Sabato Fusco

Perspectives on: PEO-PPO-PEO Triblock Copolymers and their Biomedical Applications

Thermo-reversible polymeric gels represent an interesting class of materials that can be tailored for a wide range of applications. The triblock poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) based systems, PEO-PPO-PEO, show thermoreversible gelation around body temperature and, therefore, are particularly suitable for biomedical applications such as drug delivery, gene therapy and tissue engineering. The PEO-PPO-PEO tri-block copolymers have amphiphilic characteristics and self-assemble into micelles to form a variety of close packed structures. By varying the block composition (PEO/PPO ratio) and the molecular weight, it is possible to tailor the final properties of these systems to meet the specific application needs. In this report the thermodynamic basis of micellization of PEO-PPO-PEO systems is described. The factors influencing the micelles formation are discussed along with the methods used to investigate the micellization process and morphology as well as with the main applications of these systems in biomedical fields.

Shuttle‐Mediated Nanoparticle Delivery to the Blood–Brain Barrier

Many therapeutic drugs are excluded from entering the brain due to their lack of transport through the blood-brain barrier (BBB). The development of new strategies for enhancing drug delivery to the brain is of great importance in diagnostics and therapeutics of central nervous diseases. To overcome this problem, a viral fusion peptide (gH625) derived from the glycoprotein gH of Herpes simplex virus type 1 is developed, which possesses several advantages including high cell translocation potency, absence of toxicity of the peptide itself, and the feasibility as an efficient carrier for delivering therapeutics. Therefore, it is hypothesized that brain delivery of nanoparticles conjugated with gH625 should be efficiently enhanced. The surface of fluorescent aminated polystyrene nanoparticles (NPs) is functionalized with gH625 via a covalent binding procedure, and the NP uptake mechanism and permeation across in vitro BBB models are studied. At early incubation times, the uptake of NPs with gH625 by brain endothelial cells is greater than that of the NPs without the peptide, and their intracellular motion is mainly characterized by a random walk behavior. Most importantly, gH625 peptide decreases NP intracellular accumulation as large aggregates and enhances the NP BBB crossing. In summary, these results establish that surface functionalization with gH625 may change NP fate by providing a good strategy for the design of promising carriers to deliver drugs across the BBB for the treatment of brain diseases.

Structural and Mechanical Properties of UV-Photo-Cross-Linked Poly(N-vinyl-2-pyrrolidone) Hydrogels

Biocompatible poly( N-vinyl-2-pyrrolidone) (PVP) hydrogels have been produced by UV irradiation of aqueous polymer mixtures, using a high-pressure mercury lamp. The resulting materials have been characterized by a combination of experimental techniques, including rheology, small-angle neutron scattering (SANS), electron paramagnetic resonance (EPR), and pulsed gradient spin-echo nuclear magnetic resonance (PGSE-NMR), to put in evidence the relationship between the microstructural properties and the macrofunctional behavior of the gels. Viscoelastic measurements showed that UV photo-cross-linked PVP hydrogels present a strong gel mechanical behavior and viscoelastic moduli values similar to those of biological gels. The average distance between the cross-linking points of the polymer network was estimated from the hydrogels elastic modulus. However, SANS measurements showed that the network microstructure is highly inhomogeneous, presenting polymer-rich regions more densely cross-linked, surrounded by a water-rich environment. EPR and PGSE-NMR data further support the existence of these water-rich domains. Inclusion of a third component, such as glycerol, in the PVP aqueous mixture to be irradiated has been also investigated. A small amount of glycerol (<3% w/w) can be added keeping satisfactory properties of the hydrogel, while higher amounts significantly affect the cross-linking process.

Ribonuclease/angiogenin inhibitor 1 regulates stress-induced subcellular localization of angiogenin to control growth and survival.

