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Insulin nanoparticles (NPs) a ɛwɔ adesoa a ɛkɔ soro no anya dwumadie ahodoɔ wɔ dosage forms.Saa adwuma yi botaeɛ ne sɛ wɔbɛhwɛ nkɛntɛnsoɔ a freeze-drying ne spray-drying akwan nya wɔ nhyehyɛeɛ a insulin-loaded chitosan nanoparticles, a anaasɛ enni mannitol sɛ cryoprotectant.Yɛsan nso hwɛɛ saa nanoparticles yi su denam redissolving wɔn.Before dehydration, na chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles no mu nneɛma nketenkete no kɛse yɛɛ yiye sɛ ɛbɛyɛ 318 nm, PDI no yɛ 0.18, encapsulation efficiency no yɛ 99.4%, na adesoa no yɛ 25.01%.After reconstitution, nanoparticles nyinaa, gye nea wɔyɛe denam freeze-drying kwan so a wɔmfa nni dwuma of mannitol, maintained their spherical particle structure.Sɛ wɔde toto nanoparticles a mannitol wom a nsuo afiri mu denam aduru biara a wɔde petepete so ho a, nanoparticles a wɔde petepete so a mannitol nni mu nso daa no adi sɛ ɛyɛ ketewaa koraa wɔ abɔdeɛ nketenkete kɛseɛ (376 nm) ne adesoa a ɛkorɔn sen biara (25.02%) a ɛwɔ encapsulation rate a ɛte saa ara (98.7%) ne PDI (0.20) denam drying anaa freeze-drying techniques.Nanoparticles a ayow denam spray drying a mannitol nka ho so nso maa insulin yii ntɛmntɛm sen biara na ɛyɛɛ yiye sen biara wɔ cellular uptake.Saa adwuma yi kyerɛ sɛ spray drying betumi ama nsu a ɛwɔ insulin nanoparticles mu a enhia sɛ wɔde cryoprotectants toto freeze drying akwan a wɔtaa fa so no ho a, ɛma tumi a wɔde bɛhyɛ mu kɛse, additive ahwehwɛde a ɛba fam ne adwumayɛ ɛka mfaso kɛse.
Efi bere a wohuu no wɔ afe 19221 mu no,2,3 no, insulin ne nnuru a wɔde yɛ no agye ayarefo a wɔwɔ asikreyare a ɛto so 1 (T1DM) ne asikreyare a ɛto so abien (T1DM) nkwa.Nanso, esiane sɛ ɛyɛ protein a emu duru yɛ kɛse nti, ɛnyɛ den sɛ insulin bɛboaboa ano, denam proteolytic enzymes a ɛpaapae mu, na ɛnam ɔkwan a edi kan no so yi fi hɔ effect.Nkurɔfo a wɔahu sɛ wɔwɔ asikreyare a ɛto so 1 no hia insulin a wɔde twitwiw wɔn wɔ wɔn nkwa nna nyinaa mu.Ayarefo pii a mfiase no wohui sɛ wɔwɔ asikreyare a ɛto so abien no nso hia sɛ wɔde insulin a wɔde twitwiw wɔn bere tenten.Insulin a wɔde twitwiw wɔn da biara da no yɛ ade a emu yɛ den a ɛde ɛyaw ne ɔhaw a emu yɛ den ba da biara da ma saa ankorankoro yi, na ɛde nkɛntɛnso bɔne ba adwenemyare so.Nea efi mu ba ne sɛ, insulin a wɔde ma no akwan foforo a ɛmma ɔhaw pii mma, te sɛ sɛ insulin a wɔde ma wɔ anom no, wɔreyɛ ho nhwehwɛmu kɛse5 efisɛ ɛwɔ tumi a ɛbɛma nnipa bɛyɛ ɔpepepem 5 a wɔwɔ asikreyare asetra ayɛ papa wɔ wiase nyinaa.
Nanoparticle mfiridwuma ama nkɔso kɛse aba wɔ mmɔden a wɔbɔ sɛ wɔbɛfa insulin a wɔnom mu4,6,7.Nea ɛkata insulin so yiye na ɛbɔ ho ban fi ɔsɛe ho ma wɔde kɔ nipadua no mmeae pɔtee bi.Nanso, nanoparticle nnuru a wɔde di dwuma no wɔ anohyeto ahorow pii, titiriw esiane nsɛm a ɛfa sɛnea nneɛma nketenkete a wɔde ahyɛ mu no gyina pintinn nti.Ebi a wɔaboaboa ano betumi aba bere a wɔkora so, a ɛtew bioavailability a ɛwɔ insulin-loaded nanoparticles8.Bio nso, ɛsɛ sɛ wosusuw nnuru a ɛwɔ polymer matrix a ɛwɔ nanoparticles ne insulin no mu nso ho na ama wɔahwɛ ahu sɛ insulin nanoparticles (NPs) no gyina pintinn.Mprempren, freeze-drying mfiridwuma ne sika kɔkɔɔ gyinapɛn a wɔde yɛ NPs a ɛyɛ den bere a wosiw nsakrae a wɔmpɛ bere a wɔde sie no ano
Nanso, freeze-drying hwehwɛ sɛ wɔde cryoprotectants ka ho na amma NPs kurukuruwa nhyehyɛe no annya nkɛntɛnso wɔ mfiridwuma mu nhyɛso a nsukyenee ahwehwɛ mu.Eyi brɛ insulin nanoparticles adesoa so kɛse wɔ lyophilization akyi, efisɛ cryoprotectant no gye dodow no ara mu duru ratio.Enti, wɔtaa hu sɛ insulin NPs a wɔayɛ no mfata mma a wɔyɛ nnuru a wɔde powder a ɛyɛ dry, te sɛ nnuru a wɔde ano ka ne sini a wɔde ano, esiane hia a ehia sɛ wɔyɛ nanoparticles a ɛyɛ dry pii na ama wɔanya insulin ayaresa mfɛnsere no nti.
Spray drying yɛ mfiridwuma-scale kwan a wonim no yiye na ne bo nyɛ den a wɔde yɛ dry powders fi nsu phases mu wɔ nnuruyɛ adwumayɛ mu10,11.Control wɔ particle formation nhyehyɛe no ma kwan ma encapsulation yiye bioactive compounds pii 12, 13 .Bio nso, abɛyɛ ɔkwan a etu mpɔn a wɔfa so siesie encapsulated proteins ma oral administration.During spray drying, nsuo evaporates ntɛmntɛm paa, a ɛboa ma ɔhyew a particle core low11,14, ma ne application tumi encapsulate ɔhyew-sensitive components.Ansa na spray drying, ɛsɛ sɛ coating ade no yiye homogenized ne solution a encapsulated ingredients11,14.Unlike freeze-drying, homogenization ansa encapsulation wɔ spray-drying mu ma encapsulation efficiency tu mpɔn bere a dehydration.Esiane sɛ spray-drying encapsulation nhyehyɛe no nhia cryoprotectants, wobetumi de spray-drying ayɛ NPs a ayow a loading content pii.
