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Ba nanoparticules ya insuline (NPs) oyo ezali na teneur ya chargement ya likolo ezui ba applications différentes na ba formes ya dosage différents.Mosala oyo ezali na tina ya ko évaluer effet ya ba processus ya séchage en congelation na ya séchage en pulvérisation na structure ya ba nanoparticules ya chitosan chargées na insuline, na to sans mannitol comme cryoprotectant.To évaluaki pe qualité ya ba nanoparticules oyo en redissolvant bango.Avant déshydratation, taille ya particule ya ba nanoparticules réticules chitosan/tripolyphosphate de sodium/insuline e optimisé mpo ezala 318 nm, PDI ezalaki 0,18, efficacité ya encapsulation ezalaki 99,4%, mpe chargement ezalaki 25,01%.Après reconstitution, ba nanoparticules nionso, sauf oyo esalemaki na méthode ya séchage congelé sans usage ya mannitol, esimbaki structure na bango ya particules sphérique.Soki tokokanisi yango na ba nanoparticules oyo ezali na mannitol oyo e déshydraté na soit pulvérisation, ba nanoparticules oyo ekauki na pulvérisation sans mannitol elakisaki pe taille moyenne ya particule ya moke koleka (376 nm) mpe teneur ya chargement ya likolo (25,02%) na taux ya encapsulation ya ndenge moko (98,7%) mpe PDI (0,20) na kokauka to techniques ya séchage en congelé.Ba nanoparticules oyo ekauki na séchage en pulvérisation sans mannitol esali pe que insuline ebima noki pe efficacité ya absorption cellulaire.Mosala oyo ezo lakisa que séchage en pulvérisation ekoki ko déshydrater ba nanoparticules ya insuline sans besoin ya ba cryoprotectants par rapport na ba méthodes conventionnelles ya séchage en congelé, ko créer capacité ya chargement ya munene, ba besoins ya additifs ya se pe fonctionnement esɛngaka litomba monene.
Uta bokutani na yango na 19221,2,3, insuline mpe biloko na yango ya nkisi ebikisi bomoi ya baye bazali na diabète ya lolenge ya liboso (T1DM) mpe ya diabète ya lolenge ya mibale (T1DM).Nzokande, mpo na bizaleli na yango lokola proteine ​​ya poids moléculaire ya likolo, insuline esanganaka na pete, epanzani na ba enzymes protéolytiques, mpe elongolamaka na nzela ya liboso effet.Bato oyo bazwami na diabète ya type 1 basengeli na ba injections ya insuline bomoi na bango mobimba.Ba maladi mingi oyo bazwamaki na ebandeli na diabète ya type 2 basengeli mpe kozwa ba injections ya insuline ya ntango molai.Ba injections ya insuline ya mokolo na mokolo ezali source ya mpasi makasi ya mokolo na mokolo mpe ya kozanga komiyoka malamu mpo na bato oyo, na ba effets négatifs na santé mentale.Comme résultat, ba formes mosusu ya administration ya insuline oyo epesaka inconfort moke, ya boye lokola bopesi insuline na monoko, ezali koyekola mingi5 lokola ezali na makoki ya kozongisa lolenge ya bomoi ya bato pene na miliare 5 oyo bazali na diabète na mokili mobimba.
Technologie ya nanoparticules epesi avancement ya monene na ba tentatives ya komela insuline orale4,6,7.Moko oyo e encapsuler pe ebatelaka insuline na dégradation pona livraison ciblé na ba sites spécifiques ya nzoto.Nzokande, usage ya ba formulations ya nanoparticules ezali na ba limitations ebele, surtout en raison ya ba problèmes ya stabilité ya ba suspensions ya particules.Some agrégation ekoki kosalema na tango ya stockage, oyo ekitisaka biodisponibilité ya nanoparticules chargées na insuline8.En plus, esengeli pe ko considérer stabilité chimique ya matrice polymère ya ba nanoparticules na insuline pona ko assurer stabilité ya ba nanoparticules ya insuline (NP).
Kasi, kokauka na congelation esengaka kobakisa ba cryoprotectants mpo na kopekisa structure sphérique ya ba NPs ezala affecté na stress mécanique ya ba cristaux ya glace.Yango ekitisaka mingi chargement ya ba nanoparticules ya insuline sima ya lyophilisation, lokola cryoprotectant ezuaka mingi ya rapport ya poids.Na yango, ba NPs ya insuline oyo ebimisami mbala mingi emonanaka ete ebongi te mpo na kosala ba formulations ya poudre sèche, lokola ba tablettes orales mpe ba films oraux, mpo na besoin ya ba quantités minene ya ba nanoparticules sèches mpo na kozua fenêtre thérapeutique ya insuline.
Kokauka na nzela ya pulvérisation ezali procédé ya échelle industrielle oyo eyebani malamu pe ya talo moke pona kobimisa ba poudres ya kokauka na ba phases liquides na industrie pharmaceutique10,11.Contrôle sur la procédé ya formation ya ba particules epesaka nzela ya encapsulation malamu ya ba composés bioactifs ebele 12, 13 .Lisusu, ekomi technique efficace pona préparation ya ba protéines encapsulées pona administration oral.During kokauka na pulvérisation, mayi evaporer noki mingi, oyo esalisaka kobatela température ya noyau ya particule na se11,14, ko permettre application na yango ya ko encapsuler ba composants sensibles na chaleur.Avant ya kokauka na pulvérisation, esengeli ko homogénéiser matériel ya revêtement malamu na solution oyo ezali na ba ingrédients encapsulé11,14.Unlike congelé-séchage, homogénéisation avant encapsulation na spray-drying ebongisaka efficacité ya encapsulation na tango ya déshydratation.Lokola procédé ya encapsulation na pulvérisation esengaka ba cryoprotectants te, spray-drying ekoki kosalelama pona kobimisa ba NPs ya kokauka oyo ezali na teneur ya chargement ya likolo.