Summary Angiogenin (ANG) promotes cell growth and survival. Under growth conditions, ANG undergoes nuclear translocation and accumulates in the nucleolus where it stimulates rRNA transcription. When cells are stressed, ANG mediates the production of tRNA-derived stress-induced small RNA (tiRNA), which reprograms protein translation into a survival mechanism. The ribonucleolytic activity of ANG is essential for both processes but how this activity is regulated is unknown. We report here that ribonuclease/angiogenin inhibitor 1 (RNH1) controls both the localization and activity of ANG. Under growth conditions, ANG is located in the nucleus and is not associated with RNH1 so that the ribonucleolytic activity is retained to ensure rRNA transcription. Cytoplasmic ANG is associated with and inhibited by RNH1 so that random cleavage of cellular RNA is prevented. Under stress conditions, ANG is localized to the cytoplasm and is concentrated in stress granules where it is not associated with RNH1 and thus remains enzymatically active for tiRNA production. By contrast, nuclear ANG is associated with RNH1 in stressed cells to ensure that the enzymatic activity is inhibited and no unnecessary rRNA is produced to save anabolic energy. Knockdown of RNH1 abolished stress-induced relocalization of ANG and decreased cell growth and survival.

Injectable thermally responsive mucoadhesive gel for sustained protein delivery.

Poloxamer thermoresponsive gels are widely explored in controlled drug delivery. Nevertheless, these gels possess inadequate mechanical properties, poor bioadhesiveness, and high permeability to water. To overcome these issues, we blended mucoadhesive hyaluronic acid (HA) with poloxamer analogs. This study aimed to investigate the features affecting the microscopic properties of the gels, which determine their macroscopic properties and capability to control/sustain protein release. Results showed that HA hampers water-poloxamer interactions, thus, strongly influencing physicochemical properties of poloxamer gels. This leads to gels with improved mechanical properties in which the diffusion kinetics of macromolecular active molecules are drastically slowed down. Poloxamer-HA gels can sustain the delivery of proteins, such as insulin, and may allow the modulation of its release kinetics by modifying HA content within the gels in the administration sites in which the active molecule release mechanism is mainly governed by its diffusion.

New Insights into the Mechanisms of the Interactions Between Doxorubicin and the Ion-Exchange Hydrogel DC Bead™ for Use in Transarterial Chemoembolization (TACE)

Ion-exchange microspheres (IEMs) are widely employed in controlled drug delivery of ionic drugs due to their high loading capacity and the possibility to obtain the controlled release of the loaded drug(s) at a specific site. Among IEMs, DC Bead™ are embolic microdevices (100–300 μm diameter) designed for transarterial chemoembolization (TACE) and composed of cross-linked poly(vinyl alcohol) (PVA) hydrogel, bearing anionic sulfonate moieties on the cross-links, and able to bind cationic drugs such as doxorubicin hydrochloride (Dox). Even if DC Bead™ were studied for their release and bulk characteristics, a thorough characterization of these devices is still lacking. In particular, the aim of this work was the determination of bound and free water, Dox distribution within the microdevices and drug–DC Bead™ interactions, in terms of transport features within the device. Compared with previous results, different Dox radial distributions in DC Bead™ were found, and related to bead microsctructure and ion exchange mechanism. Artifacts due to the self-quenching of Dox at high concentration were prevented and the diffusion coefficients of drug-polymer (Dox-ionic sites) evaluated in different sections of the microspheres. Furthermore, DSC results indicated that in the hydrogel either free (bulk) or bound (non-freezable) water could be found, and that no freezing-bound water was present.

Crosstalk between focal adhesions and material mechanical properties governs cell mechanics and functions.

Mechanical properties of materials strongly influence cell fate and functions. Focal adhesions are involved in the extremely important processes of mechanosensing and mechanotransduction. To address the relationship between the mechanical properties of cell substrates, focal adhesion/cytoskeleton assembly and cell functions, we investigated the behavior of NIH/3T3 cells over a wide range of stiffness (3-1000kPa) using two of the most common synthetic polymers for cell cultures: polyacrylamide and polydimethylsiloxane. An overlapping stiffness region was created between them to compare focal adhesion characteristics and cell functions, taking into account their different time-dependent behavior. Indeed, from a rheological point of view, polyacrylamide behaves like a strong gel (elastically), whereas polydimethylsiloxane like a viscoelastic solid. First, focal adhesion characteristics and dynamics were addressed in terms of material stiffness, then cell spreading area, migration rate and cell mechanical properties were correlated with focal adhesion size and assembly. Focal adhesion size was found to increase in the whole range of stiffness and to be in agreement in the overlapping rigidity region for the investigated materials. Cell mechanics directly correlated with focal adhesion lengths, whereas migration rate followed an inverse correlation. Cell spreading correlated with the substrate stiffness on polyacrylamide hydrogel, while no specific trend was found on polydimethylsiloxane. Substrate mechanics can be considered as a key physical cue that regulates focal adhesion assembly, which in turn governs important cellular properties and functions.

