Primena elektronske paramagnetne rezonancije za proučavanje albuminskih hidrogelova za kontrolisanu isporuku antikancerskih lekova i detekciju vijabilnosti ćelija

Abstract

U cilju smanjenja neželjenih dejstava i obezbeđivanja optimalne biodistribucije lekova, te ciljane terapije, mnoga istraživanja fokusirana su na alternativne načine za njihovu isporuku. Među brojnim platformama za inkapsulaciju lekova, hidrogelovi na bazi prirodnih polimera su posebno atraktivni kao nosači, pre svega zahvaljujući svojoj biokompatibilnosti i biorazgradivosti. Serumski albumin je dobro poznat po sposobnosti za vezivanje velikog broja endogenih i egzogenih supstancija, što je u ovoj studiji bio osnov za razvoj proteinskog hidrogela koji bi služio kao pogodan rezervoar citotoksičnog indolo[3,2-d]benzazepinskog liganda. Prepoznajući udeo vode u hidrogelu kao važnu karakteristiku za otpuštanje agenasa iz polimerne matrice, elektronska paramagnetna rezonancija (EPR), kao izuzetno osetljiva spektroskopska tehnika, iskorišćena je za razvoj nove metodologije za određivanje sadržaja vode u hidrogelu. Pokazano je da EPR aktivna spinska proba 3-karbamoil-proksil (3CP), koja ne interaguje sa proteinskom matricom, već je lokalizovana isključivo u vodenoj fazi, može da se koristi kao pouzdan indikator ukupne količine vode u hidrogelu. Utvrđeno je da između EPR spektralnih parametara 3CP-a i odnosa masa vode i suvog polimera postoji korelacija, koja je uspostavljena na albuminskim hidrogelovima, a potvrđena na hidrogelovima na bazi drugih biomolekula. Pokazano je da je EPR imidžingom moguće vizuelno pratiti difundovanje probe kroz polimernu matricu gela, otvarajući time mogućnost za primenu ove tehnike i za praćenje oslobađanja lekova i degradacije hidrogela. U narednoj fazi istraživanja EPR tehnika se pokazala izuzetno korisnom u in vitro ispitivanju vezivanja i oslobađanja spinski obeleženog jedinjenja iz klase indolo[3,2-d]benzazepina (HL), koji se odlikuje značajnom antikancerskom aktivnosti. Takođe je EPR protokol uspešno primenjen na hidrogelovima obeleženim probom 3CP, odnosno 5-doksil-stearatom (5-DS), u cilju izučavanja procesa difuzije, odnosno degradacije polimerne matrice, pružajući uvid u narušavanje interakcija na molekulskom nivou. S druge strane je potvrđen potencijal termalno-indukovanog hidrogela na bazi humanog serumskog albumina (HSA) da služi kao depo antikancerskih agenasa s odloženim oslobađanjem, sposoban da tokom 11 dana dijalize zadrži čak 60% citotoksičnog liganda HL, te i da je pogodniji nosač u odnosu na rastvor HSA, iz kog se 100% liganda otpusti u roku od 8 dana. Odsustvo citotoksične aktivnosti tokom 3 dana inkubacije HL/HSA hidrogela sa Colo-205 kancerskim ćelijama (adenokarcinom debelog creva) sugeriše da bi se odloženo oslobađanje citotoksičnog liganda moglo postići i u in vivo uslovima. Najzad, dijagnostički aspekt multifunkcionalnog proteinskog hidrogela ogleda se u potvrđenoj osetljivosti albuminskog hidrogela obeleženog spinskom probom 3CP na ćelijski redoks metabolizam. Kao inicijalna faza u razvoju prototipa hidrogela koji bi bio namenjen za praćenje odgovora na terapiju, hidrogel je u tom pogledu ispitan u Saccharomyces cerevisiae, koji je korišćen kao model sistem. U ovoj studiji je pokazana mogućnost da spinski obeležen albuminski hidrogel služi kao inidikator redoks okruženja, kroz smanjenje intenziteta EPR signala nitroksidne probe zavisno od broja prisutnih vitalnih ćelija u sistemu. U narednoj fazi istraživanja se očekuje da rezultati budu potvrđeni na eksperimentalnim animalnim modelima. Na osnovu sveukupnih rezultata, zaključuje se da bi ova studija mogla otvoriti vrata potencijalnoj primeni in vivo EPR molekularnog imidžinga u sprezi sa spinski obeleženim albuminskim hidrogelom, koji bi imao ulogu ne samo rezervoara antikancerskih lekova, već bi se koristio i za praćenje efekata lekova na okolna tkiva u pretkliničkim istraživanjima, te bi se otišlo i korak bliže integrisanju dijagnostike i terapije.

