Regular Article
High throughput acoustic microfluidic mixer controls self-assembly of protein nanoparticles with tuneable sizes

https://doi.org/10.1016/j.jcis.2020.11.070Get rights and content

Abstract

Hypothesis

Protein nanoparticles have attracted increased interest due to their broad applications ranging from drug delivery and vaccines to biocatalysts and biosensors. The morphology and the size of the nanoparticles play a crucial role in determining their suitability for different applications. Yet, effectively controlling the size of the nanoparticles is still a significant challenge in their manufacture. The hypothesis of this paper is that the assembly conditions and size of protein particles can be tuned via a mechanical route by simply modifying the mixing time and strength, while keeping the chemical parameters constant.

Experimental

We use an acoustically actuated, high throughput, ultrafast, microfluidic mixer for the assembly of protein particles with tuneable sizes. The performance of the acoustic micro-mixer is characterized via Laser Doppler Vibrometry and image processing. The assembly of protein nanoparticles is monitored by dynamic light scattering (DLS) and transmission electron microscopy (TEM).

Findings

By changing actuation parameters, the turbulence and mixing in the microchannel can be precisely varied to control the initiation of protein particle assembly while the solution conditions of assembly (pH and ionic strength) are kept constant. Importantly, mixing times as low as 6 ms can be achieved for triggering protein assembly in the microfluidic channel. In comparison to the conventional batch process of assembly, the acoustic microfluidic mixer approach produces smaller particles with a more uniform size distribution, promising a new way to manufacture protein particles with controllable quality.

Introduction

Protein-based nanoparticles have recently attracted considerable attention for various biomedical applications [1], [2]. They have been employed in cell imaging and nanomedicine [3], for the development of vaccines, and targeted drug delivery [4], [5]. Their use has also been proposed to form scaffolds [6], 3D cell culture [7], and biocatalysis [8], [9], [10], [11], [12], [13]. The morphology and the size of the nanoparticles play a crucial role in determining their suitability for different applications. For instance, an optimized size often results in improvement of the immune response of particles in vaccination [14]. For bio-catalysis applications, reducing the particle size is beneficial for colloid stability and further improves catalytic activity due to its higher surface area [15].

Similarly, when drug delivery is considered, a reduction in size often results in increased cell uptake [10], [11], [14], [16]. The desired size for particular applications also depends on the type of particles and their surface structure. On the other hand, relatively larger particles are more favourable in non-medical applications such as CO2 sequestration due to cost-effective and fast recovery of particles after the reaction using membranes [11]. It is thus highly desirable to have a method that can conveniently tune the size of protein particles.

Conventionally, protein nanoparticles are formed via batch methods, and the relevant conditions dictating their self-assembly have been widely studied considering protein-peptide systems. The particle formation is generally controlled via changing of assembly conditions. For instance, modifying the pH, temperature, and ionic strength of the solutions can tune the charge interaction between the protein molecules. In turn, the combination of these factors can be used to control the formation of protein particles with different sizes [10]. However, altering solution conditions may affect the functionality of protein particles and requires additional steps of buffer exchange to return to the ideal solution conditions after assembly, provided that protein particles formed are stable.

For example, encapsulating protein particles after assembly may be necessary to maintain the pH and temperature, hence the bioactivity of proteins [17], [18], [19]. Additionally, in conventional batch methods, large time-scales are needed to uniformly mix the reacting solutions. This leads to the formation of a concentration gradient in the mixing medium and affects the nucleation process and hampers the uniformity and reproducibility of particles.

We address these issues by using an acoustically actuated microfluidic mixer [20] (Fig. 1a) which can homogenize the solutions in under 6 ms. Effectively and rapidly mixing the reacting solutions prevents any concentration fluctuations and minimizes unwanted growth after assembly (Fig. 1c and d).

To demonstrate the feasibility of the microfluidic technique for manufacturing of protein nanoparticles, we select a recently developed protein-peptide candidate, BCA-P114, which consists of enzyme bovine carbonic anhydrase (BCA) fused with a self-assembling peptide (P114) through a GS-linker [11]. BCA-P114 is designed to form protein particles when subjected to a pH or metal-ion stimuli that is introduced by mixing proteins with the trigger. Conventionally, the size of the protein particles can be altered by varying the concentration of magnesium in the solution [10]. Here, we keep all solution conditions such as pH and ionic strength constant. We show that the self-assembly of the particles, their average size and uniformity can be controlled via a mechanical route by simply tuning the mixing time and strength.

Section snippets

Mixer characterization

The star shaped micro mixer (Fig. 1a) consists of a variable thickness, silicon resonator [20] with a through-etched hole, which is sandwiched between two PDMS channels. The BCAP114 and MgCl2 solutions are fed to the system via two inlets on the bottom channel. The assembled protein particles are extracted from the top channel via an outlet (Fig. 1e). When a piezoelectric transducer, attached to the substrate via an epoxy layer applies an AC voltage at MHz frequencies, the sharp features of the

Protein purification

The BCA-P114 fusion protein was expressed in BL21(DE3) competent E. coli cells (New England Biolabs) as described previously [11]. After expression, harvested cells were resuspended in the lysis buffer (50 mM Tris-HCl, 0.5 M NaCl, 0.5% Triton X, 1 mM EDTA pH 8) and sonicated for 5 mins with 10 s pulse and 20 s pause interval at an amplitude of 90% using probe sonicator (Q125 sonicator, Qsonica). After sonication, crude lysate was centrifuged and the supernatant was filtered with 0.45 µm

Conclusion

This paper reports a mechanical route for the controlled assembly of BCA-P114 protein nanoparticles with tuneable sizes. Conventionally, protein particles are assembled via batch methods, where large time-scales are needed to uniformly mix the reacting solutions. This affects the nucleation process and hampers the uniformity and reproducibility of particles. Particle size is typically controlled by tuning the physiochemical conditions of the solutions (such as pH, temperature, and ionic

CRediT authorship contribution statement

Amir Pourabed: Conceptualization, Investigation, Methodology, Formal analysis, Writing - original draft, Writing - review & editing, Visualization. Tayyaba Younas: Investigation, Resources, Validation, Writing - review & editing. Chang Liu: Investigation, Resources. Bhuvana K. Shanbhag: Validation, Writing - review & editing. Lizhong He: Writing - review & editing, Supervision, Project administration. Tuncay Alan: Writing - review & editing, Supervision, Project administration.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This work was performed in part at the Melbourne Centre for Nanofabrication (MCN) in the Victorian Node of the Australian National Fabrication Facility (ANFF). Further, we thank Monash Centre for Electron Microscopy and The Clive and Ramaciotti Centre for Structural Cryo-Electron Microscopy for TEM sample preparation and imaging. We acknowledge Dr Paulo Santos for his help in statistical analysis. LH acknowledges funding from Australian Research Council (ARC) through the ARC Research Hub for

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