Boyekoli oyo ezali kopesa lapolo ya bokeli ya ba NP chargés na insuline na nzela ya réticulation ya chitosan na tripolyphosphate de sodium na nzela ya méthode ya gel ionique.Gélation ionique ezali méthode ya préparation oyo epesaka nzela na production ya ba nanoparticules na nzela ya ba interactions électrostatiques entre deux ou plusieurs espèces ioniques sous certaines conditions.Ezala techniques ya séchage en congelé pe ya séchage en pulvérisation esalemaki pona ko déshydrater chitosan/sodium optimisé nanoparticules réticules tripolyphosphate/insuline.Après déshydratation, morphologie na bango e analysé na SEM.Makoki na bango ya recombination e évaluer na ko mesurer distribution ya taille na yango, charge ya surface, PDI, efficacité ya encapsulation, na contenus ya chargement.Qualité ya ba nanoparticules résolubilisées oyo ebimisami na ba méthodes différentes ya déshydratation e évaluer pe na ko comparer protection na bango ya insuline, comportement ya liberation, comportement ya liberation na bango mpe efficacité ya absorption cellulaire.
pH ya solution mélangée na rapport ya chitosan na insuline ezali deux facteurs clés oyo ezo affecter taille ya particule pe efficacité ya encapsulation (EE) ya ba NPs ya suka, lokola ezo affecter directement processus ya gélation ionotrope.PH ya solution mélangée elakisami kozala na corrélation makasi na taille ya particule pe efficacité ya encapsulation (Fig. 1a).Lokola elakisami na Fig. 1a, lokola pH emati depuis 4,0 kino 6,0, moyenne ya taille ya particule (nm) ekiti mpe EE emati makasi, nzokande tango pH emati na 6,5, moyenne ya taille ya particule ebandaki komata mpe EE etikalaki kobongwana te.Lokola rapport ya chitosan na insuline emati, taille moyenne ya particule pe emati.Lisusu, mbongwana moko te na EE emonanaki tango ba nanoparticules ebongisamaki na rapport ya masse ya chitosan/insuline eleki 2,5:1 (poids/poids) (Fig. 1b).Na yango, ba conditions ya préparation optimale na études oyo (pH 6,0, rapport ya masse chitosan/insuline ya 2,5:1) esalemaki pona kobongisa ba nanoparticules chargées na insuline pona études mosusu.Na condition oyo ya préparation, taille moyenne ya particules ya ba nanoparticules e optimisé pona kozala 318 nm (Fig. 1c), PDI ezalaki 0,18, efficacité ya intégration ezalaki 99,4%, potentiel zeta ezalaki 9,8 mv, mpe chargement ya insuline ezalaki 25,01% (m/m ).Na kotalela ba résultats ya microscopie électronique ya transmission (TEM), ba nanoparticules optimisées ezalaki grossièrement sphériques mpe discrètes na taille relativement uniforme (Fig. 1d).
Optimisation ya paramètre ya ba nanoparticules ya insuline : (a) effet ya pH na diamètre moyen mpe na efficacité ya encapsulation (EE) ya ba nanoparticules ya insuline (ebongisami na rapport ya masse 5:1 ya chitosan na insuline); (b) chitosan mpe Influence ya rapport masse ya insuline na diamètre moyen mpe na efficacité ya encapsulation (EE) ya ba NPs ya insuline (ebongisami na pH 6); (c) bopanzani ya bonene ya biloko ya mikemike ya ba nanoparticules ya insuline oyo ebongisami malamu; (d) Micrographie TEM ya ba NP ya insuline oyo ebongisami malamu .
Eyebani malamu ete chitosan ezali polyélectrolyte ya bolembu na pKa ya 6,5.Ezali na charge positive na milieu acide mpo groupe aminé na yango ya monene ezali protoné na ba ions hydrogène15.Na yango, mbala mingi esalelamaka lokola porteur mpo na ko encapsuler ba macromolécules chargées négatives.Na études oyo, chitosan esalelamaki mpo na ko encapsuler insuline na point isoélectrique ya 5.3.Lokola chitosan esalelamaka lokola matériel ya revêtement, na bomati ya proportion na yango, épaisseur ya couche ya libanda ya ba nanoparticules emati na correspondance, esali que taille moyenne ya particule ezala munene.En plus, niveaux ya chitosan ya likolo ekoki ko encapsuler insuline mingi.Na cas na biso, EE ezalaki likolo mingi tango rapport ya chitosan na insuline ekomaki na 2,5:1, pe changement ya significatif ezalaki te na EE ntango ratio yango ezalaki se komata.
Longola rapport ya chitosan na insuline, pH e jouaki pe rôle crucial na préparation ya ba NPs.Gan et al. 17 bayekolaki effet ya pH na taille ya particule ya ba nanoparticules ya chitosan.Bamonaki bokiti ya continu ya taille ya particule tii pH ekomaki na 6,0, mpe bomati ya monene ya taille ya particule emonanaki na pH > 6,0, oyo ezali na boyokani na observations na biso.Phénomène oyo ezali mpo na le fait que na bomati ya pH, molécule ya insuline ezuaka charge ya surface négative, donc, favorisant interactions électrostatiques na complexe chitosan/tripolyphosphate de sodium (TPP), oyo esali que taille ya particule ya muke pe EE ya likolo .Nzokande, tango ba ajusté pH na 6,5, ba groupes aminés oyo ezalaki na chitosan e déprotonés, esali que chitosan e pli.Na yango, pH ya likolo esali que ba ions aminés ezala moke na TPP na insuline, esali que ba réticules ezala moke, moyenne finale ya munene taille ya particule na EE ya se.