Many studies have focused on alternative ways for drug delivery to reduce side effects and to ensure optimal biodistribution and targeted therapy. Among various platforms for drug encapsulation, hydrogels based on natural polymers are attractive carriers, primarily due to their biocompatibility and biodegradability. Serum albumin is well known for its ability to bind a variety of endogenous and exogenous substances, which, in this study, was the foundation for the development of a protein hydrogel that is a suitable reservoir of the cytotoxic indolo[3,2-d]benzazepine ligand. Considering the importance of the water amount in the hydrogel for the active agent release from the polymeric matrix, electron paramagnetic resonance (EPR), as an extremely sensitive spectroscopic technique, was used to develop a new methodology for hydrogel water content determination. It was shown that the EPR active spin probe 3-carbamoyl-proxyl (3CP), which does not interact with the protein matrix and is entirely localized in the aqueous phase, can be used as a reliable indicator of the total hydrogel water content. A functional correlation between 3CP EPR spectral parameters and the mass ratio of water to dry polymer was established on albumin hydrogels and confirmed on hydrogels based on other biomolecules. It was shown that it is possible to visualize the diffusion of the sample through the gel polymer matrix by EPR imaging, opening the possibility for the application of this technique for the monitoring of drug release and hydrogel degradation. In the subsequent research phase, the EPR technique has proved to be extremely useful in investigating the binding and release of a spin-labeled indolo[3,2-d]benzazepine ligand (HL) which displays significant anticancer activity. The EPR protocol was also successfully applied on hydrogels labeled with probes 3CP and 5-doxyl-stearate (5-DS), to study the diffusion and polymer matrix degradation processes, providing insight into the disruption of interactions at the molecular level. In addition, it was confirmed that the human serum albumin (HSA) hydrogel has the potential to serve as an anticancer drug depot with delayed release, capable of retaining even 60% of the cytotoxic HL during 11 days of dialysis and that it is a more suitable carrier compared to HSA solution, from which 100% of the ligand is released within 8 days. The absence of cytotoxic activity during three days of HL/HSA hydrogel incubation with Colo-205 cancer cells (colon adenocarcinoma) suggests that it is possible to achieve the delayed release of the cytotoxic ligand under in vivo conditions. Finally, the diagnostic aspect of the multifunctional protein hydrogel is reflected through the observed sensitivity of the albumin hydrogel labeled with the spin probe 3CP to the cellular redox metabolism. As an initial phase in the development of a hydrogel prototype intended for monitoring the drug treatment response, the hydrogel was tested in the Saccharomyces cerevisiae, used as a model system. In this study, the possibility of the spin-labeled albumin hydrogel to serve as an indicator of the redox environment was demonstrated through the reduction of the EPR signal of the nitroxide probe, dependent on the number of vital cells present in the system. It is expected that this observation will be confirmed on experimental animal models in future experiments. Overall, it can be concluded that this study allows for the potential application of in vivo EPR molecular imaging in conjunction with the spin-labeled albumin hydrogel, which would act as an anticancer drug depot-anchoring hydrogel, also allowing for the monitoring of drug effects on surrounding tissues, thus contributing to the integration of diagnostics and therapy.