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Mkpụrụ ndụ ihe nketa insulin (NPs) nwere nnukwu ibu dị elu ahụtala ojiji dị iche iche n'ụdị usoro onunu ogwu dị iche iche. Ọrụ a na-achọ inyocha mmetụta nke usoro nkụ oyi na nkụ na-agba na nhazi nke mkpụrụ ndụ ihe nketa chitosan nke insulin nwere, ma ọ bụ na-enweghị mannitol dị ka ihe nchebe. Anyị nyochakwara ịdị mma nke mkpụrụ ndụ ihe nketa ndị a site na ịgbagharị ha ọzọ. Tupu nkụ mmiri, nha mkpụrụ ndụ ihe nketa nke chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles ka mma ka ọ bụrụ 318 nm, PDI bụ 0.18, arụmọrụ encapsulation bụ 99.4%, na ibu ya bụ 25.01%. Mgbe emechara ya, mkpụrụ ndụ ihe nketa niile, ma e wezụga ndị e ji usoro nkụ oyi na-agbapụta na-ejighị mannitol, nọgidere na-enwe nhazi mkpụrụ ndụ ihe nketa ha. Ma e jiri ya tụnyere mkpụrụ ndụ ihe nketa ndị nwere mannitol nke mmiri mmiri nke mmiri mmiri nke mannitol nke mmiri mmiri nke mannitol mere, mkpụrụ ndụ ihe nketa ndị a kpọrọ nkụ nke na-enweghị ịgbapụta gosikwara obere nha mkpụrụ ndụ ihe nketa kacha nta (376 nm) na ọdịnaya ibu kachasị elu (25.02%) yana ihe yiri ya. Ọnụego mkpuchi (98.7%) na PDI (0.20) site na usoro ịkpọ nkụ ma ọ bụ ịkpọ nkụ. Mkpụrụ ndụ ihe nketa kpọrọ nkụ site na ịkpọ nkụ na-enweghị mannitol mekwara ka e wepụta insulin ngwa ngwa na arụmọrụ kachasị elu nke nnabata mkpụrụ ndụ. Ọrụ a na-egosi na nkụ ahịhịa nwere ike ime ka mkpụrụ ndụ ihe nketa insulin ghara ịmịkọrọ mmiri na-enweghị mkpa maka cryoprotectants ma e jiri ya tụnyere ụzọ nkụ ahịhịa nkịtị, na-emepụta ikike ibu ibu ka ukwuu, obere ihe achọrọ maka mgbakwunye na ọnụ ahịa ọrụ bara uru dị ukwuu.
Kemgbe nchọpụta ya na 19221,2,3, insulin na nkwadebe ọgwụ ya azọpụtala ndụ ndị ọrịa nwere ụdị shuga 1 (T1DM) na ụdị shuga 2 (T1DM). Agbanyeghị, n'ihi ihe ndị dị na ya dị ka protein dị elu nke molekul, a na-achịkọta insulin ngwa ngwa, na-agbaji ya site na enzymes proteolytic, ma wepụ ya site na mmetụta mbụ-pass. Ndị mmadụ chọpụtara na ha nwere ụdị shuga 1 chọrọ ọgwụ insulin ruo ndụ ha niile. Ọtụtụ ndị ọrịa chọpụtara na ha nwere ụdị shuga 2 na mbụ na-achọkwa ọgwụ insulin ogologo oge. Ọgwụ insulin kwa ụbọchị bụ isi iyi dị njọ nke ihe mgbu na ahụ erughị ala kwa ụbọchị maka ndị a, yana mmetụta ọjọọ na ahụike uche. N'ihi ya, a na-amụ ọtụtụ ụdị ọgwụ insulin ndị ọzọ nke na-akpata obere ahụ erughị ala, dị ka ọgwụ insulin ọnụ, ebe ha nwere ike iweghachi ịdị mma nke ndụ nke ihe dị ka ijeri mmadụ 5 nwere ọrịa shuga n'ụwa niile.
Teknụzụ Nanoparticle enyela ọganihu dị ukwuu n'ọchịchọ iji insulin ọnụ 4,6,7. Otu nke na-ekpuchi ma na-echebe insulin site na mmebi maka nnyefe ezubere iche na ebe ahụ ụfọdụ. Agbanyeghị, ojiji nke usoro nanoparticle nwere ọtụtụ mmachi, ọkachasị n'ihi nsogbu nkwụsi ike nke nkwụsịtụ ihe. Ụfọdụ nchịkọta nwere ike ime n'oge nchekwa, nke na-ebelata nnweta nke nanoparticles nke insulin jupụtara na ya8. Na mgbakwunye, a ga-atụlekwa nkwụsi ike kemịkalụ nke matrix polymer nke nanoparticles na insulin iji hụ na insulin nanoparticles (NPs) kwụsiri ike. Ugbu a, teknụzụ ịkpọ nkụ oyi bụ ọkọlọtọ ọla edo maka ịmepụta NPs kwụsiri ike ebe ọ na-egbochi mgbanwe achọghị n'oge nchekwa9.
Agbanyeghị, ịkpọ nkụ oyi chọrọ mgbakwunye nke cryoprotectants iji gbochie nhazi okirikiri nke NPs ka nrụgide mekaniki nke kristal ice ghara imetụta ya. Nke a na-ebelata ibu nke insulin nanoparticles nke ukwuu mgbe lyophilization gasịrị, ebe cryoprotectant na-ejide ọtụtụ oke ibu. Ya mere, a na-ahụkarị insulin NPs emepụtara na-adịghị mma maka imepụta usoro ntụ ntụ akọrọ, dị ka mbadamba ọnụ na ihe nkiri ọnụ, n'ihi mkpa ọ dị maka nnukwu nanoparticles akọrọ iji nweta windo ọgwụgwọ nke insulin.
