The cellular adhesion receptor αvβ6-integrin is highly expressed in many cancers, e.g., pancreatic, lung, head-and-neck, cervical, bladder, and esophageal carcinoma. Multimerization of αvβ6-integrin-specific RGD peptides increases the target affinity and retention but affects biodistribution and pharmacokinetics. Amide formation of the terminal carboxylic acid moieties of the square-symmetrical bifunctional chelator DOTPI with 3-azidopropylamine yields derivatives with 4, 3, and 2 terminal azides and zero, 1, and 2 remaining carboxylic acids, respectively, whereby formation of the 2-cis-isomer is preferred according to NMR investigation of the Eu(III)-complexes. Cu(II)-catalyzed alkyne-azide cycloaddition (CuAAC) of the alkyne-functionalized αvβ6-integrin binding peptide cyclo[YRGDLAYp(NMe)K(pent-4-ynoic amide)] (Tyr2) yields the respective di-, tri-, and tetrameric conjugates for Lu-177-labeling. In mice bearing αvβ6-integrin-expressing xenografts of H2009 (human lung adenocarcinoma) cells, the Lu-177-labeled trimer's tumor-to-blood ratio of 112 exceeds that of the tetramer (10.4) and the dimer (54). Co-infusion of gelofusine (succinylated gelatin) reduces the renal uptake of the trimer by 89%, resulting in a 10-fold better tumor-to-kidney ratio, while no improvement of that ratio is observed with arginine/lysine, para-aminohippuric acid (PAH), and hydroxyethyl starch (HES) coinfusions. Since the Lu-177-labeled Tyr2-trimer outperforms the dimer and the tetramer, such trimers are considered the best lead structures for the ongoing development of αvβ6-integrin targeted anticancer theranostics.
- MeSH
- antigeny nádorové * metabolismus MeSH
- chelátory * chemie MeSH
- integriny * metabolismus MeSH
- lidé MeSH
- lutecium * chemie MeSH
- myši nahé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory farmakoterapie MeSH
- oligopeptidy * chemie farmakokinetika MeSH
- radiofarmaka farmakokinetika chemie terapeutické užití MeSH
- radionuklidy * chemie MeSH
- syntetická chemie okamžité shody MeSH
- teranostická nanomedicína metody MeSH
- tkáňová distribuce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antigeny nádorové * MeSH
- arginyl-glycyl-aspartic acid MeSH Prohlížeč
- chelátory * MeSH
- integrin alphavbeta6 MeSH Prohlížeč
- integriny * MeSH
- lutecium * MeSH
- Lutetium-177 MeSH Prohlížeč
- oligopeptidy * MeSH
- radiofarmaka MeSH
- radionuklidy * MeSH
Minimal immunogen vaccines are being developed to focus antibody responses against otherwise challenging targets, including human immunodeficiency virus (HIV), but multimerization of the minimal peptide immunogen on a carrier platform is required for activity. Star copolymers comprising multiple hydrophilic polymer chains ("arms") radiating from a central dendrimer unit ("core") were recently reported to be an effective platform for arraying minimal immunogens for inducing antibody responses in mice and primates. However, the impact of different parameters of the star copolymer (e.g., minimal immunogen density and hydrodynamic size) on antibody responses and the optimal synthetic route for controlling those parameters remains to be fully explored. We synthesized a library of star copolymers composed of poly[N-(2-hydroxypropyl)methacrylamide] hydrophilic arms extending from poly(amidoamine) dendrimer cores with the aim of identifying the optimal composition for use as minimal immunogen vaccines. Our results show that the length of the polymer arms has a crucial impact on the star copolymer hydrodynamic size and is precisely tunable over a range of 20-50 nm diameter, while the dendrimer generation affects the maximum number of arms (and therefore minimal immunogens) that can be attached to the surface of the dendrimer. In addition, high-resolution images of selected star copolymer taken by a custom-modified environmental scanning electron microscope enabled the acquisition of high-resolution images, providing new insights into the star copolymer structure. Finally, in vivo studies assessing a star copolymer vaccine comprising an HIV minimal immunogen showed the criticality of polymer arm length in promoting antibody responses and highlighting the importance of composition tunability to yield the desired biological effect.