Analyse ya ba propriétés morphologiques ya ba NPs oyo ekauki na congelé pe oyo ekauki na pulvérisation ekoki ko guider sélection ya ba techniques ya déshydratation ya malamu pe ya formation ya poudre.Méthode préférée esengeli kopesa stabilité ya kisi, forme ya particule uniforme, chargement ya kisi ya likolo pe solubilité ya malamu na solution originale.Na études oyo, pona ko comparer malamu ba techniques mibale, ba NPs ya insuline na to sans 1% ya mannitol esalemaki na tango ya déshydratation.Mannitol esalelamaka lokola agent bulking to cryoprotectant na ba formulations ndenge na ndenge ya poudre sèche pona séchage en congelé pe séchage en pulvérisation.Po na ba nanoparticules ya insuline lyophilisées sans mannitol, ndenge elakisami na figure 2a, structure ya poudre très poreuse na ba surfaces ya minene, irregulières pe rugueuses emonanaki na microscopie électronique à scanner (SEM).Ba particules discrètes ya Few emonanaki na poudre sima ya déshydratation (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 kasi e rides na likolo (Fig. 2c).Ba surfaces spheriques na rides elobelami lisusu na ba tests ya comportement ya liberation pe ya absorption cellulaire na se.Na kotalaka apparence visible ya ba NPs oyo ekauki, ba NPs nionso mibale oyo ekauki na pulvérisation sans mannitol pe ba NPs oyo ekauki na congelé pe oyo ekauki na pulvérisation na mannitol epesaki ba poudres ya NPs ya malamu (Fig. 2f,g,h).Soki etando ya likolo kati ya ba surfaces ya particule ezali monene, solubilité ekozala mingi mpe na yango taux ya liberation ekozala likolo.
Morphologie ya ba NP ya insuline déshydraté ndenge na ndenge : (a) Image SEM ya ba NP ya insuline lyophilisé sans mannitol; (b) Image SEM ya ba NP ya insuline lyophilisé na mannitol; (c) ba NPs ya insuline oyo ekauki na pulvérisation sans manitol SEM image ya ; (d) Elilingi ya SEM ya ba NP ya insuline oyo ekauki na mannitol na pulvérisation; (e) elilingi ya poudre ya insuline NPs lyophilisée sans mannitol; (f) elilingi ya ba NP ya insuline lyophilisée na mannitol; ( g) Elilingi ya poudre ya insuline NPs oyo ekauki na pulvérisation oyo ezangi mannitol; (h) elilingi ya poudre ya insuline NPs oyo ekauki na pulvérisation na mannitol.
Na tango ya kokauka na congelé, mannitol esalaka lokola cryoprotecteur, ebatelaka ba NP na forme amorphe mpe epekisaka kobebisama na ba cristaux ya glace19.Na bokeseni, ezali na étape moko te ya congelation na tango ya kokauka na nzela ya pulvérisation.Yango wana mannitol esengeli te na méthode oyo.En fait, ba NPs oyo ekauki na pulvérisation sans mannitol epesaki ba NPs ya malamu ndenge elobamaki liboso.Nzokande, epesaki ba NPs ya malamu koleka ndenge elobamaki liboso.Nzokande, ezali na étape ya congelation te na tango ya kokauka na nzela ya pulvérisation. mannitol ekoki kaka kosala lokola remplissage na procédé ya kokauka na pulvérisation mpo na kopesa ba NPs structure sphérique mingi20 (Fig. 2d), oyo esalisaka mpo na kozua comportement ya liberation uniforme ya ba NPs encapsulés ya boye.En plus, ezali clair que ba particules mosusu ya minene ekoki ko détecté na ba NPs ya insuline oyo ekauki na congelation mpe na pulvérisation oyo ezali na mannitol (Fig. 2b,d), oyo ekoki kozala mpo na accumulation ya mannitol na kati ya noyau ya particule elongo na insuline encapsulé. To.Couche ya chitosan.Ezali na tina ya koyeba ete na boyekoli oyo, mpo na kosala ete structure sphérique ezala intacte sima ya déshydratation, rapport ya mannitol na chitosan ezala na 5:1, po ete motango monene ya remplissage ekoki pe ko augmenter taille ya particule ya ba NPs oyo ekauki..
Spectroscopie ya réflexion totale atténuée infrarouge transformation de Fourier (FTIR-ATR) e caractériser mélange physique ya insuline libre, chitosan, chitosan, TPP na insuline.Ba NP nionso déshydraté e caractérisé na nzela ya spectroscopie FTIR-ATR.Notablement, ba intensités ya bande ya 1641, 1543 na 1412 cm-1 emonanaki na ba NP encapsulé congelé na mannitol mpe na ba NPs oyo ekauki na pulvérisation na mannitol mpe sans mannitol (Fig. 3).Ndenge elobamaki liboso, bomati wana ya makasi ezalaki na boyokani na réticulation kati na chitosan, TPP mpe insuline.Enquête ya interaction entre chitosan na insuline elakisaki que na ba spectres FTIR ya ba nanoparticules ya chitosan chargés na insuline, bande ya chitosan e se superposer na oyo ya insuline, na ko augmenter ba intensité carbonyle (1641 cm-1) mpe ceinture amine (1543 cm-1).Ba groupes tripolyphosphate ya TPP ekangami na ba groupes ammonium na chitosan, esali bande na 1412 cm-1.
