István Bányai

RGD peptide-modified multifunctional dendrimer platform for drug encapsulation and targeted inhibition of cancer cells

Development of multifunctional nanoscale drug-delivery systems for targeted cancer therapy still remains a great challenge. Here, we report the synthesis of cyclic arginine-glycine-aspartic acid (RGD) peptide-conjugated generation 5 (G5) poly(amidoamine) dendrimers for anticancer drug encapsulation and targeted therapy of cancer cells overexpressing αvβ3 integrins. In this study, amine-terminated G5 dendrimers were used as a platform to be sequentially modified with fluorescein isothiocyanate (FI) via a thiourea linkage and RGD peptide via a polyethylene glycol (PEG) spacer, followed by acetylation of the remaining dendrimer terminal amines. The developed multifunctional dendrimer platform (G5.NHAc-FI-PEG-RGD) was then used to encapsulate an anticancer drug doxorubicin (DOX). We show that approximately six DOX molecules are able to be encapsulated within each dendrimer platform. The formed complexes are water-soluble, stable, and able to release DOX in a sustained manner. One- and two-dimensional NMR techniques were applied to investigate the interaction between dendrimers and DOX, and the impact of the environmental pH on the release rate of DOX from the dendrimer/DOX complexes was also explored. Furthermore, cell biological studies demonstrate that the encapsulation of DOX within the G5.NHAc-FI-PEG-RGD dendrimers does not compromise the anticancer activity of DOX and that the therapeutic efficacy of the dendrimer/DOX complexes is solely related to the encapsulated DOX drug. Importantly, thanks to the role played by RGD-mediated targeting, the developed dendrimer/drug complexes are able to specifically target αvβ3 integrin-overexpressing cancer cells and display specific therapeutic efficacy to the target cells. The developed RGD peptide-targeted multifunctional dendrimers may thus be used as a versatile platform for targeted therapy of different types of αvβ3 integrin-overexpressing cancer cells.

Fabrication and morphology control of electrospun poly(γ-glutamic acid) nanofibers for biomedical applications

We report the fabrication of water-stable electrospun γ-polyglutamic acid (γ-PGA) nanofibers with morphology control for biomedical applications. In this study, the processing variables including polymer concentration, flow rate, applied voltage, collection distance, and ambient humidity were systematically optimized to generate uniform γ-PGA nanofibers with a smooth morphology. By changing the trifluoroacetic acid concentration in the electrospinning solution, the diameter of the γ-PGA nanofibers can be controlled within the range of 186-603 nm. To render the γ-PGA nanofibers with good water stability, cystamine was employed as a crosslinking agent to amidate the carboxyl groups of γ-PGA. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay in conjunction of cell morphology observation reveals that the obtained γ-PGA nanofibers have an excellent biocompatibility to promote the cell adhesion and proliferation. We anticipate that the fabricated electrospun γ-PGA nanofibers with controllable morphology and good water stability may find extensive applications in future development of tissue engineering scaffold materials, drug delivery systems, environmental remediation, and sensing.

Time‐Dependent Solution Speciation of the AlIII−Citrate System: Potentiometric and NMR Studies

Time-dependent potentiometric and NMR spectroscopic measurements were carried out in the AlIII−citric acid system in an equimolar solution and at an excess of ligand in order to monitor the changes in speciation as a function of time. In fresh solutions, the mononuclear 1:1 species [AlLH]+, [AlL], [AlLH−1]− and [AlLH−2]2− and the 1:2 complexes [AlL2]3−, [AlL2H−1]4− and [AlL2H−2]5− are formed. In agreement with earlier findings (Inorg. Chem. 1988, 27, 2565), these complexes are converted to a large extent into a thermodynamically more stable trinuclear species as the solutions age. Depending on the composition of the initially formed mononuclear species, the formation of the trinuclear species is accompanied either by the consumption or by the liberation of hydroxide ions. NMR spectroscopy was used to confirm the considerable time dependence of the species distribution. The 1H, 27Al and 13C NMR spectra indicated that at equilibrium the trinuclear species [Al3(LH−1)3(OH)]4− predominates in the pH range 4−7. Oligomerization of the mononuclear species takes place through at least one intermediate complex. At equilibrium, the ligand-exchange reactions between the trinuclear species, the mononuclear species and free citrate are slow on the NMR time scale.

