Antibody-mediated targeting is an efficient strategy to enhance the specificity and selectivity of polymer nanomedicines towards the target site, typically a tumor. However, direct covalent coupling of an antibody with a polymer usually results in a partial damage of the antibody binding site accompanied with a compromised biological activity. Here, an original solution based on well-defined non-covalent interactions between tris-nitrilotriacetic acid (trisNTA) and hexahistidine (His-tag) groups, purposefully introduced to the structure of each macromolecule, is described. Specifically, trisNTA groups were attached along the chains of a hydrophilic statistical copolymer based on N-(2-hydroxypropyl)methacrylamide (HPMA), and at the end or along the chains of thermo-responsive di-block copolymers based on N-isopropylmethacrylamide (NIPMAM) and HPMA; His-tag was incorporated to the structure of a recombinant single chain fragment of an anti-GD2 monoclonal antibody (scFv-GD2). Static and dynamic light scattering analyses confirmed that mixing of polymer with scFv-GD2 led to the formation of polymer/scFv-GD2 complexes; those prepared from thermo-responsive polymers formed stable micelles at 37 °C. Flow cytometry and fluorescence microscopy clearly demonstrated antigen-specific binding of the prepared complexes to GD2 positive murine T-cell lymphoma cells EL-4 and human neuroblastoma cells UKF-NB3, while no interaction with GD2 negative murine fibroblast cells NIH-3T3 was observed. These non-covalent polymer protein complexes represent a new generation of highly specific actively targeted polymer therapeutics or diagnostics.
Polymer nanomedicines with anti-tumor activity should exhibit sufficient stability during systemic circulation to the target tissue; however, they should release the active drug selectively in the tumor. Thus, choice of a tumor-specific stimuli-sensitive spacer between the drug and the carrier is critical. Here, a series of polymer conjugates of anti-cancer drugs doxorubicin and pirarubicin covalently bound to copolymers based on N-(2-hydroxypropyl)methacrylamide via various enzymatically cleavable oligopeptide spacers were prepared and characterized. The highest rate of the drug release from the polymer carriers in presence of the lysosomal protease cathepsin B was determined for the copolymers with Val-Cit-Aba spacer. Copolymers containing pirarubicin were more cytotoxic and showed higher internalization rate than the corresponding doxorubicin counterparts. The conjugates containing GFLG and Val-Cit-Aba spacers exhibited the highest anti-tumor efficacy in vivo against murine sarcoma S-180, the highest rate of the enzymatically catalyzed drug release, and the highest cytotoxicity in vitro.
- MeSH
- doxorubicin farmakologie chemie MeSH
- lidé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory * farmakoterapie MeSH
- nanomedicína MeSH
- nosiče léků chemie MeSH
- polymery chemie MeSH
- protinádorové látky * farmakologie terapeutické užití chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A BCL1 leukemia-cell-targeted polymer-drug conjugate with a narrow molecular weight distribution consisting of an N-(2-hydroxypropyl)methacrylamide copolymer carrier and the anticancer drug pirarubicin is prepared by controlled radical copolymerization followed by metal-free click chemistry. A targeting recombinant single chain antibody fragment (scFv) derived from a B1 monoclonal antibody is attached noncovalently to the polymer carrier via a coiled coil interaction between two complementary peptides. Two pairs of coiled coil forming peptides (abbreviated KEK/EKE and KSK/ESE) are used as linkers between the polymer-pirarubicin conjugate and the targeting protein. The targeted polymer conjugate with the coiled coil linker KSK/ESE exhibits 4× better cell binding activity and 2× higher cytotoxicity in vitro compared with the other conjugate. Treatment of mice with established BCL1 leukemia using the scFv-targeted polymer conjugate leads to a markedly prolonged survival time of the experimental animals compared with the treatment using the free drug and the nontargeted polymer-pirarubicin conjugate.