- MeSH
- dendrimery * chemie MeSH
- lidé MeSH
- myši MeSH
- nosiče léků chemie MeSH
- polyaminy MeSH
- polymery chemie MeSH
- vakcíny proti AIDS imunologie chemie aplikace a dávkování MeSH
- vakcíny imunologie chemie aplikace a dávkování MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- dendrimery * MeSH
- nosiče léků MeSH
- Poly(amidoamine) MeSH Prohlížeč
- polyaminy MeSH
- polymery MeSH
- vakcíny proti AIDS MeSH
- vakcíny MeSH
Targeted protein degradation or TPD, is rapidly emerging as a treatment that utilizes small molecules to degrade proteins that cause diseases. TPD allows for the selective removal of disease-causing proteins, including proteasome-mediated degradation, lysosome-mediated degradation, and autophagy-mediated degradation. This approach has shown great promise in preclinical studies and is now being translated to treat numerous diseases, including neurodegenerative diseases, infectious diseases, and cancer. This review discusses the latest advances in TPD and its potential as a new chemical modality for immunotherapy, with a special focus on the innovative applications and cutting-edge research of PROTACs (Proteolysis TArgeting Chimeras) and their efficient translation from scientific discovery to technological achievements. Our review also addresses the significant obstacles and potential prospects in this domain, while also offering insights into the future of TPD for immunotherapeutic applications.
- MeSH
- chiméra pro cílenou proteolýzu MeSH
- imunoterapie * metody MeSH
- lidé MeSH
- nádory terapie farmakoterapie imunologie metabolismus MeSH
- proteasomový endopeptidasový komplex * metabolismus MeSH
- proteolýza * účinky léků MeSH
- ubikvitin * metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- chiméra pro cílenou proteolýzu MeSH
- proteasomový endopeptidasový komplex * MeSH
- ubikvitin * MeSH
Surface functionalization with biological macromolecules is an important task for the development of sensor materials, whereby the interaction with other biological materials should be suppressed. In this work, we developed a novel multifunctional poly(2-ethyl-2-oxazoline)-dithiolane conjugate as a versatile linker for gold surface immobilization of amine-containing biomolecules, containing poly(2-ethyl-2-oxazoline) as antifouling polymer, dithiolane for surface immobilization, and activated esters for protein conjugation. First, a well-defined carboxylic acid containing copoly(2-ethyl-2-oxazoline) was synthesized by cationic ring-opening copolymerization of 2-ethyl-2-oxazoline with a methyl ester-containing 2-oxazoline monomer, followed by postpolymerization modifications. The side-chain carboxylic groups were then converted to amine-reactive pentafluorophenyl (PFP) ester groups. Part of the PFP groups was used for the attachment of the dithiolane moiety, which can efficiently bind to gold surfaces. The final copolymer contained 1.4 mol% of dithiolane groups and 4.5 mol% of PFP groups. The copolymer structure was confirmed by several analytical techniques, including NMR spectroscopy and size-exclusion chromatography. The kinetics of the PFP ester aminolysis and hydrolysis demonstrated significantly faster amidation compared to hydrolysis, which is essential for subsequent protein conjugation. Successful coating of gold surfaces with the polymer was confirmed by spectroscopic ellipsometry, showing a polymer brush thickness of 4.77 nm. Subsequent modification of the coated surfaces was achieved using bovine serum albumin as a model protein. This study introduces a novel reactive polymer linker for gold surface functionalization and offers a versatile polymer platform for various applications including biosensing and surface functionalization.
A series of 2'-deoxyribonucleoside triphosphates (dNTPs) bearing 2- or 4-linked trans-cyclooctene (TCO) or bicyclononyne (BCN) tethered through a shorter propargylcarbamate or longer triethyleneglycol-based spacer were designed and synthesized. They were found to be good substrates for KOD XL DNA polymerase for primer extension enzymatic synthesis of modified oligonucleotides. We systematically tested and compared the reactivity of TCO- and BCN-modified nucleotides and DNA with several fluorophore-containing tetrazines in inverse electron-demand Diels-Alder (IEDDA) click reactions to show that the longer linker is crucial for efficient labeling. The modified dNTPs were transported into live cells using the synthetic transporter SNTT1, incubated for 1 h, and then treated with tetrazine conjugates. The PEG3-linked 4TCO and BCN nucleotides showed efficient incorporation into genomic DNA and good reactivity in the IEDDA click reaction with tetrazines to allow staining of DNA and imaging of DNA synthesis in live cells within time periods as short as 15 min. The BCN-linked nucleotide in combination with TAMRA-linked (TAMRA = carboxytetramethylrhodamine) tetrazine was also efficiently used for staining of DNA for flow cytometry. This methodology is a new approach for in cellulo metabolic labeling and imaging of DNA synthesis which is shorter, operationally simple, and overcomes several problems of previously used methods.
