hiku ti chela eka mati ya DD eka 25°C.Morphology na ku hangalasiwa ka vukulu swi tiveka hi Hitachi H7600 transmission electron microscope (TEM) (Hitachi, Tokyo, Japan), naswona swifaniso endzhaku swikamberiwile hiku tirhisa software ya swifaniso swa Hitachi (Hitachi, Tokyo, Japan).Ku kambela vuswikoti bya encapsulation (EE) na vuswikoti byo layicha (LC) bya ti insulin NPs, ti NPs ti pipetted eka ti tubes ta ultrafiltration letingana molecular weight cut-off ya 100 kDa naswona ti centrifuged eka 500 xg kuringana 30 min.Insulin leyi nga pfaleriwangiki eka filtrate yi pimiwe hi ku tirhisa sisiteme ya Agilent 1100 Series HPLC (Agilent, Santa Clara, California, USA) leyi vumbiwaka hi pompo ya quaternary, autosampler, column heater, na DAD detector.Insulin yi kamberiwile hi kholomo ya C18 (Zorbax, 3.5 μm, 4.6 mm × 150 mm, Agilent, USA) naswona yi kumiwile eka 214 nm.Xiphemu lexi fambafambaka a xi ri acetonitrile na mati, lexi nga na 0.1% TFA, gradient ratios ku suka eka 10/90 ku ya eka 100/0, naswona xi famba ku ringana timinete ta 10. Xiphemu lexi fambafambaka xi pomperiwe hi mpimo wa ku khuluka wa 1.0 ml/min.Mahiselo ya kholomo ya vekiwile eka 20 °C.Hlela tiphesente ta EE na LC hi ku tirhisa swiringaniso.(1) na Eq.(2) ya xiyimo xa le henhla.
Ti ratios tohambana hambana ta CS/insulin kusuka eka 2.0 kuya eka 4.0 ti kamberiwile kuva ti antswisa insulin NP.Tinhlayo tohambana hambana ta CS solution ti cheriwile hi nkarhi wa ku lulamisiwa, kasi nkatsakanyo wa insulin/TPP wu hlayisiwile wu nga cinci.Ti insulin NPs ti lulamisiwile eka pH range ya 4.0 kuya eka 6.5 hiku lawula hi vukheta pH ya nkatsakanyo endzhaku ko cheriwa ti solutions hinkwato (insulin, TPP na CS).EE na vukulu bya ti particle ta ti nanoparticles ta insulin swi kamberiwile eka mimpimo yo hambana ya pH na CS/insulin mass ratios ku antswisa ku vumbiwa ka ti insulin NPs.
Ti insulin NPs leti antswisiweke ti vekiwile eka xigwitsirisi xa aluminiyamu naswona ti funengetiwile hi tissue leyi tiyisiweke hi thepi yin’wana.Endzhaku ka sweswo, swibye leswi nga na swikwirikiri swi vekiwile eka Labconco FreeZone freeze dryer (Labconco, Kansas City, MO, USA) leyi hlomisiweke hi tray dryer.Mahiselo na ntshikelelo wa vacuum swi vekiwile eka -10 °C, 0.350 Torr eka 2 h yo sungula, na 0 °C na 0.120 Torr eka 22 h leyinga sala ya 24 h kuva ku kumiwa ti dry insulin NPs.
Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Switzerland) yitirhisiwile ku humelerisa insulin leyi encapsulated.Ti parameters to omisa leti hlawuriweke akuri: mahiselo ya 100 °C, ku khuluka ka swakudya 3 L/min, na ku khuluka ka gasi 4 L/min.
Ti insulin NPs emahlweni na le ndzhaku ka ku pfumaleka ka mati ti hlawuriwile hi ku tirhisa FTIR-ATR spectroscopy.Ti nanoparticles leti nga riki na mati xikan’we na insulin ya mahala na chitosan ti kamberiwile hi ku tirhisa Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA) leyi hlomisiweke hi xitirhisiwa xa universal ATR sampling accessory (PerkinElmer, Waltham, Massachusetts, USA). swiringaniso swikumeke kusuka eka 16 wa swikeni eka resolution ya 4 cm2 eka frequency range ya 4000-600 cm2.
Mofoloji ya ti NP ta insulin yo oma yi kamberiwile hi swifaniso swa SEM swa ti insulin NP leti omisiweke hi freeze na leti omisiweke hi ku fafazela leti tekiweke hi Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, USA). Parameter leyikulu leyi tirhisiweke akuri voltage 5 keV na current 30 mA.
