Elastin-based biopolymers for biomedical and biotechnological applications

Perhaps the most appealing opportunity is represented by gaining inspiration from nature for the precise tailoring of biomaterials with finely tuned unique functional properties. A very promising model is represented by Human Elastin-Like Polypeptides (HELP), repetitive artificial polypeptides based on penta- or hexa- peptidic motifs that characterize elastin. These protein polymers retain several peculiar biophysical properties as, the reversible inverse phase transition, changing the solubility and aggregation state in response to temperature variation. The smart nature of this class of compounds makes them attractive for many applications in the biomedical and biotechnological fields, in particular for biomaterial development. The Trans2Care project aiming to translating scientific and technological expertise into products for the biomedical field represents an exciting and challenging environment to best exploit the potential these biomimetic macromolecules

Biomimetic poypeptides: a new strategy for muscle tissue regeneration

Despite recent progress in regenerative medicine, functional muscle tissue restoration still represent a challenge, being unable to self- restore significant tissue loss, as consequence of trauma, congenital defects, tumor ablation or denervation. The creation of new muscle through tissue engineering represents an alternative for the replacement of tissue after severe damage. Among the many materials available, those of natural origin are preferable for their biocompatibility and their capacity to resemble the native physiologic environment of cells. A new opportunity is represented by the adoption of a biomimetic approach that allows to realize compounds that are in –between natural and synthetic origin with limited variability and controlled features. Human elastin-like polypeptides that have been designed and produced in our laboratory represent an innovative and promising tool to be exploited in muscle tissue engineering

PROTEINE ASSOCIATE COL DNA: STRUTTURA E FUNZIONE CON RIFERIMENTO ALL'ATTIVAZIONE/REPRESSIONE DELL'ESPRESSIONE GENICA IN SISTEMI NORMALI E NEOPLASTICI

1993/1994VI Ciclo1965Versione digitalizzata della tesi di dottorato cartacea

Macromolecular and Solution Properties of the Recombinant Fusion Protein HUG

The recombinant fusion protein HELP-UnaG (HUG) is a bifunctional product that exhibits human elastin-like polypeptide (HELP)-specific thermal behavior, defined as a reverse phase transition, and UnaG-specific bilirubin-dependent fluorescence emission. HUG provides an interesting model to understand how its two domains influence each other's properties. Turbidimetric, calorimetric, and light scattering measurements were used to determine different parameters for the reverse temperature transition and coacervation behavior. This shows that the UnaG domain has a measurable but limited effect on the thermal properties of HELP. Although the HELP domain decreased the affinity of UnaG for bilirubin, HUG retained the property of displacing bilirubin from bovine serum albumin and thus remains one of the strongest bilirubin-binding proteins known to date. These data demonstrate that HELP can be used to create new bifunctional fusion products that pave the way for expanded technological applications

Epidermal Growth Factor \u2013 based adhesion substrates elicit myoblast scattering, proliferation, differentiation and promote satellite cell myogenic activation

The biochemical properties of muscle extracellular matrix are essential for stem cell adhesion, motility, proliferation and myogenic development. Recombinant elastin-like polypeptides are synthetic polypeptides that, besides maintaining some properties of the native protein, can be tailored by fusing bioactive sequences to their C-terminal. Our laboratory synthesized several Human Elastin-Like Polypeptides (HELP) derived from the sequence of human tropoelastin. Here, we developed a novel HELP family member by fusing the elastin-like backbone to the sequence of human Epidermal Growth Factor. We employed this synthetic protein, named HEGF, either alone or in combination with other proteins of the HELP family carrying RGD-integrin binding sites, as adhesion substrate for C2C12 myoblasts and satellite cells primary cultures. Adhesion of myoblasts to HEGF-based substrates induced scattering, decreased adhesion and cytoskeleton assembly; the concomitant presence of the RGD motifs potentiated all these effects. Recombinant substrates induced myoblasts proliferation, differentiation and the development of multinucleated myotubes, thus favoring myoblasts expansion and preserving their myogenic potential. The effects induced by adhesion substrates were inhibited by AG82 (Tyrphostin 25) and herbimycin A, indicating their dependence on the activation of both the EGF receptor and the tyrosine kinase c-src. Finally, HEGF increased the number of muscle stem cells (satellite cells) derived from isolated muscle fibers in culture, thus highlighting its potential as a novel substrate for skeletal muscle regeneration strategies

3D-printed scaffold composites for the stimuli-induced local delivery of bioactive adjuncts

Polysaccharide scaffolds have been successfully employed to reconstruct environments that sustain skin tissue regeneration after injuries. Three-dimensional (3D) advanced additive manufacturing technologies allow creating scaffolds with controlled and reproducible macro- and micro-structure that improve the quality of the restored tissue to favor spontaneous repair. However, when persistent inflammation occurs, the physiological tissue healing capacity is reduced, like in the presence of pathologies like diabetes, vascular diseases, chronic infection, and others. In these circumstances, the bioavailability of therapeutic adjuncts like the growth factors in addition to the standard treatments represents undoubtedly a promising strategy to accelerate the healing of skin lesions. Precisely designed polysaccharide scaffolds obtained by 3D printing represent a robust platform that can be further implemented with the controlled delivery of bioactive adjuncts. Human elastin-like polypeptides (HELPs) are stimuli-responsive biopolymers. Their structure allows the integration of domains endowed with biological functionality, making them attractive compounds to prepare composites with smart properties. In the present study, 3D-printed alginate and chitosan scaffolds were combined with the HELP components. The HELP biopolymer was fused to the epidermal growth factor (EGF) as the bioactive domain. Different constructs were prepared and the stimuli-responsive behavior as well as the biological activity were evaluated, suggesting that these smart bioactive composites are suitable to realize multifunctional dressings that sustain the local release of therapeutic adjuncts