Saa nhwehwɛmu yi bɔ amanneɛ sɛ wɔyɛ NPs a insulin ahyɛ mu ma denam chitosan ne sodium tripolyphosphate a wɔde cross-linking di dwuma denam ion gel kwan so.Ion gelation yɛ ahosiesie kwan a ɛma kwan ma wɔyɛ nanoparticles denam electrostatic nkitahodi a ɛda ionic ahorow abien anaa nea ɛboro saa ntam wɔ tebea horow bi mu so.Wɔde freeze-drying ne spray-drying akwan nyinaa dii dwuma de maa nsu fi chitosan/sodium a wɔayɛ no yiye no mu tripolyphosphate/insulin cross-linked nanoparticles.After dehydration, wɔn morphology yɛɛ nhwehwɛmu denam SEM.Wɔn recombination tumi no yɛɛ nhwehwɛmu denam susuw wɔn kɛse kyekyɛ, surface charge, PDI, encapsulation efficiency, ne loading content.The quality of resolubilized nanoparticles produced by different dehydration methods was also evaluated by toto their insulin protection, release behavior, . ne nkwammoaa a wɔde gye nneɛma a etu mpɔn.
pH a ɛwɔ aduru a wɔadi afra no mu ne chitosan ne insulin nsusuwii yɛ nneɛma atitiriw abien a ɛka NP ahorow a etwa to no mu nneɛma nketenkete kɛse ne encapsulation efficiency (EE), efisɛ ɛka ionotropic gelation nhyehyɛe no tẽẽ.Wɔkyerɛe sɛ aduru a wɔde afrafra no pH ne abɔde nketenkete kɛse ne encapsulation efficiency wɔ abusuabɔ kɛse (Mfonini 1a).Sɛnea wɔada no adi wɔ Mfonini 1a mu no, sɛnea pH kɔɔ soro fi 4.0 kosi 6.0, sɛ wɔkyekyem pɛpɛɛpɛ a, nneɛma nketenkete no kɛse (nm) so tew na EE kɔɔ soro kɛse, bere a bere a pH kɔɔ soro koduu 6.5 no, sɛ wɔkyekyem pɛpɛɛpɛ a, nneɛma nketenkete no kɛse fii ase kɔɔ soro na EE no ansakra.Bere a chitosan ne insulin ntam nsonsonoe kɔ soro no, nneɛma nketenkete no kɛse nso kɔ soro.Bio nso, wɔanhu nsakrae biara wɔ EE mu bere a wosiesiee nanoparticles wɔ mass ratio a ɛyɛ chitosan/insulin a ɛkorɔn sen 2.5:1 (w/w) (Mfonini 1b).Enti, wɔde ahosiesie tebea horow a eye sen biara wɔ nhwehwɛmu yi mu (pH 6.0, chitosan/insulin mass ratio a ɛyɛ 2.5:1) dii dwuma de siesiee insulin-loaded nanoparticles maa adesua foforo.Wɔ saa ahosiesie tebea yi ase no, na wɔmaa nanoparticles no mu nneɛma nketenkete no kɛse yɛ nea eye sen biara 318 nm (Mfonini 1c), na PDI no yɛ 0.18, na embedding efficiency no yɛ 99.4%, zeta tumi no yɛ 9.8 mv, na insulin loading no yɛ 25.01% (m / m ).Wɔgyina transmission electron microscopy (TEM) aba so no, na nanoparticles a wɔayɛ no yiye no yɛ bɛyɛ kurukuruwa ne discrete a ne kɛse yɛ pɛ (Mfonini 1d).
Parameter optimization of insulin nanoparticles: (a) nkɛntɛnso a pH nya wɔ mean diameter ne encapsulation efficiency (EE) a insulin nanoparticles (a wɔasiesie wɔ 5:1 mass ratio chitosan ne insulin); (b) chitosan ne Nkɛntɛnso a insulin dodow nsusuwii nya wɔ insulin NPs (a wɔasiesie wɔ pH 6) no ntwemu ne encapsulation efficiency (EE) so; (c) insulin nanoparticles a wɔayɛ no yiye no mu nneɛma nketenkete kɛse kyekyɛ; (d) TEM micrograph a ɛkyerɛ insulin NP ahorow a wɔayɛ no yiye .
Ɛyɛ nea wonim yiye sɛ chitosan yɛ polyelectrolyte a ɛyɛ mmerɛw a ne pKa yɛ 6.5.Ɛwɔ ahoɔden pa wɔ acidic media mu efisɛ ne amino kuw titiriw no yɛ protonated denam hydrogen ions15.Enti, wɔtaa de di dwuma sɛ ade a wɔde fa so de kata macromolecules a ahoɔden bɔne wom so.Wɔ nhwehwɛmu yi mu no, wɔde chitosan dii dwuma de kataa insulin a isoelectric point of 5.3.Esiane sɛ wɔde chitosan di dwuma sɛ ade a wɔde kata so nti, bere a ne fã no kɔ soro no, nanoparticles no akyi fã no mu duru kɔ soro sɛnea ɛfata, na ɛde abɔde nketenkete kɛse a ɛyɛ kɛse ba.Nea ɛka ho no, chitosan dodow a ɛkɔ soro betumi akata insulin pii so.Wɔ yɛn asɛm no mu no, na EE korɔn sen biara bere a chitosan ne insulin nsusuwii duu 2.5:1, na nsakrae kɛse biara amma wɔ EE mu bere a dodow no kɔɔ so kɔɔ soro no.
Sɛ chitosan ne insulin nsusuwii da nkyɛn a, pH nso dii dwuma titiriw wɔ NP ahorow a wɔyɛe no mu.Gan et al. 17 suaa nkɛntɛnso a pH nya wɔ chitosan nanoparticles.Wɔhunuu sɛ nneɛma nketenkete no kɛseɛ so tew kɔ so kosii sɛ pH duu 6.0, na wɔhunuu sɛ nneɛma nketenkete no kɛseɛ kɔ soro kɛseɛ wɔ pH > 6.0, a ɛne yɛn nhwɛsoɔ hyia.Saa adeyɛ yi firi nokwasɛm a ɛyɛ sɛ sɛ pH kɔ soro a, insulin molecule no nya surface charge a enye, ma enti, ɛyɛ papa electrostatic nkitahodi ne chitosan/sodium tripolyphosphate (TPP) complex, a ɛde nneɛma nketenkete kɛse ba ne EE a ɛkɔ soro .Nanso, bere a wɔyɛɛ nsakrae wɔ pH no mu kɔɔ 6.5 no, wɔde protonated amino akuw a ɛwɔ chitosan so no fii mu, na ɛde chitosan folding bae.Enti, pH a ɛkɔ soro no ma amino ions no ntumi nkɔ TPP ne insulin mu kakraa bi, na ɛde cross-linking a ɛba fam ba, sɛ wɔkyekyem pɛpɛɛpɛ a, ɛyɛ kɛse particle kɛse ne EE a ɛba fam.
