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Ma nanoparticles a insulin (NPs) okhala ndi katundu wambiri apeza ntchito zosiyanasiyana m'njira zosiyanasiyana. Ntchitoyi ikufuna kuwunika momwe njira zowumitsira ndi kupopera zimakhudzira kapangidwe ka chitosan nanoparticles yodzazidwa ndi insulin, yokhala ndi kapena yopanda mannitol ngati cryoprotectant. Tinayesanso ubwino wa nanoparticles izi mwa kuzisungunulanso. Tisanathe madzi m'thupi, kukula kwa tinthu ta chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles kunakonzedwa kukhala 318 nm, PDI inali 0.18, mphamvu ya encapsulation inali 99.4%, ndipo katunduyo anali 25.01%. Pambuyo pokonzanso, tinthu tating'onoting'ono tonse, kupatula tomwe timapangidwa ndi njira yowumitsira popanda kugwiritsa ntchito mannitol, tinasunga kapangidwe ka tinthu tating'onoting'ono tozungulira. Poyerekeza ndi tinthu tating'onoting'ono tomwe tili ndi mannitol tomwe timatha madzi ndi kupopera, tinthu tating'onoting'ono topanda kupopera tomwe tili ndi mannitol tinawonetsanso kukula kwa tinthu tating'onoting'ono kwambiri (376 nm) komanso kuchuluka kwakukulu kwa katundu (25.02%). ndi kuchuluka kofanana kwa encapsulation (98.7%) ndi PDI (0.20) mwa njira zowumitsa kapena zowumitsa mufiriji. Tinthu tating'onoting'ono touma pogwiritsa ntchito spray dryer popanda mannitol tinapangitsanso kuti insulin itulutsidwe mwachangu komanso kuti maselo azitha kuyamwa bwino kwambiri. Ntchitoyi ikuwonetsa kuti kuuma mufiriji kumatha kuchepetsa madzi a insulin popanda kugwiritsa ntchito cryoprotectants poyerekeza ndi njira zachikhalidwe zowumitsa mufiriji, zomwe zimapangitsa kuti katundu azitha kunyamula bwino, kuchepetsa zofunikira zowonjezera komanso ndalama zogwirira ntchito.
Kuyambira pamene idapezeka mu 1922, 2, 3, insulin ndi mankhwala ake apulumutsa miyoyo ya odwala matenda a shuga amtundu woyamba (T1DM) ndi matenda a shuga amtundu wachiwiri (T1DM). Komabe, chifukwa cha mphamvu zake monga mapuloteni olemera kwambiri, insulin imasonkhanitsidwa mosavuta, kusweka ndi ma enzymes a proteolytic, ndikuchotsedwa ndi zotsatira zoyamba. Anthu omwe amapezeka ndi matenda a shuga amtundu woyamba amafunika jakisoni wa insulin kwa moyo wawo wonse. Odwala ambiri omwe adapezeka ndi matenda a shuga amtundu wachiwiri poyamba amafunikanso jakisoni wa insulin kwa nthawi yayitali. Jakisoni wa insulin tsiku lililonse ndi gwero lalikulu la ululu ndi kusasangalala tsiku ndi tsiku kwa anthu awa, zomwe zimakhudza thanzi la maganizo. Zotsatira zake, mitundu ina ya insulin yomwe imayambitsa kusasangalala pang'ono, monga kupereka insulin pakamwa, ikuphunziridwa kwambiri5 chifukwa imatha kubwezeretsa moyo wa anthu pafupifupi 5 biliyoni omwe ali ndi matenda a shuga padziko lonse lapansi.
Ukadaulo wa nanoparticle wapereka kupita patsogolo kwakukulu pakuyesa kumwa insulin yakumwa4,6,7. Ukadaulo womwe umateteza bwino ndikuteteza insulin kuti isawonongeke kuti iperekedwe kumalo enaake a thupi. Komabe, kugwiritsa ntchito ma nanoparticle formulations kuli ndi zoletsa zingapo, makamaka chifukwa cha mavuto okhazikika a tinthu tomwe timayimitsidwa. Kusonkhana kwina kumatha kuchitika panthawi yosungira, zomwe zimachepetsa kupezeka kwa nanoparticles zodzaza ndi insulin8. Kuphatikiza apo, kukhazikika kwa mankhwala a polymer matrix a nanoparticles ndi insulin kuyeneranso kuganiziridwa kuti zitsimikizire kukhazikika kwa insulin nanoparticles (NPs). Pakadali pano, ukadaulo wozizira ndi wofunikira kwambiri popanga ma NP okhazikika pomwe akuletsa kusintha kosafunikira panthawi yosungira9.
Komabe, kuumitsa mufiriji kumafuna kuwonjezera zinthu zoteteza khungu kuti zisakhudzidwe ndi kupsinjika kwa makina a makhiristo a ayezi. Izi zimachepetsa kwambiri kudzaza kwa tinthu tating'onoting'ono ta insulin pambuyo pa lyophilization, chifukwa cryoprotectant imatenga gawo lalikulu la kulemera. Chifukwa chake, ma NP a insulin opangidwa nthawi zambiri amapezeka kuti si oyenera kupanga ufa wouma, monga mapiritsi omwa ndi mafilimu omwa, chifukwa chosowa tinthu tating'onoting'ono touma kuti tipeze njira yochiritsira ya insulin.
Kuumitsa kwa spray ndi njira yodziwika bwino komanso yotsika mtengo yamafakitale yopanga ufa wouma kuchokera ku magawo amadzimadzi mumakampani opanga mankhwala10,11. Kulamulira njira yopangira tinthu tating'onoting'ono kumalola kuyika bwino zinthu zingapo zomwe zimagwira ntchito 12, 13. Kuphatikiza apo, yakhala njira yothandiza yokonzekera mapuloteni otsekedwa kuti aperekedwe pakamwa. Pakuuma kwa spray, madzi amasanduka nthunzi mwachangu kwambiri, zomwe zimathandiza kuti kutentha kwa tinthu tating'onoting'ono kukhale kochepa11,14, zomwe zimathandiza kuti igwiritsidwe ntchito poyika zinthu zomwe zimakhudzidwa ndi kutentha. Musanaumitse spray, zinthu zokutira ziyenera kusakanikirana bwino ndi yankho lomwe lili ndi zosakaniza zomwe zatsekedwa11,14. Mosiyana ndi kuumitsa mufiriji, kuyika bwino musanayambe kuyika mu spray-dryer kumathandizira kuti kuyika bwino mu capsular panthawi yotaya madzi. Popeza njira yoyika mu spray-dryer sikufuna cryoprotectives, kuuma kwa spray kungagwiritsidwe ntchito kupanga ma NP ouma okhala ndi katundu wambiri.
