Green synthesis, characterization, and anticancer activity of hyaluronan/zinc oxide nanocomposite

Figures & data

Figure 1 FTIR spectra of (a) hyaluronan, (b) Sargassum muticum, and (c) HA/ZnO nanocomposite.

Abbreviations: FTIR, Fourier transform infrared; HA, hyaluronan; ZnO, zinc oxide.

Figure 2 FESEM image of the HA/ZnO nanocomposite.

Abbreviations: FESEM, field emission scanning electron microscopy; HA/ZnO, hyaluronan/zinc oxide.

Figure 3 Morphological characteristic of HA/ZnO nanocomposite.

Notes: (A) TEM image and (B) particle size distribution of the HA/ZnO nanocomposite.
Abbreviations: TEM, transmission electron microscope; HA/ZnO, hyaluronan/zinc oxide; Std dev, standard deviation.

Figure 4 X-ray diffraction pattern of the HA/ZnO nanocomposite.

Abbreviations: HA, hyaluronan; ZnO, zinc oxide; S. muticum, Sargassum muticum.

Figure 5 UV–vis absorption spectrum of the HA/ZnO nanocomposite.

Abbreviations: UV, ultraviolet; HA, hyaluronan; ZnO, zinc oxide; S. muticum, Sargassum muticum.

Figure 6 The polydispersity index of the HA (1) and HA/ZnO (2) nanocomposite.

Abbreviations: HA, hyaluronan; ZnO, zinc oxide.

Figure 7 Cytotoxic effect of HA/ZnO nanocomposite and Cisplatin using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide on various human cell lines.

Notes: (A) Cytotoxic effects of HA/ZnO nanocomposite on various human cancer cells at 72 h of treatment were evaluated using MTT assay. Each point is the mean value of three replicates. PANC-1: pancreatic adenocarcinoma cell line, CaOV-3: ovarian adenocarcinoma cell line, COLO-205: colonic adenocarcinoma cell line, HL-60: acute promyelocytic leukemia cells. (B) Cytotoxic effects of HA/ZnO nanocomposite on normal human lung fibroblast cell line (MRC-5) at 72 h of treatment were evaluated using MTT assay. Each point is the mean value of three replicates. (C) Cytotoxic effects of cisplatin on HL-60 cells at 24 h, 48 h, and 72 h of treatment were evaluated through mitochondrial activity using the MTT assay. Each point is the mean value of three replicates.
Abbreviations: HA/ZnO, hyaluronan/zinc oxide; h, hours; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Figure 8 Fluorescent micrograph of AO/PI double-stained HL-60 cells that were treated with HA/ZnO nanocomposite.

Notes: (A) Untreated cells showing normal cell structure. (B) Treated cells after 24 h showing membrane blebbing, nuclear margination, and chromatin condensation. (C) Early apoptotic cells showing blebbing and nuclear fragmentation after 48 h treatment. (D) Late apoptotic cells showing nuclear fragmentation and apoptotic body formation after 72 h treatment (magnification 400×).
Abbreviations: AO/PI, acridine orange/propidium iodide; HL-60, acute promyelocytic leukemia cells; HA/ZnO, hyaluronan/zinc oxide; VC, viable cells; BL, membrane blebbing; CC, chromatin condensation; MN, marginated nucleus; EA, early apoptotic cells; FN, fragmented nucleus; LA, late apoptotic cells; AB, apoptotic body.

Figure 9 Flow cytometric analysis of apoptosis induction by HA/ZnO nanocomposite in HL-60 cells after staining with FITC-conjugated annexin-V and PI.

Notes: (A1–C1) Untreated (control) HL-60 cells at 6 h, 12 h, and 24 h incubation, respectively. (A2–C2) Effects of 6 h, 12 h, and 24 h HA/ZnO nanocomposite treatment, respectively.
Abbreviations: HA/ZnO, hyaluronan/zinc oxide; HL-60, acute promyelocytic leukemia cells; FITC, fluorescein isothiocyanate; PI, propidium iodide; h, hours.

Table 1 Flow cytometric analysis of HL-60 cells treated with HA/ZnO nanocomposite

Cell condition	Cells (%)
	Control 6 h	Treated 6 h	Control 12 h	Treated 12 h	Control 24 h	Treated 24 h
Viable cells	97.4±0.74	80.29±0.65	92.30±0.55	75.29±0.15	90.7±0.25	73.5±0.13
Early apoptosis	1.87±0.15	10.5±0.99*	4.95±0.70	15.00±0.30*	6.1±0.45	9.5±0.22*
Late apoptosis/necrosis	0.69±0.35	8.65±0.95**	2.62±0.50	10.7±0.80**	3.8±0.25	16.9±0.10**

Notes: The cells were stained with FITC-conjugated annexin-V and PI and incubated at 37°C for 6 h, 12 h, and 24 h. Values are expressed as mean ± SD of three different experiments. Data have been analyzed using post hoc comparison test, one-way ANOVA, and means compared by Tukey’s b-test.

