Materials
Bulk crystals of black phosphorus (BP) were obtained from Smart-elements (Vienna, Austria). Sodium hyaluronate (HA, MW = 10 kDa) was acquired from Lifecore Biomedical (Chaska, MN, USA). 1-Methyl-2-pyrrolidinone (NMP), 1,6-diaminohexane (DAH), dimethyl sulfoxide (DMSO), and N-hydroxysulfosuccinimide (NHS) sodium salt were purchased from Sigma Aldrich (St. Louis, MO). 5-Aminofluorescein (FITC) and 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) hydrochloride were purchased from Tokyo Chemical Industry (Tokyo, Japan). Cell counting kit-8 (CCK-8) was acquired from Dojindo Molecular Technologies (Kumamoto, Japan). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotics were purchased from Gibco (Grand Island, NY). HepG2 cells were purchased from Korean Cell Line Bank (Seoul, Korea).
Preparation of BP nanosheets
BP nanosheets were prepared by liquid exfoliation technique. In brief, 30 mg of BP was dispersed in 30 mL NMP solution with sodium hydroxide. Then, the solution was sonicated for 10 h (amplifier: 20%, on/off: 6 s / 4 s). The non-exfoliated BP nanosheets were removed using centrifugation for 10 min at 5000 rpm and the supernatant was centrifuged again for 15 min at 12,500 rpm to re-disperse BP nanosheets in distilled (DI) water.
Preparation of HA-DAH/BP complexes
HA-DAH was synthesized by the EDC chemistry between HA (10 kDa, 100 mg) and DAH (578 mg, 10 M ratios to HA) in DI water at pH 4.8 for 24 h. Then, HA-DAH was dialyzed against 15% ethanol and DI water, respectively (MWCO = 3500 Da). HA-DAH was freeze-dried for 3 days. After that, BP nanosheets were mixed with HA-DAH at an equal content and stirred for 1 h to form HA-DAH/BP complexes. The solution was centrifuged for 15 min at 12,500 rpm to remove excess HA-DAH and obtained HA-DAH/BP complexes were dispersed in DI water.
Characterization of BP nanosheets and HA-DAH/BP complexes
The prepared BP nanosheets and HA-DAH/BP complexes were analyzed by dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Instruments Co., Malvern, UK), UV/vis spectrophotometry (S-3100, Scinco Co., Seoul, Korea), Fourier transform - infrared spectroscopy (FT-IR, Cary 600, Agilent Technologies), and transmission electron microscopy (TEM, JEM-1011, JEOL Co., Akishima, Japan). The physical structure of BP nanosheets and HA-DAH/BP complexes was analyzed by TEM, and the surface modification of BP nanosheets with HA was assessed by DLS and FT-IR.
Stability and biodegradation tests
BP nanosheets and HA-DAH/BP complexes were suspended in DI water at an equal concentration and then the absorbance spectrum of each solution was measured for 7 days by UV/vis spectrophotometry.
In vitro release test of FITC
First, HA-FITC conjugates were synthesized by the EDC chemistry between FITC and HA-DAH with a DAH content of 50 mol%. FITC/BP complexes were prepared by simple mixing of BP nanosheets and FITC, and HA-FITC/BP complexes were also prepared by simple mixing of BP nanosheets and HA-FITC conjugates via electrostatic interaction. Solutions were centrifuged for 15 min at 12,500 rpm to remove excess FITC or HA-FITC. After 0, 48, 96, and 144 h, fluorescent intensity of FITC/BP and HA-FITC/BP complexes was analyzed using a microplate fluorometer (Fluoroskan ascent FL, Thermo Scientific). The released amount of FITC was calculated from the decrease of the fluorescent intensity.
Determination of cu(Cu) content
CuSO4 aqueous solution (10 mL, 40 μM) was mixed with BP nanosheets or HA-DAH/BP complexes solution (10 mL, 20 μg mL− 1) for 1 h. BP nanosheets and HA-DAH/BP complexes were prepared at the desired degradation time point from day 0 to 6. After centrifugation for 15 min at 12,500 rpm, the concentration of metal ions in the supernatants was measured using an atomic absorption spectrometer (TAS-990, Puxi, China).
Binding capacity of BP nanosheets and HA-DAH/BP complexes to metal ions
Each metal ion stock solution was prepared from CaCl2, Mg (NO3)2, ZnCl2, FeCl3·6H2O and CuSO4·5H2O, respectively. BP nanosheets or HA-DAH/BP complexes solution (10 mL, 20 μg mL− 1) was mixed with 10 mL of the metal ion stock solution for 1 h. After centrifugation for 15 min at 12,500 rpm, the concentration of metal ions in the supernatants was measured using an atomic absorption spectrometer (TAS-990, Puxi, China). The binding capacity was calculated by the following equation as previously reported elsewhere [9]:
$$ \mathrm{Binding}\ \mathrm{capacity}=\frac{{\mathrm{C}}_{\mathrm{T}}-{\mathrm{C}}_{\mathrm{s}}}{{\mathrm{C}}_{\mathrm{T}}}\times 100 $$
where CT represents the total concentration of metal ions in the mixture and Cs represents the concentration of metal ions in the supernatant.
Characterization of BP-Cu and HA-DAH/BP-Cu complexes
The energy dispersive spectroscopy mapping (EDS mapping) of BP, BP-Cu, HA-DAH/BP, HA-DAH/BP-Cu was performed with a field emission - scanning electron microscope (FE-SEM, JSM-7401F, JEOL, Akishima, Japan).
In vitro biocompatibility test
Liver cancer cells, HepG2, in DMEM were maintained in a humidified 5% CO2 incubator at 37 °C. HepG2 cells were seeded at a density of 2 × 104 onto 96-well plates and treated with different concentrations of BP nanosheets and HA-DAH/BP complexes for 24 h. Serum-free medium and CCK-8 were added to the cells after washing with PBS. The relative cell viability (n = 4) was assessed by the microplate reader (EMax microplate reader, Bucher Biotec AG, Basel, Switzerland).
In vitro cellular uptake
HepG2 cells were seeded at a density of 1 × 104 cells onto the confocal dish. After incubating for a day, the cells were pre-incubated with excess HA for 2 h to confirm HA receptor-mediated endocytosis of HA-FITC/BP complexes. FITC/BP or HA-FITC/BP complexes were added, incubated for 2 h, and washed with PBS. The cells were fixed with 4% paraformaldehyde solution and stained with DAPI. Images were acquired by confocal microscopy (TCS SP5 Ltd., Leica Korea).
Statistical analysis
Statistical comparison was performed using the software SigmaPlot 10.0 (Systat Software Inc. San Jose, CA). Values for *P < 0.05 and ***P < 0.001 were considered significant.