Ịkpọcha ihe n'ime mmiri bụ usoro a ma ama ma dị ọnụ ala maka imepụta ntụ ntụ akọrọ site na usoro mmiri mmiri na ụlọ ọrụ ọgwụ10,11. Njikwa usoro nhazi ihe n'ime mmiri na-enye ohere maka mkpuchi kwesịrị ekwesị nke ọtụtụ ihe ndị na-arụ ọrụ n'ime mmiri 12, 13. Ọzọkwa, ọ ghọwo usoro dị irè maka nkwadebe nke protein ndị a kpuchiri ekpuchi maka ojiji ọnụ. N'oge nkụcha ihe n'ime mmiri, mmiri na-apụ ngwa ngwa, nke na-enyere aka idobe okpomọkụ nke isi ihe n'ime mmiri dị ala11,14, na-eme ka ojiji ya nwee ike itinye ihe ndị na-emetụta okpomọkụ. Tupu nkụcha ihe n'ime mmiri, a ga-eme ka ihe mkpuchi ahụ yie nke ọma na ngwọta nwere ihe ndị a kpuchiri ekpuchi11,14. N'adịghị ka nkụcha ihe n'ime mmiri oyi, nhazi tupu itinye ihe n'ime mmiri na nkụcha ihe na-eme ka arụmọrụ nchekwa dịkwuo mma n'oge nkụcha ihe. Ebe ọ bụ na usoro mkpuchi nkụcha ihe n'ime mmiri anaghị achọ ihe nchebe, enwere ike iji nkụcha ihe n'ime mmiri mepụta NP kpọrọ nkụ nwere nnukwu ibu.
Nnyocha a na-akọ maka mmepụta nke NPs nke insulin jupụtara site na njikọ chitosan na sodium tripolyphosphate site na iji usoro ion gel. Gelation Ion bụ usoro nkwadebe nke na-enye ohere mmepụta nke nanoparticles site na mmekọrịta electrostatic n'etiti ụdị ionic abụọ ma ọ bụ karịa n'okpuru ọnọdụ ụfọdụ. E jiri usoro ịkpọ nkụ oyi na-akpọ nkụ na-agba mmiri iji mee ka mmiri dị na chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles kachasị mma. Mgbe nkụchara ahụ gasịrị, SEM nyochachara ọdịdị ha. A tụlere ikike njikọta ha site na ịtụ nkesa nha ha, chajị elu, PDI, arụmọrụ encapsulation, na ọdịnaya ibu. A tụlekwara ịdị mma nke nanoparticles resolubilized nke usoro mmiri dị iche iche mepụtara site na ịtụnyere nchebe insulin ha, omume ntọhapụ, na ịdị irè nke nnabata sel.
pH nke ngwọta agwakọtara na oke nke chitosan na insulin bụ ihe abụọ dị mkpa na-emetụta nha na arụmọrụ encapsulation (EE) nke NPs ikpeazụ, ebe ha na-emetụta usoro gelation ionotropic ozugbo. Egosiri na pH nke ngwọta agwakọtara nwere njikọ dị ukwuu na nha na arụmọrụ encapsulation (Fig. 1a). Dịka egosiri na Fig. 1a, ka pH mụbara site na 4.0 ruo 6.0, nkezi nha mkpụrụ (nm) belatara na EE mụbara nke ukwuu, ebe mgbe pH mụbara ruo 6.5, nkezi nha mkpụrụ ahụ malitere ịbawanye na EE ka na-agbanweghị agbanwe. Ka oke chitosan na insulin na-abawanye, nkezi nha mkpụrụ ahụ na-abawanyekwa. Ọzọkwa, a hụghị mgbanwe na EE mgbe a kwadebere nanoparticles na oke oke nke chitosan/insulin karịa 2.5:1 (w/w) (Fig. 1b). Ya mere, ejiri ọnọdụ nkwadebe kacha mma na ọmụmụ ihe a (pH 6.0, oke oke chitosan/insulin nke 2.5:1) kwadebe Nanoparticles ndị nwere insulin maka ọmụmụ ihe ọzọ. N'okpuru ọnọdụ nkwadebe a, a haziri nkezi nha nke insulin nanoparticles ka ọ bụrụ 318 nm (Foto 1c), PDI bụ 0.18, arụmọrụ itinye ihe bụ 99.4%, ikike zeta bụ 9.8 mv, ibu insulin bụkwa 25.01% (m/m). Dabere na nsonaazụ transmission electron microscopy (TEM), nanoparticles kachasị mma bụ ihe dị ka gburugburu na nke dị iche na nha nha (Foto 1d).
Nhazi paramita nke insulin nanoparticles: (a) mmetụta nke pH na dayameta nkezi na arụmọrụ encapsulation (EE) nke insulin nanoparticles (akwadoro na oke nha 5:1 nke chitosan na insulin); (b) chitosan na Mmetụta nke oke oke nke insulin na dayameta nkezi na arụmọrụ encapsulation (EE) nke insulin NPs (akwadoro na pH 6); (c) nkesa nha obere nke insulin nanoparticles kachasị mma; (d) TEM micrograph nke insulin NPs kachasị mma.
A maara nke ọma na chitosan bụ polyelectrolyte na-adịghị ike nke nwere pKa nke 6.5. A na-enweta ya nke ọma na acidic media n'ihi na isi amino otu ya bụ proton site na hydrogen ions15. Ya mere, a na-ejikarị ya eme ihe dị ka ihe na-ebu ibu iji kpuchie macromolecules na-adịghị mma. N'ọmụmụ ihe a, ejiri chitosan kpuchie insulin na isi isoelectric nke 5.3. Ebe ọ bụ na a na-eji chitosan eme ihe dị ka ihe mkpuchi, na mmụba nke nha ya, ọkpụrụkpụ nke oyi akwa mpụta nke nanoparticles na-abawanye n'otu aka ahụ, na-ebute nha nkezi nkezi. Na mgbakwunye, ọkwa dị elu nke chitosan nwere ike itinye insulin karịa. N'ọnọdụ anyị, EE kachasị elu mgbe oke chitosan na insulin ruru 2.5:1, enweghịkwa mgbanwe dị ukwuu na EE mgbe oke ahụ nọgidere na-abawanye.
E wezụga oke nke chitosan na insulin, pH rụkwara ọrụ dị mkpa na nkwadebe nke NPs.Gan et al. 17 mụrụ mmetụta nke pH na nha nke chitosan nanoparticles. Ha chọpụtara mbelata na-aga n'ihu na nha nke ihe ruru ruo mgbe pH ruru 6.0, a hụkwara mmụba dị ukwuu na nha nke ihe ruru na pH > 6.0, nke kwekọrọ na ihe anyị hụrụ. Ihe a bụ n'ihi eziokwu ahụ bụ na ka pH na-abawanye, molekul insulin na-enweta ụgwọ elu na-adịghị mma, si otú a na-akwado mmekọrịta electrostatic na mgbagwoju anya chitosan/sodium tripolyphosphate (TPP), na-ebute obere nha nke ihe ruru na EE dị elu. Agbanyeghị, mgbe agbanwere pH ka ọ bụrụ 6.5, e wepụrụ otu amino dị na chitosan, nke butere mpịachi chitosan. Ya mere, pH dị elu na-eme ka obere ikpughe nke amino ions na TPP na insulin, na-ebute obere njikọ, nnukwu nha nkezi ikpeazụ na obere EE.
