Redox Properties and in Vivo Magnetic Resonance Imaging of Cyclodextrin-Polynitroxides Contrast Agents.

This paper reports the synthesis, characterization and in vivo application of water-soluble supramolecular contrast agents (Mw: 5-5.6 kDa) for MRI obtained from ß-cyclodextrin functionalized with different kinds of nitroxide radicals, both with piperidine structure (CD2 and CD3) and with pyrrolidine structure (CD4 and CD5). As to the stability of the radicals in presence of ascorbic acid, CD4 and CD5 have low second order kinetic constants (≤0.05 M-1 s-1 ) compared to CD2 (3.5 M-1 s-1 ) and CD3 (0.73 M-1 s-1 ). Relaxivity (r1 ) measurements on compounds CD3-CD5 were carried out at different magnetic field strength (0.7, 3, 7 and 9.4 T). At 0.7 T, r1 values comprised between 1.5 mM-1 s-1 and 1.9 mM-1 s-1 were found while a significant reduction was observed at higher fields (r1 ≈0.6-0.9 mM-1 s-1 at 9.4 T). Tests in vitro on HEK293 human embryonic kidney cells, L929 mouse fibroblasts and U87 glioblastoma cells indicated that all compounds were non-cytotoxic at concentrations below 1 µmol mL-1 . MRI in vivo was carried out at 9.4 T on glioma-bearing rats using the compounds CD3-CD5. The experiments showed a good lowering of T1 relaxation in tumor with a retention of the contrast for at least 60 mins confirming improved stability also in vivo conditions.

3D Bioprinting of Pectin-Cellulose Nanofibers Multicomponent Bioinks.

Pectin has found extensive interest in biomedical applications, including wound dressing, drug delivery, and cancer targeting. However, the low viscosity of pectin solutions hinders their applications in 3D bioprinting. Here, we developed multicomponent bioinks prepared by combining pectin with TEMPO-oxidized cellulose nanofibers (TOCNFs) to optimize the inks' printability while ensuring stability of the printed hydrogels and simultaneously print viable cell-laden inks. First, we screened several combinations of pectin (1%, 1.5%, 2%, and 2.5% w/v) and TOCNFs (0%, 0.5%, 1%, and 1.5% w/v) by testing their rheological properties and printability. Addition of TOCNFs allowed increasing the inks' viscosity while maintaining shear thinning rheological response, and it allowed us to identify the optimal pectin concentration (2.5% w/v). We then selected the optimal TOCNFs concentration (1% w/v) by evaluating the viability of cells embedded in the ink and eventually optimized the writing speed to be used to print accurate 3D grid structures. Bioinks were prepared by embedding L929 fibroblast cells in the ink printed by optimized printing parameters. The printed scaffolds were stable in a physiological-like environment and characterized by an elastic modulus of E = 1.8 ± 0.2 kPa. Cells loaded in the ink and printed were viable (cell viability >80%) and their metabolic activity increased in time during the in vitro culture, showing the potential use of the developed bioinks for biofabrication and tissue engineering applications.

Cyclodextrin-Based Organic Radical Contrast Agents for in†vivo Imaging of Gliomas.

Cyclodextrins (CDs), a class of cyclic oligosaccharides formed by α-(1,4) linked glucopyranose units, were functionalized with (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) radicals to prepare water soluble supramolecular organic radical contrast agents (ORCAs) for the in vivo detection of glioma tumor in animal models. A first set of molecules (CDn1, n=6,7,8 is the number of both TEMPO and glucopyranose units) was studied by superconducting quantum interference devices (SQUID) magnetometry in order to define the role of the CD macrocycle on the effective magnetic moment (µeff ). The µeff value increased from 3.982 µB (CD61) to 4.522 µB (CD81) but was limited by intramolecular antiferromagnetic (AF) interactions. A set of water-soluble ORCAs (CDn8, n=6,7,8) was prepared by a sequence of thiol-ene and Cu(I)-catalyzed alkyne-azide "click" reactions. Their 1 H water relaxivities r1 of these ORCAs were between 0.739 mM-1 s-1 (CD68) to 1.047 mM-1 s-1 (CD88) in D2 O/H2 O 9 : 1 (v : v) at 300 K. One of them (CD78) was tested on glioma-bearing rats with reduced side effects and good relaxivity in vivo.

