Alzheimer´s disease, Parkinson´s disease, type II Diabetes Mellitus and more than 50 other disorders known as amyloidoses are incurable protein misfolding diseases [1]. Magnetic nanoparticles (MNPs) gained massive attention in the last decades. Amino acid functionalization of their surface provides a more extensive surface area to interact with macromolecules allowing them to be used as anti-amyloid agents [2].
The aim of the presented work was to study the anti-amyloid effect of MNPs functionalized by proline (Pro-MNPs), cysteine (Cys-MNPs), and poly-L-lysine (PLL-MNPs) on the amyloid aggregation of α-lactalbumin (α-LA) in vitro. α-LA, a small globular, acidic and Ca2+-binding protein present in the mammalian milk, can be used as a model protein for amyloid aggregation in vitro [3]. The interaction of amino acid-functionalized magnetic nanoparticles (aa-MNPs) with amyloid fibrils can be influenced by their physico-chemical properties and type of functionalized aa [2]; therefore, the aa-MNPs were analyzed by dynamic light scattering (DLS) technique to obtain their hydrodynamic diameter (DH) and by laser Doppler velocimetry to characterize their zeta potential (ζ) and isoelectric point (pI). The results showed that the biggest Cys-MNPs have the lowest ζ and pI; the smallest Pro-MNPs have lower ζ and pI; and smaller PLL-MNPs have the lowest ζ and pI.
Thioflavin T (ThT) fluorescence assay was used to investigate destroying and inhibitory capability of aa-MNPs on α-LA amyloid aggregation. Our results revealed that aa-MNPs destroy α-LA fibrils (α-LAF) and inhibit the formation of α-LAF in a concentration-dependent manner. The efficiency of destroying and inhibitory ability of aa-MNPs were quantified by DC50 and IC50 values (concentration of aa-MNPs causing a 50% decline in fluorescence intensities corresponding to 50% destruction or inhibition of fibrils). Results showed no correlation between destroying ability of aa-MNPs and their physico-chemical properties. However, the inhibitory effect of aa-MNPs negatively correlates with their DH (the smallest Pro-MNPs had the highest inhibitory activity (the lowest IC50), the lower inhibition was observed for bigger PLL-MNPs followed by Cys-MNPs). Atomic force microscopy was used to visualize the effects of aa-MNPs on the morphology of α-LA. There were no morphology changes of fibrils observed after their interaction with aa-MNPs. On the other hand, shorter fibrils and aggregates were formed due to the inhibition of α-LAF fibrillization by aa-MNPs. Moreover, aa-MNPs´ cytotoxicity was tested on human kidney (HEK293) cells by lactate dehydrogenase assay and optical microscopy. We have found that PLL-MNPs were the most cytotoxic.
To conclude, we studied aa-MNPs and their anti-amyloid potential and we found out that Cys-MNPs with a low DC50 value and relatively low cytotoxicity effect are a suitable candidate for amyloid fibrils destruction. Instead, Pro-MNPs may serve as a potential inhibitor of amyloid fibrils formation due to their low IC50 and relatively low cytotoxicity.
This work was supported by the Slovak Research and Development Agency under the Contract no. APVV-18-0284; Slovak Grant Agency VEGA 02/0176/21, VEGA 02/0164/22, VEGA 02/0033/19; Italian MIUR grant (PRIN 20173L7W8K). This work is also the result of the project implementation BIOVID-19 (ITMS2014+ 313011AVG3) and NANOVIR (ITMS2014+ 313011AUW7), supported by the Operational Programme Integrated Infrastructure (OPII) funded by the ERDF. Microscopy was carried out at the SPM@ISMN facility.
[1] S. Kumar Chaturvedi, M. K. Siddiqi, P. Alam, R. H. Khan, Process Biochemistry, 51 (2016), 1183–1192.
[2] A. Antosova et al., Journal of Magnetism and Magnetic Materials 471 (2019), 169–176.
[3] E. A. Permyakov, Biomolecules 10 (2020), 1–50.