- Publikační typ
- časopisecké články MeSH
Nucleosides and 2'-deoxyribonucleoside triphosphates (dNTPs) bearing 3,3'-dimethoxy-2,2'-diphenyl-6-(4-hydroxyphenyl)-bodipy fluorophore attached through a propargyl or propargyl-triethylene glycol linker to position 5 of 2'-deoxycytidine were designed and synthesized. They exerted bright red fluorescence and good sensitivity to viscosity changing their lifetime from 1.6 to 4.5 ns. The modifed dNTPs were substrates for DNA polymerases and were used in enzymatic synthesis of labeled DNA through primer extension. The modified DNA probes served as viscosity sensors responding to protein binding by changes of lifetime. The nucleotide with longer linker (dCpegMOBTP) was transported to live cells and incorporated into the genomic DNA, which can be useful for staining of DNA and imaging of DNA synthesis.
UNLABELLED: As angiogenesis plays a key role in tumor growth and metastasis, the angiogenic process has attracted scientific interest as a target for diagnostic and therapeutic agents. Factors influencing angiogenesis include the vascular endothelial growth factor (VEGF) family and the two associated receptor types (VEGFR-1 and VEGFR-2). VEGFR-1/-2 detection and quantification in cancer lesions are essential for tumor process management. As a result of the advantageous pharmacokinetics and image contrast, peptides radiolabeled with PET emitters have become interesting tools for the visualization of VEGFR-1/-2-positive tumors. In this study, we prepared 68Ga-labeled peptides containing 15 (peptide 1) and 23 (peptide 2) amino acids as new PET tracers for tumor angiogenic process imaging. METHODS: The peptides were conjugated with NODAGA-tris(t-Bu ester) and subsequently radiolabeled with [68Ga]Ga-chloride. The prepared [68Ga]Ga-NODAGA-peptide 1 and [68Ga]Ga-NODAGA-peptide 2 were tested for radiochemical purity and saline/plasma stability. Consequently, the binding affinity toward VEGFRs was assessed in vitro on human glioblastoma and kidney carcinoma cells. The found peptide receptor affinity was compared with the calculated values in the PROtein binDIng enerGY prediction (PRODIGY) server. Finally, the biodistribution study was performed on BALB/c female mice to reveal the basic pharmacokinetic behavior of radiopeptides. RESULTS: The in vitro affinity testing of [68Ga]Ga-NODAGA-peptides 1 and 2 showed retained receptor binding as characterized by equilibrium dissociation constant (KD) values in the range of 0.5-1.2 μM and inhibitory concentration 50% (IC50) values in the range of 3.0-5.6 μM. Better binding properties of peptide 2 to VEGFR-1/-2 were found in the PRODIGY server. The biodistribution study on mice showed remarkable accumulation of both peptides in the kidneys and urinary bladder with a short half-life after intravenous application. The in vitro plasma stability of [68Ga]Ga-NODAGA-peptide 2 was superior to that of [68Ga]Ga-NODAGA-peptide 1. CONCLUSIONS: The obtained results demonstrated a high radiolabeling yield with no need for purification and preserved binding potency of 68Ga-labeled peptides 1 and 2 toward VEGFRs in cancer cells. The peptide-receptor protein interaction assessed in protein-peptide docking determined the strongest interaction of peptide 2 with domain 2 of VEGFR-2 in addition to a more acceptable plasma stability (t1/2 = 120 min) than that for peptide 1. We found both radiolabeled peptides very potent in their receptor binding, which makes them suitable imaging agents. The rapid transition of the radiopeptides into the urinary tract indicates suitable pharmacokinetic characteristics.
