Elastin-like Polymers as Nanovaccines: Protein Engineering of Self-Assembled, Epitope-Exposing Nanoparticles.

In this chapter we describe two unconventional strategies for the formulation of new nanovaccines. Both strategies are based on obtaining chimeric genes that code for proteins in which the major antigens of the pathogens are fused to an elastin-like recombinamer (ELR) as carrier. ELRs are a family of synthetic protein biopolymers obtained using DNA recombinant techniques. The ELRs employed in the present chapter are block copolymers that are able to assemble, under controlled conditions, into nanoparticles similar to virus-like particles and to provoke an immune response. We describe the biosynthesis of ELRs genetically fused to an antigenic sequence from Mycobacterium tuberculosis and a simple procedure for obtaining stable nanoparticles displaying the antigen in the first strategy. The second approach describes the production of a DNA vaccine library consisting of plasmids codifying for major antigens from Rift Valley fever virus fused to different ELR-based block copolymer architectures.The procedures described can be adapted for the production of other chimeric DNA-protein vaccines based on protein polymer carriers.

Elastin-like recombinamer-based devices releasing Kv1.3 blockers for the prevention of intimal hyperplasia: An in vitro and in vivo study.

Coronary artery disease (CAD) is the most common cardiovascular disorder. Vascular surgery strategies for coronary revascularization (either percutaneous or open) show a high rate of failure because of restenosis of the vessel, due to phenotypic switch of vascular smooth muscle cells (VSMCs) leading to proliferation and migration. We have previously reported that the inhibition of Kv1.3 channel function with selective blockers represents an effective strategy for the prevention of restenosis in human vessels used for coronary angioplasty procedures. However, delivery systems for controlled release of these drugs have not been investigated. Here we tested the efficacy of several formulations of elastin like recombinamers (ELRs) hydrogels to deliver the Kv1.3 blocker PAP-1 in various restenosis models. The dose and time course of PAP-1 release from ELRs click hydrogels was able to inhibit human VSMC proliferation in vitro as well as remodeling of human vessels in organ culture and restenosis in in vivo models. We conclude that this combination of active compound and advanced delivery method could improve the outcomes of vascular surgery in patients. STATEMENT OF SIGNIFICANCE: Vascular surgery strategies for coronary revascularization show a high rate of failure, because of occlusion (restenosis) of the vessel, due to vascular smooth muscle cells proliferation and migration. We have previously reported that blockers of Kv1.3 channels represent an effective anti-restenosis therapy, but delivery systems for their controlled release have not being explored. Here we tested the efficacy of several formulations of elastin like recombinamers (ELRs) hydrogels to deliver the Kv1.3 blocker PAP-1 in various restenosis models, both in vivo and in vitro, and also in human vessels. We demonstrated that combination of active compound and advanced delivery method could improve the outcomes of vascular surgery in patients.

Functional characterization of an enzymatically degradable multi-bioactive elastin-like recombinamer.

One of the main goals in both tissue engineering and regenerative medicine is to design innovative synthetic scaffolds that can simulate and control the communication pathways between cells and the extracellular matrix (ECM). In this context, we describe herein the characterization of protein polymer, a recombinant elastin-like recombinamer (ELR) designed for developing tissue-engineered devices for use in vascular regeneration. This ELR is composed of an elastin-like backbone that contains a fibronectin domain, which provides specific, endothelial cell adhesion, and a protease target domain directed towards specific proteases involved in ECM remodeling. We also compare the specific response of endothelial and fibroblast cells to ELR scaffolds and show that cell adhesion and spreading on this ELR is significantly higher for endothelial cells than for fibroblasts. The reactivity of this polymer and its hydrogels to specific enzymatic degradation is demonstrated in vitro. As with natural elastin, enzymatic hydrolysis of the ELR produces elastin-derived peptides, or "matrikines", which, in turn, are potentially able to regulate important cell activities.

A DNA Vaccine Delivery Platform Based on Elastin-Like Recombinamer Nanosystems for Rift Valley Fever Virus.

This work analyzes the immunogenicity of six genetically engineered constructs based on elastin-like recombinamers (ELRs) fused to the Gn glycoprotein from Rift Valley fever virus (RVFV). Upon transfection, all constructs showed no effect on cell viability. While fusion constructs including ELR blocks containing hydrophobic amino acids (alanine or isoleucine) did not increase the expression of viral Gn in eukaryotic cells, glutamic acid- or valine-rich fusion proteins showed enhanced expression levels compared with the constructs encoding the viral antigen alone. However, in vivo DNA plasmid immunization assays determined that the more hydrophobic constructs reduced viremia levels after RVFV challenge to a higher extent than glutamic- or valine-rich encoding plasmids and were better inducers of cellular immunity as judged by in vitro restimulation experiments. Although the Gn-ELR fusion constructs did not surpass the protective efficacy of a plasmid vaccine expressing nonfused Gn, our results warrant further experiments directed to take advantage of the immunomodulatory potential of ELR biomaterials for improving vaccines against infectious diseases.

