A novel design of injectable porous hydrogels with in situ pore formation

The use of injectable porous hydrogels is of great interest in biomedical applications due to their excellent permeability and ease of integration into sites of surgical intervention. By implementing a method that enables the formation in situ of pores with controllable porosity and pore size, it is possible to synthesize bioactive hydrogels that are tailor-made for specific biomedical applications. An emulsion-templating technique was used to encapsulate oil droplets, which are subsequently leached out of the hydrogel to create the porous structure. Pore size and porosity were manipulated by changing oil-to-water ratios and the surfactant concentrations. Highly swellable porous hydrogels were obtained with control over mechanical strength and diffusive properties. The relationship between porosity, pore size, and the hydrogel’s physical and mechanical characteristics was analyzed, and the potential of this material as a protein drug delivery system was demonstrated.     

Ligand accessibility as means to control cell response to bioactive bilayer membranes

We report a new method to create a biofunctional surface in which the accessibility of a ligand is used as a means to influence the cell behavior. Supported bioactive bilayer membranes were created by Langmuir-Blodgett (LB) deposition of either a pure poly(ethylene glycol) (PEG) lipid, having PEG head groups of various lengths, or 50 mol % binary mixtures of a PEG lipid and a novel collagen-like peptide amphiphile on a hydrophobic surface. The peptide amphiphile contains a peptide synthetically lipidated by covalent linkage to hydrophobic dialkyl tails. The amphiphile head group lengths were determined using neutron reflectivity. Cell adhesion and spreading assays showed that the cell response to the membranes depends on the length difference between head groups of the membrane components. Cells adhere and spread on mixtures of the peptide amphiphile with the PEG lipids having PEG chains of 120 and 750 molecular weight (MW). In contrast, cells adhered but did not spread on the mixture containing the 2000 MW PEG. Cells did not adhere to any of the pure PEG lipid membranes or to the mixture containing the 5000 MW PEG. Selective masking of a ligand on a surface is one method of controlling the surface bioactivity.     

Phloroglucinol-based biomimetic adhesives for medical applications

An adhesive that functions well under moist conditions could facilitate many surgical procedures. In recent studies we designed novel biomimetic glues which mimic the adhesion mechanism of algae, renowned for their remarkable adherence to wet surfaces. Here we extend our previous studies and propose biomimetic formulations, composed of alginate gel and native phloroglucinol, that do not induce cell cytotoxicity. Characterization of the adherence to tissues showed that adhesion was directly related to the mechanical strength of the cross-linked alginate. Therefore the adhesion strength can be altered by changing the source of the calcium cross-linker, the alginate G-content or the molecular weight of the alginate. The adhesion strength was comparable to that of Tisseel^?, a commercial tissue adhesive.     

Physical and structural characteristics of acrylated poly(ethylene glycol)-alginate conjugates

Transmucosal delivery of therapeutic agents is a non-invasive approach that utilizes human entry paths such as the nasal, buccal, rectal and vaginal routes. Mucoadhesive polymers have the ability to adhere to the mucus layer covering those surfaces and by that promote drug release, targeting and absorption. We have recently demonstrated that acrylated polymers display enhanced mucoadhesive properties due to their ability to covalently attach to mucus type glycoproteins. We have synthesized an acrylated poly(ethylene glycol)-alginate conjugate (alginate-PEGAc), a molecule which combines the gelation ability of alginate with the mucoadhesion properties arising from both the characteristics of poly(ethylene glycol) and the acrylate functionality. In the current investigation we introduce an in-depth characterization of the thermal, mechanical and structural properties of alginate-PEGAc aimed at gaining a better knowledge of its structure-function relations. The thermal stability, evaluated by thermal gravimetric analysis and differential scanning calorimetry, was compared with that of alginate and the intermediate product thiolated alginate. Dehydration at temperatures up to 200 °C was detected for all samples, followed by distinctive decomposition steps arising from the decomposition of the polymer backbone and side-chains. The nanostructure of the solutions and gels was evaluated from small angle X-ray scattering patterns, to which the "broken rod linked by flexible chain" model was fitted, and from rheology measurements. The maxima arising from electrostatic repulsion between the highly charged alginate chains was diminished for both modified alginate samples, suggesting that modification led to electrostatic screening. Alginate, thiolated alginate and alginate-PEGAc cross-linked with calcium ions demonstrated similar scattering patterns. However, different scattering intensities, gel strengths, and gelation kinetics were observed, suggesting a decrease in the cross-linking density in the order alginate>thiolated alginate>alginate-PEGAc. These results were attributed to the increased size of the grafted side groups, which interfere with the gelation process. Examining the effect of the method of alginate-PEGAc gelation (physical or chemical) has shown that additional UV irradiation of calcium cross-linked gels did not cause a significant change in the network structure and strength. It seems that the concentration of the acrylated end group is not high enough to create a chemically cross-linked network.     