Kafukufukuyu akufotokoza za kupanga kwa ma NP okhala ndi insulin polumikiza chitosan ndi sodium tripolyphosphate pogwiritsa ntchito njira ya ion gel. Ion gelation ndi njira yokonzekera yomwe imalola kupanga ma nanoparticles kudzera mu mgwirizano wamagetsi pakati pa mitundu iwiri kapena kuposerapo ya ionic pansi pa mikhalidwe ina. Njira zonse ziwiri zozizira-kuumitsa ndi kupopera-kuumitsa zinagwiritsidwa ntchito kupumitsa madzi a chitosan/sodium tripolyphosphate/insulin cross-linked nanoparticles. Pambuyo pa kutaya madzi m'thupi, mawonekedwe awo adawunikidwa ndi SEM. Mphamvu yawo yobwezeretsanso idawunikidwa poyesa kufalikira kwawo, mphamvu ya pamwamba, PDI, mphamvu ya encapsulation, ndi kuchuluka kwa katundu. Ubwino wa ma nanoparticles osungunuka omwe amapangidwa ndi njira zosiyanasiyana zochotsera madzi m'thupi unawunikidwanso poyerekeza chitetezo chawo cha insulin, momwe amatulutsira, komanso mphamvu ya maselo.
PH ya yankho losakanikirana ndi chiŵerengero cha chitosan ndi insulin ndi zinthu ziwiri zofunika kwambiri zomwe zimakhudza kukula kwa tinthu tating'onoting'ono ndi mphamvu ya encapsulation (EE) ya ma NP omaliza, chifukwa zimakhudza mwachindunji njira ya ionotropic gelation. PH ya yankho losakanikirana yawonetsedwa kuti ikugwirizana kwambiri ndi kukula kwa tinthu tating'onoting'ono ndi mphamvu ya encapsulation (Chithunzi 1a). Monga momwe zasonyezedwera pa Chithunzi 1a, pamene pH inakwera kuchokera pa 4.0 mpaka 6.0, kukula kwa tinthu tating'onoting'ono (nm) kunachepa ndipo EE inakwera kwambiri, pomwe pH inakwera kufika pa 6.5, kukula kwa tinthu tating'onoting'ono kunayamba kuwonjezeka ndipo EE sinasinthe. Pamene chiŵerengero cha chitosan ndi insulin chikukwera, kukula kwa tinthu tating'onoting'ono kumawonjezekanso. Kuphatikiza apo, palibe kusintha kwa EE komwe kunawonedwa pamene nanoparticles zinakonzedwa pa chiŵerengero cha chitosan/insulin choposa 2.5:1 (w/w) (Chithunzi 1b). Chifukwa chake, mikhalidwe yabwino kwambiri yokonzekera mu kafukufukuyu (pH 6.0, chiŵerengero cha chitosan/insulin mass cha 2.5:1) chinagwiritsidwa ntchito Konzani tinthu tating'onoting'ono tomwe tili ndi insulin kuti muphunzire zambiri. Pansi pa izi, kukula kwa tinthu tating'onoting'ono ta insulin tinakonzedwa kuti tikhale 318 nm (Chithunzi 1c), PDI inali 0.18, mphamvu yolumikizira inali 99.4%, zeta potential inali 9.8 mv, ndipo insulin inakwezedwa inali 25.01% (m/m ). Kutengera zotsatira za transmission electron microscopy (TEM), tinthu tating'onoting'ono tomwe tinakwezedwa tinali tozungulira komanso tosiyana ndi kukula kofanana (Chithunzi 1d).
Kukonza bwino magawo a tinthu tating'onoting'ono ta insulin: (a) momwe pH imakhudzira kukula kwa pakati ndi kuchuluka kwa insulin (EE) (yokonzedwa pa chiŵerengero cha 5:1 cha chitosan ndi insulin); (b) chitosan ndi mphamvu ya kuchuluka kwa insulin pa kukula kwa pakati ndi kuchuluka kwa insulin (EE) ya insulin NPs (yokonzedwa pa pH 6); (c) kugawa kwa tinthu tating'onoting'ono ta insulin nanoparticles yokonzedwa bwino; (d) TEM micrograph ya insulin NPs yokonzedwa bwino.
Ndizodziwika bwino kuti chitosan ndi polyelectrolyte yofooka yokhala ndi pKa ya 6.5. Imayikidwa bwino mu acidic media chifukwa gulu lake lalikulu la amino limapangidwa ndi ma hydrogen ions15. Chifukwa chake, nthawi zambiri imagwiritsidwa ntchito ngati chonyamulira kuti isunge ma macromolecules omwe ali ndi negative charge. Mu kafukufukuyu, chitosan idagwiritsidwa ntchito posunga insulin ndi isoelectric point ya 5.3. Popeza chitosan imagwiritsidwa ntchito ngati chinthu chophikira, ndi kuwonjezeka kwa gawo lake, makulidwe akunja kwa nanoparticles amawonjezeka mofanana, zomwe zimapangitsa kuti tinthu tating'onoting'ono tikhale tambiri. Kuphatikiza apo, kuchuluka kwa chitosan kumatha kusunga insulin yambiri. Kwa ife, EE inali yapamwamba kwambiri pamene chiŵerengero cha chitosan ndi insulin chinafika pa 2.5:1, ndipo panalibe kusintha kwakukulu mu EE pamene chiŵerengerocho chinapitirira kukwera.
