Immobilization of the recombinant, plant-derived Petroselinum crispum phenylalanine ammonia lyase (PcPAL) in electrospun matrices have the potential to create promising, easy-to-use biocatalysts. Polylactic acid (PLA) a biologically inert, commercial biopolymer, was chosen as the material of the carrier system. PLA could be electrospun properly only from water-immiscible organic solvents, which limits its application as a carrier of sensitive biological objects. The emulsion electrospinning is a proper solution to overcome this issue using non-ionic emulsifiers with different hydrophilic-lipophilic balance (HLB) values. The stabilized emulsion could protect the sensitive PcPAL dissolved in the aqueous buffer phase and improve fiber formation, plus help to keep the biocatalytic activity of enzymes. In this study, the first approach is described to produce PLA nanofibers containing PcPAL enzymes by emulsion electrospinning and to use the resulted biocatalyst in the ammonia elimination reaction from l-phenylalanine.
The application of enzyme-based therapies has received significant attention in modern drug development. Lipases are one of the most versatile enzymes that can be used as therapeutic agents in basic skin care and medical treatment related to excessive sebum production, acne, and inflammation. The traditional formulations available for skin treatment, such as creams, ointments or gels, are widely applied; however, their use is not always accompanied by good drug penetration properties, stability, or patient adherence. Nanoformulated drugs offer the possibility of combining enzymatic and small molecule formulations, making them a new and exciting alternative in this field. In this study polymeric nanofibrous matrices made of polyvinylpyrrolidone and polylactic acid were developed, entrapping lipases from Candida rugosa and Rizomucor miehei and antibiotic compound nadifloxacin. The effect of the type of polymers and lipases were investigated, and the nanofiber formation process was optimized to provide a promising alternative in topical treatment. Our experiments have shown that entrapment by electrospinning induced two orders of magnitude increase in the specific enzyme activity of lipases. Permeability investigations indicated that all lipase-loaded nanofibrous masks were capable of delivering nadifloxacin to the human epidermis, confirming the viability of electrospinning as a formulation method for topical skin medications.
The increasing application of recombinant enzymes demands not only effective and sustainable fermentation, but also highly efficient downstream processing and further stabilization of the enzymes by immobilization. In this study, a novel approach for the isolation and immobilization of His-tagged transaminase from Chromobacterium violaceum (CvTA) has been developed. A recombinant of CvTA was simultaneously isolated and immobilized by binding on silica nanoparticles (SNPs) with metal affinity linkers and additionally within poly(lactic acid) (PLA) nanofibers. The linker length and the nature of the metal ion significantly affected the enzyme binding efficiency and biocatalytic activity of CvTA-SNPs. The formation of PLA nanofibers by electrospinning enabled rapid embedding of CvTA-SNPs biocatalysts and ensured enhanced stability and activity. The developed advanced immobilization method reduces the time required for enzyme isolation, purification and immobilization by more than fourfold compared to a classical stepwise technique.
In vitro non-cellular permeability models such as the parallel artificial membrane permeability assay (PAMPA) are widely applied tools for early-phase drug candidate screening. In addition to the commonly used porcine brain polar lipid extract for modeling the blood–brain barrier’s permeability, the total and polar fractions of bovine heart and liver lipid extracts were investigated in the PAMPA model by measuring the permeability of 32 diverse drugs. The zeta potential of the lipid extracts and the net charge of their glycerophospholipid components were also determined. Physicochemical parameters of the 32 compounds were calculated using three independent forms of software (Marvin Sketch, RDKit, and ACD/Percepta). The relationship between the lipid-specific permeabilities and the physicochemical descriptors of the compounds was investigated using linear correlation, Spearman correlation, and PCA analysis. While the results showed only subtle differences between total and polar lipids, permeability through liver lipids highly differed from that of the heart or brain lipid-based models. Correlations between the in silico descriptors (e.g., number of amide bonds, heteroatoms, and aromatic heterocycles, accessible surface area, and H-bond acceptor–donor balance) of drug molecules and permeability values were also found, which provides support for understanding tissue-specific permeability.
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