Antitumor activities of carboplatin–doxorubicin–ZnO complexes in different human cancer cell lines (breast, cervix uteri, colon, liver and oral) under UV exposition

Figures & data

Table 1. Reported studies on therapeutic agents used for cancer treatment in the past 5 years.

Research group	Target cell	Drug carrier	Therapeutic agents
Smith et al. [1]	Human retinoblastoma	–	Carboplatin = 50 mg/mL
Galam et al. [2]	Human breast adenocarcinoma (MCF-7)	Silk fibroin	Carboplatin = 10–200 μg/mL
Ouyang et al. [3]	Human hepatocellular carcinoma (HepG-2) Human breast adenocarcinoma (MCF-7)	Pectin	Doxorubicin = 1–5 μg/mL
Locharoenrat [4]	Human breast adenocarcinoma (MCF-7)	Diamond	Doxorubicin = 0.4 mg/mL
Xia et al. [5,6]	Cervical cancer cells (HeLa) Colorectal cancer cells (HT-29)	RGDfC-Se	Derlin1-siRNA = 200 nM
Xia et al. [7]	Human liver cancer cells (HepG2)	Galactose-Se	Doxorubicin = 4 μg/mL
Xia et al. [8]	Cervical cancer cells (HeLa)	Hyaluronic acid-Se	Doxorubicin = 4 μg/mL
Vu et al. [9]	Human myeloid leukaemia	–	Doxorubicin = 0.5–1 μg/mL
Malky et al. [10]	Human breast adenocarcinoma (MCF-7)	–	Doxorubicin = 0.25–1 μg/mL
Christowitz et al. [11]	Murine breast tumour (E0771)	–	Doxorubicin = 6–15 mg/mL
Al-Harthy et al. [12]	Human breast adenocarcinoma (MCF-7)	–	Doxorubicin = 13–36 μg/mL
Matcovschii et al. [13]	Human glioblastoma	–	Doxorubicin = 2–20 μg/mL
Allen et al. [14]	Mouse gonads	–	Carboplatin = 0.1–1 μg/mL Cisplatin = 0.5–5 μg/mL
Kuittinen et al. [15]	Ovarian cancer cells	–	Carboplatin = 0.1–50 μg/mL Paclitaxel = 1–10 ng/mL
Corn et al. [16]	Human prostate cancer	–	Carboplatin = 3–4 mg/mL Cabazitaxel = 20–25 mg/mL
Lopes et al. [17]	Human and mouse ovary	–	Doxorubicin = 1–2 μg/mL Cisplatin = 5–10 μg/mL
Yu et al. [18]	Bladder cancer (MBT-2)	–	Doxorubicin = 0.62 μg/mL Lipodoxorubicin = 130 μg/mL
Eroglu et al. [19]	Human breast adenocarcinoma (MCF-7)	–	Doxorubicin = 0.01 mg/mL 5-Fluorouracil = 6 mg/mL Propranolol = 30 mg/mL
Escobar-Resendiz et al. [20]	Human lung carcinoma cell (A549)	–	Carboplatin = 515 μg/mL Cisplatin = 10 μg/mL AMB = 29 μg/mL A21 derivative = 67 μg/mL

Table 2. Detailed preparation of chemo drug under various binding conditions.

ZnO (µM)	DOX (µM)	CP (µM)	Binding ratio of CP:DOX = CP:1	Loading capacity^a (%)	Loading efficiency^a (%)
388.95	5.39	8.42	1	66.15	81.13
388.95	2.69	12.63	3	74.98	94.55
388.95	1.35	14.73	7	76.24	97.52
388.95	0.67	15.78	15	77.43	98.19
388.95	0.34	16.31	31	77.81	99.05

CP: carboplatin; DOX: doxorubicin; ZnO: zinc oxide nanoparticles.

^a See Ref. [4] for detailed calculations.

