Tomasz M. Goszczyński

Hydroxyethyl starch as an effective methotrexate carrier in anticancer therapy

At present, effective anticancer therapy remains one of the most challenging tasks facing the scientific community. A major limitation to most conventional low‐molecular weight anticancer chemotherapeutics is their unfavourable uptake by healthy tissue, fast metabolism and lack of tumour cell selectivity. One way to solve this problem is the application of hybrid nanoparticles containing widely known therapeutic substances. This study was performed with the aim of investigating the potential of use hydroxyethyl starch (HES) as a high‐molecular weight carrier for anticancer drug (methotrexate, MTX). HES‐MTX conjugates were characterized in terms of MTX content, hydrodynamic size, zeta potential, and drug release kinetics. In vitro biological characteristics were determined using different cancer cell lines. The antitumor effect in vivo was tested in NOD/SCID mice subcutaneously inoculated with MV‐4‐11 human leukaemia cells and CDF1 mice intraperitoneally inoculated with P388 murine leukaemia cells. The in vivo experiments revealed the considerably higher antitumor efficacy of HES‐MTX conjugates in comparison to unconjugated drug. The results presented in this article demonstrate that the application of HES as an anticancer drug carrier can improve the treatment efficacy and have significant implications for the future design and implementation of drug‐carrier conjugates. The study should help create new opportunities in the design of HES‐based innovative drug‐carrier conjugates.

Solid state, thermal synthesis of site-specific protein–boron cluster conjugates and their physicochemical and biochemical properties

BACKGROUND Boron clusters represent a vast family of boron-rich compounds with extraordinary properties that provide the opportunity of exploitation in different areas of chemistry and biology. In addition, boron clusters are clinically used in boron neutron capture therapy (BNCT) of tumors. In this paper, a novel, in solid state (solvent free), thermal method for protein modification with boron clusters has been proposed. METHODS The method is based on a cyclic ether ring opening in oxonium adduct of cyclic ether and a boron cluster with nucleophilic centers of the protein. Lysozyme was used as the model protein, and the physicochemical and biological properties of the obtained conjugates were characterized. RESULTS The main residues of modification were identified as arginine-128 and threonine-51. No significant changes in the secondary or tertiary structures of the protein after tethering of the boron cluster were found using mass spectrometry and circular dichroism measurements. However, some changes in the intermolecular interactions and hydrodynamic and catalytic properties were observed. CONCLUSIONS To the best of our knowledge, we have described the first example of an application of cyclic ether ring opening in the oxonium adducts of a boron cluster for protein modification. In addition, a distinctive feature of the proposed approach is performing the reaction in solid state and at elevated temperature. GENERAL SIGNIFICANCE The proposed methodology provides a new route to protein modification with boron clusters and extends the range of innovative molecules available for biological and medical testing.

Interactions of Boron Clusters and their Derivatives with Serum Albumin

Boron clusters are polyhedral boron hydrides with unique properties, and they are becoming increasingly widely used in biology and medicine, including for boron neutron capture therapy (BNCT) of cancers and in the design of novel bioactive molecules and potential drugs. Among boron cluster types, icosahedral boranes, carboranes, and metallacarboranes are particularly interesting, and there is a need for basic studies on their interaction with biologically important molecules, such as proteins. Herein, we report studies on the interaction of selected boron clusters and their derivatives with serum albumin, the most abundant protein in mammalian blood. The interaction of boron clusters with albumin was examined by fluorescence quenching, circular dichroism, dynamic and static light scattering measurements and MALDI-TOF mass spectrometry. Our results showed that metallacarboranes have the strongest interaction with albumin among the tested clusters. The observed strength of boron cluster interactions with albumin decreases in order: metallacarboranes [M(C2B9H11)2]− > carboranes (C2B10H12) >> dodecaborate anion [B12H12]2−. Metallacarboranes first specifically interact with the binding cavity of albumin and then, with increasing compound concentrations, interact non-specifically with the protein surface. These findings can be of importance and are useful in the development of new bioactive compounds that contain boron clusters.

β-Cyclodextrin/protein conjugates as a innovative drug systems: synthesis and MS investigation

The design of proteins whose structure and function can be manipulated by binding with specific ligands such as cyclodextrins, has been of great interest in the field of protein engineering and also could be used as drug delivery systems in targeted cancer therapy (Loftsson and Duchêne, Int. J. Pharm. 329:1–11, 1; Loftsson et al., Expert. Opin. Drug Deliv. 2:335–351, 2). CD/proteins conjugates are synthesized using original high temperature method in which mono-6-O-formyl-β-CD reacts with two proteins: basic pancreatic trypsin inhibitor and lysozyme. The proposed synthesis method has a high reproducibility which makes it useful for pharmaceutical purposes. That method allows to obtain the conjugate without losing protein’s biological and enzymatic activity which will used in the reaction, and without violating the chemical structure of cyclodextrin molecules.

Synthesis of lysozyme-metallacarborane conjugates and the effect of boron cluster modification on protein structure and function.

Two complementary methods, “in solution” and “in solid state”, for the synthesis of lysozyme modified with metallacarborane (cobalt bis(dicarbollide), Co(C2B9H11)22−) were developed. As metallacarborane donors, oxonium adducts of cobalt bis(dicarbollide) and 1,4‐dioxane or tetrahydropyran were used. The physicochemical and biochemical properties of the obtained lysozyme–metallacarborane conjugates were studied for changes in secondary and tertiary structure, aggregation behavior, and biological activity. Only minor changes in primary, secondary, and tertiary protein structure were observed, caused by the single substitution of metallacarborane on lysozyme. However, the modification produced significant changes in lysozyme enzymatic activity and a tendency toward time‐ and temperature‐dependent aggregation.

