Electrospun Nanofibers as enzyme carrier

Electrospinning is known to be a very versatile technique as part of incorporating functional additives to nanofibers. Enzymes are useful in accelerating reactions of targeted molecules under mild conditions are used in various industries. However, they are generally sensitive to the environmental conditions and costly. For the use of enzymes in an economically viable manner, they have to be protected from detrimental conditions until the point of use and retained for subsequent usage where possible. This is where electrospinning and its nanofibers come in. Electrospinning may be used as a protective carrier for enzymes and their large surface area allows rapid release of the enzymes when needed. Enzymes may also be immobilized on electrospun nanofibers for repeated use and this is where their high surface area compared to film carrier gives it an advantage.

Enzymes, being proteins, can be sensitive to the environment and the processing conditions. Using electrospinning, the enzyme will be subjected to high voltage and high shearing force. Since most enzymes have low molecular weight, a carrier polymer is usually needed to enable electrospinning into fibers and organic solvents may also be used to dissolve the polymer. Hence the processing and solution conditions may denature the enzyme or affect its activity after it has been electrospun. Onyekuru et al (2021) investigated the enzymatic activity of alkaline phosphatase (ALP) with poly(ethylene oxide) (PEO) after electrospinning and electrospraying. The ALP activity can be maintained at 100% for both blended and core-shell electrospun fibers. For electrospraying, the ALP activity can be maintained at 100% for ALP blended with PEO but the activity drops by 40% for core-shell particles. This reduction in activity has been attributed to the voltage used. Where the ALP activity was maintained at 100%, the voltage used was below 16 kV. However, for electrospraying core-shell particles, the voltage used was 22.5 kV. Other conditions were not found to affect ALP activity.

Since some biological agents may be sensitive to organic solvents used in the preparation of polymer solution for electrospinning, emulsion electrospinning offers a way to encapsulate the biological agent and shield it from the organic solvents for electrospinning. Koplányi et al (2021) was able to prepare an emulsion containing Petroselinum crispum phenylalanine ammonia lyase (PcPAL) enzyme and biodegradable polylactic acid (PLA) for electrospinning. The addition of emulsifiers was demonstrated to generate more uniform, and mainly spherical droplets in the emulsion as compared to polymorphic droplets in emulsifier-free precursor mixtures. Electrospun fibers from emulsifiers with lower HLB values (< 9.7) resulted in thinner and more uniform nanofibers. However, the lowest HLB values and the corresponding smallest diameter fiber did not result in the best enzymatic activity. Electrospun fibers from emulsifier-free precursor mixtures showed low enzymatic activity while significantly higher enzymatic activity were found in emulsion formed with PLA, PcPAL and Brij 30 emulsifier (HLB 9.7). PLA/Brij 30 with 0.15% PcPAL loading has a specific enzyme activity (U_E) of 117 Ug^-1 compared to non-immobilized PcPAL with U_E of 96 Ug^-1.

Electrospun nanofibers are a good carrier for quick release of enzyme for applications such as microfluidic detection chip. High surface area of the nanofibers allows rapid dissolution in appropriate water or solvents. Dai et al (2012) used water soluble electrospun polyvinyl pyrrolidone nanofibers as carrier for horseradish peroxidises (HRP). Placed in a microfluidic chip, PVP nanofibers loaded with HRP readily dissolve to release HRP when the aqueous sample passed through it.

Enzymes immobilized in electrospun fibers have been shown to retain its functionality and even after exposure to adverse conditions. Wang et al (2008) compared lipase activity of those encapsulated in electrospun polyvinyl alcohol (PVA) through blending and crude lipase after storage in different conditions for the digestion of olive oil. Their studies showed that lipase in PVA was able to retain 85% of the original activity when stored at 40°C and 50% relative humidity for 4 h compared to 70% loss of crude lipase activity after storage for 1 h under the same condition. It is also possible to cross-link PVA to render it insoluble in water using glutaraldehyde (GA) with optimized condition at 0.1 M GA/EthOH at pH 3 to give the highest activity of 360 U/g of lipase in 10% lipase loaded fibers.

The enzyme's ability to withstand the electrospinning process is not universal across all enzymes. Kim et al (2020) showed that glucose oxidase (GOx) blended into poly(vinyl alcohol) (PVA) aqueous solution followed by the cross linking agent 1,2,3,4-butanetetracarboxylic acid (BTCA), when electrospun produced nanofibers with very low enzyme activity. However, when GOx was allowed to form complexes with β-cyclodextrin (β-CD) prior to blending with PVA and BTCA for electrospinning and post spinning heat treatment, the enzymatic activity improved significantly to 59.3%. The enzyme activity was further increased to 76.3% with the addition of Au nanoparticles into the PVA/BTCA/β-CD/GOx hydrogel, possibly due to better conductivity.

