FRET Sensor-Modified Synthetic Hydrogels for Real-Time Monitoring of Cell-Derived Matrix Metalloproteinase Activity using Fluorescence Lifetime Imaging.

Matrix remodeling plays central roles in a range of physiological and pathological processes and is driven predominantly by the activity of matrix metalloproteinases (MMPs), which degrade extracellular matrix (ECM) proteins. Our understanding of how MMPs regulate cell and tissue dynamics is often incomplete as in vivo approaches are lacking and many in vitro strategies cannot provide high-resolution, quantitative measures of enzyme activity in situ within tissue-like 3D microenvironments. Here, we incorporate a Förster resonance energy transfer (FRET) sensor of MMP activity into fully synthetic hydrogels that mimic many properties of the native ECM. We then use fluorescence lifetime imaging to provide a real-time, fluorophore concentration-independent quantification of MMP activity, establishing a highly accurate, readily adaptable platform for studying MMP dynamics in situ. MCF7 human breast cancer cells encapsulated within hydrogels highlight the detection of MMP activity both locally, at the sub-micron level, and within the bulk hydrogel. Our versatile platform may find use in a range of biological studies to explore questions in the dynamics of cancer metastasis, development, and tissue repair by providing high-resolution, quantitative and in situ readouts of local MMP activity within native tissue-like environments.

Mechanisms of growth and homeostasis in the Drosophila wing.

Animal shape and size is controlled with amazing precision during development. External factors such as nutrient availability and crowding can alter overall animal size, but individual body parts scale reproducibly to match the body even with challenges from a changing environment. How is such precision achieved? Here, we review selected research from the last few years in Drosophila--arguably the premier genetic model for the study of animal growth--that sheds light on how body and tissue size are regulated by forces intrinsic to individual organs. We focus on two topics currently under intense study: the influence of pattern regulators on organ and tissue growth and the role of local competitive interactions between cells in tissue homeostasis and final size.

ILC1 drive intestinal epithelial and matrix remodelling.

Organoids can shed light on the dynamic interplay between complex tissues and rare cell types within a controlled microenvironment. Here, we develop gut organoid cocultures with type-1 innate lymphoid cells (ILC1) to dissect the impact of their accumulation in inflamed intestines. We demonstrate that murine and human ILC1 secrete transforming growth factor ß1, driving expansion of CD44v6+ epithelial crypts. ILC1 additionally express MMP9 and drive gene signatures indicative of extracellular matrix remodelling. We therefore encapsulated human epithelial-mesenchymal intestinal organoids in MMP-sensitive, synthetic hydrogels designed to form efficient networks at low polymer concentrations. Harnessing this defined system, we demonstrate that ILC1 drive matrix softening and stiffening, which we suggest occurs through balanced matrix degradation and deposition. Our platform enabled us to elucidate previously undescribed interactions between ILC1 and their microenvironment, which suggest that they may exacerbate fibrosis and tumour growth when enriched in inflamed patient tissues.

Ambipolar Resistive Switching in an Ultrathin Surface-Supported Metal-Organic Framework Vertical Heterojunction.

Memristors (MRs) are considered promising devices with the enormous potential to replace complementary metal-oxide-semiconductor (CMOS) technology, which approaches the scale limit. Efforts to fabricate MRs-based hybrid materials may result in suitable operating parameters coupled to high mechanical flexibility and low cost. Metal-organic frameworks (MOFs) arise as a favorable candidate to cover such demands. The step-by-step growth of MOFs structures on functionalized surfaces, called surface-supported metal-organic frameworks (SURMOFs), opens the possibility for designing new applications in strategic fields such as electronics, optoelectronics, and energy harvesting. However, considering the MRs architecture, the typical high porosity of these hybrid materials may lead to short-circuited devices easily. In this sense, here, it is reported for the first time the integration of SURMOF films in rolled-up scalable-functional devices. A freestanding metallic nanomembrane provides a robust and self-adjusted top mechanical contact on the SURMOF layer. The electrical characterization reveals an ambipolar resistive switching mediated by the humidity level with low-power consumption. The electronic properties are investigated with density functional theory (DFT) calculations. Furthermore, the device concept is versatile, compatible with the current parallelism demands of integration, and transcends the challenge in contacting SURMOF films for scalable-functional devices.

