Targeting transdifferentiated hepatic stellate cells and monitoring the hepatic fibrogenic process by means of IGF2R-specific peptides designed in silico.

The lack of accurate and easily applicable methods for the diagnosis of liver fibrosis, a disease characterized by an accumulation of the extracellular matrix released by activated hepatic stellate cells (HSCs), has been a major limitation for the clinical management of liver diseases. The identification of biomarkers specific to liver microstructure alterations, combined with a non-invasive optical imaging modality, could guide clinicians towards a therapeutic strategy. In this study, structural information of the insulin-like growth factor 2 receptor (IGF2R), an overexpressed protein on activated HSCs, was used for in silico screening of novel IGF2R-specific peptide ligands. Molecular dynamics simulations, followed by computational alanine scanning of the IGF2R/IGF2 complex, led to the identification of a putative peptide sequence containing the most relevant amino acids for the receptor-ligand interaction (IGF2 E12-C21). The Residue Scan tool, implemented in the MOE software, was then used to optimize the binding affinity of this sequence by amino acid mutations. The designed peptides and their associated scrambled sequences were fluorescently labelled and their binding affinity to LX-2 cells (model for activated human HSCs) was tested using flow cytometry and confocal microscopy. In vitro binding was verified for all sequences (KD ≤ 13.2 µM). With respect to the putative binding sequence, most mutations led to an increased affinity. All sequences have shown superior binding compared to their associated scrambled sequences. Using HPLC, all peptides were tested in vitro for their proteolytic resistance and showed a stability of ≥60% intact after 24 h at 37 °C in 50% v/v FBS. In view of their prospective diagnostic application, a comparison of binding affinity was performed in perpetuated and quiescent-like LX-2 cells. Furthermore, the IGF2R expression for different cell phenotypes was analysed by a quantitative mass spectrometric approach. Our peptides showed increased binding to the perpetuated cell state, indicating their good selectivity for the diagnostically relevant phenotype. In summary, the increased binding affinity of our peptides towards perpetuated LX-2 cells, as well as the satisfactory proteolytic stability, proves that the in silico designed sequences offer a new potential strategy for the targeting of hepatic fibrosis.

Synergy of Phospholipid-Drug Formulations Significantly Deactivates Profibrogenic Human Hepatic Stellate Cells.

The pivotal role of hepatic stellate cells (HSCs) in orchestrating the bidirectional process of progression and regression of liver fibrosis makes them an ideal target for exploring new antifibrotic therapies. Essential phospholipids (EPLs), with their polyenylphosphatidylcholine (PPC) fraction, either alone or combined with other hepatoprotective substances such as silymarin, are recommended in hepatic impairment, but a scientific rationale for their use is still lacking. Herein, we compared the ability of EPLs to restore quiescent-like features in HSCs with that of dilinoleoylphosphatidylcholine (DLPC), PPC fraction's main component. Specifically, we screened at the cellular level the antifibrotic effects of PPC formulations in the presence and absence of silymarin, by using LX-2 cells (pro-fibrogenic HSCs) and by assessing the main biochemical hallmarks of the activated and deactivated states of this cell line. We also proved the formulations' direct effect on the motional order of cell membranes of adherent cells. LX-2 cells, examined for lipid droplets as a quiescence marker, showed that PPCs led to a more prominent deactivation than DLPC. This result was confirmed by a reduction of collagen and α-SMA expression, and by a profound alteration in the cell membrane fluidity. PPC-silymarin formulations deactivated HSCs with a significant synergistic effect. The remarkable bioactivity of PPCs in deactivating fibrogenic HSCs paves the way for the rational design of new therapeutics aimed at managing hepatic fibrosis.

Bio-reduction of redox-sensitive albumin conjugates in FcRn-expressing cells.

Disulfide-containing IgG-, Fc-, or albumin-based prodrugs that rely on FcRn-trafficking by endothelial cells for prolonged circulation in the body might be hampered by premature bio-reduction processes during FcRn-mediated recycling events. A detailed bio-reduction analysis of redox-sensitive albumin conjugates in two FcRn-expressing cell lines has been performed. The obtained results indicate that the FcRn-mediated recycling pathway is not (or is only poorly) bio-reducing.

Disulfide-containing parenteral delivery systems and their redox-biological fate.

Exploiting the redox-sensitivity of disulfide bonds is an increasingly popular means to trigger drug release at a target location in the body. The bio-reducible linker (containing a disulfide) can be cleaved when the drug delivery system in which it is incorporated passes from the poorly reducing extra-cellular biological environments to the strongly reducing intra-cellular spaces. This phenomenon has been characterized for a variety of drug carriers (e.g. antibody-drug conjugates and nucleic acid carriers) and made use of not only for intra-cellular drug release, but also to provide a mechanism of biodegradation. However, successful therapeutic application of redox-sensitive drug delivery systems, which are mostly investigated in the treatment of cancer, depends on timely cleavage of the disulfide in the body. As a result, an accurate and detailed understanding of the biological redox stimulus and the properties of the redox-sensitive moiety is of importance. This review introduces a number of currently relevant reducing agents and redox enzymes, and provides an overview of the redox environments a disulfide-containing drug delivery system encounters upon parenteral administration. Furthermore, the current state of knowledge regarding the behavior and responsiveness of disulfides in these redox-biological compartments is discussed.

Broad control of disulfide stability through microenvironmental effects and analysis in complex redox environments.

Disulfide bonds stabilize the tertiary- and quaternary structure of proteins. In addition, they can be used to engineer redox-sensitive (bio)materials and drug-delivery systems. Many of these applications require control of the stability of the disulfide bond. It has recently been shown that the charged microenvironment of the disulfide can be used to alter their stability by ∼3 orders of magnitude in a predictable and finely tunable manner at acidic pH. The aim of this work is to extend these findings to physiological pH and to demonstrate the validity of this approach in complex redox milieu. Disulfide microenvironments were manipulated synergistically with steric hindrance herein to control disulfide bond stability over ∼3 orders of magnitude at neutral pH. Control of disulfide stability through microenvironmental effects could also be observed in complex redox buffers (including serum) and in the presence of cells. Such fine and predictable control of disulfide properties is not achievable using other existing approaches. These findings provide easily implementable and general tools for controlling the responsiveness of biomaterials and drug delivery systems toward various local endogenous redox environments.

Tracking the bioreduction of disulfide-containing cationic dendrimers.

Disulfides enhance the transfection efficacy and reduce the toxicity of cationic gene delivery polymers. A quantitative analysis is provided of the bioreduction of a dynamic bioreducible dendritic polycationic probe in four cell lines. Such knowledge is indispensible for understanding and optimizing bioreducible drug and gene delivery systems.