Optimized dispersion of nanoparticles for biological in vitro and in vivo studies

Abstract

Background: The aim of this study was to establish and validate a practical method to disperse nanoparticles in physiological solutions for biological in vitro and in vivo studies.

Results: TiO2 (rutile) dispersions were prepared in distilled water, PBS, or RPMI 1640 cell culture medium. Different ultrasound energies, various dispersion stabilizers (human, bovine, and mouse serum albumin, Tween 80, and mouse serum), various concentrations of stabilizers, and different sequences of preparation steps were applied. The size distribution of dispersed nanoparticles was analyzed by dynamic light scattering and zeta potential was measured using phase analysis light scattering. Nanoparticle size was also verified by transmission electron microscopy. A specific ultrasound energy of 4.2 x 105 kJ/m3 was sufficient to disaggregate TiO2 (rutile) nanoparticles, whereas higher energy input did not further improve size reduction. The optimal sequence was first to sonicate the nanoparticles in water, then to add dispersion stabilizers, and finally to add buffered salt solution to the dispersion. The formation of coarse TiO2 (rutile) agglomerates in PBS or RPMI was prevented by addition of 1.5 mg/ml of human, bovine or mouse serum albumin, or mouse serum. The required concentration of albumin to stabilize the nanoparticle dispersion depended on the concentration of the nanoparticles in the dispersion. TiO2 (rutile) particle dispersions at a concentration lower than 0.2 mg/ml could be stabilized by the addition of 1.5 mg/ml albumin. TiO2 (rutile) particle dispersions prepared by this method were stable for up to at least 1 week. This method was suitable for preparing dispersions without coarse agglomerates (average diameter < 290 nm) from nanosized TiO2 (rutile), ZnO, Ag, SiOx, SWNT, MWNT, and diesel SRM2975 particulate matter.

Conclusion: The optimized dispersion method presented here appears to be effective and practicable for preparing dispersions of nanoparticles in physiological solutions without creating coarse agglomerates.

Figure 1

Effect of intensity of sonication on TiO₂ (rutile) particle size. TiO₂ (rutile) dispersed in distilled water at a concentration of 0.02 mg/ml was sonicated with different specific energies, and the average of the hydrodynamic diameter of the particles was measured. The experiments were carried out in triplicates (*, p < 0.05).

Figure 2

Role of the sequence of preparation steps. TiO₂ (rutile) dispersed in distilled water at a concentration of 0.02 mg/ml was sonicated with 4.2 × 10⁵ kJ/m³ specific energy (US) or not sonicated (no US). Tween 80 0.1% (T), HSA 1.5 mg/ml (A) or distilled water (W) was given to the dispersion before (disp, PBS) or after (PBS, disp) addition of concentrated PBS. The average hydrodynamic diameter of the particles was measured (n = 4; *, p < 0.05 vs. dispersion in distilled water (W) without sonication (no US), #, p < 0.05 vs. dispersion in distilled water (W) with sonication (US), § p < 0.05).

Figure 3

Size distribution by volume of a TiO₂ (rutile) dispersion measured after each preparation step. TiO₂ (rutile) was dispersed in distilled water and sonicated (red), then HSA (blue) and finally concentrated PBS (green) was given to the dispersion. TiO₂ (rutile) was also prepared on the same way but without HSA (black).

Figure 4

Albumin from different species and serum as dispersion stabilizer. TiO₂ (rutile) dispersed in distilled water at a concentration of 0.02 mg/ml was sonicated, and HSA, MSA, BSA or mouse serum was given to the dispersion before the addition of concentrated PBS. The average hydrodynamic diameter of the particles was measured. The experiments were carried out in triplicates (*, p < 0.05 vs. dispersion without albumin in PBS; #, p < 0.05 vs. dispersion without albumin in RPMI).

Figure 5

TiO₂ (rutile) particle size in dispersions with different HSA concentrations. TiO₂ (rutile) dispersed in distilled water at a concentration of 0.02 mg/ml was sonicated and HSA at concentrations ranging from 0.0015 to 15 mg/ml was given to the dispersion prior to addition of concentrated PBS. The average hydrodynamic diameter of the particles was measured. The experiments were carried out in triplicates (*, p < 0.05 vs. dispersion with 0.0015 mg/ml HSA).