Analysis of the morphological properties of freeze-dried ne spray-dried NPs betumi akyerɛ kwan ma wɔapaw dehydration ne powder formation techniques a eye.Ɛsɛ sɛ ɔkwan a wɔpɛ no ma nnuru no gyina pintinn, uniform particle shape, high drug loading na solubility pa wɔ mfitiase solution no mu.Wɔ saa nhwehwɛmu yi mu no, sɛ yɛde akwan abien no bɛtoto ho yiye a, wɔde insulin NPs a ɛwɔ anaasɛ enni 1% mannitol dii dwuma bere a dehydration.Mannitol na wɔde dii dwuma sɛ a bulking agent anaa cryoprotectant wɔ dry powder formulations ahorow mu ma freeze drying ne spray drying.Wɔ lyophilized insulin nanoparticles a enni mannitol, sɛnea wɔada no adi wɔ Mfonini 2a mu no, wohuu powder nhyehyɛe a ɛyɛ porous kɛse a ɛwɔ soro, a ɛnyɛ pɛpɛɛpɛ na ɛyɛ mmerɛw wɔ scanning electron microscopy (SEM) ase.Few discrete particles na wohuu wɔ powder no mu bere a nsu asɛe akyi (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 nanso wrinkled wɔ soro (Mfonini 2c).Wɔaka kurukuruwa ne wrinkled surfaces ho asɛm bio wɔ release suban ne cellular uptake sɔhwɛ ahorow a ɛwɔ ase ha.Wɔgyina sɛnea wotumi hu NPs a ayow no so, NPs a wɔde petepete so a enni mannitol ne NPs nyinaa freeze-dried ne spray-dried ne mannitol maa NPs powders a ɛyɛ fɛ (Mfonini. 2f,g,h).Dodow a asase a ɛda nsunsuansoɔ no ntam no yɛ kɛseɛ no, dodoɔ no ara na nsuo a ɛtumi nwura mu no yɛ kɛseɛ na ɛno nti dodoɔ no ara na ɛtumi gyae.
Insulin NP ahorow a nsu nnim no nsɛso: (a) SEM mfonini a ɛkyerɛ insulin NP ahorow a wɔde lyophilized a mannitol nni mu; (b) SEM mfonini a ɛkyerɛ insulin NP ahorow a wɔde mannitol ayɛ no lyophilized; (c) insulin NPs a wɔde aduru apetepete so a mannitol SEM mfonini a ɛwɔ ; (d) SEM mfonini a ɛkyerɛ insulin NPs a wɔde mannitol apetepete so; (e) insulin NPs powder a wɔde lyophilized ayɛ a mannitol nni mu no mfonini; (f) insulin NP ahorow a wɔde mannitol ayɛ no mfonini; ( g) Insulin NPs powder a wɔde aduru apetepete so a mannitol nnim mfonini; (h) mfonini a ɛkyerɛ insulin NPs powder a wɔde petepete so a wɔde mannitol ayɛ.
Wɔ freeze-drying mu no, mannitol yɛ adwuma sɛ cryoprotectant, ɛma NPs kɔ so yɛ amorphous na esiw ice crystals sɛe19.Nea ɛne eyi bɔ abira no, freezing anammɔn biara nni hɔ bere a spray drying.Enti mannitol ho nhia wɔ saa kwan yi so.Nokwarem no, NPs a spray-dried a mannitol nni mu no maa NPs a ɛyɛ fɛ sen sɛnea wɔadi kan aka ho asɛm no.Nanso, nanso, freezing step biara nni hɔ bere a spray drying. mannitol da so ara tumi yɛ adwuma sɛ ade a ɛhyɛ ma wɔ aduru a wɔde petepete so no mu ma ɛma NPs yɛ kurukuruwa nhyehyɛe20 (Mfonini 2d), a ɛboa ma wonya NPs a wɔde ahyɛ mu a ɛte saa no a wɔde gyae nneɛma a ɛyɛ pɛ.Nea ɛka ho no, ɛda adi pefee sɛ wobetumi ahu nneɛma nketenkete bi wɔ insulin NPs a wɔayam no nwini ne nea wɔde petepete mu a mannitol wom nyinaa mu (Mfonini 2b,d), a ebetumi aba sɛ efi mannitol a ɛwɔ abɔde nketenkete no mu a ɛka insulin a wɔde ahyɛ mu no ho. To.Chitosan layer.Ɛfata sɛ yɛhyɛ no nsow sɛ wɔ saa nhwehwɛmu yi mu no, sɛnea ɛbɛyɛ a wɔbɛhwɛ sɛ kurukuruwa nhyehyɛe no bɛkɔ so ayɛ sɛnea ɛte wɔ nsu a ɛho ayɛ fĩ akyi no, wɔma mannitol ne chitosan nsusuwii no kɔ so yɛ 5:1, sɛnea ɛbɛyɛ a filler dodow bi nso betumi ama NP ahorow a ayow no mu nneɛma nketenkete no ayɛ kɛse.
Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy kyerɛɛ nipadua mu afrafra a ɛyɛ insulin a enni mu, chitosan, chitosan, TPP ne insulin.Wɔde FTIR-ATR spectroscopy na ɛkyerɛɛ NPs a nsuo nni mu nyinaa.Nea ɛda nsow ne sɛ, wɔhunuu band ahoɔden a ɛyɛ 1641, 1543 ne 1412 cm-1 wɔ NPs a wɔde ahyɛ mu no mu freeze-dried with mannitol and in NPs spray-dried with and without mannitol (Fig. 3).Sɛnea wɔadi kan abɔ amanneɛ no, na ahoɔden a ɛkɔ soro yi ne cross-linking a ɛda chitosan, TPP ne insulin ntam no wɔ abusuabɔ.Nhwehwɛmu a wɔyɛe wɔ nkitahodi a ɛda chitosan ne insulin ntam no kyerɛe sɛ wɔ FTIR spectra a ɛwɔ insulin-loaded chitosan nanoparticles mu no, chitosan band no ne insulin de no hyia, wɔ increasing the carbonyl ahoɔden (1641 cm-1) ne amine (1543 cm-1) belt.TPP mu tripolyphosphate akuw no ne ammonium akuw a ɛwɔ chitosan mu no wɔ abusuabɔ, na ɛyɛ band wɔ 1412 cm-1.
FTIR-ATR spectra a ɛkyerɛ insulin a wontua hwee, chitosan, honam fam afrafra a ɛyɛ chitosan/TPP/insulin ne NPs a wɔde akwan horow ayɛ nsuo.