TEMPO-Nanocellulose/Ca2+ Hydrogels: Ibuprofen Drug Diffusion and In Vitro Cytocompatibility.

Stable hydrogels with tunable rheological properties were prepared by adding Ca2+ ions to aqueous dispersions of 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)-oxidized and ultra-sonicated cellulose nanofibers (TOUS-CNFs). The gelation occurred by interaction among polyvalent cations and the carboxylic units introduced on TOUS-CNFs during the oxidation process. Both dynamic viscosity values and pseudoplastic rheological behaviour increased by increasing the Ca2+ concentration, confirming the cross-linking action of the bivalent cation. The hydrogels were proved to be suitable controlled release systems by measuring the diffusion coefficient of a drug model (ibuprofen, IB) by high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. IB was used both as free molecule and as a 1:1 pre-formed complex with ß-cyclodextrin (IB/ß-CD), showing in this latter case a lower diffusion coefficient. Finally, the cytocompatibility of the TOUS-CNFs/Ca2+ hydrogels was demonstrated in vitro by indirect and direct tests conducted on a L929 murine fibroblast cell line, achieving a percentage number of viable cells after 7 days higher than 70%.

Molecular Dynamics and Hyperpolarization Performance of Deuterated ß-Cyclodextrins.

We discuss the temperature dependence of the 1H and 13C nuclear spin-lattice relaxation rate 1/ T1 and dynamic nuclear polarization (DNP) performance in ß-cyclodextrins with deuterated methyl groups. It is shown that 13C DNP-enhanced polarization is raised up to 10%. The temperature dependence of the buildup rate for nuclear spin polarization and of 1/ T1, below 4.2 K, is analyzed in the framework of the thermal mixing regime and the origin of the deviations from the theoretical behavior discussed. 13C 1/ T1 is determined at low temperature by the glassy dynamics and at high temperature by the rotational molecular motions of the deuterated methyl groups. Thanks to deuteration, relaxation times approaching 30 s are achieved at room temperature, making this material interesting for molecular imaging applications. The effect of molecular dynamics on the line width of the NMR spectra is also discussed.

Naked-Eye Heterogeneous Sensing of Fluoride Ions by Co-Polymeric Nanosponge Systems Comprising Aromatic-Imide-Functionalized Nanocellulose and Branched Polyethyleneimine.

Heterogeneous colorimetric sensors for fluoride ions were obtained by cross-linking TEMPO-oxidized cellulose nanofibers (TOCNF) with chemically modified branched polyethyleneimine 25 kDa (bPEI). Functionalization of bPEI primary amino groups with aromatic anhydrides led to the formation of the corresponding mono- and bis-imides on the grafted polymers (f-bPEI). A microwave-assisted procedure allowed the optimization of the synthetic protocol by reducing reaction time from 17 h to 30 minutes. Hydrogels obtained by mixing different ratios of TOCNF, bPEI and f-bPEI were lyophilized and thermally treated at about 100 °C to promote the formation of amide bonds between the amino groups of poly-cationic polymers and the carboxylic groups of cellulose nanofibers. This approach generated a series of cellulose nanosponges S1-S3 which were characterized by FT-IR and by solid state 13 C CPMAS NMR. These sponge materials can act as colorimetric sensors for the selective naked-eye recognition of fluoride ions over chloride, phosphate and acetate ions at concentrations of up to 0.05 M in DMSO. Moreover, when the sponges were functionalized with perylene tetracarboxylic diimide, successful naked-eye detection was achieved with only 0.02 % w/w of chromophore units per gram of material.

