doktorska disertacija
Ana Miklavžin (Avtor), Julijana Kristl (Mentor), Mateja Cegnar (Komentor)

Povzetek

Razvoj rekombinantne tehnologije DNK in biotehnologije je omogočil proizvodnjo proteinov v industrijskem merilu ter jih povzdignil na nov nivo uporabe. Postali so učinkovine izbora za zdravljenje številnih bolezni zaradi njihove selektivnosti in terapevtske učinkovitosti. Vnos proteinskih učinkovin v telo je danes omejen predvsem na parenteralno dajanje, saj je absorpcija proteinov iz prebavil zaradi strukturnih lastnosti (velikosti molekul, hidrofilnosti, fizikalne, kemijske in proteolitske nestabilnosti) skoraj nična. S tega vidika in stališča pacientov postajata razvoj in uporaba naprednih dostavnih sistemov za peroralno dajanje proteinskih učinkovin vse bolj pomembna. Ena izmed naprednih dostavnih oblik za proteinske učinkovine so nanodelci, ki zaradi svojih specifičnih lastnosti predstavljajo obetaven sistem za izboljšanje stabilnosti proteinskih učinkovin in biološke uporabnosti. Mehanizem prehajanja proteinskih učinkovin na ravni membran še ni v celoti pojasnjen, pogledi raziskovalcev pa se razlikujejo zlasti glede na pomen nosilnih materialov za vgrajevanje in posledično na lastnosti in permeabilnost nanodelcev, kar je področje v okviru disertacije. Uvodni del disertacije predstavlja osnovne lastnosti proteinov, načrtovanje, izdelavo ter in vitro in in vivo vrednotenje različnih proteinskih učinkovin in nanodelcev, nato sledijo eksperimenti in rezultati. Proučevali smo humani rekombinantni eritropoietin (EPO), njegov sintezno modificiran konjugat s kaprojsko kislino (mC6EPO), rekombinantni granulocitne kolonije stimulirajoči dejavnik (GCSF), pa tudi ovalbumin v procesih sušenja. Izbrani biokompatibilni polielektroliti v naših raziskavah so hitosan (CS), trimetilhitosan (TMC), kopolimer polimlečne in asparginske kisline, novo sintetiziran in ovrednoten kemijsko modificiran hitosan-graft-poli(L-glutamat) (CS-g-PGlu), alginat in hondroitin sulfat. Nanodelce smo pripravljali s kompleksiranjem proteinskih učinkovin in polielektrolitov v vodnem mediju, ki predstavlja najblažje pogoje in spontan nastanek. Z optimiranim postopkom smo dobili disperzije ND s povprečnim hidrodinamskim premerom med 200 in 300 nm, polidisperznim indeksom pod 0,3 in pri tem ohranili biološko aktivnost vgrajenih proteinov. V predformulacijskih študijah smo sprva proučili površinsko hidrofobnost prostega in v nanodelce vgrajenega EPO in njen vpliv na transepitelno prehajanje. Površinske lastnosti EPO smo ugotavljali s spreminjanjem intenzitete fluorescence spojine bis-ANS. Dokazali smo, da se hidrofobnost površin proteinov spreminja z variiranjem pH okolja in kompleksiranjem s polielektrolitom(i). Najvišjo površinsko hidrofobnost so izkazovali izdelani CS nanodelci z vgrajenim EPO, sledil je prost EPO, najnižjo pa CS-TMC ND z vgrajenim EPO. Največji permeabilnostni koeficient za EPO skozi epitelijski monosloj Caco-2 celic smo izmerili za CS nanodelce, ki so imeli tudi najvišjo hidrofobnost. Dokazali smo, da se pri kompleksiranju hidrofilnih proteinov s pH-občutljivimi polielektroliti zmanjša gostota nabojev in s tem poveča površinska hidrofobnost. Primerljivo obnašanje so pokazali nanodelci hitosan/mC6EPO/tripolifosfat, pa tudi hitosan/ovalbumin/kopolimer polimlečne in asparginske kisline in alginat/ovalbumin/hitosan. Novo sintetizirane hitosan-graft-poli(L-glutamat) kopolimere s 5, 10 ali 15 molekul dolgimi verigami smo uporabili za pripravo ND z GCSF in jih obložili s TMC. Dokazali smo, da dolžina pripetih verig statistično značilno poveča kapaciteto polielektrolita za kompleksiranje GCSF. Odkrili smo, da je za medsebojne interakcije proteinskih učinkovin s polielektroliti pomembna gostota naboja in strukturna dostopnost skupin. V izdelane ND se vgradi do 45 % GCSF, nanodelci pa so bili stabilni v temperaturnem območju med 25° - 39°C in pH vrednostih pod izoelektrično točko GCSF (pI 6,1) daljši čas. S pripravo EPO konjugata s kaprojsko kislino (mC6EPO) smo izboljšali naslednje lastnosti proteina kot so višja vgradnja v ND, delna zaščita pred kislinsko in encimsko razgradnjo ter povečano prehajanje skozi monosloj Caco-2 celic v primerjavi s prostim EPO. Z dodatkom pospeševalca absorpcije CYMAL v ND smo še izboljšali prehajanje tako za EPO kot mC6EPO glede na prost EPO v primerjavi brez pospeševalca absorpcije (prehajanje EPO se izboljša za 1186 krat in mC6EPO za 927 krat). Izdelani nanodelci z vključenim EPO in mC6EPO ter CYMAL so zaščitili protein pred encimsko razgradnjo, ohranila se je biološka aktivnost vgrajenih proteinov, kar smo dokazali s farmakološkim odzivom EPO in mC6EPO