INTRODUCTION: The present study reports on examination of the effects of encapsulating the tyrosine kinase inhibitors (TKIs) vandetanib and lenvatinib into a biomacromolecular ferritin-based delivery system. METHODS: The encapsulation of TKIs was performed via two strategies: i) using an active reversible pH-dependent reassembly of ferritin´s quaternary structure and ii) passive loading of hydrophobic TKIs through the hydrophobic channels at the junctions of ferritin subunits. After encapsulation, ferritins were surface-functionalized with folic acid promoting active-targeting capabilities. RESULTS: The physico-chemical and nanomechanical analyses revealed that despite the comparable encapsulation efficiencies of both protocols, the active loading affects stability and rigidity of ferritins, plausibly due to their imperfect reassembly. Biological experiments with hormone-responsive breast cancer cells (T47-D and MCF-7) confirmed the cytotoxicity of encapsulated and folate-targeted TKIs to folate-receptor positive cancer cells, but only limited cytotoxic effects to healthy breast epithelium. Importantly, the long-term cytotoxic experiments revealed that compared to the pH-dependent encapsulation, the passively-loaded TKIs exert markedly higher anticancer activity, most likely due to undesired influence of harsh acidic environment used for the pH-dependent encapsulation on the TKIs' structural and functional properties. CONCLUSION: Since the passive loading does not require a reassembly step for which acids are needed, the presented investigation serves as a solid basis for future studies focused on encapsulation of small hydrophobic molecules.

• Klíčová slova
• drug delivery, lenvatinib, nanomedicine, vandetanib,
• MeSH
• antitumorózní látky farmakologie   MeSH
• biokompatibilní materiály chemie   MeSH
• buněčná smrt účinky léků   MeSH
• buněčné klony MeSH
• buněčné linie MeSH
• chinazoliny chemie   farmakologie   MeSH
• chinoliny chemie   farmakologie   MeSH
• difuze MeSH
• fenylmočovinové sloučeniny chemie   farmakologie   MeSH
• ferritin chemie   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• koncentrace vodíkových iontů MeSH
• koně MeSH
• kyselina listová chemie   MeSH
• lidé MeSH
• nosiče léků chemie   MeSH
• piperidiny chemie   farmakologie   MeSH
• pohyb buněk účinky léků   MeSH
• povrchové vlastnosti MeSH
• systémy cílené aplikace léků * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antitumorózní látky MeSH
• biokompatibilní materiály MeSH
• chinazoliny MeSH
• chinoliny MeSH
• fenylmočovinové sloučeniny MeSH
• ferritin MeSH
• inhibitory proteinkinas MeSH
• kyselina listová MeSH
• lenvatinib MeSH Prohlížeč
• nosiče léků MeSH
• piperidiny MeSH
• vandetanib MeSH Prohlížeč

BACKGROUND: Currently, the diagnosis and treatment of neuroblastomas-the most frequent solid tumors in children-exploit the norepinephrine transporter (hNET) via radiolabeled norepinephrine analogs. We aim to develop a nanomedicine-based strategy towards precision therapy by targeting hNET cell-surface protein with hNET-derived homing peptides. RESULTS: The peptides (seq. GASNGINAYL and SLWERLAYGI) were shown to bind high-resolution homology models of hNET in silico. In particular, one unique binding site has marked the sequence and structural similarities of both peptides, while most of the contribution to the interaction was attributed to the electrostatic energy of Asn and Arg (< - 228 kJ/mol). The peptides were comprehensively characterized by computational and spectroscopic methods showing ~ 21% β-sheets/aggregation for GASNGINAYL and ~ 27% α-helix for SLWERLAYGI. After decorating 12-nm ferritin-based nanovehicles with cysteinated peptides, both peptides exhibited high potential for use in actively targeted neuroblastoma nanotherapy with exceptional in vitro biocompatibility and stability, showing minor yet distinct influences of the peptides on the global expression profiles. Upon binding to hNET with fast binding kinetics, GASNGINAYLC peptides enabled rapid endocytosis of ferritins into neuroblastoma cells, leading to apoptosis due to increased selective cytotoxicity of transported payload ellipticine. Peptide-coated nanovehicles significantly showed higher levels of early apoptosis after 6 h than non-coated nanovehicles (11% and 7.3%, respectively). Furthermore, targeting with the GASNGINAYLC peptide led to significantly higher degree of late apoptosis compared to the SLWERLAYGIC peptide (9.3% and 4.4%, respectively). These findings were supported by increased formation of reactive oxygen species, down-regulation of survivin and Bcl-2 and up-regulated p53. CONCLUSION: This novel homing nanovehicle employing GASNGINAYLC peptide was shown to induce rapid endocytosis of ellipticine-loaded ferritins into neuroblastoma cells in selective fashion and with successful payload. Future homing peptide development via lead optimization and functional analysis can pave the way towards efficient peptide-based active delivery of nanomedicines to neuroblastoma cells.