- MeSH
- aminokyseliny MeSH
- chloridy MeSH
- estery MeSH
- heterocyklické sloučeniny monocyklické chemie MeSH
- lidé MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- peptidy chemie MeSH
- pozitronová emisní tomografie metody MeSH
- radioizotopy galia * chemie MeSH
- receptor 1 pro vaskulární endoteliální růstový faktor MeSH
- receptor 2 pro vaskulární endoteliální růstový faktor MeSH
- receptory vaskulárního endoteliálního růstového faktoru MeSH
- tkáňová distribuce MeSH
- vaskulární endoteliální růstový faktor A * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane MeSH Prohlížeč
- aminokyseliny MeSH
- chloridy MeSH
- estery MeSH
- heterocyklické sloučeniny monocyklické MeSH
- peptidy MeSH
- radioizotopy galia * MeSH
- receptor 1 pro vaskulární endoteliální růstový faktor MeSH
- receptor 2 pro vaskulární endoteliální růstový faktor MeSH
- receptory vaskulárního endoteliálního růstového faktoru MeSH
- vaskulární endoteliální růstový faktor A * MeSH
Although triple labeling of molecular beacons has been documented to improve quenching efficiencies and studies generally assume similar benefits at long TaqMan probes, a limited number of works have studied this issue in TaqMan probes. We therefore prepared a series of long triple-labeled oligodeoxynucleotide probes with 6-carboxyfluorescein as a fluorophore at the 5'-end and BlackBerry (BBQ-650) or azaphthalocyanine quenchers at the 3'-end and in the intrastrand position and systematically compared their quenching efficiencies with those of the corresponding double-labeled probes including important control probes. A model polymerase chain reaction (PCR) assay enabled the determination of the quenching efficiencies of static and Förster resonance energy transfer (FRET) quenching in the target probes. The type of probe had no effect on the static quenching ability. Importantly, FRET quenching of double-labeled probes with a quencher at the 3'-end showed a statistically insignificant difference from the control probe without any quencher, indicating the need to shift the quencher closer to the fluorophore in long probes. Shortening the distance between the fluorophore and the quencher played a key role in FRET quenching, whereas the introduction of an additional quencher only slightly improved the quenching efficiency. BBQ-labeled probes had lower quenching efficiencies than azaphthalocyanine probes. The methodologies and relationships described above seem, however, to be universal and applicable to any quencher.
Preexisting serum albumin-polymer bioconjugates have been formed either through covalent conjugation or supramolecular interactions. However, the viability of producing a bioconjugate where both covalent conjugation and supramolecular interactions have been adopted is yet to be explored. In this work, the noncovalent interaction of two polymers bearing fatty acid-based end-functionalities were compared and the superior binder was carried forward for testing with serum albumin that possessed a polymer conjugated to its Cys34 residue. The studies demonstrated that an albumin-polymer bioconjugate equipped with polymers via both covalent and supramolecular interactions can be successfully achieved.
- MeSH
- molekulární struktura MeSH
- polymery * chemie MeSH
- sérový albumin * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- polymery * MeSH
- sérový albumin * MeSH
Protein corona formation has been regarded as an obstacle to developing diagnostic and therapeutic nanoparticles for in vivo applications. Serum proteins that assemble around nanoparticles can hinder their targeting efficiency. Virus-based nanoparticles should be naturally predisposed to evade such barriers in host organisms. Here, we demonstrate that virus-like particles derived from mouse polyomavirus do not form a rich protein corona. These particles can be efficiently targeted to cells that overproduce transferrin receptors, e.g., cancer cells, by conjugating transferrin to the particle surface. In this study, we provide evidence that the interaction of virus-like particles with their newly assigned target receptor is not obstructed by serum proteins. The particles enter target cells via a clathrin-dependent endocytic pathway that is not naturally used by the virus. Our results support the notion that the natural properties of virus-like particles make them well-suited for development of nanosized theranostic tools resistant to detargeting by protein coronas.
- MeSH
- krevní proteiny chemie metabolismus MeSH
- lidé MeSH
- myši MeSH
- nanočástice chemie MeSH
- Polyomavirus chemie MeSH
- proteinová korona chemie metabolismus MeSH
- receptory transferinu metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- krevní proteiny MeSH
- proteinová korona MeSH
- receptory transferinu MeSH
