Designing Microreactors Resembling Cellular Microenvironment via

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Designing microreactors resembling cellular microenvironment via polyamine-mediated nanoparticleassembly for tuning Glucose oxidase kinetics Gousia Begum, shikha Lalwani, and Rohit Kumar Rana Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00303 • Publication Date (Web): 06 Jul 2018 Downloaded from http://pubs.acs.org on July 7, 2018

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Bioconjugate Chemistry

Designing Microreactors Resembling Cellular Microenvironment via Polyamine-Mediated Nanoparticle-Assembly for Tuning Glucose Oxidase Kinetics Gousia Begum,* Shikha Lalwani and Rohit Kumar Rana*

Nanomaterials Laboratory, CSIR-Indian Institute of Chemical Technology, Hyderabad-500 007, India. E-mail: [email protected] & [email protected]

ABSTRACT: Spatial confinement of Glucose oxidase (GOx) in the hollow interior of a bioinspired matrix via polyamine mediated silica nanoparticle assembly under environmentally benign conditions is demonstrated herein. In a similarity to the biosilicification processes in Diatoms, we use polyallylamine hydrochloride (PAH) to direct the assembly of silica nanoparticles on CaCO3 spheres as the removable core. When this assembly process is performed on the CaCO3 spheres, which are pre-loaded with GOx in a post-synthesis method, microspheres encapsulating GOx are formed. Interestingly, the encapsulated GOx in these microreactors exhibits activity with a Michaelis-Menten constant (KM) that is 2 to 3-fold less compared to the free enzyme in the solution. While the microenvironment of the organic (PAH)-inorganic (silica) hybrid system can be advantageous for the substrate to interact with enzyme, the effective pH in the vicinity of the entrapped enzyme may also be accountable for the improved activity resulting in the lower apparent KM and enhanced specificity constant (kcat/KM). A 2-fold higher thermal stability of the encapsulated GOx compared to free GOx in solution further demonstrates the efficacy of the integrated architecture. Additionally, the PAH by virtue of its buffering capability allows the microspheres in imparting pH stability to

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the encapsulated GOx. Therefore, the method is not only a greener process for performing enzyme immobilization, but also anticipated to aid in designing microreactors for enhanced enzyme activity. INTRODUCTION Nanostructured reactors, in which enzymes are immobilized, have attracted ever increasing attention due to their potential applications in industrially important biocatalysis besides proteomic analysis, biofuel cells, tissue engineering etc.1-2 Particularly as a biocatalyst, its performance including stability is mainly dependent on the local environment of the immobilized enzyme, which in turn has a consequence on the process sustainability.3 Enzymes are primarily immobilized either covalently on surfaces or entrapped inside various solid matrices, which often requires harsh conditions leading to denaturation of enzymes, or lacks conducive environment or structural robustness resulting in loss in enzymatic activity.4 In this context, as an analogue to the cells in the living systems, microcapsule structures which can provide optimal space mimicking biocompartments for enzymatic reactions would be desirable.5 Inside the living cells, the compartmentalization allows a better control over the biological processes including the enzymatic reactions. The confined space is believed to provide conducive environment for the reactions, controlled permeability through the shell membrane, while the processes are protected from undesired influences that can affect the stability and activity of the enzymes inside the cell.6-7 Hence, there have been efforts to prepare various microcapsule structures for encapsulation of enzymes in their micro- or nanodimension space.

For example, polymer capsules, fabricated either with the aid of a

sacrificial template or via the self-assembly of block copolymers (polymersomes), lipids and surfactants have been used for encapsulation of enzymes.5,8 Although, the polyelectrolyte capsules contains many aspects found in natural cells, such as self-assembly,