Nnyocha nke njirimara ọdịdị nke NP ndị a kpọnwụrụ akpọnwụ na ndị a gbara mmiri nwere ike iduzi nhọrọ nke usoro ka mma nke ịkpọnwụ akpọnwụ na nhazi ntụ ntụ. Usoro a họọrọ kwesịrị inye nkwụsi ike ọgwụ, ọdịdị otu nke ihe dị iche iche, ibu ọgwụ dị elu na ezigbo mgbaze na ngwọta mbụ. N'ọmụmụ ihe a, iji tụnyere usoro abụọ ahụ nke ọma, ejiri NP insulin nwere ma ọ bụ na-enweghị 1% mannitol n'oge akpọnwụ akpọnwụ. A na-eji Mannitol dị ka ihe na-eme ka ihe dị arọ ma ọ bụ ihe nchebe na ụdị ntụ ntụ dị iche iche maka ịkpọnwụ akpọnwụ na ịkpọnwụ akpọnwụ. Maka nanoparticles insulin lyophilized na-enweghị mannitol, dịka egosiri na Foto 2a, a hụrụ nhazi ntụ ntụ dị oke oghere nke nwere nnukwu elu, nke na-adịghị agbanwe agbanwe na nke siri ike n'okpuru nyocha electron microscopy (SEM). Achọpụtara obere ihe dị iche iche na ntụ ahụ mgbe akpọnwụchara akpọnwụ (Fig. 2e). Nsonaazụ ndị a gosiri na ọtụtụ NPs gbawara n'oge akpọnwụ akpọnwụ na-enweghị cryoprotectant ọ bụla. Maka nanoparticles insulin ndị a kpọnwụrụ akpọnwụ na ndị a gbara mmiri nke nwere 1% mannitol, a hụrụ nanoparticles gburugburu nwere elu dị ire ụtọ (Fig. 2b,d,f,h). Insulin nanoparticles a na-agba nkụ na-enweghị mannitol nọgidere bụrụ okirikiri mana ọ gbagọrọ agbagọ n'elu (Foto 2c). A na-atụle elu dị gburugburu na nke gbagọrọ agbagọ n'omume ntọhapụ na nnwale nnabata sel n'okpuru. Dabere na ọdịdị a na-ahụ anya nke NPs akpọnwụrụ akpọnwụ, ma NPs a na-agba nkụ na-enweghị mannitol na NPs a kpọnwụrụ akpọnwụ na mannitol a kpọnwụrụ akpọnwụ na-enye ntụ ntụ NPs dị mma (Foto 2f,g,h). Ka oke elu dị n'etiti elu ihe ndị ahụ buru ibu, otú ahụ ka ọ na-agbaze elu ma ya mere ọnụego ntọhapụ dị elu.
Usoro nke insulin NPs dị iche iche a gbahapụrụ agbahapụ: (a) Foto SEM nke insulin NPs a gbahapụrụ agbahapụ na-enweghị mannitol; (b) Foto SEM nke insulin NPs a gbahapụrụ agbahapụ na mannitol; (c) insulin NPs a gbahapụrụ agbahapụ na-enweghị mannitol SEM Foto nke ; (d) Foto SEM nke insulin NPs a gbahapụrụ agbahapụ na mannitol; (e) onyonyo nke insulin NPs a gbahapụrụ agbahapụ na-enweghị mannitol; (f) onyonyo nke insulin NPs a gbahapụrụ agbahapụ na mannitol; (g) Foto nke insulin NPs a gbahapụrụ agbahapụ na-enweghị mannitol; (h) onyonyo nke insulin NPs a gbahapụrụ agbahapụ na mannitol.
N'oge a na-akpọ nkụ na friza, mannitol na-arụ ọrụ dị ka ihe nchebe, na-edebe NPs n'ụdị amorphous ma na-egbochi mmebi site na kristal ice19. N'ụzọ dị iche, ọ dịghị usoro oyi n'oge a na-akpọ nkụ. Ya mere, ọ dịghị mkpa mannitol n'ụzọ a. N'ezie, NPs a kpọrọ nkụ na-enweghị mannitol na-enye NPs ka mma dịka akọwara na mbụ. Agbanyeghị, mannitol ka nwere ike ịrụ ọrụ dị ka ihe na-emeju usoro nkụ iji nye NPs nhazi gburugburu karịa20 (Foto 2d), nke na-enyere aka inweta omume ntọhapụ otu nke NPs a kpọchiri akpọchi. Na mgbakwunye, o doro anya na enwere ike ịchọpụta ụfọdụ obere ihe na ma insulin NPs a kpọchiri akpọchi na nke a kpọchiri akpọchi nwere mannitol (Foto 2b,d), nke nwere ike ịbụ n'ihi mkpokọta mannitol na isi ihe ahụ yana insulin a kpọchiri akpọchi. Iji. Chitosan oyi akwa. Ọ dị mkpa ịmara na n'ọmụmụ ihe a, iji hụ na nhazi okirikiri ahụ nọgidere na-adịgide mgbe mmiri gwụchara, a na-edebe oke mannitol na chitosan na 5:1, ka nnukwu ihe na-ejupụtakwa wee nwee ike ibuli nha nke NP kpọrọ nkụ.
Nlegharị anya Fourier transform infrared attenuated total reflection (FTIR-ATR) gosiri ngwakọta anụ ahụ nke insulin efu, chitosan, chitosan, TPP na insulin. E ji FTIR-ATR spectroscopy mara NP niile gbawara agbawa. N'ụzọ dị mkpa, a hụrụ ike band nke 1641, 1543 na 1412 cm-1 na NPs e tinyere n'ime oyi kpọnwụrụ akpọnwụ na mannitol na NPs e tinyere n'ime oyi kpọnwụrụ akpọnwụ na mannitol na-agba ya na-enweghị mannitol (Foto 3). Dịka akọwara na mbụ, mmụba ike ndị a jikọtara ya na njikọ dị n'etiti chitosan, TPP na insulin. Nnyocha nke mmekọrịta dị n'etiti chitosan na insulin gosiri na na FTIR spectra nke nanoparticles chitosan nke insulin jupụtara, eriri chitosan jikọtara ya na nke insulin, na-eme ka ike carbonyl (1641 cm-1) na eriri amine (1543 cm-1) dịkwuo elu. Otu tripolyphosphate nke TPP jikọtara na otu ammonium na chitosan, na-emepụta eriri na 1412 cm-1.