Methotrexate and epirubicin conjugates as potential antitumor drugs

INTRODUCTION The use of hybrid molecules has become one of the most significant approaches in new cytotoxic drug design. This study describes synthesis and characterization of conjugates consisting of two well-known and characterized chemotherapeutic agents: methotrexate (MTX) and epirubicin (EPR). The synthesized conjugates combine two significant anticancer strategies: combinatory therapy and targeted therapy. These two drugs were chosen because they have different mechanisms of action, which can increase the anticancer effect of the obtained conjugates. MTX, which is a folic acid analog, has high cytotoxic properties and can serve as a targeting moiety that can reach folate receptors (FRs) overexpresing tumor cells. Combination of nonselective drugs such as EPR with MTX can increase the selectivity of the obtained conjugates, while maintaining the high cytotoxic properties. MATERIALS AND METHODS Conjugates were purified by RP-HPLC and the structure was investigated by MS and MS/MS methods. The effect of the conjugates on proliferation of LoVo, LoVo/Dx, MCF-7 and MV-4-11 human cancer cell lines was determined by SRB or MTT assay. RESULTS The conjugation reaction results in the formation of monosubstituted (α, γ) and disubstituted MTX derivatives. In vitro proliferation data demonstrate that the conjugates synthesized in our study show lower cytotoxic properties than both chemotherapeutics used alone. DISCUSSION Epirubicin cytotoxicity was not observed in obtained conjugates. Effective drugs release after internalization needs further investigation.

Design, Synthesis, and Evaluation of ω-(Isothiocyanato)alkylphosphinates and Phosphine Oxides as Antiproliferative Agents

A series of 21 novel, structurally diverse ω‐(isothiocyanato)alkylphosphinates and phosphine oxides (ITCs) were designed and synthesized in moderate to good yields. The synthesized compounds were evaluated for in vitro antiproliferative activity using LoVo and LoVo/DX cancer cell lines. The biological activity of the synthesized compounds was higher than that of natural isothiocyanates such as benzyl isothiocyanate or sulforaphane. The antiproliferative activity of selected ITCs was also tested on selected cancer cell lines: A549, MESSA and MESSA/DX‐5, HL60 and HL60MX2, BALB/3T3, and 4T1. These compounds were assessed for their mechanism of action as inducers of cell‐cycle arrest and apoptosis. Ethyl (6‐isothiocyanatohexyl)(phenyl)phosphinate (71) was tested in vivo on the 4T1 cell line and demonstrated moderate antitumor activity, similar to that benzyl isothiocyanate and cyclophosphamide.

Synthesis of β-cyclodextrin-lysozyme conjugates and their physicochemical and biochemical properties

Recently a great interest in the field of protein engineering and the design of innovative drug delivery systems employing specific ligands such as cyclodextrins is observed. The paper reports the solid state, thermal method for protein coupling with β-cyclodextrin and the physicochemical and biological properties of the obtained conjugates. The structure of the obtained conjugates was investigated via liquid chromatography-mass spectrometry, dynamic light scattering and circular dichroism analysis. The presented conjugates were biologically active and covalently bound β-cyclodextrin preserved the ability to form inclusion complexes with the model compound. This report demonstrates the great potential of cyclodextrin as a modifying unit that can be used to modulate the properties of therapeutic proteins, additionally giving such conjugates the possibility to transport many therapeutic substances in the form of inclusion complexes. In addition, the paper presents the potential of protein-cyclodextrin conjugates to construct innovative bioactive molecules for biological and medical applications.

Methods for preliminary determination of pemetrexed in macromolecular drug-carrier systems

Abstract Pemetrexed (PMX) is an antifolate drug utilized in the treatment of non-small cell lung cancer. For studies of potential macromolecular carriers for PMX, fast and precise methods were developed to determine the bound and free drug contained in investigated conjugate preparations. The analysis of the total amount of PMX in conjugates was based on absorption spectrophotometry. The linearity was found in the range of 4.697–46.97 μmol L−1 PMX. The limit of quantitation was 1.070 μmol L−1. The method for the analysis of unbound PMX was based on size-exclusion chromatography and detection at 225 nm. This method shows linear range of 2.230–223.0 μmol L−1. LOQ was 0.539 μmol L−1. The proposed methods can be used both for the characterization of the polysaccharide based conjugates of PMX and for the determination of conjugate drug release profiles.

Icosahedral boron clusters as modifying entities for biomolecules

ABSTRACT Introduction: Icosahedral boron clusters have unique properties useful in medicinal chemistry: rigidity, chemical stability, and three-dimensional aromaticity. Furthermore, these abiotic compounds have low toxicity and are stable in the biological environment. All these features ultimately give them the ability to interact with biological molecules in a different mode than organic compounds. Areas covered: In the present article, we aim to introduce boron clusters as a class of entities suitable for modifications of biomolecules to obtain a specific biological effect. We will focus on icosahedral boron clusters, as well as metallacarboranes, and their biological activity and interaction with the biological environment. Expert opinion: Boron clusters are suitable for altering structural and functional features of biomolecules and can be used in the development of new drugs and drug delivery systems. The high affinity of boron clusters, especially metallacarboranes, to albumin creates a new possibility to use them to optimize the pharmacokinetics of biologically active peptides. Boron clusters have high potential in biological and medicinal applications. Due to their peculiar properties, they can be used to optimize parameters critical for the biological activity of therapeutic substances and their affinity toward biological targets.