Ďakujeme za veľmi zaujímavý
Ďakujeme za veľmi zaujímavý príspevok - opäť téma, ktorá už dve dekády vo výskume rezonuje, pretože s predlžovaním doby dožitia je v centre záujmu zvýšiť šance dožitia bez zdravotného obmedzenia. Ochorenia spomínané v abstrakte, ktoré patria do skupiny amyloidóz, výrazne znižujú kvalitu života nielen pacientov, ale aj ich okolia, preto je výskum v tejto oblasti mimoriadne aktuálny. Moja otázka sa týka magnetických vlastností študovaných nanočastíc, kde a akým spôsobom sa tieto vlastnosti uplatňujú?
Thank you for a very interesting contribution – once again, a topic that has resonated in research in recent two decades, because, as life expectancy increases, the aim is to increase the chances of living without disability or long-term illness. The diseases mentioned in the abstract, which belong to the group of amyloidoses, significantly deteriorates the quality of life not only in patients but also in their caregivers and family environment, therefore, research in this area is extremely valuable. My question concerns the magnetic properties of the studied nanoparticles, where and how do these properties apply?
iwa
Ďakujem pekne za zaujímavú
Ďakujem pekne za zaujímavú otázku.
Magnetické nanočastice majú široké využitie, najmä v priemysle a biomedicíne. Čo sa týka priameho využitia ich magnetických vlastností v praxi, tak ide hlavne o terapeutické a diagnostické aplikácie v nanomedicíne, napríklad pri diagnostike Alzheimerovej chorobe sa využíva značenie amyloidných plakov a ich zobrazenie pomocou MRI alebo selektívne značenie feritínu v miestach s vysokou akumuláciou amyloidných plakov (Fernandéz et al., 2018). Ďalej sa magnetické vlastnosti využívajú aj na magnetickú separáciu protilátok, a sú aj súčasťou hypertermie pri liečbe nádorových ochorení (Akbarzadeh et al., 2012). V nanomedicíne sa využívajú aj ako próby na sledovanie prognózy ochorenia, na prípravu biosenzorov, na dizajn fluorescenčných-magnetických biozobrazovacích prób ako aj na syntézu nosičov liečiv (Hepel et al., 2020).
Nanočastice použité v našej práci sú superparamagnetické. Táto vlastnosť podporuje relaxáciu protónov počas MRI, čo znamená, že v porovaní s paramagnetickými nanočasticami je potrebná nižšia dávka superparamagnetických. Z tejto vlastnosti vyplýva aj ich malá veľkosť (niekoľko nm) a veľký povrch, čo umožňuje omnoho účinnejšiu separáciu a purifikáciu buniek a biomolekúl v porovnaní s konvenčnými metódami (Akbarzadeh et al., 2012). Samotné magnetické vlastnosti však nestačia, na to aby boli použité v klinickej praxi je dôležitá aj ich biokompatibilita, cytotoxicita a biodegradovateľnosť.
Magnetic nanoparticles are widely used, especially in industry and biomedicine. Concerning the magnetic properties, the diagnostic and therapeutic applications are the most prevalent, for example, marking of amyloid plaques and their imaging by MRI or selective marking of ferritin in areas with high accumulation of amyloid plaques in Alzheimer's disease diagnostics (Fernandéz et al., 2018). Moreover, magnetic properties of nanoparticles are used for antibodies separation or hyperthermy in cancer treatment (Akbarzadeh et al., 2012). The other uses of nanoparticles in nanomedicine include probing the status of the disease, biosensor preparation, fluorescent-magnetic bioimaging probe design, and synthesis of drug delivery nanocarriers (Hepel et al., 2020).
Nanoparticles used in our work are superparamagnetic. This property supports the relaxation of protons during MRI giving the advantage of using a smaller dose compared to the paramagnetic nanoparticles. The property also accounts for their small size (several nm) and extensive surface area that enables more effective separation and purification of cells and biomolecules compared to the conventional methods (Akbarzadeh et al., 2012). Magnetic property alone is not sufficient for clinical applications, biocompatibility, cytotoxicity, and biodegradability are also important.
Ďakujem za odpoveď, oceňujem
Ďakujem za odpoveď, oceňujem komplexný pohľad na problematiku.
Thank you for your reply, I appreciate the comprehensive view to what makes a successful application.
iwa