Bio nso, saa nsunsuansoɔ yi ne deɛ wɔada no adi wɔ SEM mu no hyia, a ɛkyerɛɛ sɛ NPs a wɔde ahyɛ mu no kɔɔ so yɛɛ adwuma berɛ a wɔde mannitol petepeteeɛ ne freeze-dried nyinaa, nanso sɛ mannitol nni hɔ a, spray-drying nko ara na ɛma encapsulated particles.Nea ɛne no bɔ abira no, FTIR-ATR spectral aba a ɛfiri NPs freeze-dried a mannitol nni mu no di nsɛ paa of chitosan, TPP, and insulin.Saa aba yi kyerɛ sɛ cross-links a ɛda chitosan, TPP ne insulin ntam no nni NPs a wɔde freeze-dried a mannitol nni mu no mu bio.Wɔsɛee NPs nhyehyɛe no bere a wɔde freeze-drying a enni cryoprotectant, a wobetumi ahu wɔ SEM aba no mu (Mfonini 2a).Wɔgyina morphology ne FTIR aba a efii insulin a nsu nnim mu bae no so, nkutoo wɔde lyophilized, spray-dried, ne mannitol-free NPs dii dwuma de yɛɛ reconstitution experiments ne NPs a mannitol nnim esiane NPs a mannitol nnim a ɛporɔw bere a nsu nni mu nti. pɛnsɛpɛnsɛ mu.
Wɔde nsuo a ɛtɔ gu nipadua mu no di dwuma de sie bere tenten na wɔsan yɛ no foforo kɔ nnuru afoforo mu.Tumi a NP a ayow no tumi san yɛ bio bere a wɔde asie akyi no ho hia kɛse ma wɔde di dwuma wɔ nnuru ahorow te sɛ tablets ne films mu.Yɛhyɛɛ no ​​nsow sɛ sɛ wɔkyekyem pɛpɛɛpɛ a, insulin NPs a wɔde aduru a wɔde petepete so no mu nneɛma nketenkete kɛse bere a mannitol nni mu no kɔɔ soro kakra wɔ nea wɔasan ayɛ no akyi.Ɔkwan foforo so no, nneɛma nketenkete a wɔde petepete so no kɛse na freeze-dried insulin nanoparticles with mannitol kɔɔ soro kɛse (Table 1).PDI ne EE ansakra kɛse (p > 0.05) akyi recombination of all NPs wɔ nhwehwɛmu yi mu (Table 1).Saa aba yi kyerɛ sɛ dodow no ara particles kɔɔ so yɛɛ intact akyi redissolving.Nanso, mannitol a wɔde kaa ho no maa insulin loading a lyophilized ne spray-dried mannitol nanoparticles (Table 1).Nea ɛne eyi bɔ abira no, insulin adesoa a ɛwɔ NPs a wɔde spray-dried a mannitol nka ho no kɔɔ so te sɛ kan no (Table 1).
Ɛyɛ nea wonim yiye sɛ nanoparticles a wɔde hyɛ mu no ho hia bere a wɔde di dwuma de yɛ nnuru a wɔde ma no.Wɔ NP ahorow a wɔde nneɛma a ɛba fam no ho no, ɛho hia sɛ wɔde nneɛma pii di dwuma na ama wɔadu ayaresa aboboano no ho.Nanso, NP dodow a ɛkɔ soro saa no mu nsu a ɛyɛ den no de ɔhaw ne ɔhaw ba wɔ anom a wɔde ma ne nnuru a wɔde twitwiw mu, sɛnea ɛte biara 22 .Nea ɛka ho no, NPs betumi nso wɔde bɛyɛ tablets ne viscous biofilms23, 24, a ɛhwehwɛ sɛ wɔde NPs pii di dwuma wɔ loading levels a ɛba fam mu, na ɛde tablets akɛse ne biofilms a ɛyɛ den a ɛnyɛ nea ɛfata sɛ wɔde di dwuma wɔ anom ba.Enti, dehydrated NPs a insulin adesoa kɛse wom no yɛ nea wɔpɛ kɛse.Yɛn aba no kyerɛ sɛ insulin adesoa a ɛkɔ soro a ɛwɔ mannitol-free spray-dried NPs mu no betumi de pii ama mfaso horow a ɛyɛ anigye a ɛwɔ akwan foforo a wɔfa so de nneɛma ma yi so.
Wɔde NPs a nsuo nni mu nyinaa guu frigye mu asram mmiɛnsa.SEM aba no kyerɛɛ sɛ NPs a nsuo nni mu nyinaa nsɛsoɔ ansakra kɛseɛ wɔ asram mmiɛnsa a wɔde siee no mu (Mfonini 4).Bere a wɔsan hyehyɛɛ no ​​wɔ nsuo mu akyi no, NPs no nyinaa kyerɛɛ sɛ EE so tew kakra na ɛyii insulin bɛyɛ kakraa bi (~5%) adi wɔ asram mmiɛnsa a wɔde siee no mu (Table 2).Nanso, sɛ wɔkyekyem pɛpɛɛpɛ a, nanoparticles nyinaa mu nneɛma nketenkete kɛse kɔɔ soro.NPs a wɔde aduru apetepete so a mannitol nka ho no mu nneɛma nketenkete kɛse kɔɔ soro koduu 525 nm, bere a NPs a wɔde petepete so a wɔde petepete so ne nea wɔde mannitol ayɛ no freeze-dried no kɔɔ soro koduu 872 ne 921 nm, sɛnea ɛte biara (Table 2).
Insulin NP ahorow a nsu nnim a wɔde asie asram abiɛsa no nsɛso: (a) SEM mfonini a ɛkyerɛ insulin NP ahorow a wɔde mannitol ayɛ no lyophilized; (b) SEM mfonini a ɛkyerɛ insulin nanoparticles a wɔde aduru a wɔde petepete so a mannitol nni mu; (c) a mannitol SEM mfonini ahorow a ɛfa insulin NP ahorow a wɔde aduru a wɔde petepete so ho nni mu.
Afei nso, wohuu nsuo a ɛtɔ gu insulin nanoparticles a wɔasan ayɛ a wɔde mannitol apetepete na wɔde freeze-dried (Mfonini S2).Eyi betumi afi nsunsuanso akɛse a ɛnsensɛn nsu no mu yiye.Nea afi atifi hɔ aba no nyinaa kyerɛ sɛ ɔkwan a wɔfa so petepete insulin nanoparticles no betumi abɔ insulin nanoparticles ho ban afi nsu a ɛbɛtɔ mu na wobetumi anya insulin nanoparticles a ɛdɔɔso a enni fillers biara anaasɛ nnuru a ɛbɔ cryoprotectants ho ban.