Kupatula chiŵerengero cha chitosan ndi insulin, pH idachitanso gawo lofunika kwambiri pakukonzekera NPs.Gan et al. 17 adaphunzira momwe pH imakhudzira kukula kwa tinthu tating'onoting'ono ta chitosan. Adapeza kuchepa kosalekeza kwa kukula kwa tinthu mpaka pH itafika pa 6.0, ndipo kuwonjezeka kwakukulu kwa kukula kwa tinthu tating'onoting'ono kunawonedwa pa pH > 6.0, zomwe zikugwirizana ndi zomwe tawona. Izi zimachitika chifukwa chakuti pH ikawonjezeka, molekyulu ya insulin imakhala ndi mphamvu yoipa pamwamba, motero, imalimbikitsa kuyanjana kwa electrostatic ndi chitosan/sodium tripolyphosphate (TPP) complex, zomwe zimapangitsa kuti tinthu tating'onoting'ono tikhale tating'onoting'ono komanso EE yayikulu. Komabe, pH itasinthidwa kukhala 6.5, magulu a amino pa chitosan adachotsedwa, zomwe zimapangitsa kuti chitosan ipindike. Chifukwa chake, pH yayikulu imapangitsa kuti ma amino ions asamawonekere kwambiri ku TPP ndi insulin, zomwe zimapangitsa kuti pakhale kutsika kwa cross-linking, kukula kwakukulu kwa tinthu tating'onoting'ono komanso EE yotsika.
Kusanthula kwa mawonekedwe a ma NP ouma ndi opopera kungathandize kusankha njira zabwino zopangira madzi m'thupi komanso kupanga ufa. Njira yabwino iyenera kupereka kukhazikika kwa mankhwala, mawonekedwe ofanana a tinthu tating'onoting'ono, kuchuluka kwa mankhwala komanso kusungunuka bwino mu yankho loyambirira. Mu kafukufukuyu, kuti tiyerekeze bwino njira ziwirizi, ma NP a insulin okhala ndi kapena opanda 1% mannitol adagwiritsidwa ntchito panthawi ya madzi m'thupi. Mannitol imagwiritsidwa ntchito ngati chowonjezera kapena choteteza cryoprotectant m'njira zosiyanasiyana za ufa wouma kuti awume ndi kuuma. Pa ma nanoparticles a insulin omwe ali ndi lyophilized opanda mannitol, monga momwe zasonyezedwera pa Chithunzi 2a, kapangidwe ka ufa wokhala ndi mapopoma ambiri okhala ndi malo akuluakulu, osakhazikika komanso okhwima adawonedwa pansi pa scanning electron microscopy (SEM). Tinthu tating'onoting'ono tochepa tapezeka mu ufawo pambuyo pa madzi m'thupi (Chithunzi 2e). Zotsatirazi zikusonyeza kuti ma NP ambiri adawonongeka panthawi ya kuuma popanda cryoprotectant. Pa ma nanoparticles a insulin ouma ndi opopera okhala ndi 1% mannitol, ma nanoparticles ozungulira okhala ndi malo osalala adawonedwa (Chithunzi 1). 2b,d,f,h). Ma insulin nanoparticles oumitsidwa ndi spray popanda mannitol anakhalabe ozungulira koma okwinya pamwamba (Chithunzi 2c). Malo ozungulira ndi okwinya akukambidwanso mu machitidwe otulutsa ndi mayeso otengedwa ndi maselo pansipa. Kutengera mawonekedwe owoneka a ma NP ouma, ma NP onse oumitsidwa ndi spray opanda mannitol ndi NPs adawuma ndi kuzizira komanso oumitsidwa ndi spray ndi mannitol adatulutsa ufa wa NPs wabwino (Chithunzi 2f,g,h). Malo akuluakulu pakati pa tinthu tating'onoting'ono, kusungunuka kumakhala kwakukulu ndipo motero kuchuluka kwa kutulutsa kumakhala kwakukulu.
Kapangidwe ka ma NP osiyanasiyana a insulin omwe alibe madzi m'thupi: (a) Chithunzi cha SEM cha ma NP a insulin omwe ali ndi lyophilized opanda mannitol; (b) Chithunzi cha SEM cha ma NP a insulin omwe ali ndi lyophilized okhala ndi mannitol; (c) chithunzi cha ma NP a insulin ouma popanda mannitol SEM cha ; (d) Chithunzi cha SEM cha ma NP a insulin ouma popanda mannitol; (e) chithunzi cha ufa wa ma NP a insulin omwe ali ndi lyophilized opanda mannitol; (f) chithunzi cha ma NP a insulin omwe ali ndi lyophilized okhala ndi mannitol; (g) Chithunzi cha ufa wa ma NP a insulin ouma popanda mannitol; (h) chithunzi cha ufa wa ma NP a insulin ouma popanda mannitol.
Pa nthawi youma mufiriji, mannitol imagwira ntchito ngati cryoprotectant, kusunga ma NP mu mawonekedwe osasinthika ndikuletsa kuwonongeka ndi ma crystals a ayezi19. Mosiyana ndi zimenezi, palibe sitepe yozizira panthawi youma mufiriji. Chifukwa chake mannitol sikofunikira mu njira iyi. Ndipotu, ma NP ouma mufiriji opanda mannitol adapereka ma NP abwino kwambiri monga momwe tafotokozera kale. Komabe, mannitol ikhoza kugwirabe ntchito ngati chodzaza mu njira youma mufiriji kuti ipatse ma NP kapangidwe kozungulira20 (Chithunzi 2d), zomwe zimathandiza kupeza mawonekedwe ofanana a ma NP omangika. Kuphatikiza apo, n'zoonekeratu kuti tinthu tating'onoting'ono tating'onoting'ono tating'onoting'ono titha kupezeka mu ma NP a insulin ouma mufiriji komanso ouma mufiriji okhala ndi mannitol (Chithunzi 2b,d), zomwe zitha kukhala chifukwa cha kuchuluka kwa mannitol m'chimake cha tinthu pamodzi ndi insulin yomangika. Ku. Chitosan layer. Ndikofunika kudziwa kuti mu kafukufukuyu, kuti titsimikizire kuti kapangidwe kake kamakhalabe kolimba pambuyo poti madzi atha, chiŵerengero cha mannitol ndi chitosan chimasungidwa pa 5:1, kotero kuti kuchuluka kwa zodzaza kumathanso kukulitsa kukula kwa tinthu ta NPs zouma.
Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy inasonyeza kusakaniza kwa insulin yaulere, chitosan, chitosan, TPP ndi insulin. Ma NP onse osowa madzi m'thupi anadziwika pogwiritsa ntchito FTIR-ATR spectroscopy. Chodziwika bwino n'chakuti, mphamvu ya band ya 1641, 1543 ndi 1412 cm-1 inawonedwa mu ma NP otsekedwa omwe anali ouma ndi mannitol ndipo mu ma NP ouma ndi mannitol ndi popanda mannitol (Chithunzi 3). Monga momwe tafotokozera kale, kuwonjezeka kwa mphamvu kumeneku kunagwirizanitsidwa ndi kulumikiza pakati pa chitosan, TPP ndi insulin. Kafukufuku wa kuyanjana pakati pa chitosan ndi insulin adawonetsa kuti mu ma FTIR spectra a chitosan nanoparticles omwe ali ndi insulin, chitosan band inalumikizana ndi ya insulin, zomwe zimawonjezera mphamvu ya carbonyl (1641 cm-1) ndi amine (1543 cm-1). Magulu a tripolyphosphate a TPP amalumikizidwa ndi magulu a ammonium mu chitosan, ndikupanga mkanda pa 1412 cm-1.