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compartmentalization and in few cases provides protection against externally added proteases, these methods have limitations, such as difficulty in handling the materials, their storage and stability towards pH and temperature.9 On the other hand, inclusion of inorganic components to create hybrid compositions have been shown to be beneficial in addressing the stability issues. There are a few examples, which mainly involve silica based hollow structures, owing to their non-toxicity, good biocompatibility, tunable pore size and ease of surface functionalization.10 Cao et al. immobilized glucose oxidase (GOx) in hierarchical hollow silica. Immobilization of glucose oxidase (GOx) in a hierarchical hollow silica sphere consisting of a shell-in-shell structure was shown to improve enzyme loading capacity and mechanical stability with reduced enzyme leaching compared with that for single-shell hollow silica strcuture.11 Hollow silica nanospheres prepared via the microemulsion templating method for confining horseradish peroxidase in its cavity was found to be beneficial in achieving better enzyme activity compared with HRP supported on solid silica spheres.12 In another report, amphiphilic silica Janus particles used for the encapsulation of lipase resulted in capsules with a shell comprising a monolayer of silica particles at the oil/water interface.13 The higher activity of the encapsulated lipase in organic media compared to free enzyme was attributed to large interfacial area and low mass transfer resistance. However, the significance of free space of the micro- or nanoreactors and its impact on the kinetic properties of enzymes encapsulated in silica hollow spheres is rarely studied. Moreover, the use of harsh reaction conditions, such as basic medium, high temperatures and organic solvents have been detrimental to the advancement in this field. In the present work, we report a bio-inspired method of nanoparticle-assembly for an effective confinement of the enzyme. Similar to the role of long chain polyamines in the biomineralization of Diatoms involving silica condensation and their assembly, herein we use polyallylamine hydrochloride (PAH) to assemble silica nanoparticles.14 While the 3 ACS Paragon Plus Environment

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environmentally benign synthesis conditions are conducive for the encapsulation of GOx, the organic-inorganic

hybrid

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providing

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like

microenvironment. RESULTS AND DISCUSSION Synthesis and Characterization of GOx loaded microspheres The encapsulation method involves addition of GOx on to the surface of PAH functionalized CaCO3 microspheres (Scheme 1). As the isoelectric point of GOx is 4.2, at neutral pH it exists in its anionic form.15 So, the PAH coating to CaCO3 microspheres is advantageous to make the surface positive, which in turn is favourable for immobilization of GOx through electrostatic interaction.

Scheme 1. Illustration of the nanoparticle-assembly strategy utilized for encapsulation of GOx to generate GOx loaded silica-PAH hollow (GOx@SPH) spheres. The colour indications are: light orange: CaCO3; green: enzyme; yellow lines: PAH; light blue: SiO2 nanoparticles. The assembly process was monitored by measuring the change in surface charge (Zeta potential, ζ) of the particles at each step using DLS technique (Figure 1). The zeta potential of CaCO3 microspheres was -9.1 mV and after the PAH coating, the surface charge became positive with a potential of 12.1 mV. This positive surface charge of the PAH coated spheres was essential for the immobilization of negatively charged GOx on their surface. Consequently with deposition of GOx, there was a change in the surface charge toward a


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negative ζ potential (-6.7 mV). The spheres were coated again with PAH so as to facilitate the assembly silica nanoparticles forming a shell structure. Finally, the assembled silica nanoparticles imparted a negative surface charge (-26.2 mV). Similarly for the three repetitive steps of the PAH-silica nanoparticle assembly, the zeta potential varied between 2.7 to -26.2 mV depicting the successive changes in the surface charge as desirable for the assembly process.

Figure 1. Change in Zeta potential of the microspheres during the PAH-coating, GOx addition and silica nanoparticle-assembly processes. The GOx loaded CaCO3 (GOx@PAH-CaCO3) particles as further characterized by Field emission scanning electron microscopy (FESEM), revealed formation of micron sized spherical particles in the range of 1-3 µm (Figure 2a). Analysis by dynamic light scattering (DLS) confirmed the size distribution of the particles in the range of 1-3 µm (Figure S1a) in agreement with the FESEM data. Energy dispersive X-ray spectroscopy (EDS) for elemental analysis revealed presence of calcium, carbon, oxygen and nitrogen (Figure 2b & S1 (c-f)). From the X-ray diffraction (XRD) analysis, the formed crystalline phases were identified as both vaterite (51%) and calcite (49%) (Figure 2c). The Fourier transform infrared (FT-IR) spectrum (Figure 2d) also displayed characteristic band for calcite phase at 710 cm-1 in addition to the bands at 746 and 877 cm-1 assignable to the vaterite phase.16-17 Furthermore, a band at 1090 cm-1 together with the split band at 1460 cm-1 indicated the presence of



