Ferritins are globular proteins with an internal cavity that enables the encapsulation of a plethora of low-mass compounds. Unfortunately, the overall negative surface charge of ferritin's internal cavity hampers efficient loading of negatively charged molecules. Therefore, we produced a genetically engineered human H-chain ferritin containing a cationic RKRK domain, reversing the natural net charge of the cavity to positive, thus allowing for efficient encapsulation of negatively charged siRNA. Due to the reversed, positive charge mediated by RKRK domains, the recombinant ferritin produced in E. coli inherently carries a load of bacterial RNA inside its cavity, turning the protein into an effective sponge possessing high affinity for DNA/RNA-binding substances that can be loaded with markedly higher efficiency compared to the wildtype protein. Using doxorubicin as payload, we show that due to its loading through the RNA sponge, doxorubicin is released in a sustained manner, with a cytotoxicity profile similar to the free drug. In summary, this is the first report demonstrating a ferritin/nucleic acid hybrid delivery vehicle with a broad spectrum of properties exploitable in various fields of biomedical applications.

• MeSH
• apoferritiny * genetika   MeSH
• doxorubicin farmakologie   chemie   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• ferritiny genetika   chemie   MeSH
• lidé MeSH
• RNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• apoferritiny * MeSH
• doxorubicin MeSH
• ferritiny MeSH
• RNA * MeSH

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.

• Klíčová slova
• doxorubicin, drug delivery, ellipticine, ferritin, hydrophobic drugs, nanoparticle, protein engineering, tumor cells,
• 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
• Názvy látek
• apoferritiny MeSH
• doxorubicin MeSH
• elipticiny * MeSH
• ferritiny MeSH
• nosiče léků MeSH
• protinádorové látky * MeSH
• tryptofan 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.

• Klíčová slova
• apoferritin, cancer targeting, medullary thyroid cancer, neuroblastoma, vandetanib,
• 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
• Názvy látek
• apoferritiny MeSH
• chinazoliny MeSH
• piperidiny MeSH
• vandetanib MeSH Prohlížeč

BACKGROUND: Hereditary hyperferritinemia-cataract syndrome (HHCS) is an autosomal dominant disorder manifesting with high serum ferritin levels and the formation of early-onset cataracts, with numerous small opacities, predominantly in the lens cortex. HHCS is caused by mutations in the iron-responsive element of the FTL gene. The aim of this study was to establish a molecular diagnosis in three Czech probands with suspected HHCS. METHODS: A complex ocular and systemic evaluation, including ferritin and iron measurements, was performed. The 5' untranslated region of FTL was directly sequenced in all available family members, followed by paternity testing in one family. RESULTS: Three different FLT pathogenic variants (c.-161C>T, c.-167C>T, and c.-168G>C) present in the heterozygous state were detected in each of the 3 probands. Two segregated with the disease phenotype within the families, but c.-167C>T occurred de novo (confirmed by paternity testing). Prior to establishing molecular diagnosis, two probands were misdiagnosed with hemochromatosis. One individual, aged 43 years, underwent phlebotomy; another, aged 8.5 years, was treated with the iron chelator deferasirox, leading to life-threatening acute hyperammonemia, without severe liver injury. CONCLUSIONS: Lack of family history does not exclude HHCS, because the pathogenic variant can arise de novo. Noncoding regions are often omitted from diagnostic gene panels, thus evading detection. Careful clinical evaluations and targeted genetic screening are important for avoiding potentially harmful treatments.

• MeSH
• apoferritiny genetika   MeSH
• hyperferritinemie * MeSH
• katarakta * diagnóza   genetika   MeSH
• lidé MeSH
• molekulární biologie MeSH
• mutace MeSH
• rodokmen MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• apoferritiny MeSH

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 film 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. This work was supported by COST European Cholangiocarcinoma Network CA18122 and International Collaboration Project of The European Technology Platform for Nanomedicine. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 21. 3. 2019 Accepted: 14. 5. 2019.

• Klíčová slova
• apoferritin nanotransporter, gold nanoparticles, malignant tumors, nanomedicine, prostate tumors, silver nanoparticles, targeted therapy, transferin receptors,
• MeSH
• apoferritiny chemie   MeSH
• doxorubicin analogy a deriváty   chemie   farmakologie   MeSH
• koncentrace vodíkových iontů MeSH
• kovové nanočástice chemie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• protinádorová antibiotika chemie   farmakologie   MeSH
• receptory transferinu metabolismus   MeSH
• stříbro chemie   MeSH
• uvolňování léčiv MeSH
• zlato chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• apoferritin doxorubicin MeSH Prohlížeč
• apoferritiny MeSH
• doxorubicin MeSH
• protinádorová antibiotika MeSH
• receptory transferinu MeSH
• stříbro MeSH
• zlato 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.

