A Conditionally Activated Cytosol-Penetrating Antibody for TME-Dependent Intracellular Cargo Delivery.

Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody was serendipitously discovered, which eventually localizes to the cytosol of target cells. Functional characterization revealed that the tested antibody has beneficial cytosol-penetrating capabilities and can deliver cargo proteins (up to 70 kDa) to the cytosol. To achieve tumor-specific cell targeting and cargo delivery through conditional activation of the cell-penetrating antibody in the tumor microenvironment, a single-chain Fc fragment (scFv) and a VL domain were isolated as masking units. Several in vitro assays demonstrated that fusing the masking protein with a cleavable linker to the cell penetration antibody results in the inactivation of antibody cell binding and internalization. Removal of the mask via MMP-9 protease cleavage, a protease that is frequently overexpressed in the tumor microenvironment (TME), led to complete regeneration of binding and cytosol-penetrating capabilities. Masked and conditionally activated cytosol-penetrating antibodies have the potential to serve as a modular platform for delivering protein cargoes addressing intracellular targets in tumor cells.

Effect of calcium phosphate-based fillers on the structure and bonding strength of novel gelatin-alginate bioadhesives.

Interest in soft and hard tissue adhesives as alternatives for conventional wound closing and bone fixation applications has increased in recent decades as a result of numerous possible advantages such as better comfort and lower cost. A novel bioadhesive based on the natural polymers GA has recently been developed and studied in our laboratory. Hydroxyapatite and tricalcium phosphate are two bioactive ceramics known for their ability to enhance bone regeneration. In the current study, these two bioactive fillers were incorporated into the bioadhesive at concentrations of 0.125, 0.25 and 0.5% w/v, and their effects on the resulting adherence properties to soft and hard tissues were studied. Porcine skin and cortical portions of bovine femurs were used as soft and hard tissue specimens, respectively. The bonding strength was evaluated using an Instron universal testing machine in tensile mode, and the microstructure analysis was based on environmental scanning electron microscope observations. Both bioactive fillers were found to have a reinforcing effect on the adhesives, significantly improving their adhesion to soft tissues in certain concentrations. The best bonding strength results were obtained for 0.25% hydroxyapatite and 0.5% w/v tricalcium phosphate-18.1 ± 4.0 and 15.2 ± 2.6 kPa, respectively, compared with 8.4 ± 2.3 kPa for adhesive with no fillers. The improved adherence is probably related to the stiffness of the insoluble hydroxyapatite and tricalcium phosphate particles which reinforce the adhesive. These particles can clearly be observed in the environmental scanning electron microscope analysis. The potential of these fillers to increase the bonding strength of the adhesive to hard tissues was also demonstrated. Hydroxyapatite and tricalcium phosphate thus improve our new gelatin-alginate bioadhesives, which can be used for both soft and hard tissue adhesive applications.

Novel membrane-bound reporter molecule for sorting high producer cells by flow cytometry.

We developed a membrane bound reporter and selection molecule for sorting by fluorescence activated cell sorting (FACS) of cells producing a protein of interest. This molecule is composed of a transmembrane (TM) domain, fused on its extracellular end to a biotin mimetic peptide (BMP) and on its intracellular side to puromycin N-acetyl transferase (PAC). In this format BMP is displayed on the cell membrane surface and PAC faces the cell cytoplasm. BMP was detected and quantified on the cell surface by fluorescently labelled streptavidin, allowing cell sorting by FACS, according to the reporter expression level. The reporter and a gene of interest (GOI) were connected on the same transcript via an internal ribosomal entry site (IRES). The reporter expression level was found to correlate with that of the GOI, enabling sorting of high producer cells by FACS. Thus, the highest fluorescent cells sorted had also the highest protein of interest (POI) productivity level.

Novel gelatin/alginate soft tissue adhesives loaded with drugs for pain management: structure and properties.

Interest in tissue adhesives as alternatives for conventional wound closing applications, such as sutures and staples, has increased in the last few decades due to numerous possible advantages, including less discomfort and lower cost. Novel tissue adhesives based on gelatin, with alginate as a polymeric additive and crosslinked by carbodiimide were developed and loaded with two types of drugs for pain relief, bupivacaine and ibuprofen, in order to improve the therapeutic effect. The release of the drugs from the adhesive matrix was found to be controlled mainly by the adhesive's characteristics, i.e. swelling and hydrophilic group concentration. The drug characteristics, i.e. hydrophilicity and electrical interactions between the drug and the polymeric components, were also found to have some effect. Incorporation of bupivacaine was found to improve the bonding strength of the adhesive due to its inert nature and the reinforcing effect of its fibrous crystals, whereas incorporation of ibuprofen was found to have an adverse effect on the bonding strength, probably due to its reaction with the other adhesive components which increased the crosslinking density. Overall, the novel drug-eluting gelatin-based bioadhesives investigated in this research, especially those loaded with bupivacaine, demonstrated a promising potential for use in wound closing applications.

Gelatin-alginate novel tissue adhesives and their formulation-strength effects.

Interest in tissue adhesives as alternatives for conventional wound-closing applications such as sutures and staples has increased in the last few decades due to numerous possible advantages, including less discomfort and lower cost. Novel tissue adhesives based on gelatin, with alginate as a polymeric additive and crosslinked by carbodiimide, were recently developed by our research group. The effects of the formulation parameters on the adhesives' function were investigated in the current study. We examined the effects of gelatin and alginate concentrations and their viscosities on the ability of the bioadhesives to bind soft tissues. The effect of the crosslinking agent's concentration was studied as well. A qualitative model describing these effects in terms of adherence mechanisms was developed. Our results show that the adherence properties of our new bioadhesives are achieved by a combination of two main mechanisms: mechanical interlocking and chemical adsorption. The former mechanism is probably more dominant. The polymer's molecular weight and concentration affect the mechanical interlocking through mobility and penetration ability, entanglement of the three-dimensional structure and crosslinking density. The crosslinking agent's concentration as well as the polymer's concentration affect the crosslinking density and contribute to higher strength, achieved through both the mechanical interlocking and the chemical adsorption mechanisms. Understanding the effects of the adhesives' components and their viscosities on the bonding strength enabled us to elucidate the bonding strength mechanisms. This can lead to proper selection of the adhesive formulation and may enable tailoring the bioadhesives to the desired applications.