A double safety lock tumor-specific device for suicide gene therapy in breast cancer.

The complexity and continuous evolution of cancer make the design of novel strategies of treatment a constant challenge in biomedicine. Moreover, most of cancer treatments are still not tumor-specific and provoke high systemic toxicity. Herein we have developed a novel selective nanodevice to eliminate tumor cells while leaving healthy ones intact. To achieve this objective, a polyplex carrier, comprising an elastin like-recombinamer covalently conjugated to an aptamer and complexed with therapeutic DNA, was tested. This carrier forms a double-lock multifunctional device due to specific binding to a tumor cell marker and the selective expression of therapeutic DNA inside human breast-cancer cells. Due to the stability provided by ELRs, the homogeneous population of polyplexes obtained showed selective toxicity against cancer cells in in vitro and in vivo assay. Inhibition of tumor progression was detected early being very significant at the end point, with a dose-dependent reduction in tumor mass. Histological studies revealed a specific reduction in tumor parenchyma and in specific tumor cell markers. These results represent an important step toward the rational development of an efficient, safe and more specialized gene-delivery device for tumor therapy.

Self-Assembling ELR-Based Nanoparticles as Smart Drug-Delivery Systems Modulating Cellular Growth via Akt.

This work investigates the physicochemical properties and in vitro accuracy of a genetically engineered drug-delivery system based on elastin-like block recombinamers. The DNA recombinant techniques allowed us to create this smart complex polymer containing bioactive sequences for internalization, lysosome activation under acidic pH, and blockage of cellular growth by a small peptide inhibitor. The recombinant polymer reversibly self-assembled when the temperature was increased above 15 °C into nanoparticles with a diameter of 72 nm and negative surface charge. Furthermore, smart nanoparticles were shown to enter in the cells via clathrin-dependent endocytosis and properly blocked phosphorylation and consequent activation of Akt kinase. This system provoked apoptosis-mediated cell death in breast and colorectal cancer cells, which possess higher expression levels of Akt, whereas noncancerous cells, such as endothelial cells, fibroblasts, and mesenchymal stem cells, were not affected. Hence, we conclude that the conformational complexity of this smart elastin-like recombinamer leads to achieving successful drug delivery in targeted cells and could be a promising approach as nanocarriers with bioactive peptides to modulate multiple cellular processes involved in different diseases.

A novel lipase-catalyzed method for preparing ELR-based bioconjugates.

Herein we present a novel one-pot method for the chemical modification of elastin-like recombinamers (ELRs) in a mild and efficient manner involving enzymatic catalysis with Candida antarctica lipase B. The introduction of different functionalities into such ELRs could open up new possibilities for the development of advanced biomaterials for regenerative medicine and, specifically, for controlled drug delivery given their additional ability to respond to stimuli other than pH or temperature, such as glucose concentration or electromagnetic radiation. Candida antarctica lipase B immobilized on a macroporous acrylic resin (Novozym 435) was used to enzymatically couple different aminated substrates to a recombinamer containing carboxylic groups along its amino acid chain by way of an amidation reaction. A preliminary study of the kinetics of this amidation in response to different reaction conditions, such as solvent, temperature or reagent ratio, was carried out using a phenylazobenzene derivative (azo-NH2) as a model. The optimal amidation conditions were used to couple other amine reagents, such as phenylboronic acid (FB-NH2) or polyethylene glycol (PEG-NH2), thus allowing us to obtain photoresponsive, glucose-responsive or PEGylated ELRs that could potentially be useful as sensors in devices for controlled drug delivery.

Elastin-Like Recombinamers As Smart Drug Delivery Systems.