High surface area of nanofibers also exposes more enzymes for reaction. Nakane et al (2007) showed that lipase immobilized on polyvinyl alcohol (PVA) gave rise to high activity for esterification compared to film immobilized lipase and lipase powder. Lower activity in film may be attributed to lower surface area. Lower performance of lipase powder may be attributed to agglomeration of the powder which decreases its surface area. In nanofibers, the interconnected fibers and high porosity maintains open channel for the participation of the enzymes throughout the membrane. This gives it better activity compared to the other forms. Moreover, recovery of the membrane immobilized with the enzyme was much easier compared to lipase powder and commercial form powders. Surface immobilization of enzymes is a good way to maintain the reactivity of the enzyme and increase its stability. Pan et al (2015) used electrospun poly(styrene-co-methacrylic acid) (PSMAA) random copolymer nanofibers which has surface carboxyl groups for surface adhesion of horseradish peroxidise (HRP) which has amine groups. Comparing the stability and performance between immobilized HRP on nanofibers and free HRP, the immobilized HRP showed higher thermal stability, pH stability and storage stability. The activity of immobilized HRP on nanofiber was 87% while free HRP was only 45% after 5 days of storage. This has been attributed to reduced freedom of conformational change in the immobilized enzymes.

Covalent bonding of enzymes to the surface of electrospun nanofibers provides a more secure way of immobilizing the enzymes. Uzun et al (2014) fabricated nylon 6,6/multi-walled carbon nanotube (MWCNT) with a coating of conductive polymer, (poly-4-(4,7-di(thiophen-2-yl)-1H-benzo[d]imidazol-2-yl)benzaldehyde) (PBIBA). Glucose oxidase (GOx) was covalently bonded on the surface of the composite nanofiber using glutaraldehyde (GA). The glucose biosensor was found to display consistent results for 78 measurements its performance did not decrease after 44 days. N-ethyl-NO-(3-dimethylaminopropyl carbondiimide) and N-hydroxysuccinimide (EDC-NHS) is another cross-linking agent commonly used to covalently bond enzyme to fibers. Mondal et al (2014) fabricated electrospun TiO₂ nanofibers and used plasma treatment to introduce various functional groups (eg. -COOH, -CHO, -OH) on the surface. These functional groups were used to form covalent bond with -NH₂ groups on the enzymes cholesterol esterase and cholesterol oxidase (ChEt-ChOx) molecules for cholesterol.

The performance of catalytic activity of immobilized enzyme compared to free enzymes depends on whether the fiber material is able to complement the process. Encapsulation and immobilization of papain on electrospun polyvinyl alcohol (PVA) using GA showed catalytic activity that was only 88% that of free enzyme [Moreno-Cortez et al 2015]. However, the removal of o-Methoxyphenol by the immobilized HRP at 80% was better than free HRP. This has been attributed to PSMMA nanofiber for facilitating adsorption of the harmful chemical and bringing the chemical closer to the enzyme for deactivation. Dai et al (2013) also made similar observation using laccase immobilized in different electrospun fibers for the removal of polycyclic aromatic hydrocarbons (PAH). The degradation efficiency of the laccase immobilized nanofibers depends on the hydrophobic-hydrophilic properties of the fiber material which influenced its ability to absorb the PAHs. By concentrating the PAH molecules around the enzyme molecules on the nanofibers, it brings about greater degradation efficiency which was better than free laccase. Using electrospun poly(acrylonitrile-comaleic acid) (PANCAA) for covalent bonding of lipase, Wang et al (2006) showed that the addition of multi-walled carbon nanotube (MWCNT) into the electrospun PANCAA matrix was able to increase the activity of the enzyme. The MWCNT facilitate the activity by increasing electron transfer in the reaction.

Degradation kinetics of o-Methoxyphenol by PSMAA, free HRP and HRP-PSMAA. [Pan et al 2015. Journal of Nanomaterials, vol. 2015, Article ID 616879. This work is licensed under a Creative Commons Attribution 3.0 Unported License.]

While a greater catalytic efficiency of the immobilized enzyme on the electrospun fibers will increase the activity of the membrane or scaffold, another obvious method of increasing its activity is to load more enzymes on the fibrous structure. Nair et al (2007) showed that by opening up the pores of electrospun polystyrene-poly(styrene-co-maleic anhydride) (PSPSMA) fibers, the amount of enzyme loading can be increased up to 8 times. This significantly increases the enzymatic activity per unit mass of the nanofibers.

Electrospun fibers may be used to create a favorable environment to increase the performance and activity of enzymes. Most lipases perform catalytic action at hydrophilic-hydrophobic interfaces. Zhang et al (2023) demonstrated the use of electrospun fibers to create numerous hydrophilic-hydrophobic interfaces to achieve high catalytic efficiency. Two construction strategies were tested using electrospun fibers. The first method was to electrospin hydrophobic polyurethane (PU) and hydrophilic polyacrylic acid (PAA) fibers using separate nozzles but simultaneously on a collector. This will result in a nonwoven, evenly distributed mix of hydrophobic and hydrophilic fibers (PUf/PAAf). The second method was to blend hydrophobic ethyl cellulose (EC) and hydrophilic PAA polymers into a single solution followed by electrospinning into a EC/PAA fibrous membrane. The constructed membranes were subsequently heat treated for thermal cross-linking. Lipase was introduced into the membranes by soaking them in a lipase solution. The membranes would swell as it absorbed the water and took in the lipase. Hybrid membrane reactors (HMRs) were assembled by stacking the membranes and placing it in p-NPP isooctane solution. Both PUf/PAAf membrane and EC/PAA blended membrane performed much better than conventional macroscopic "oil-up/water-down" systems and emulsion systems. PUf/PAAf membrane and EC/PAA blended membrane showed a respective 2.56-fold and 2.12-fold improvement in specific activity of lipases over macroscopic "oil-up/water-down" systems and respective 1.93-fold and 2.01-fold over emulsion systems.

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