Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential.

BACKGROUND: Cutinases are serine hydrolases that degrade cutin, a polyester of fatty acids that is the main component of plant cuticle. These biocatalysts have recently attracted increased biotechnological interest due to their potential to modify and degrade polyethylene terephthalate (PET), as well as other synthetic polymers. METHODS: A cutinase from the mesophilic fungus Fusarium oxysporum, named FoCut5a, was expressed either in the cytoplasm or periplasm of Escherichia coli BL21. Its X-ray structure was determined to 1.9Å resolution using molecular replacement. The activity of the recombinant enzyme was tested on a variety of synthetic esters and polyester analogues. RESULTS: The highest production of recombinant FoCut5a was achieved using periplasmic expression at 16°C. Its crystal structure is highly similar to previously determined Fusarium solani cutinase structure. However, a more detailed comparison of the surface properties and amino acid interactions revealed differences with potential impact on the biochemical properties of the two enzymes. FoCut5a showed maximum activity at 40°C and pH 8.0, while it was active on three p-nitrophenyl synthetic esters of aliphatic acids (C(2), C(4), C(12)), with the highest catalytic efficiency for the hydrolysis of the butyl ester. The recombinant cutinase was also found capable of hydrolyzing PET model substrates and synthetic polymers. CONCLUSIONS: The present work is the first reported expression and crystal structure determination of a functional cutinase from the mesophilic fungus F. oxysporum with potential application in surface modification of PET synthetic polymers. GENERAL SIGNIFICANCE: FoCut5a could be used as a biocatalyst in industrial applications for the environmentally-friendly treatment of synthetic polymers.

A microneedle vaccine printer for thermostable COVID-19 mRNA vaccines.

Decentralized manufacture of thermostable mRNA vaccines in a microneedle patch (MNP) format could enhance vaccine access in low-resource communities by eliminating the need for a cold chain and trained healthcare personnel. Here we describe an automated process for printing MNP Coronavirus Disease 2019 (COVID-19) mRNA vaccines in a standalone device. The vaccine ink is composed of lipid nanoparticles loaded with mRNA and a dissolvable polymer blend that was optimized for high bioactivity by screening formulations in vitro. We demonstrate that the resulting MNPs are shelf stable for at least 6 months at room temperature when assessed using a model mRNA construct. Vaccine loading efficiency and microneedle dissolution suggest that efficacious, microgram-scale doses of mRNA encapsulated in lipid nanoparticles could be delivered with a single patch. Immunizations in mice using manually produced MNPs with mRNA encoding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain stimulate long-term immune responses similar to those of intramuscular administration.

A Machine Learning-optimized system for on demand, pulsatile, photo- and chemo-therapeutic treatment using near-infrared responsive MoS 2 -based microparticles in a breast cancer model.

Cancer therapy research is of high interest because of the persistence and mortality of the disease and the side effects of traditional therapeutic methods, while often multimodal treatments are necessary based on the patient's needs. The development of less invasive modalities for recurring treatment cycles is thus of critical significance. Herein, a light-activatable microparticle system was developed for localized, pulsatile delivery of anticancer drugs with simultaneous thermal ablation, by applying controlled ON-OFF thermal cycles using near-infrared laser irradiation. The system is composed of poly(caprolactone) microparticles of 200 µm size with incorporated molybdenum disulfide (MoS 2 ) nanosheets as the photothermal agent and hydrophilic doxorubicin or hydrophobic violacein, as model drugs. Upon irradiation the nanosheets heat up to ≥50 °C leading to polymer matrix melting and release of the drug. MoS 2 nanosheets exhibit high photothermal conversion efficiency and allow for application of low power laser irradiation for the system activation. A Machine Learning algorithm was applied to acquire optimal laser operation conditions; 0.4 W/cm 2 laser power at 808 nm, 3-cycle irradiation, for 3 cumulative minutes. In a mouse subcutaneous model of 4T1 triple-negative breast cancer, 25 microparticles were intratumorally administered and after 3-cycle laser treatment the system conferred synergistic phototherapeutic and chemotherapeutic effect. Our on-demand, pulsatile synergistic treatment resulted in increased median survival up to 40 days post start of treatment compared to untreated mice, with complete eradication of the tumors at the primary site. Such a system could have potential for patients in need of recurring cycles of treatment on subcutaneous tumors.

Co-Encapsulation of Violacein and Iron Oxide in Poly(lactic acid) Nanoparticles for Simultaneous Antibacterial and Anticancer Applications.

To date, the possibility of drug-resistant bacterial infections in hospitals and intensive care units comprises a significant concern especially for immunocompromised cancer patients. In the current study, violacein and superparamagnetic iron oxide nanoparticles were co-encapsulated in polylactic acid nanoparticles (vio-Fe3O4-PLA) and tested for their antimicrobial and anticancer activity. The loaded nanoparticles presented efficient saturation magnetization that rendered this nanosystem a promising candidate for magnetic targeting. Moreover, violacein released from the nanoparticles at 500 µg/mL successfully inhibited the growth of the "superbug" methicillin-resistant Staphylococcus aureus (MRSA) with an IC50 value of 595.8 µg/mL, while it did not prove effective against multi-drug-resistant Escherichia coli at concentrations of 10-1000 µg/mL. Finally, a concentration of 500 µg/mL of drug loaded magnetic nanoparticles induced an over 80% growth inhibition of glioblastoma and melanoma cancer cell lines with IC50 values of 221.30 and 201.60 µg/mL, respectively. Since bacterial infections are a key clinical problem for cancer patients due to their compromised immune systems, the presented results suggest that our system should be further studied for its simultaneous anti-bacterial and anti-cancer properties, as it comprises a promising strategy for combating bacterial infections and providing anticancer activity through magnetic-targeted delivery.