BACKGROUND: Drug delivery systems that are able to control the release of bioactive molecules and designed to carry drugs to target sites are of particular interest for tissue therapy. Moreover, systems comprising materials that can respond to environmental stimuli and promote self-assembly and higher order supramolecular organization are especially useful in the biomedical field. Objetive: This review focuses on biomaterials suitable for this purpose and that include elastin-like recombinamers (ELRs), a class of proteinaceous polymers bioinspired by natural elastin, designed using recombinant technologies. The self-assembly and thermoresponsive behaviour of these systems, along with their biodegradability, biocompatibility and well-defined composition as a result of their tailormade design, make them particularly attractive for controlled drug delivery. RESULTS: ELR-based delivery systems that allow targeted delivery are reviewed, especially ELR-drug recombinant fusion constructs, ELR-drug systems chemically bioconjugated in their monomeric and soluble forms, and drug encapsulation by nanoparticle-forming ELRs. Subsequently, the review focuses on those drug carriers in which smart release is triggered by pH or temperature with a particular focus on cancer treatments. Systems for controlled drug release based on depots and hydrogels that act as both a support and reservoir in which drugs can be stored will be described, and their applications in drug delivery discussed. Finally, smart drug-delivery systems not based on ELRs, including those comprising proteins, synthetic polymers and non-polymeric systems, will also be briefly discussed. CONCLUSION: Several different constructions based on ELRs are potential candidates for controlled drug delivery to be applied in advanced biomedical treatments.

Self-Assembling Elastin-Like Hydrogels for Timolol Delivery: Development of an Ophthalmic Formulation Against Glaucoma.

This work focuses on improving the effectiveness of current therapies against glaucoma by incorporating self-assembled polymers into the ophthalmic formulation. To that end, we first studied the influence of the dispersing medium on the mechanical performance of self-assembling elastin-like (EL) and silk-elastin-like (SEL) hydrogels by conducting rheological tests. These polymers were subsequently incorporated into the antiglaucoma formulation, which contains timolol maleate (TM) as active ingredient, and in vivo tests, namely adhesion tests and intraocular pressure measurements (IOP), were performed in New Zealand rabbits. An enhanced reduction in IOP due to the presence of the polymers was observed. Moreover, differences in the effectiveness between both EL- and SEL-hydrogels, which can be explained on the basis of the different rheological properties displayed by these two systems, were also encountered. The results point to the potential of this system as a basis for the development of an ophthalmic formulation against glaucoma.

Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

Endoglin (CD105) is an accessory component of the TGF-ß receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

Biocompatible ELR-Based Polyplexes Coated with MUC1 Specific Aptamers and Targeted for Breast Cancer Gene Therapy.

The search for new and biocompatible materials with high potential for improvement is a challenge in gene delivery applications. A cell type specific vector made of elastin-like recombinamer (ELR) and aptamers has been specifically designed for the intracellular delivery of therapeutic material for breast cancer therapy. A lysine-enriched ELR was constructed and complexed with plasmid DNA to give positively charged and stable polyplexes. Physical characterization of these polyplexes showed a particle size of around 140 nm and a zeta potential of approximately +40 mV. The incorporation of MUC1-specific aptamers into the polyplexes resulted in a slight decrease in zeta potential but increased cell transfection specificity for MCF-7 breast cancer cells with respect to a MUC1-negative tumor line. After showing the transfection ability of this aptamer-ELR vector which is facilitated mainly by macropinocytosis uptake, we demonstrated its application for suicide gene therapy using a plasmid containing the gene of the toxin PAP-S. The strategy developed in this work about using ELR as polymeric vector and aptamers as supplier of specificity to deliver therapeutic material into MUC1-positive breast cancer cells shows promising potential and continues paving the way for ELRs in the biomedical field.

Amphiphilic Elastin-Like Block Co-Recombinamers Containing Leucine Zippers: Cooperative Interplay between Both Domains Results in Injectable and Stable Hydrogels.

Many biological processes are regulated by reversible binding events, with these interactions between macromolecules representing the core of dynamic chemistry. As such, any attempt to gain a better understanding of such interactions, which would pave the way to the extrapolation of natural designs to create new advanced systems, is clearly of interest. This work focuses on the development of a leucine zipper-elastin-like recombinamer (ZELR) in order to elucidate the behavior of such domains when coexisting along the same molecule and to engineer reversible, injectable and stable hydrogels. The unique propensity of the Z-moiety selected to dimerize, together with the thermosensitive behavior of the ELR, which has been constructed as a thermosensitive amphiphilic tetrablock, has been engineered into a single recombinant molecule. In this molecular design, the Z-moieties are unable to form a network, while the ELR is below its Tt, thus, guaranteeing the liquid-like state of the system. However, this situation changes rapidly as the temperature increases above Tt, where a stable hydrogel is formed, as demostrated by rheological tests. The inability of the ELR molecule (without Z-domains) to form such a stable hydrogel above Tt clearly points to a positive cooperative effect between these two domains (Z and EL), and no conformational changes in the former are involved, as demonstrated by circular dichroism analysis. AFM shows that Z-motifs seem to induce the aggregation of micelles, which supports the enhanced stability displayed by ZELRs when compared to ELR at the macroscale level. To the best of our knowledge, this is the first time that such an interplay between these two domains has been reported. Furthermore, the cytocompatibility of the resulting hydrogels opens the door to their use in biomedical applications.