FTIR-ATR spectra nke insulin efu, chitosan, ngwakọta anụ ahụ nke chitosan/TPP/insulin na NPs nke usoro dị iche iche kpọrọ nkụ.
Ọzọkwa, nsonaazụ ndị a kwekọrọ na nke egosiri na SEM, nke gosiri na NP ndị e tinyere n'ime ya nọgidere na-adị mma ma mgbe a fesara ya ma kpọchie ya na mannitol, mana na enweghị mannitol, naanị nkụ mmiri na-emepụta ihe ndị e tinyere n'ime ya. N'ụzọ dị iche, nsonaazụ FTIR-ATR nke NP ndị e tinyere n'ime ya kpọchiri akpọchi na-enweghị mannitol yiri nke chitosan, TPP, na insulin. Nsonaazụ a na-egosi na njikọ dị n'etiti chitosan, TPP na insulin adịghịzi na NP ndị e tinyere n'ime ya kpọchiri akpọchi na-enweghị mannitol. E bibiri nhazi NPs n'oge nkụ mmiri na-enweghị cryoprotectant, nke enwere ike ịhụ na nsonaazụ SEM (Foto 2a). Dabere na ọdịdị na nsonaazụ FTIR nke insulin NP ndị e tinyere n'ime ya kpọchiri akpọchi, e jiri naanị NP ndị e tinyere n'ime ya kpọchiri akpọchi, ndị e tinyere n'ime ya kpọchiri akpọchi, na ndị na-enweghị mannitol mee ihe maka nnwale nhazigharị na NP ndị na-enweghị mannitol n'ihi mmebi nke NP ndị na-enweghị mannitol n'oge nkụ mmiri. tụlee.
A na-eji mmiri agwụcha maka nchekwa ogologo oge na nhazigharị n'ime usoro ndị ọzọ. Ike nke NP kpọrọ nkụ ịhazigharị mgbe echekwara dị oke mkpa maka ojiji ha na usoro dị iche iche dịka mbadamba na ihe nkiri. Anyị hụrụ na nkezi nha nke insulin NPs a na-agba mmiri na-enweghị mannitol mụbara naanị ntakịrị mgbe agwakọtachara ya. N'aka nke ọzọ, nha nke insulin nanoparticles a na-agba mmiri na nke a na-akpọ mannitol mụbara nke ukwuu (Tebụl 1). Agbanweghị PDI na EE nke ukwuu (p > 0.05) mgbe ejikọtara NPs niile na ọmụmụ ihe a (Tebụl 1). Nsonaazụ a na-egosi na ọtụtụ n'ime ihe ndị ahụ nọgidere na-adị ọcha mgbe agbanyụchara ya ọzọ. Agbanyeghị, mgbakwunye nke mannitol mere ka ibu insulin belata nke ukwuu nke mannitol nanoparticles a na-akpọ lyophilized na nke a na-akpọ (Tebụl 1). N'ụzọ dị iche, ọdịnaya insulin nke NPs a na-agba mmiri na-enweghị mannitol nọgidere otu ihe ahụ na mbụ (Tebụl 1).
A maara nke ọma na ibu nke nanoparticles dị oke mkpa mgbe ejiri ya maka ebumnuche nnyefe ọgwụ. Maka NPs nwere obere ibu, a chọrọ nnukwu ihe iji ruo n'ókè ọgwụgwọ. Agbanyeghị, oke viscosity nke oke NP dị otú ahụ na-eduga na nsogbu na ihe isi ike na ojiji ọnụ na usoro ọgwụ a na-agba n'ime, n'otu n'otu 22. Na mgbakwunye, enwere ike iji insulin NPs mee mbadamba na viscous biofilms23, 24, nke chọrọ iji nnukwu NPs na ọkwa ibu dị ala, na-ebute nnukwu mbadamba na biofilm dị arọ nke na-adabaghị maka ojiji ọnụ. Ya mere, NPs ndị gbazere agbaze nwere nnukwu ibu insulin dị oke mma. Nsonaazụ anyị na-egosi na nnukwu ibu insulin nke NPs ndị a kpọrọ nkụ na-enweghị mannitol nwere ike inye ọtụtụ uru mara mma maka ụzọ nnyefe ndị ọzọ a.
E debere NP niile e tinyere n'ime friji ruo ọnwa atọ. Nsonaazụ SEM gosiri na ọdịdị nke NP niile e tinyere n'ime mmiri agbanweghị nke ukwuu n'oge nchekwa ọnwa atọ (Foto 4). Mgbe e megharịrị ha na mmiri, NP niile gosipụtara obere mbelata na EE ma wepụta ihe dị ka obere ego (~5%) nke insulin n'oge nchekwa ọnwa atọ (Tebụl 2). Agbanyeghị, nkezi nha nke nanoparticles niile mụbara. Nha nke NP ndị a na-agba n'ime mmiri na-enweghị mannitol mụbara ruo 525 nm, ebe nke NP ndị a kpọrọ nkụ na ndị a kpọnwụrụ akpọnwụ na mannitol mụbara ruo 872 na 921 nm, n'otu n'otu (Tebụl 2).
Usoro nke ụdị insulin NP dị iche iche a gbahapụrụ agbahapụ echekwara ruo ọnwa atọ: (a) Foto SEM nke insulin NPs a gbahapụrụ agbahapụ nwere mannitol; (b) Foto SEM nke insulin nanoparticles a gbahapụrụ agbahapụ na-enweghị mannitol; (c) na-enweghị mannitol Foto SEM nke insulin NPs a gbahapụrụ agbahapụ.