Ma spectra a FTIR-ATR a insulin yaulere, chitosan, zosakaniza zakuthupi za chitosan/TPP/insulin ndi NPs zomwe zimachotsedwa madzi m'thupi m'njira zosiyanasiyana.
Kuphatikiza apo, zotsatirazi zikugwirizana ndi zomwe zawonetsedwa mu SEM, zomwe zawonetsa kuti ma NP omwe ali mkati mwake adakhalabe osawonongeka akamapopera ndi kuzizira ndi mannitol, koma popanda mannitol, kupopera kokha komwe kumapanga tinthu tomwe timapopera. Mosiyana ndi zimenezi, zotsatira za FTIR-ATR spectral za ma NP omwe amazizira popanda mannitol zinali zofanana kwambiri ndi chitosan, TPP, ndi insulin. Zotsatirazi zikusonyeza kuti maulalo pakati pa chitosan, TPP ndi insulin sapezekanso mu ma NP omwe amazizira popanda mannitol. Kapangidwe ka ma NP kanawonongeka panthawi yozizira popanda cryoprotectant, zomwe zitha kuwoneka mu zotsatira za SEM (Chithunzi 2a). Kutengera mawonekedwe ndi zotsatira za FTIR za ma NP a insulin omwe alibe madzi, ma NP omwe ali ndi lyophilized, spray-dryed, ndi mannitol okha ndi omwe adagwiritsidwa ntchito poyesa kukonzanso ndi ma NP omwe alibe mannitol chifukwa cha kuwonongeka kwa ma NP omwe alibe mannitol panthawi yotaya madzi. kambiranani.
Kusowa madzi m'thupi kumagwiritsidwa ntchito posungira kwa nthawi yayitali ndikukonzanso kukhala mitundu ina. Kuthekera kwa ma NP ouma kuti akhazikikenso pambuyo posungira ndikofunikira kwambiri kuti agwiritsidwe ntchito m'njira zosiyanasiyana monga mapiritsi ndi mafilimu. Tinaona kuti kukula kwa tinthu tating'onoting'ono ta insulin NPs youma popanda mannitol kunawonjezeka pang'ono pambuyo pokonzanso. Kumbali ina, kukula kwa tinthu ta insulin nanoparticles youma ndi youma ndi mannitol kunawonjezeka kwambiri (Table 1). PDI ndi EE sizinasinthe kwambiri (p > 0.05) pambuyo pokonzanso ma NPs onse mu kafukufukuyu (Table 1). Zotsatirazi zikusonyeza kuti tinthu tating'onoting'ono tambiri tinakhalabe tomwe titasungunukanso. Komabe, kuwonjezera kwa mannitol kunapangitsa kuti insulin ichepetse kwambiri kuchuluka kwa ma mannitol nanoparticles omwe ali ndi lyophilized ndi spray (Table 1). Mosiyana ndi zimenezi, kuchuluka kwa insulin mu ma NPs ouma popanda mannitol kunakhalabe komweko monga kale (Table 1).
Ndizodziwika bwino kuti kudzaza kwa nanoparticles ndikofunikira kwambiri pogwiritsidwa ntchito popereka mankhwala. Kwa ma NP okhala ndi katundu wochepa, zinthu zambiri zimafunika kuti zifike pamlingo wochiritsira. Komabe, kukhuthala kwakukulu kwa kuchuluka kwa NP kotereku kumabweretsa zovuta komanso zovuta pakumwa ndi jakisoni, motsatana. Kuphatikiza apo, ma NP a insulin angagwiritsidwenso ntchito popanga mapiritsi ndi ma biofilms okhuthala23, 24, zomwe zimafuna kugwiritsa ntchito ma NP ambiri pamlingo wotsika wonyamula, zomwe zimapangitsa kuti mapiritsi akuluakulu ndi ma biofilms okhuthala omwe sayenera kugwiritsidwa ntchito pakamwa. Chifukwa chake, ma NP ouma omwe ali ndi insulin yambiri ndi ofunikira kwambiri. Zotsatira zathu zikusonyeza kuti kuchuluka kwa insulin komwe kumapangidwa ndi ma NPs ouma opanda ma mannitol kungapereke zabwino zambiri zokopa njira zina zoperekera mankhwalawa.
NP zonse zopanda madzi m'thupi zinasungidwa mufiriji kwa miyezi itatu. Zotsatira za SEM zinasonyeza kuti mawonekedwe a NP zonse zopanda madzi m'thupi sanasinthe kwambiri panthawi yosungira miyezi itatu (Chithunzi 4). Pambuyo pokonzanso m'madzi, NP zonse zinasonyeza kuchepa pang'ono kwa EE ndipo zinatulutsa pafupifupi pang'ono (~5%) ya insulin panthawi yosungira miyezi itatu (Table 2). Komabe, kukula kwapakati kwa tinthu tating'onoting'ono ta nanoparticles kunawonjezeka. Kukula kwa tinthu tating'onoting'ono ta NPs zouma popanda mannitol kunawonjezeka kufika pa 525 nm, pomwe za NPs zouma ndi zouma ndi mannitol zinawonjezeka kufika pa 872 ndi 921 nm, motsatana (Table 2).
Kapangidwe ka ma NP osiyanasiyana a insulin omwe alibe madzi okwanira omwe amasungidwa kwa miyezi itatu: (a) Chithunzi cha SEM cha ma NP a insulin omwe ali ndi lyophilized okhala ndi mannitol; (b) Chithunzi cha SEM cha tinthu tating'onoting'ono ta insulin touma popanda mannitol; (c) zithunzi za SEM zopanda mannitol za ma NP a insulin ouma popanda spray.