Spectres FTIR-ATR ya insuline ya ofele, chitosan, ba mélanges physiques ya chitosan/TPP/insuline na ba NPs déshydratées na ba méthodes différentes.
Lisusu ya chitosan, TPP, mpe insuline.Résultat oyo ezali kolakisa ete ba réticulations entre chitosan, TPP na insuline ezali lisusu te na ba NPs oyo ekauki na congelé sans mannitol.Sstructure ya ba NPs ebebisamaki na tango ya séchage congelé sans cryoprotectant, oyo ekoki komonana na ba résultats ya SEM (Fig. 2a).Na kotalela morphologie mpe ba résultats ya FTIR ya ba NPs ya insuline déshydraté, kaka ba NPs lyophilisées, spray-dried, mpe sans mannitol esalemaki mpo na ba expériences ya reconstitution mpe ba NPs sans mannitol mpo na décomposition ya ba NPs sans mannitol na tango ya déshydratation. kolobela.
Déshydratation esalelamaka pona kobombama na tango molayi pe ko retrait yango na ba formulations misusu.Makoki ya ba NPs ya kokauka ya ko reconstituer sima ya kobomba ezali critique pona usage na yango na ba formulations différentes lokola ba tablettes pe ba films.To remarqué que taille moyenne ya ba particules ya ba NPs ya insuline oyo ekauki na pulvérisation na absence ya mannitol emati kaka mua moke sima ya reconstitution.Na ngambo mosusu, taille ya ba particules ya ba NPs oyo ekauki na pulvérisation pe ba nanoparticules ya insuline oyo ekauki na congelé na mannitol emati makasi (Tableau 1).PDI na EE ebongwanaki mingi te (p > 0.05) sima ya recombination ya ba NP nionso na études oyo (Tableau 1).Resultat oyo elakisaka que mingi ya ba particules etikalaki intact sima ya ko rédissoudre.Nzokande, kobakisa mannitol esali que charge ya insuline ya lyophilisé mpe nanoparticules ya mannitol oyo ekauki na pulvérisation (Tableau 1).Na bokeseni, charge ya insuline ya ba NP oyo ekauki na pulvérisation sans mannitol etikalaki ndenge moko na liboso (Tableau 1).
Eyebani malamu ete chargement ya ba nanoparticules ezali critique tango esalemi mpo na ba buts ya livraison ya kisi.Po na ba NPs oyo ezali na ba chargements ya nse, esengeli ba quantités ya matériel ya minene mingi mpo na kokoma na seuil thérapeutique.Nzokande, viscosité ya likolo ya ba concentrations ya NP ya likolo boye ememaka na inconvénient mpe na difficulté na administration oral mpe na ba formulations injectables, respectivement 22 .En plus, ba NPs ekoki esalelamaka mpe mpo na kosala ba tablettes mpe ba biofilms visqueux23, 24, oyo esengaka kosalela ba NPs ebele na niveau ya chargement ya nse, oyo esali que ba tablettes ya minene mpe ba biofilms ya minene oyo ebongi te mpo na ba applications orales.Na yango, ba NPs déshydratés oyo ezali na charge ya insuline mingi ezali très désirable.Ba résultats na biso ezo lakisa que charge ya insuline ya likolo ya ba NPs sans mannitol oyo ekauki na pulvérisation ekoki kopesa mingi matomba ya kobenda likebi mpo na mayele mosusu wana ya kopesa.
Ba NP nionso déshydraté ebombamaki na frigo na boumeli ya sanza misato.Ba résultats ya SEM elakisaki que morphologie ya ba NP nionso déshydraté ebongwanaki mingi te na tango ya bobateli ya sanza misato (Fig. 4).Nsima ya reconstitution na mayi, ba NP nionso elakisaki mwa bokiti ya EE mpe ebimisaki pene na mwa ndambo moke (~5%) ya insuline na eleko ya bobateli ya sanza misato (Tableau 2).Nzokande, moyenne ya taille ya particules ya ba nanoparticules nionso emati.Taille ya particules ya ba NPs oyo ekauki na pulvérisation sans mannitol emati na 525 nm, alors que oyo ya ba NPs oyo ekauki na pulvérisation pe oyo ekauki na congelation na mannitol emati na 872 pe 921 nm, respectivement (tableau 2).
Morphologie ya ba NP ya insuline déshydraté ndenge na ndenge oyo ebombami na boumeli ya sanza misato : (a) image SEM ya ba NP ya insuline lyophilisé na mannitol; (b) Elilingi ya SEM ya ba nanoparticules ya insuline oyo bakaukisi na pulvérisation oyo ezangi mannitol; (c) kozanga bililingi ya SEM ya manitol ya ba NP ya insuline oyo bakaukisi na pulvérisation.
Lisusu, ba précipités emonanaki na ba nanoparticules ya insuline reconstituées oyo ekauki na pulvérisation na mannitol mpe ekauki na congelé (Fig. S2).Oyo ekoki kozala mpo na ba particules minene oyo ekangami malamu te na mayi.Ba résultats nionso oyo ezali likolo elakisaka que technique ya séchage na pulvérisation ekoki kobatela ba nanoparticules ya insuline na déshydratation mpe que ba charges ya likolo ya ba nanoparticules ya insuline ekoki kozuama sans aucune remplisseur to biloko oyo ebatelaka na cryoprotecteur.