pri poskusnih živalih. Shranjevanje nanodelcev z občutljivimi proteinskimi učinkovinami za daljše časovno obdobje predstavlja raziskovalcem velik izziv. Z raziskovanjem liofilizacije tekočih disperzij nanodelcev s proteinsko učinkovino smo dokazali, da lahko z izborom ustreznih krio- in lio- protektantov (kot optimalna sta se izkazala trehaloza in manitol) uspešno izdelamo suhe nanodelce, ki tvorijo po redispergiranju v vodi disperzijo s podobnimi lastnostmi kot jih imajo pred sušenjem. Kot prvi smo v postopek sušenja disperzije nanodelcev vpeljali sušenje z razprševanjem disperzije ND v tekoči dušik (spray-freeze drying) in dobili sipek prašek. Rezultati eksperimentov v okviru doktorske disertacije nedvoumno dokazujejo odločilen pomen izbranih polielektrolitov za izdelavo nanodelcev s proteinskimi učinkovinami. Blagi tehnološki pogoji, nanometrska velikost delcev, visoka učinkovitost vgrajevanja proteina v komplekse, spremenjena površinska polarnost nanodelcev in pH-občutljivo sproščanje so obetavne možnosti za razvoj ustreznega dostavnega sistema za peroralni vnos proteinskih učinkovin. Ugotavljamo, da polielektrolitni nanodelci prinašajo preobrat v oblikovanje nosilnih sistemov za proteinske učinkovine v primerjavi s konvencionalnimi farmacevtskimi oblikami in omejitvami znanih tehnoloških pristopov. Temeljni cilj raziskav v okviru disertacije je bil torej dopolnitev znanja o mehanizmu vgrajevanja terapevtskih proteinov v polimerne nanodelce in njihovem vplivu na prehajanje bioloških membran, pa tudi pretvorba tekočih disperzij nanodelcev v suho, stabilno stanje, kar skupno predstavlja osnovo za učinkovitejšo terapevtsko uporabo.

Ključne besede

farmacevtska tehnologija;tehnologija rekombinantne DNA;zdravilne učinkovine;rekombinantni proteini;terapevtski proteini;peroralna aplikacija;dostavni sistemi;nanodelci;nanodostavni sistemi;rekombinantni humani eritropoetin;disertacije;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.08 - Doktorska disertacija
Organizacija: UL FFA - Fakulteta za farmacijo
Založnik: [A. Miklavžin]
UDK: 615.015.14:620.3(043.3)
COBISS: 298838528 Povezava se bo odprla v novem oknu
Št. ogledov: 108
Št. prenosov: 0
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Design, formulation and evaluation of polyelectrolyte nanoparticles for peroral delivery of therapeutic proteins
Sekundarni povzetek: The development of recombinant DNA technology and biotechnology has allowed for the industrial-scale production of proteins, which has elevated their use to a new level. Indeed, due to their selectivity and therapeutic efficacy, protein drugs have become the first choice of treatment for numerous diseases. The route of administration of protein drugs into the body is mainly limited to parenteral delivery, as there is little or no protein absorption from the gastrointestinal tract due to their structural properties (i.e., molecular size, hydrophilicity, and physical, chemical and proteolytic instability). On this basis, and from the patient perspective, the development and use of advanced delivery systems for oral administration of protein drugs have become increasingly important. Due to their specific properties, nanoparticles (NPs) offer a promising way forward for the delivery of protein drugs. As an advanced delivery system, NPs offer a promising way forward to improve the stability and bioavailability of protein drugs. The mechanism of protein drug permeability at the membrane level is not yet fully understood, and opinions in the field differ in terms of the importance of the carrier materials on drug loading, and consequently on the properties and permeability of NPs therefore this was also the aim of the research carried out in the framework of this Doctoral thesis. The Introduction of this thesis presents the basic characteristics, design, preparation and in-vitro and in-vivo evaluation of various protein drugs and NPs. This is followed by the study of human recombinant erythropoietin (EPO), its synthesised modified conjugate with caproic acid (mC6EPO), recombinant granulocyte colony stimulating factor (GCSF), and ovalbumin. The selected biocompatible polyelectrolytes used in this study were: chitosan (CS); trimethylchitosan (TMC); a copolymer of polylactic and aspartic acid; a newly synthesised and evaluated chemically modified chitosan -graft-poly(L-glutamate) (CS-g-PGlu); alginate; and chondroitin sulphate. The crosslinker used was tripolyphosphate (TPP). The NPs were prepared by complexation of the protein drugs with the polyelectrolytes in an aqueous medium, which allowed spontaneous formation of NPs under mild preparation