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Alshehri, et al.
fixation was done by 1% (w/v) osmium tetroxide in 0.1 allows improved gas exchange and nutrient diffusion.
M PBS for 1 h in the dark followed by washing 3 times The size of the gas vesicles is also ideal for biomedical
by ddH O. This was followed by serial dehydration with applications. GVNPs are small enough to fit within the
2
10 mL of H O (twice), 25% ethanol, 50% ethanol, 75% pores of the fibrous extracellular matrix (ECM) while
2
ethanol, 80% ethanol, 90% ethanol, and 100% (v/v) still too large to cross the blood-brain barrier and cell
ethanol (twice). Samples in ethanol were then critically membrane .
[52]
point dried using liquid CO (Sorvall Critical Point
2
Drying System). 3.2. Hexapeptide characterization
2.12. Statistical analysis The IK amphiphilic peptide was designed with a
6
hydrophilic head group at the C terminus followed
All experimental approaches were executed in triplicates by a series of hydrophobic residues, increasing in
to allow for statistical testing. Results are represented hydrophobicity to the N terminus at the tail end of the
as mean ± standard deviation, n ≥ 3. The differences peptide. The peptide in this study readily formed a
observed in HEK293 cell behavior for conditions with hydrogel in PBS with the aforementioned method, and
and without GVNPs were compared. Statistical analysis was selected for its rapid self-assembly, ease of use, and
was performed by a Student’s t-test, and values with biocompatibility. As a bioink, this peptide demonstrated
P < 0.05 were considered statistically significant. excellent gelling properties as it formed quickly enough to
ensure the structural integrity of the construct and printed
3. Results and discussion smoothly to avoid clumping or inconsistencies as some
3.1. Characterization of GVNPs other materials have. We have previously reported fibrous
peptide network formation, characterizing the structures
Haloarchaeal GVNPs are attractive for biotechnological with circular dichroism (CD) and X-ray diffraction . The
[53]
and biomedical applications and have already found peptide IK form similar network as recently described
6
use as drug delivery systems and contrast agents for self-assembling ultrashort tera peptide compound [54,55] .
ultrasound and magnetic resonance imaging [30-35] . While In this case, the peptide was characterized with SEM
the biotechnological tools for the expression of GVNPs after printing with our printing system (Figure 3, Figure
are now established, production so far suffers from the S3). This imaging showed evidence of the hydrogel’s
slow growth rates, the inconsistent induction system, and formation of a fibrous network present throughout the
the genetic instability of the Halobacterium expression construct. These morphological characteristics are similar
host. Therefore, we developed a new plasmid construct to those observed in hydrogel samples prepared manually.
allowing fast and high-yielding GVNPs production in The samples prepared for imaging were at a concentration
H. volcanii without the requirement of antibiotics. The of approximately 16 mM, which is the minimum gelation
completely sequenced and clean genetic background concentration, and this concentration of peptide was used
with respect to gas vesicle genes makes H. volcanii a for the remainder of the printing experiments with the cells
suitable and attractive organism of choice for expressing and GVNPs. This ensured that the printing in subsequent
Halobacterium GVNPs. H. volcanii is a nonpathogenic experiments occurred smoothly and consistently, and
halophile that grows to high density in the presence of high further highlights both the versatility of our printing
concentrations of salt, which precludes contamination by system and the strong case for the use of ultrashort self-
less halophilic microorganisms [47,51] . assembling peptide as bioink material.
Expression of the gas vesicle operon in H. volcanii
led to spindle- or cylinder-shaped gas vesicles with 3.3. Effect of GVNPs on cell viability in 3D
conical tips. Transmission electron microscopy (FEI construct
Titan CT microscope) equipped with a 4 k × 4 k CCD
camera (Gatan, Pleasanton, CA, USA) and dynamic light Halobacterium sp. NRC-1 gas vesicle cytotoxicity
scattering (DLS, Zetasizer Nano, Malvern Inc., Malvern, was assessed through the 2D culture of HEK cells in a
[30]
U.K.) measurements of purified gas vesicles nanoparticle previous study . The results showed nearly 100% cell
suspensions range from 30 to 250 nm in width and from viability even at the highest concentrations of GVNPs
40 nm to 1.5 μm in length with a mean diameter of 255 nm tested, approximately 500 μg/mL . Cytotoxicity of gas
[30]
(Figure 2). The straightforward protein expression vesicles expressed in H. volcanii was assessed in this
system and efficient purification process produced a work by testing cell growth and proliferation in 2D culture
substantial number of gas vesicles that can be scaled (Figure S4) and 3D culture with varying concentrations
(Figure S2). In addition, the spindle or cylindrical shape of GVNPs.
of these GVNPs (Figure 2) is of particular interest in this Bright-field microscopy imaging and cell viability
study because the increased volume to surface area ratio (live/dead assay) were performed to examine the HEK

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