Ferritin, a naturally occurring iron storage protein, has gained significant attention as a drug delivery platform due to its inherent biocompatibility and capacity to encapsulate therapeutic agents. In this study, we successfully genetically engineered human H ferritin by incorporating 4 or 6 tryptophan residues per subunit, strategically oriented towards the inner cavity of the nanoparticle. This modification aimed to enhance the encapsulation of hydrophobic drugs into the ferritin cage. Comprehensive characterization of the mutants revealed that only the variant carrying four tryptophan substitutions per subunit retained the ability to disassemble and reassemble properly. As a proof of concept, we evaluated the loading capacity of this mutant with ellipticine, a natural hydrophobic indole alkaloid with multimodal anticancer activity. Our data demonstrated that this specific mutant exhibited significantly higher efficiency in loading ellipticine compared to human H ferritin. Furthermore, to evaluate the versatility of this hydrophobicity-enhanced ferritin nanoparticle as a drug carrier, we conducted a comparative study by also encapsulating doxorubicin, a commonly used anticancer drug. Subsequently, we tested both ellipticine and doxorubicin-loaded nanoparticles on a promyelocytic leukemia cell line, demonstrating efficient uptake by these cells and resulting in the expected cytotoxic effect.

• MeSH
• apoferritiny genetika   MeSH
• doxorubicin farmakologie   chemie   MeSH
• elipticiny * MeSH
• ferritiny genetika   chemie   MeSH
• hydrofobní a hydrofilní interakce MeSH
• lékové transportní systémy MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nanočástice * chemie   MeSH
• nosiče léků chemie   MeSH
• protinádorové látky * farmakologie   chemie   MeSH
• tryptofan MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

A tyrosine kinase inhibitor, vandetanib (Van), is an anticancer drug affecting the signaling of VEGFR, EGFR and RET protooncogenes. Van is primarily used for the treatment of advanced or metastatic medullary thyroid cancer; however, its usage is significantly limited by side effects, particularly cardiotoxicity. One approach to minimize them is the encapsulation or binding of Van in- or onto a suitable carrier, allowing targeted delivery to tumor tissue. Herein, we constructed a nanocarrier based on apoferritin associated with Van (ApoVan). Based on the characteristics obtained by analyzing the average size, the surface ζ-potential and the polydispersive index, ApoVan nanoparticles exhibit long-term stability and maintain their morphology. Experiments have shown that ApoVan complex is relatively stable during storage. It was found that Van is gradually released from its ApoVan form into the neutral environment (pH 7.4) as well as into the acidic environment (pH 6.5). The effect of free Van and ApoVan on neuroblastoma and medullary thyroid carcinoma cell lines revealed that both forms were toxic in both used cell lines, and minimal differences between ApoVan and Van were observed. Thus, we assume that Van might not be encapsulated into the cavity of apoferritin, but instead only binds to its surface.

• MeSH
• apoferritiny chemie   farmakokinetika   MeSH
• chinazoliny chemie   farmakokinetika   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nanočástice chemie   MeSH
• piperidiny chemie   farmakokinetika   MeSH
• stabilita léku MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Cíl: Cílem projektu je navrhnout a ověřit nanokonstrukt jako inovativní nástroj pro efektivní zacílení na bakteriální buňku. Metodika: Konstrukt SPIONs/ Au/NPs/AB1/GF/AgNPsGS/ APO/ATB je tvořen: A – stříbrná nanočástice, která byla připravena zelenou syntézou z rostlinných extraktů (AgNPsGS); B – apoferitin (APO) s enkapsulovaným antibiotikem (ATB); C – superparamagnetická zlatá nanočástice modifikovaná grafenovým listem a protilátkou (SPIONs/Au/NPs/AB1/GF). Výsledky: Nejlepší antibakteriální efekt k S. aureus byl pozorován u AgNPsGS4 (Thymus serpyllum): 0,4 μg/ ml. Efektivita enkapsulace antibiotika do nanometrické struktury apoferitinu byla okolo 15 % aplikované koncentrace. V přítomnosti nanokonstruktu byla zaznamenána dramatická inhibice S. aureus. Biologický efekt nanotransportérů spočívá ve dvou hlavních mechanismech. AgNPsGS indukují vznik reaktivních kyslíkových radikálů (ROS reactive oxygen species), což následně vede k poškození buněčné membrány bakteriální buňky a k destrukci prokaryotické nukleové kyseliny. Kromě efektu AgNPsGS se uplatňuje i účinek enkapsulovaného ATB, který se podílí na léčbě bakteriální infekce a který lze vhodně zvolit podle typu infekčního onemocnění. Dále svou roli také sehrává významný synergický efekt AgNPsGS a ATB. Závěr: Díky inovativnímu a funkčnímu spojení účinku AgNPsGS a ATB dochází ke vzniku jedinečného terapeutického nanosystému pro cílenou terapii vážných bakteriálních infekcí.