Ọzọkwa, a hụrụ ihe ndị na-eme ka mmiri ghara ịdị n'ime mmiri insulin nanoparticles e ji mannitol mee ma kpọnwụọ (Foto S2). Nke a nwere ike ịbụ n'ihi nnukwu ihe ndị na-anaghị ejide nke ọma n'ime mmiri. Nsonaazụ niile dị n'elu na-egosi na usoro nkụcha mmiri nwere ike ichebe insulin nanoparticles site na mmiri ịkpọ nkụ nakwa na enwere ike nweta nnukwu ibu nke insulin nanoparticles na-enweghị ihe mgbakwunye ma ọ bụ cryoprotectants ọ bụla.
A nwalere njide insulin na pH = 2.5 nke etiti site na iji pepsin, trypsin, na α-chymotrypsin iji gosipụta ikike nchekwa nke NPs megide mgbaze enzymatic mgbe akpọnwụchara. A tụnyere njigide insulin nke NPs akpọnwụrụ akpọnwụ na nke NPs a kwadebere ọhụrụ, a na-ejikwa insulin efu dị ka njikwa na-adịghị mma. Na nnyocha a, insulin efu gosipụtara mwepụ insulin ngwa ngwa n'ime awa 4 na ọgwụgwọ enzymatic atọ niile (Fig. 5a–c). N'ụzọ dị iche, nnwale mwepụ insulin nke NPs akpọnwụrụ akpọnwụ na mannitol na NPs a gbara nkụ ma ọ bụ na-enweghị mannitol gosipụtara nchebe dị elu nke NPs ndị a megide mgbaze enzymatic, nke yiri nke insulin NPs a kwadebere ọhụrụ (Foto 1).5a-c). Site n'enyemaka nke nanoparticles na pepsin, trypsin, na α-chymotrypsin, enwere ike ichebe ihe karịrị 50%, 60%, na 75% nke insulin n'ime awa 4, n'otu n'otu (Foto 5a–c). Ike nchekwa insulin a nwere ike ime ka ohere nke insulin dị elu dịkwuo elu. Ntinye n'ime ọbara25. Nsonaazụ ndị a na-egosi na ịgba mmiri nkụ ma ọ bụ na-enweghị mannitol na ịkpọ nkụ na mannitol nwere ike ichekwa ikike nchekwa insulin nke NPs mgbe akpọnwụchara.
Nchedo na omume ntọhapụ nke insulin NPs gbawara agbawa: (a) nchekwa insulin na mmiri pepsin; (b) nchekwa insulin na mmiri trypsin; (c) nchekwa insulin site na mmiri α-chymotrypsin; (d) Omume ntọhapụ nke NPs gbawara agbawa na mmiri pH = mmiri 2.5; (e) omume ntọhapụ nke NPs gbawara agbawa na mmiri pH = mmiri 6.6; (f) omume ntọhapụ nke NPs gbawara agbawa na mmiri pH = mmiri 7.0.
E tinyere NPs nke insulin kpọrọ nkụ e kwadebere ọhụrụ ma kpụgharịa n'ime ọtụtụ ihe nchekwa (pH = 2.5, 6.6, 7.0) na 37 °C, na-eme ka gburugburu pH nke afọ, duodenum, na eriri afọ dị elu, iji lelee mmetụta insulin nwere na iguzogide insulin. Omume mwepụta n'ebe dị iche iche. Nkewa nke eriri afọ. Na pH = 2.5, NP ndị nwere insulin na insulin kpọrọ nkụ NP ndị a gbanyere mkpọrọgwụ gosipụtara mwepụta mbụ n'ime otu awa mbụ, wee soro ya nwayọ nwayọ n'ime awa ise sochirinụ (Foto 5d). Mwepụta ngwa ngwa a na mbido bụ ihe ga-esi na mwepụ ngwa ngwa nke molekul protein n'elu nke na-anaghị emegharị ahụ kpamkpam n'ime nhazi nke ihe ahụ. Na pH = 6.5, NP ndị nwere insulin na insulin kpọrọ nkụ NP ndị a gbanyere mkpọrọgwụ gosipụtara mwepụta dị nro ma dị nwayọ n'ime awa 6, ebe pH nke ngwọta nnwale ahụ yiri nke ngwọta NP ndị a kwadebere (Foto 5e). Na pH = 7, NP ndị a adịghị akwụsi ike ma fọrọ nke nta ka ọ gbazee kpamkpam n'ime awa abụọ mbụ (Foto 5f). Nke a bụ n'ihi na mwepụ nke chitosan na-eme na pH dị elu, nke na-ebute netwọk polymer na-adịghị obere na ntọhapụ nke insulin ebugoro.
Ọzọkwa, ọgwụ insulin NPs a na-agba n'enweghị mannitol gosiri na ọ na-ewepụta ngwa ngwa karịa ọgwụ ndị ọzọ a gbahapụrụ agbahapụ (Foto 5d–f). Dịka akọwara na mbụ, ọgwụ insulin NPs a kpọgharịrị akpọnwụghị na-enweghị mannitol gosiri obere nha mkpụrụ ahụ. Obere mkpụrụ ahụ na-enye nnukwu oke elu, yabụ ọtụtụ ọgwụ ndị dị mkpa ga-adị n'elu mkpụrụ ahụ ma ọ bụ nso ya, nke na-eme ka ọgwụ ahụ dị ngwa ngwa26.
E jiri nyocha MTT nyochaa nsí nke NPs. Dịka egosiri na Foto S4, achọpụtara na NPs niile akpọnwụrụ akpọnwụ enweghị mmetụta dị ukwuu na ndụ mkpụrụ ndụ na njupụta nke 50-500 μg/ml, nke na-egosi na enwere ike iji NPs niile akpọnwụrụ akpọnwụ ruo na windo ọgwụgwọ n'enweghị nsogbu.