Kuphatikiza apo, madontho amadzimadzi adawoneka mu tinthu tating'onoting'ono ta insulin tomwe tidapangidwanso kuti tiume ndi mannitol ndikuuma mufiriji (Chithunzi S2). Izi zitha kuchitika chifukwa cha tinthu tating'onoting'ono tomwe sitinayime bwino m'madzi. Zotsatira zonse zomwe zili pamwambapa zikuwonetsa kuti njira yowumitsira madontho imatha kuteteza tinthu tating'onoting'ono ta insulin kuti tisamafe komanso kuti tinthu tating'onoting'ono ta insulin tomwe timapezeka titha kupezeka popanda zodzaza kapena zoteteza ma cryoprotectants.
Kusunga insulin kunayesedwa mu pH = 2.5 medium ndi pepsin, trypsin, ndi α-chymotrypsin kuti awonetse mphamvu yoteteza ya NPs ku kugaya kwa enzymatic pambuyo pa kutaya madzi m'thupi. Kusunga insulin kwa NPs zopanda madzi m'thupi kunayerekezeredwa ndi kwa NPs zokonzedwa kumene, ndipo insulin yaulere idagwiritsidwa ntchito ngati njira yoletsa. Mu kafukufukuyu, insulin yaulere idawonetsa kuchotsedwa kwa insulin mwachangu mkati mwa maola 4 mu ma enzyme onse atatu (Chithunzi 5a–c). Mosiyana ndi izi, kuyesa kuchotsa insulin kwa NPs zouma ndi mannitol ndi NPs zouma ndi kapena popanda mannitol kunawonetsa chitetezo chapamwamba kwambiri cha NPs izi ku kugaya kwa enzymatic, zomwe zinali zofanana ndi za NPs zokonzedwa kumene za insulin (chithunzi 1).5a-c). Mothandizidwa ndi nanoparticles mu pepsin, trypsin, ndi α-chymotrypsin, insulin yoposa 50%, 60%, ndi 75% ikhoza kutetezedwa mkati mwa maola 4, motsatana (Chithunzi 5a–c). Mphamvu yoteteza insulini iyi ikhoza kuwonjezera mwayi wokhala ndi insulin yambiri. kuyamwa m'magazi25. Zotsatirazi zikusonyeza kuti kuumitsa ndi mankhwala opopera kapena opanda mannitol komanso kuumitsa ndi mannitol mufiriji kungasunge mphamvu yoteteza insulin ya NPs pambuyo poti madzi atha.
Chitetezo ndi kumasula machitidwe a insulin NPs youma: (a) kuteteza insulin mu pepsin solution; (b) kuteteza insulin mu trypsin solution; (c) kuteteza insulin ndi α-chymotrypsin solution; (d) Kutulutsa machitidwe a NPs youma mu pH = 2.5 solution; (e) kutulutsa machitidwe a NPs youma mu pH = 6.6 solution; (f) kutulutsa machitidwe a NPs youma mu pH = 7.0 solution.
Ma NP a insulin ouma omwe adakonzedwanso ndi kukonzedwanso adayikidwa mu ma buffers osiyanasiyana (pH = 2.5, 6.6, 7.0) pa 37 °C, kutsanzira pH ya m'mimba, duodenum, ndi matumbo ang'onoang'ono, kuti awone momwe insulin imakhudzira kukana kwa insulin. Kachitidwe kotulutsa m'malo osiyanasiyana. Chidutswa cha njira ya m'mimba. Pa pH = 2.5, ma NP okhala ndi insulin ndi ma NP ouma omwe asungunukanso adawonetsa kutulutsidwa koyamba mkati mwa ola limodzi loyamba, kutsatiridwa ndi kutulutsidwa pang'onopang'ono m'maola 5 otsatira (Chithunzi 5d). Kutulutsidwa mwachangu kumeneku pachiyambi mwina kumachitika chifukwa cha kuchotsedwa mwachangu kwa mamolekyu a mapuloteni omwe sanakhazikike bwino mkati mwa kapangidwe ka mkati mwa tinthu tating'onoting'ono. Pa pH = 6.5, ma NP okhala ndi insulin ndi ma NP ouma omwe adapangidwanso adawonetsa kutulutsidwa kosalala komanso pang'onopang'ono mkati mwa maola 6, popeza pH ya yankho loyesera inali yofanana ndi ya yankho lokonzedwa ndi NPs (Chithunzi 5e). Pa pH = 7, ma NP anali osakhazikika ndipo pafupifupi anawola kwathunthu mkati mwa maola awiri oyamba (Chithunzi 5f). Izi zili choncho chifukwa kuchotsedwa kwa chitosan kumachitika pa pH yokwera, zomwe zimapangitsa kuti pakhale netiweki yochepa ya polymer ndikutulutsidwa kwa insulin yodzaza.
Kuphatikiza apo, ma NP a insulin omwe amaumitsidwa popanda mannitol adawonetsa kutulutsidwa mwachangu kuposa ma NP ena omwe alibe madzi (Chithunzi 5d–f). Monga tafotokozera kale, ma NP a insulin omwe adaumitsidwanso popanda mannitol adawonetsa kukula kwa tinthu tating'onoting'ono. Tinthu tating'onoting'ono timapereka malo akuluakulu, kotero mankhwala ambiri oyenera amakhala pamwamba pa tinthu tating'onoting'ono kapena pafupi ndi pamwamba pake, zomwe zimapangitsa kuti mankhwala azitulutsidwa mwachangu26.
Kuopsa kwa poizoni wa ma NP kunafufuzidwa pogwiritsa ntchito njira ya MTT. Monga momwe zasonyezedwera pa Chithunzi S4, ma NP onse osowa madzi m'thupi adapezeka kuti alibe mphamvu yaikulu pa moyo wa maselo pamlingo wa 50–500 μg/ml, zomwe zikusonyeza kuti ma NP onse osowa madzi m'thupi angagwiritsidwe ntchito mosamala kuti akafike pawindo lochiritsira.