- MeSH
- akrylamidy chemie MeSH
- cílená molekulární terapie MeSH
- click chemie MeSH
- cyklin D1 antagonisté a inhibitory imunologie MeSH
- imunoglobuliny - fragmenty aplikace a dávkování imunologie MeSH
- imunokonjugáty aplikace a dávkování chemie MeSH
- lékové transportní systémy MeSH
- leukemie imunologie patologie terapie MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- monoklonální protilátky chemie imunologie MeSH
- myši MeSH
- nosiče léků aplikace a dávkování chemie MeSH
- peptidy chemie imunologie MeSH
- polymery aplikace a dávkování chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Coiled coils are a common structural motif in many natural proteins that can also be utilized in the design and preparation of drug delivery systems for the noncovalent connection of two macromolecules. In this work, two different pairs of peptides forming coiled coil hetero-oligomers were designed, synthesized, and characterized. While the peptide sequences (VAALEKE)4 and (VAALKEK)4 predominantly form coiled coil heterodimers with randomly orientated peptide chains, (IAALESE)2-IAALESKIAALESE and IAALKSKIAALKSE-(IAALKSK)2 tend to form higher hetero-oligomers with an antiparallel orientation of their peptide chains. The associative behavior of these peptides was studied in aqueous solutions using circular dichroism spectroscopy, size-exclusion chromatography, isothermal titration calorimetry and sedimentation analyses. The orientation of the peptide chains in the coiled coil heterodimers was assessed using fluorescence spectroscopy with fluorescence resonance energy transfer labels attached to the ends of the peptides. The formation of the heterodimer can be used as a general method for the selective noncovalent conjugation of a specific targeting moiety with various drug carrier systems; this process involves simple self-assembly in a physiological solution before drug administration. The preparation of targeted macromolecular therapeutics consisting of a synthetic polymer drug carrier and a recombinant protein targeting ligand is discussed.
The coiled coil is a superhelical structural protein motif that has been thoroughly investigated in recent years. Because of the relatively well-understood principles that determine the properties of coiled coil peptides and proteins, macromolecular systems containing the coiled coil motif have been suggested for various applications. This short review focuses on hybrid polymer coiled coil systems designed for drug delivery purposes. After a short introduction, the most important features of the coiled coils (stability, association number, oligomerization selectivity and orientation of helices) are described, and the factors influencing these characteristics are discussed. Several examples of the most interesting biomedical applications of the polymer-coiled coil systems (according to the authors' opinion) are presented.
- MeSH
- aminokyselinové motivy * MeSH
- hydrogely MeSH
- konformace proteinů MeSH
- lékové transportní systémy metody MeSH
- molekulární modely MeSH
- rekombinantní proteiny aplikace a dávkování genetika farmakologie MeSH
- rozpustnost MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
We have designed, synthesized, and characterized peptides containing four repeats of the sequences VAALEKE (peptide E) or VAALKEK (peptide K). While the peptides alone adopt in aqueous solutions a random coil conformation, their equimolar mixture forms heterodimeric coiled coils as confirmed by CD spectroscopy. 5-Azidopentanoic acid was connected to the N-terminus of peptide E via a short poly(ethylene glycol) spacer. The terminal azide group enabled conjugation of the peptide with a synthetic drug carrier based on the N-(2-hydroxypropyl)methacrylamide copolymer containing propargyl groups using "click" chemistry. When incorporated into the polymer drug carrier, peptide E formed a stable noncovalent complex with peptide K belonging to a recombinant single-chain fragment (scFv) of the M75 antibody. The complex thereby mediates a noncovalent linkage between the polymer drug carrier and the protein. The recombinant scFv antibody fragment was selected as a targeting ligand against carbonic anhydrase IX-a marker overexpressed by tumor cells of various human carcinomas. The antigen binding affinity of the polymer-scFv complex was confirmed by ELISA. This approach offers a well-defined, specific, and nondestructive universal method for the preparation of protein (antibody)-targeted polymer drug and gene carriers designed for cell-specific delivery.