Engineering a naturally derived hemostatic sealant for sealing internal organs.

Controlling bleeding from a raptured tissue, especially during the surgeries, is essentially important. Particularly for soft and dynamic internal organs where use of sutures, staples, or wires is limited, treatments with hemostatic adhesives have proven to be beneficial. However, major drawbacks with clinically used hemostats include lack of adhesion to wet tissue and poor mechanics. In view of these, herein, we engineered a double-crosslinked sealant which showed excellent hemostasis (comparable to existing commercial hemostat) without compromising its wet tissue adhesion. Mechanistically, the engineered hydrogel controlled the bleeding through its wound-sealing capability and inherent chemical activity. This mussel-inspired hemostatic adhesive hydrogel, named gelatin methacryloyl-catechol (GelMAC), contained covalently functionalized catechol and methacrylate moieties and showed excellent biocompatibility both in vitro and in vivo. Hemostatic property of GelMAC hydrogel was initially demonstrated with an in vitro blood clotting assay, which showed significantly reduced clotting time compared to the clinically used hemostat, Surgicel®. This was further assessed with an in vivo liver bleeding test in rats where GelMAC hydrogel closed the incision rapidly and initiated blood coagulation even faster than Surgicel®. The engineered GelMAC hydrogel-based seaalant with excellent hemostatic property and tissue adhesion can be utilized for controlling bleeding and sealing of soft internal organs.

Experimental and computational understanding of pulsatile release mechanism from biodegradable core-shell microparticles.

Next-generation therapeutics require advanced drug delivery platforms with precise control over morphology and release kinetics. A recently developed microfabrication technique enables fabrication of a new class of injectable microparticles with a hollow core-shell structure that displays pulsatile release kinetics, providing such capabilities. Here, we study this technology and the resulting core-shell microstructures. We demonstrated that pulsatile release is governed by a sudden increase in porosity of the polymeric matrix, leading to the formation of a porous path connecting the core to the environment. Moreover, the release kinetics within the range studied remained primarily independent of the particle geometry but highly dependent on its composition. A qualitative technique was developed to study the pattern of pH evolution in the particles. A computational model successfully modeled deformations, indicating sudden expansion of the particle before onset of release. Results of this study contribute to the understanding and design of advanced drug delivery systems.

Paving the way for successful islet encapsulation.

Type 1 diabetes mellitus (T1DM) is a disorder that decimates pancreatic ß-cells which produce insulin. Direct pancreatic islet transplantation cannot serve as a widespread therapeutic modality owing to the need for lifelong immunosuppression and donor shortage. Therefore, several encapsulation techniques have been developed to enclose the islets in semipermeable vehicles that will allow oxygen and nutrient input as well as insulin, other metabolites and waste output, while accomplishing immunoisolation. Although encapsulation technology continues to face significant obstacles, recent advances in material science, stem cell biology and immunology potentially serve as pathways to success. This review summarizes the accomplishments of the past 5 years.

Microbial Production of Violacein and Process Optimization for Dyeing Polyamide Fabrics With Acquired Antimicrobial Properties.

In the present study, crude bacterial extract containing violacein is investigated for the preparation of antimicrobial polyamide fabrics. The optimal culture conditions of Janthinobacterium lividum (JL) for maximum biomass and violacein production were found to be 25°C, pH 7.0, while the addition of ampicillin of 0.2 mg mL-1 in the small scale increased violacein production 1.3-fold. In scale-up trials, the addition of 1% (v/v) glycerol in a fed-batch bioreactor, resulted in fivefold extracted crude violacein increase with final concentration of 1.828 g L-1. Polyamide 6.6 fabrics were dyed following three different processes; through simultaneous fermentation and dyeing (SFD), by incubating the fabric in the sonicated bacterial culture after fermentation and by using cell-free extract containing violacein. Maximum color change (ΔE) and color strength (K/S) obtained for SFD fabrics were 74.81 and 22.01, respectively, while no alteration of fastness and staining of dye at acid and alkaline perspiration or at water was indicated. The dyed fabrics presented significant antifungal activity against Candida albicans, C. parapsilosis, and C. krusei, as well as antibacterial properties against Escherichia coli, Staphylococcus aureus, and the S. aureus MRSA. We have shown that J. lividum cultures can be successfully used for violacein production and for simultaneous dying of fabrics resulting in dyed fabrics with antimicrobial properties without utilization of organic solvents.

Acylation of soluble polysaccharides in a biphasic system catalyzed by a CE2 acetyl esterase.

Within the last decade, acylated polysaccharides have drawn attention, since they find applications in numerous fields as biocompatible and biodegradable amphiphilic compounds. The ability of a CE2 acetyl esterase from Clostridium thermocellum to catalyze acyl transfer to ß-glucan and manno-polysaccharides of significant interest was investigated. Initially, screening tests were conducted on aldohexose monosaccharides and disaccharides, exploiting the enzyme's strict regioselectivity at O-6 position. Modified monosaccharides acquired acylation yields from 11 up to 65%, showing preference for small chain acyl donors, while disaccharides exhibited conversion yields from 23 up to 58%, with preference to monoacylation. The transesterification reactions were carried out in two-phase mixtures consisted of water/vinyl esters. Acylation of polysaccharides were confirmed by TLC and FT-IR, while the degree of acylation was determined via an indirect method, estimating a range of acylation from 0.022 to 1.083mmolacylgroup*gpolysaccharide-1 depending on the structure and composition of the target polysaccharide.