Chiwindi ndi chiwalo chachikulu chomwe insulin imagwiritsa ntchito pochita ntchito zake za thupi. Maselo a HepG2 ndi mzere wa maselo a hepatoma a munthu omwe amagwiritsidwa ntchito kwambiri ngati chitsanzo cha in vitro hepatocyte uptake. Pano, maselo a HepG2 adagwiritsidwa ntchito poyesa kuyamwa kwa ma NP omwe ali ndi madzi m'thupi pogwiritsa ntchito njira zoziziritsira ndi kupopera. Kutengedwa kwa maselo pogwiritsa ntchito confocal laser scanning pogwiritsa ntchito flow cytometry ndi masomphenya pambuyo pa maola angapo a incubation ndi free FITC insulin pa concentration ya 25 μg/mL, FITC insulin-loaded NPs yatsopano yokonzedwa ndi FITC insulin-loaded NPs yopanda madzi m'thupi pa equal insulin concentrations Quantitative microscopy (CLSM) experiments idachitika. Lyophilized NPs popanda mannitol idawonongeka panthawi ya madzi m'thupi ndipo sinayesedwe mu mayeso awa. Mphamvu ya intracellular fluorescence ya NPs yatsopano yokonzedwa ndi insulin, lyophilized NPs ndi mannitol, ndi spray-dryed NPs yokhala ndi ndi yopanda mannitol (Chithunzi 6a) inali 4.3, 2.6, 2.4, ndi Kukwera nthawi 4.1 kuposa kwaulere. Gulu la FITC-insulin, motsatana (Chithunzi 6b). Zotsatirazi zikusonyeza kuti insulin yolumikizidwa ndi yamphamvu kwambiri pakuyamwa kwa maselo kuposa insulin yaulere, makamaka chifukwa cha kukula kochepa kwa tinthu tating'onoting'ono tomwe timapangidwa mu kafukufukuyu.
Kutengedwa kwa maselo a HepG2 pambuyo pa maola 4 opangidwa ndi ma NP atsopano komanso ma NP ouma: (a) Kufalikira kwa FITC-insulin uptake ndi maselo a HepG2. (b) Kuchuluka kwa mphamvu ya fluorescence komwe kumayesedwa ndi flow cytometry (n = 3), *P < 0.05 poyerekeza ndi insulin yaulere.
Momwemonso, zithunzi za CLSM zinawonetsa kuti mphamvu ya FITC fluorescence ya FITC-insulin-loaded NPs yokonzedwa kumene ndi FITC-insulin-loaded spray-dryed NPs (yopanda mannitol) inali yamphamvu kwambiri kuposa ya zitsanzo zina (Chithunzi 6a). Kuphatikiza apo, ndi kuwonjezera kwa mannitol, kukhuthala kwakukulu kwa yankho kunawonjezera kukana kwa kuyamwa kwa maselo, zomwe zinapangitsa kuti insulin ichuluke. Zotsatirazi zikusonyeza kuti NPs zouma zopanda spray-dryed mannitol zinawonetsa mphamvu yayikulu kwambiri yoyamwa kwa maselo chifukwa kukula kwa tinthu tawo kunali kochepa kuposa kwa NPs zouma zozizira zitasungunukanso.
Chitosan (kulemera kwapakati kwa mamolekyulu 100 KDa, 75–85% deacetylated) idagulidwa ku Sigma-Aldrich. (Oakville, Ontario, Canada). Sodium tripolyphosphate (TPP) idagulidwa ku VWR (Radnor, Pennsylvania, USA). Insulin ya anthu yophatikizidwa yomwe idagwiritsidwa ntchito mu kafukufukuyu idachokera ku Fisher Scientific (Waltham, MA, USA). Fluorescein isothiocyanate (FITC)-labeled human insulin ndi 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) zidagulidwa ku Sigma-Aldrich. (Oakville, Ontario, Canada). Mzere wa maselo a HepG2 udapezeka ku ATCC (Manassas, Virginia, USA). Ma reagents ena onse anali a analytical kapena chromatographic grade.
Konzani yankho la 1 mg/ml la CS polisungunula m'madzi osungunuka kawiri (madzi a DD) okhala ndi 0.1% acetic acid. Konzani mayankho a 1 mg/ml a TPP ndi insulin powasungunula m'madzi a DD ndi 0.1% acetic acid, motsatana. Pre-emulsion idakonzedwa ndi polytron PCU-2-110 high speed homogenizer (Brinkmann Ind. Westbury, NY, USA). Njira yokonzekera ndi iyi: choyamba, 2ml ya yankho la TPP imawonjezeredwa ku 4ml ya yankho la insulin, ndipo chisakanizocho chimasakanizidwa kwa mphindi 30 ndikusakanikirana bwino. Kenako, yankho losakanikirana linawonjezedwa pang'onopang'ono ku yankho la CS kudzera mu sirinji pansi pa kusakaniza kwachangu (10,000 rpm). Zosakanizazo zinasungidwa pansi pa kusakaniza kwachangu (15,000 rpm) mu bafa la ayezi kwa mphindi 30, ndipo zinasinthidwa kukhala pH inayake kuti zipeze ma NP a insulin olumikizidwa. Kuti zigwirizanenso bwino ndikuchepetsa kukula kwa tinthu ta insulin NPs, zidasinthidwa kuti zigwiritsidwe ntchito. Kwa mphindi 30 mu bafa la ayezi pogwiritsa ntchito sonicator ya mtundu wa probe (UP 200ST, Hielscher Ultrasonics, Teltow, Germany).
Insulin NPS idayesedwa kuti ione ngati pali diameter ya Z-average, polydispersity index (PDI) ndi zeta potential pogwiritsa ntchito muyeso wa dynamic light scattering (DLS) pogwiritsa ntchito Litesizer 500 (Anton Paar, Graz, Austria) powasakaniza m'madzi a DD pa 25°C. Kapangidwe kake ndi kugawa kwa kukula kwake kunadziwika ndi Hitachi H7600 transmission electron microscope (TEM) (Hitachi, Tokyo, Japan), ndipo zithunzizo zidasanthulidwa pambuyo pake pogwiritsa ntchito pulogalamu ya Hitachi imaging (Hitachi, Tokyo, Japan). Kuti aone momwe encapsulation efficiency (EE) ndi loading capacity (LC) ya insulin NPs, ma NP adalowetsedwa m'machubu a ultrafiltration okhala ndi molecular weight cut-off ya 100 kDa ndikuyikidwa centrifuge pa 500 xg kwa mphindi 30. Insulin yosalowa mu filtrate idayesedwa pogwiritsa ntchito Agilent 1100 Series HPLC system (Agilent, Santa Clara, California, USA) yokhala ndi quaternary pump, autosampler, column. chotenthetsera, ndi chowunikira cha DAD. Insulin idawunikidwa ndi column ya C18 (Zorbax, 3.5 μm, 4.6 mm × 150 mm, Agilent, USA) ndipo idapezeka pa 214 nm. Gawo loyenda linali acetonitrile ndi madzi, okhala ndi 0.1% TFA, ma gradient ratios kuyambira 10/90 mpaka 100/0, ndipo idayendetsedwa kwa mphindi 10. Gawo loyenda linapopedwa pamlingo woyenda wa 1.0 ml/min. Kutentha kwa column kunayikidwa pa 20 °C. Werengani maperesenti a EE ndi LC pogwiritsa ntchito ma equation.(1) ndi Eq.(2).