Composite of elastin-based matrix and electrospun poly(L-lactic acid) fibers: A potential smart drug delivery system

Stimuli-responsive hydrogel matrices are inspiring manifold applications in controlled delivery of bioactive compounds. Elastin-derived polypeptides form hydrogel matrices that may release bioactive moieties as a function of local increase of active elastases, as it would occur in several processes like inflammation. In view of the development of a patch for healing wounds, recombinant elastin-based polypeptides were combined with a proteolysis-resistant scaffold, made of electrospun poly-L-lactic acid (PLLA) fibers. The results of this study demonstrated the compatibility of these two components. An efficient procedure to obtain a composite material retaining the main features of each component was established. The release of the elastin moiety was monitored by means of a simple protocol. Our data showed that electrospun PLLA can form a composite with fusion proteins bound to elastin-derived polypeptides. Therefore, our approach allows designing a therapeutic agent delivery platform to realize devices capable of responding and interacting with biological systems at the molecular level

Alginate/human elastin-like polypeptide composite films with antioxidant properties for potential wound healing application

In this contribution we describe the preparation and characterization of a series of cross-linked films based on the combination of an elastin-derived biomimetic polypeptide (Human elastin-like polypeptide (HELP)) with alginate (ALG) to obtain a composite with enhanced properties. ALG/HELP composite films loaded with the hydrophobic natural antioxidant curcumin were prepared by solvent casting method followed by the cross-linking with calcium chloride. The compatibility between the two components as well as the final properties was evaluated. The micro-morphological study of films showed a homogeneous structure, but the film tensile strength decrease with HELP content and elongation at break was adversely affected by biopolymer addition. Spectroscopic and thermal analyses confirmed an interaction between ALG and HELP which also causes a modification in swelling kinetics and faster degradation. Moreover, the study of curcumin release showed a controlled delivery up to 10\u202fdays with a faster release rate in the presence of HELP. Human Dermal Fibroblasts (hDF) were used to test the in vitro cytocompatibility. The antioxidant activity correlated to the increase of HELP content suggested the applicability of these composites to develop smart biomaterials. Overall, these features indicated how this composite material has considerable potential as customizable platforms for various biomedical applications

Identification of stably expressed reference small non-coding RNAs for microRNA quantification in high-grade serous ovarian carcinoma tissues

MicroRNAs (miRNAs) belong to a family of small non‐coding RNAs (sncRNAs) playing important roles in human carcinogenesis. Multiple investigations reported miRNAs aberrantly expressed in several cancers, including high‐grade serous ovarian carcinoma (HGS‐OvCa). Quantitative PCR is widely used in studies investigating miRNA expression and the identification of reliable endogenous controls is crucial for proper data normalization. In this study, we aimed to experimentally identify the most stable reference sncRNAs for normalization of miRNA qPCR expression data in HGS‐OvCa. Eleven putative reference sncRNAs for normalization (U6, SNORD48, miR‐92a‐3p, let‐7a‐5p, SNORD61, SNORD72, SNORD68, miR‐103a‐3p, miR‐423‐3p, miR‐191‐5p, miR‐16‐5p) were analysed on a total of 75 HGS‐OvCa and 30 normal tissues, using a highly specific qPCR. Both the normal tissues considered to initiate HGS‐OvCa malignant transformation, namely ovary and fallopian tube epithelia, were included in our study. Stability of candidate endogenous controls was evaluated using an equivalence test and validated by geNorm and NormFinder algorithms. Combining results from the three different statistical approaches, SNORD48 emerged as stably and equivalently expressed between malignant and normal tissues. Among malignant samples, considering groups based on residual tumour, miR‐191‐5p was identified as the most equivalent sncRNA. On the basis of our results, we support the use of SNORD48 as best reference sncRNA for relative quantification in miRNA expression studies between HGS‐OvCa and normal controls, including the first time both the normal tissues supposed to be HGS‐OvCa progenitors. In addition, we recommend miR‐191‐5p as best reference sncRNA in miRNA expression studies with prognostic intent on HGS‐OvCa tissues

Evaluation of a novel human IgG1 anti-claudin3 antibody that specifically recognizes its aberrantly localized antigen in ovarian cancer cells and that is suitable for selective drug delivery

Membrane protein claudin3 has been recently suggested as a marker for biologically aggressive tumors and a possible target for the therapeutic delivery of active anti-cancer compounds. Claudin3-binding molecules such as the Clostridium perfringens enterotoxin (CPE), CPE-related molecules, and murine and chimeric antibodies have shown promising antitumor efficacy in preclinical oncological settings. We first engineered a fully human anti-claudin3 IgG1 antibody (IgGH6) by fusing the human IgG1 Fc-domain to the anti-claudin3 scFvH6 previously isolated from a pre-immune phage display library. The construct was expressed in mammalian cells and specifically targeted claudin3 endogenously expressed on the surface of different human ovarian cancer cell lines. No detectable cross-reactivity with other homologous claudins was observed. The epitope recognized by IgGH6 is located within the minor extracellular domain of claudin3 and becomes accessible only in tumor cells characterized by incomplete junction formation. Confocal microscopy experiments demonstrated that IgGH6 was actively internalized in tumor cells after binding to native claudin3 and co-localized, likely within intracellular vesicles, with the C-CPE peptide. Preliminary results indicate that IgGH6 accumulated in vivo in free claudin3 ovarian carcinoma xenografts. For its selective uptake in tumor cells and its human nature, IgGH6 represents a valuable candidate for antibody-drug conjugate therapeutic applications in ovarian cancer patients