Ba testaki rétention ya insuline na milieu pH = 2,5 na pepsine, trypsine, na α-chymotrypsine pona kolakisa makoki ya protection ya ba NPs contre digestion enzymatique après déshydratation.Ba comparaison ya rétention ya insuline ya ba NPs déshydratés na oyo ya ba NPs déshydratés, pe insuline ya ofele esalelamaki lokola contrôle négatif.Na études oyo, insuline ya ofele elakisaki élimination rapide ya insuline na kati ya 4 h na ba traitements enzymatiques nionso misato (Fig. 5a–c).Na bokeseni, test ya elimination ya insuline ya ba NPs oyo ekauki na congelé na mannitol mpe ba NPs oyo ekauki na pulvérisation na mannitol to sans mannitol elakisaki protection ya likolo mingi ya ba NPs oyo contre digestion enzymatique, oyo ekokanaki na oyo ya ba NPs ya insuline oyo ewutaki kobongisama ( figure 1).5a-c).Na lisalisi ya ba nanoparticules na pepsine, trypsine, mpe α-chymotrypsine, koleka 50%, 60%, mpe 75% ya insuline ekokaki kobatelama na kati ya 4 h, respectivement (Fig. 5a–c).Makoki oyo ya kobatela insuline ekoki komatisaka chance ya absorption ya insuline ya likolo na makila25.Ba résultats oyo ezo lakisa que kokauka na pulvérisation na to sans mannitol mpe kokauka na congelé na mannitol ekoki kobatela ba makoki ya kobatela insuline ya ba NP nsima ya kozanga mai na nzoto.
Bobateli mpe bizaleli ya kobimisama ya ba NP ya insuline oyo ezangi mai: (a) bobateli ya insuline na solution ya pepsine; (b) kobatela insuline na kati ya solution ya trypsine; (c) kobatela insuline na nzela ya solution α-chymotrypsine; ( d) Comportement ya liberation ya ba NP déshydraté na solution ya pH = 2,5; (e) comportement ya liberation ya ba NP déshydraté na solution ya pH = 6,6; (f) comportement ya liberation ya ba NP déshydraté na solution ya pH = 7,0.
Ba NP ya insuline ya kokauka oyo ebongisamaki sika mpe oyo ezongisamaki sika ekɔtisamaki na ba tampons ndenge na ndenge (pH = 2,5, 6,6, 7,0) na 37 °C, kosala lokola esika ya pH ya libumu, ya duodénum, ​​mpe ya intestin grêle ya likolo, mpo na kotala bopusi ya insuline na résistance na insuline. Comportement ya liberation na ba environnements différents.Fragment ya traitement gastro-intestinal.Na pH = 2.5, ba NPs chargées na insuline na ba NPs ya insuline ya kokauka oyo e résolubilisé elakisaki sortie ya explosion ya liboso na kati ya heure moko ya liboso, elandami na sortie ya malembe na 5 heures oyo elandi (Fig. 5d).Libération oyo ya noki na ebandeli ezali plus probablement résultat ya désorption rapide ya ba molécules ya surface ya proteine ​​oyo ezali entièrement te immobilisé na structure interne ya particule.Na pH = 6.5, ba NPs chargées na insuline na ba NPs ya insuline ya sec reconstitués elakisaki liberation ya lisse mpe ya malembe na 6 h, lokola pH ya solution ya test ekokanaki na oyo ya solution préparée na NPs (Fig. 5e).Na pH = 7, ba NPs ezalaki instable mpe presque entièrement décomposées na kati ya deux heures ya liboso (Fig.. 5f).Yango ezali mpo déprotonation ya chitosan esalemaka na pH ya likolo, oyo epesaka réseau polymère moins compact mpe kobimisa insuline chargée.
Lisusu.
Cytotoxicité ya ba NPs e investigamaki na test MTT.Ndenge elakisami na figure S4, ba NPs nionso déshydratés emonanaki kozala na effet ya significatif te na viability ya ba cellules na concentrations ya 50–500 μg/ml, elakisaka que ba NPs nionso déshydratés ekoki kosalelama sans danger pona kokoma na fenêtre thérapeutique.
Foie ezali organe principal na nzela na yango insuline esalaka ba fonctions physiologiques na yango.Ba cellules HepG2 ezali ligne cellulaire ya hépatome ya moto oyo esalelamaka mingi lokola modèle ya absorption ya ba hépatocytes in vitro.Awa, ba cellules HepG2 esalelamaki pona ko évaluer absorption cellulaire ya ba NPs déshydratés en utilisant ba méthodes ya congelé-séchage na pulvérisation.Apportation cellulaire par scanning laser confocale en utilisant flux cytométrie mpe vision sima ya ba heures ebele ya incubation na insuline FITC ya ofele na concentration ya 25 μg/mL, ba NPs chargés na insuline FITC oyo euti kobongisama mpe ba NPs chargés na insuline FITC déshydratées na concentrations égales ya insuline Ba observations ya microscopie quantitative (CLSM) esalemaki.Ba NPs lyophilisées sans mannitol ebebisamaki na tango ya déshydratation mpe evaluer te na oyo test.Ba intensités ya fluorescence intracellulaire ya ba NPs chargés na insuline oyo ebongisami sika, ba NPs lyophilisés na mannitol, mpe ba NPs oyo ekauki na pulvérisation na mannitol mpe sans mannitol (Fig. 6a) ezalaki 4,3, 2,6, 2,4, mpe 4,1 mbala koleka oyo ya ofele.FITC-insuline group, respectivement (Fig. 6b).Ba résultats oyo epesi likanisi ete insuline encapsulé ezali makasi mingi na absorption cellulaire koleka insuline ya ofele, mingi mingi mpo na taille moke ya ba nanoparticules chargées na insuline oyo esalemi na études.