An NMR and DFT Investigation on the Conformational Properties of Lanthanide(III) 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetate Analogues Containing Methylenephosphonate Pendant Arms

The conformational properties of lanthanide(III) complexes with the mono- and biphosphonate analogues of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA) are investigated by means of density functional theory (DFT) calculations and NMR spectroscopy. Geometry optimizations performed at the B3LYP/6-31G(d) level and using a 46 + 4f(n) effective core potential for lanthanides provide two energy minima corresponding to the square-antiprismatic (SAP) and twisted square-antiprismatic (TSAP) geometries. Our calculations give relative free energies between the SAP and TSAP isomers in fairly good agreement with the experimental values. The SAP isomer presents the highest binding energy of the ligand to the metal ion, which further increases with respect to that of the TSAP isomer across the lanthanide series as the charge density of the metal ion increases. The stabilization of the TSAP isomer upon substitution of the acetate arms of DOTA by methylenephosphonate ones is attributed to the higher steric demand of the phosphonate groups and the higher strain of the ligand in the SAP isomer. A (1)H NMR band-shape analysis performed on the [Ln(DO2A2P)](3-) (Ln = La and Lu) complexes provided the activation parameters for enantiomerization of the TSAP form of the complexes. The TSAP isomerization process was also investigated by using DFT calculations on the [Lu(DOTA)](-) and [Ln(DO2A2P)](3-) (Ln = La and Lu) systems. Our results confirm that enantiomerization requires both rotation of the pendant arms and inversion of the four five-membered chelate rings formed upon coordination of the macrocyclic unit. According to our calculations, the arm rotation pathway in [Lu(DOTA)](-) is a one-step process involving the simultaneous rotation of the four acetate arms, while in the DO2A2P analogue, the arm-rotation process is a multistep path involving the stepwise rotation of each of the four pendant arms. The calculated activation free energies are in reasonably good agreement with the experimental data. A comparison of the experimental (13)C NMR shifts of [Ln(DO2A2P)](3-) (Ln = La and Lu) complexes and those calculated by using the GIAO method confirms that the major isomer observed in solution for these complexes corresponds to the TSAP isomer.

Impact of dendrimer surface functional groups on the release of doxorubicin from dendrimer carriers

Generation 5 (G5) poly(amidoamine) dendrimers with acetyl (G5.NHAc), glycidol hydroxyl (G5.NGlyOH), and succinamic acid (G5.SAH) terminal groups were used to physically encapsulate an anticancer drug doxorubicin (DOX). Both UV-vis spectroscopy and multiple NMR techniques including one-dimensional NMR and two-dimensional NMR were applied to investigate the interactions between different dendrimers and DOX. The influence of the surface functional groups of G5 dendrimers on the DOX encapsulation, release kinetics, and cancer cell inhibition effect was investigated. We show that all three types of dendrimers are able to effectively encapsulate DOX and display therapeutic inhibition effect to cancer cells, which is solely associated with the loaded DOX. The relatively stronger interactions of G5.NHAc or G5.NGlyOH dendrimers with DOX than that of G5.SAH dendrimers with DOX demonstrated by NMR techniques correlate well with the slow release rate of DOX from G5.NHAc/DOX or G5.NGlyOH/DOX complexes. In contrast, the demonstrated weak interaction between G5.SAH and DOX causes a fast release of DOX, suggesting that the G5.SAH/DOX complex may not be a proper option for further in vivo research. Our findings suggest that the dendrimer surface functional groups are crucial for further design of multifunctional dendrimer-based drug delivery systems for various biomedical applications.

INTERACTIONS OF AL(III) WITH PHOSPHORYLATED AMINO ACIDS

Abstract In order to assess the Al(III)-binding abilities of phosphorylated proteins and peptides, the interactions of Al(III) with the building blocks O-phosphoserine (PSer) and O-phosphotyrosine (PTyr) were studied. pH-metric and 31 P NMR measurements were carried out to determine the stoichiometries and stability constants of the complexes formed, and to establish the most probable binding sites of the metal ion. PSer was found to bind Al(III) in a monodentate manner at the phosphate moiety, and in a tridentate manner with the simultaneous coordination of all donor groups. PTyr binds Al(III) only at the separate phosphate function. Citric acid (Cit) proved to be a stronger Al(III) binder at the physiological pH, though, it was able to displace PSer only in a rather slow process through formation of the trinuclear species Al 3 (Cit) 3 (OH). The results were used to evaluate the potential role of Al(III) in inducing the formation of neurofilamentous aggregates in neurological disorders.

