Page 74 - IJB-9-3

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

compounds such as growth factors or antimicrobial agents. The degree of functionalization (DoF) was determined
PLGA (lactic-co-glycolic acid) is a polymer used extensively as previously described . OPA reacts in the presence of
[33]
for drug delivery formulations and is approved by the primary amine or thiol groups to generate a fluorescent
U.S. Food and Drug Administration (FDA) . Previous product. The OPA reagent was prepared by dissolving 2.5 g
[34]
works studied PLGA nanoparticles (NPs) encapsulating of OPA in 560 mL of sodium borate 40 mM (pH 10.4),
different antimicrobial compounds and their effectivity in 65.5 mL of methanol, and 3 mL of 2-mercaptoethanol.
preventing or treating bacterial infections [35,36] . Gelatin or GelMA were dissolved at 1% (w/v) in

Therefore, this study aimed to develop a 3D-printed deionized water, mixed with OPA reagent in a volume
GelMA-PLGA NPs multilayer structure that allows local ratio 1:2 (sample:OPA), and fluorescence was measured
and sustained delivery of two antibiotics, i.e., Rif and Van, at 340/455 nm (microplate reader Fluostar Omega, BMG
to prevent the selection of antibiotic-resistant bacterial Labtech, Jozefow, Poland). Butylamine was used as a
strains and allow the eradication of even the antibiotic- standard for the calibration curve (Figure S1). The DoF
resistant strains. The formulation developed in this was calculated according to the following equation:
work may be applied mainly as a 3D-printed coating on free amine molgelatin − free amine molGelMA
orthopedic implants or as a bioink for tissue engineering DoF =
solutions. This technique may offer a personalized therapy free amine molgelatiin solution
based on the patient’s needs where the physician will have (I)
the option to combine (a wide range of) different antibiotic
inks with a personalized drug loading and controlled 2.3. Preparation of Rif-PLGA nanoparticles
release. NPs loaded with Rif (Rif-NPs) were synthesized
following the single emulsion and evaporation process,
[37]
2. Materials and methods as previously described (Figure 1b.1). PLGA with
three different molecular weights (LMW, MMW, and
2.1. Materials HMW) was used. First, 500 mg of PLGA were dissolved
Type A gelatin from porcine skin (G1890), methacrylic in 20 mL of DCM. Rif was dissolved in the solution at
anhydride (94%), 2-Hydroxy-4-(2-hydroxyethoxy)- 15% (w/w). The solution was poured into 200 mL of PVA
2-methylpropiophenone (Irgacure 2959, 98%), 2% (w/v) and sonicated for 1 min at 40% amplitude.
O-Phthaldialdehyde (OPA), butylamine, Span 80, The solution was left under stirring overnight to allow
rifampicin (Rif), vancomycin (Van), and sodium evaporation of DCM. The obtained Rif-NPs were washed
borate were ordered from Sigma-Aldrich (St. Louis, with deionized water by centrifugation at 6,000 rpm for
MO, United States. Poly (D,L-lactide-co-glycolide) 30 min. The washing process was repeated three times.
(PLGA; acid endcap) low (LMW; AP081) and medium The supernatants were stored for further analysis, and
(MMW; AP041) molecular weight was purchased from the washed Rif-NPs were freeze-dried and sterilized by
Akina, Inc. (West Lafayette, IN, United States) and high gamma radiation (25 kGy dose). Rif-NPs were stored at
molecular weight (HMW) Purasorb PDLG 5004A was 4°C for further use.
purchased from Corbion (Amsterdam, the Netherlands).
Phosphate-buffered saline (PBS) tablets were purchased 2.4. Preparation of Van-PLGA nanoparticles
from Carl Roth Gmbh (Karlsruhe, Germany). Polyvinyl NPs loaded with Van (Van-NPs) were synthesized by the
alcohol (PVA) 87%–89% hydrolyzed 13–23 kDa was double emulsion and evaporation process, as described
[38]
purchased from Acros Organics (Geel, Belgium). Sorbitan previously (Figure 1b.2). Van-NPs with three different
Monooleate (Span 80) was purchased from Glentham Life molecular weight PLGA polymers were synthesized, i.e.,
Science (Corsham, United Kingdom). Dichloromethane with LMW, MMW, and HMW. First, 500 mg of PLGA were
(DCM) was purchased from Stanlab (Lublin, Poland). dissolved in 20 mL of DCM. Then, Span80 was added to
the solution at 1% (w/v). Two milliliter of Van at 4.5% (w/v)
2.2. Synthesis of GelMA in PBS were added to the solution and sonicated for 1 min
[33]
GelMA was synthesized as previously described . In and 40% amplitude (Vibra-cell, Sonics & Materials Inc.,
short, type A porcine gelatin was dissolved at 10% (w/v) Newton, CT, United States). The emulsion was poured into
in PBS at 60°C. About 0.8 mL of methacrylic anhydride 200 mL PVA 2% solution and sonicated at 40% amplitude
per gram of gelatin was added dropwise under constant for 1 min. The evaporation, cleaning, freeze-drying, and
stirring (Figure 1a). After 3 h, the solution was dialyzed sterilization processes were performed as described above
against deionized water at 37°C for 5 days to remove the for the Rif-NPs. Non-loaded NPs (C-NPs) were prepared
methacrylic acid and anhydride. The solution was freeze- by following the same method as Van-NPs, but without
dried for 72 h and stored at -20°C until further use. Van in the PBS.

Volume 9 Issue 3 (2023) 66 https://doi.org/10.18063/ijb.683