Wɔde pepsin, trypsin, ne α-chymotrypsin sɔɔ insulin a ɛkora so hwɛe wɔ pH = 2.5 medium mu de kyerɛɛ tumi a NPs tumi bɔ ho ban fi enzymatic digestion ho wɔ nsuo a ɛho ayɛ fĩ akyi.Wɔde insulin a wɔkora so wɔ NPs a nsuo nni mu no totoo NPs a wɔasiesie no foforɔ no ho, na wɔde insulin a wɔde ma kwa dii dwuma sɛ aduru a ɛnyɛ papa.Wɔ nhwehwɛmu yi mu no, insulin a wɔde ma kwa no daa insulin a wɔayi afiri mu ntɛmntɛm adi wɔ 4 mu h wɔ enzymatic ayaresa abiɛsa no nyinaa mu (Mfonini 5a–c).Nea ɛne eyi bɔ abira no, insulin yiyi sɔhwɛ a wɔyɛe wɔ NPs a wɔde mannitol ayɛ freeze-dried ne NPs a wɔde mannitol apetepete so anaasɛ wɔmfa nni dwuma no daa ahobammɔ a ɛkorɔn kɛse wɔ saa NPs yi ho wɔ enzymatic digestion ho, a na ɛte sɛ nea ɛwɔ insulin NPs a wɔasiesie no foforo ( mfonini 1).5a-c).Wɔde nanoparticles a ɛwɔ pepsin mu no mmoa so no, ɛdenam nanoparticles a ɛwɔ pepsin mu no mmoa so, trypsin, ne α-chymotrypsin, na wobetumi abɔ insulin bɛboro 50%, 60%, ne 75% ho ban wɔ h 4 mu, sɛnea ɛte biara (Mfonini 5a–c).Saa tumi a ɛbɔ insulin ho ban yi betumi ama hokwan a ɛwɔ hɔ sɛ insulin bɛkɔ mogya no mu kɛse no ayɛ kɛse25.Saa nea efi mu ba yi kyerɛ sɛ sɛ wɔde petepete mannitol ka ho anaasɛ wɔmfa nka ho na wɔde mannitol yɛ freeze-drying betumi akora tumi a NP ahorow tumi bɔ insulin ho ban bere a nsu a ɛho ayɛ fĩ akyi.
Insulin NPs a nsuo nni mu no ho banbɔ ne ne gyae suban: (a) insulin a ɛwɔ pepsin solution mu no ho banbɔ; (b) insulin a ɛwɔ trypsin aduru mu a wɔbɔ ho ban; (c) insulin a wɔde α-chymotrypsin aduru bɔ ho ban; ( d) Suban a NPs a nsuo nni mu no gyae wɔ pH = 2.5 solution mu; (e) NPs a nsuo nni mu a wɔayi no adi wɔ pH = 6.6 solution mu no suban; (f) NPs a nsuo nni mu a wɔayi no adi wɔ pH = 7.0 solution mu no suban.
Wɔde insulin NP a ɛyɛ dry a wɔasiesie no foforo na wɔasan ayɛ no guu buffers ahorow mu (pH = 2.5, 6.6, 7.0) wɔ 37 °C, de yɛɛ pH tebea a ɛwɔ yafunu, duodenum, ne dwensɔtwaa ketewaa a ɛwɔ soro no ho mfonini, de hwehwɛɛ nkɛntɛnso a insulin nya wɔ insulin a wɔko tia so. Release behavior in different environments.Fragment of the gastrointestinal tract.Wɔ pH = 2.5 mu no, NPs a insulin ahyɛ mu ma ne insulin NPs a ɛyɛ dry a wɔasan ayɛ no daa no adi sɛ mfiase no ɛpaapae wɔ dɔnhwerew biako a edi kan no mu, na ɛno akyi no, wɔayi no adi brɛoo wɔ nnɔnhwerew 5 a edi hɔ no mu (Mfonini 5d).Saa a woyi fi adi ntɛmntɛm wɔ mfiase yi yɛ nea ɛda adi kɛse sɛ efi protein molecule ahorow a enni soro a wɔpopa ntɛmntɛm no immobilized in the internal structure of the particle.Wɔ pH = 6.5 no, NPs a wɔde insulin ahyɛ mu ma ne insulin NPs a ɛyɛ dry a wɔasan ayɛ no daa no adi sɛ wɔayi no adi ntɛmntɛm na ɛyɛ brɛoo wɔ 6 h mu, efisɛ na pH a ɛwɔ sɔhwɛ aduru no mu no te sɛ nea ɛwɔ NPs-asiesie aduru no mu (Mfonini 5e).Wɔ pH = 7 mu no, na NPs no ntumi nnyina na ɛkame ayɛ sɛ ɛporɔw koraa wɔ nnɔnhwerew abien a edi kan no mu (Mfonini. 5f).Eyi te saa efisɛ chitosan deprotonation ba wɔ pH a ɛkorɔn mu, na ɛma polymer network a ɛnyɛ den pii na ɛma insulin a wɔde ahyɛ mu ma no fi adi.
Bio nso, insulin NPs a wɔde petepete so a mannitol nka ho no daa no adi sɛ wɔayi no ntɛmntɛm sen NP afoforo a nsuo nni mu no (Mfonini 5d–f).Sɛdeɛ yɛadi kan aka ho asɛm no, insulin NPs a wɔasan ayɛ a wɔayam a mannitol nka ho no daa nsunsuansoɔ ketewaa bi adi.Nneɛma nketenkete ma nsuo a ɛwɔ soro kɛseɛ, enti aduru a ɛfa ho no mu dodoɔ no ara bɛba anaasɛ ɛbɛbɛn aduru no ani ntɛmntɛm
Wɔnam MTT nhwehwɛmu so hwehwɛɛ NPs cytotoxicity mu.Sɛnea wɔada no adi wɔ Mfonini S4 mu no, wohui sɛ NPs a nsuo nni mu nyinaa nni nkɛntɛnsoɔ kɛseɛ biara wɔ nkwammoaa nkwa mu wɔ dodoɔ a ɛyɛ 50–500 μg/ml, a ɛkyerɛ sɛ wɔbɛtumi de NPs a nsuo nni mu nyinaa adi dwuma dwoodwoo de adu ayaresa mfɛnsere no mu.