* Significant (P<0.05) increase of early apoptotic cells in HA/ZNO nanocomposite-treated groups compared to that of untreated control.

** Significant (P<0.05) increase of late apoptotic/necrotic cells in HA/ZnO nanocomposite-treated groups compared to that of untreated control.

Abbreviations: HL-60, acute promyelocytic leukemia cells; HA/ZnO, hyaluronan/zinc oxide; h, hours; FITC, fluorescein isothiocyanate; PI, propidium iodide; ANOVA, analysis of variance; SD, standard deviation.

Figure 10 Cell cycle analysis of HL-60 cells treated with HA/ZnO nanocomposite after staining with PI.

Notes: (A1–C1) Untreated HL-60 cells for 24 h, 48 h, and 72 h, respectively. (A2–C2) Effects of 24 h, 48 h, and 72 h, respectively, exposure of HL-60 cells to HA/ZnO nanocomposite. G0/G1, G2/M, and S indicate the cell phase, and sub-G0/G1 refers to the portion of apoptotic cells.
Abbreviations: HL-60, acute promyelocytic leukemia cells; HA/ZnO, hyaluronan/zinc oxide; PI, propidium iodide; h, hours.

Table 2 Flow cytometric analysis of HL-60 cells treated with HA/ZnO nanocomposite

Cell cycle phases	Cells (%)
	Control 24 h	Treated 24 h	Control 48 h	Treated 48 h	Control 72 h	Treated 72 h
G0/G1	55.25±0.06	43.40±0.45	52.10±0.29	42.61±0.52	40.64±0.32	35.68±0.68
G2/M	16.85±0.76	20.00±0.41*	19.16±0.26	26.29±0.35*	22.30±0.22	27.06±0.93*
Synthesis	24.91±0.06	26.30±0.33	26.24±0.06	20.93±0.12	31.50±0.61	25.35±0.18
Sub-G0/G1	1.9±0.23	10.50±0.28*	2.50±0.34	10.00±0.20*	6.20±0.46	13.85±0.56*

Notes: The cells were stained with PI and incubated at 37°C for 24 h, 48 h, and 72 h. Values are expressed as mean ± SD of three different experiments. Data have been analyzed using post hoc comparison test, one-way ANOVA, and means compared by Tukey’s b-test.

* Significant (P<0.05) increase of cells in sub-G0/G1 phase with accumulation and G2/M cell cycle arrest in HA/ZNO nanocomposite-treated groups compared to that of untreated control.

Abbreviations: HL-60, acute promyelocytic leukemia cells; HA/ZnO, hyaluronan/zinc oxide; h, hours; PI, propidium iodide; ANOVA, analysis of variance; SD, standard deviation.

Figure 11 Effects of HA/ZnO nanocomposite treatment on HL-60 cell caspase-3 and -9.

Notes: The values are mean ± SD of three independent experiments. Significant differences (P<0.05) between treated and control groups for caspase-3 and -9 are found.
Abbreviations: HA/ZnO, hyaluronan/zinc oxide; HL-60, acute promyelocytic leukemia cells; h, hours; SD, standard deviation.

Table 3 Caspases spectrophotometric analysis of HL-60 cells treated with HA/ZnO nanocomposite for 24 h, 48 h, and 72 h

Caspase	Cells (%)
	Control 24 h	Treated 24 h	Control 48 h	Treated 48 h	Control 72 h	Treated 72 h
Caspase-3	0.060±0.0012	0.15±0.001*	0.077±0.005	0.18±0.0043*	0.099±0.0032	0.22±0.006*
Caspase-9	0.069±0.007	0.19±0.003*	0.080±0.001	0.20±0.0025*	0.12±0.002	0.24±0.0035*

Notes: Values are expressed as mean ± SD of three different experiments. Data have been analyzed using post hoc comparison test, one-way ANOVA, and means compared using Tukey’s b-test.

* Significant (P<0.05) increase of apoptotic cells in HA/ZnO nanocomposite-treated groups compared to that of untreated control.

Abbreviations: HL-60, acute promyelocytic leukemia cells; HA/ZnO, hyaluronan/zinc oxide; h, hours; ANOVA, analysis of variance; SD, standard deviation.