Imeju bụ akụkụ ahụ bụ isi nke insulin na-arụ ọrụ ya n'ahụ. Mkpụrụ ndụ HepG2 bụ ahịrị mkpụrụ ndụ hepatoma mmadụ nke a na-ejikarị eme ihe dị ka ụdị nnabata hepatocyte in vitro. N'ebe a, e jiri mkpụrụ ndụ HepG2 nyochaa nnabata mkpụrụ ndụ nke NPs ndị gbazere agbaze site na iji usoro ịkpọ nkụ na ịgba mmiri. Ntinye sel site na nyocha laser confocal site na iji cytometry flow na ọhụụ mgbe awa ole na ole nke incubation na insulin FITC n'efu na mkpokọta nke 25 μg/mL, FITC edoziri ọhụrụ na NPs ndị gbazere agbaze na FITC insulin-loaded na mkpokọta insulin hà nhata E mere nlele microscopy quantitative (CLSM). E bibiri NPs ndị gbazere agbaze na-enweghị mannitol n'oge nkụ mmiri ma enyochaghị ha na nnwale a. Ike fluorescence nke NPs ndị e tinyere insulin ọhụrụ, NPs ndị e tinyere lyophilized na mannitol, na NPs ndị a gbara nkụ na-enweghị mannitol (Fig. 6a) dị okpukpu 4.3, 2.6, 2.4, na okpukpu 4.1 karịa nke efu. otu. FITC-insulin, n'otu n'otu (Foto 6b). Nsonaazụ ndị a na-egosi na insulin e tinyere n'ime akpa nwere ike karịa n'ịnara mkpụrụ ndụ karịa insulin n'efu, ọkachasị n'ihi obere nha nke nanoparticles insulin jupụtara na ya nke emere na ọmụmụ ihe ahụ.
Ntinye mkpụrụ ndụ HepG2 mgbe awa anọ gachara site na iji NPs ndị a kwadebere ọhụrụ na NP ndị akpọnwụrụ akpọnwụ: (a) Nkesa nke nnabata FITC-insulin site na mkpụrụ ndụ HepG2.(b) Nkezi geometric nke ike fluorescence nyochachara site na cytometry flow (n = 3), *P < 0.05 ma e jiri ya tụnyere insulin efu.
N'otu aka ahụ, onyonyo CLSM gosiri na ike ọkụ ọkụ FITC nke NPs FITC-insulin-loaded ọhụrụ na FITC-insulin-loaded spray-fire (na-enweghị mannitol) siri ike karịa nke ihe atụ ndị ọzọ (Foto 6a). Ọzọkwa, site na mgbakwunye nke mannitol, oke viscosity nke ngwọta ahụ mụbara iguzogide nnabata sel, na-ebute mbelata mmụba insulin. Nsonaazụ ndị a na-egosi na NPs akpọnwụrụ akpọnwụ nke na-enweghị mannitol gosipụtara arụmọrụ nnabata sel kachasị elu n'ihi na nha ihe ha dị obere karịa nke NPs akpọnwụrụ akpọnwụ mgbe agbazechara ọzọ.
A zụtara Chitosan (nkezi ibu molekul 100 KDa, 75–85% deacetylated) site na Sigma-Aldrich. (Oakville, Ontario, Canada). A zụtara Sodium tripolyphosphate (TPP) site na VWR (Radnor, Pennsylvania, USA). Insulin mmadụ ejiri mee ihe n'ọmụmụ ihe a sitere na Fisher Scientific (Waltham, MA, USA). A zụtara Fluorescein isothiocyanate (FITC)-labeled human insulin na 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) site na Sigma-Aldrich. (Oakville, Ontario, Canada). E nwetara ahịrị mkpụrụ ndụ HepG2 site na ATCC (Manassas, Virginia, USA). Ihe ndị ọzọ niile bụ ọkwa nyocha ma ọ bụ chromatographic.
Kwadebe ihe ngwọta CS nke 1 mg/ml site na ịgbaze ya na mmiri a gwakọtara agwakọta okpukpu abụọ (mmiri DD) nke nwere 0.1% acetic acid. Kwadebe ihe ngwọta TPP na insulin 1 mg/ml site na ịgbaze ha na mmiri DD na 0.1% acetic acid, n'otu n'otu. Ejiri polytron PCU-2-110 high speed homogenizer (Brinkmann Ind. Westbury, NY, USA) kwadebe ihe ngwọta tupu emulsion ahụ. Usoro nkwadebe dị ka ndị a: nke mbụ, a na-agbakwunye 2ml nke ihe ngwọta TPP na 4ml nke ihe ngwọta insulin, a na-akpali ngwakọta ahụ ruo nkeji 30 ma gwakọta ya nke ọma. Mgbe ahụ, a na-agbakwunye ihe ngwọta agwakọtara na mmiri CS site na sirinji n'okpuru mkpali ọsọ (10,000 rpm). A na-edebe ngwakọta ndị ahụ n'okpuru mkpali ọsọ ọsọ (15,000 rpm) na ice bat maka nkeji 30, a na-agbanwekwa ha na pH ụfọdụ iji nweta insulin NPs jikọtara ọnụ. Iji mee ka ọ dịkwuo mma ma belata nha nha nke insulin NPs, a na-etinye ha sonicated maka tinyekwuo nkeji iri atọ n'ime mmiri ịsa ahụ ice site na iji sonicator ụdị probe (UP 200ST, Hielscher Ultrasonics, Teltow, Germany).
A nwalere Insulin NPS maka dayameta Z-average, polydispersity index (PDI) na zeta potential site na iji nha dynamic light scattering (DLS) site na iji Litesizer 500 (Anton Paar, Graz, Austria) site na ịgwakọta ha na mmiri DD na 25°C. Ejiri igwe mikroskopu elektrọn nnyefe Hitachi H7600 (TEM) (Hitachi, Tokyo, Japan) mara morphology na nkesa nha, e mesịa jiri ngwanrọ onyonyo Hitachi (Hitachi, Tokyo, Japan) nyochaa onyonyo. Iji nyochaa arụmọrụ encapsulation (EE) na ikike ibu (LC) nke insulin NPs, a na-etinye NPs na tubes ultrafiltration nwere mbelata ibu molekul nke 100 kDa ma centrifuge na 500 xg maka nkeji 30. Ejiri sistemụ Agilent 1100 Series HPLC (Agilent, Santa Clara, California, USA) nke nwere ọkpọkọ quaternary, tụọ insulin na-enweghị mkpuchi na filtrate ahụ. ihe nlele akpaaka, ihe na-ekpo ọkụ kọlụm, na ihe nchọpụta DAD. Ejiri kọlụm C18 (Zorbax, 3.5 μm, 4.6 mm × 150 mm, Agilent, USA) nyochaa insulin ma chọpụta ya na 214 nm. Usoro mkpanaka bụ acetonitrile na mmiri, nwere 0.1% TFA, oke gradient site na 10/90 ruo 100/0, ma gbaa ọsọ ruo nkeji 10. A na-apịpụta usoro mkpanaka na ọnụego mmiri nke 1.0 ml/min. Atọrọ okpomọkụ kọlụm ahụ na 20 °C. Gbakọọ pasentị nke EE na LC site na iji nha nha.(1) na Eq.(2).