Ma CS/insulin ratios osiyanasiyana kuyambira 2.0 mpaka 4.0 adayesedwa kuti akwaniritse bwino insulin NP. Kuchuluka kosiyanasiyana kwa yankho la CS kudawonjezedwa panthawi yokonzekera, pomwe kusakaniza kwa insulin/TPP kudasungidwa kosasintha. Ma Insulin NPs adakonzedwa mu pH range ya 4.0 mpaka 6.5 mwa kuwongolera mosamala pH ya kusakaniza pambuyo powonjezera mayankho onse (insulin, TPP ndi CS). Kukula kwa EE ndi tinthu tating'onoting'ono ta insulin nanoparticles kudayesedwa pamitundu yosiyanasiyana ya pH ndi ma CS/insulin mass ratios kuti akwaniritse bwino mapangidwe a insulin NPs.
Ma insulin NP okonzedwa bwino anaikidwa pa chidebe cha aluminiyamu ndipo anaphimbidwa ndi minofu yolimba ndi tepi. Kenako, ma screwed bottle anaikidwa mu Labconco FreeZone freezer dryer (Labconco, Kansas City, MO, USA) yokhala ndi thireyi dryer. Kutentha ndi vacuum pressure zinayikidwa pa -10 °C, 0.350 Torr kwa maola awiri oyamba, ndi 0 °C ndi 0.120 Torr kwa maola 22 otsala a maola 24 kuti mupeze ma insulin NP ouma.
Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Switzerland) idagwiritsidwa ntchito popanga insulin yozungulira. Magawo osankhidwa owumitsa anali: kutentha 100 °C, kuyenda kwa chakudya 3 L/min, ndi kuyenda kwa mpweya 4 L/min.
Ma NP a insulin asanayambe komanso atamaliza kutaya madzi m'thupi adadziwika pogwiritsa ntchito FTIR-ATR spectroscopy. Tinthu tating'onoting'ono tomwe tinasowa madzi komanso insulin yaulere ndi chitosan zinawunikidwa pogwiritsa ntchito Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, Massachusetts, USA) yokhala ndi chowonjezera cha ATR sampling (PerkinElmer, Waltham, Massachusetts, USA). Avereji ya chizindikiro idapezeka kuchokera ku ma scan 16 pa resolution ya 4 cm2 pa frequency range ya 4000-600 cm2.
Kapangidwe ka ma NP a insulin ouma kanayesedwa ndi zithunzi za SEM za ma NP a insulin ouma ouma ndi opopera omwe anajambulidwa ndi Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, USA). Chigawo chachikulu chomwe chinagwiritsidwa ntchito chinali voltage 5 keV ndi current 30 mA.
Ma NP onse a insulin omwe sanamwe madzi anasungunukanso m'madzi a dd. Kukula kwa tinthu tating'onoting'ono, PDI, EE ndi LC zinayesedwanso pogwiritsa ntchito njira yomweyi yomwe yatchulidwa kale kuti aone ubwino wawo atangotaya madzi. Kukhazikika kwa ma NP a anhydroinsulin kunayesedwanso poyesa makhalidwe a ma NP atasungidwa kwa nthawi yayitali. Mu kafukufukuyu, ma NP onse atataya madzi anasungidwa mufiriji kwa miyezi itatu. Pambuyo pa miyezi itatu yosungidwa, ma NP anayesedwa kukula kwa tinthu tating'onoting'ono, PDI, EE ndi LC.
Sungunulani 5 mL ya ma NP obwezeretsedwa mu 45 mL yokhala ndi madzi am'mimba oyeserera (pH 1.2, okhala ndi 1% pepsin), madzi am'mimba (pH 6.8, okhala ndi 1% trypsin) kapena yankho la chymotrypsin (100 g/mL, mu phosphate buffer, pH 7.8) kuti muwone momwe insulin imagwirira ntchito poteteza ma NPs atatha kutaya madzi m'thupi. Anayikidwa pa 37°C ndi liwiro la 100 rpm. 500 μL ya yankho idasonkhanitsidwa nthawi zosiyanasiyana ndipo kuchuluka kwa insulin kudatsimikiziridwa ndi HPLC.
Khalidwe la kutulutsa insulin NPs yokonzedwa kumene komanso yopanda madzi m'thupi linayesedwa pogwiritsa ntchito njira ya dialysis bag (molecular weight cut-off 100 kDa, Spectra Por Inc.). Ma NPs ouma omwe adakonzedwanso ndi kukonzedwanso adayikidwa mu dialysis m'madzi pa pH 2.5, pH 6.6, ndi pH 7.0 (0.1 M phosphate-buffered saline, PBS) kuti ayerekezere malo a pH m'mimba, duodenum, ndi m'matumbo ang'onoang'ono apamwamba, motsatana. Zitsanzo zonse zidayikidwa mu incubation pa 37 °C ndikugwedezeka kosalekeza pa 200 rpm. Thirani madziwo kunja kwa thumba la dialysis la 5 mL nthawi zotsatirazi: 0.5, 1, 2, 3, 4, ndi 6 maola, ndikudzaza nthawi yomweyo ndi dialysate yatsopano. Kuipitsidwa kwa insulin m'madzi kunawunikidwa ndi HPLC, ndipo kuchuluka kwa insulin yotulutsidwa kuchokera ku nanoparticles kunawerengedwa kuchokera ku chiŵerengero cha insulin yaulere yotulutsidwa ku insulin yonse yomwe ili mu nanoparticles (Equation 3).
Maselo a HepG2 a khansa ya chiwindi cha munthu adakulitsidwa m'mbale za mainchesi 60 pogwiritsa ntchito Dulbecco's Modified Eagle's Medium (DMEM) yokhala ndi 10% fetal bovine serum, 100 IU/mL penicillin, ndi 100 μg/mL streptomycin29. Ma cultures adasungidwa pa 37°C, 95% chinyezi, ndi 5% CO2. Pa mayeso otengedwa, maselo a HepG2 adalimidwa pa 1 × 105 cells/ml pa 8-well Nunc Lab-Tek chamber slide system (Thermo Fisher, NY, USA). Pa mayeso a cytotoxicity, adalimidwa m'ma plates a 96-well (Corning, NY, USA) pa kuchuluka kwa 5 × 104 cells/ml.