Glycosaminoglycan-Inspired Biomaterials for the Development of Bioactive Hydrogel Networks.

Glycosaminoglycans (GAG) are long, linear polysaccharides that display a wide range of relevant biological roles. Particularly, in the extracellular matrix (ECM) GAG specifically interact with other biological molecules, such as growth factors, protecting them from proteolysis or inhibiting factors. Additionally, ECM GAG are partially responsible for the mechanical stability of tissues due to their capacity to retain high amounts of water, enabling hydration of the ECM and rendering it resistant to compressive forces. In this review, the use of GAG for developing hydrogel networks with improved biological activity and/or mechanical properties is discussed. Greater focus is given to strategies involving the production of hydrogels that are composed of GAG alone or in combination with other materials. Additionally, approaches used to introduce GAG-inspired features in biomaterials of different sources will also be presented.

Hybrid nanomembrane-based capacitors for the determination of the dielectric constant of semiconducting molecular ensembles.

Considerable advances in the field of molecular electronics have been achieved over the recent years. One persistent challenge, however, is the exploitation of the electronic properties of molecules fully integrated into devices. Typically, the molecular electronic properties are investigated using sophisticated techniques incompatible with a practical device technology, such as the scanning tunneling microscopy. The incorporation of molecular materials in devices is not a trivial task as the typical dimensions of electrical contacts are much larger than the molecular ones. To tackle this issue, we report on hybrid capacitors using mechanically-compliant nanomembranes to encapsulate ultrathin molecular ensembles for the investigation of molecular dielectric properties. As the prototype material, copper (II) phthalocyanine (CuPc) has been chosen as information on its dielectric constant (k CuPc) at the molecular scale is missing. Here, hybrid nanomembrane-based capacitors containing metallic nanomembranes, insulating Al2O3 layers, and the CuPc molecular ensembles have been fabricated and evaluated. The Al2O3 is used to prevent short circuits through the capacitor plates as the molecular layer is considerably thin (<30 nm). From the electrical measurements of devices with molecular layers of different thicknesses, the CuPc dielectric constant has been reliably determined (k CuPc = 4.5 ± 0.5). These values suggest a mild contribution of the molecular orientation on the CuPc dielectric properties. The reported nanomembrane-based capacitor is a viable strategy for the dielectric characterization of ultrathin molecular ensembles integrated into a practical, real device technology.

Post-assembly functionalization of supramolecular nanostructures with bioactive peptides and fluorescent proteins by native chemical ligation.

Post-assembly functionalization of supramolecular nanostructures has the potential to expand the range of their applications. We report here the use of the chemoselective native chemical ligation (NCL) reaction to functionalize self-assembled peptide amphiphile (PA) nanofibers. This strategy can be used to incorporate specific bioactivity on the nanofibers, and as a model, we demonstrate functionalization with the RGDS peptide following self-assembly. Incorporation of bioactivity is verified by the observation of characteristic changes in fibroblast morphology following NCL-mediated attachment of the signal to PA nanofibers. The NCL reaction does not alter the PA nanofiber morphology, and biotinylated RGDS peptide was found to be accessible on the nanofiber surface after ligation for binding with streptavidin-conjugated gold nanoparticles. In order to show that this strategy is not limited to short peptides, we utilized NCL to conjugate yellow fluorescent protein and/or cyan fluorescent protein to self-assembled PA nanofibers. Förster resonance energy transfer and fluorescence anisotropy measurements are consistent with the immobilization of the protein on the PA nanofibers. The change in electrophoretic mobility of the protein upon conjugation with PA molecules confirmed the formation of a covalent linkage. NCL-mediated attachment of bioactive peptides and proteins to self-assembled PA nanofibers allows the independent control of self-assembly and bioactivity while retaining the biodegradable peptide structure of the PA molecule and thus can be useful in tailoring design of biomaterials.

Injectable PEG Hydrogels with Tissue-Like Viscoelasticity Formed through Reversible Alendronate-Calcium Phosphate Crosslinking for Cell-Material Interactions.