Environmentally Sustainable and Ecosafe Polysaccharide-Based Materials for Water Nano-Treatment: An Eco-Design Study.

Nanoremediation, which is the use of nanoparticles and nanomaterials for environmental remediation, is widely explored and proposed for preservation of ecosystems that suffer from the increase in human population, pollution, and urbanization. We herein report a critical analysis of nanotechnologies for water remediation by assessing their sustainability in terms of efficient removal of pollutants, appropriate methods for monitoring their effectiveness, and protocols for the evaluation of any potential environmental risks. Our purpose is to furnish fruitful guidelines for sustainable water management, able to promote nanoremediation also at European level. In this context, we describe new nanostructured polysaccharide-based materials obtained from renewable resources as alternative efficient and ecosafe solutions for water nano-treatment. We also provide eco-design indications to improve the sustainability of the production of these materials, based on life-cycle assessment methodology.

Microwave-assisted synthesis of TEMPO-labeled hydrogels traceable with MRI.

Polymer functionalization strategies have recently attracted considerable attention for several applications in biomaterials science. In particular, technological advancements in medical imaging have focused on the design of polymeric matrices to improve non-invasive approaches and diagnostic accuracy. In this scenario, the use of microwave irradiation of aqueous solutions containing appropriate combinations of polymers is gaining increasing interest in the synthesis of sterile hydrogels without using monomers, eliminating the need to remove unreacted species. In this study, we developed a method for the in situ fabrication of TEMPO-labeled hydrogels based on a one-pot microwave reaction that can then be tracked by magnetic resonance imaging (MRI) without using toxic compounds that could be hostile for the target tissue. Click chemistry was used to link TEMPO to the polymeric scaffold. In an in vivo model, the system was able to preserve its TEMPO paramagnetic activity up to 1 month after hydrogel injection, showing a clear detectable signal on T1-weighted MRI with a longitudinal relaxivity value of 0.29 mM s-1, comparable to a value of 0.31 mM s-1 characteristic of TEMPO application. The uncleavable conjugation between the contrast agent and the polymeric scaffold is a leading point to record these results: the use of TEMPO only physically entrapped in the polymeric scaffold did not show MRI traceability even after few hours. Moreover, the use of TEMPO-labeled hydrogels can also help to reduce the number of animals sacrificed being a longitudinal non-invasive technique.

Proton and Carbon-13 Dynamic Nuclear Polarization of Methylated ß-Cyclodextrins.

1H and 13C dynamic nuclear polarizations have been studied in 13C-enriched ß-cyclodextrins doped with (2,2,6,6-tetramethylpiperidin-1-yl)oxyl free radical. 1H and 13C polarizations raised above 7.5 and 7%, respectively, and for both nuclear species, the transfer of polarization from the electron spins appears to be consistent with a thermal mixing scenario for a concentration of 9 13C nuclei per molecule. When the concentration is increased to 21 13C nuclei per molecule, a decrease in the spin-lattice relaxation and polarization buildup rates is observed. This reduction is associated with the bottleneck effect induced by the decrease in the number of electron spins per nucleus when both the nuclear spin-lattice relaxation and the polarization occur through the electron non-Zeeman reservoir. 13C nuclear spin-lattice relaxation has been studied in the 1.8-340 K range, and the effects of internal molecular motions and of the free radicals on the relaxation are discussed. 13C hyperpolarization performances and room-temperature spin-lattice relaxation times show that these are promising materials for future biomedical applications.

Lipophilic N-Hydroxyphthalimide Catalysts for the Aerobic Oxidation of Cumene: Towards Solvent-Free Conditions and Back.

