A novel actively targeted polymer carrier for anticancer drugs based on an N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) is proposed. An oligopeptide sequence GE7, attached to the polymer, is a specific ligand for the EGF receptor overexpressed on most tumor cells. Co-attachment of selected chemotherapeutics will therefore lead to formation of tumor-specific polymer therapeutics, further enhanced by the EPR effect. FACS measurements prove elevated binding activity of the fluorescently labeled PHPMA/GE7 conjugate in EGFR-rich cells (FaDu, MCF-7), compared to conjugates of scrambled peptides. Cell lines with low EGFR level (SW620, B16F10) bind the GE7 conjugate significantly less.
- MeSH
- erbB receptory metabolismus MeSH
- kyseliny polymethakrylové chemická syntéza chemie terapeutické užití MeSH
- lékové transportní systémy metody MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- oligopeptidy genetika metabolismus MeSH
- pozitronová emisní tomografie MeSH
- protinádorové látky chemie metabolismus MeSH
- průtoková cytometrie MeSH
- spektrofotometrie ultrafialová MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Herein, new biodegradable star polymer-doxorubicin conjugates designed for passive tumor targeting were investigated, and their synthesis, physico-chemical characterization, drug release, biodegradation, biodistribution and in vivo anti-tumor efficacy are described. In the conjugates, the core formed by poly(amidoamine) (PAMAM) dendrimers was grafted with semitelechelic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers bearing doxorubicin (Dox) attached by hydrazone bonds, which enabled intracellular pH-controlled drug release. The described synthesis facilitated the preparation of biodegradable polymer conjugates in a broad range of molecular weights (200-1000g/mol) while still maintaining low polydispersity (~1.7). The polymer grafts were attached to the dendrimers through either stable amide bonds or enzymatically or reductively degradable spacers, which enabled intracellular degradation of the high-molecular-weight polymer carrier to excretable products. Biodegradability tests in suspensions of EL4 T-cell lymphoma cells showed that the rate of degradation was much faster for reductively degradable conjugates (close to completion within 24h of incubation) than for conjugates linked via an enzymatically degradable oligopeptide GFLG sequence (slow degradation taking several days). This finding was likely due to the differences in steric hindrance in terms of the accessibility of the small molecule glutathione and the bulky enzyme cathepsin B to the polymer substrate. Regarding drug release, the conjugates were fairly stable in buffer at pH 7.4 (model of blood stream) but released doxorubicin under mild acidic conditions that model the tumor cell microenvironment. The star polymer-Dox conjugates exhibited significantly prolonged blood circulation and enhanced tumor accumulation in tumor-bearing mice, indicating the important role of the EPR effect in its anti-cancer activity. The star polymer conjugates showed prominently higher in vivo anti-tumor activities than the free drug or linear polymer conjugate when tested in mice bearing EL4 T-cell lymphoma, with a significant number of long-term surviving (LTS). Based on the results, we conclude that a M(w) of HPMA copolymers of 200,000 to 600,000g/mol is optimal for polymer carriers designed for the efficient passive targeting to solid tumors. In addition, an expressive therapy-dependent stimulation of the immune system was observed.
- MeSH
- biokompatibilní materiály chemie metabolismus MeSH
- dendrimery chemie metabolismus MeSH
- doxorubicin chemie metabolismus farmakokinetika terapeutické užití MeSH
- lidé MeSH
- lymfom farmakoterapie MeSH
- methakryláty chemie metabolismus MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- protinádorová antibiotika chemie metabolismus farmakokinetika terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Radioactive decay of some radionuclides produces a shower of Auger electrons, potent ionizing radiation within a very short range in living tissue (typically ca. 100 nm). Therefore, they must be brought to DNA-containing cell compartments and preferentially directly to DNA to be fully biologically effective. They may be used for a triple-targeting approach (first targeting, polymer-based system targeting into tumor tissue due to EPR effect; second targeting, pH-controlled release of intercalator-bound Auger electron emitter in slightly acidic tumor tissue or endosome; third targeting, into DNA in cell nucleus by the intercalator) minimizing radiation burden of healthy tissues. We describe a first system of this type, an ellipticine derivative-bound iodine-125 attached to hydrazide moieties containing poly[N-(2-hydroxypropyl)methacrylamide]. The system is stable at pH 7.4 (0% intercalator released after 24 h incubation), while iodine-containing biologically active intercalator is released upon decrease of pH (25% intercalator released after 24 h incubation at pH 5.0-model of late endosomes). Both 2-N-(2-oxobutyl)-9-iodoellipticinium bromide and the noniodinated 2-N-(2-oxobutyl)ellipticinium bromide are potent intercalators, as proven by direct titration with DNA and ethidium displacement assay, and readily penetrate into cell nuclei, as proven by confocal microscopy. They retain chemotherapeutical antiproliferative properties of ellipticine against Raji, EL-4, and 4T1cells with IC(50) in the range 0.27-8.8 μmol/L. Polymer conjugate of 2-N-(2-oxobutyl)-9-iodoellipticinium bromide is internalized into endosomes, releases active drug, possesses cytotoxic activity, and the drug accumulates in cell nuclei.
