Alessandra Girotti

Synthesis and Characterization of Macroporous Thermosensitive Hydrogels from Recombinant Elastin-Like Polymers

Multifunctional bioactive chemically cross-linked elastin-like polymers (ELPs) have been prepared as three-dimensional scaffolds for tissue engineering. The salt-leaching/gas-foaming technique was found suitable to prepare highly porous biodegradable hydrogels based on this novel material type. The porosity can be controlled by the amount of sodium hydrogen carbonate incorporated during the cross-linking reaction, whereas the mean pore size is determined by the salt particle size. The gas-foaming process, which involves immersion in a citric acid solution after the cross-linking, facilitates pore interconnectivity and allows a grooved surface essential for cell colonization. Due to the thermoresponsive nature of the ELPs, their physical properties are strongly influenced by the temperature of the aqueous medium. The feasibility to obtain tridimensional scaffolds for tissue engineering has been studied by testing the adhesion and spreading of endothelial cells into the porous ELP hydrogels. The methods and structures described herein provide a starting point for the design and synthesis of macroporous multifunctional elastin-like hydrogels with potential broad applicability.

Genetically Engineered Elastin-Like Polymer as a Substratum to Culture Cells from the Ocular Surface

Purpose: To investigate epithelial cell adhesion and proliferation on a newly developed elastin-like polymer (ELP) that mimics the functional characteristics of extracellular matrices. Materials and Methods: A genetically engineered ELP with cell attachment sequences was adsorbed onto glass coverslips as 1, 2, or 3 molecular films. Conjunctival epithelial cells from a human cell line and human skin fibroblast cells (as controls) were plated onto coverslips with three different substrata: plain glass, Thermanox®, and ELP-coated. Cells (104) were plated after EDTA- or trypsin-based detachment. To test adhesion, epithelial and fibroblast cells were incubated for 4 hr, stained with hematoxylin, and counted. To study proliferation, Ki-67-positive epithelial cells were counted after 1, 3, and 5 days in culture. Immunostaining for conjunctival and adhesion markers was performed. Results: Epithelial cell, but not fibroblast, adhesion on ELP was significantly enhanced compared to that of control substrata. Epithelial cells detached with EDTA alone adhered significantly better than those detached with trypsin. By day 5, epithelial cell proliferation on ELP was significantly greater than that on plain glass. Epithelial cells grown on ELP expressed conjunctival and adhesion markers. Conclusions: The recombinant ELP resembling the ocular surface extracellular matrix was a suitable substratum to sustain epithelial cell attachment and growth. This type of polymer may be suitable for tissue engineering to restore vision by reconstructing the ocular surface.

Genetic Engineering of Protein-Based Polymers: The Example of Elastinlike Polymers

In spite of the enormous possibilities of macromolecules as key elements in developing advancedmaterials with increased functionality and complexity, the success in this development is often limitedby the randomness associated with polymer synthesis and the exponential increase in technical difficultiescaused by the attempt to reach a sufficiently high degree of complexity in the molecular design.This paper describes a new approach in the design of complex and highly functional macromolecules,the genetic engineering of protein-based macromolecules. The exploitation of the efficient machineryof protein synthesis in living cells opens a path to obtain extremely well-defined and complexmacromolecules. Different molecular designs are presented, with increasing degree of complexity,showing how the controlled increase in their complexity yields (multi)functional materials with moreselect and sophisticated properties. The simplest designs show interesting properties already, butthe adequate introduction of given chemical functions along the polymer chain presents an opportunityto expand the range of properties to enhanced smart behavior and self-assembly. Finally, examplesare given where those molecular designs further incorporate selected bioactivities in order to developmaterials for the most cutting-edge applications in the field of biomedicine and nano(bio)technology.

Dynamic cell culturing and its application to micropatterned, elastin-like protein-modified poly(N-isopropylacrylamide) scaffolds

In this study a tissue engineering scaffold was constructed from poly(N-isopropylacrylamide) (pNIPAM) to study the influence of strain on cell proliferation and differentiation. The effect of surface chemistry and topography on bone marrow mesenchymal stem cells was also investigated. Micropatterned pNIPAM films (channels with 10 microm groove width, 2 microm ridge width, 20 microm depth) were prepared by photopolymerization. The films were chemically modified by adsorption of a genetically engineered and temperature sensitive elastin-like protein (ELP). Dynamic conditions were generated by repeated temperature changes between 29 degrees C and 37 degrees C. ELP presence on the films enhanced initial cell attachment two fold (Day 1 cell number on films with ELP and without ELP were 27.6 x 10(4) and 13.2 x 10(4), respectively) but had no effect on proliferation in the long run. ELP was crucial for maintaining the cells attached on the surface in dynamic culturing (Day 7 cell numbers on the films with and without ELP were 81.4 x 10(4) and 12.1 x 10(4), respectively) and this enhanced the ability of pNIPAM films to transfer mechanical stress on the cells. Dynamic conditions improved cell proliferation (Day 21 cell numbers with dynamic and with static groups were 180.4 x 10(4) and 157.7 x 10(4), respectively) but decreased differentiation (Day 14 specific ALP values on the films of static and dynamic groups were 6.6 and 3.5 nmol/min/cell, respectively). Thus, a physically and chemically modified pNIPAM scaffold had a positive influence on the population of the scaffolds under dynamic culture conditions.