amorphous phase of CaCO3 as well. The band at 2927 cm-1 corresponded to the asymmetric stretching vibrations of methylene groups from PAH and GOx. A broad band around 3445 cm-1 was attributed to O-H stretching vibrations of adsorbed water molecules. The Raman spectrum presented lattice modes of vibration at 284 and 301 cm-1 indicating the presence of calcite and vaterite phases, respectively (Figure 2e).18 The symmetric stretching vibration (ʋ1) modes of the carbonate group in vaterite structure were revealed at 1074 and 1090 cm-1. Additionally, the band at 750 cm-1 (ʋ4, in-plane bending mode of carbonate) showed the existence of a vaterite phase of CaCO3.

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120 100 1074

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Figure 2 (a) FESEM image (inset: higher magnification image indicating the porous architecture of the surface), (b) EDS (c) XRD pattern (inset: pie diagram indicating the percentage of vaterite and calcite phases of CaCO3), (d) FT-IR, and (e) Micro-Raman spectra of GOx@PAH-CaCO3 microspheres. The GOx loaded PAH-CaCO3 microspheres were then used as the template for the polyamine mediated assembly of silica nanoparticles (GOx@SiO2-PAH-CaCO3). The microscopic analysis illustrated the presence of ~12 nm silica nanoparticles assembled on the surface 6 ACS Paragon Plus Environment

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(Figure 3a). As analyzed by DLS, the nanoparticle-assembly resulted in increase in the particle size and the size distribution was in the range of 2-3 µm (Figure S2a). The element mapping by EDS confirmed the presence of silicon in addition to calcium, carbon, oxygen and nitrogen. The detection of silicon corroborated the presence of silica nanoparticles (Figure 3b & S2 (c-g)). The X-ray diffraction (XRD) analysis (Figure 3c) revealed the presence of vaterite (41%) and calcite phases (59%) of CaCO3.The crystalline phases of CaCO3 were further identified by FT-IR and Micro-Raman analyses (Figure 3d & e). The FTIR and Raman spectra in addition to bands for calcite, vaterite and amorphous phases of CaCO3 exhibited asymmetric and symmetric stretching modes of the Si-O-Si vibrations at 1115 and 802 cm-1 respectively (in FT-IR spectrum) and the Raman band at about 480 cm-1 associated with Si–O–Si linkages (in Raman spectrum).19-21

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Figure 3 (a) FESEM image (inset: higher magnification image indicating the presence of ~12 nm silica nanoparticles on the surface) (b) EDS spectrum (c) XRD pattern (inset: pie diagram indicating the percentage of vaterite and calcite phases of CaCO3) (d) FT-IR and (e) MicroRaman spectra of GOx@SiO2-PAH-CaCO3 microspheres.



The removal of CaCO3 core with sodium acetate buffer generated GOx loaded hybrid silicaPAH hollow (GOx@SPH) spheres. The FESEM analysis of the GOx@SPH showed spherical morphology of the particles of 1-3 µm sizes (Figure 4a). The DLS study confirmed the size distribution of the particle in the same range (Figure S3a). The particles were similar in size and shape to that of the particles before removal of the CaCO3 core. This clearly indicates that the structural integrity of the particles was not disturbed during the synthesis process. Additionally, it was observed that the shell of the hollow sphere consisted of silica nanoparticles resulting in a shell thickness of ~200 nm (Figure S3b & S4). The hollow architecture of the spheres was further determined from the TEM analysis, wherein the presence of ~12 nm sized silica particles could be clearly seen (Figure 4b). The XRD pattern (Figure S3c) and FT-IR spectrum (Figure 4c) did not reveal any characteristic peak for calcium carbonate phase and this was well corroborated with the EDS data (Figure 4a (inset) & S3 (e-h)) indicating the removal of the CaCO3 core (Note: The band at 1631 cm-1 in the FT-IR is due to O–H bending vibrations of adsorbed water molecules). In order to assess the surface area and porosity, the synthesized microspheres were analyzed by N2-sorption studies. The result showed a specific surface area of 238.3 m2g-1 and pore volume of 0.5 cm3g-1 with a broad pore-size distribution (

Designing Microreactors Resembling Cellular Microenvironment via

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