Amphiphilic chitosan-polyaminoxyls loaded with daunorubicin: Synthesis, antioxidant activity, and drug delivery capacity.

The binding of aminoxyls to polymers extends their potential use as antioxidants and EPR-reporting groups and opens up new horizons for tailoring new smart materials. In this work, we synthesized and characterized non-sulfated and N-sulfated water-soluble amphiphilic chitosans with a critical micelle concentration of 0.02-0.05 mg/mL that contain 13-18% of aminoglycosides bound with various aminoxyls. Chitosan-polyaminoxyls (CPAs) formed micelles with hydrodynamic radii Rh of ca. 100 nm. The EPR spectra of CPAs were found to depend on the rigidity of the aminoxyl-polymer bond and structural changes caused by sulfation. CPAs demonstrated antioxidant capacity/activity in three tests against reactive oxygen species (ROS) of various nature. The charge of micelles and structure of aminoxyls significantly affected their antioxidant properties. CPAs were low toxic against tumor (HepG2, HeLa, A-172) and non-cancerous (Vero) cells (IC50 > 0.8 mM of aminoglycosides). Sulfated CPAs showed better water solubility and the ability of binding and retaining the anti-tumor antibiotic daunorubicin (DAU). DAU-loaded micelles of CPAs (CPAs-DAU) demonstrated a 1.5-4-fold potentiation of DAU cytotoxicity against several cell lines. CPAs-DAU micelles were found to affect the cell cycle in a manner markedly different from that of free DAU. Our results demonstrated the ability of CPAs to act as bioactive drug delivery vehicles.

In Vitro Nephrotoxicity Studies of Established and Experimental Platinum-Based Compounds.

Cisplatin is one of the most commonly used drugs for the treatment of various solid cancers. However, its efficacy is restricted by severe side effects, especially dose-limiting nephrotoxicity. New platinum-based compounds are designed to overcome this limitation. Previous investigations showed that the platinum(IV)-nitroxyl complex PN149 is highly cytotoxic in various tumor cell lines. In the present study, investigations with PN149 were extended to normal human kidney tubule epithelia. Coincident with higher intracellular platinum accumulation, the cytotoxicity of PN149 in the proximal tubule epithelial cell line ciPTEC was more pronounced compared to the established platinum chemotherapeutics cisplatin, carboplatin and oxaliplatin. Quantitative gene expression profiling revealed the induction of ROS-inducible and anti-oxidative genes, suggesting an oxidative stress response by PN149. However, in contrast to cisplatin, no pro-inflammatory response was observed. Genes coding for distinct DNA damage response factors and genes related to apoptosis were up-regulated, indicating the activation of the DNA damage response system and induction of the apoptotic cascade by PN149. Altogether, a comparable transcriptional response was observed for PN149 and the platinum chemotherapeutics. However, the lack of inflammatory activity, which is a possible cause contributing to toxicity in human renal proximal tubule epithelia, might indicate the reduced nephrotoxic potential of PN149.

Oxygen Atom Transfer in the Oxidation of Dimethyl Sulfoxide by Oxoammonium Cations.

Cyclic oxoammonium salts and DMSO are known as important reagents for their diverse and unique reactivity. In the present work, we have studied the reaction of six- and five-membered oxoammonium salts with DMSO. The reaction includes ∼100% selective transfer of the O atom from the >N+═O group to the S atom of DMSO and structural rearrangement of the remaining cationic framework, leading to the formation of hydrolytically unstable iminium salts. The logarithms of the bimolecular rate constants k of the reaction correlated linearly with the reduction potentials E>N+═O/>N-O•, a relationship known for other electrophile-nucleophile combinations. The kinetic data and results of the DFT calculations allow for the suggestion that the studied process proceeds via the prereactive charge-transfer complex >N+═O···S (O)Me2 and its direct concerted rearrangement to the iminium salts. An alternative mechanism that includes intermediate steps with discrete nitrenium cations can be ruled out on the basis of product analysis and DFT computations. The obtained results allow a deeper understanding of the redox chemistry of a pair of nitroxide radicals-oxoammonium cations.

Activity profile of the cisplatin analogue PN149 in different tumor cell lines.