- MeSH
- akrylamidy chemie MeSH
- antigeny nádorové imunologie metabolismus MeSH
- bakteriální transformace MeSH
- cirkulární dichroismus MeSH
- click chemie metody MeSH
- dimerizace MeSH
- ELISA MeSH
- Escherichia coli MeSH
- imunokonjugáty chemie imunologie farmakologie MeSH
- karboanhydrasy imunologie metabolismus MeSH
- karcinom farmakoterapie enzymologie imunologie patologie MeSH
- klonování DNA MeSH
- lidé MeSH
- molekulární konformace MeSH
- monoklonální protilátky chemie genetika imunologie MeSH
- nádorové biomarkery imunologie metabolismus MeSH
- nosiče léků chemická syntéza farmakologie MeSH
- oligopeptidy chemická syntéza imunologie farmakologie MeSH
- plazmidy MeSH
- polyethylenglykoly chemie MeSH
- rekombinantní proteiny chemie genetika imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Polymer therapeutics including polymer-drug conjugates, polymer-protein conjugates and polymer-modified gene delivery vectors are addressed in this review. Brief history of the polymer therapeutics is described with a focus on the pioneering work accomplished at the Institute of Macromolecular Chemistry in Prague. The advantages of polymer therapeutics compared with lowmolecular- weight drugs are outlined. Polymer cancerostatics, polymer-protein conjugates with anti-cancer activity and polymer-modified viruses based on N-(2-hydroxypropyl) methacrylamide copolymers and biodegradable multiblock poly(ethylene glycol) polymers are chosen as examples. The current status of clinical evaluation of the polymer therapeutics is also mentioned.
- MeSH
- biopolymery chemie klasifikace terapeutické užití MeSH
- cytostatické látky MeSH
- ethylenglykoly chemie terapeutické užití MeSH
- financování organizované MeSH
- genetická terapie metody MeSH
- léčivé přípravky MeSH
- lidé MeSH
- maleinanhydridy terapeutické užití MeSH
- methakryláty chemie terapeutické užití MeSH
- polymery dějiny chemie terapeutické užití MeSH
- polystyreny terapeutické užití MeSH
- zinostatin terapeutické užití MeSH
- Check Tag
- lidé MeSH
Short polypeptides with four pentad repeats, (VPGVG)(4) and (VPAVG)(4), were synthesised by manual fluorenylmethoxycarbonyl/tert-butyl (Fmoc/t-Bu) solid phase peptide synthesis using a convergent approach. In the next step, the peptides were coupled via their N-terminus with activated semi-telechelic poly(ethylene glycol) O-(N-Fmoc-2-aminoethyl)-O'-(2-carboxyethyl)undeca(ethylene glycol) (Fmoc-PEG-COOH) to yield monodisperse Fmoc-PEG-peptide diblock copolymer. Both the presence of the terminal hydrophobic Fmoc group and the hydrophilic PEG chain in the copolymers were shown to play a crucial role in their self-associative behaviour, leading to reversible formation of supramolecular thermoresponsive assemblies. The peptides and their PEG derivatives were characterised by HPLC, NMR and MALDI-TOF MS. The associative behaviour of the peptides and their PEG derivatives was studied by dynamic light scattering, MAS NMR and phase contrast microscopy. [image: see text].
- MeSH
- elastin chemie MeSH
- financování organizované MeSH
- konformace proteinů MeSH
- magnetická rezonanční spektroskopie MeSH
- mikroskopie fázově kontrastní MeSH
- molekulární modely MeSH
- oligopeptidy chemická syntéza chemie MeSH
- peptidy chemie MeSH
- polyethylenglykoly chemie MeSH
- radiační rozptyl MeSH
- sekvence aminokyselin MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- termodynamika MeSH
- vysokoúčinná kapalinová chromatografie MeSH