Nanostructuring biosynthetic hydrogels for tissue engineering: a cellular and structural analysis

The nanostructuring of hydrogel scaffolds used in tissue engineering provides the ability to control cellular fate and tissue morphogenesis through cell-matrix interactions. Here we describe a method to provide nanostructure to a biosynthetic hydrogel scaffold made from crosslinked poly(ethylene glycol)-fibrinogen conjugates (PEG-fibrinogen), by modifying them with the block-copolymer Pluronic® F127. The copolymeric additive self-assembled into micelles at certain concentrations and temperatures, thereby creating nanostructures within the crosslinked hydrogel. Small-angle X-ray scattering (SAXS) and transmission electron microscopy at cryogenic temperature were used to detect Pluronic® F127 micelles embedded within the crosslinked PEG-fibrinogen hydrogels. The density and order of the micelles within the hydrogel matrix increased as the relative Pluronic® F127 concentration was raised. The transient stability of the micelles within the hydrogel network was analyzed using time-dependent swelling and Pluronic® F127 release measurements. These characterizations revealed that most of the Pluronic® F127 molecules diffuse out of the hydrogels after 4 days in aqueous buffer and SAXS analysis confirmed a significant change in the structure and interactions of the micelles during this time. Cell culture experiments evaluating the three-dimensional fibroblast morphology within the matrix indicated a strong correlation between cell spreading and the hydrogel’s characteristic mesh size. The present research thereby provides a more quantitative understanding of how structural features in an encapsulating hydrogel environment can affect cell morphogenesis towards tissue regeneration.     

Mucoadhesive Hybrid Polymer/Liposome Pastes Based on Modified Polysaccharides

Mucoadhesive hybrid polymer/liposome paste is a new drug delivery system presenting controllable and tailorable delivery mechanism. By using mucoadhesive material, the delivery can be more specific and local. Here, we present a study investigating the effect of polymer type, concentration, functional end group, and cross-linking on the release profile of nanoliposomes from polymer pastes. Polymer pastes can be expected to combine the mucoadhesion mechanisms of dry and wet dosage forms but have not been studied extensively. To better understand the mucoadhesion of pastes, we investigated a series of pastes based on the same polymer and used different chemical modifications that can produce interactions at different levels. Native and thiolated polymers presented enhanced mucoadhesion in a wet environment in comparison to acrylated polymers which dissolved rapidly because of the enhanced solubility of PEG chains in water. Paste cross-linking resulted in a sustained release profile compared to non–cross-linked pastes. Pectin-SH pastes, especially 3% (w/v), showed a linear liposomal release profile which is ascribed to the combination of ionic cross-linking and disulfide bridging. By configuring the polymer type or concentration, we can control the release mechanisms and achieve distinct inherent properties which can be applied for diverse medical applications.     

