Razvoj nanosistemov za povečanje hitrosti raztapljanja težko topnih učinkovin in aktivno ciljanje tumorskih celic

• Other Authors: Kristl, Julijana
• Format: Doctoral or Postdoctoral Thesis
• Language: Slovenian
• Published: P. Kocbek 2021
• Subjects: nanosuspenzije; nanodelci; nanosistemi; dostavni sistem; ciljanje rakavih celic s PLGA; imunonanodelci; info:eu-repo/classification/udc/66.017; sami
• Online Access: https://repozitorij.uni-lj.si/IzpisGradiva.php?id=127017; https://repozitorij.uni-lj.si/Dokument.php?id=143215&dn=; https://plus.si.cobiss.net/opac7/bib/238696192?lang=sl

Nanosistemi predstavljajo danes zelo aktualno področje raziskav, saj omogočajo vnos tako nizko kot visokomolekularnih učinkovin, kot so proteini in geni v organizem. Izbiro ustreznega nanodostavnega sistema in metode njegove izdelave pogojujejo predvsem lastnosti učinkovine. Namen našega raziskovalnega dela je bil pripraviti in ovrednotiti nanosuspenzije in polimerne nanodelce za vnos težko topnih učinkovin v organizem ter oblikovati imunonanodelce za ciljano dostavo proteinskih učinkovin v tumorske celice. Razvili smo emulzijsko-difuzijsko metodo in metodo emulgiranja taline za izdelavo nanosuspenzij ibuprofena kot modelne težko topne učinkovine. Z nadzorom tehnoloških parametrov smo pripravili nanosuspenzije s povprečno velikostjo delcev od 70 do 450 nm. S primerjavo raztapljanja vzorcev smo dokazali povečano hitrost raztapljanja nanosuspenzije v primerjavi z mikronizirano učinkovino. V raziskave smo vključili dve novi, težko topni učinkovini, ki sta inhibitorja 17β- hidroksisteroid dehidrogenaze tipa 1 in potencialni učinkovini za zdravljenje raka, saj povečana aktivnost omenjenega encima povzroči povečano proliferacijo celic dojke, kar povzroči nastanek in napredovanje raka. Sami učinkovini v raztopini oz. suspenziji v in vitro testih nista izkazovali biološkega učinka poleg tega je bila stabilnost učinkovin v vodnem mediju majhna. Po vgrajevanju učinkovin v nanodelce iz poli(ε-kaprolaktona) in testiranju na celični liniji T-47D, ki izraža tarčni encim, smo ugotovili zmanjšano proliferacijo in spremenjeno morfologijo celic, kar je bil odraz biološkega učinka vgrajenih inhibitorjev. Ti rezultati kažejo, da lahko z oblikovanjem nanodelcev pripravimo terapevtsko uporabno farmacevtsko obliko za vnos težko topne učinkovine, ki sama v dovolj veliki koncentraciji ne doseže mesta delovanja in zato ne sproži farmakološkega učinka. Aktivno ciljanje tumorskih celic je cilj razvoja novih farmacevtskih oblik za zdravljenje raka, saj na tak način povečamo učinkovitost zdravljenja in hkrati zmanjšamo stranske učinke. Z dvojno emulzijsko-difuzijsko metodo smo izdelali nanodelce iz kopolimera mlečne in glikolne kisline in vanje vgradili modelno proteinsko učinkovino tj.kokošji cistatin, ki je inhibitor cisteinskih proteaz. Na površino nanodelcev smo z adsorpcijo ali kovalentno vezali monoklonsko protitelo CDI 315, ki prepozna specifični antigen na površini celic raka dojke. Ugotovili smo, da je ustreza metoda izdelave imunonanodelcev le z adsorpcijo, saj je vgrajeni protein ohranil biološko aktivnost, protitelo pa sposobnost prepoznavanja in vezave na tarčni antigen. V kokulturi celic raka dojke MCF-10A neoT s celicami Caco-2 ali z diferenciranimi celicami U-937 so imunonanodelci selektivno vstopili le v tarčne celic in povzročili inhibicijo znotrajcelične proteolizne aktivnosti. S temi rezultati smo potrdili uspešnost aktivnega ciljanja tumorskih celic in selektivnega vnosa biološko aktivne proteinske učinkovine z imunonanodelci. Na podlagi rezultatov lahko zaključimo, da z oblikovanjem nanosuspenzij povečamo hitrost raztapljanja in tako biološko uporabnost težko topnih učinkovin. V določenih primerih pa to ni dovolj, da bi dosegli biološki učinek, kar velja zlasti za učinkovine, ki so nestabilne v fiziološkem okolju. V takšnih primerih smo dokazali, da je ustrezen pristop oblikovanje nanodelcev, kjer ogrodje ščiti vgrajeno učinkovino pred vplivi okolja in hkrati nadzorovano sprošča učinkovino na mestu