Page 359 - IJB-10-2

International Journal of Bioprinting 3D bioprinting for vascular regeneration

characteristics for 3D printing applications. It is our imaging analyses of NPs were performed using a Bio-TEM
hypothesis that these specially designed NPs offer distinct (Talos L120C TEM, Thermo Fisher Scientific, Waltham,
advantages, primarily through their unique capability USA) operating at 200 kV, and the size distribution was
in controlled and sustained drug release. Rapamycin, investigated. A diluted NP suspension was dropped onto
a potent immunosuppressant and mTOR inhibitor, a carbon-coated mesh transmission electron microscopy
exhibits immense potential for preventing restenosis. 31-34 (TEM) grid, and the solvent was left to evaporate at room
By encapsulating rapamycin within NPs, it is possible to temperature. The NP size distributions were analyzed from
precisely deliver the therapeutic agent to targeted areas, the TEM images using ImageJ software (National Institutes
facilitating controlled release and sustained effects. This of Health, USA).
approach offers a unique advantage in maintaining the
patency and functionality of engineered blood vessels, 2.3. Rapamycin release profiling from rapamycin-
further enhancing their therapeutic potential. nanoparticles
The drug release amount of rapamycin in NPs was
2. Materials and methods measured using a liquid chromatography-mass
spectrometry system (QTRAP 6500 plus; SCIEX, USA)
2.1. Preparation of nanoparticles and rapamycin- according to the previously described method. 36,37 NP-R
nanoparticles was diluted in distilled water and harvested on indicated
The NPs were prepared using the previously reported days. Rapamycin released from the NPs was centrifuged at
method with a few modifications. First, 0.5 g of 10,000 × g for 10 min, and supernatants were analyzed. The
35
cetyltrimethylammonium bromide (CTAB) in 1 L of quantification of the data was performed by setting the 5
deionized water was mixed with 2 mL NaOH (1 M) while days as the 100% reference point and relatively quantifying
stirring continuously; 1 mL tetraethyl orthosilicate and 0.1 the data for the 1 and 3 days.
g N-(2-aminoethyl)-3-aminopropyltrimethoxy silane were
then added after separately dissolving them in ethanol 2.4. Drug loading efficiency of rapamycin-
at a 1:5 volume ratio and 1:5 weight ratio, respectively. nanoparticles
A white precipitate was formed, which was filtered out, Drug loading efficiency of rapamycin in NPs was measured
washed, and dried at 80°C for 48 h. The CTAB from the using an ultraviolet-visible spectrometer (Evolution 300,
precipitate was removed by dispersing the dried precipitate Thermo Fisher Scientific, USA). Rapamycin was dissolved
in 100 mL of ethanol solvent; 0.3 g of NH NO was added in methanol and added to the NPs at various ratios: 1:2,
4
3
to the solution and stirred at 60°C for several hours, after 1:5, and 1:10. Subsequently, the supernatant was collected,
which the precipitate was collected by filtration, followed diluted 1/100 in methanol, and placed in a cuvette before
by drying at 60°C for 12 h. Thereafter, 0.2 g ZnCl was measuring its absorbance.
2
added to deionized water, and the precipitate was added
to the zinc solution. The solution was dried using a rotary 2.5. Preparation of 3D-bioprinted artificial blood
evaporator; the obtained powder was further centrifuged, vessel
washed with water and ethanol several times, dried at 80°C To prepare the bioink, sodium alginate (viscosity, >2,000 cP
for 12 h, and calcinated at 55°C for >5 h. The resultant at 25°C; Sigma-Aldrich; St. Louis, MO, USA) was stirred into
products were referred to as NPs. After dissolving 80 mg Dulbecco’s phosphate-buffered saline (DPBS; Gibco, USA)
of NPs in 80 mL ethanol, they were dispersed by ultrasonic for 6 h at 37°C. Subsequently, an atelocollagen solution (pH
waves for 3 min; simultaneously, 20 mg of rapamycin was 4.0; Baobab Healthcare, Republic of Korea) was combined
dissolved in 20 mL ethanol and mixed for 3 min at a speed with reconstituted buffer (132 mM Na HPO ) at a 1:1 volume
2
4
of 300 rpm. The above solutions were depressurized for 1 h ratio for neuralization. The 3D bioprinter (Root 1; Baobab
and maintained at 100 BAR in a vacuum state. The pressure Healthcare, Republic of Korea) equipped with a coaxial
was then lowered to 60 BAR, maintained for 30 min, and nozzle (inner needle: 28 G, outer needle: 20 G; Ramé-hart,
then dried in a vacuum condition at room temperature for USA) was used for vascular printing. For the shell material,
6 h; this product was termed NP-rapamycin (NP-R). a mixture of 3% w/v neutralized atelocollagen and 3% w/v
alginate was prepared at a 4:1 ratio for the bioink. NP-R
2.2. Characterization of nanoparticles and or NP only was then incorporated into the shell bioinks to
rapamycin-nanoparticles achieve a final concentration of 2 mg/mL. Regarding the
The zeta potential of the NPs and rapamycin-loaded NPs core material, a sacrificial material was prepared using
were measured using a Zetasizer Nano ZSP (Malvern 40% w/v Pluronic F-12 (Sigma-Aldrich) with 100 mM
Panalytical, Malvern, UK) according to the manufacturer’s calcium chloride. Vascular cell printing was performed by
instructions. Transmission electron microscopy (TEM) blending the shell material with endothelial progenitor cells

Volume 10 Issue 2 (2024) 351 doi: 10.36922/ijb.1465