“Advanced nanomedicine and cancer: challenges and opportunities in clinical translation“

  J. Gonzalez-Valdivieso, A. Girotti, J. Schneider, F.J. Arias
International Journal of Pharmaceutics, 599, (2021), 120438.
Impact Factor (JCR): 5.875   DOI: 10.1016/j.ijpharm.2021.120438

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.

“Production of elastin-like recombinamer-based nanoparticles for Docetaxel encapsulation and use as smart drug-delivery systems using a supercritical anti-solvent process”

  R. Vallejo, J. Gonzalez-Valdivieso, M. Santos, S. Rodriguez-Rojo, F.J. Arias.
Journal of Industrial and Engineering Chemistry, 93 (2021), 361-374.
Impact Factor (JCR): 6.064   DOI: 10.1016/j.jiec.2020.10.013

This study presents a new groundbreaking methodology for integrating innovative concepts to develop novel drug-delivery strategies. This methodology combines genetically engineered elastin-like recombinamers (ELRs) with supercritical fluid (SCF) techniques to encapsulate a poorly water-soluble drug in a one-step process. The chemotherapeutic agent docetaxel (DTX) is encapsulated with a block copolymer ELR containing the RGD peptide, a specific target sequence for cancer cells, using the supercritical anti-solvent (SAS) technique in a high process yield of up to 70%. SEM studies show spherical microparticles of 10 μm after encapsulation. After dispersion under physiological conditions, microparticles disaggregate into stable monodisperse nanoparticles of 40 nm size and −30 mV ζ-potential. This protects the drug, as confirmed by NMR analysis, thereby increasing the water solubility of DTX up to fifty orders of magnitude. The delivery process is governed by the Fick diffusion mechanism and indicates that the presence of DTX on the particles surface is practically negligible. Cellular assays showed that, due to the presence of the cancer target sequence RGD, breast cancer cells were more affected than human endothelial cells, thus meaning that the strategy developed in this work opens the way to new controlled release systems more precise than non-selective chemotherapeutic drugs.


“Aptamer-Functionalized Natural Protein-based Polymers as Innovative Biomaterials”

  A. Girotti, S. Escalera Anzola, I. Alonso Sampedro, J. Gonzalez Valdivieso, F.J. Arias.
Pharmaceutics, 12, (2020), 1115.
Impact Factor (JCR): 6.321   DOI:10.3390/pharmaceutics12111115

Biomaterials science is one of the most rapidly evolving fields in biomedicine. However, although novel biomaterials have achieved well-defined goals, such as the production of devices with improved biocompatibility and mechanical properties, their development could be more ambitious. Indeed, the integration of active targeting strategies has been shown to allow spatiotemporal control of cell–material interactions, thus leading to more specific and better-performing devices. This manuscript reviews recent advances that have led to enhanced biomaterials resulting from the use of natural structural macromolecules. In this regard, several structural macromolecules have been adapted or modified using biohybrid approaches for use in both regenerative medicine and therapeutic delivery. The integration of structural and functional features and aptamer targeting, although still incipient, has already shown its ability and wide-reaching potential. In this review, we discuss aptamer-functionalized hybrid protein-based or polymeric biomaterials derived from structural macromolecules, with a focus on bioresponsive/bioactive systems. 

“Functional characterization of an enzymatically degradable multi-bioactive Elastin-Like Recombinamer” 

    A. Girotti, J. Gonzalez-Valdivieso, M. Santos, L. Martin, F.J. Arias
International Journal of Biological Macromolecules, 164, 1640-1648 (2020)
Impact Factor (JCR): 6.953    DOI: 10.1016/j.ijbiomac.2020.08.004

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.

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

  S. Moreno-Estar; S. Serrano; M.C. Arevalo-Martinez; P. Cidad; J.R López-López; M. Santos; M. T. Perez-Garcia and F. J. Arias.
Acta Biomaterialia, 115, 264-274 (2020)
Impact Factor (JCR): 8.947     DOI: 10.1016/j.actbio.2020.07.053

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.

“A DNA vaccine delivery platform based on Elastin-Like Recombinamer nanosystems for Rift Valley fever virus”

J. Gonzalez-Valdivieso, B. Borrego, A. Girotti, S. Moreno, A. Brun, J. Bermejo-Martin, and F.J. Arias
Molecular Pharmaceutics, 17, 1608-1620 (2020)
Impact Factor (JCR): 4.939   DOI: 10.1021/acs.molpharmaceut.0c00054

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”

M.J. Piña, A. Girotti, S. Serrano, R. Muñoz, J. C. Rodriguez-Cabello and F.J. Arias.
Cancer Letters, 470, 43-53 (2020)
Impact Factor (JCR): 8.679   DOI: 10.1016/j.canlet.2019.11.031

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”

J. Gonzalez-Valdivieso, A. Girotti, R. Muñoz, J. C. Rodriguez-Cabello and F.J. Arias.
Biomacromolecules, 20, 1996-2007 (2019)
Impact Factor (JCR): 6.092  DOI: 10.1021/acs.biomac.9b00206

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.