Synthetic hydrogels provide controllable 3D environments, which can be used to study fundamental biological phenomena. The growing body of evidence that cell behavior depends upon hydrogel stress relaxation creates a high demand for hydrogels with tissue-like viscoelastic properties. Here, a unique platform of synthetic polyethylene glycol (PEG) hydrogels in which star-shaped PEG molecules are conjugated with alendronate and/or RGD peptides, attaining modifiable degradability as well as flexible cell adhesion, is created. Novel reversible ionic interactions between alendronate and calcium phosphate nanoparticles, leading to versatile viscoelastic properties with varying initial elastic modulus and stress relaxation time, are identified. This new crosslinking mechanism provides shear-thinning properties resulting in differential cellular responses between cancer cells and stem cells. The novel hydrogel system is an improved design to the other ionic crosslink platforms and opens new avenues for the development of pathologically relevant cancer models, as well as minimally invasive approaches for cell delivery for potential regenerative therapies.

Customizing 3D Structures of Vertically Aligned Carbon Nanotubes to Direct Neural Stem Cell Differentiation.

Neural tissue-related illnesses have a high incidence and prevalence in society. Despite intensive research efforts to enhance the regeneration of neural cells into functional tissue, effective treatments are still unavailable. Here, a novel therapeutic approach based on vertically aligned carbon nanotube forests (VA-CNT forests) and periodic VA-CNT micropillars produced by thermal chemical vapor deposition is explored. In addition, honeycomb-like and flower-like morphologies are created. Initial viability testing reveals that NE-4C neural stem cells seeded on all morphologies survive and proliferate. In addition, free-standing VA-CNT forests and capillary-driven VA-CNT forests are created, with the latter demonstrating enhanced capacity to stimulate neuritogenesis and network formation under minimal differentiation medium conditions. This is attributed to the interaction between surface roughness and 3D-like morphology that mimics the native extracellular matrix, thus enhancing cellular attachment and communication. These findings provide a new avenue for the construction of electroresponsive scaffolds based on CNTs for neural tissue engineering.

Impact of atomic layer deposited TiO2 on the photocatalytic efficiency of TiO2/w-VA-CNT nanocomposite materials.

Titanium oxide (TiO2) has been widely investigated as a photocatalytic material, and the fact that its performance depends on its crystalline structure motivates further research on the relationship between preparation methods and material properties. In this work, TiO2 thin films were grown on non-functionalized wave-like patterned vertically aligned carbon nanotubes (w-VA-CNTs) via the atomic layer deposition (ALD) technique. Grazing incidence X-ray diffraction (GIXRD) analysis revealed that the structure of the TiO2/VA-CNT nanocomposites varied from amorphous to a crystalline phase with increasing deposition temperature, suggesting a "critical deposition temperature" for the anatase crystalline phase formation. On the other hand, scanning transmission electron microscopy (STEM) studies revealed that the non-functionalized carbon nanotubes were conformally and homogeneously coated with TiO2, forming a nanocomposite while preserving the morphology of the nanotubes. X-ray photoelectron spectroscopy (XPS) provided information about the surface chemistry and stoichiometry of TiO2. The photodegradation experiments under ultraviolet (UV) light on a model pollutant (Rhodamine B, RhB) revealed that the nanocomposite comprised of anatase crystalline TiO2 grown at 200 °C (11.2 nm thickness) presented the highest degradation efficiency viz 55% with an illumination time of 240 min. Furthermore, its recyclability was also demonstrated for multiple cycles, showing good recovery and potential for practical applications.

Directed self-assembly of spheroids into modular vascular beds for engineering large tissue constructs.

Spheroids can be used as building-blocks for bottom-up generation of artificial vascular beds, but current biofabrication strategies are often time-consuming and complex. Also, pre-optimization of single spheroid properties is often neglected. Here, we report a simple setup for rapid biomanufacturing of spheroid-based patch-like vascular beds. Prior to patch assembly, spheroids combining mesenchymal stem/stromal cells (MSCs) and outgrowth endothelial cells (OECs) at different ratios (10:1; 5:1; 1:1; 1:5) were formed in non-adhesive microwells and monitored along 7 d. Optimal OEC retention and organization was observed at 1:1 MSC/OEC ratio. Dynamic remodelling of spheroids led to changes in both cellular and extracellular matrix components (ECMs) over time. Some OEC formed internal clusters, while others organized into a peripheral monolayer, stabilized by ECM and pericyte-like cells, with concomitant increase in surface stiffness. Along spheroid culture, OEC switched from an active to a quiescent state, and their endothelial sprouting potential was significantly abrogated, suggesting that immature spheroids may be more therapeutically relevant. Non-adhesive moulds were subsequently used for triggering rapid, one-step, spheroid formation/fusion into square-shaped patches, with spheroids uniformly interspaced via a thin cell layer. The high surface area, endothelial sprouting potential, and scalability of the developed spheroid-based patches make them stand out as artificial vascular beds for modular engineering of large tissue constructs.