Absorption ya ba cellules HepG2 sima ya 4 h ya incubation na ba NPs oyo esalemi sika pe ba NPs déshydratées : (a) Distribution ya absorption ya FITC-insuline na ba cellules HepG2.(b) Moyenne géométrique ya intensités ya fluorescence analysé na cytométrie ya flux (n = 3), *P < 0.05 par rapport na insuline ya ofele.
Ndenge moko pe, ba images ya CLSM elakisaki que ba intensités ya fluorescence ya FITC ya ba NPs chargés na insuline ya FITC oyo ebongisami sika pe ba NPs chargés na pulvérisation chargés na insuline ya FITC (sans mannitol) ezalaki makasi mingi koleka oyo ya ba échantillons misusu (Fig. 6a).Lisusu, na kobakisa mannitol, viscosité ya likolo ya solution ematisaki résistance na absorption cellulaire, esali que prolifération ya insuline ekita.Ba résultats oyo ezo lakisa que ba NPs sans mannitol oyo ekaukisi na pulvérisation elakisaki efficacité ya absorption cellulaire ya likolo mpo taille ya particules na yango ezalaki moke koleka oyo ya ba NPs oyo ekauki na congelation sima ya ré-dissolution.
Chitosan (poids moléculaire moyen 100 KDa, 75–85% désacétylé) esombamaki na Sigma-Aldrich.(Oakville, Ontario, Canada).Tripolyphosphate de sodium (TPP) esombamaki na VWR (Radnor, Pennsylvanie, USA).Insulin humain recombinant oyo esalelamaki na boyekoli oyo ezalaki na Fisher Scientific (Waltham, MA, USA).Fluorescein isothiocyanate insuline humaine marquée (FITC) mpe dihydrochlorure de 4′,6-diamidino-2-phenylindole (DAPI) esombamaki na Sigma-Aldrich.(Oakville, Ontario, Canada).Ligne cellulaire HepG2 ezwamaki na ATCC (Manassas, Virginia, USA).Ba réactifs mosusu nyonso ezalaki na grade analytique to chromatographique.
Bongisa solution ya CS ya 1 mg/ml na ko dissoudre yango na mayi double distillé (mai ya DD) oyo ezali na acide acétique 0,1%.Bongisa ba solutions ya 1 mg/ml ya TPP na insuline na ko dissoudre yango na mayi ya DD na acide acétique 0,1%, respectivement.Pré-emulsion esalemaki na homogénizer ya polytron PCU-2-110 ya vitesse makasi (Brinkmann Ind. Westbury, NY, USA).Nzela ya kobongisa ezali boye: ya liboso, 2ml ya solution ya TPP ebakisami na 4ml ya solution ya insuline, mpe basangisaka mélange na boumeli ya 30min mpe basangisaka yango mobimba.Na nsima, babakisaki solution oyo esangani goutte goutte na solution ya CS na nzela ya seringue na nse ya koningana ya mbangu mingi (10.000 rpm).Ba mélanges ebombamaki na vitesse makasi stirring (15.000 rpm) na bain glace pendant 30 min, mpe ba ajusté na pH moko boye mpo na kozua ba NPs ya insuline réticulés.Po na ko homogénéiser lisusu mpe ko réduire taille ya particule ya ba NPs ya insuline, ba soniqué bango pendant 30 min ya kobakisa na bain glace en utilisant sonicateur ya type sonde (UP 200ST, Hielscher Ultrasonics, Teltow, Allemagne).
NPS ya insuline emekamaki mpo na diamètre moyen Z, indice ya polydispersité (PDI) mpe potentiel ya zeta na kosalelaka ba mesures ya dispersion dynamique ya lumière (DLS) na nzela ya Litesizer 500 (Anton Paar, Graz, Autriche) na ko diluer yango na mayi ya DD na 25°C.Morphologie mpe distribution ya taille e caractérisé na microscope électronique ya transmission Hitachi H7600 (TEM) (Hitachi, Tokyo, Japon), mpe na sima ba analysé bilili na nzela ya logiciel ya imagerie Hitachi (Hitachi, Tokyo, Japon).Mpo na kotala efficacité ya encapsulation (EE) mpe capacité ya chargement (LC) ya ba NPs ya insuline, ba NPs e pipetté na ba tubes ya ultrafiltration na poids moléculaire ya coupe ya 100 kDa mpe e centrifugé na 500 xg pendant 30 min.Insuline non encapsulé na filtrat e quantifiamaki na nzela ya système HPLC ya Série Agilent 1100 (Agilent, Santa Clara, Californie, USA) oyo ezali na pompe quaternaire, autoéchantillonnage, chauffe- colonne, mpe détecteur DAD.Insuline e analysé na colonne C18 (Zorbax, 3,5 μm, 4,6 mm × 150 mm, Agilent, USA) mpe ezwamaki na 214 nm.Fase mobile ezalaki acétonitrile mpe mayi, oyo ezalaki na 0,1% ya TFA, ba rapports ya gradient kobanda 10/90 kino 100/0, mpe ekendeke na boumeli ya miniti 10. Phase mobile e pompamaki na débit ya 1,0 ml/min.Température ya colonne etiamaki na 20 °C.Kosala calcul ya ba pourcentages ya EE mpe LC na kosalelaka ba équations.(1) mpe Eq.(2) oyo ezali.
