CD44-targeted nanoparticles for co-delivery of docetaxel and an Akt inhibitor against colorectal cancer.

New strategies to develop drug-loaded nanocarriers with improved therapeutic efficacy are needed for cancer treatment. Herein we report a novel drug-delivery nanosystem comprising encapsulation of the chemotherapeutic drug docetaxel (DTX) and recombinant fusion of a small peptide inhibitor of Akt kinase within an elastin-like recombinamer (ELR) vehicle. This combined approach is also precisely targeted to colorectal cancer cells by means of a chemically conjugated DNA aptamer specific for the CD44 tumor marker. This 53 nm dual-approach nanosystem was found to selectively affect cell viability (2.5 % survival) and proliferation of colorectal cancer cells in vitro compared to endothelial cells (50 % survival), and to trigger both apoptosis- and necrosis-mediated cell death. Our findings also show that the nanohybrid particles remain stable under physiological conditions, trigger sustained drug release and possess an adequate pharmacokinetic profile after systemic intravenous administration. In vivo assays showed that these dual-approach nanohybrids significantly reduced the number of tumor polyps along the colorectal tract in a murine colorectal cancer model. Furthermore, systemic administration of advanced nanohybrids induced tissue recovery by improving the morphology of gastrointestinal crypts and the tissue architecture. Taken together, these findings indicate that our strategy of an advanced dual-approach nanosystem allows us to achieve successful controlled release of chemotherapeutics in cancer cells and may have a promising potential for colorectal cancer treatment.

Metronomic Anti-Cancer Therapy: A Multimodal Therapy Governed by the Tumor Microenvironment.

The concept of cancer as a systemic disease, and the therapeutic implications of this, has gained special relevance. This concept encompasses the interactions between tumor and stromal cells and their microenvironment in the complex setting of primary tumors and metastases. These factors determine cellular co-evolution in time and space, contribute to tumor progression, and could counteract therapeutic effects. Additionally, cancer therapies can induce cellular and molecular responses in the tumor and host that allow them to escape therapy and promote tumor progression. In this study, we describe the vascular network, tumor-infiltrated immune cells, and cancer-associated fibroblasts as sources of heterogeneity and plasticity in the tumor microenvironment, and their influence on cancer progression. We also discuss tumor and host responses to the chemotherapy regimen, at the maximum tolerated dose, mainly targeting cancer cells, and a multimodal metronomic chemotherapy approach targeting both cancer cells and their microenvironment. In a combination therapy context, metronomic chemotherapy exhibits antimetastatic efficacy with low toxicity but is not exempt from resistance mechanisms. As such, a better understanding of the interactions between the components of the tumor microenvironment could improve the selection of drug combinations and schedules, as well as the use of nano-therapeutic agents against certain malignancies.

Smart Nanoparticles as Advanced Anti-Akt Kinase Delivery Systems for Pancreatic Cancer Therapy.

Pancreatic cancer is one of the deadliest cancers partly due to late diagnosis, poor drug delivery to the target site, and acquired resistance to therapy. Therefore, more effective therapies are urgently needed to improve the outcome of patients. In this work, we have tested self-assembling genetically engineered polymeric nanoparticles formed by elastin-like recombinamers (ELRs), carrying a small peptide inhibitor of the protein kinase Akt, in both PANC-1 and patient-derived pancreatic cancer cells (PDX models). Nanoparticle cell uptake was measured by flow cytometry, and subcellular localization was determined by confocal microscopy, which showed a lysosomal localization of these nanoparticles. Furthermore, metabolic activity and cell viability were significantly reduced after incubation with nanoparticles carrying the Akt inhibitor in a time- and dose-dependent fashion. Self-assembling 73 ± 3.2 nm size nanoparticles inhibited phosphorylation and consequent activation of Akt protein, blocked the NF-κB signaling pathway, and triggered caspase 3-mediated apoptosis. Furthermore, in vivo assays showed that ELR-based nanoparticles were suitable devices for drug delivery purposes with long circulating time and minimum toxicity. Hence, the use of these smart nanoparticles could lead to the development of more effective treatment options for pancreatic cancer based on the inhibition of Akt.

Soft Hydrogel Inspired by Elastomeric Proteins.

Elastin polypeptides based on -VPGVG- repeated motifs are widely used in the production of biomaterials because they are stimuli-responsive systems. On the other hand, glycine-rich sequences, mainly present in tropoelastin terminal domains, are responsible for the elastin self-assembly. In a previous study, we have recombinantly expressed a chimeric polypeptide, named resilin, elastin, and collagen (REC), inspired by glycine-rich motifs of elastin and containing resilin and collagen sequences as well. Herein, a three-block polypeptide, named (REC)3, was expressed starting from the previous monomer gene by introducing key modifications in the sequence. The choice was mandatory because the uneven distribution of the cross-linking sites in the monomer precluded the hydrogel production. In this work, the cross-linked polypeptide appeared as a soft hydrogel, as assessed by rheology, and the linear un-cross-linked trimer self-aggregated more rapidly than the REC monomer. The absence of cell-adhesive sequences did not affect cell viability, while it was functional to the production of a material presenting antiadhesive properties useful in the integration of synthetic devices in the body and preventing the invasion of cells.