Self-organized ECM-mimetic model based on an amphiphilic multiblock silk-elastin-like corecombinamer with a concomitant dual physical gelation process.

Although significant progress has been made in the area of injectable hydrogels for biomedical applications and model cell niches, further improvements are still needed, especially in terms of mechanical performance, stability, and biomimicry of the native fibrillar architecture found in the extracellular matrix (ECM). This work focuses on the design and production of a silk-elastin-based injectable multiblock corecombinamer that spontaneously forms a stable physical nanofibrillar hydrogel under physiological conditions. That differs from previously reported silk-elastin-like polymers on a major content and predominance of the elastin-like part, as well as a more complex structure and behavior of such a part of the molecule, which is aimed to obtain well-defined hydrogels. Rheological and DSC experiments showed that this system displays a coordinated and concomitant dual gelation mechanism. In a first stage, a rapid, thermally driven gelation of the corecombinamer solution takes place once the system reaches body temperature due to the thermal responsiveness of the elastin-like (EL) parts and the amphiphilic multiblock design of the corecombinamer. A bridged micellar structure is the dominant microscopic feature of this stage, as demonstrated by AFM and TEM. Completion of the initial stage triggers the second, which is comprised of a stabilization, reinforcement, and microstructuring of the gel. FTIR analysis shows that these events involve the formation of ß-sheets around the silk motifs. The emergence of such ß-sheet structures leads to the spontaneous self-organization of the gel into the final fibrous structure. Despite the absence of biological cues, here we set the basis of the minimal structure that is able to display such a set of physical properties and undergo microscopic transformation from a solution to a fibrous hydrogel. The results point to the potential of this system as a basis for the development of injectable fibrillar biomaterial platforms toward a fully functional, biomimetic, artificial extracellular matrix, and cell niches.

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.

Recent contributions of elastin-like recombinamers to biomedicine and nanotechnology.

The emergence of the new scientific field known as nanomedicine is being catalyzed by multiple improvements in nanoscience techniques and significant progress in materials science, especially as regards the testing of novel and sophisticated biomaterials. This conjuncture has furthered the development of promising instruments in terms of detection, bioanalysis, therapy, diagnostics and imaging. Some of the most innovative new biomaterials are protein-inspired biomimetic materials in which modern biotechnology and genetic-engineering techniques complement the huge amount of information afforded by natural protein evolution to create advanced and tailor-made multifunctional molecules. Amongst these protein-based biomaterials, Elastin-like Recombinamers (ELRs) have demonstrated their enormous potential in the fields of biomedicine and nanoscience in the last few years. This broad applicability derives from their unmatched properties, particularly their recombinant and tailor-made nature, the intrinsic characteristics derived from their elastin-based origin (mainly their mechanical properties and ability to self-assemble as a result of their stimuli-responsive behavior), their proven biocompatibility and biodegradability, as well as their versatility as regards incorporating advanced chemical or recombinant modifications into the original structure that open up an almost unlimited number of multifunctional possibilities in this developing field. This article provides an updated review of the recent challenges overcome by using these recombinant biomaterials in the fields of nano- and biomedicine, ranging from nanoscale applications in surface modifications and self-assembled nanostructures to drug delivery and regenerative medicine.

Layer-by-layer film growth using polysaccharides and recombinant polypeptides: a combinatorial approach.

Nanostructured films consisting of polysaccharides and elastin-like recombinamers (ELRs) are fabricated in a layer-by-layer manner. A quartz-crystal microbalance with dissipation monitoring (QCM-D) is used to follow the buildup of hybrid films containing one polysaccharide (chitosan or alginate) and one of several ELRs that differ in terms of amino acid content, length, and biofunctionality in situ at pH 4.0 and pH 5.5. The charge density of the ingredients at each pH is determined by measuring their ζ-potential, and the thickness of a total of 36 different films containing five bilayers is estimated using the Voigt-based viscoelastic model. A comparison of the values obtained reveals that thicker films can be obtained when working at a pH close to the acidity constant of the polysaccharide used (near-pKa conditions), suggesting that the construction of such films is more favorable when based on the presence of hydrophobic interactions between ELRs and partially neutralized polysaccharides. Further analysis shows that the molecular weight of the ELRs plays only a minor role in defining the growth tendency. When taken together, these results point to the most favorable conditions for constructing nanostructured films from natural and distinct recombinant polypeptides that can be tuned to exhibit specialized biofunctionality for tissue-engineering, drug-delivery, and biotechnological applications.