A new class of lipophilic N-hydroxyphthalimide (NHPI) catalysts designed for the aerobic oxidation of cumene in solvent-free conditions was synthesized and tested. The specific strategy proposed for the introduction of lipophilic tails on the NHPI moiety leads to lipophilic catalysts that-while completely preserving the activity of the precursor-allow the catalytic oxidation to be conducted in neat cumene, for the first time. The corresponding cumyl hydroperoxide is obtained in good yields (28-52 %) and with high selectivity (95-97 %), under mild conditions. Importantly, the presence of a polar solvent is no longer required to guarantee complete solubilization of the catalyst. On the other hand, the oxidation conducted in neat cumene reveals the unexpected necessity of using small amounts of acetonitrile to fully promote the hydrogen atom transfer process and prevent the catalyst from detrimental hydrogen-bond (HB) driven aggregation.

TEMPO-mediated oxidation of polysaccharides: An ongoing story.

The oxidation of natural polysaccharides by TEMPO has become by now an "old chemical reaction" which led to numerous studies mainly conducted on cellulose. This regioselective oxidation of primary alcohol groups of neutral polysaccharides has generated a new class of polyuronides not identified before in nature, even if the discovery of enzymes promoting an analogous oxidation has been more recently reported. Around the same time, the scientific community discovered the surprising biological and techno-functional properties of these anionic macromolecules with a high potential of application in numerous industrial fields. The objective of this review is to establish the state of the art of TEMPO chemistry applied to polysaccharide oxidation, its history, the resulting products, their applications and the associated modifying enzymes.

Dynamic Nuclear Polarization of ß-Cyclodextrin Macromolecules.

1H dynamic nuclear polarization and nuclear spin-lattice relaxation rates have been studied in amorphous complexes of ß-cyclodextrins doped with different concentrations of the TEMPO radical. Nuclear polarization increased up to 10% in the optimal case, with a behavior of the buildup rate (1/TPOL) and of the nuclear spin-lattice relaxation rate (1/T1n) consistent with a thermal mixing regime. The temperature dependence of 1/T1n and its increase with the radical concentration indicate a relaxation process arising from the modulation of the electron-nucleus coupling by the glassy dynamics. The high-temperature relaxation is driven by molecular motions, and 1/T1n was studied at room temperature in liquid solutions for dilution levels close to the ones typically used for in vivo studies.

Dynamics and interactions of ibuprofen in cyclodextrin nanosponges by solid-state NMR spectroscopy.

Two different formulations of cyclodextrin nanosponges (CDNS), obtained by polycondensation of ß-cyclodextrin with ethylenediaminetetraacetic acid dianhydride (EDTAn), were treated with aqueous solutions of ibuprofen sodium salt (IbuNa) affording hydrogels that, after lyophilisation, gave two solid CDNS-drug formulations. 1H fast MAS NMR and 13C CP-MAS NMR spectra showed that IbuNa was converted in situ into its acidic and dimeric form (IbuH) after freeze-drying. 13C CP-MAS NMR spectra also indicated that the structure of the nanosponge did not undergo changes upon drug loading compared to the unloaded system. However, the 13C NMR spectra collected under variable contact time cross-polarization (VCT-CP) conditions showed that the polymeric scaffold CDNS changed significantly its dynamic regime on passing from the empty CDNS to the drug-loaded CDNS, thus showing that the drug encapsulation can be seen as the formation of a real supramolecular aggregate rather than a conglomerate of two solid components. Finally, the structural features obtained from the different solid-state NMR approaches reported matched the information from powder X-ray diffraction profiles.

Mechanical and Drug Release Properties of Sponges from Cross-linked Cellulose Nanofibers.