Ba ratios ndenge na ndenge ya CS/insuline oyo ebandi na 2,0 kino 4,0 emekamaki mpo na ko optimiser NP ya insuline.Ba quantités différentes ya solution ya CS ebakisami na tango ya préparation, alors que mélange insuline/TPP e gardaki constant.Ba NPs ya insuline ebongisamaki na intervalle ya pH ya 4,0 à 6,5 na ko contrôler na bokebi pH ya mélange sima ya kobakisa ba solutions nionso (insuline, TPP na CS).Ee mpe taille ya particule ya ba nanoparticules ya insuline evaluer na ba valeurs ya pH différentes mpe na ba rapports ya masse CS/insuline mpo na ko optimiser formation ya ba NPs ya insuline.
Ba NPs ya insuline optimisé etiamaki na récipient ya aluminium mpe ezipamaki na tissus serré na mua bande.Na sima, ba récipients visé etiamaki na sécheur congelé Labconco FreeZone (Labconco, Kansas City, MO, USA) équipé na sécheur ya plateau.Ba température na pression ya vide etiamaki na -10 °C, 0,350 Torr mpo na 2 h ya liboso, mpe 0 °C mpe 0,120 Torr mpo na 22 h oyo etikali ya 24 h mpo na kozwa ba NP ya insuline ya kokauka.
Buchi Mini Spray Dryer B-290 (BÜCHI, Flawil, Suisse) esalemaki mpo na kobimisa insuline encapsulé.Ba paramètres ya kokauka oyo eponami ezalaki : température 100 °C, débit ya alimentation 3 L/min, mpe débit ya gaz 4 L/min.
Ba NP ya insuline liboso pe sima ya déshydratation e caractérisé na nzela ya spectroscopie FTIR-ATR.Ba nanoparticules déshydratées lokola pe insuline ya ofele na chitosan e analysé na nzela ya spectrophotomètre Spectrum 100 FTIR (PerkinElmer, Waltham, Massachusetts, USA) équipé na accessoire universel ya échantillonnage ATR (PerkinElmer, Waltham, Massachusetts, USA). ba moyennes ezuami na ba scans 16 na résolution ya 4 cm2 na intervalle ya fréquence ya 4000-600 cm2.
Morphologie ya ba NP ya insuline ya kokauka etalemaki na bilili ya SEM ya ba NP ya insuline oyo ekauki na congelé mpe oyo ekauki na pulvérisation oyo ezuami na Microscope électronique Helios NanoLab 650 Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) (FEI, Hillsboro, Oregon, USA). Paramètre principal oyo basalelaki ezalaki tension 5 keV na courant 30 mA.
Ba NP nionso ya insuline déshydraté epanzani lisusu na mayi dd.Taille ya particule, PDI, EE na LC emekamaki lisusu na nzela ya méthode moko oyo tolobelaki liboso pona kotala qualité na yango sima ya déshydratation.Stabilité ya ba NP ya anhydroinsuline emekamaki pe na komeka ba propriétés ya ba NPs sima ya kobombama tango molayi.Na boyekoli oyo, ba NP nionso sima ya déshydratation ebombamaki na frigo pendant misato sanza.Nsima ya sanza misato ya kobombama, ba NPs emekamaki mpo na bonene ya particule morphologique, PDI, EE mpe LC.
Dissolve 5 mL ya ba NPs reconstitués na 45 mL oyo ezali na fluide gastrique simulé (pH 1,2, oyo ezali na 1% ya pepsine), fluide intestinal (pH 6,8, oyo ezali na 1% ya trypsine) to solution ya chymotrypsine (100 g/mL, na tampon phosphate, pH 7,8) mpo na kotala efficacité ya na kobatela ba NPs sima déshydratation.Ba incuber na 37°C na vitesse ya agitation ya 100 rpm.500 μL ya solution ezuami na ba points de temps différents mpe concentration ya insuline ezuami na HPLC.
Comportement ya liberation in vitro ya ba NPs ya insuline oyo euti kobongisama sika mpe oyo esili ko déshydraté emekamaki na nzela ya méthode ya sac ya dialyse (poids moléculaire cut-off 100 kDa, Spectra Por Inc.).Ba NPs ya kokauka oyo ebongisamaki sika mpe ezongisamaki na dialyse na ba fluides na pH 2,5, pH 6,6, mpe pH 7,0 (0,1 M saline tamponné na phosphate PBS) mpo na ko simuler environnement ya pH ya libumu, ya duodénum, ​​mpe ya intestin grêle ya likolo, respectivement.Ba échantillons nionso e incuber na 37 °C na koningana ya continu na 200 rpm.Aspirater fluide libanda ya sac ya dialyse ya 5 mL na ba temps oyo elandi: 0,5, 1, 2, 3, 4, na 6 h, mpe mbala moko kotondisa volume na ya sika dialysate.Contamination ya insuline na kati ya fluide e analysé na HPLC, mpe taux ya sortie ya insuline na ba nanoparticules e calculer na rapport ya insuline ya ofele oyo ebimaki na insuline totale encapsulé na ba nanoparticules (Equation 3).
Ligne cellulaire ya carcinome hépatocellulaire ya moto Ba cellules HepG2 ekolisama na ba plats ya diamètre 60 mm na nzela ya Dulbecco’s Modified Eagle’s Medium (DMEM) oyo ezalaki na 10% ya sérum bovin ya fœtus, 100 UI/mL ya pénicilline, mpe 100 μg/mL ya streptomycine29.Ba cultures e maintenir na 37°C, 95% relative humidité, mpe 5% ya CO2.Mpo na ba tests ya absorption, ba cellules HepG2 e semer na 1 × 105 cellules/ml na système ya diapositives ya chambre Nunc Lab-Tek ya 8 puits (Thermo Fisher, NY, USA).Po na ba tests ya cytotoxicité, e semer na ba plaques ya 96 puits (Corning, NY, USA) na densité ya 5 × 104 cellules/ml.
