Vial sonication and ultrasonic immersion probe sonication to generate stable dispersions of multiwall carbon nanotubes for physico-chemical characterization and biological testing

Figures & data

Table 1. Description of the sonication conditions used.

Sonication method	Sonicator	Amplitude (%)	Cycle time (%)	Instantaneous sonication power (W/ml)	Time (h)	Energy delivered to suspension (J/ml)	MWCNT concentration (mg/mL)
PSa	probe	50	50	0.97	0.5	1746	0.2
PSb	probe	50	50	0.97	0.5	1746	1.0
VSc	vial	50 + 75	50	0.85 (50% amplitude) and 1.6 (75% amplitude)	1 + 0.5	3060 + 2880 = 5940	0.2
VSd	vial	75	50	1.6	6	34560	0.2
VSe	vial	75	50	1.6	12	69120	0.2

Table 2. Summary of features of probe and vial sonicator sonication.

Probe sonication	Vial sonicator sonication
- Direct sonication Variable geometry decreases reproducibility Shorter sonication time Larger treatment volumes possible Active cooling possible	- Indirect sonication Fixed sample holder’s geometry improves reproducibility Longer sonication times (pulsed) may be necessary to allow heat dispersion No contamination by probe and reduce risk of bio-contamination

Figure 1. TEM images of 0.2 mg/mL MWCNT material stock suspension in tannic acid sonicated in a vial sonicator by the sonication methods: (a) VSc (b) VSd, (c) VSd, (d) variation in the % of fiber length with time (1< fiber length (µm)<5). (e–f) length size distribution after 6 (VSd) and 12 h (VSe) of sonication.

Table 3. Mean fiber length, corresponding standard deviation and % of total individual fibers counted: “short: 0.15 µm Length 0.9 µm” and “long: 1 µm Length 5 µm” of samples subject to probe sonication and vial sonicator sonication (PSa: probe 0.2 mg/mL, PSb: probe 1 mg/mL, and VSc: vial sonicator 0.2 mg/mL), persistent vial sonicator sonication for 6 (VSd) and 12 h (VSe) and subsequent storage for 1 month after sonication (PSa, PSb and VSc).

Sonication method	MWCNT sample	Length (µm)	% of fibers between 0.15 µm Length 0.9 µm	% of fibers between 1 µm Length 5 µm
PSa	Probe sonicated stock (0.2 mg/mL)	0.27 ± 0.19	87.3	12.7
PSa	Probe sonicated stock (0.2 mg/mL) (1 month storage)	0.27 ± 0.17	86.8	13.2
PSb	Probe sonicated stock (1.0 mg/mL)	0.28 ± 0.16	91.3	8.7
PSb	Probe sonicated stock (1.0 mg/mL) (1 month storage)	0.28 ± 0.18	87.2	12.7
VSc	Vial sonicated stock (0.2 mg/mL)	0.28 ± 0.17	88.8	11.2
VSc	Vial sonicated stock (0.2 mg/mL) (1 month storage)	0.29 ± 0.17	90.2	9.8
VSd	Vial sonicated stock (0.2 mg/mL) (6 hours sonication)	0.25 ± 0.16	96.5	3.5
VSe	Vial sonicated stock (0.2 mg/mL) (12 hours sonication)	0.25 ± 0.16	97.6	2.4

Figure 2. Temporal variation of UV-Vis absorption at 800 nm of MWCNTs dispersed with Tannic acid (300 mg/L).

Figure 3. TEM images of MWCNT material stock suspension in tannic acid as prepared (a,c,d) and after 48 h in culture medium (b,d,f) as follows: (a,b) PSb, (c,d) PSa, (e,f) VSc. Corresponding graphs of stocks suspensions show the length measured and the Gaussian distribution with mean length calculated.

Figure 4. A549 in vitro toxicity experiments using CFE (A,C) and HCI (B,D). Cells exposed to different concentrations of probe and vial sonicated MWCNTs from stock 0.2 mg/mL (PSa and VSc) and the corresponding solvent controls (A,B). Cells exposed to probe-sonicated MWNCTs from stock 1 mg/mL (PSb) and its solvent control (C,D). Cell viability was calculated by counting colonies formed after exposure as compared with the negative control for CFE assay and by determining the number of nuclei in the exposed conditions as compared to the number of nuclei in the negative control for HCl test. Results are expressed in cell viability as percentage of negative control. The tannic acid concentrations corresponding to each MWCNT concentration tested are expressed in µg/mL.

Figure 5. Nuclei-stained A549 cells exposed to MWCNTs for in vitro toxicity experiments using HCI. After incubation for 48 h with the corresponding controls or MWCNTs, cells were fixed, stained with Hoechst and imaged using INCell Analyzer. Negative control (A), solvent control for 0.2 mg/mL (B), solvent control for 1.0 mg/mL (C), PSb stock at 200 µg/mL. (D), PSa stock at 40 µg/mL concentration (E), VSc stock at 40 µg/mL concentration (F).

Table 4. IC50 estimated in vitro values for MWCNTs dispersed using a probe and a vial sonicator and corresponding solvent control (tannic acid).

Sonication method	MWCNT samples	IC50 (µg/mL) by CFE		IC50 (µg/mL) by HCl
		MWCNTs	Tannic acid	MWCNTs	Tannic acid
	Solvent control (0.2 mg/mL)	15.9	23.9	11.4	17.1
PSa	Probe sonicated (0.2 mg/mL)	37.8	–	29.8	–
VSc	Vial sonicated (0.2 mg/mL)	12.1	–	25.7	–
	Solvent control (1 mg/mL)	95.7	28.7	82.1	24.63
PSb	Probe sonicated (1 mg/mL)	668.6*	–	517.6*	–

* The IC50 values given for sample PS_b were not reached experimentally; they were estimated using non-linear regression with an inhibitory dose-response model.

Cell viability was calculated using CFE and HCI. PSa: probe 0.2 mg/mL, PSb: probe 1 mg/mL, and VSc: vial sonicator 0.2 mg/mL.

Figure 6. (a) UV-Vis spectra of tannic acid solution (300 mg/L) before and after Amicon 10 KDa filtration, 0.2 µm filtration and filtrate of sonicated MWCNTs (PSb), (b) Cell viability percentage by CFE of A549 exposed to tannic acid after filtration and corresponding to 60 µg/mL of tannic acid.

Figure 7. Uptake mechanisms observed after A549 exposure to MWCNTs; this material is clearly internalized by cells mainly by 2 different mechanisms: (i) directly passing through the membrane for single MWCNTs of around 300 nm length, that are later found inside lysosomes or late endosomes (a,b) or (ii) via endocytic pathway after membrane invagination mainly for MWCNT bundles (c) or both mechanisms together (d). (a) PSb; (b) VSc; (c,d) PSa.