Male courtship song drives escape responses that are suppressed for successful mating.

Persuasion is a crucial component of the courtship ritual needed to overcome contact aversion. In fruit flies, it is well established that the male courtship song prompts receptivity in female flies, in part by causing sexually mature females to slow down and pause, allowing copulation. Whether the above receptivity behaviours require the suppression of contact avoidance or escape remains unknown. Here we show, through genetic manipulation of neurons we identified as required for female receptivity, that male song induces avoidance/escape responses that are suppressed in wild type flies. First, we show that silencing 70A09 neurons leads to an increase in escape, as females increase their walking speed during courtship together with an increase in jumping and a reduction in pausing. The increase in escape response is specific to courtship, as escape to a looming threat is not intensified. Activation of 70A09 neurons leads to pausing, confirming the role of these neurons in escape modulation. Finally, we show that the escape displays by the female result from the presence of a courting male and more specifically from the song produced by a courting male. Our results suggest that courtship song has a dual role, promoting both escape and pause in females and that escape is suppressed by the activity of 70A09 neurons, allowing mating to occur.

Selectively Cross-Linked Tetra-PEG Hydrogels Provide Control over Mechanical Strength with Minimal Impact on Diffusivity.

Synthetic hydrogels formed from poly(ethylene glycol) (PEG) are widely used to study how cells interact with their extracellular matrix. These in vivo-like 3D environments provide a basis for tissue engineering and cell therapies but also for research into fundamental biological questions and disease modeling. The physical properties of PEG hydrogels can be modulated to provide mechanical cues to encapsulated cells; however, the impact of changing hydrogel stiffness on the diffusivity of solutes to and from encapsulated cells has received only limited attention. This is particularly true in selectively cross-linked "tetra-PEG" hydrogels, whose design limits network inhomogeneities. Here, we used a combination of theoretical calculations, predictive modeling, and experimental measurements of hydrogel swelling, rheological behavior, and diffusion kinetics to characterize tetra-PEG hydrogels' permissiveness to the diffusion of molecules of biologically relevant size as we changed polymer concentration, and thus hydrogel mechanical strength. Our models predict that hydrogel mesh size has little effect on the diffusivity of model molecules and instead predicts that diffusion rates are more highly dependent on solute size. Indeed, our model predicts that changes in hydrogel mesh size only begin to have a non-negligible impact on the concentration of a solute that diffuses out of hydrogels for the smallest mesh sizes and largest diffusing solutes. Experimental measurements characterizing the diffusion of fluorescein isothiocyanate (FITC)-labeled dextran molecules of known size aligned well with modeling predictions and suggest that doubling the polymer concentration from 2.5% (w/v) to 5% produces stiffer gels with faster gelling kinetics without affecting the diffusivity of solutes of biologically relevant size but that 10% hydrogels can slow their diffusion. Our findings provide confidence that the stiffness of tetra-PEG hydrogels can be modulated over a physiological range without significantly impacting the transport rates of solutes to and from encapsulated cells.

Temperature- and strain-independent torsion sensor using a fiber loop mirror based on suspended twin-core fiber.

In this Letter, we present a fiber loop mirror configuration based on a suspended twin-core fiber for sensing applications. Using the suspended twin-core fiber, the fringe pattern is due to the differential optical patch of the light in the two cores associated with a refractive index difference of approximately 10(-3), which indicates an advantage of this approach compared with those based on high-birefringent fibers, namely, the possibility of using a small length of fiber. The sensing configuration was characterized for torsion, temperature, and strain. Using the fast Fourier transform technique, it is possible to obtain measurand-induced amplitude variations of the fringe pattern. The results obtained indicate the viability of a temperature- and strain-independent torsion sensor.

Hydrophobic-electrostatic balance driving the LCST offset aggregation-redissolution behavior of N-alkylacrylamide-based ionic terpolymers.

