Protein materials as sustainable non- and minimally invasive strategies for biomedical applications

Protein-based materials have found applications in a wide range of biomedical fields because of their biocompatibility, biodegradability and great versatility. Materials of different physical forms including particles, hydrogels, films, fibers and microneedles have been fabricated e.g. as carriers for drug delivery, factors to promote wound healing and as structural support for the generation of new tissue. This review aims at providing an overview of the current scientific knowledge on protein-based materials, and selected preclinical and clinical studies will be reviewed in depth as examples of the latest progress within the field of protein-based materials, specifically focusing on non- and minimally invasive strategies mainly for topical application

metabolic approach to the enhancement of antitumor effect of chemotherapy a key role of acetyl l carnitine

Purpose: Acetyl-l-carnitine (ALC) plays a relevant role in energy metabolism and stress response because of its function in the complex metabolic system regulating the acetyl-CoA levels that provide a source of acetyl groups for metabolic and acetylation-regulated processes. Because acetylation may influence p53 activity/stability and, therefore, the response to platinum compounds, this study was designed to investigate the effect of ALC in combination with platinum compounds. Experimental Design: The antiproliferative and antitumor activity studies were done in a panel of human tumor cell lines with functional or defective p53. The antimetastatic drug efficacy was investigated in the s.c. growing H460/M tumor subline, which is able to generate lung metastases. Results: ALC enhanced the sensitivity to cisplatin of tumor cells with functional p53. The sensitization by ALC was reflected in an improved in vivo antitumor efficacy of the combination over cisplatin alone in wild-type p53 lung tumors. ALC did not increase the cisplatin efficacy in the p53-mutant SW620 tumor. ALC exhibited a significant antimetastatic activity, and this effect was better exploited in combination with the histone deacetylase inhibitor, ST3595. The in vivo ALC/cisplatin combination caused the activation of p53, associated with protein acetylation and induction of target genes. Conclusions: ALC was effective in enhancing the antitumor potential of platinum compounds in wild-type p53 tumors. ALC, alone and in combination with a histone deacetylase inhibitor, exhibited an outstanding antimetastatic activity. Both effects, likely mediated by protein acetylation, may have implications for platinum-based therapy and combinations with histone deacetylase inhibitors. Clin Cancer Res; 16(15); 3944–53. ©2010 AACR

Increased carrier peptide stability through ph adjustment improves insulin and pth(1-34) delivery in vitro and in vivo rather than by enforced carrier peptide-cargo complexation

Oral delivery of therapeutic peptides is hampered by their large molecular size and labile nature, thus limiting their permeation across the intestinal epithelium. Promising approaches to overcome the latter include co-administration with carrier peptides. In this study, the cell-penetrating peptide penetratin was employed to investigate effects of co-administration with insulin and the pharmacologically active part of parathyroid hormone (PTH(1-34)) at pH 5, 6.5, and 7.4 with respect to complexation, enzymatic stability, and transepithelial permeation of the therapeutic peptide in vitro and in vivo. Complex formation between insulin or PTH(1-34) and penetratin was pH-dependent. Micron-sized complexes dominated in the samples prepared at pH-values at which penetratin interacts electrostatically with the therapeutic peptide. The association efficiency was more pronounced between insulin and penetratin than between PTH(1-34) and penetratin. Despite the high degree of complexation, penetratin retained its membrane activity when applied to liposomal structures. The enzymatic stability of penetratin during incubation on polarized Caco-2 cell monolayers was pH-dependent with a prolonged half-live determined at pH 5 when compared to pH 6.5 and 7.4. Also, the penetratin-mediated transepithelial permeation of insulin and PTH(1-34) was increased in vitro and in vivo upon lowering the sample pH from 7.4 or 6.5 to 5. Thus, the formation of penetratin-cargo complexes with several molecular entities is not prerequisite for penetratin-mediated transepithelial permeation a therapeutic peptide. Rather, a sample pH, which improves the penetratin stability, appears to optimize the penetratin-mediated transepithelial permeation of insulin and PTH(1-34)

α-casein micelles-membranes interaction: Flower-like lipid protein coaggregates formation

Background: Environmental conditions regulate the association/aggregation states of proteins and their action in cellular compartments. Analysing protein behaviour in presence of lipid membranes is fundamental for the comprehension of many functional and dysfunctional processes. Here, we present an experimental study on the interaction between model membranes and α-casein. α-casein is the major component of milk proteins and it is recognised to play a key role in performing biological functions. The conformational properties of this protein and its capability to form supramolecular structures, like micelles or irreversible aggregates, are key effectors in functional and pathological effects. Methods: By means of quantitative fluorescence imaging and complementary spectroscopic methods, we were able to characterise α-casein association state and the course of events induced by pH changes, which regulate the interaction of this molecule with membranes. Results: The study of these complex dynamic events revealed that the initial conformation of the protein critically regulates the fate of α-casein, size and structure of the newly formed aggregates and their effect on membrane structures. Disassembly of micelles due to modification in electrostatic interactions results in increased membrane structure rigidity which accompanies the formation of protein lipid flower-like co-aggregates with protein molecules localised in the external part. General significance: These results may contribute to the comprehension of how the initial state of a protein establishes the course of events that occur upon changes in the molecular environment. These events which may occur in cells may be essential to functional, pathological or therapeutical properties specifically associated to casein proteins

Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly

Ensembles of protein aggregates are characterized by a nano- and micro-scale heterogeneity of the species. This diversity translates into a variety of effects that protein aggregates may have in biological systems, both in connection to neurodegenerative diseases and immunogenic risk of protein drug products. Moreover, this naturally occurring variety offers unique opportunities in the field of protein-based biomaterials. In the above-mentioned fields, the isolation and structural analysis of the different amyloid types within the same ensemble remain a priority, still representing a significant experimental challenge. Here we address such complexity in the case of insulin for its relevance as biopharmaceutical and its involvement in insulin-derived amyloidosis. By combining Fourier Transform Infrared Microscopy (micro-FTIR) and fluorescence lifetime imaging microscopy (FLIM) we show the occurrence, within the same ensemble of insulin protein aggregates, of a variable β-structure architecture and content not only dependent on the species analyzed (spherulites or fibrils), but also on the position within a single spherulite at submicron scale. We unambiguously reveal that the surface of the spherulites are characterized by β-structures with an enhanced H-bond coupling compared to the core. This information, inaccessible via bulk methods, allows us to relate the aggregate structure at molecular level to the overall morphology of the aggregates. Our findings robustly solve the problem of probing the ensemble and single particle heterogeneity of amyloid samples. Furthermore, we offer a unique, scalable and ready-to-use screening methodology for in-depth characterization of self-assembled structures, being this translatable to material sciences, drug quality control and clinical imaging of amyloid-affected tissues