delovanja. Z vezavo specifičnih monoklonskih protiteles na nanodelce smo dosegli aktivno ciljanje na celičnem nivoju tj. selektivni vstop imunonanodelcev in dostavo proteinske učinkovine do ciljnega mesta, kar prepreči pojav stranskih učinkov, ki so rezultat delovanja učinkovine na zdrave celice. Nanosystems represent a very promising research area nowadays, since they enable delivery of high and low molecular weight drugs into the body, such as proteins and genes. The choice of a suitable nanodelivery system and preparation method mainly depends on drug properties. The aim of our research work was to prepare and evaluate nanosuspensions and polymeric nanoparticles for delivery of poorly soluble drugs into the body and to formulate immuno-nanoparticles for targeted delivery of protein drug into tumour cells. Emulsion diffusion method and melt emulsification method were developed for preparation of nanosuspensions with ibuprofen as a model drug. Control of technological parameters allowed formulation of nanosuspensions with mean particle size ranging from 70 to 450 nm. Increased dissolution rate was confirmed for nanosuspesion compared to the dissolution of the micronized drug.Two new poorly soluble drugs with potential anticancer activity were included in our research. The drugs are inhibitors of type 1 17β-hydroxysteroid dehydrogenase, an enzyme which is involved in increased breast cell proliferation and therefore development and progression of cancer. The suspension of the inhibitors did not exert any biological effect in vitro. Besides that, stability of inhibitors in aqueous medium was low. Evaluation of formulated poly(ε-caprolactone) nanoparticles on T-47D cell line, which expresses target enzyme, showed reduced cell proliferation and changes in cell morphology due to biological effect of incorporated inhibitors. Poorly soluble drugs do not reach the site of action in concentration high enough to exert pharmacological effect, however, these results show that we can prepare therapeutically useful dosage form of poorly soluble drugs using nanoparticle formulation. The aim of development of new dosage forms for cancer treatment is active targeting of tumour cells, because this can lead to increased treatment effectiveness and reduced side effects. Cystatin is an inhibitor of cysteine proteases and was chosen as our model protein drug. Poly(lactide-co-glycolide) nanoparticles with incorporated cystatin were formulated using double emulsion solvent diffusion method. Monoclonal antibody, which recognizes specific antigen on breast tumour cells, was bound by adsorption or covalently to the nanoparticle surface. Only adsorption was suitable for binding of monoclonal antibody, since biological activity of incorporated protein as well as recognition and binding properties of adsorbed antibody were preserved. In co-culture of MCF-10A neoT breast cancer cells and Caco-2 cells or differentiated U-937 cells immunonanoparticles selectively entered target MCF-10A neoT cells and caused inhibition of intracellular proteolytic activity in those cells. With these results successful active targeting of tumor cells and selective delivery of biologically active protein drug using immuno-nanoparticles was confirmed. Based on our results it can be concluded that dissolution rate can be increased using nanosuspension formulations and consequently increased bioavailability of poorly soluble drugs can be expected. Due to instability of some drugs in physiological environment increased drug dissolution rate alone is not enough to achieve biological effect. For such drugs nanoparticle formulation can provide protection from environment and enables controlled drug release at the site of action as shown in our work. Active targeting on cellular level was achieved with specific monoclonal antibody bound to nanoparticles. Active targeting means selective uptake of immuno-nanoparticles and delivery of protein drug to target site which can reduce side effects caused by drug action on healthy cells.