Aim: The aim of the project was to design and verify the nanoconstruct as an innovative tool for effective targeting to the bacterial cell. Methods: The SPIONs/Au/ NPs/AB1/GF/AgNPsGS/APO/ ATB construct consists of: A – a silver nanoparticle prepared by green synthesis using plant extracts (AgNPsGS); B – apoferritin (APO) with the encapsulated antibiotic (ATB); and C – a superparamagnetic gold nanoparticle modified with graphene sheet and antibody (SPIONs/Au/NPs/AB1/GF). Results: The highest antibacterial effect (Staphylococcus aureus) was observed in AgNPsGS4 (Thymus serpyllum) at a concentration of 0.4 μg/mL. The effectiveness of encapsulation of the antibiotic into the nanometric structure of apoferritin was about 15% of the applied concentration. Dramatic inhibition of S. aureus was observed in the presence of the nanoconstruct. The biological effect of nanotransporter consists in two main mechanisms. AgNPsGS induce the formation of ROS, which subsequently leads to damage to the bacterial cell membrane and destruction of prokaryotic nucleic acid. In addition to the effect of AgNPsGS, the effect of encapsulated ATB is involved in the treatment of bacterial infection. This antibiotic can be appropriately selected according to the type of infectious disease. Furthermore, a significant synergistic effect of AgNPsGS and ATB also plays a role. Conclusion: Due to the innovative and functional combination of the effects of AgNPsGS and ATB, a unique therapeutic nanosystem was created.

• MeSH
• antibakteriální látky MeSH
• apoferritiny chemie   terapeutické užití   MeSH
• bakteriální infekce * farmakoterapie   MeSH
• fotosyntéza MeSH
• kovové nanočástice * chemie   MeSH
• lidé MeSH
• rostlinné extrakty MeSH
• stříbro chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Východiska: Ferritin je globulární vnitrobuněčný protein, který slouží jako hlavní zásobárna železa. U nádorových onemocnění je plazmatická koncentrace ferritinu zvýšená. V řadě studií bylo zjištěno, že nádorové buňky exprimují ve zvýšené míře transferinové receptory (TfR). Zvýšená exprese TfR byla pozorována i u karcinomu prostaty. Apoferritin (APO) lze využít jako proteinový nanotransportér, do kterého je možné enkapsulovat vhodnou léčivou látku. Bylo zjištěno, že nanočástice zvyšují prostupnost nanotransportérů do nádorové buňky a vykazují fototermální efekt. Cílem projektu bylo enkapsulovat doxorubicin (DOX) do APO a vytvořený APO/DOX modifikovat zlatými (gold nanoparticles - AuNPs) a stříbrnými (silver nanoparticles prepared by green synthesis - AgNPsGS) nanočásticemi. Metody: Pro charakterizaci APO byla použita 10% gelová elektroforéza (sodium dodecylsulphate polyacrylamide gel electrophoresis - SDS-PAGE) - 120 V, 60 min, 24 mM Tris, 0,2 M glycin, 3 mM SDS. Fluorescence DOX (Ex 480 nm, Em 650 nm) s typickým absorpčním maximem v 560 nm. Elektrochemické měření bylo provedeno v Brdičkově roztoku (tříelektrodové zapojení). AgNPsGS byly připraveny zelenou syntézou z jetele lučního (Trifolium pratense L.). Výsledky: Byla provedena elektroforetická studie APO a APO/DOX (5- 100 μg//ml). Bylo sledováno chování APO a APO/DOX (10 μM) v závislosti na pH. APO v kyselém prostředí tvoří subjednotky o velikosti asi 20 kDa a v neutrálním a zásaditém prostředí se zformuje na globulární protein o velikosti asi 450 kDa. U APO/DOX byla pozorována změna mobility (asi o 10 %). Na povrchu APO/DOX byl vytvořen film z AuNPs. APO/DOX/AuNPs byl následně promyt ultračistou vodou. Bylo sledováno uvolnění DOX v závislosti na pH. Množství analyzovaného DOX se zvýšilo až o 50 %. Dále byl navržen a připraven komplex AgNPsGS-DOX (1 mg AgNPsGS/100 μM DOX). Takto připravený komplex AgNPsGS-DOX byl uzavřen do APO. Pro další zlepšení terapeutické účinnosti byl připravený komplex APO/AgNPsGS-DOX pokryt vrstvou AuNPs. Vytvořený APO/AgNPsGS-DOX/AuNPs prokázal svoji stabilitu a při změně fyzikálních parametrů byl z komplexu uvolněn DOX. Závěr: Byly připraveny a modifikovány nanokomplexy APO pro zvýšení terapeutické účinnosti protinádorové léčby. Zacílení k nádorové buňce bylo na TfR nebo efektem zvýšené propustnosti a retence. Uvolnění léčiva bylo možné změnou pH nebo fototermální aktivací, která bude nyní testována.