Slow dynamics of aluminium-citrate complexes studied by 1H- and 13C-NMR spectroscopy

Abstract Inter- and intra-molecular exchange reactions of the major Al(III)-citrate (Cit) complexes have been studied under equilibrium conditions in aqueous solution by 1H- and 13C-NMR using band shape analysis and magnetization transfer methods. Rate equations and activation parameters have been evaluated from concentration, pH and temperature dependent studies. The lability with respect to ligand exchange was: Al ( Cit ) 2 3− (k 298 =1.1±0.1 s −1 )> Al 3 ( H −1 Cit ) 3 ( OH ) 4 7− (Sy; k 298 =0.08±0.01 s −1 )≫ Al 3 ( H −1 Cit ) 3 ( OH ) 4− (As) . The variation in lability appears to be related to the different coordination modes of the citrate ligands in the complexes. The ligand exchange reactions show an Ia mechanism for Al(Cit)23− and Sy. These two complexes are fluxional (k 298 Sy =230 s −1 ) , the intra-molecular rearrangement of Sy goes through a bond rupture mechanism. As is neither fluxional nor labile for ligand exchange, kAs≤0.03 s−1 at 353 K.

Calcium binding of transglutaminases: A 43Ca NMR study combined with surface polarity analysis

Abstract Transglutaminases (TGases) form cross-links between glutamine and lysine side-chains of polypeptides in a Ca2+-dependent reaction. The structural basis of the Ca2+-effect is poorly defined. 43Ca NMR, surface polarity analysis combined with multiple sequence alignment and the construction of a new homology model of human tissue transglutaminase (tTGase) were used to obtain structural information about Ca2+ binding properties of factor XIII-A2, tTGase and TGase 3 (each of human origin). 43Ca NMR provided higher average dissociation constants titrating on a wide Ca2+-concentration scale than previous studies with equilibrium dialysis performed in shorter ranges. These results suggest the existence of low affinity Ca2+ binding sites on both FXIII-A and tTGase in addition to high affinity ones in accordance with our surface polarity analysis identifying high numbers of negatively charged clusters. Upon increasing the salt concentration or activating with thrombin, FXIII-A2 partially lost its original Ca2+ affinity; the NMR data suggested different mechanisms for the two activation processes. The NMR provided structural evidence of GTP-induced conformational changes on the tTGase molecule diminishing all of its Ca2+ binding sites. NMR data on the Ca2+ binding properties of the TGase 3 are presented here; it binds Ca2+ the most tightly, which is weakened after its proteolytic activation. The investigated TGases seem to have very symmetric Ca2+ binding sites and no EF-hand motifs.

1H- and 13C-NMR as tools to study aluminium coordination chemistry—aqueous Al(III)–citrate complexes

Abstract Based on the use thermodynamic and solid state structure data, 1H- and 13C-NMR is a very useful tool to understand the conformational and dynamic behavior of complexes containing organic ligands in solution. In this paper we describe shortly the possibilities of the assignation of the spectra by means of modern NMR techniques. From the assigned spectra the scalar and dipolar couplings make it possible to determine the orientation of the ligand around the metal ion and the distances between hydrogen atoms in space. Aluminium–citrate complexes are reviewed as examples. It is shown that with the armory of correlation NMR spectroscopy unique insight can be obtained in the behavior of Al–citrate species even if oligomers are present in the solution.

Glyphosate complexation to aluminium(III). An equilibrium and structural study in solution using potentiometry, multinuclear NMR, ATR-FTIR, ESI-MS and DFT calculations

The stoichiometries and stability constants of a series of Al(3+)-N-phosponomethyl glycine (PMG/H(3)L) complexes have been determined in acidic aqueous solution using a combination of precise potentiometric titration data, quantitative (27)Al and (31)P NMR spectra, ATR-FTIR spectrum and ESI-MS measurements (0.6M NaCl, 25 degrees C). Besides the mononuclear AlH(2)L(2+), Al(H(2)L)(HL), Al(HL)(2)(-) and Al(HL)L(2-), dimeric Al(2)(HL)L(+) and trinuclear Al(3)H(5)L(4)(2+) complexes have been postulated. (1)H and (31)P NMR data show that different isomers co-exist in solution and the isomerization reactions are slow on the (31)P NMR time scale. The geometries of monomeric and dimeric complexes likely double hydroxo bridged and double phosphonate bridged isomers have been optimized using DFT ab initio calculations starting from rational structural proposals. Energy calculations using the PCM solvation method also support the co-existence of isomers in solutions.