Test ya MTT esalelamaki pona kotala cytotoxicité ya ba NPs30 ya insuline oyo euti kobongisama sika pe déshydraté.Ba cellules HepG2 esimbamaki na ba plaques ya mabulu 96 na densité ya 5 × 104 cellules/mL pe e cultivé mikolo 7 avant ya test.Ba NPs ya insuline e dilué na ba concentrations ndenge na ndenge (50 à 500 μg/mL) na milieu ya culture pe sima epesamaki na ba cellules.Après 24 heures ya incubation, ba cellules e sukolamaki mbala 3 na PBS mpe e incuber na milieu oyo ezalaki na 0,5 mg/ml ya MTT pendant 4 heures ya kobakisa.Cytotoxicité e évaluer na ko mesurer réduction enzymatique ya MTT ya tétrazolium jaune na formazan violet na 570 nm na nzela ya prospectrophotomètre M200 infini ya Tecan motángi ya bapalaki (Tecan, Männedorf, Suisse).
Efficacité ya absorption cellulaire ya ba NPs emekamaki na microscopie ya scanner laser confocale mpe na analyse ya cytométrie ya flux.Libulu moko na moko ya système ya diapositives ya chambre Nunc Lab-Tek esalemaki na FITC-insuline ya ofele, ba NPs chargés na FITC-insuline, mpe e reconstituer 25 μg/mL ya ba NPs FITC-insuline déshydratées na concentration moko mpe e incuber mpo na 4 heures.Ba cellules esukolamaki mbala 3 na PBS mpe ekangamaki na 4% ya paraformaldehyde.Ba noyaux e taché na 4′,6-diamidino-2-phenylindole (DAPI).Localisation ya insuline emonanaki na nzela ya microscope confocal Olympus FV1000 scanning laser/deux photons (Olympus, Shinjuku City, Tokyo, Japon).Po na écoulement analyse ya cytométrie, ba concentrations moko ya 10 μg/mL ya FITC-insuline libre, NPs chargés na FITC-insuline, mpe ba NP FITC-insuline déshydraté résolubilisées ebakisami na ba plaques ya 96 puits oyo elonamaki na ba cellules HepG2 mpe e incuber pendant 4 heures .Après 4 h ya incubation, ba cellules elongolamaki mpe esukolamaki mbala 3 na Ba cellules FBS.5 × 104 na échantillon moko e analysé na cytomètre ya écoulement BD LSR II (BD, Franklin Lakes, New Jersey, Etats-Unis).
Ba valeurs nionso elakisami lokola moyenne ± écart standard.Bokokanisi kati ya bituluku nionso etalemaki na kosalelaka ANOVA ya nzela moko to t-test na IBM SPSS Statistics 26 pona Mac (IBM, Endicott, New York, USA) pe p < 0.05 etalemaki lokola statistiquement significatif.
Boyekoli oyo ezali kolakisa flexibilité mpe makoki ya séchage na pulvérisation mpo na ko déshydrater ba nanoparticules ya chitosan/TPP/insuline réticulations na reconstitution ya malamu koleka soki tokokanisi yango na ba méthodes standard ya séchage en congelé oyo esalelaka capacité ya ba agents bulking to cryoprotectants mpe capacité ya charge ya likolo.Ba nanoparticules ya insuline optimisées epesaki taille moyenne ya particules ya 318 nm mpe efficiency ya encapsulation ya 99.4%.Ba résultats ya SEM na FTIR sima ya déshydratation elakisaki que structure sphérique esimbamaki kaka na ba NPs oyo ekauki na pulvérisation na mannitol pe sans mannitol pe e lyophilisé na mannitol, kasi ba NPs lyophilisées sans mannitol e décomposé na tango ya déshydratation.Na test ya makoki ya reconstitution, ba nanoparticules ya insuline oyo ekauki na pulvérisation sans mannitol elakisaki taille ya particule moyenne ya moke pe charge ya likolo upon reconstitution.Ba comportements ya liberation ya ba NPs nionso oyo déshydraté elakisaki que ebimisamaki noki na ba solutions ya pH = 2,5 na pH = 7, mpe très stable na solution ya pH = 6.5.Comparé na ba NPs déshydratés misusu rédissous, ba NPs oyo ekauki na pulvérisation sans mannitol elakisaki liberation ya mbangu koleka.Resultat oyo ezali na boyokani na oyo emonanaki na assay ya absorption cellulaire, lokola ba NPs oyo bakaukisi na pulvérisation na absence ya mannitol e maintenir presque entièrement efficacité ya absorption cellulaire ya ba NPs oyo ewutaki kobongisama.Ba résultats oyo ezo lakisa que ba nanoparticules ya insuline ya kokauka oyo ebongisami na séchage ya pulvérisation sans mannitol ebongi mingi pona ko traité lisusu na ba formes misusu ya dosage anhydre, lokola ba tablettes orales to ba films bioadhésifs.
Na tina ya makambo ya bozwi mayele, ba ensembles ya ba données oyo esalemi pe/to oyo etalelami na tango ya boyekoli ya lelo ezali na miso ya bato te, kasi ezwami na bakomi oyo etali bango soki basengi yango na ndenge ya malamu.
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Singh, AP, Guo, Y., Singh, A., Xie, W. & Jiang, P. Bokoli ya encapsulation ya insuline: administration orale ezali sikoyo possible?J. Pharmacie.bio-pharmacie.réservoir.1, 74–92 (2019).
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