“Genetically Engineered Elastin-based Biomaterials for Biomedical Applications”

  M. Santos, S. Serrano-Dúcar, J. González-Valdivieso, R. Vallejo, A. Girotti, P. Cuadrado and F.J. Arias
Current Medicinal Chemistry, 26, 7117-7146 (2019)
Impact Factor (JCR): 4.184  DOI: 10.2174/0929867325666180508094637  

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.


“Elastin-like recombinamers as smart drug delivery systems”

F.J. Arias, M. Santos, A. Ibáñez-Fonseca, MªJ. Piña and S. Serrano-Dúcar
Current Drug Targets, 19, 360-379 (2018)
Impact Factor (JCR): 2.642   DOI: 10.2174/1389450117666160201114617 

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. 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. 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”

A. Fernández-Colino, D. Quinteros, D. Allemandi, A. Girotti, S. Palma and F.J. Arias
Molecular Pharmaceutics, 14, 4498-4508 (2017)
Impact Factor (JCR): 4.556   DOI: 10.1021/acs.molpharmaceut.7b00615

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.

“Advanced systems for controlled drug delivery from chemically modified Elastin-like Recombinamers”

M. Santos, C. González-Obeso, D. Orbanic, E. Pérez del Río and F.J. Arias
Current Organic Chemistry, 21: 21-33 (2017)
Impact Factor (JCR): 2.193   DOI: 10.2174/1385272820666160511120925

Targeted drug delivery is a new multidisciplinary field that aims to develop innovative nanomaterials, tools and devices to deploy a therapeutic agent to specific parts of the body where there is solely diseased tissue, thereby avoiding interaction with healthy tissue. Advanced drug-delivery systems attempt to control the site of action and release rate and act by means of either a physiological or a chemical trigger. In this sense, stimuliresponsive biomaterials are of special interest for application as components of drug-delivery devices. This review discusses the use of elastin-like recombinamers (ELR) in drug-delivery systems. These biopolymers possess special properties that encompass biodegradability, bioactivity and stimuli-responsiveness. Their tailormade design using recombinant DNA technologies allows an absolute control of their amino acid sequence and design of the most appropriate macromolecule for each application. Firstly, devices based on monomeric elastinlike recombinamers which have been chemically modified to attach functionalities that enable us to follow or direct their distribution or anticancer drugs in an attempt to improve drug-conjugate uptake are described. Secondly, ELRs that form part of nanoparticles as drug carriers will be studied in their different versions, including nanoparticles chemically reinforced by interchain cross-linking, nanoparticles formed by self-assembly of chemically modified ELRs to achieve amphiphilic properties and multifunctional composites made up of nanoparticles coated with ELRs. Finally, recent advances in the area of 3D platforms for drug delivery, comprising interconnected hydrogels and ELR-based coacervates in the form of depots, will be reviewed.


“Anti-human Endoglin (hCD105) Immunotoxin Containing Recombinant Single Chain Ribosome-inactivating Protein Musarmin 1”

B. Barriuso, P. Antolín, F. J. Arias, A. Girotti, P. Jiménez, M. Córdoba-Diaz, D. Córdoba-Diaz, T. Girbés
Toxins, 8: E184 (2016)
Impact Factor (JCR): 3.273   DOI: 10.3390/toxins8060184

Endoglin (CD105) is an accessory component of the TGF-β 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”

M.J. Piña, A. Girotti, M. Santos, J.C. Rodríguez-Cabello and F.J. Arias
Molecular Pharmaceutics, 13: 795-808 (2016)
Impact Factor (JCR): 4.440   DOI: 10.1021/acs.molpharmaceut.5b00712

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.

“Development of a mechanism and an accurate and simple mathematical model for the description of drug release: application to a relevant example of acetazolamide-controlled release from a bio-inspired elastin-based hydrogel”

A. Fernández Colino, J. M. Bermúdez, F. J. Arias, D. A. Quinteros and E. E. Gonzo
Materials Science & Engineering C-Materials for Biological Applications, 61: 286-292 (2016)
Impact Factor (JCR): 4.164   DOI: 10.1016/j.msec.2015.12.050t.5b00712

Transversality between mathematical modeling, pharmacology, and materials science is essential in order to achieve controlled-release systems with advanced properties. In this regard, the area of biomaterials provides a platform for the development of depots that are able to achieve controlled release of a drug, whereas pharmacology strives to find new therapeutic molecules and mathematical models have a connecting function, providing a rational understanding by modeling the parameters that influence the release observed. Herein we present a mechanism which, based on reasonable assumptions, explains the experimental data obtained very well. In addition, we have developed a simple and accurate “lumped” kinetics model to correctly fit the experimentally observed drug-release behavior. This lumped model allows us to have simple analytic solutions for the mass and rate of drug release as a function of time without limitations of time or mass of drug released, which represents an important step-forward in the area of in vitro drug delivery when compared to the current state of the art in mathematical modeling. As an example, we applied the mechanism and model to the release data for acetazolamide from a recombinant polymer. Both materials were selected because of a need to develop a suitable ophthalmic formulation for the treatment of glaucoma. The in vitro release model proposed herein provides a valuable predictive tool for ensuring product performance and batch-to-batch reproducibility, thus paving the way for the development of further pharmaceutical devices.