A nwalere oke CS/insulin dị iche iche site na 2.0 ruo 4.0 iji mee ka insulin NP ka mma. A gbakwunyere oke CS dị iche iche n'oge nkwadebe ahụ, ebe ngwakọta insulin/TPP nọgidere na-adị. A kwadebere NP insulin na oke pH nke 4.0 ruo 6.5 site na iji nlezianya na-achịkwa pH nke ngwakọta ahụ mgbe etinyere ngwọta niile (insulin, TPP na CS). A tụlere nha EE na nha nke insulin nanoparticles na ụkpụrụ pH dị iche iche na oke CS/insulin iji mee ka mmepụta insulin NPs ka mma.
E tinyere insulin NPs kachasị mma n'ime akpa aluminom ma jiri teepu kpuchie ya. Mgbe nke ahụ gasịrị, e tinyere akpa ndị a kpụkọrọ akpụkọ n'ime igwe nkụnye osisi Labconco FreeZone (Labconco, Kansas City, MO, USA) nke nwere igwe nkụnye tray. E tinyere okpomọkụ na nrụgide vacuum na -10 °C, 0.350 Torr maka awa abụọ mbụ, na 0 °C na 0.120 Torr maka awa 22 fọdụrụ n'ime awa 24 iji nweta insulin NPs akọrọ.
E jiri Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Switzerland) mepụta insulin e tinyere n'ime akpa. Ihe ndị a họọrọ bụ: okpomọkụ 100 °C, mmiri nri 3 L/min, na mmiri gas 4 L/min.
E ji FTIR-ATR spectroscopy chọpụta NPs nke insulin tupu na mgbe mmiri gwụchara. E jiri Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA) nyochaa nanoparticles ndị gbawara agbawa yana insulin na chitosan n'efu nke nwere ihe mgbakwunye ATR zuru ụwa ọnụ (PerkinElmer, Waltham, Massachusetts, USA). E nwetara nkezi mgbaàmà site na nyocha 16 na mkpebi nke 4 cm2 na ugboro ole nke 4000-600 cm2.
E jiri onyonyo SEM nke insulin kpọrọ nkụ ma kpọnwụọ akpọnwụ nke Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, USA) chọpụta usoro nke insulin kpọrọ nkụ NPs. Isi ihe e ji mee ya bụ voltaji 5 keV na ugbu a 30 mA.
E tinyere insulin NP niile gbapụrụ agbahapụ n'ime mmiri dd. E jiri otu ụzọ ahụ e kwuru na mbụ nwalee nha mkpụrụ, PDI, EE na LC iji chọpụta ịdị mma ha mgbe mmiri gwụchara. A tụkwara nkwụsi ike nke anhydroinsulin NPs site na ịnwale njirimara nke NPs mgbe nchekwa ogologo oge gasịrị. N'ọmụmụ ihe a, echekwara NP niile mgbe mmiri gwụchara n'ime friji ruo ọnwa atọ. Mgbe ọnwa atọ nke nchekwa gasịrị, a nwalere NPs maka nha mkpụrụ, PDI, EE na LC.
Gbazee 5 mL nke NPs e megharịrị n'ime 45 mL nke nwere mmiri afọ e mere ka ọ dị nro (pH 1.2, nke nwere 1% pepsin), mmiri eriri afọ (pH 6.8, nke nwere 1% trypsin) ma ọ bụ mmiri chymotrypsin (100 g/mL, na phosphate buffer, pH 7.8) iji nyochaa irè insulin n'ichebe NPs mgbe mmiri gwụchara. E tinyere ha na 37°C na ọsọ mkpali nke 100 rpm.500 μL nke mmiri ahụ ka anakọtara n'oge dị iche iche ma HPLC kpebiri itinye insulin n'ime ahụ.
A nwalere omume mwepụta in vitro nke insulin NPs a kwadebere ọhụrụ ma kpọnwụọ akpọnwụ site na iji usoro akpa dialysis (mbepụ oke molekul 100 kDa, Spectra Por Inc.). E tinyere NPs akọrọ a kwadebere ọhụrụ ma gbanwee n'ime mmiri na pH 2.5, pH 6.6, na pH 7.0 (0.1 M phosphate-buffered saline, PBS) iji mee ka gburugburu pH nke afọ, duodenum, na eriri afọ elu dị ka ọ dị. A na-etinye ihe nlele niile na 37 °C na ịma jijiji na-aga n'ihu na 200 rpm. Tinye mmiri ahụ n'èzí akpa dialysis 5 mL n'oge ndị a: 0.5, 1, 2, 3, 4, na 6 awa, wee tinye dialysate ọhụrụ ozugbo. HPLC nyochachara mmetọ insulin na mmiri ahụ, a gbakọọkwa ọnụego mwepụta insulin site na nanoparticles site na oke nke insulin efu ewepụtara na insulin zuru oke nke e tinyere na nanoparticles (Nhazi 3).
A zụlitere mkpụrụ ndụ HepG2 n'ime efere dị sentimita 60 site na iji Dulbecco's Modified Eagle's Medium (DMEM) nke nwere serum ehi fetal 10%, penicillin 100 IU/mL, na 100 μg/mL streptomycin29. A na-ejigide omenala na 37°C, iru mmiri dị pasent 95, na 5% CO2. Maka nnwale nnabata, a kụrụ mkpụrụ ndụ HepG2 na mkpụrụ ndụ 1 × 105/ml na sistemụ slide ụlọ Nunc Lab-Tek nke nwere olulu 8 (Thermo Fisher, NY, USA). Maka nnwale cytotoxicity, a kụrụ ha n'ime efere olulu 96 (Corning, NY, USA) na njupụta nke mkpụrụ ndụ 5 × 104/ml.