• Klíčová slova
• Apoferritin nanoparticles, Cytochrome P450-mediated metabolism, Cytotoxicity, DNA adducts, Ellipticine, Neuroblastoma,
• 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
• Názvy látek
• adukty DNA MeSH
• apoferritiny MeSH
• CYP3A4 protein, human MeSH Prohlížeč
• cytochrom P-450 CYP3A MeSH
• elipticiny MeSH
• ellipticine MeSH Prohlížeč
• H2AX protein, human MeSH Prohlížeč
• histony MeSH
• nosiče léků * MeSH
• protinádorové látky MeSH

Herein, we describe the in vivo effects of doxorubicin (DOX) encapsulated in ubiquitous protein apoferritin (APO) and its efficiency and safety in anti-tumor treatment. APODOX is both passively (through Enhanced Permeability and Retention effect) and actively targeted to tumors through prostate-specific membrane antigen (PSMA) via mouse antibodies conjugated to the surface of horse spleen APO. To achieve site-directed conjugation of the antibodies, a HWRGWVC heptapeptide linker was used. The prostate cancer-targeted and non-targeted nanocarriers were tested using subcutaneously implanted LNCaP cells in athymic mice models, and compared to free DOX. Prostate cancer-targeted APODOX retained the high potency of DOX in attenuation of tumors (with 55% decrease in tumor volume after 3 weeks of treatment). DOX and non-targeted APODOX treatment caused damage to liver, kidney and heart tissues. In contrast, no elevation in liver or kidney enzymes and negligible changes were revealed by histological assessment in prostate cancer-targeted APODOX-treated mice. Overall, we show that the APO nanocarrier provides an easy encapsulation protocol, reliable targeting, high therapeutic efficiency and very low off-target toxicity, and is thus a promising delivery system for translation into clinical use.

• MeSH
• antigeny povrchové imunologie   MeSH
• apoferritiny škodlivé účinky   terapeutické užití   MeSH
• doxorubicin škodlivé účinky   analogy a deriváty   terapeutické užití   MeSH
• glutamátkarboxypeptidasa II imunologie   MeSH
• heterografty MeSH
• imunokonjugáty terapeutické užití   MeSH
• játra účinky léků   MeSH
• ledviny účinky léků   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty farmakoterapie   terapie   MeSH
• nanokonjugáty terapeutické užití   MeSH
• srdce účinky léků   MeSH
• výsledek terapie MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové MeSH
• apoferritin doxorubicin MeSH Prohlížeč
• apoferritiny MeSH
• doxorubicin MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• imunokonjugáty MeSH
• nanokonjugáty MeSH

Due to many adverse effects of conventional chemotherapy, novel methods of targeting drugs to cancer cells are being investigated. Nanosize carriers are a suitable platform for this specific delivery. Herein, we evaluated the long-term stability of the naturally found protein nanocarrier apoferritin (Apo) with encapsulated doxorubicin (Dox). The encapsulation was performed using Apo's ability to disassemble reversibly into its subunits at low pH (2.7) and reassemble in neutral pH (7.2), physically entrapping drug molecules in its cavity (creating ApoDox). In this study, ApoDox was prepared in water and phosphate-buffered saline and stored for 12 weeks in various conditions (-20°C, 4°C, 20°C, and 37°C in dark, and 4°C and 20°C under ambient light). During storage, a very low amount of prematurely released drug molecules were detected (maximum of 7.5% for ApoDox prepared in PBS and 4.4% for ApoDox prepared in water). Fourier-transform infrared spectra revealed no significant differences in any of the samples after storage. Most of the ApoDox prepared in phosphate-buffered saline and ApoDox prepared in water and stored at -20°C formed very large aggregates (up to 487% of original size). Only ApoDox prepared in water and stored at 4°C showed no significant increase in size or shape. Although this storage caused slower internalization to LNCaP prostate cancer cells, ApoDox (2.5 μM of Dox) still retained its ability to inhibit completely the growth of 1.5×104 LNCaP cells after 72 hours. ApoDox stored at 20°C and 37°C in water was not able to deliver Dox inside the nucleus, and thus did not inhibit the growth of the LNCaP cells. Overall, our study demonstrates that ApoDox has very good stability over the course of 12 weeks when stored properly (at 4°C), and is thus suitable for use as a nanocarrier in the specific delivery of anticancer drugs to patients.