Advanced nanomedicine and cancer: Challenges and opportunities in clinical translation.

Cancer has reached pandemic dimensions in the whole world. Although current medicine offers multiple treatment options against cancer, novel therapeutic strategies are needed due to the low specificity of chemotherapeutic drugs, undesired side effects and the presence of different incurable types of cancer. Among these new strategies, nanomedicine arises as an encouraging approach towards personalized medicine with high potential for present and future cancer patients. Therefore, nanomedicine aims to develop novel tools with wide potential in cancer treatment, imaging or even theranostic purposes. Even though numerous preclinical studies have been published with successful preliminary results, promising nanosystems have to face multiple obstacles before adoption in clinical practice as safe options for patients with cancer. In this MiniReview, we provide a short overview on the latest advances in current nanomedicine approaches, challenges and promising strategies towards more accurate cancer treatment.

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 transferrin receptor-binding mucoadhesive elastin-like recombinamer: In vitro and in vivo characterization.

The development of mucoadhesive materials is of great interest and is also a major challenge. Being adsorption sites, mucosae are suitable targets for drug delivery, but as defensive barriers they are complex biological surfaces to interact with, mainly due to their protective mucus layer. As such, first- and second-generation mucoadhesives focused on material-mucus interactions, whereas the third generation of mucoadhesives introduced structural motifs that are able to interact with the cells beneath the mucus layer. The combination of different prerequisites (water solubility, soft gel formation at body temperature and able to interact with the mucus) in a single molecule is easily achieved using elastin-like recombinamers (ELRs) given their multiple block design. Moreover, we have been able to introduce a short amino-acid sequence known as T7 that is able to bind to transferrin receptors in the epithelial cell layer. The T7 sequence enhances the cell-binding properties of the mucoadhesive ELR (MELR), as demonstrated using a Caco-2 epithelial cell model. In vivo experiments confirmed the mucoadhesive properties found in vitro. STATEMENT OF SIGNIFICANCE: The development of a mucoadhesive material is a major challenge. Mucosae are suitable targets for drug delivery, but as defense barriers, they are complex surfaces to interact with. In this work we report the first ELR that combines different functional blocks, in a single molecule, which provide it with the properties of soft-gel forming at body temperature and being able of efficiently adhering to the mucus layer of mucosas, as well as to the underlying epithelial cell layer, as demonstrated in vitro and in vivo. The rationally designed materials presented in this work sets the basis for developing ELR-based, mucosa-directed drug delivery systems, which could improve patient's compliance, enhancing drug retention at the mucosal site.

Genetically Engineered Elastin-based Biomaterials for Biomedical Applications.

Protein-based polymers are some of the most promising candidates for a new generation of innovative biomaterials as recent advances in genetic-engineering and biotechnological techniques mean that protein-based biomaterials can be designed and constructed with a higher degree of complexity and accuracy. Moreover, their sequences, which are derived from structural protein-based modules, can easily be modified to include bioactive motifs that improve their functions and material-host interactions, thereby satisfying fundamental biological requirements. The accuracy with which these advanced polypeptides can be produced, and their versatility, self-assembly behavior, stimuli-responsiveness and biocompatibility, means that they have attracted increasing attention for use in biomedical applications such as cell culture, tissue engineering, protein purification, surface engineering and controlled drug delivery. The biopolymers discussed in this review are elastin-derived protein-based polymers which are biologically inspired and biomimetic materials. This review will also focus on the design, synthesis and characterization of these genetically encoded polymers and their potential utility for controlled drug and gene delivery, as well as in tissue engineering and regenerative medicine.

Regeneration of hyaline cartilage promoted by xenogeneic mesenchymal stromal cells embedded within elastin-like recombinamer-based bioactive hydrogels.