Ti insulin NP hinkwato letinga na mati titlhele ti pfanganisiwa eka mati ya dd.Vukulu bya ti particle, PDI, EE na LC ti kamberiwile nakambe hiku tirhisa ndlela leyi fanaka leyi boxiweke ekusunguleni ku kambela khwalithi ya tona endzhaku ka ku pfumaleka ka mati.Ku tshamiseka ka ti anhydroinsulin NPs switlhele swi pimiwa hiku kambela swivumbeko swa ti NPs endzhaku ka ku hlayisiwa nkarhi woleha.Eka ndzavisiso lowu, ti NPs hinkwato endzhaku ka ku pfumaleka ka mati ti hlayisiwile efrijini kuringana tinharhu tinhweti.Endzhaku ka tinhweti tinharhu ta vuhlayiselo, ti NPs ti kamberiwile eka vukulu bya morphological particle, PDI, EE na LC.
N’okisela 5 mL ya ti NPs leti vumbiweke nakambe eka 45 mL leyingana mati ya le khwirini lawa ya tekeleriweke (pH 1.2, lawa yanga na 1% pepsin), mati ya marhumbu (pH 6.8, lawa yanga na 1% trypsin) kumbe chymotrypsin solution (100 g/mL, eka phosphate buffer, pH 7.8) ku kambela ku tirha kahle ka eka ku sirhelela ti NPs endzhaku dehydration.Ti pfaleriwile eka 37°C hi rivilo ra agitation ra 100 rpm.500 μL ya solution yi hlengeletiwile eka tindzhawu to hambana ta nkarhi naswona nhlayo ya insulin yikumiwile hi HPLC.
Mahanyelo ya ku humesiwa ka in vitro ya ti insulin NPs letinga lulamisiwa na letinga pfumala mati ya kamberiwile hi ndlela ya dialysis bag (molecular weight cut-off 100 kDa, Spectra Por Inc.).Ti NPs letinga lulamisiwile naku vumbiwa nakambe letinga oma ti dialyzed eka swihalaki eka pH 2.5, pH 6.6, na pH 7.0 (0.1 M phosphate-buffered saline, phosphate-buffered saline PBS) ku tekelela ndzhawu ya pH ya khwiri, duodenum, na marhumbu yale henhla, hiku landzelelana.Tisampulu hinkwato ti pfaleriwile eka 37 °C hiku tsekatseka loku yaka emahlweni hi 200 rpm.Aspirate the fluid ehandle ka 5 mL dialysis bag hi minkarhi leyi landzelaka: 0.5, 1, 2, 3, 4, na 6 h, naswona hiku hatlisa u tata volume hi fresh dialysate.Ku thyakisa ka insulin eka mati ku kamberiwile hi HPLC, naswona mpimo wa ku humesiwa ka insulin ku suka eka ti nanoparticles wu hlayiwile ku suka eka mpimo wa insulin ya mahala leyi humesiweke eka insulin hinkwayo leyi pfaleriweke eka ti nanoparticles (Equation 3).
Human hepatocellular carcinoma cell line Ti cells ta HepG2 ti byarisiwile eka swibya swa 60 mm diameter hiku tirhisa Dulbecco’s Modified Eagle’s Medium (DMEM) leyingana 10% fetal bovine serum, 100 IU/mL penicillin, na 100 μg/mL streptomycin29.Mindzhavuko yi hlayisiwile eka 37°C, 95% relative humidity, na 5% CO2.Eka swikambelo swa uptake, ti cells ta HepG2 ti byariwile eka 1 × 105 cells/ml eka 8-well Nunc Lab-Tek chamber slide system (Thermo Fisher, NY, USA).Eka swikambelo swa cytotoxicity, ti byariwile eka 96-well plates (Corning, NY, USA) eka density ya 5 × 104 cells/ml.
MTT assay yitirhisiwile ku kambela cytotoxicity ya insulin NPs30 leyinga lulamisiwa na leyi nga hava mati.Ti cells ta HepG2 ti byariwile eka 96-well plates eka density ya 5 × 104 cells/mL naswona ti byariwile kuringana 7 wa masiku loko kungase kamberiwa.Insulin NPs yi cheriwile kuya eka ti concentrations to hambana hambana (50 kuya eka 500 μg/mL) eka culture medium kutani yi nyikiwile eka ti cells.Endzhaku ka 24 wa tiawara ta ku pfaleriwa, ti cells ti hlantswiwile minkarhi ya 3 hi PBS naswona ti pfaleriwile na medium leyingana 0.5 mg/ml MTT kuringana 4 wa tiawara to engetela.Cytotoxicity yi kamberiwile hiku pima kuhunguteka ka enzymatic ka yellow tetrazolium MTT kuya eka purple formazan eka 570 nm hiku tirhisa Tecan infinite M200 pro spectrophotometer xihlaya xa pulati (Tecan, Männedorf, Switzerland).