E jiri nnwale MTT nyochaa nsí nke insulin NPs30 a kwadebere ọhụrụ ma kpọnwụọ akpọnwụ. A kụrụ mkpụrụ ndụ HepG2 n'ime efere olulu 96 na njupụta nke mkpụrụ ndụ 5 × 104/mL ma kụọ ha ruo ụbọchị asaa tupu nnwale ahụ. A gwakọtara NPs insulin n'ọtụtụ njupụta (50 ruo 500 μg/mL) na usoro ọdịbendị wee nye ha na mkpụrụ ndụ. Mgbe awa 24 nke incubation gasịrị, a sachara mkpụrụ ndụ ahụ ugboro atọ na PBS ma tinye ya na usoro nwere 0.5 mg/ml MTT maka awa anọ ọzọ. A tụlere nsí site n'ịtụ mbelata enzymatic nke tetrazolium MTT odo odo gaa na formazan odo odo na 570 nm site na iji ihe na-agụ efere Tecan infinite M200 pro spectrophotometer (Tecan, Männedorf, Switzerland).
A nwalere arụmọrụ nnabata nke NPs site na iji igwe nyocha laser confocal na nyocha cytometry flow. E jiri FITC-insulin n'efu, FITC-insulin-loaded NPs gwọọ olulu mmiri ọ bụla nke Nunc Lab-Tek chamber slab, ma tinye 25 μg/mL nke FITC-insulin NPs nke mmiri mmiri na-agbaze n'otu mkpokọta ahụ ma tinye ya na mkpuchi ruo awa anọ. E jiri PBS saa mkpụrụ ndụ ugboro atọ ma dozie ha na 4% paraformaldehyde. E ji 4′, 6-diamidino-2-phenylindole (DAPI) mee ka nuclei dị ọcha. A hụrụ ebe insulin dị site na iji nyocha laser Olympus FV1000/microscope confocal abụọ (Olympus, Shinjuku City, Tokyo, Japan). Maka nyocha cytometry flow, etinyere otu mkpokọta nke 10 μg/mL nke FITC-insulin na-enweghị, FITC-insulin-loaded NPs, na FITC-insulin NPs nke mmiri mmiri na-agbaze na efere olulu mmiri 96 nke ejiri mkpụrụ ndụ HepG2 na mkpụrụ ndụ. a kpọchiri akpọchi ruo awa anọ. Mgbe awa anọ nke incubation gasịrị, ewepụrụ mkpụrụ ndụ ma saa ha ugboro atọ na FBS. Ejiri BD LSR II flow cytometer (BD, Franklin Lakes, New Jersey, United States) nyochaa mkpụrụ ndụ 5 × 104 kwa sample.
A na-egosipụta ụkpụrụ niile dị ka nkezi ± nkewa ọkọlọtọ. E jiri otu ụzọ ANOVA ma ọ bụ ule t nyochaa ntụnyere dị n'etiti otu niile site na IBM SPSS Statistics 26 maka Mac (IBM, Endicott, New York, USA) ma were p < 0.05 dị ka ihe dị mkpa na ọnụọgụgụ.
Ọmụmụ ihe a na-egosi mgbanwe na ikike nke nkụ mmiri iji mee ka mmiri ghara ịdị n'ime mmiri na-akpọnwụ chitosan/TPP/insulin nanoparticles nke jikọtara ya na mmiri na-eme ka mmiri ghara ịdị n'ime mmiri, ma e jiri ya tụnyere ụzọ ndị a na-akpọ nkụ oyi nkịtị site na iji ihe ndị na-eme ka mmiri ghara ịdị n'ime mmiri ma ọ bụ ikike cryoprotectants, yana ikike ibu dị elu. Mkpụrụ ndụ ihe nketa insulin kachasị mma nwetara nha mkpụrụ ndụ ihe nketa nke 318 nm, arụmọrụ encapsulation nke 99.4%. Nsonaazụ SEM na FTIR mgbe nkụ mmiri gasịrị gosiri na a na-edobe nhazi okirikiri naanị na NP ndị a kpọrọ nkụ nwere mannitol na enweghị lyophilized, ma lyophilized NPs na-enweghị mannitol gbawara n'oge nkụ mmiri. Na nnwale ikike nhazigharị, mkpụrụ ndụ ihe nketa insulin nke a na-akpọ nkụ na-enweghị mannitol gosiri obere nha mkpụrụ ndụ ihe nketa na ibu kachasị elu mgbe emegharịrị ya. Omume ntọhapụ nke mkpụrụ ndụ ihe nketa ndị a niile gosiri na ewepụtara ha ngwa ngwa na ngwọta nke pH = 2.5 na pH = 7, ma kwụsie ike na ngwọta nke pH = 6.5. Ma e jiri ya tụnyere mkpụrụ ndụ ihe nketa ndị ọzọ a na-akpọ nkụ, mkpụrụ ndụ ihe nketa ndị a na-akpọ nkụ na-enweghị mannitol gosiri mwepụta kachasị ọsọ. Nsonaazụ a kwekọrọ na nke a hụrụ na nnwale nnabata mkpụrụ ndụ, ebe NP ndị a na-agba mmiri na-enweghị mannitol fọrọ nke nta ka ọ nọgide na-enwe arụmọrụ nnabata mkpụrụ ndụ nke NP ndị a kwadebere ọhụrụ. Nsonaazụ ndị a na-egosi na nanoparticles insulin akọrọ nke a na-eji mannitol eme ihe kacha mma maka nhazi ọzọ n'ime ụdị ọgwụ ndị ọzọ na-anaghị anabata mmiri, dị ka mbadamba ọnụ ma ọ bụ ihe nkiri bioadhesive.
N'ihi nsogbu ihe onwunwe ọgụgụ isi, data ndị e mepụtara na/ma ọ bụ nyochachara n'oge ọmụmụ ihe a adịghị n'ihu ọha, mana ha dị n'aka ndị dere ya ma ọ bụrụ na ha rịọ ya.
Kagan, A. Ọrịa shuga nke ụdị nke abụọ: mmalite mmekọrịta mmadụ na ibe ya na sayensị, nsogbu ahụike, na ihe ọ pụtara nye ndị ọrịa na ndị ọzọ. (McFarlane, 2009).
Singh, AP, Guo, Y., Singh, A., Xie, W. & Jiang, P. Mmepe nke mkpuchi insulin: ojiji ọnụ ò kwere omume ugbu a? J. Pharmacy.bio-pharmacy.reservoir.1, 74–92 (2019).
Wong, CY, Al-Salami, H. & Dass, CR Mmepe ndị a na-eme n'oge na-adịbeghị anya na sistemụ nnyefe liposome nke nwere insulin n'ọnụ maka ọgwụgwọ ọrịa shuga.Interpretation.J. Pharmacy.549, 201–217 (2018).