Sinteza i karakterizacija biokompatibilnih optički aktivnih fluorida retkih zemalja

Abstract

Optički aktivne nanočestice fluorida elemenata retkih zemalja privlače veliku pažnju poslednjih godina zbog svoje uspešne primene u biološkim snimanjima, odnosno u vizualizaciji i karakterizaciji bioloških procesa na ćelijskom i molekulskom nivou. Optička aktivnost ovih materijala posledica je prisustva dopanata koji omogućavaju „Up“- konvertorsku (anti-štoks) emisiju svetlosti nakon pobude infracrvenim izvorom zračenja. U ovoj tezi, optički aktivne biokompatibilne čestice NaYF4 dopiranog iterbijumom i erbijumom, sintetisane su hidro/solvo-termalnim procesiranjem nitrata elemenata retkih zemalja uz upotrebu NaF kao precipitatora. Ispitan je uticaj koncentracije polaznih reaktanata, rastvarača (vode, etanola, etilen glikola i acetona) i prisustva različitih modifikatora površine (poletilenglikola - PEG, polivilinpirolidona - PVP, polilaktid-ko-glikolida - PLGA, hitozana - CS, poliakrilne kiseline - PAA, i etilendiamintetrasirćetne kiseline (etilendiamintetraacetat - EDTA) na proces nukleacije kubne i/ili heksagonalne NaY0.8Yb0.17Er0.03F4 faze, morfologiju i hemijske karakteristike površine čestica. U slučaju dobijanja čestica niske kristaliničnosti vršena je i dodatna kalcinacija praha u atmosferi argona, pri čemu je praćen proces konverzije kubne (α) u heksagonalnu (β) NaY0.8Yb0.17Er0.03F4 fazu. Efikasnost „up“- konverzije (UC), odnosno optička aktivnost čestica u vidljivom delu spektra, utvrđena je merenjem intenziteta i vremena emisije plave (na 408 nm, usled 2H9/2 →4I11/2 prelaza), zelene (na 520 i 540 nm, usled 2H11/2 →4I15/2 i 4S3/2 → 4I15/2 prelaza) i crvene (na 655 nm, usled 4F9/2 → 4I15/2 prelaza) svetlosti nakon pobuđivanja infracrvenim izvorom zračenja (λ ~ 980 nm), definisanjem njihovog međusobnog odnosa, kao i izračunavanjem vrednosti hromatičnih koordinata x i y u CIE dijagramu. Rendgeno strukturna analiza praha pokazala je da korišćenje navedenih modifikatora površine u rastvorima niže koncentracije (5 i 10 mmol) dovodi primarno do kristalizacije α- NaY0.8 Yb0.17Er0.03F4 faze u sferičnim česticama nanometarskih dimenzija (<100 nm), osim u slučaju korišćenja PVP gde je postignuta in situ kristalizacija heksagonalne β faze. Utvrđeno je da na stabilizaciju heksagonalne kristalne strukture tokom procesa sinteze, pored izbora modifikatora površine, utiče i vreme hidro/solvo-termalnog tretmana, vrsta rastvarača i koncentracija polaznog rastvora. Kako heksagonalnu kristalnu strukturu odlikuje anizotropni rast kristala, ostvarena je i in situ kristalizacija monodisperznih heksagonalnih poliedarskih mikročestica u rastvorima veće koncentracije (100 mmol) korišćenjem EDTA modifikatora. U radu je pokazano da je dodatnim termičkim tretmanom nanočestica kubne kristalne strukture moguće dobiti čestice heksagonalne kristalne strukture, pri čemu čestice gube svoj sferičan oblik. Sve sintetisane čestice odlikuje visoka čistoća i hidrofilnost. Homogena raspodela jona dopanta u česticama pokazana je skenirajućom elektronskom mikroskopijom u kombinaciji sa energetskom disperzionom analizom X-zracima (SEM/EDAX). Prisustvo amino, karboksilnih ili hidroksilnih grupa na površini sintetisanih čestica, potvrđeno infracrvenom spektroskopijom sa Furijeovom transformacijom, što je ukazalo na formiranje biokompatibilnog sloja na površini čestica. U cilju ispitivanja mogućnosti njihovog korišćenja u biomedicinske svrhe antibakterijska aktivnost čestica je testirana prema Gram-negativnoj bakteriji Escherichia coli, Gram-pozitivnoj bakteriji Staphylococcus aureus i gljivici Candida albicans. Dodatno, za čestice koje se odlikuju najboljim morfološko-strukturnim karakteristikama (nanočestice α- NaY0.8Yb0.17Er0.03F4 faze dobijene u prisustvu PLGA i CS), ispitana je i citotoksičnost na primarnim ćelijama skvamoznog karcinoma jezika (eng. oral squamous cell carcinoma OSCC) i zdravim gingivalnim ćelijama korena pulpe (eng. human gingival cell, HGC) korišćenjem 3-(4,5-dimetiltiazol-2-il)-2,5-difenil tetrazolijum bromid (MTT) testa u opsegu koncentracija od 10-50 μg/mL. U oba slučaja, utvrđena je niža toksičnost nanočestica za HGC nego za OSCC te je dalje ispitana i mogućnost in vitro vizuelizacije ovih ćelija primenom nelinearne skenirajuće mikroskopije. Na osnovu dobijenih rezultata je utvrđeno da je vizuelizacija ćelija moguća i pri najnižim koncentracijama sintetisanih nanočestica, jer je njihovo prisustvo u citoplazmi potvrđeno jasnim svetlosnim odzivom pri pobudi laserom talasne dužine od 980 nm. Ovim istraživanjima je pokazano da je in situ sintezu biokompaktibilnih optički aktivnih nanočestica sastava NaY0.8Yb0.17Er0.03F4 moguće izvesti u kontrolisanim uslovima hidro/solvo-termalne sinteze, kao i da dobijene čestice poseduju karakteristike potencijalnih biomarkera nove generacije.