Mmerɛbo ne akwaa titiriw a insulin nam so yɛ ne nipadua mu dwumadi ahorow.HepG2 nkwammoaa yɛ nnipa hepatoma nkwammoaa a wɔtaa de di dwuma sɛ in vitro hepatocyte uptake model.Ɛha yi, wɔde HepG2 nkwammoaa dii dwuma de hwɛɛ nkwammoaa mu a wɔde NPs a nsu afi mu no fa freeze-drying ne spray-drying akwan so.Cellular uptake by confocal laser scanning using flow cytometry ne anisoadehu akyi nnɔnhwerew pii a wɔde FITC insulin a wontua hwee a ne dodow yɛ 25 μg/mL, NPs a wɔasiesie no foforo a wɔde FITC insulin ahyɛ mu ma ne NPs a nsu afra mu a FITC insulin ahyɛ mu ma wɔ insulin dodow a ɛyɛ pɛ mu akyi no, wɔyɛɛ quantitative microscopy (CLSM) nhwehwɛmu.Wɔsɛee NPs a wɔayam a enni mannitol bere a nsu retu mu na wɔansusuw ho wɔ eyi mu test.Nkwammoaa mu fluorescence ahoɔden a ɛwɔ NPs a wɔasiesie no foforo a wɔde insulin ahyɛ mu, NPs a wɔde mannitol ahyɛ mu ma, ne NPs a wɔde petepete mu a wɔde mannitol ahyɛ mu ne nea enni mu (Mfonini 6a) no boro nea wɔde ma kwa no so mpɛn 4.3, 2.6, 2.4, ne 4.1.FITC-insulin kuw no, sɛnea ɛte biara (Mfonini 6b).Nea efii mu bae yi kyerɛ sɛ insulin a wɔde ahyɛ mu no yɛ nea tumi wom kɛse wɔ nkwammoaa a wɔfa mu sen insulin a wontua hwee, titiriw esiane sɛnea insulin ahyɛ mu ma nanoparticles a wɔyɛe wɔ nhwehwɛmu no mu no kɛse sua nti.
HepG2 nkwammoaa a wɔfa wɔ 4 h a wɔde NP a wɔasiesie no foforo ne NP a nsu nnim ahyɛ mu akyi: (a) FITC-insulin a HepG2 nkwammoaa gye no kyekyɛ.(b) Geometric mean of fluorescence intensities analyzed by flow cytometry (n = 3), *P < 0.05 sɛ wɔde toto insulin a wontua hwee ho a.
Saa ara nso na CLSM mfonini ahorow no kyerɛe sɛ FITC fluorescence ahoɔden a ɛwɔ NPs a wɔasiesie no foforo a wɔde FITC-insulin ahyɛ mu ne NPs a wɔde petepete mu a wɔde insulin ahyɛ mu (a mannitol nni mu) mu no mu yɛ den kɛse sen nea ɛwɔ nhwɛsode afoforo no mu (Mfonini 6a).Bio nso, bere a wɔde mannitol kaa ho no, aduru no mu ntini a ɛkɔ soro no maa nea ɛko tia no yɛɛ kɛse to cellular uptake, resulting in decreased insulin proliferation.Saa nea efii mu bae yi kyerɛ sɛ NP ahorow a wɔde petepete a wɔde petepete so a mannitol nnim no daa nkwammoaa mu ahoɔden a ɛkorɔn sen biara adi efisɛ na wɔn nneɛma nketenkete kɛse sua sen NP ahorow a wɔde nwini ayow bere a wɔasan apete akyi no.
Wɔtɔɔ chitosan (sɛ wɔkyekyɛ mu a, molecule mu duru yɛ 100 KDa, 75–85% deacetylated) fii Sigma-Aldrich.(Oakville, Ontario, Canada).Wɔtɔɔ sodium tripolyphosphate (TPP) fii VWR (Radnor, Pennsylvania, USA).Nnipa insulin a wɔasan ayɛ a wɔde dii dwuma wɔ nhwehwɛmu yi mu no fi Fisher Scientific (Waltham, MA, USA).Fluorescein isothiocyanate (FITC)-a wɔakyerɛw so sɛ nnipa insulin ne 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) na wɔtɔɔ fii Sigma-Aldrich.(Oakville, Ontario, Canada).Wɔnyaa HepG2 nkwammoaa no fii ATCC (Manassas, Virginia, USA).Nneɛma afoforo nyinaa yɛ nhwehwɛmu anaa chromatographic grade.
Siesie CS aduru a ɛyɛ 1 mg/ml denam nsuo a wɔde ahyɛ mu mmienu (DD nsuo) a acetic acid 0.1% wom mu.Siesie TPP ne insulin aduru 1 mg/ml denam DD nsuo ne acetic acid 0.1% a wode bɛpete so.Wɔde polytron PCU-2-110 high speed homogenizer (Brinkmann) na ɛyɛɛ pre-emulsion no Ind. Westbury, NY, USA).Ahosiesie kwan no te sɛ nea edidi so yi: nea edi kan no, wɔde TPP aduru 2ml gu insulin aduru 4ml mu, na wɔde afrafrade no kankan simma 30 na wɔde fra koraa.Afei, wɔde aduru a wɔde afrafra no guu CS aduru no mu nsuo a ɛtɔ gu fam denam syringe so wɔ ahoɔhare a wɔde kanyan no mu (10,000 rpm).Wɔde afrafrade no siee ntɛmntɛm stirring (15,000 rpm) wɔ nsukyenee aguaree mu simma 30, na wɔyɛɛ nsakrae wɔ pH pɔtee bi mu sɛnea ɛbɛyɛ a wobenya cross-linked insulin NPs.To kɔ so homogenize na wɔatew insulin NPs abɔde nketenkete kɛse so no, wɔde sonicated foforo simma 30 wɔ nsukyenee aguaree mu denam probe-type sonicator (UP 200ST, Hielscher Ultrasonics, Teltow, Germany) so.
Wɔde Litesizer 500 (Anton Paar, Graz, Austria) susuw a wɔde Litesizer 500 (Anton Paar, Graz, Austria) dii dwuma de yɛɛ Z-average diameter, polydispersity index (PDI) ne zeta tumi hwɛe sɛ ɛwɔ Z-average diameter, polydispersity index (PDI) ne zeta tumi. Tokyo, Japan), na akyiri yi wɔde Hitachi imaging software (Hitachi, Tokyo, Japan) yɛɛ mfonini ahorow mu nhwehwɛmu.Sɛnea ɛbɛyɛ na wɔasusuw encapsulation efficiency (EE) ne loading capacity (LC) a insulin NPs wɔ mu no, wɔde pipet guu NPs no mu guu ultrafiltration tubes a molecule weight cut-off yɛ 100 kDa na wɔde centrifuged wɔ 500 xg ma 30 min.Wɔde Agilent 1100 Series HPLC nhyehyɛe (Agilent, Santa Clara, California, USA) a ɛyɛ quaternary pump, autosampler, column heater, ne DAD detector na ɛkyerɛɛ insulin dodow a ɛwɔ filtrate no mu.Wɔde C18 column (Zorbax, 3.5 μm, 4.6 mm × 150 mm, Agilent, USA) yɛɛ insulin mu nhwehwɛmu na wɔhunuu no wɔ 214 nm.Na mobile phase no yɛ acetonitrile ne nsuo, a 0.1% TFA wom, gradient ratios firi 10/90 kɔsi 100/0, na ɛtu mmirika simma 10. Wɔde mobile phase no pumped wɔ flow rate a ɛyɛ 1.0 ml/min.The column temperature was set to 20 °C.Calculate the percentages of EE and LC using the equations.(1) and Eq.(2) no yɛ nea ɛfata.
