Protein-imprinted polymers: how far have “plastic antibodies” come?

Antibodies are highly selective and sensitive, making them the gold standard for recognition affinity tools. However, their production cost is high and their downstream processing is time-consuming. Molecularly imprinted polymers (MIPs) are tailor-made by incorporating specific molecular recognition sites in their structure, thus translating into receptor-like activity mode of action. The interest in molecular imprinting technology, applied to biomacromolecules, has increased in the past decade. MIPs, produced using biomolecules as templates, commonly referred to as “plastic antibodies” or “artificial receptors”, have been considered as suitable cheaper and easy to produce alternatives to antibodies. Research on MIPs, designed to recognize proteins or peptides is particularly important, with potential contributions towards biomedical applications, namely biosensors and targeted drug delivery systems. This mini review will cover recent advances on (bio)molecular imprinting technology, where proteins or peptides are targeted or mimicked for sensing and therapeutic applications. Polymerization methods are reviewed elsewhere, being out of the scope of this review. Template selection and immobilization approaches, monomers and applications will be discussed, highlighting possible drawbacks and gaps in research.Peer ReviewedPostprint (author's final draft

Dendritic self-assembled structures from therapeutic charged pentapeptides

CRENKA [Cys-Arg-(NMe)Glu-Lys-Ala, where (NMe)Glu refers to N-methyl-Glu], an anti-cancer pentapeptide that induces prostate tumor necrosis and significant reduction in tumor growth, was engineered to increase the resistance to endogenous proteases of its parent peptide, CREKA (Cys-Arg-Glu-Lys-Ala). Considering their high tendency to aggregate, the self-assembly of CRENKA and CREKA into well-defined and ordered structures has been examined as a function of peptide concentration and pH. Spectroscopic studies and atomistic molecular dynamics simulations reveal significant differences between the secondary structures of CREKA and CRENKA. Thus, the restrictions imposed by the (NMe)Glu residue reduce the conformational variability of CRENKA with respect to CREKA, which significantly affects the formation of well-defined and ordered self-assembly morphologies. Aggregates with poorly defined morphology are obtained from solutions with low and moderate CREKA concentrations at pH 4, whereas well-defined dendritic microstructures with fractal geometry are obtained from CRENKA solutions with similar peptide concentrations at pH 4 and 7. The formation of dendritic structures is proposed to follow a two-step mechanism: (1) pseudo-spherical particles are pre-nucleated through a diffusion-limited aggregation process, pre-defining the dendritic geometry, and (2) such pre-nucleated structures coalesce by incorporating conformationally restrained CRENKA molecules from the solution to their surfaces, forming a continuous dendritic structure. Instead, no regular assembly is obtained from solutions with high peptide concentrations, as their dynamics is dominated by strong repulsive peptide–peptide electrostatic interactions, and from solutions at pH 10, in which the total peptide charge is zero. Overall, results demonstrate that dendritic structures are only obtained when the molecular charge of CRENKA, which is controlled through the pH, favors kinetics over thermodynamics during the self-assembly process.Peer ReviewedPostprint (published version

Hydroxyapatite-filled osteoinductive and piezoelectric nanofibers for bone tissue engineering

In this study entitled “Hydroxyapatite-filled osteoinductive and piezoelectric nanofibers for bone tissue engineering”, we describe the development of novel hydroxyapatite (HAp)-filled osteoinductive piezoelectric poly(vinylidene fluoride-cotetrafluoroethylene) (PVDF-TrFE) electrospun nanofibers as a potential strategy for supporting bone repair in delayed-union and non-union osteoporotic-related fractures, for which current clinical techniques have proven to be largely inadequate and scaffold-based tissue engineering approaches hold significant promise. While the piezoelectric properties of native bone tissue have been extensively discussed in the literature, including their key role in preserving tissue homeostasis and promoting tissue repair, they have been widely neglected in the design of scaffolds for bone tissue engineering (BTE) applications. Piezoelectric scaffolds can be used not only for mimicking the native piezoelectric features of bone but also to provide a platform for applying electrical or mechanical stimuli to damaged tissue, contributing to an accelerated regeneration process. The nanofibrous scaffolds generated in this study were capable of replicating the main electrical, structural and compositional properties of bone extracellular matrix (ECM). To the best of our knowledge, this was the first time that the combination of HAp with the piezoelectric polymer PVDF-TrFE was found to induce key shifts in the chemical structure of the polymer and promote ß phase nucleation, not only enhancing the piezoelectric features of the constructs but also improving their surface properties, including their ability to support mineralization in vitro. The HAp nanoparticles also provided meaningful bone-like biological cues (osteoinduction), enhancing the osteogenic differentiation of seeded human mesenchymal stem/stromal cells (hMSCs), which was confirmed by an increased ALP activity, cellderived calcium deposition and expression of important osteogenic gene markers. Overall, our findings highlight, for the first time, the potential of combining PVDFTrFE and HAp for developing electroactive and osteoinductive nanofibrous constructs with improved piezoelectric properties, surface features and osteogenic potential capable of improving bone tissue regeneration.Peer ReviewedPostprint (published version

Electroresponsive and pH-sensitive hydrogel as carrier for controlled chloramphenicol release

Multiresponsive hydrogels, which are smart soft materials that respond to more than one external stimulus, have emerged as powerful tools for biomedical applications, such as drug delivery. Within this context and with the aim of eliminating the systematic administration of antibiotics, special attention is being paid to the development of systems for controlled delivery of antibiotic for topical treatment of bacterial infections. In this work, an electro-chemo responsive hydrogel able to release chloramphenicol (CAM), a broad spectrum antibiotic also used for anticancer therapy, is proposed. This has been prepared by grafting poly(acrylic acid) (PAA) to sodium alginate (Alg) and in situ encapsulation of poly(3,4-ethylenedioxythiophene) nanoparticles loaded with CAM (PEDOT/CAM NPs), which were obtained by emulsion polymerization. Although the response to electrical stimuli of PEDOT was the main control for the release of CAM from PEDOT/CAM NPs, the release by passive diffusion had a relatively important contribution. Conversely, the passive release of antibiotic from the whole engineered hydrogel system, Alg-g-PAA/PEDOT/CAM, was negligible, whereas significant release was achieved under electrostimulation in an acid environment. Bacterial tests and assays with cancer cells demonstrated that the biological activity of CAM remained after release by electrical stimulation. Notably, the successful dual-response of the developed hydrogel to electrical stimuli and pH changes evidence the great prospect of this smart material in the biomedical field, as a tool to fight against bacterial infections and to provide local cancer treatment.Peer ReviewedPostprint (author's final draft