Background: Ferritin is a globular intracellular protein that acts as the main reservoir for iron. Malignancies are associated with increased plasma ferritin concentrations. A number of studies show that tumor cells express high levels of transferrin receptors (TfR). Increased TfR expression was observed in prostate carcinoma. Apoferritin (APO) can be used as a protein nanotransporter into which a suitable medicinal substance can be encapsulated. Nanoparticles increase the permeability of tumor cells to nanotransporters and have a photothermal effect. The aim of this study was to encapsulate doxorubicin (DOX) into APO and to modify the resulting APO/DOX with gold (AuNPs) and silver nanoparticles prepared by green synthesis (AgNPsGS). Methods: APO was characterized using 10% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS- -PAGE) – 120 V, 60 min, 24 mM Tris, 0.2 M glycine, 3 mM SDS. DOX fluorescence (Ex 480 nm; Em 650 nm) was observed, with a typical absorption maximum at 560 nm. Electrochemical measurement was performed in Brdicka solution (three-electrode setup). AgNPsGS were prepared by green synthesis using clover (Trifolium pratense L.). Results: An electrophoretic study of APO and APO/DOX (5–100 μg/mL) was performed and the behavior of APO and APO/DOX (10 μM) as a function of pH was monitored. In an acidic environment, APO forms subunits of about 20 kDa; in an alkaline medium, it forms a globular protein of about 450 kDa. A change in APO/DOX mobility (about by 10%) was observed. A fi lm of gold nanoparticles was applied to the APO/DOX surface. APO/DOX-AuNPs were washed with ultra-pure water. pH-dependent release of DOX a was monitored. The amount of DOX analyzed was increased by up to 50%. Furthermore, an AgNPsGS-DOX complex (1 mg AgNPsGS/100 μM DOX) was generated and prepared. Subsequently, the AgNPsGS-DOX complex was encapsulated into APO. To further improve therapeutic efficacy, the APO/AgNPsGS-DOX complex was coated with an Au layer. APO/AgNPsGS-DOX/AuNPs were stable and DOX was released from the complex after physical parameters had changed. Conclusion: APO nanocomplexes were prepared and modified to increase therapeutic efficacy against tumors. Tumor cell targeting was achieved by binding to TfR and via increased tumor cell permeability and retention. Release of the drug was made possible due to a pH change and photothermal activation that will now be tested.

• Klíčová slova
• nanotransportér, poferritinový nanotransportér, transferinové receptory, stříbrné nanočástice, zlaté nanočástice,
• MeSH
• apoferritiny farmakologie   MeSH
• kovové nanočástice MeSH
• lékové transportní systémy * metody   MeSH
• lidé MeSH
• nádory prostaty * farmakoterapie   MeSH
• nanomedicína * metody   MeSH
• objevování léků MeSH
• stříbro MeSH
• zlato MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH

Although ellipticine (Elli) is an efficient anticancer agent, it exerts several adverse effects. One approach to decrease the adverse effects of drugs is their encapsulation inside a suitable nanocarrier, allowing targeted delivery to tumour tissue whereas avoiding healthy cells. We constructed a nanocarrier from apoferritin (Apo) bearing ellipticine, ApoElli, and subsequently characterized. The nanocarrier exhibits a narrow size distribution suggesting its suitability for entrapping the hydrophobic ellipticine molecule. Ellipticine was released from ApoElli into the water environment under pH 6.5, but only less than 20% was released at pH 7.4. The interaction of ApoElli with microsomal membrane particles containing cytochrome P450 (CYP) biotransformation enzymes accelerated the release of ellipticine from this nanocarrier making it possible to be transferred into this membrane system even at pH 7.4 and facilitating CYP-mediated metabolism. Reactive metabolites were formed not only from free ellipticine, but also from ApoElli, and both generated covalent DNA adducts. ApoElli was toxic in UKF-NB-4 neuroblastoma cells, but showed significantly lower cytotoxicity in non-malignant fibroblast HDFn cells. Ellipticine either free or released from ApoElli was concentrated in the nuclei of neuroblastoma cells, concentrations of which being significantly higher in nuclei of UKF-NB-4 than in HDFn cells. In HDFn the higher amounts of ellipticine were sequestrated in lysosomes. The extent of ApoElli entering the nuclei in UKF-NB-4 cells was lower than that of free ellipticine and correlated with the formation of ellipticine-derived DNA adducts. Our study indicates that the ApoElli form of ellipticine seems to be a promising tool for neuroblastoma treatment.