• Klíčová slova
• anticancer therapy, doxorubicin-loaded apoferritin, encapsulation, long-term stability, protein nanocarriers,
• MeSH
• apoferritiny aplikace a dávkování   chemie   farmakokinetika   MeSH
• doxorubicin aplikace a dávkování   chemie   MeSH
• koncentrace vodíkových iontů MeSH
• lékové transportní systémy MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty farmakoterapie   patologie   MeSH
• nosiče léků aplikace a dávkování   chemie   farmakokinetika   MeSH
• protinádorové látky aplikace a dávkování   farmakokinetika   farmakologie   MeSH
• screeningové testy protinádorových léčiv MeSH
• stabilita léku MeSH
• uvolňování léčiv MeSH
• voda chemie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• apoferritiny MeSH
• doxorubicin MeSH
• nosiče léků MeSH
• protinádorové látky MeSH
• voda MeSH

Herein, we describe a novel approach for targeting of ubiquitous protein apoferritin (APO)-encapsulating doxorubicin (DOX) to prostate cancer using antibodies against prostate-specific membrane antigen (PSMA). The conjugation of anti-PSMA antibodies and APO was carried out using HWRGWVC heptapeptide, providing their site-directed orientation. The prostate-cancer-targeted and nontargeted nanocarriers were tested using LNCaP and HUVEC cell lines. A total of 90% of LNCaP cells died after treatment with DOX (0.25 μM) or DOX in nontargeted and prostate-cancer-targeted APO, proving that the encapsulated DOX toxicity for LNCaP cells remained the same. Free DOX showed higher toxicity for nonmalignant cells, whereas the toxicity was lower after treatment with the same dosage of APO-encapsulated DOX (APODOX) and even more in prostate-cancer-targeted APODOX. Hemolytic assay revealed exceptional hemocompatibility of the entire nanocarrier. The APO encapsulation mechanism ensures applicability using a wide variety of chemotherapeutic drugs, and the presented surface modification enables targeting to various tumors.

• Klíčová slova
• antibodies, apoferritin, doxorubicin, nanomedicine, targeted drug delivery,
• MeSH
• apoferritiny metabolismus   MeSH
• doxorubicin aplikace a dávkování   MeSH
• lékové transportní systémy metody   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty farmakoterapie   MeSH
• protilátky metabolismus   MeSH
• protinádorové látky aplikace a dávkování   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• apoferritiny MeSH
• doxorubicin MeSH
• protilátky MeSH
• protinádorové látky MeSH

Increased oxidative stress is indisputably an important mechanism of doxorubicin side effects, especially its cardiotoxicity. To prevent impairment of non-tumorous tissue and to improve the specificity in targeting the tumor tissue, new drug nanotransporters are developed. In many cases preclinical therapeutic advantage has been shown when compared with the administration of conventional drug solution. Three forms of doxorubicin--conventional (DOX), encapsulated in liposomes (lipoDOX) and in apoferritin (apoDOX) were applied to Wistar rats. After 24 h exposition, the plasma level of 4-hydroxy-2-nonenal (4-HNE) as a marker of lipoperoxidation and tissue gene expression of thioredoxin reductase 2 (TXNRD2) and aldehyde dehydrogenase 3A1 (ALDH3A1) as an important part of antioxidative system were determined. Only conventional DOX significantly increases the level of 4-HNE; encapsulated forms on the other hand show significant decrease in plasma levels of 4-HNE in comparison with DOX. They also cause significant decrease in gene expression of ALDH3A1 and TXNRD2 in liver as a main detoxification organ, and a mild influence on the expression of these enzymes in left heart ventricle as a potential target of toxicity. Thus, 4-HNE seems to be a good potential biomarker of oxidative stress induced by various forms of doxorubicin.

• MeSH
• aldehyddehydrogenasa genetika   metabolismus   MeSH
• aldehydy krev   MeSH
• apoferritiny aplikace a dávkování   chemie   toxicita   MeSH
• biologické markery krev   MeSH
• down regulace MeSH
• doxorubicin aplikace a dávkování   analogy a deriváty   chemie   toxicita   MeSH
• farmaceutická chemie MeSH
• játra účinky léků   enzymologie   MeSH
• oxidační stres účinky léků   MeSH
• peroxidace lipidů účinky léků   MeSH
• polyethylenglykoly aplikace a dávkování   chemie   toxicita   MeSH
• potkani Wistar MeSH
• protinádorová antibiotika aplikace a dávkování   chemie   toxicita   MeSH
• regulace genové exprese enzymů MeSH
• thioredoxinreduktasa 2 genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• 4-hydroxy-2-nonenal MeSH Prohlížeč
• aldehyddehydrogenasa MeSH
• aldehydy MeSH
• apoferritin doxorubicin MeSH Prohlížeč
• apoferritiny MeSH
• biologické markery MeSH
• doxorubicin MeSH
• liposomal doxorubicin MeSH Prohlížeč
• polyethylenglykoly MeSH
• protinádorová antibiotika MeSH
• thioredoxinreduktasa 2 MeSH
• Txnrd2 protein, rat MeSH Prohlížeč