Figure 1. (a) Absorbance and (b) calibration curve at the various concentrations of DPPH*. A, B, C, D, E, F, G, H, I and J represent 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µM of DPPH*, respectively.

Figure 2. TEM images of the formulated chemo drugs: (a) ZnO, (b) DOX + ZnO, (c) CP + ZnO and (d) CP + DOX + ZnO. CP: carboplatin; DOX: doxorubicin; ZnO: zinc oxide nanoparticles.

Table 3. Quantities of chemo drug in DPPH*.

Chemo drug formulation	Reaction kinetics	IC50 (mole drug/mole DPPH*)	Number of reduced DPPH*	Zeta potential (mV)	Zeta average (nm)	Fluorescence quantum yield
ZnO	Intermediate	0.0765	6.536	35.9667	48.133	–
CP	Intermediate	0.0817	6.120	–	–	–
CP + ZnO	Intermediate	0.0644	7.764	17.3667	68.600	–
DOX	Intermediate	0.0516	9.689	–	–	0.170–0.302
DOX + ZnO	Intermediate	0.0254	19.685	21.8667	73.967	0.241–0.403
CP + DOX	Intermediate	0.0447	11.185	–	–	0.226–0.642
CP + DOX + ZnO	Slow	0.0052	96.153	24.4333	81.600	0.399–0.860
CP + ZnO + UV	Slow	0.0184	27.173	–	–	–
DOX + ZnO + UV	Slow	0.0127	39.370	–	–	–
CP + DOX +  ZnO  + UV	Slow	0.0037	135.135	–	–	–

CP: carboplatin; DOX: doxorubicin; ZnO: zinc oxide nanoparticles; IC₅₀: inhibitory concentration at 50% inhibition.

Number of reduced DPPH* was calculated by the inverse value of inhibitory concentration multiplied by 0.5 (p<.05).

Figure 3. FTIR spectra of the formulated chemo drugs before UV-irradiation on DPPH*.

Figure 4. (a) Absorbance, (b) fluorescence and (c) fluorescence quantum yield of DOX, before and after ZnO adsorption at a reaction time of 0 min. A, B, C, D and E represent 5.39, 2.69, 1.35, 0.67 and 0.34 µM of DOX, respectively. (d) Absorbance, (e) fluorescence and (f) fluorescence quantum yield of CP + DOX, before and after ZnO adsorption at a reaction time of 0 min. Notably, DOX or CP + DOX remained in the supernatant after facilitating adsorption to the drug-loaded nanoparticle complexes. Absorbance and fluorescence peaks are centred at 485 and 594 nm, respectively. See Ref. [24] for detailed equations of fluorescence quantum yield.

Table 4. Representative drug release rate of CP + DOX + ZnO + UV under different binding ratios (pH = 6.0).

Binding ratio of CP:DOX = CP:1	% Drug release rate at different time intervals (min)
	10	30	60	90	180	360	720
1	20.15	29.41	45.2	58.76	68.94	78.15	80.18
3	22.08	35.05	49.98	64.20	71.98	78.48	80.05
7	30.05	41.22	54.81	68.94	74.52	78.01	80.01
15	36.25	48.82	64.32	74.94	77.64	79.55	80.06
31	41.02	52.70	68.48	78.19	79.89	80.06	80.12

CP: carboplatin; DOX: doxorubicin.

Figure 5. Integrated absorption intensities of DPPH* caused by (a) DOX, (b) DOX + ZnO and (c) DOX + ZnO + UV as a function of reaction time from 0 to 720 min at different concentrations of DOX. Disappearance of DPPH* caused by (d) DOX, (e) DOX + ZnO and (f) DOX + ZnO + UV as a function of reaction time from 0 to 720 min at different concentrations of DOX. Molar ratio of ZnO to DOX is 5:1. A, B, C, D and E represent 5.39, 2.69, 1.35, 0.67 and 0.34 µM of DOX, respectively.