Kuyesa kwa MTT kunagwiritsidwa ntchito poyesa kuopsa kwa insulini NPs30 yokonzedwa kumene komanso yopanda madzi m'thupi. Maselo a HepG2 anabzalidwa m'mabolo 96 okhala ndi kachulukidwe ka maselo 5 × 104/mL ndipo anabzalidwa kwa masiku 7 asanayesedwe. Ma insulin NPs anachepetsedwa kufika pamlingo wosiyanasiyana (50 mpaka 500 μg/mL) mu culture medium kenako anaperekedwa ku maselo. Pambuyo pa maola 24 a incubation, maselo anatsukidwa katatu ndi PBS ndipo anabzalidwa ndi 0.5 mg/ml MTT kwa maola ena 4. Cytotoxicity inayesedwa poyesa kuchepa kwa enzymatic kwa yellow tetrazolium MTT kukhala purple formazan pa 570 nm pogwiritsa ntchito Tecan infinite M200 pro spectrophotometer plate reader (Tecan, Männedorf, Switzerland).
Kugwira ntchito bwino kwa ma NPs m'maselo kunayesedwa pogwiritsa ntchito microscopy ya confocal laser scanning ndi flow cytometry analysis. Chitsime chilichonse cha Nunc Lab-Tek chamber slide system chinachiritsidwa ndi free FITC-insulin, FITC-insulin-loaded NPs, ndikubwezeretsanso 25 μg/mL ya FITC-insulin NPs youma pamlingo womwewo ndikusungidwa kwa maola 4. Maselo anatsukidwa katatu ndi PBS ndikukhazikika ndi 4% paraformaldehyde. Nuclei inapakidwa utoto ndi 4′,6-diamidino-2-phenylindole (DAPI). Kupezeka kwa insulin kunawonedwa pogwiritsa ntchito Olympus FV1000 laser scanning/two-photon confocal microscope (Olympus, Shinjuku City, Tokyo, Japan). Pa kusanthula kwa flow cytometry, kuchuluka komweko kwa 10 μg/mL free FITC-insulin, FITC-insulin-loaded NPs, ndi resolubilized dehydrated FITC-insulin. Ma NP anawonjezedwa m'mabolo 96 omwe anabzalidwa ndi maselo a HepG2 ndipo anaikidwa mu incubation kwa maola 4. Pambuyo pa maola 4 a incubation, maselo anachotsedwa ndikutsukidwa katatu ndi FBS. Maselo 5 × 104 pa chitsanzo chilichonse anawunikidwa ndi BD LSR II flow cytometer (BD, Franklin Lakes, New Jersey, United States).
Ma mtengo onse amafotokozedwa ngati apakati ± kupotoka kokhazikika. Kuyerekeza pakati pa magulu onse kunayesedwa pogwiritsa ntchito njira imodzi ya ANOVA kapena t-test ndi IBM SPSS Statistics 26 ya Mac (IBM, Endicott, New York, USA) ndipo p < 0.05 idawonedwa ngati yofunika kwambiri pa ziwerengero.
Kafukufukuyu akuwonetsa kusinthasintha ndi kuthekera kwa kuuma kwa spray kuti achotse madzi m'thupi la chitosan/TPP/insulin nanoparticles zomwe zimalumikizidwa ndi cross-linked ndi reconstruction yabwino poyerekeza ndi njira zozizira zouma pogwiritsa ntchito zinthu zokulungira kapena cryoprotectants komanso mphamvu yayikulu yonyamula. Ma insulin nanoparticles opangidwa bwino adapereka kukula kwa tinthu tating'onoting'ono ta 318 nm komanso mphamvu yolumikizira ya 99.4%. Zotsatira za SEM ndi FTIR pambuyo pa kutaya madzi m'thupi zidawonetsa kuti kapangidwe kake kamakhalabe mu ma NP ouma opopera ndi opanda mannitol ndipo adasinthidwa ndi mannitol, koma ma NP opupera popanda mannitol adawonongeka panthawi ya kutaya madzi m'thupi. Mu mayeso a reconstitution ability, ma insulin nanoparticles opupera popanda mannitol adawonetsa kukula kwa tinthu tating'onoting'ono kwambiri komanso katundu wapamwamba kwambiri akasinthidwa. Makhalidwe otulutsa ma NP onse opuperawa adawonetsa kuti adatulutsidwa mwachangu mu mayankho a pH = 2.5 ndi pH = 7, komanso okhazikika kwambiri mu yankho la pH = 6.5. Poyerekeza ndi ma NP ena opupera osungunuka, ma NPs opupera ouma popanda Mannitol inatulutsa mofulumira kwambiri. Zotsatirazi zikugwirizana ndi zomwe zinapezeka mu kuyesa kwa ma cell, chifukwa ma NP oumitsidwa ndi spray popanda mannitol adasunga bwino momwe ma NP okonzedwa mwatsopano amagwirira ntchito. Zotsatirazi zikusonyeza kuti tinthu tating'onoting'ono ta insulin touma tokonzedwa ndi mannitol-free spray drying ndi toyenera kwambiri kuti tigwiritsidwe ntchito mu mitundu ina ya anhydrous dosing, monga mapiritsi omwa kapena mafilimu omatira.
Chifukwa cha mavuto a umwini wa nzeru, ma data omwe apangidwa ndi/kapena kufufuzidwa panthawi ya kafukufukuyu sapezeka pagulu, koma akupezeka kwa olemba omwewo ngati apempha moyenera.
Kagan, A. Matenda a shuga amtundu wachiwiri: chiyambi cha chikhalidwe ndi sayansi, zovuta zachipatala, ndi zotsatira zake kwa odwala ndi ena. (McFarlane, 2009).
Singh, AP, Guo, Y., Singh, A., Xie, W. & Jiang, P. Kukula kwa insulin encapsulation: kodi kumwa pakamwa tsopano n'kotheka? J. Pharmacy.bio-pharmacy.reservoir.1, 74–92 (2019).
Wong, CY, Al-Salami, H. & Dass, CR Kupita patsogolo kwaposachedwa kwa njira zoperekera liposome zodzaza ndi insulin mkamwa pochiza matenda a shuga. Interpretation.J. Pharmacy.549, 201–217 (2018).