Wɔsɔɔ CS/insulin nsusuiɛ ahodoɔ a ɛfiri 2.0 kɔsi 4.0 hwɛeɛ sɛdeɛ ɛbɛyɛ a insulin NP bɛyɛ papa.Wɔde CS aduru dodoɔ ahodoɔ kaa ho wɔ berɛ a wɔresiesie no, berɛ a wɔmaa insulin/TPP afrafradeɛ no kɔɔ so daa.Wɔsiesiee insulin NPs wɔ pH a ɛyɛ 4.0 kɔsi 6.5 denam ahwɛyie a wɔde hwɛɛ afrafradeɛ no pH so berɛ a wɔde aduru no nyinaa (insulin, TPP ne CS).Wɔhwɛɛ EE ne insulin nanoparticles no kɛseyɛ wɔ pH gyinapɛn ahorow ne CS/insulin mass ratios mu de yɛɛ insulin NPs a wɔhyehyɛ no yiye.
Wɔde insulin NPs a wɔayɛ no yiye no guu aluminium ahina no so na wɔde ntini a wɔde teyp bi akyekye mu den kataa so.Ɛno akyi no, wɔde nsukorade a wɔde nkɔnsɔnkɔnsɔn ayɛ no guu Labconco FreeZone freeze dryer (Labconco, Kansas City, MO, USA) a wɔde tray dryer ahyɛ mu.Wɔde ɔhyew ne vacuum nhyɛso no sii hɔ sɛ -10 °C, 0.350 Torr wɔ nnɔnhwerew 2 a edi kan no mu, na 0 °C ne 0.120 Torr ma 22 h a aka wɔ 24 h no mu na ama wɔanya insulin NPs a ɛyɛ dry.
Wɔde Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Switzerland) dii dwuma de yɛɛ insulin a wɔde ahyɛ mu.Nneɛma a wɔpaw sɛ wɔde bɛma ayow ne: ɔhyew 100 °C, aduan a ɛsen 3 L/min, ne gas a ɛsen 4 L/min.
Wɔde FTIR-ATR spectroscopy na ɛkyerɛɛ insulin NPs ansa na nsuo a ɛho ayɛ fĩ ne akyire.Wɔde Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA) a wɔde amansan ATR sampling accessory (PerkinElmer, Waltham, Massachusetts, USA) ahyɛ mu ma na ɛyɛɛ nanoparticles a nsuo nni mu ne insulin ne chitosan a ɛnyɛ hwee mu nhwehwɛmu.Signal wɔnyaa nkyɛmu firii scan 16 a ɛwɔ resolution a ɛyɛ 4 cm2 wɔ frequency range a ɛyɛ 4000-600 cm2 mu.
Wɔnam SEM mfonini ahorow a ɛfa insulin NPs a wɔayam no nwini ne nea wɔde petepete so a wɔde Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, U.S.A.) kyeree no so na ɛhwɛɛ insulin NP a ɛyɛ dry no nsɛso. Parameter titiriw a wɔde dii dwuma ne voltage 5 keV ne current 30 mA.
Wɔsan guu insulin NPs a nsuo nni mu nyinaa guu nsuo dd mu.Wɔsan de ɔkwan korɔ no ara a yɛadi kan aka ho asɛm no sɔɔ nneɛma nketenkete no kɛseɛ, PDI, EE ne LC hwɛeɛ de hwɛɛ wɔn su wɔ nsuo a ɛho ayɛ fĩ akyi.Wɔsan nso susuw sɛnea anhydroinsulin NPs no gyina pintinn wɔ NPs no su a wɔsɔɔ hwɛe wɔ bere tenten a wɔde asie akyi no so.Wɔ saa nhwehwɛmu yi mu no, wɔde NPs a nsuo atɔ mu akyi nyinaa siee frigye mu mprɛnsa asram.Asram abiɛsa akyi a wɔde sie no, wɔsɔɔ NP ahorow hwɛe sɛ ebia morphological particle size, PDI, EE ne LC.
Fa 5 mL NPs a wɔasan ayɛ no gu 45 mL a ɛwɔ yafunu mu nsu a wɔayɛ no sɛnea ɛte (pH 1.2, a pepsin 1% wom), dwensɔtwaa mu nsu (pH 6.8, a trypsin 1% wom) anaa chymotrypsin aduru (100 g/mL, wɔ phosphate buffer mu, pH 7.8) mu de ahwɛ sɛnea insulin yɛ adwuma yiye wɔ NPs a wɔbɔ ho ban akyi dehydration.Wɔde wɔn too 37°C mu a ahoɔhare a ɛtwetwe adwene a ɛyɛ 100 rpm.Wɔboaboaa ano aduru no ano 500 μL wɔ bere ahorow mu na wɔde HPLC kyerɛɛ insulin dodow.
Wɔnam dialysis bag kwan so (molecular weight cut-off 100 kDa, Spectra Por Inc.) sɔɔ insulin NPs a wɔasiesie no foforo ne nea nsu nnim no suban a wɔde ma wɔ vitro mu hwɛe.Wɔde dialyzed NPs a ɛyɛ dry a wɔasiesie no foforo na wɔasan ayɛ no wɔ nsu a ɛwɔ pH 2.5, pH 6.6, ne pH 7.0 (0.1 M phosphate-buffered saline, 0.1 M phosphate-buffered saline PBS) de yɛ pH tebea a ɛwɔ yafunu, duodenum, ne dwensɔtwaa ketewa a ɛwɔ soro no ho mfonini dialysate.Wɔde HPLC yɛɛ insulin a efĩ a ɛwɔ nsu no mu no mu nhwehwɛmu, na wɔbuu insulin a efi nanoparticles no mu no ntɛmntɛm fii insulin a wɔayi no adi a wontua hwee ne insulin a wɔde ahyɛ nanoparticles no mu nyinaa nsusuwii mu (Equation 3).
Wɔde Dulbecco’s Modified Eagle’s Medium (DMEM) a ɛwɔ awotwaa mu nantwi mogya 10%, 100 IU/mL penicillin, ne 100 μg/mL streptomycin29 na enyinii HepG2 nkwammoaa wɔ nkuku a ne kɛse yɛ mm 60 mu humidity, ne 5% CO2.Wɔ uptake assays mu no, wɔde HepG2 nkwammoaa no guu 1 × 105 cells/ml so wɔ 8-well Nunc Lab-Tek chamber slide system (Thermo Fisher, NY, USA) so.Wɔ cytotoxicity nhwehwɛmu mu no, wɔde aba guu 96-well plates (Corning, NY, USA) mu wɔ density a ɛyɛ 5 × 104 cells/ml.
