Regular ArticleHigh throughput acoustic microfluidic mixer controls self-assembly of protein nanoparticles with tuneable sizes
Graphical abstract
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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