Ester Chiessi

Poly(vinyl alcohol) as versatile biomaterial for potential biomedical applications

In this paper, we present some new case examples where the chemical versatility of poly (vinyl alcohol) (PVA) can be used for potential biomedical applications. PVA, the polymeric material used for designing new nanostructured devices, is water soluble, biocompatible and has excellent physical properties. We point out the possibility of obtaining wall-to-wall chemical hydrogels as well as microgels without diminishing the biocompatibility available in the starting PVA material. Injectability is another important factor to take into account in controlled drug delivery for gene therapy. In this respect, in this paper, established and more innovative methods are prospected in order to obtain particles with dimensions suitable for these applications.

STRUCTURAL FLEXIBILITY MODULATES THE ACTIVITY OF HUMAN GLUTATHIONE TRANSFERASE P1-1 : ROLE OF HELIX 2 FLEXIBILITY IN THE CATALYTIC MECHANISM

Presteady-state and steady-state kinetic studies performed on human glutathione transferase P1-1 (EC) with 1-chloro-2,4-dinitrobenzene as co-substrate indicate that the rate-determining step is a physical event that occurs after binding of the two substrates and before the σ-complex formation. It may be a structural transition involving the ternary complex. This event can be related to diffusion-controlled motions of protein portions as k°cat/kcat linearly increases by raising the relative viscosity of the solution. Similar viscosity dependence has been observed for KGSHm, while KCDNBm is independent. No change of the enzyme structure by viscosogen has been found by circular dichroism analysis. Thus, kcat and KGSHm seem to be related to the frequency and extent of enzyme structural motions modulated by viscosity. Interestingly, the reactivity of Cys-47 which can act as a probe for the flexibility of helix 2 is also modulated by viscosity. Its viscosity dependence parallels that observed for kcat and KGSHm, thereby suggesting a possible correlation between kcat, KGSHm, and diffusion-controlled motion of helix 2. The viscosity effect on the kinetic parameters of C47S and C47S/C101S mutants confirms the involvement of helix 2 motions in the modulation of KGSHm, whereas a similar role on kcat cannot be ascertained unequivocally. The flexibility of helix 2 modulates also the homotropic behavior of GSH in these mutants. Furthermore, fluorescence experiments support a structural motion of about 4 Å occurring between helix 2 and helix 4 when GSH binds to the G-site.

Novel PVA-based hydrogel microparticles for doxorubicin delivery.

Micro- and nanoparticles are considered suitable drug delivery systems for their unique features, such as a large surface to volume ratio, and for the possibility to tune their size and hydrophobicity. A polymer/polymer/water emulsion method was used for producing a chemically cross-linked hydrogel made of poly(vinyl alcohol) and of poly(methacrylate) moieties. Mesoscopic investigation of the microparticles was accomplished by laser scanning confocal microscopy. Dynamics of confined water within the gel meshes was studied by quasi-elastic incoherent neutron scattering. Succinoylation of these particles allowed an efficient loading with a maximum doxorubicin payload of about 50% (w/w) of dry microparticles. To evaluate the potentials of such a microdevice for drug delivery, LoVo colon cancer cells have been exposed to doxorubicin loaded microparticles to study the in vitro efficiency of the payload release and the consequent cytotoxic effect.

Polymer Shelled Microparticles for a Targeted Doxorubicin Delivery in Cancer Therapy

Targeting is a main feature supporting any controlled drug delivery modality. Recently we developed poly(vinyl alcohol), PVA, based microbubbles as a potential new ultrasound contrast agent featuring an efficient ultrasound backscattering and a good shelf stability. The chemical versatility of the polymeric surface of this device offers a vast variety of coupling modalities useful for coating and specific targeting. We have designed a conjugation strategy on PVA shelled microbubbles to enable the localization and the drug delivery on tumor cells by modifying the surface of this polymeric ultrasound contrast agent (UCA) with oxidized hyaluronic acid (HAox). After the conversion of the microbubbles into microcapsules, the kinetics of the release of doxorubicin, a well-known antitumor drug, from uncoated and HAox-coated PVA microbubbles and microcapsules was investigated. Cytocompatibility and bioadhesive properties of the HA-modified microparticles were then tested on the HT-29 tumor cell line. Cytotoxicity to HT-29 tumor cells of microcapsules after loading with doxorubicin was studied, evidencing the efficacy of the HAox coating for the delivery of the drug to cells. These features are a prerequisite for a theranostic, that is, diagnostic and therapeutic, use of polymer-based UCAs.