- MeSH
- buněčné jádro účinky léků metabolismus MeSH
- DNA chemie MeSH
- elektrony MeSH
- elipticiny chemie farmakologie MeSH
- hydraziny chemie MeSH
- koncentrace vodíkových iontů MeSH
- kyseliny polymethakrylové chemická syntéza chemie farmakologie MeSH
- lidé MeSH
- molekulární struktura MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- organely chemie účinky léků MeSH
- radioizotopy jodu MeSH
- stereoizomerie MeSH
- tkáňová distribuce MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE: In vivo efficacy and safety of HPMA-based copolymers armed with doxorubicin via a spacer containing pH-sensitive linkage that can be prepared within a broad range of attached drug contents (1) was tested in murine tumor models. METHODS: Mice bearing T cell lymphoma EL4 or B cell lymphoma 38C13 were treated with a single dose of the conjugate (15, 25, and 75 mg Dox eq./kg i.v.) in a therapeutic regime. Anti-tumor resistance of the cured animals was proved by a second challenge with a lethal dose of tumor cells without additional treatment. RESULTS: The content of drug bound to the polymer is an important parameter in relation to the conjugate therapeutic efficacy. The best anti-tumor effects were produced by conjugates with 10 - 13 wt% of bound doxorubicin. Free doxorubicin up to 4.6% relative to total drug content had no impact on the treatment efficacy and acute toxicity. The conjugates induced a complete cure of mice and regular treatment-dependent development of specific anti-tumor resistance. No myelosuppression or organ damage was observed. CONCLUSIONS: A well-defined HPMA copolymer-doxorubicin conjugate with pH-sensitive drug release is a good candidate for clinical trials as it has remarkable anti-tumor efficacy and a favorable safety profile.
- MeSH
- doxorubicin analogy a deriváty chemická syntéza farmakokinetika farmakologie MeSH
- imunomodulace účinky léků MeSH
- koncentrace vodíkových iontů MeSH
- kyseliny polymethakrylové chemická syntéza farmakokinetika farmakologie MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nosiče léků chemická syntéza farmakokinetika farmakologie MeSH
- polymery chemická syntéza farmakokinetika farmakologie MeSH
- proliferace buněk účinky léků MeSH
- protinádorová antibiotika farmakokinetika farmakologie MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
There is a wide range of techniques utilizing fluorescence of doxorubicin (Dox) commonly used for analysis of intracellular accumulation and destiny of various drug delivery systems containing this anthracycline antibiotic. Unfortunately, results of these studies can be significantly influenced by doxorubicin degradation product, 7,8-dehydro-9,10-desacetyldoxorubicinone (D*) forming spontaneously in aqueous environment, whose fluorescence strongly interfere with that of doxorubicin. Here, we define two microscopy techniques enabling to distinguish and separate Dox and D* emission based either on its spectral properties or on fluorescence lifetime analysis. To analyze influx and nuclear accumulation of Dox (free or polymer-bound) by flow cytometry, we propose using an indirect method based on its DNA intercalation competition with Hoechst 33342 rather than a direct measurement of doxorubicin fluorescence inside the cells.
- MeSH
- buněčné jádro metabolismus MeSH
- buňky 3T3 MeSH
- DNA metabolismus MeSH
- doxorubicin analogy a deriváty metabolismus farmakologie MeSH
- fluorescenční spektrometrie MeSH
- hydrofobní a hydrofilní interakce MeSH
- lékové transportní systémy MeSH
- lidé MeSH
- lymfom T-buněčný farmakoterapie metabolismus MeSH
- myši MeSH
- nádorové buňky kultivované MeSH
- polymery metabolismus MeSH
- protinádorová antibiotika metabolismus farmakologie MeSH
- průtoková cytometrie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To avoid the side effects of the anti-cancer drug doxorubicin (Dox), we conjugated this drug to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone. Dox was conjugated via an amide bond (Dox-HPMA(AM), PK1) or a hydrazone pH-sensitive bond (Dox-HPMA(HYD)). In contrast to Dox and Dox-HPMA(HYD), Dox-HPMA(AM) accumulates within the cell's intracellular membranes, including those of the Golgi complex and endoplasmic reticulum, both involved in protein glycosylation. Flow cytometry was used to determine lectin binding and cell death, immunoblot to characterize the presence of CD7, CD43, CD44, and CD45, and high-performance anion exchange chromatography with pulsed amperometric detector analysis for characterization of plasma membrane saccharide composition. Incubation of EL4 cells with Dox-HPMA(AM) conjugate, in contrast to Dox or Dox-HPMA(HYD), increased the amounts of membrane surface-associated glycoproteins, as well as saccharide moieties recognized by peanut agglutinin, Erythrina cristagalli, or galectin-1 lectins. Only Dox-HPMA(AM) increased expression of the highly glycosylated membrane glycoprotein CD43, while expression of others (CD7, CD44, and CD45) was unaffected. The binding sites for galectin-1 are present on CD43 molecule. Furthermore, we present that EL4 treated with Dox-HPMA(AM) possesses increased sensitivity to galectin-1-induced apoptosis. In this study, we demonstrate that Dox-HPMA(AM) treatment changes glycosylation of the EL4 T cell lymphoma surface and sensitizes the cells to galectin-1-induced apoptosis.