Nanostructuring of PEG–fibrinogen polymeric scaffolds

Recent studies have shown that nanostructuring of scaffolds for tissue engineering has a major impact on their interactions with cells. The current investigation focuses on nanostructuring of a biocompatible, biosynthetic polymeric hydrogel scaffold made from crosslinked poly(ethylene glycol)–fibrinogen conjugates. Nanostructuring was achieved by the addition of the block copolymer Pluronic^® F127, which self-assembles into nanometric micelles at certain concentrations and temperatures. Cryo-transmission electron microscopy experiments detected F127 micelles, both embedded within PEGylated fibrinogen hydrogels and in solution. The density of the F127 micelles, as well as their ordering, increased with increasing block copolymer concentration. The mechanical properties of the nanostructured hydrogels were investigated using stress-sweep rheological testing. These tests revealed a correlation between the block copolymer concentration and the storage modulus of the composite hydrogels. In vitro cellular assays confirmed that the increased modulus of the hydrogels did not limit the ability of the cells to form extensions and become spindled within the three-dimensional (3-D) hydrogel culture environment. Thus, altering the nanostructure of the hydrogel may be used as a strategy to control cellular behavior in 3-D through changes in mechanical properties of the environment.     

Composite alginate hydrogels: An innovative approach for the controlled release of hydrophobic drugs

We present an innovative methodology for the sustained delivery of hydrophobic drugs using composite hydrogels, prepared by embedding oil-in-water microemulsions in hydrophilic hydrogels. The hydrophobic nature of the microemulsion core enhances the solubilization of hydrophobic drugs, while the crosslinked matrix could be readily used as a solid controlled delivery vehicle. A microemulsion was formulated from pharmaceutical accepted components; the droplets diameter was shown to be about 10 nm by dynamic light scattering, cryo-transmission electron microscopy and small-angle X-ray scattering (SAXS). Combining the microemulsion with alginate solution and crosslinking with calcium ions resulted in a clear hydrogel. A model hydrophobic drug, Ketoprofen, precipitated from the alginate hydrogel, but the drug–containing composite hydrogel was clear and macroscopically homogeneous. The nanostructure was investigated by SAXS; scattering plots indicate that oil droplets exist in the composite hydrogel. Release profiles of the drug from the composite hydrogel with various concentrations of polymer and crosslinker demonstrate the applicability of this system as a controlled delivery vehicle, and suggest that the release rate is governed not by the microemulsion structure but, rather, by the network properties. Furthermore, it was demonstrated that the release rate could be tailored for a specific application utilizing different alginate and calcium concentrations. The generalization of the methodology of including hydrophobic drugs in composite gels is discussed.     

Nanostructuring PEG-fibrinogen hydrogels to control cellular morphogenesis

The nanostructuring of hydrogel scaffolds used in tissue engineering aims to provide an ability to control cellular morphogenesis through defined cell-matrix interactions. Toward this objective, we developed a method that alters the molecular network structure of biosynthetic hydrogel scaffolds made from crosslinked poly(ethylene glycol)-fibrinogen conjugates (PEG-fibrinogen, PF). The modifications were based on Pluronic(?) F127 micelles that were formed in the hydrogel precursor solution and that altered the hydrogel network assembly during photopolymerization crosslinking. Two variations of the cell-encapsulating hydrogels (high and low crosslinking density) were prepared with three concentrations of Pluronic(?) F127 (3%, 7%, 10% w/v). Quantitative morphometrics were used to characterize fibroblast shape parameters (both transient and stable) in all hydrogels, and rheological characterizations were used to measure the elastic (storage) component of the complex shear modulus of these hydrogels. The morphometric data was then correlated to both the nanostructure and modulus of the hydrogels for day 1 and day 4 in culture. These correlations revealed that structural features imparted by the Pluronic(?) F127 micelles were able to reverse the normally strong correlations found between indicators of cell spreading and the hydrogel's mechanical properties. Therefore, the data supports the conclusion that nanostructural features in the encapsulating hydrogel culture environment can facilitate better cell spreading in a dense hydrogel milieu, simply by introducing imperfections into the network structure. This research also provides further prospective regarding biocompatible approaches toward making structural modifications to hydrogel scaffolds for the purpose of 3-D cell culture and tissue engineering.