Page 77 - IJB-9-3

International Journal of Bioprinting 3DP hydrogels to combat antibiotic-resistant bacteria

500 μL of PBS, vortexed for 30 s and sonicated at 35 kHz for with a 4-nm platinum–palladium layer using a Leica EM
15 min in a water bath sonicator (Elma Transsonic T460, ACE600 sputter coater (Microsystems, Wetzlar, Germany).
Elma) to dislodge adherent bacteria. The suspensions Images were acquired at 3 kV using a Zeiss Sigma 300 SEM
and sonicates were serially diluted and the numbers of (Zeiss, Oberkochen, Germany) at the Electron Microscopy
viable bacteria were determined by quantitative culture Center Amsterdam (Amsterdam UMC, Amsterdam, the
on blood agar plates. An MIC/MBC assay was performed, Netherlands). Of each hydrogel, six fields were viewed and
as described in section 2.12, to determine if the bacteria photographed at magnifications of 1,000×.
retrieved from the suspension and detached from the
hydrogels had developed resistance. 2.17. Statistical analysis
All statistical analyses were performed with GraphPad
2.15. Detection of mutation on the rpoB gene Prism 9. The statistical analysis for the adhesion assay
Genomic DNA from selected S. aureus Rif-resistant isolates was performed with a Dunn’s multiple comparison test
was extracted and used as a template for amplification to evaluate the differences between the groups compared
to assess the presence of mutation(s) in the rpoB gene to the control group. For all tests, P-values of ≤0.05 were
that could explain the resistance. The Rif-resistant S. considered significant.
aureus isolates were: S. aureus RN4220, S. aureus AMC
201, S. aureus Rif , S. aureus RN4220 after contact with 2.18. 3D modeling software
R
GelMA-C-NPs hydrogel, S. aureus RN4220 after contact 3D concepts of hydrogels dual antibiotic-loaded NPs
with GelMA-Rif-NPs hydrogel, S. aureus AMC 201 after were designed and rendered in 3ds Max (Autodesk Inc.)
contact with GelMA-C-NPs, and S. aureus AMC 201 after (Figure 4f–i). BioRender was used to make some of the
contact with GelMA-Rif-NPs hydrogel. A Wizard kit was figures in this article.
used for the genomic DNA extraction (Promega, Madison,
WI, USA). A 702-bp fragment from nucleotide positions 3. Results
441–673 corresponding to the Rif resistance-determining 3.1. PLGA NPs characterization
region of the rpoB gene was amplified with rpoB forward Non-loaded control PLGA NPs (C-NPs), Rif-NPs and
(5ʹ-AGTCTATCACACCTCAACAA-3ʹ; Tm 50°C) and Van-NPs were successfully synthesized with three different
reverse (5ʹ-TAATAGCCGCACCAGAATCA-3ʹ; Tm 53°C, molecular weight polymers. C-NPs were prepared with
1°C) primers using the high-fidelity Phusion polymerase LMW PLGA, whereas Rif-NPs and Van-NPs were prepared
[42]
kit (Invitrogen, Waltham, MA, USA) and an annealing with LMW, MMW, or HMW PLGA. SEM characterization
temperature of 50°C. The amplified fragments were was performed to analyze the mean size and the shape
purified from agarose gels with the GeneJET Purification of the PLGA NPs. Spherical shape was observed in the
kit (Thermo Fisher) and sequenced with the rpoB forward C-NPs, Van-NPs, and Rif-NPs (Figure 2). A correlation
and reverse primers at the Core Genomic Facility of the between MW and mean size was observed, since PLGA
Amsterdam UMC, Amsterdam, the Netherlands. The LMW produced the smallest NPs in both the Van- and
nucleotide sequences obtained were aligned to the S. aureus Rif-loaded NPs (Table 1). PDI values below 0.2 were
RN4220 sequence obtained from NCBI (NZ_CP076105.1) considered monodisperse (homogeneous population),
by using Benchling. while PDI values above 0.2 were considered polydisperse

2.16. Biofilm imaging (heterogeneous population). The non-loaded LMW NPs
SEM was performed to visually confirm the bacterial showed a mean size of 228 nm and a PDI of 0.29, which
attachment and biofilm formation on and in the 3D-printed were a significantly higher mean size and a PDI when
hydrogels. The setup was the same as in section 2.14 until compared to the Van- or Rif-loaded LMW NPs (Table 1).
the two washing steps with demineralized water. Before The mean size of LMW Van-NPs and LMW Rif-NPs were
SEM, hydrogels were fixed in 4% (v/v) paraformaldehyde 207 nm and 164 nm, which were significantly lower than
and 1% (v/v) glutaraldehyde (Merck, Kenilworth, NJ, control. The PDIs for both Van-NPs and Rif-NPs were 0.09
United States) overnight at room temperature. The and 0.17, respectively, indicating a narrow size distribution.
hydrogels were then rinsed twice with distilled water for The drug loading of Rif in the NPs was 68.86, 29.80, and
10 min and dehydrated in a graded ethanol concentration 16.60 mg/mg for LMW, MMW, and HMW, respectively,
series from 50% to 100% of ethanol. The hydrogels were showing an inverse correlation between drug loading and
immersed in hexamethyldisilazane (Polysciences Inc., molecular weight of the polymer. For Van-loaded NPs, the
Warrington, FL, United States) overnight and air-dried to drug loading was 59.98, 40.61, and 68.53 mg/mg for LMW,
reduce the surface tension. Before imaging, the hydrogels MMW, and HMW, respectively, without a correlation
were mounted on aluminum SEM stubs and sputter-coated between drug loading and polymer MW.

Volume 9 Issue 3 (2023) 69 https://doi.org/10.18063/ijb.683