The efficacy of the anticancer drug cisplatin is restricted by tumor cell resistance and occurrence of severe side effects. One strategy to overcome these limitations is the development of new, improved platinum drugs. Previous investigations showed that platinum(IV)-nitroxyl complexes are able to circumvent cisplatin resistance in bladder cancer cells. In the present study the mode of action of the platinum(IV)-nitroxyl complex PN149 was investigated in the bladder cancer cell line RT112 and the renal cell carcinoma cell line A498 on the molecular and cellular level. Gene expression analysis showed that PN149 induced genes related to DNA damage response (RRM2B, GADD45A), cell cycle regulation (CDKN1A, PLK3, PPM1D) as well as those coding for the pro-apoptotic factors PUMA and Noxa. These findings on the transcriptional level were confirmed on the functional level revealing that PN149 treatment increased levels of p53 and resulted in cell cycle arrest and drug-induced cytotoxicity via induction of apoptosis. Regarding the expression of oxidative-stress sensitive genes, PN149 induced FTH1, GCLC, HMOX1 and TXNRD1 but relevant effects were restricted to RT112 cells treated with 50 µM. The pro-inflammatory IL-8 was induced by PN149 in RT112 but not A498 cells indicating a cell-type specific activation. Taken together, PN149 possessed promising activity in different tumor cell lines rendering it an interesting alternative to cisplatin in chemotherapy.

Platinum(IV)-nitroxyl complexes as possible candidates to circumvent cisplatin resistance in RT112 bladder cancer cells.

The therapeutic efficacy of the anticancer drug cisplatin is limited by the development of resistance. We therefore investigated newly synthesized platinum-nitroxyl complexes (PNCs) for their potential to circumvent cisplatin resistance. The complexes used were PNCs with bivalent cis-PtII(R·NH2)(NH3)Cl2 and cis-PtII(DAPO)Ox and four-valent platinum cis,trans,cis-PtIV(R·NH2)(NH3)(OR)2Cl2 and cis,trans,cis-PtIV(DAPO)(OR)2Ox, where R· are TEMPO or proxyl nitroxyl radicals, DAPO is trans-3,4-diamino-2,2,6,6-tetramethylpiperidine-1-oxyl, and OR and Ox are carboxylato and oxalato ligands, respectively. The complexes were characterized by spectroscopic methods, HPLC, log P ow data and elemental analysis. We studied intracellular platinum accumulation, DNA platination and cytotoxicity upon treatment with the PNCs in a model system of the bladder cancer cell line RT112 and its cisplatin-resistant subline RT112-CP. Platinum accumulation and DNA platination were similar in RT112 and RT112-CP cells for both bivalent and four-valent PNCs, in contrast to cisplatin for which a reduction in intracellular accumulation and DNA platination was observed in the resistant subline. The PNCs were found to platinate DNA in relation to the length of their axial RO-ligands. Furthermore, the PNCs were increasingly toxic in relation to the elongation of their axial RO-ligands, with similar toxicities in RT112 and its cisplatin-resistant subline. Using a cell-free assay, we observed induction of oxidative DNA damage by cisplatin but not PNCs suggesting that cisplatin exerts its toxic action by platination and oxidative DNA damage, while cells treated with PNCs are protected against oxidatively induced lesions. Altogether, our study suggests that PNCs may provide a more effective treatment for tumors which have developed resistance toward cisplatin.

Heparin-polynitroxides: synthesis and preliminary evaluation as cardiovascular EPR/MR imaging probes and extracellular space-targeted antioxidants.

We report here the synthesis of heparin-polynitroxide derivatives (HPNs) in which nitroxide moieties are linked either to uronic acid or glycosamine residues of the heparin macromolecule. HPNs have low anticoagulant activity, possess superoxide scavenging properties, bind to the vascular endothelium/extra-cellular matrix and can be detected by EPR and MRI techniques. As the vascular wall-targeted redox-active paramagnetic compounds, HPNs may have both diagnostic (molecular MRI) and therapeutic (ecSOD mimics) applications.

Spin-labeled heparins as polarizing agents for dynamic nuclear polarization.

A potentially biocompatible class of spin-labeled macromolecules, spin-labeled (SL) heparins, and their use as nuclear magnetic resonance (NMR) signal enhancers are introduced. The signal enhancement is achieved through Overhauser-type dynamic nuclear polarization (DNP). All presented SL-heparins show high (1)H DNP enhancement factors up to E=-110, which validates that effectively more than one hyperfine line can be saturated even for spin-labeled polarizing agents. The parameters for the Overhauser-type DNP are determined and discussed. A striking result is that for spin-labeled heparins, the off-resonant electron paramagnetic resonance (EPR) hyperfine lines contribute a non-negligible part to the total saturation, even in the absence of Heisenberg spin exchange (HSE) and electron spin-nuclear spin relaxation (T(1ne)). As a result, we conclude that one can optimize the use of, for example, biomacromolecules for DNP, for which only small sample amounts are available, by using heterogeneously distributed radicals attached to the molecule.