Optically active rare earth doped flouride nanoparticles have attracted great attention in recent years because of their successful application in biological imaging, including visualization and characterization of biological processes at the cellular and molecular level. Optical activity of these materials is due to the presence of dopants that allow Up- converting (Anti-Stokes) light emission after excitation by infrared radiation. In this work, optically active biocompatible NaYF4 particles doped with ytterbium and erbium were synthesized by hydro/solo-thermal processing of rare earth nitrates and NaF. The influence of precursor concentration, solvents (water, ethanol, ethylene glycol and acetone) and surface modifiers (polyethylene glycol-PEG, polyvinylpyrrolidone-PVP, polylactid-co-glycolide acid-PLGA, chitosan-CS, polyacrylic acid-PAA, and ethylenediamintetraacetate - EDTA) on the nucleation process of cubic and hexagonal NaY0.8Yb0.17Er0.03F4 phase, particle morphology and surface chemical characteristics was examined. In the case of obtaining low crystalline particles, additional calcination in argon atmosphere was carried out, whereby the conversion process of the cubic (α) into the hexagonal (β) NaY0.8Yb0.17Er0.03F4 phase was monitored. Up-conversion efficiency, i.e optical activity of the particles in the visible part of the spectra was determined by measuring the intensity and decay time of the blue (at 408 nm, due to 2H9/2 →4I11/2 transition), green (at 520 i 540 nm, due to 2H11/2 →4I15/2 i 4S3/2 → 4I15/2 transition) and red (at 655 nm, due to 4F9/2 → 4I15/2 transition) emission after excitation by NIR (near infrared, λ =980 nm) light, defining their mutual relationship and calculating chromatic coordinates (X,Y) in the CIE diagram. XRPD analysis showed that crystalization from the low-concentrated solutions (5 and 10 mmol) led to the α- NaY0.8Yb0.17Er0.03F4 phase generation in nanometric spherical particles (<100 nm) in the presence of all surface modifiers except PVP which enables in situ crystallization of the hexagonal β- NaY0.8Yb0.17Er0.03F4 phase. Stabilization of the hexagonal crystal structure is additionally achieved by extension of the hydro/solvo-thermal treatment, ethanol usage and with rise of the precursor concentration. As the growth of this structure is anisotropic, in situ crystallization of monodisperse hexagonal polyhedral particles is achieved during hydrothermal treating of 100 mmol precursor in the presence of EDTA. All of the synthesized particles are characterized by high purity and hydrophilicity. The homogeneous distribution of dopant ions in the particles, was demonstrated by scanning electron microscopy in combination with energy dispersive analysis of X-rays (SEM/EDAX). The presence of amino, carboxylic or hydroxyl groups on the particles surface is confirmed by infrared spectroscopy with Furie's transformation, indicating their biocompatibility. In order to examine their antibacterial activity, the particles was tested using Gram-negative bacterium Escherichia coli, Gram-positive bacterium Staphylococcus aureus and Candida albicans fungus. Additionaly, cytotoxicity of NaY0.8Yb0.17Er0.03F4 nanoparticles obtained in the presence of PLGA and CS (in a concentration range of 10-50 μg/mL) was determined against oral squamous cell carcinoma (OSCC) and healthy non-cancerous human gingival cells (HGC) using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT). Since lower cytotoxicity against HGC than against OSCC was determined for both kind of nanoparticles, the possibility of their in vitro visualization was further investigated using non-linear scanning microscopy. It was shown that cell visualization is possible using lowest nanoparticle concentrations of 10 μg/mL. Clear light responce of the nanoparticles present in cells cytoplasm was obtained under excitation at 980 nm. Obtained results show that in situ functionalized biocompatible optically active NaY0.8Yb0.17Er0.03F4 nanoparticles synthetised in a controlled manner by hydro/solvo-thermal synthesis could be used as biomarkers of the new generation.