A series of random terpolymers composed of N-isopropylacrylamide (NIPAAm), 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), and N-tert-butylacrylamide (NTBAAm) monomers were synthesized by free radical polymerization. The molar fraction of the negatively charged monomer (AMPS) was maintained constant (0.05) for all studied terpolymer compositions. Turbidity measurements were used to evaluate the influence of the relative amount of NIPAAm and NTBAAm, polymer concentration, and solution ionic strength on the cloud point and redissolution temperatures (macroscopic phase separation). Dynamic light scattering (DLS) was employed to elucidate some aspects regarding the molecular scale mechanism of the temperature-induced phase separation and to determine the low critical solution temperature (LCST). The aqueous solutions of terpolymers remained clear at all studied temperatures; turbidity was only observed in the presence of NaCl. The cloud point temperature (CPT) determined by turbidimetry was found to be systematically much higher than the LCST determined by DLS; nanosized aggregates were observed at temperatures between the LCST and the CPT. Both CPT and LCST decreased when increasing the molar ratio of NTBAAm (increased hydrophobicity). It was found that above a critical molar fraction of NTBAAm (0.25-0.30) the aggregation rate suddenly decreased. Polymers with NTBAAm content lower than 0.25 showed a fast macroscopic phase separation, but the formed large aggregates are disaggregating during the cooling ramp at temperatures still higher than the LCST. On the contrary, polymers with NTBAAm contents above 0.30 showed a slow macroscopic phase separation, and the formed large aggregates only redissolved when LCST was reached. These differences were explained on the basis of a delicate balance between the electrostatic repulsion and the hydrophobic attractive forces, which contribute cooperatively to the formation of metastable nanosized aggregates.

Temperature-Independent Polarization of Ultrathin Phthalocyanine-Based Hybrid Organic/Inorganic Heterojunctions.

The combination of organic and inorganic materials at the nanoscale to form functional hybrid structures is a powerful strategy to develop novel electronic devices. The knowledge on semiconductor thin-film polarization brings direct benefits to the hybrid organic/inorganic electronics, becoming primordial for the development of devices such as electromechanical logic gates, solar cells, miniaturized valves, organic diodes, and molecular supercapacitors, among others. Here, we report on the dielectric polarization of ultrathin organic semiconducting films-ca. 5 nm thick metal phthalocyanine ensembles (viz., CuPc, CoPc, F16CuPc)-employed to build up hybrid metal/oxide/molecule heterojunctions. Such hybrid heterostructures are fully integrated into self-rolled nanomembrane-based capacitors and further investigated by impedance spectroscopy measurements as a function of temperature (from 6 to 300 K). The dielectric polarization of the metal phthalocyanines is found to be thermally activated above a specific threshold temperature, which depends on the molecular structure. Below this threshold, the current leakage across the system is suppressed, thus evidencing intrinsic-like polarization mechanisms. The temperature-independent permittivities of the ultrathin molecular films are found to be strongly dependent on the organic/inorganic hybrid interfaces, while the calculated relaxation times are more likely related to each single-molecule polarization. Beyond the advances in determining the temperature dependence of the permittivity for ultrathin phthalocyanine films integrated within solid-state electronics, our results also support the deterministic design of novel functional devices based on nanoscale hybrid organic/inorganic heterojunctions.

Adhesive Hydrogels for Maxillofacial Tissue Regeneration Using Minimally Invasive Procedures.

Minimally invasive surgical procedures aiming to repair damaged maxillofacial tissues are hampered by its small, complex structures and difficult surgical access. Indeed, while arthroscopic procedures that deliver regenerative materials and/or cells are common in articulating joints such as the knee, there are currently no treatments that surgically place cells, regenerative factors or materials into maxillofacial tissues to foster bone, cartilage or muscle repair. Here, hyaluronic acid (HA)-based hydrogels are developed, which are suitable for use in minimally invasive procedures, that can adhere to the surrounding tissue, and deliver cells and potentially drugs. By modifying HA with both methacrylate (MA) and 3,4-dihydroxyphenylalanine (Dopa) groups using a completely aqueous synthesis route, it is shown that MA-HA-Dopa hydrogels can be applied under aqueous conditions, gel quickly using a standard surgical light, and adhere to tissue. Moreover, upon oxidation of the Dopa, human marrow stromal cells attach to hydrogels and survive when encapsulated within them. These observations show that when incorporated into HA-based hydrogels, Dopa moieties can foster cell and tissue interactions, ensuring surgical placement and potentially enabling delivery/recruitment of regenerative cells. The findings suggest that MA-HA-Dopa hydrogels may find use in minimally invasive procedures to foster maxillofacial tissue repair.

