This study analyzes the physico-chemical properties of interpenetrated polymer networks (IPNs) and semi-IPN resulting from cross-linking chitosan with glutaraldehyde and alginate with Ca²⁺ cations, as a function of the order in which the cross-linking agents are added to the polymer mixture. Three physico-chemical methods were used to assess the differences between systems: rheology, IR spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy. While rheology and IR spectroscopy are commonly used to characterize gel materials, EPR spectroscopy is rarely used, but has the advantage of providing local information about the dynamics of a system. The rheological parameters, which describe the global behavior of the samples, show that semi-IPN systems have a weaker gel behavior and the order of introducing the cross-linker in the polymer systems plays a role. The IR spectra of samples resulting by adding only Ca²⁺ or Ca²⁺ as the first cross-linker are similar to that of the alginate gel, while the spectra of samples in which glutaraldehyde is firstly added resemble the chitosan gel spectrum. Using spin-labeled alginate and spin-labeled chitosan, we monitored the changes occurring in the dynamic of the spin labels due to the formation of IPN and semi-IPN. The results show that the order of adding the cross-linking agents influences the dynamic of the IPN network, and that the formation of the alginate network determines the characteristics of the entire IPN system. The EPR data were correlated with the rheological parameters and IR spectra of the analyzed samples.

The emergence and spread of drug-resistant Plasmodium falciparum parasites shed a serious concern on the worldwide control of malaria, the most important tropical disease in terms of mortality and morbidity. This situation has led us to consider the use of peptide-alkoxyamine derivatives as new antiplasmodial prodrugs that could potentially be efficient in the fight against resistant malaria parasites. Indeed, the peptide tag of the prodrug has been designed to be hydrolysed by parasite digestive proteases to afford highly labile alkoxyamines drugs, which spontaneously and instantaneously homolyse into two free radicals, one of which is expected to be active against P. falciparum. Since the parasite enzymes should trigger the production of the active drug in the parasite’s food vacuoles, our approach is summarized as “to dig its grave with its fork”. However, despite promising sub-micromolar IC₅₀ values in the classical chemosensitivity assay, more in-depth tests evidenced that the anti-parasite activity of these compounds could be due to their cytostatic activity rather than a truly anti-parasitic profile, demonstrating that the antiplasmodial activity cannot be based only on measuring antiproliferative activity. It is therefore imperative to distinguish, with appropriate tests, a genuinely parasiticidal activity from a cytostatic activity.

The expansion of drug resistant parasites sheds a serious concern on several neglected parasitic diseases. Our recent results on cancer led us to envision the use of peptide-alkoxyamines as a highly selective and efficient new drug against schistosome adult worms, the etiological agents of schistosomiasis. Indeed, the peptide tag of the hybrid compounds can be hydrolyzed by worm’s digestive enzymes to afford a highly labile alkoxyamine which homolyzes spontaneously and instantaneously into radicals—which are then used as a drug against Schistosome adult parasites. This approach is nicely summarized as digging their graves with their forks. Several hybrid peptide-alkoxyamines were prepared and clearly showed an activity: two of the tested compounds kill 50% of the parasites in two hours at a concentration of 100 µg/mL. Importantly, the peptide and alkoxyamine fragments that are unable to generate alkyl radicals display no activity. This strong evidence validates the proposed mechanism: a specific activation of the prodrugs by the parasite proteases leading to parasite death through in situ alkyl radical generation.

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Nitroreductase-mediated alkoxyamine activation: a selective approach for prodrugs.

The complexation of two diastereoisomers of a β-phosphorylated cyclic nitroxide radical (2,5-dimethyl-5-hydroxymethylene-2-diethoxyphosphonyl-pyrrolidin-N-oxyl) with cyclodextrins (β-CD and γ-CD) was investigated using electron paramagnetic resonance (EPR) spectroscopy and cyclic voltammetry. The two isomers have different EPR signatures due to the differences in their phosphorus and nitrogen hyperfine splitting constants. The cis stereoisomer (1c˙) promotes intramolecular hydrogen bonding, involving the HO group attached to the methylene group and O atom from the phosphorus moiety, while in the case of the trans stereoisomer (1t˙), this intramolecular bonding is not possible. The EPR spectra indicate the higher affinity of the 1t˙ isomer for γ-CD, highlighting the different EPR parameters of the free radical and complexed radical. The binding constants determined based on the EPR and cyclic voltammetry data show stronger affinity for γ-CD compared with β-CD, particularly for 1t˙. An increasing potassium chloride concentration enhances the stability of the complex linearly by modulating the solvation and electrostatic interactions, as indicated by the thermodynamic parameters obtained from EPR measurements. Electrochemical measurements demonstrate decreased diffusion coefficients and anodic peak shifts upon complexation, supporting the spectroscopic results. These findings highlight the important role of molecular stereochemistry and ionic strength in modulating host–guest interactions between cyclodextrins and nitroxides.