Figure 6

Polydispersity index of TiO₂ (rutile) in dispersions with different HSA concentrations. TiO₂ (rutile) dispersed in distilled water at a concentration of 0.02 mg/ml was sonicated and HSA at concentrations ranging from 0.0015 to 15 mg/ml were given to the dispersion prior to addition of concentrated PBS. Polydispersity index (PdI) of the particles was measured. The experiments were carried out in triplicates (*, p < 0.05 vs. dispersion with 0.0015 mg/ml HSA).

Figure 7

TiO₂ (rutile) particle size in dispersions with different TiO₂ concentrations. TiO₂ (rutile) dispersed in distilled water at concentrations ranging from 0.002 to 2 mg/ml was sonicated and 1.5 mg/ml HSA was given to the dispersion before adding concentrated PBS to the dispersion. TiO₂ (rutile) dispersions at a concentration of 2 mg/ml were also prepared in the same way but with addition of 10 times more (15 mg/ml) HSA (hatched bar). The average hydrodynamic diameter of the particles was measured. The experiments were carried out in triplicates (*, p < 0.05 vs. dispersion with 2 mg/ml TiO₂ (rutile)).

Figure 8

Polydispersity index of TiO₂ (rutile) dispersions with different TiO₂ concentrations. TiO₂ (rutile) dispersed in distilled water at concentrations ranging from 0.002 to 2 mg/ml was sonicated and 1.5 mg/ml HSA was given to the dispersion before adding concentrated PBS to the dispersion. TiO₂ (rutile) dispersions at a concentration of 2 mg/ml were also prepared in the same way but with addition of 10 times more (15 mg/ml) HSA (hatched bar). Polydispersity index (PdI) of the particles was measured. The experiments were carried out in triplicates (*, p < 0.05 vs. dispersion with 2 mg/ml TiO₂ (rutile)).

Figure 9

Stability of TiO₂ (rutile) dispersion. TiO₂ (rutile) dispersions were prepared in distilled water at a concentration of 0.02 mg/ml with (closed circles) or without (open circles) addition of HSA before giving concentrated PBS to the dispersion. The average hydrodynamic diameter of the particles was measured at different time points for up to one week. The experiments were carried out in triplicates (*, p < 0.05, dispersions with vs. without HSA at the same time point).

Figure 10

Electron microscopy of titanium dioxide nanoparticles. Electron microscopic image at 100,000 times magnification (950 × 950 nm) from TiO₂ (rutile) and TiO₂ (anatase) nanoparticle dispersions prepared in distilled water at a concentration of 0.02 mg/ml without stabilizer (PBS) or with addition of human serum albumin (+HSA) or mouse serum (+Serum) before giving concentrated PBS to the dispersion.

Figure 11

Electron microscopy of zinc oxide, silicon oxide and silver nanoparticles. Electron microscopic image at 100,000 times magnification (950 × 950 nm) from ZnO, SiO_x and silver nanoparticle dispersions prepared in distilled water at a concentration of 0.02 mg/ml without stabilizer (PBS) or with addition of human serum albumin (+HSA) or mouse serum (+Serum) before giving concentrated PBS to the dispersion.

Figure 12

Electron microscopy of nanotubes and diesel particles. Electron microscopic image at 100,000 times magnification (950 × 950 nm) from SWNT, MWNT and diesel (SRM 2975) particle dispersions prepared in distilled water at a concentration of 0.02 mg/ml without stabilizer (PBS) or with addition of mouse serum (+Serum) or human serum albumin (+HSA) before giving concentrated PBS to the dispersion.

Figure 13

Preparation steps of nanoparticle dispersion. 1. Sonicate the nanoparticles in distilled water (power consumption: 7 W, 1 mL dispersion, 60 sec sonication, = 4.2 × 10⁵ kJ/m³). 2. Add stabilizer (1.5 mg/ml HSA for dispersions with less than 0.2 mg/ml nanoparticle concentration or serum with a similar albumin concentration). 3. Add PBS to achieve physiological buffer and salt concentration.