Fabrication of electrospun poly(lactic acid) nanoporous membrane loaded with niobium pentoxide nanoparticles as a potential scaffold for biomaterial applications.

Tissue engineering aims to regenerate and restore damaged human organs and tissues using scaffolds that can mimic the native tissues. The requirement for modern and efficient biomaterials that are capable of accelerating the healing process has been considerably increased. In this work, a novel electrospun poly(lactic acid) (PLA) nanoporous membrane incorporated with niobium pentoxide nanoparticles (Nb2 O5 ) for biomaterial applications was developed. Nb2 O5 nanoparticles were obtained by microwave-assisted hydrothermal synthesis, and different concentrations (0, 1, 3, and 5% wt/wt) were tested. Chemical, morphological, mechanical, and biological properties of membranes were evaluated. Cell viability results demonstrated that the membranes presented nontoxic effects. The incorporation of Nb2 O5 improved cell proliferation without impairing the wettability, porosity, and mechanical properties of membranes. Membranes containing Nb2 O5 nanoparticles presented biocompatible properties with suitable porosity, which facilitated cell attachment and proliferation while allowing diffusion of oxygen and nutrients. This study has demonstrated that Nb2 O5 nanoparticle-loaded electrospun PLA nanoporous membranes are potential candidates for drug delivery and wound dressing applications.

Poly(N-isopropylacrylamide) surface-grafted chitosan membranes as a new substrate for cell sheet engineering and manipulation.

The immobilization of poly(N-isopropylacrylamide) (PNIPAAm) on chitosan membranes was performed in order to render membranes with thermo-responsive surface properties. The aim was to create membranes suitable for cell culture and in which confluent cell sheets can be recovered by simply lowering the temperature. The chitosan membranes were immersed in a solution of the monomer that was polymerized via radical initiation. The composition of the polymerization reaction solvent, which was a mixture of a chitosan non-solvent (isopropanol) and a solvent (water), provided a tight control over the chitosan membranes swelling capability. The different swelling ratio, obtained at different solvent composition of the reaction mixture, drives simultaneously the monomer solubility and diffusion into the polymeric matrix, the polymerization reaction rate, as well as the eventual chain transfer to the side substituents of the pyranosyl groups of chitosan. A combined analysis of the modified membranes chemistry by proton nuclear magnetic resonance ((1)H-NMR), Fourier transform spectroscopy with attenuated total reflection (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) showed that it was possible to control the chitosan modification yield and depth in the solvent composition range between 75% and 100% of isopropanol. Plasma treatment was also applied to the original chitosan membranes in order to improve cell adhesion and proliferation. Chitosan membranes, which had been previously subjected to oxygen plasma treatment, were then modified by means of the previously described methodology. A human fetal lung fibroblast cell line was cultured until confluence on the plasma-treated thermo-responsive chitosan membranes and cell sheets were harvested lowering the temperature.

Transport of small anionic and neutral solutes through chitosan membranes: dependence on cross-linking and chelation of divalent cations.

Chitosan membranes were prepared by solvent casting and cross-linked with glutaraldehyde at several ratios under homogeneous conditions. The cross-linking degree, varying from 0 to 20%, is defined as the ratio between the total aldehyde groups and the amine groups of chitosan. Permeability experiments were conducted using a side-by-side diffusion cell to determine the flux of small molecules of similar size but with different chemical moieties, either ionized (benzoic acid, salicylic acid, and phthalic acid) or neutral (2-phenylethanol) at physiological pH. The permeability of the different model molecules revealed to be dependent on the affinity of those structurally similar molecules to chitosan. The permeability of the salicylate anion was significantly enhanced by the presence of metal cations commonly present in biological fluids, such as calcium and magnesium, but remained unchanged for the neutral 2-phenylethanol. This effect could be explained by the chelation of metal cations on the amine groups of chitosan, which increased the partition coefficient. The cross-linking degree was also correlated with the permeability and partition coefficient. The change in the permeation properties of chitosan to anionic solutes in the presence of these metallic cations is an important result and should be taken into consideration when trying to make in vitro predictions of the drug release from chitosan-based controlled release systems.