Search Results (6)

The development of biomaterials with gradient surface modification capable of spatially controlled cell adhesion and migration is of great importance for tissue engineering and regeneration. In this study, we proposed a method for the covalent modification of PLA-based materials with a cationic polypeptide (polylysine, PLys) via a thiol-ene click reaction carried out under a light gradient. With this aim, PLA-based films were fabricated and modified with 2–aminoethyl methacrylate (AEMA) as a double bond source. The latter was introduced by reacting pre-formed and activated surface carboxyl groups with the amino group of AEMA. The success of the modification was confirmed by ¹H NMR, Raman and X-ray photoelectron spectroscopy data. A further photoinduced thiol-ene click reaction in the presence of a photosensitive initiator as a radical source was further optimized using cysteine. For grafting of PLys via the thiol-ene click reaction, PLys with a terminal thiol group was synthesized by ring-opening polymerization using Cys(Acm) as an amine initiator. Deprotection of the polypeptide resulted in the formation of free thiol groups of Cys-PLys. Successful gradient grafting of Cys-PLys was evidenced by covalent staining with the fluorescent dye Cy3-NHS. In addition, PLys gradient-dependent adhesion and migration of HEK 293 cells on PLys-PLA-based surfaces was confirmed. Full article

Nanosilver applications, including sensing and water treatment, have significantly increased in recent years, although safety for humans and the environment is still under debate. Here, we tested the environmental safety of a novel formulation of silver nanoparticles functionalized with citrate and L-cysteine (AgNPcitLcys) on freshwater cnidarian Hydra vulgaris as an emerging ecotoxicological model for the safety of engineered nanomaterials. AgNPcitLcys behavior was characterized by dynamic light scattering (DLS), while Ag release was measured by inductively coupled plasma mass spectrometry (ICP-MS). H. vulgaris (n = 12) subjects were evaluated for morphological aberration after 96 h of exposure and regeneration ability after 96 h and 7 days of exposure, after which the predatory ability was also assessed. The results show a low dissolution of AgNPcitLcys in Hydra medium (max 0.146% of nominal AgNPcitLcys concentration) and highlight a lack of ecotoxicological effects, both on morphology and regeneration, confirming the protective role of the double coating against AgNP biological effects. Predatory ability evaluation suggests a mild impairment of the entangling capacity or of the functionality of the tentacles, as the number of preys killed but not ingested was higher than the controls in all exposed animals. While their long-term sub-lethal effects still need to be further evaluated on H. vulgaris, AgNPcitLcys appears to be a promising tool for environmental applications, for instance, for water treatment and sensing. Full article

In this study, a hydrogel composite wound dressing with antibacterial and self-healing ability was prepared using cysteine-modified carboxymethyl chitosan, sodium oxidized alginate, and but-3-yn-2-one base on Schiff base and thiol-alkynone double cross-links. The structure and properties of the hydrogel were characterized by scanning electron microscope, Fourier-transform infrared, and rheological test, followed by antibacterial and in vivo biocompatibility tests. The results showed that the hydrogel exhibited good self-healing, mechanical properties, good antibacterial effect, and in vivo biocompatibility, and can inhibit inflammation and promote skin tissue regeneration in mice. This novel self-healing hydrogel dressing has a broad application prospect in skin tissue engineering. Full article

Site-specific antibody conjugations generate homogeneous antibody-drug conjugates with high therapeutic index. However, there are limited examples for producing the site-specific conjugates with a drug-to-antibody ratio (DAR) greater than two, especially using engineered cysteines. Based on available Fc structures, we designed and introduced free cysteine residues into various antibody CH2 and CH3 regions to explore and expand this technology. The mutants were generated using site-directed mutagenesis with good yield and properties. Conjugation efficiency and selectivity were screened using PEGylation. The top single cysteine mutants were then selected and combined as double cysteine mutants for expression and further investigation. Thirty-six out of thirty-eight double cysteine mutants display comparable expression with low aggregation similar to the wild-type antibody. PEGylation screening identified seventeen double cysteine mutants with good conjugatability and high selectivity. PEGylation was demonstrated to be a valuable and efficient approach for quickly screening mutants for high selectivity as well as conjugation efficiency. Our work demonstrated the feasibility of generating antibody conjugates with a DAR greater than 3.4 and high site-selectivity using THIOMAB^TM method. The top single or double cysteine mutants identified can potentially be applied to site-specific antibody conjugation of cytotoxin or other therapeutic agents as a next generation conjugation strategy. Full article

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others. Full article

Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg⁻¹) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg⁻¹), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase. Full article