- MeSH
- amidy chemie MeSH
- antigeny CD43 metabolismus MeSH
- apoptóza MeSH
- doxorubicin analogy a deriváty farmakologie MeSH
- endoplazmatické retikulum metabolismus MeSH
- galektin 1 metabolismus MeSH
- glykosylace MeSH
- Golgiho aparát metabolismus MeSH
- kyseliny polymethakrylové farmakologie MeSH
- lymfom T-buněčný farmakoterapie metabolismus patologie MeSH
- myši MeSH
- nádorové buněčné linie účinky léků MeSH
- nosiče léků MeSH
- proliferace buněk MeSH
- protinádorová antibiotika farmakologie MeSH
- průtoková cytometrie MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Synthesis, physicochemical and biological properties and preliminary anticancer activity of new star-shaped polymer-doxorubicin (DOX) conjugates targeted with anti-CD20 monoclonal antibody were investigated. Mild reduction of antibody (Ab) with dithiothreitol (DTT) resulted in introduction of thiol groups into Ab. Polymer precursors used for the synthesis of the conjugates were based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with a functional group at the polymer chain end. The copolymers were linked to the thiol groups of the reduced Ab via one-point attachment forming a star-shaped structure with central antibody surrounded by hydrophilic polymer chains. Neither reduction nor polymer modification of Ab influenced binding activity of the Ab to its specific cancer cell membrane antigen as it was confirmed in vitro by standard flow cytometry. The anticancer drug DOX was attached to the HPMA copolymer chain in an Ab-polymer system via a pH-labile hydrazone linkage or via an oligopeptide sequence degradable by lysosomal enzymes. Such Ab-polymer-DOX conjugates were fairly stable in aqueous solution at pH 7.4 and the drug was readily released in mildly acid environment at pH 5-5.5 by hydrolysis of hydrazone bond or more slowly by enzymolysis with lysosomal enzymes. The cytostatic activity of the anti-CD20 monoclonal Ab-targeted conjugates tested on several CD20-positive or negative human and mouse cancer cell lines confirmed considerable targeting capacity of the monoclonal Ab after its binding to the polymer carrier. New method of synthesis of star antibody-targeted polymer-drug conjugates with pH-controlled drug release described in this paper opens new perspectives for development of new therapeutics intended for cancer therapy.
- MeSH
- akrylamidy chemie MeSH
- Burkittův lymfom farmakoterapie metabolismus MeSH
- chemické modely MeSH
- cytostatické látky metabolismus MeSH
- doxorubicin chemie MeSH
- koncentrace vodíkových iontů MeSH
- lékové transportní systémy MeSH
- lidé MeSH
- lymfom T-buněčný farmakoterapie metabolismus MeSH
- methakryláty chemie MeSH
- molekulová hmotnost MeSH
- monoklonální protilátky imunologie MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nosiče léků chemická syntéza chemie MeSH
- polymery chemie MeSH
- preklinické hodnocení léčiv MeSH
- protinádorová antibiotika chemie MeSH
- rozpustnost MeSH
- voda chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- hodnotící studie MeSH
- práce podpořená grantem MeSH
The successful treatment of cancer with conventional drugs is frequently complicated by the resistance of tumor cells to such a non-specific therapy. Over the last few years, immunotherapy has gained attention as a tumor-specific approach. Recent findings demonstrated that some conventional cytostatics stimulate local anticancer responses. New anticancer drugs, including their polymeric derivatives, are currently being developed with the aim of destroying tumors more effectively and more specifically. Among these, the water-soluble conjugates of doxorubicin with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carrier have emerged as efficient therapeutics because they are able to not only directly destroy cancer cells but also elicit systemic tumor-specific anticancer responses. Here, we discuss new insights into their mechanisms of immune surveillance, which could suggest novel approaches to cancer therapy.
- MeSH
- adjuvancia imunologická aplikace a dávkování MeSH
- biologické modely MeSH
- cytotoxiny aplikace a dávkování MeSH
- financování organizované MeSH
- imunologické faktory aplikace a dávkování MeSH
- lidé MeSH
- nádory farmakoterapie imunologie MeSH
- polymery aplikace a dávkování MeSH
- protinádorové látky aplikace a dávkování MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