All-organic porous sponges were obtained throughout the direct and solvent-free (oven 105 °C, time>6 h) crosslinking of TEMPO-oxidized cellulose nanofibers (TOCNF) with 25 kDa branched polyethyleneimine (bPEI) in the presence of different amounts of citric acid (CA) as co-crosslinker. The chemical and mechanical stability of these materials was provided by the formation of amide bonds between the carboxylic moieties of TOCNF and CA with the primary amines of bPEI. The mechanical properties were investigated under static and dynamic loads with both dry and wet samples. The materials had the interesting capability to recover their shape with reduced losses in mechanical resistance, while their Young's modulus progressively increased with the content of CA. In work toward developing possible applications of bPEI-TOCNF sponges in drug delivery, amoxicillin (AM) and ibuprofen (IB) were considered as model drugs. All materials showed very good performance in adsorbing both AM and IB (ca. 200 mg g-1 ) from methanol solution. In particular, an increased adsorption of IB was observed in parallel to the increase of citrate moieties in the samples. Moreover, samples crosslinked in presence of CA showed slower kinetic release in aqueous environments than materials obtained without CA.

Tuning structural parameters for the optimization of drug delivery performance of cyclodextrin-based nanosponges.

INTRODUCTION: In light of the recent development of new soft materials, nanostructured self-assembled systems have attracted attention in a variety of technological fields of high social impact. Cyclodextrin nanosponges (CDNS) represent a new and highly versatile class of cross-linked cyclodextrin (CD)-based nanoporous polymers. Their intriguing properties, including safety, biodegradability, negligible toxicity, marked swelling behavior, superior inclusion capability with respect to native CD, are the bases for potential for applications in drug delivery, tissue engineering and regenerative medicine. Areas covered: We report on the state-of-art concerning a detailed characterization of structural and dynamical features of CDNS explored by the combined use of different and complementary techniques, such as Fourier transform infrared absorption in attenuated total reflectance geometry (FTIR-ATR) and Raman spectroscopies, and high resolution magic angle spinning (HR-MAS) NMR spectroscopy. The ambitious objective is to furnish an exhaustive survey of the role played by hydrophobic and hydrophilic groups within the cross-linked network, in dry and swollen states, in determining the macroscopic functional features of CDNS. Expert opinion: The reported results may significantly contribute in the rational design and optimization of new stimuli-responsive systems exhibiting tunable inclusion/release properties, adapted to the therapeutic demands of pathology.

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study.

The chemical cross-linking of ß-cyclodextrin (ß-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as cyclodextrin nanosponges (CDNSEDTA). Two different preparations are described with 1:4 and 1:8 CD-EDTA molar ratios. The corresponding cross-linked polymers were contacted with 0.27 M aqueous solution of ibuprofen sodium salt (IP) leading to homogeneous, colorless, drug loaded hydrogels. The systems were characterized by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Pulsed field gradient spin echo (PGSE) NMR spectroscopy was used to determine the mean square displacement (MSD) of IP inside the polymeric gel at different observation times td. The data were further processed in order to study the time dependence of MSD: MSD = f(td). The proposed methodology is useful to characterize the different diffusion regimes that, in principle, the solute may experience inside the hydrogel, namely normal or anomalous diffusion. The full protocols including the polymer preparation and purification, the obtainment of drug-loaded hydrogels, the NMR sample preparation, the measurement of MSD by HR-MAS NMR spectroscopy and the final data processing to achieve the time dependence of MSD are here reported and discussed. The presented experiments represent a paradigmatic case and the data are discussed in terms of innovative approach to the characterization of the transport properties of an encapsulated guest within a polymeric host of potential application for drug delivery.

An aerogel obtained from chemo-enzymatically oxidized fenugreek galactomannans as a versatile delivery system.

We describe a new aerogel obtained from laccase-oxidized galactomannans of the leguminous plant fenugreek (Trigonella foenum-graecum) and suggest its potential practical use. Laccase/TEMPO oxidation of fenugreek in aqueous solution caused a viscosity increase of over 15-fold. A structured, elastic, stable hydrogel was generated, due to formation of carbonyl groups from primary OH of galactose side units and subsequent establishment of hemiacetalic bonds with available free hydroxyl groups. Upon lyophilization of this hydrogel, a water-insoluble aerogel was obtained (EOLFG), capable of uptaking aqueous or organic solvents over 20 times its own weight. The material was characterized by scanning electron microscopy, FT-IR, elemental analysis and (13)C CP-MAS NMR spectroscopy and its mechanical properties were investigated. To test the EOLFG as a delivery system, the anti-microbial enzyme lysozyme was used as model active principle. Lysozyme was added before or after formation of the aerogel, entrapped or absorbed in the gel, retained and released in active form, as proven by its hydrolytic glycosidase activity on lyophilized Micrococcus lysodeikticus cells wall peptidoglycans. This new biomaterial, composed of a chemo-enzymatically modified plant polysaccharide, might represent a versatile, biocompatible "delivery system" of active principles in food and non-food products.