• Klíčová slova
• Ferritin, Homing peptide, Neuroblastoma, Norepinephrine transporter, Targeted therapy,
• MeSH
• antitumorózní látky chemie   farmakokinetika   farmakologie   MeSH
• endocytóza genetika   MeSH
• ferritin chemie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nanomedicína MeSH
• nanostruktury chemie   MeSH
• neuroblastom metabolismus   MeSH
• peptidy chemie   genetika   metabolismus   MeSH
• proteiny přenášející noradrenalin přes plazmatickou membránu * chemie   genetika   metabolismus   MeSH
• systémy cílené aplikace léků metody   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antitumorózní látky MeSH
• ferritin MeSH
• peptidy MeSH
• proteiny přenášející noradrenalin přes plazmatickou membránu * 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 antitumorózní 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

BACKGROUND: Suitable fluorophores are the core of fluorescence imaging. Among the most exciting, yet controversial, labels are quantum dots (QDs) with their unique optical and chemical properties, but also considerable toxicity. This hinders QDs applicability in living systems. Surface chemistry has a profound impact on biological behavior of QDs. This study describes a two-step synthesis of QDs formed by CdTe core doped with Schiff base ligand for lanthanides [Ln (Yb3+, Tb3+ and Gd3+)] as novel cytocompatible fluorophores. RESULTS: Microwave-assisted synthesis resulted in water-soluble nanocrystals with high colloidal and fluorescence stability with quantum yields of 40.9-58.0%. Despite induction of endocytosis and cytoplasm accumulation of Yb- and TbQDs, surface doping resulted in significant enhancement in cytocompatibility when compared to the un-doped CdTe QDs. Furthermore, only negligible antimigratory properties without triggering formation of reactive oxygen species were found, particularly for TbQDs. Ln-doped QDs did not cause observable hemolysis, adsorbed only a low degree of plasma proteins onto their surface and did not possess significant genotoxicity. To validate the applicability of Ln-doped QDs for in vitro visualization of receptor status of living cells, we performed a site-directed conjugation of antibodies towards immuno-labeling of clinically relevant target-human norepinephrine transporter (hNET), over-expressed in neuroendocrine tumors like neuroblastoma. Immuno-performance of modified TbQDs was successfully tested in distinct types of cells varying in hNET expression and also in neuroblastoma cells with hNET expression up-regulated by vorinostat. CONCLUSION: For the first time we show that Ln-doping of CdTe QDs can significantly alleviate their cytotoxic effects. The obtained results imply great potential of Ln-doped QDs as cytocompatible and stable fluorophores for various bio-labeling applications.

• Klíčová slova
• Cellular labeling, Cytotoxicity, Inorganic fluorophore, Nanocrystal, Surface dopant,
• MeSH
• analýza jednotlivých buněk metody   MeSH
• fluorescenční barviva toxicita   MeSH
• kvantové tečky toxicita   MeSH
• lanthanoidy chemie   MeSH
• lidé MeSH
• mikrovlny MeSH
• nádorové buněčné linie MeSH
• optické zobrazování metody   MeSH
• povrchové vlastnosti MeSH
• Schiffovy báze chemie   MeSH
• sloučeniny kadmia toxicita   MeSH
• telur toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cadmium telluride MeSH Prohlížeč
• fluorescenční barviva MeSH
• lanthanoidy MeSH
• Schiffovy báze MeSH
• sloučeniny kadmia MeSH
• telur 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
• antitumorózní látky aplikace a dávkování   farmakokinetika   farmakologie   MeSH
• apoferritiny aplikace a dávkování   chemie   farmakokinetika   MeSH
• doxorubicin aplikace a dávkování   chemie   MeSH
• koncentrace vodíkových iontů MeSH
• léky antitumorózní - screeningové testy MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty farmakoterapie   patologie   MeSH
• nosiče léků aplikace a dávkování   chemie   farmakokinetika   MeSH
• stabilita léku MeSH
• systémy cílené aplikace léků MeSH
• uvolňování léčiv MeSH
• voda chemie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antitumorózní látky MeSH
• apoferritiny MeSH
• doxorubicin MeSH
• nosiče léků MeSH
• voda MeSH