Structural flexibility modulates the activity of human glutathione transferase P1-1. Influence of a poor co-substrate on dynamics and kinetics of human glutathione transferase.

Presteady-state and steady-state kinetics of human glutathione transferase P1-1 (EC) have been studied at pH 5.0 by using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a poor co-substrate for this isoenzyme. Steady-state kinetics fits well with the simplest rapid equilibrium random sequential bi-bi mechanism and reveals a strong intrasubunit synergistic modulation between the GSH-binding site (G-site) and the hydrophobic binding site for the co-substrate (H-site); the affinity of the G-site for GSH increases about 30 times at saturating co-substrate and vice versa. Presteady-state experiments and thermodynamic data indicate that the rate-limiting step is a physical event and, probably, a structural transition of the ternary complex. Similar to that observed with 1-chloro-2,4-dinitrobenzene (Ricci, G., Caccuri, A. M., Lo Bello, M., Rosato, N., Mei, G., Nicotra, M., Chiessi, E., Mazzetti, A. P., and Federici, G. (1996) J. Biol. Chem. 271, 16187-16192), this event may be related to the frequency of enzyme motions. The observed low, viscosity-independent kcat value suggests that these motions are slow and diffusion-independent for an increased internal viscosity. In fact, molecular modeling suggests that the hydroxyl group of Tyr-108, which resides in helix 4, may be in hydrogen bonding distance of the oxygen atom of this new substrate, thus yielding a less flexible H-site. This effect might be transmitted to the G-site via helix 4. In addition, a new homotropic behavior exhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole is found in Cys-47 mutants revealing a structural intersubunit communication between the two H-sites.

Copper complexes immobilized to chitosan.

Polymeric ligands, such as 2-substituted pentanedioic acid (2), 2-substituted propanoic acid (3), and deoxylactit-1-yl (4) derivatives of chitosan (1), were used to prepare copper complexes that are widely soluble in aqueous solution. EPR results (100 K) show that all association complexes basically have a tetragonal symmetry. Visible CD spectra suggest, however, that the order of increasing departure from this geometry is Cu-(1) approximately Cu-(3) less than Cu-(2) less than or equal to Cu-(4), the lack of sterically constraining side-chains in (1) and (3) allowing a more symmetric arrangement of ligands around the central metal ion. Results on the catalytic activity of the association complexes for air oxidation of catechol derivatives are also presented.

Gel-Like Structure of a Hexadecyl Derivative of Hyaluronic Acid for the Treatment of Osteoarthritis

Hyaluronic acid is a polysaccharide with viscoelastic and mechanical properties that are crucial for the normal functioning of osteoarticular junctions. It is demonstrated that introduction of a hexadecyl side chain into HA yields an injectable polysaccharide capable of forming physical hydrogels, which are stable at very low polymer concentrations, whereas native hyaluronic acid forms viscous solutions at concentrations that are ten times higher. Characterization of this system showed that the driving force for its gel-like behavior is the occurrence of hydrophobic interactions involving aliphatic side chains, despite the low degree of substitution, as confirmed by molecular dynamics simulations of HYADD4 and HA hydrogels.