Over the last decades, novel therapeutic tools for osteochondral regeneration have arisen from the combination of mesenchymal stromal cells (MSCs) and highly specialized smart biomaterials, such as hydrogel-forming elastin-like recombinamers (ELRs), which could serve as cell-carriers. Herein, we evaluate the delivery of xenogeneic human MSCs (hMSCs) within an injectable ELR-based hydrogel carrier for osteochondral regeneration in rabbits. First, a critical-size osteochondral defect was created in the femora of the animals and subsequently filled with the ELR-based hydrogel alone or with embedded hMSCs. Regeneration outcomes were evaluated after three months by gross assessment, magnetic resonance imaging and computed tomography, showing complete filling of the defect and the de novo formation of hyaline-like cartilage and subchondral bone in the hMSC-treated knees. Furthermore, histological sectioning and staining of every sample confirmed regeneration of the full cartilage thickness and early subchondral bone repair, which was more similar to the native cartilage in the case of the cell-loaded ELR-based hydrogel. Overall histological differences between the two groups were assessed semi-quantitatively using the Wakitani scale and found to be statistically significant (p < 0.05). Immunofluorescence against a human mitochondrial antibody three months post-implantation showed that the hMSCs were integrated into the de novo formed tissue, thus suggesting their ability to overcome the interspecies barrier. Hence, we conclude that the use of xenogeneic MSCs embedded in an ELR-based hydrogel leads to the successful regeneration of hyaline cartilage in osteochondral lesions.

3D silicon doped hydroxyapatite scaffolds decorated with Elastin-like Recombinamers for bone regenerative medicine.

The current study reports on the manufacturing by rapid prototyping technique of three-dimensional (3D) scaffolds based on silicon substituted hydroxyapatite with Elastin-like Recombinamers (ELRs) functionalized surfaces. Silicon doped hydroxyapatite (Si-HA), with Ca10(PO4)5.7(SiO4)0.3(OH)1.7h0.3 nominal formula, was surface functionalized with two different types of polymers designed by genetic engineering: ELR-RGD that contain cell attachment specific sequences and ELR-SNA15/RGD with both hydroxyapatite and cells domains that interact with the inorganic phase and with the cells, respectively. These hybrid materials were subjected to in vitro assays in order to clarify if the ELRs coating improved the well-known biocompatible and bone regeneration properties of calcium phosphates materials. The in vitro tests showed that there was a total and homogeneous colonization of the 3D scaffolds by Bone marrow Mesenchymal Stromal Cells (BMSCs). In addition, the BMSCs were viable and able to proliferate and differentiate into osteoblasts. STATEMENT OF SIGNIFICANCE: Bone tissue engineering is an area of increasing interest because its main applications are directly related to the rising life expectancy of the population, which promotes higher rates of several bone pathologies, so innovative strategies are needed for bone tissue regeneration therapies. Here we use the rapid prototyping technology to allow moulding ceramic 3D scaffolds and we use different bio-polymers for the functionalization of their surfaces in order to enhance the biological response. Combining the ceramic material (silicon doped hydroxyapatite, Si-HA) and the Elastin like Recombinamers (ELRs) polymers with the presence of the integrin-mediate adhesion domain alone or in combination with SNA15 peptide that possess high affinity for hydroxyapatite, provided an improved Bone marrow Mesenchymal Stromal Cells (BMSCs) differentiation into osteoblastic linkage.

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.

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.

Biocompatible elastin-like click gels: design, synthesis and characterization.

Elastin-like recombinamer click gels (ELR-CGs) for biomedical applications, such as drug delivery or tissue engineering, have been developed by taking advantage of the click reaction (CuAAC) in the absence of traditional crosslinking agents. ELRs are functionalized with alkyne and azide groups using conventional chemical techniques to introduce the reactivity required to carry out the 1,3-dipolar cycloaddition under mild biocompatible conditions, with no toxic by-products and in short reaction times. Hydrogels with moduli in the range 1,000-10,000 Pa have been synthesized, characterized, and tested in vitro against several cell types. The cells embedded into ELR-CGs possessed high viability and proliferation rate. The mechanical properties, porosity and swelling of the resulting ELR-CGs can easily be tuned by adjusting the ELR concentration. We also show that it is possible to replicate different patterns on the hydrogel surface, thus allowing the use of this type of hydrogel to improve applications that require cell guidance or even differentiation depending on the surface topography.

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.

Synthesis of genetically engineered protein polymers (recombinamers) as an example of advanced self-assembled smart materials.

In this chapter, we describe two methods for bio-producing recombinant repetitive polypeptide polymers for use in biomedical devices. These polymers, known as elastin-like recombinamers (ELRs), are derived from the repetition of selected amino acid domains of extracellular matrix proteins with the aim of recreating their mechanical and physiological features. The proteinaceous nature of ELRs allows us to make use of the natural biosynthetic machinery of heterologous hosts to express advanced and large polymers or "recombinamers." Despite the essentially unlimited possibilities for designing recombinamers, the production of synthetic genes to encode them should allow us to overcome the difficulties surrounding bioproduction of these non-natural monotonous DNA and protein sequences. The aim of this work is to supply the biotechnologist with fine-tuning methods to biosynthesize advanced self-assembled smart materials.

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 Bruch's membrane prosthesis and carrier for transplantation of RPE cells in patients suffering with AMD.

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.

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 (10(4)) 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.