• MeSH
• adukty DNA genetika   metabolismus   MeSH
• apoferritiny chemie   farmakologie   MeSH
• cytochrom P-450 CYP3A metabolismus   MeSH
• elipticiny chemie   farmakologie   MeSH
• fosforylace MeSH
• histony metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nanočástice * MeSH
• neuroblastom farmakoterapie   enzymologie   genetika   patologie   MeSH
• nosiče léků * MeSH
• příprava léků MeSH
• protinádorové látky chemie   farmakologie   MeSH
• uvolňování léčiv MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakt z konference MeSH

Magnetoferritin is a metalloprotein composed of a protein coat (apoferritin) surrounding the magnetic iron oxide nanoparticles. Physicochemical characterization of magnetic, structural and morphological properties, size distribution and stability of magnetoferritin have been extensively investigated. Magneto-optical properties of magnetic nanoparticles in magnetoferritin can be applied in in vivo diagnosis of various diseases associated with the formation of magnetite in pathological processes in tissues. In addition, the confirmed peroxidase activity enables magnetoferritin to be used in important bioapplications.

• Klíčová slova
• magnetoferritin, magnetit, magneto-optické vlastnosti,
• MeSH
• apoferritiny * farmakologie   chemie   MeSH
• nanočástice MeSH
• oxidace-redukce MeSH
• oxidy farmakologie   chemie   MeSH
• železo farmakologie   chemie   MeSH
• Publikační typ
• práce podpořená grantem MeSH

The nanoparticle based drugs, mostly in liposomes, are already approved for clinical use or under clinical investigation. Many attempts are made to improve nanoparticles sizes, shapes and surface modifications that lead to prolongation of drug circulation in blood stream and targeting to cancer cells. Thus small molecules like polyethylene glycol and targeting ligands like folic acid, peptides, antibodies, aptamers and nucleic acids are bound on the surface of nanoparticles with the aim to increase specific cell uptake. Very promising are multifunctional nanoparticles that combine both diagnostic as well as delivery role together.

• Klíčová slova
• stealth liposomes,
• MeSH
• apoferritiny terapeutické užití   MeSH
• ferritiny terapeutické užití   MeSH
• genetická terapie MeSH
• genetické vektory MeSH
• grafit MeSH
• lékové transportní systémy MeSH
• lidé MeSH
• liposomy MeSH
• magnetická rezonanční tomografie MeSH
• magnetické nanočástice chemie   terapeutické užití   MeSH
• magnetismus MeSH
• nádory diagnóza   terapie   MeSH
• nanočástice * chemie   terapeutické užití   MeSH
• nanomedicína MeSH
• nanotrubičky uhlíkové MeSH
• nosiče léků * chemie   terapeutické užití   MeSH
• protinádorové látky * chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

Nanomedicine as a continuously evolving discipline is still looking for a structure with perfect properties that is usable as a multifunctional transporter. Great potential is attributed to synthetic materials such as fullerenes, porous hollow silica nanoparticles and single-wall nanotubes, among others. However, materials that are natural to the human body are more acceptable by the organism, and thus become an attractive approach in this field of research. Ferritins are proteins that naturally occur in most living organisms throughout evolution and may be a possible transporter choice. Numerous applications have demonstrated the possibilities of iron-free ferritins, called apoferritins, serving as platforms for various nanomedical purposes This article summarizes the advantages and disadvantages of these proteins and discusses their practical applications and future perspectives.

• MeSH
• apoferritiny chemie   terapeutické užití   MeSH
• biosenzitivní techniky MeSH
• genetická terapie MeSH
• konformace proteinů MeSH
• lidé MeSH
• nanomedicína * MeSH
• nosiče léků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Routine cancer treatment often causes damage to whole human organism, because cytostatic drugs, which are used during the treatment and whose function is to stop growth of cancer cells, affect also healthy cells and untargeted tissues. Due to this fact, nanocarriers and targeted drug delivery have been recently studied. Side effects and organ damage can be reduced by using nanocarriers and targeted drug delivery. The aim of this assay was to monitor and characterize apoferritin as a nanocarrier for targeted drug delivery. In this work the surface of nanocarrier composed of apoferritin with encapsulated doxorubicin was modified with gold nanoparticles or chloroauric acid, conjugated with linker (HWR peptide) because of correct binding of the antibody and labeled with specific antibody. In this assay the resulting nanocarrier was studied with gel electrophoresis and fluorescent methods.

• Klíčová slova
• nanotransportér, teranostika,
• MeSH
• apoferritiny * MeSH
• doxorubicin MeSH
• elektroforéza MeSH
• ELISA MeSH
• fluorescenční protilátková technika MeSH
• imunoglobulin G MeSH
• kovové nanočástice * MeSH
• lékové transportní systémy * MeSH
• nanomedicína MeSH
• protilátky MeSH
• zlato MeSH
• Publikační typ
• práce podpořená grantem MeSH