Elastin-like recombinamers: Biosynthetic strategies and biotechnological applications

The past few decades have witnessed the development of novel naturally inspired biomimetic materials, such as polysaccharides and proteins. Likewise, the seemingly exponential evolution of genetic‐engineering techniques and modern biotechnology has led to the emergence of advanced protein‐based materials with multifunctional properties. This approach allows extraordinary control over the architecture of the polymer, and therefore, monodispersity, controlled physicochemical properties, and high sequence complexity that would otherwise be impossible to attain. Elastin‐like recombinamers (ELRs) are emerging as some of the most prolific of these protein‐based biopolymers. Indeed, their inherent properties, such as biocompatibility, smart nature, and mechanical qualities, make these recombinant polymers suitable for use in numerous biomedical and nanotechnology applications, such as tissue engineering, “smart” nanodevices, drug delivery, and protein purification. Herein, we present recent progress in the biotechnological applications of ELRs and the most important genetic engineering‐based strategies used in their biosynthesis.

Amplified photoresponse of a p-phenylazobenzene derivative of an elastin-like polymer by α-cyclodextrin: The amplified ΔTt mechanism

α-Cyclodextrin (α-CD) acts as a photoamplifier if coupled into a photoresponsive system with an elastin-like polypeptide, wherein p-(phenylazo)phenylalanine acts as a light-sensitive switch (see Figure for mechanism). α-CD promotes a tunable offset, gain, and inversion of the photoresponse, and allows operation of micromachines at body or room temperature.

Elastin-like recombinamers as substrates for retinal pigment epithelial cell growth

The aim of this study is to investigate the use of elastin-like recombinamers (ELRs) as a substrate that can maintain the growth, phenotype, and functional characteristics of retinal pigment epithelial (RPE) cells efficiently and as a suitable carrier for the transplantation of autologous RPE cells for treatment of age-related macular degeneration (AMD). ELR films containing a bioactive sequence, RGD (ELR-RGD), and one with no specific sequence (ELR-IK) as control, were obtained by solvent-casting onto glass and subsequent cross-linking. ARPE19 cells were seeded on sterilized ELR films as well as on the control surfaces. Cells were analysed after 4, 24, 72, and 120 h to study cell adhesion, proliferation, cell viability, morphology, and specificity by staining with Trypan blue, DAPI, Rhodamin-Phalloidin and RPE65, ZO-1 antibodies and observing under fluorescence as well as electron microscope. ARPE19 cells seeded on both ELR films and controls were 100% viable and maintained their morphology and set of characteristics at the different time points studied. Cell proliferation on ELR-RGD was significantly higher than that found on ELR-IK at all time points, although it was less than the growth rate on polystyrene. ARPE19 cells grow well on ELR-RGD maintaining their phenotype. These results should be extended to further studies with fresh human RPE cells and in vivo studies to determine whether this ELR-RGD matrix could be used as a Bruchs membrane prosthesis and carrier for transplantation of RPE cells in patients suffering with AMD.

Elastin-like polypeptides in drug delivery ☆

The use of recombinant elastin-like materials, or elastin-like recombinamers (ELRs), in drug-delivery applications is reviewed in this work. Although ELRs were initially used in similar ways to other, more conventional kinds of polymeric carriers, their unique properties soon gave rise to systems of unparalleled functionality and efficiency, with the stimuli responsiveness of ELRs and their ability to self-assemble readily allowing the creation of advanced systems. However, their recombinant nature is likely the most important factor that has driven the current breakthrough properties of ELR-based delivery systems. Recombinant technology allows an unprecedented degree of complexity in macromolecular design and synthesis. In addition, recombinant materials easily incorporate any functional domain present in natural proteins. Therefore, ELR-based delivery systems can exhibit complex interactions with both their drug load and the tissues and cells towards which this load is directed. Selected examples, ranging from highly functional nanocarriers to macrodepots, will be presented.

A comparative study of cell behavior on different energetic and bioactive polymeric surfaces made from elastin-like recombinamers

This work explores cell behavior as a function of the topographic (fibers vs. films) and bioactive (RGD inclusion) design of multifunctional surfaces obtained from elastin-like recombinamers (ELRs). The surfaces have been analyzed for their differential roughness, wettability, and surface free-energy, as their precise contribution and importance of controlling critical aspects of cell behavior were investigated. The results suggest that the highest proliferative capacity of cells on the highly hydrophilic surfaces is more closely related to the surface properties than to the presence of adhesion sequences, although they act as an accelerating factor. However, on energetically unfavorable surfaces, bioactivity acquires decisive significance in ensuring cell adhesion and proliferation.

Cellular uptake of multilayered capsules produced with natural and genetically engineered biomimetic macromolecules.

Multilayered microcapsules of chitosan and biomimetic elastin-like recombinamers (ELRs) were prepared envisaging the intracellular delivery of active agents. Two ELRs containing either a bioactive RGD sequence or a scrambled non-functional RDG were used to construct two types of functionalized polymeric microcapsules, both of spherical shape ∼4μm in diameter. Cell viability studies with human mesenchymal stem cells (hMSCs) were performed using microcapsule/cell ratios between 5:1 and 100:1. After 3 and 72h of co-incubation, no signs of cytotoxicity were found, but cells incubated with RGD-functionalized microcapsules exhibited higher viability values than RDG cells. The internalization efficacy and bioavailability of encapsulated DQ-ovalbumin were assessed by monitoring the fluorescence changes in the cargo. The data show that surface functionalization did not significantly influence internalization by hMSCs, but the bioavailability of DQ-ovalbumin degraded faster when encapsulated within RGD-functionalized microcapsules. The microcapsules developed show promise for intracellular drug delivery with increased drug efficacy.