Ku tirha kahle ka ku tekiwa ka ti cellular ka ti NPs swikamberiwile hi confocal laser scanning microscopy na flow cytometry analysis.Xihlovo xin’wana na xin’wana xa Nunc Lab-Tek chamber slide system xi tshunguriwile hi free FITC-insulin, FITC-insulin-loaded NPs, naswona xi vumbiwile nakambe 25 μg/mL ya dehydrated FITC-insulin NPs eka nhlayo leyi fanaka naswona yi pfaleriwile kuva yi pfaleriwa 4 wa tiawara.Tisele ti hlantswiwile minkarhi ya 3 hi PBS naswona ti lulamisiwile hi 4% paraformaldehyde.Ti nuclei ti pfaleriwile hi 4′,6-diamidino-2-phenylindole (DAPI).Ku vekiwa ka insulin endhawini ya kona ku voniwile hi ku tirhisa Olympus FV1000 laser scanning/two-photon confocal microscope (Olympus, Shinjuku City, Tokyo, Japan).Ku khuluka cytometry analysis, nhlayo leyi fanaka ya 10 μg/mL free FITC-insulin, FITC-insulin-loaded NPs, na resolubilized dehydrated FITC-insulin NPs swi engeteriwile eka 96-well plates leswi byariweke hi ti cells ta HepG2 naswona swi pfaleriwile kuringana 4 wa tiawara .Endzhaku ka 4 h ya ku pfaleriwa, ti cells ti susiwile kutani ti hlantswiwa 3 times hi FBS.5 × 104 wa ti cells hi xikombiso ti kamberiwile hi BD LSR II flow cytometer (BD, Franklin Lakes, New Jersey, United States).
Mimpimo hinkwayo yi kombisiwile tanihi xiringaniso ± ku hambuka ka ntolovelo.Ku pimanisiwa exikarhi ka mintlawa hinkwayo ku kamberiwile hi ku tirhisa ANOVA ya ndlela yin’we kumbe t-test hi IBM SPSS Statistics 26 for Mac (IBM, Endicott, New York, USA) naswona p < 0.05 yi tekiwile yi ri ya nkoka hi tlhelo ra tinhlayo.
Dyondzo leyi yi kombisa ku olova na vuswikoti bya ku omisa hi ku fafazela ku susa mati eka ti cross-linked chitosan/TPP/insulin nanoparticles leti nga na ku vumbiwa nakambe ko antswa loko ku pimanisiwa na tindlela ta ntolovelo ta freeze-drying hi ku tirhisa ti bulking agents kumbe vuswikoti bya cryoprotectants na vuswikoti bya le henhla bya ndzhwalo.The optimized insulin nanoparticles yielded an average particle size of 318 nm and an encapsulation efficiency of 99.4%.Mbuyelo wa SEM na FTIR endzhaku ka ku pfumaleka ka mati wu kombisile leswaku xivumbeko xa xirhendzevutani xi hlayisiwile ntsena eka ti NPs leti omisiweke hi ku fafazela leti nga na na na leti nga riki na mannitol naswona ti lyophilized hi mannitol, kambe ti lyophilized NPs leti nga riki na mannitol ti bola hi nkarhi wa ku pfumaleka ka mati.Eka xikambelo xa vuswikoti byo vumbiwa nakambe, ti nanoparticles ta insulin leti omisiweke hi ku fafazela handle ka mannitol ti kombisile vukulu bya swiphemuphemu swa mean leswintsongo swinene na ndzhwalo wa le henhla swinene upon reconstitution.Mahanyelo ya ku humesiwa ka ti NP leti hinkwato letinga na mati ya kombisile leswaku ti humesiwa hiku hatlisa eka swintshuxo swa pH = 2.5 na pH = 7, naswona ti tshamisekile swinene eka solution ya pH = 6.5.Loko ti pimanisiwa na ti NPs tin’wana letinga pfanganisiwa nakambe letinga na mati, ti NPs letinga fafazeriwa hiku fafazeriwa handle ka mannitol ti kombisile ku humesiwa hiku hatlisa swinene.Mbuyelo lowu wu fambisana na leswi voniweke eka cellular uptake assay, tani hi ti NPs leti omisiweke hi ku fafazela loko ku nga ri na mannitol ti lava ku hlayisa hi ku helela vuswikoti byo amukela tisele bya ti NPs leti ha ku lulamisiwaka.Mimbuyelo leyi yi ringanyeta leswaku ti nanoparticles ta insulin leti omeke leti lulamisiweke hi ku omisiwa ka ku fafazela loku nga riki na mannitol ti lulamerile swinene ku ya emahlweni ti lulamisiwa eka swivumbeko swin’wana swa mpimo lowu nga riki na mati, ku fana na tiphilisi ta nomu kumbe tifilimi ta bioadhesive.
Hikwalaho ka timhaka ta nhundzu ya vutlhari, tidathaseti leti endliweke na/kumbe leti xopaxopiweke hi nkarhi wa dyondzo ya sweswi a ti kumeki erivaleni, kambe ti kumeka eka vatsari lava faneleke loko va komberiwa hi ndlela leyi twalaka.
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