conditions. The optimised NP preparation process allowed formation of NPs with hydrodynamic diameters of 200 nm to 300 nm and a polydispersity index <0.3, accompanied by preserved biological activity of the loaded protein drugs. In the preformulation study, the surface properties of free EPO and NP-loaded EPO were investigated, to monitor the relative hydrophobicity for correlation with membrane permeability. The surface properties of EPO were determined by varying the fluorescence intensity of the 4,4’-bis-1-anilinonaphtalene-8-sulfonate (bis-ANS) dye. This demonstrated that the surface hydrophobicity of the protein drug EPO was changed by varying the pH of its environment and after its complexation with the polyelectrolyte(s). EPO-loaded CS NPs showed the highest surface hydrophobicity, followed by free EPO, and EPO-loaded CS-TMC NPs. The highest permeability coefficient of EPO for permeation through a monolayer of Caco-2 epithelial cells was seen for the CS NPs, which also had the highest hydrophobicity. We show that polyelectrolyte complexation of hydrophilic proteins with pH-sensitive polyelectrolytes reduces the protein charge density, which thus increases the protein surface hydrophobicity. Comparable behaviour was seen for CS/ mC6EPO/ TPP NPs, CS/ ovalbumin/ poly(lactic-co-aspartic acid) NPs, and alginate/ ovalbumin/ CS NPs. Newly synthesised CS-g-PGlu copolymers where CS was modified with 5-, 10- or 15-molecule-long chains of glutamic acid were used to prepare NPs with GCSF, and were coated with TMC. We show that the length of the attached glutamic acid chains significantly increased the capacity of the polyelectrolyte for complexation with GCSF. We show that for the interaction of protein drugs with polyelectrolytes, the charge density and the structural accessibility of the functional groups on the polyelectrolytes are important. Loading of GCSF into these NPs of up to 45% was obtained, and the NPs were thermally stable from 25 °C to 39 °C, and showed longer pH-dependent stability below the isoelectric point of GCSF (pI, 6.1). The modified EPO conjugate that was prepared, mC6EPO, increased its loading into the NPs, partially protected mC6EPO against acid and enzymatic degradation, and increased its permeability through the Caco-2 cell monolayers, all compared to free EPO. The addition of the penentration enhancer CYMAL into NPs improved the permeability of both proteins, (i.e., EPO, mC6EPO) further, where the enhancement ratio of Papp of the NPs relative to that of free EPO in the some medium were 1186 for CS/ CYMAL-EPO/ TPP and 927 for CS/ CYMAL-mC6EPO/ TPP). We can conclude that these NPs protected EPO against enzymatic degradation and preserved its biological activity, which was confirmed by the pharmacological responses of EPO and mC6EPO in tests on rats after oral administration. Storage of NPs with sensitive protein drugs over long periods of time presents a challenge for researchers. It has been demonstrated that the selection of suitable cryo/lyoprotectants (those optimal were trehalose and mannitol) allows the production of NPs with characteristics that are comparable to freshly prepared dispersions of NPs. As a first, we introduced the spraying of freshly prepared NP dispersions into liquid nitrogen before lyophilisation (known as spray-freeze drying) to prepare dry bulk powders of NPs with loaded protein drugs with preserved biological activities. The results in the framework of this Doctoral thesis demonstrate the crucial importance of polyelectrolyte selection for the preparation of NPs for selected protein drugs. Mild preparation conditions, NP size, high protein drug entrapment efficiency into complexes, altered surface polarity of NPs, and pH-dependent release all provide promising opportunities for development of an appropriate delivery system for per-oral administration of protein drugs. Such polyelectrolyte NPs have positive influences in the formation of protein drug delivery systems, compared to conventional dosage forms and the limitations of known technological approaches. The aim of the research carried out in the framework of this Doctoral thesis was to integrate our knowledge about the mechanisms of protein drug loading into polymeric NPs, the influence of such polymeric NPs on protein permeability across biological membranes, and the conversion of liquid NP dispersions into a stable and dry state. Together, these aspects constitute thebasis for more effective design of therapeutic proteins in NPs.
Vrsta dela (COBISS): Doktorska disertacija
Komentar na gradivo: Univ. v Ljubljani, Fak. za farmacijo
Strani: 244 str.
ID: 15595304