Surface-Functionalization of Nanostructured Cellulose Aerogels by Solid State Eumelanin Coating.

Bioinspired aerogel functionalization by surface modification and coating is in high demand for biomedical and technological applications. In this paper, we report an expedient three-step entry to all-natural surface-functionalized nanostructured aerogels based on (a) TEMPO/NaClO promoted synthesis of cellulose nanofibers (TOCNF); (b) freeze-drying for aerogel preparation; and (c) surface coating with a eumelanin thin film by ammonia-induced solid state polymerization (AISSP) of 5,6-dihydroxyindole (DHI) or 5,6-dihydroxyindole-2-carboxylic acid (DHICA) previously deposited from an organic solution. Scanning electron microscopy showed uniform deposition of the dark eumelanin coating on the template surface without affecting porosity, whereas solid state (13)C NMR and electron paramagnetic resonance (EPR) spectroscopy confirmed the eumelanin-type character of the coatings. DHI melanin coating was found to confer to TOCNF templates a potent antioxidant activity, as tested by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays as well as strong dye adsorption capacity, as tested on methylene blue. The unprecedented combination of nanostructured cellulose and eumelanin thin films disclosed herein implements an original all-natural multifunctional aerogel biomaterial realized via an innovative coating methodology.

Functionalization of Cyclodextrins with N-Hydroxyphthalimide Moiety: A New Class of Supramolecular Pro-Oxidant Organocatalysts.

N-hydroxyphthalimide (NHPI) is an organocatalyst for free-radical processes able to promote the aerobic oxidation of a wide range of organic substrates. In particular, NHPI can catalyze the hydroperoxidation of polyunsaturated fatty acids (PUFA). This property could be of interest for biological applications. This work reports the synthesis of two ß-cyclodextrin derivatives (CD5 and CD6) having a different degree of methylation and bearing a NHPI moiety. These compounds, having different solubility in water, have been successfully tested for the hydroperoxidation of methyl linoleate, chosen as the PUFA model molecule.

Toward an understanding of the thermosensitive behaviour of pH-responsive hydrogels based on cyclodextrins.

The molecular mechanism responsible for the thermosensitive behaviour exhibited by pH-responsive cyclodextrin-based hydrogels is explored here with the twofold aim of clarifying some basic aspects of H-bond interactions in hydrogel phases and contributing to a future engineering of cyclodextrin hydrogels for targeted delivery and release of bioactive agents. The degree of H-bond association of water molecules entrapped in the gel network and the extent of intermolecular interactions involving the hydrophobic/hydrophilic moieties of the polymer matrix are probed by UV Raman and IR experiments, in order to address the question of how these different and complementary aspects combine to determine the pH-dependent thermal activation exhibited by these hydrogels. Complementary vibrational spectroscopies are conveniently employed in this study with the aim of safely disentangling the spectral response arising from the two main components of the hydrogel systems, i.e. the polymer matrix and water solvent. The experimental evidence suggests that the dominant effects in the mechanism of solvation of cyclodextrin-based hydrogels are due to the changes occurring, upon increasing of temperature, in the hydrophobicity character of specific chemical moieties of the polymer, as triggered by pH variations. The achievements of this work corroborate the potentiality of the UV Raman scattering technique, in combination with more conventional IR experiments, to provide a "molecular view" of complex macroscopic phenomena exhibited in hydrogel phases.