Multifunctional compartmentalized capsules with a hierarchical organization from the nano to the macro scales.

Inspired by the cells' structure, we present compartmentalized capsules with temperature and magnetic-based responsiveness and hierarchical organization ranging from the nano- to the visible scales. Liquefied alginate macroscopic beads coated with a layer-by-layer (LbL) chitosan/alginate shell served as containers both for model fluorophores and microcapsules, which in their turn encapsulated either another fluorophore or magnetic nanoparticles (MNPs). The microcapsules were coated with a temperature-responsive chitosan/elastin-like recombinamer (ELR) nanostructured shell. By varying the temperature from 25 to 37 °C, the two-hour release of rhodamine encapsulated within the microcapsules and its diffusion through the external compartment decreased from 84% and 71%. The devices could withstand handling and centrifugal stress, with 50% remaining intact at a rotation speed of 2000g. MNPs attributed magnetic responsiveness toward external magnetic fields. Such a customizable system can be envisaged to transport bioactive agents and cells in tissue engineering applications.

Immunomodulatory nanoparticles from elastin-like recombinamers: single-molecules for tuberculosis vaccine development.

This study investigates both the physicochemical properties and immunogenicity of a genetically engineered elastin-like block corecombinamer (ELbcR) containing a major membrane protein sequence from Mycobacterium tuberculosis. The recombinant production of this ELbcR allows the production of large quantities of safe, antigenic particle-based constructs that directly and reversibly self-assemble into highly biocompatible, multivalent, monodisperse, and stable nanovesicles with a diameter of 55 nm from the same gene product using a highly efficient and cost-effective inverse transition cycling (ITC) procedure. The compositional complexity of these vesicles is retained after secondary processes such as endotoxin removal, sterilization, and lyophilization. An initial pro-chemotactic cytokine response (IL-1ß) followed by a pro-Th2/IL-5 response was observed in mice plasma following subcutaneous administration of the antigen-loaded nanovesicles in mice. This biphasic model of cytokine production was coupled with humoral isotype switching from IgM- to IgG-specific antibodies against the antigen, which was only observed in the presence of both the antigen and the polymer in the same construct and in the absence of additional adjuvants.

Biomimetic calcium phosphate mineralization with multifunctional elastin-like recombinamers.

Biomimetic hybrid materials based on a polymeric and an inorganic component such as calcium phosphate are potentially useful for bone repair. The current study reports on a new approach toward biomimetic hybrid materials using a set of recombinamers (recombinant protein materials obtained from a synthetic gene) as crystallization additive for calcium phosphate. The recombinamers contain elements from elastin, an elastic structural protein, and statherin, a salivary protein. Via genetic engineering, the basic elastin sequence was modified with the SN(A)15 domain of statherin, whose interaction with calcium phosphate is well-established. These new materials retain the biocompatibility, "smart" nature, and desired mechanical behavior of the elastin-like recombinamer (ELR) family. Mineralization in simulated body fluid (SBF) in the presence of these recombinamers reveals surprising differences. Two of the polymers inhibit calcium phosphate deposition (although they contain the statherin segment). In contrast, the third polymer, which has a triblock structure, efficiently controls the calcium phosphate formation, yielding spherical hydroxyapatite (HAP) nanoparticles with diameters from 1 to 3 nm after 1 week in SBF at 37 °C. However, at lower temperatures, no precipitation is observed with any of the polymers. The data thus suggest that the molecular design of ELRs containing statherin segments and the selection of an appropriate polymer structure are key parameters to obtain functional materials for the development of intelligent systems for hard tissue engineering and subsequent in vivo applications.

Emerging applications of multifunctional elastin-like recombinamers.

Elastin-like recombinamers have grown in popularity in the field of protein-inspired biomimetic materials and have found widespread use in biomedical applications. Modern genetic-engineering techniques have allowed the design of multifunctional materials with an extraordinary control over their architecture and physicochemical properties, such as stimuli-responsiveness, monodispersity, biocompatibility or self-assembly, amongst others. Indeed, these materials are playing an increasingly important role in a diverse range of applications, such as drug delivery, tissue engineering and 'smart' systems. Herein, we review some of the most interesting examples of recent advances and progressive applications of elastin-like recombinamers in biomaterial and nano-engineering sciences in recent years.