Biocompatibility, uptake and endocytosis pathways of polystyrene nanoparticles in primary human renal epithelial cells.

Recent years have witnessed an unprecedented growth in the number of applications—such as drug delivery, nutraceuticals and production of improved biocompatible materials—in the areas of nanoscience and nanotechnology. Engineered nanoparticles (NPs) are an important tool for the development of quite a few of these applications. Despite intense research activity, mechanisms regulating the uptake of NPs into cells are not completely defined, being the phenomenon dramatically influenced by physico-chemical properties of NPs and cell-specific differences. Since the cellular uptake of NPs is a prerequisite for their use in nanomedicine, the definition of their internalization pathway is crucial. For this reason, we used 44 nm polystyrene NPs as a model to analyze the uptake and endocytosis pathways in primary human renal cortical epithelial (HRCE) cells, which play a key role in the clearance of drugs. NPs were found not to affect the viability and cell cycle progression of HRCE cells. Distinct internalization pathways were analyzed by the use of drugs known to inhibit specific endocytosis routes. Analyses, performed by confocal microscopy in combination with quantitative spectrofluorimetric assays, indicated that NPs enter HRCE cells through multiple mechanisms, either energy-dependent (endocytosis) or energy-independent.

Investigation of the mechanisms governing doxorubicin and irinotecan release from drug-eluting beads: mathematical modeling and experimental verification.

Drug-eluting beads (DEBs) are embolising devices in clinical use for the treatment of liver cancer by transarterial chemoembolisation. In this study, release kinetics of doxorubicin (DOX) and irinotecan (IRI) were investigated by experimental evaluations and mathematical modeling, based on Langmuir isotherm and two phenomenological models (Boyd/Bhaskar) developed to determine the actual mechanisms controlling drug release rate. The model was validated through release studies, in particular by assessing how drug loading, ionic strength of the release medium and device swelling during release influence drug release kinetics. Results demonstrated that IRI is released much faster than DOX, and that DEB volume strongly depends upon drug loading and fractional release. This effect was properly taken into account in developing the mathematical model. Experimental results were well fit by numerical simulations, and two different rate-controlling mechanisms were found to govern DOX and IRI delivery.

Ligand engagement on material surfaces is discriminated by cell mechanosensoring

Peptide or protein ligands can be used for molecular decoration to enhance the functionality of synthetic materials. However, some skepticism has arisen about the efficacy of such strategy in practical contexts since serum proteins largely adsorb. To address this issue, it is crucial to ascertain whether a chemically conjugated integrin-binding peptide is fully recognized by a cell even if partially covered by a physisorbed layer of serum protein; in more general terms, if competitive protein fragments physisorbed onto the surface are distinguishable from those chemically anchored to it. Here, we engraft an RGD peptide on poly-ε-caprolactone (PCL) surfaces and follow the dynamics of focal adhesion (FA) and cytoskeleton assembly at different times and culture conditions using a variety of analytical tools. Although the presence of serum protein covers the bioconjugated RGD significantly, after the first adhesion phase cells dig into the physisorbed layer and reach the submerged signal to establish a more stable adhesion structure (mature FAs). Although the spreading area index is not substantially affected by the presence of the RGD peptide, cells attached to chemically bound signals develop a stronger adhesive interaction with the materials and assemble a mechanically stable cytoskeleton. This demonstrates that cells are able to discriminate, via mechanosensoring, between adhesive motives belonging to physisorbed proteins and those firmly anchored on the material surface.