The Impact of Biomaterial Surface Properties on Engineering Neural Tissue for Spinal Cord Regeneration.

Tissue engineering for spinal cord injury (SCI) remains a complex and challenging task. Biomaterial scaffolds have been suggested as a potential solution for supporting cell survival and differentiation at the injury site. However, different biomaterials display multiple properties that significantly impact neural tissue at a cellular level. Here, we evaluated the behavior of different cell lines seeded on chitosan (CHI), poly (ε-caprolactone) (PCL), and poly (L-lactic acid) (PLLA) scaffolds. We demonstrated that the surface properties of a material play a crucial role in cell morphology and differentiation. While the direct contact of a polymer with the cells did not cause cytotoxicity or inhibit the spread of neural progenitor cells derived from neurospheres (NPCdn), neonatal rat spinal cord cells (SCC) and NPCdn only attached and matured on PCL and PLLA surfaces. Scanning electron microscopy and computational analysis suggested that cells attached to the material's surface emerged into distinct morphological populations. Flow cytometry revealed a higher differentiation of neural progenitor cells derived from human induced pluripotent stem cells (hiPSC-NPC) into glial cells on all biomaterials. Immunofluorescence assays demonstrated that PCL and PLLA guided neuronal differentiation and network development in SCC. Our data emphasize the importance of selecting appropriate biomaterials for tissue engineering in SCI treatment.

Generation of recombinant antibodies against human tissue kallikrein 7 to treat skin diseases.

Human tissue kallikreins (KLKs) constitute a family of 15 serine proteases that are distributed in various tissues and implicated in several pathological disorders. KLK7 is an unusual serine protease that presents both trypsin-like and chymotrypsin-like specificity and appears to be upregulated in pathologies that are related to skin desquamation processes, such as atopic dermatitis, psoriasis and Netherton syndrome. In recent years, various groups have worked to develop specific inhibitors for this enzyme, as KLK7 represents a potential target for new therapeutic procedures for diseases related to skin desquamation processes. In this work, we selected nine different single-chain variable fragment antibodies (scFv) from a human naïve phage display library and characterized their inhibitory activities against KLK7. The scFv with the lowest IC50 against KLK7 was affinity maturated, which resulted in the generation of four new scFv-specific antibodies for the target protease. These new antibodies were expressed in the scFv-Fc format in HEK293-6E cells, and the characterization of their inhibitory activities against KLK7 showed that three of them presented IC50 values lower than that of the original antibody. The cytotoxicity analysis of these recombinant antibodies demonstrated that they can be safely used in a cellular model. In conclusion, our research showed that in our case, a phage-display methodology in combination with enzymology assays can be a very suitable tool for the development of inhibitors for KLKs, suggesting a new strategy to identify therapeutic protease inhibitors for diseases related to uncontrolled kallikrein activity.

Multifunctional alginate nanoparticles containing nitric oxide donor and silver nanoparticles for biomedical applications.

Nitric oxide (NO) is an endogenous molecule with antimicrobial activity. Silver nanoparticles (AgNPs) are also well known for its antimicrobial properties. In this work, we prepared, characterized, evaluated the cytotoxicity and the antibacterial activity of alginate nanoparticles (alginate NPs) containing S-nitroso-mercaptosuccinic acid (S-nitroso-MSA, a NO donor) and/or AgNPs. AgNPs were obtained by plant mediated synthesis using green tea (Camellia sinensis), according to the green principles. Alginate NPs were obtained by ionotropic gelation with calcium ions (Ca2+) and used to incorporate the NO donor and/or AgNPs. The obtained nanoparticles containing S-nitroso-MSA and/or AgNPs have hydrodynamic size distribution in the range of 103.3 ± 2.9-414.2 ± 22.6 nm with moderate polydispersity index and negative zeta potentials. Kinetic measurements showed a sustained NO release from alginate NPs, at the mili molar range. Both S-nitroso-MSA and AgNPs were able to diffuse from the nanoparticles following a Fickian diffusion mathematical model. The cytotoxicity of the nanoparticles was evaluated towards Vero cells, and a concentration dependent toxicity was observed. The combination of the NO donor and green tea AgNPs enhanced the toxicity to the tested cells. Finally, the antibacterial activity of the nanoparticles was demonstrated against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Streptococcus mutans UA159. Interestingly, the combination of the NO donor and AgNPs into alginate NPs had a superior antibacterial activity, compared with bacteria treated with alginate NPs containing S-nitroso-MSA or AgNPs individually. Moreover, antibacterial effects were observed at nanoparticle concentrations not toxic to Vero cell. To the best of knowledge, this is the first report to demonstrate the ability of alginate NPs releasing NO and AgNPs with potent antimicrobial activity.