Wɔde MTT nhwehwɛmu no dii dwuma de hwɛɛ cytotoxicity a ɛwɔ insulin NPs30 a wɔasiesie no foforo na nsuo nni mu no mu.Wɔde HepG2 nkwammoaa guu mprɛte a abura 96 ​​wom mu wɔ density a ɛyɛ 5 × 104 cells/mL mu na wɔde yɛɛ aduru nnafua 7 ansa na wɔresɔ ahwɛ.Wɔde insulin NPs guu nsuo mu dodoɔ ahodoɔ (50 kɔsi 500 μg/mL) wɔ culture medium mu na afei administered to cells.Wɔ nnɔnhwerew 24 akyi no, wɔde PBS hohoroo nkwammoaa no mpɛn 3 na wɔde aduru a 0.5 mg/ml MTT wom guu mu nnɔnhwerew 4 foforo.Wɔhwɛɛ cytotoxicity denam enzymatic reduction a ɛwɔ yellow tetrazolium MTT to purple formazan mu wɔ 570 nm a wɔde Tecan infinite M200 pro spectrophotometer dii dwuma so mprɛte akenkanfo (Tecan, Männedorf, Switzerland).
Wɔnam confocal laser scanning microscopy ne flow cytometry analysis so sɔɔ nkwammoaa mu a wɔfa NPs no hwɛe.Wɔde free FITC-insulin, FITC-insulin-loaded NPs yɛɛ Nunc Lab-Tek chamber slide system no abura biara ho adwuma, na wɔsan yɛɛ 25 μg/mL a nsuo nni mu FITC-insulin NPs wɔ dodoɔ korɔ no ara mu na wɔde too mu maa 4 hours.Wɔhohoroo nkwammoaa mpɛn 3 ne PBS na wɔde 4% paraformaldehyde sii hɔ.Wɔde 4′,6-diamidino-2-phenylindole (DAPI) yɛɛ nuclei.Wɔde Olympus FV1000 laser scanning/two-photon confocal microscope (Olympus, Shinjuku City, Tokyo, Japan) hwɛɛ insulin localization cytometry nhwehwɛmu no, wɔde 10 μg/mL free FITC-insulin, FITC-insulin-loaded NPs, ne resolubilized dehydrated FITC-insulin NPs dodow koro no ara guu mprɛte a abura 96 ​​a wɔde HepG2 nkwammoaa ahyɛ mu no mu na wɔde too mu nnɔnhwerew 4 .Afei 4 h a wɔde ahyɛ mu akyi no, woyii nkwammoaa no na wɔde hohoroo ho mpɛn 3 Wɔde BD LSR II flow cytometer (BD, Franklin Lakes, New Jersey, United States) yɛɛ FBS.5 × 104 nkwammoaa mu nhwehwɛmu wɔ nhwɛsode biara mu.
Wɔde botaeɛ nyinaa akyerɛ sɛ ɛyɛ mfimfini ± standard deviation.Wɔde ɔkwan baako ANOVA anaa t-test a IBM SPSS Statistics 26 for Mac (IBM, Endicott, New York, USA) na ɛsɔɔ ntotoho a ɛda akuo no nyinaa ntam no hwɛeɛ na wɔbuu p < 0.05 sɛ ɛyɛ akontabuo mu adeɛ a ɛho hia.
Saa nhwehwɛmu yi kyerɛ sɛnea spray drying tumi yɛ dehydrate cross-linked chitosan/TPP/insulin nanoparticles a eye reconstitution sɛ wɔde toto standard freeze-drying akwan a wɔde bulking agents anaa cryoprotectants tumi ne adesoa tumi a ɛkorɔn di dwuma.The optimized insulin nanoparticles yielded an average particle size of 318 nm and an encapsulation efficiency of 99.4%.SEM ne FTIR aba wɔ nsuo a wɔayi afiri mu akyi no kyerɛɛ sɛ wɔkuraa kurukuruwa nhyehyɛeɛ no mu wɔ NPs a wɔde aduru apetepete mu a mannitol wom ne deɛ enni mu nko ara mu na wɔde mannitol ayɛ no, nanso NP a wɔde aduru apetepete so a mannitol nni mu no porɔeɛ berɛ a nsuo a ɛwɔ mu no rekɔ mu.Wɔ reconstitution tumi sɔhwɛ no mu no, insulin nanoparticles a wɔde petepete mu a wɔamfa mannitol nka ho no kyerɛɛ sɛ mean particle kɛseɛ sua koraa na ɛwɔ adesoa a ɛkorɔn sen biara upon reconstitution.The release suban a saa dehydrated NPs yi nyinaa kyerɛ sɛ wɔayi no ntɛmntɛm wɔ solutions a pH = 2.5 ne pH = 7, na ɛyɛ den yiye wɔ solution of pH = 6.5.Sɛ wɔde toto afoforo redissolved dehydrated NPs, NPs spray-dried a mannitol kyerɛe sɛ gyae ntɛmntɛm.This aba no ne nea wohui wɔ cellular uptake assay, sɛnea Ɛkame ayɛ sɛ NPs a wɔde aduru apetepete so a mannitol nni hɔ no kuraa nkwammoaa mu a wɔde gye NP ahorow a wɔasiesie no foforo no mu koraa.Saa nea efii mu bae yi kyerɛ sɛ insulin nanoparticles a ɛyɛ dry a wɔde petepete a mannitol nnim a wɔde petepete so asiesie no fata kɛse ma wɔkɔ so yɛ ho adwuma ma ɛbɛyɛ nnuru afoforo a nsu nnim, te sɛ nnuru a wɔde ano anom anaasɛ bioadhesive films.
Esiane adwene mu agyapadeɛ ho nsɛm nti, datasets a wɔayɛ ne/anaasɛ wɔayɛ mu nhwehwɛmu wɔ mprempren nhwehwɛmu no mu no, ɔmanfoɔ ntumi nhunu, nanso sɛ wɔbisa a ɛfata a, wɔbɛtumi anya bi afiri akyerɛwfoɔ a ɛfa ho no hɔ.
Kagan, A. Asikreyare a ɛtɔ so mmienu: asetena ne nyansahu mu mfitiaseɛ, aduruyɛ mu nsɛnnennen, ne nea ɛkyerɛ ma ayarefoɔ ne afoforɔ.(McFarlane, 2009).
Singh, AP, Guo, Y., Singh, A., Xie, W. & Jiang, P. Insulin a wɔde hyɛ nipadua mu no nkɔso: so mprempren wobetumi de anom?J. Nnuruyɛ.bio-nnuruyɛ.akoraeɛ.1, 74–92 (2019).
Wong, CY, Al-Salami, H. & Dass, CR Nnansa yi nkɔso a aba wɔ liposome a wɔde insulin anom ahyɛ mu ma a wɔde sa asikreyare ho.Nkyerɛase.J. Nnuruyɛ.549, 201–217 (2018).