Conformation and Dynamics of Poly(N-isopropyl acrylamide) Trimers in Water: A Molecular Dynamics and Metadynamics Simulation Study

Conformational features, structure, and dynamic properties of the trimer of poly(N-isopropyl acrylamide), (NIPAAm)(3), in aqueous solution at 293 and 323 K were investigated by a double simulation approach. The free energy behavior as a function of backbone conformation was obtained by metadynamics-umbrella sampling simulations. The structural characteristics, the intramolecular and water hydrogen bonding, and the torsional dynamics were analyzed by molecular dynamics simulations. The four stereoisomers of (NIPAAm)(3), representing syndiotactic, isotactic, and atactic sequences, were studied to highlight the tacticity effect on the system properties. The simulation results indicate that the experimentally observed lower hydrophilicity of isotactic poly(N-isopropyl acrylamide), in comparison with the syndiotactic one, is related to a lower conformational entropy. The atactic stereoisomers display the highest intramolecular hydrogen bond capability, at both studied temperatures, due to formation of hydrogen bonds between external amide groups. The mobility of the backbone in the syndiotactic trimer is more homogeneous than in other stereoisomers. The temperature increase was found mainly to affect the conformation of N-isopropyl amide side chains, and a structural rearrangement was observed for the atactic stereoisomers, in agreement with their experimental solution behavior. Simulation results are discussed in relation with available experimental data on solution properties and reactivity of poly(N-isopropyl acrylamide).

Toward Modeling Thermoresponsive Polymer Networks: A Molecular Dynamics Simulation Study of N-Isopropyl Acrylamide Co-Oligomers

Polymer microgels of poly(vinyl alcohol)/poly(methacrylate-co-N-isopropyl acrylamide) showed a thermoresponsive behavior, suitable for application in drug delivery (Biomacromolecules 2009, 10, 1589). In this work molecular dynamics (MD) methods were used to explain which structural aspects are determining for thermoresponsivity and how water properties in the hydrogel are influenced. Two topologically different models of the junction domain in the hydrogel at the experimental hydration degree were studied at 293 and 323 K, below and above the transition temperature. MD simulations of the corresponding oligomers of poly(N-isopropyl acrylamide) were also performed for a comparison. Simulation results provided an atomic detailed description of the temperature induced modifications in the microgel network and of water dynamics, in agreement with available experimental findings.

Structure and Dynamics of a Thermoresponsive Microgel around Its Volume Phase Transition Temperature

Sustained drug delivery requires the use of multifunctional devices with enhanced properties. These properties include responsiveness to external stimuli (such as temperature, pH, ionic strength), ability to deliver suitably designed ligands to specific receptors, enhanced bioadhesion to cells, and cytocompatibility. Microgels represent one of such multifunctional drug delivery devices. Recently, we described the fabrication of a stable colloidal aqueous suspension of cytocompatible microgel spheres based on a poly(vinyl alcohol)/poly(methacrylate-co-N-isopropylacrylamide) network ( Ghugare, S. Mozetic, P. Paradossi, G. Biomacromolecules 2009 , 10 , 1589 ). These microgel spheres undergo an entropy-driven volume phase transition around the physiological temperature, this phase transition being driven by the incorporation of NiPAAm residues in the network. In that study, the microgel was loaded with the anticancer drug doxorubicin. As the microgel shrank, a marked increase in the amount of doxorubicin released was noted. Indeed, dynamic light scattering measurements showed the diameter reduction to be about 50%. In the present paper, we focus on some fundamental issues regarding modifications of the hydrogel architecture at a nanoscopic level as well as of the diffusive behavior of water associated with the polymer network around the volume phase transition temperature (VPTT). Sieving and size exclusion effects were studied by laser scanning confocal microscopy with the microgel exposed to fluorescent probes with different molecular weights. Confocal microscopy observations at room temperature and at 40 degrees C (i.e., below and above the VPTT) provided an evaluation of the variation of the average pore size (from 5 nm to less than 3 nm). Using quasielastic neutron scattering (QENS) with the IRIS spectrometer at ISIS, UK, the diffusive behavior of water molecules closely associated to the polymer network around the VPTT was investigated. A clear change in the values of diffusion coefficient of bound water was observed at the transition temperature. In addition, the local dynamics of the polymer itself was probed using the QENS spectrometer SPHERES at FRM II, Germany. For this study, the microgel was swollen in D(2)O. An average characteristic distance of about 5 A for the localized chain motions was evaluated from the elastic incoherent structure factor (EISF) and from the Q-dependence of the Lorentzian width.