Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches.

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity.

Lipid-Coated Hybrid Nanoparticles for Enhanced Bacterial Biofilm Penetration and Antibiofilm Efficacy.

Up to 80% of all infections are biofilm-mediated and they are often challenging to treat as the underlying bacterial cells can become 100- to 1000-fold more tolerant toward antibiotics. Antibiotic-loaded nanoparticles have gained traction as a potential drug delivery system to treat biofilm infections. In particular, lipid-coated hybrid nanoparticles (LCHNPs) were investigated on their capability to deliver antibiotics into biofilms. In this study, LCHNPs composed of a poly(lactic-co-glycolic acid) (PLGA) core and dioleoyl-3-trimethylammonium propane (DOTAP) lipid shell were developed and loaded with vancomycin (Van). In vitro antibacterial and antibiofilm tests were performed to evaluate the antimicrobial efficacy of the LCHNPs. LCHNPs were successfully fabricated with high vancomycin encapsulation and loading efficiencies, and exhibited enhanced antibacterial effects against planktonic Staphylococcus aureus USA300 when compared against Free-Van and Van-PLGANPs. When used to treat USA300 biofilms, Van-LCHNPs eradicated up to 99.99% of the underlying biofilm cells, an effect which was not observed for Free-Van and Van-PLGANPs. Finally, we showed that by possessing a robust DOTAP shell, LCHNPs were able to penetrate deeply into the biofilms.

Lipid-Polymer Hybrid Nanoparticles Enhance the Potency of Ampicillin against Enterococcus faecalis in a Protozoa Infection Model.

Enterococcus faecalis (E. faecalis) biofilms are implicated in endocarditis, urinary tract infections, and biliary tract infections. Coupled with E. faecalis internalization into host cells, this opportunistic pathogen poses great challenges to conventional antibiotic therapy. The inability of ampicillin (Amp) to eradicate bacteria hidden in biofilms and intracellular niches greatly reduces its efficacy against complicated E. faecalis infections. To enhance the potency of Amp against different forms of E. faecalis infections, Amp was loaded into Lipid-Polymer hybrid Nanoparticles (LPNs), a highly efficient nano delivery platform consisting of a unique combination of DOTAP lipid shell and PLGA polymeric core. The antibacterial activity of these nanoparticles (Amp-LPNs) was investigated in a protozoa infection model, achieving a much higher multiplicity of infection (MOI) compared with studies using animal phagocytes. A significant reduction of total E. faecalis was observed in all groups receiving 250 µg/mL Amp-LPNs compared with groups receiving the same concentration of free Amp during three different interventions, simulating acute and chronic infections and prophylaxis. In early intervention, no viable E. faecalis was observed after 3 h LPNs treatment whereas free Amp did not clear E. faecalis after 24 h treatment. Amp-LPNs also greatly enhanced the antibacterial activity of Amp at late intervention and boosted the survival rate of protozoa approaching 400%, where no viable protozoa were identified in the free Amp groups at the 40 h postinfection treatment time point. Prophylactic effectiveness with Amp-LPNs at a concentration of 250 µg/mL was exhibited in both bacteria elimination and protozoa survival toward subsequent infections. Using protozoa as a surrogate model for animal phagocytes to study high MOI infections, this study suggests that LPN-formulated antibiotics hold the potential to significantly improve the therapeutic outcome in highly complicated bacterial infections.

Valorizing okara waste into nutritionally rich polysaccharide/protein-extracts for co-encapsulation of ß-carotene and ferrous sulphate as a potential approach to tackle micronutrient malnutrition.

Colossal amounts of food waste are generated and discarded daily at the expense of financial resources and at a detriment to the environment. One such food waste, okara - a soybean by-product, is valorized in this study by upcycling it into nutritional extracts for micronutrients encapsulation. Micronutrient malnutrition, particularly in the developing world, is a major public health challenge. Herein, okara extracts were obtained through a low-cost extraction process and was subsequently developed as an encapsulant material for micronutrients ß-carotene, and ferrous sulphate encapsulation, using zein as an excipient. Spray-drying, as a scalable technique, was employed to produce various formulations which were assessed for release profiles, shelf-life, ß-carotene antioxidant activity and cell cytotoxicity. Finally, an optimized dual-micronutrient formulation displayed a sequential release with ferrous sulphate releasing in simulated gastric fluid, and ß-carotene releasing predominantly in simulated intestinal fluid. This sequential release profile favors the absorption of both the micronutrients and could potentially enhance their bioavailability.

Hollow Microparticles as a Superior Delivery System over Solid Microparticles for the Encapsulation of Peptides.

PURPOSE: Peptides are gaining significant interests as therapeutic agents due to their high targeting specificity and potency. However, their low bioavailability and short half-lives limit their massive potential as therapeutics. The use of dense, solid particles of biodegradable polymer as a universal carrier for peptides also has its challenges, such as inefficient peptide release and low bioactivity. In this paper, it was established that hollow microparticles (h-MPs) instead of solid microparticles (s-MPs), as peptide carriers, could improve the release efficiency, while better preserving their bioactivity. METHODS: Glucagon like Peptide-1 (GLP-1) was encapsulated as a model peptide. Mass loss, average molecular weight changes, intraparticle pH, polymer-peptide interaction and release studies, together with bioactivity assessment of the peptide for s-MPs and h-MPs were systematically analyzed and evaluated for efficacy. RESULTS: The intraparticle pH of s-MPs was as low as 2.64 whereas the pH of h-MPs was 4.99 by day 7. Consequently, 93% of the peptide extracted from h-MPs was still bioactive while only 58% of the peptide extracted from s-MPs was bioactive. Likewise, the cumulative release of GLP-1 by day 14 from h-MPs showed a cumulative amount of 88 ± 8% as compared to 33 ± 6% for s-MPs. CONCLUSIONS: The cumulative release of peptide can be significantly improved, and the bioactivity can be better preserved by simply using h-MPs instead of s-MPs as carriers.

Osmogen-Mediated One-Step Technique of Fabricating Hollow Microparticles for Encapsulation and Delivery of Bioactive Molecules.

Microparticulate systems composed of biodegradable polymers, such as poly(d,l-lactic-co-glycolic acid) (PLGA), are widely used for controlled release of bioactive molecules. However, the acidic microenvironment within these microparticles, as they degrade, has been reported to perturb the configuration of most encapsulated proteins. In addition, these polymer particles are also reported to suffer from unrealistically slow and incomplete release of proteins. To address these drawbacks, hollow PLGA microparticles are fabricated through a novel one-step oil-in-water emulsion solvent evaporation technique, by capitalizing on the osmotic property of an osmogen. The effects of fabrication para-meters on particle size and morphology, i.e., volume space of hollow cavity and shell thickness, are also studied. These hollow microparticles are subsequently loaded with bovine insulin microcrystals. It is shown that insulin release profiles can be tuned by simply changing the amount of osmogen in the formulation. At the same time, these hollow microparticles are shown to be effective in maintaining the bioactivity of the encapsulated protein.