Cytotoxicity and Antibacterial Activity of Alginate Hydrogel Containing Nitric Oxide Donor and Silver Nanoparticles for Topical Applications.

Nitric oxide (NO) and silver nanoparticles (AgNPs) are well-known for their antibacterial activity. In this work, S-nitroso-mercaptosuccinic acid (S-nitroso-MSA), a NO donor, and green tea synthesized AgNPs were individually or simultaneously incorporated into alginate hydrogel for topical antibacterial applications. The obtained hydrogels were characterized and the NO release and diffusion of AgNPs and S-nitroso-MSA from alginate hydrogels were investigated. The hydrogels showed a concentration dependent toxicity toward Vero cells. The potent antibacterial effect of the hydrogels was demonstrated toward Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Streptococcus mutans UA159. Interestingly, the combination of S-nitroso-MSA and AgNPs into alginate hydrogels had a superior antibacterial effect, compared with hydrogels containing S-nitroso-MSA or AgNPs individually. This is the first report to describe the synthesis, cytotoxicity, and antibacterial effects of alginate hydrogel containing NO donor and AgNPs. These hydrogels might find important local applications in the combat of bacterial infections.

Antimicrobial Activity and Cytotoxicity to Tumor Cells of Nitric Oxide Donor and Silver Nanoparticles Containing PVA/PEG Films for Topical Applications.

Because of their antibacterial activity, silver nanoparticles (AgNPs) have been explored in biomedical applications. Similarly, nitric oxide (NO) is an important endogenous free radical with an antimicrobial effect and toxicity toward cancer cells that plays pivotal roles in several processes. In this work, biogenic AgNPs were prepared using green tea extract and the principles of green chemistry, and the NO donor S-nitrosoglutathione (GSNO) was prepared by the nitrosation of glutathione. To enhance the potentialities of GSNO and AgNPs in biomedical applications, the NO donor and metallic nanoparticles were individually or simultaneously incorporated into polymeric solid films of poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG). The resulting solid nanocomposites were characterized by several techniques, and the diffusion profiles of GSNO and AgNPs were investigated. The results demonstrated the formation of homogeneous PVA/PEG solid films containing GSNO and nanoscale AgNPs that are distributed in the polymeric matrix. PVA/PEG films containing AgNPs demonstrated a potent antibacterial effect against Gram-positive and Gram-negative bacterial strains. GSNO-containing PVA/PEG films demonstrated toxicity toward human cervical carcinoma and human prostate cancer cell lines. Interestingly, the incorporation of AgNPs in PVA/PEG/GSNO films had a superior effect on the decrease of cell viability of both cancer cell lines, compared with cells treated with films containing GSNO or AgNPs individually. To our best knowledge, this is the first report to describe the preparation of PVA/PEG solid films containing GSNO and/or biogenically synthesized AgNPs. These polymeric films might find important biomedical applications as a solid material with antimicrobial and antitumorigenic properties.

Biocompatible and Antibacterial Nitric Oxide-Releasing Pluronic F-127/Chitosan Hydrogel for Topical Applications.

Nitric oxide (NO) is involved in physiological processes, including vasodilatation, wound healing and antibacterial activities. As NO is a free radical, designing drugs to generate therapeutic amounts of NO in controlled spatial and time manners is still a challenge. In this study, the NO donor S-nitrosoglutathione (GSNO) was incorporated into the thermoresponsive Pluronic F-127 (PL)-chitosan (CS) hydrogel, with an easy and economically feasible methodology. CS is a polysaccharide with known antimicrobial properties. Scanning electron microscopy, rheology and differential scanning calorimetry techniques were used for hydrogel characterization. The results demonstrated that the hydrogel has a smooth surface, thermoresponsive behavior and good mechanical stability. The kinetics of NO release and GSNO diffusion from GSNO-containing PL/CS hydrogel demonstrated a sustained NO/GSNO release, in concentrations suitable for biomedical applications. The GSNO-PL/CS hydrogel demonstrated a concentration-dependent toxicity to Vero cells, and antimicrobial activity to Pseudomonas aeruginosa (minimum inhibitory concentration and minimum bactericidal concentration values of 0.5 µg·mL-1 of hydrogel, which corresponds to 1 mmol·L-1 of GSNO). Interestingly, the concentration range in which the NO-releasing hydrogel demonstrated an antibacterial effect was not found to be toxic to the Vero mammalian cell. Thus, the GSNO-PL/CS hydrogel is a suitable biomaterial for topical NO delivery applications.