Figure 6. Integrated absorption intensities of DPPH* caused by (a) CP, (b) CP + ZnO and (c) CP + ZnO + UV as a function of reaction time from 0 to 720 min at different concentrations of CP. Disappearance of DPPH* caused by (d) CP, (e) CP + ZnO and (f) CP + ZnO + UV as a function of reaction time from 0 to 720 min at different concentrations of CP. Molar ratio of ZnO to CP is 5:1. A, B, C, D and E represent 16.31, 15.78, 14.73, 12.63 and 8.42 µM of CP, respectively.

Figure 7. Integrated absorption intensities of DPPH* caused by (a) CP + DOX, (b) CP + DOX + ZnO and (c) CP + DOX + ZnO + UV as a function of reaction time from 0 to 720 min at different ratios of CP and DOX. Disappearance of DPPH* caused by (d) CP + DOX, (e) CP + DOX + ZnO and (f) CP + DOX + ZnO + UV as a function of reaction time from 0 to 720 min at different ratios of DOX and CP. Molar ratio of ZnO to (CP + DOX) is 5:1. A, B, C, D and E represent CP:DOX = 1:1, 3:1, 7:1, 15:1 and 31:1, respectively.

Figure 8. Disappearance of DPPH* at a steady state with respect to drug-DPPH*. (a) Molar ratio of ZnO to DOX is 5:1. (b) Molar ratio of ZnO to CP is 5:1. (c) Molar ratio of ZnO to (CP + DOX) is 5:1. IC₅₀ value of each formulated chemo drug is also presented.

Table 5. Comparative cytotoxicity of chemo drug in DPPH* in comparison to human cancer cell lines from triplicate experiments, p<.05 using ANOVA followed by the Bonferroni post hoc test.

Chemo drug formulation	IC50 (µg/mL)						Cell viability (%)
	DPPH*	HT-29	KB	MCF-7	HeLa	HepG-2	HaCat
UV (% cytotoxicity)	–	– (7.175)	– (8.982)	– (8.984)	– (7.011)	– (7.899)	91.73 (8.20)
ZnO	2.515	1.979	2.014	2.002	2.349	2.025	71.25
CP	2.410	1.924	2.019	1.975	2.311	1.950	70.05
CP + ZnO	2.390	1.828	1.963	1.944	2.162	1.885	71.84
DOX	1.895	1.594	1.661	1.528	1.484	1.375	71.55
DOX + ZnO	1.473	1.173	1.361	1.210	1.140	1.364	71.05
CP + DOX	1.278	1.065	1.258	1.111	1.075	1.129	71.05
CP + DOX + ZnO	0.301	0.241	0.224	0.208	0.221	0.209	71.06
CP + ZnO + UV	0.683	0.590	0.551	0.390	0.492	0.456	83.77
DOX + ZnO + UV	0.736	0.749	0.650	0.516	0.619	0.594	84.08
CP + DOX + ZnO + UV	0.214	0.173	0.150	0.137	0.144	0.140	88.11

HT-29: human colon adenocarcinoma; KB: human oral cavity carcinoma; MCF-7: human breast adenocarcinoma; HeLa: human cervical adenocarcinoma; HepG-2: human hepatocarcinoma.

Human keratinocyte immortal cells (HaCat) is the normal human cell line.

Figure 9. (a) Phase contrast images of human normal cell lines (HaCat) after incubation for 24 h under UV light. (b) Fluorescence image of human normal cell lines (HaCat) after incubation for 24 h in the presence of CP + DOX + ZnO + UV. Cytoplasm of live cells is shown in red. Scale bar indicates 50 µm.

Figure 11. Representative median fluorescence intensity (MFI) and the percentages of cellular apoptosis treated by CP + DOX + ZnO + UV from three independent experiments analysed by flow cytometry, p<.05 using ANOVA followed by the Bonferroni post hoc test.

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Data availability statement

All data and material used to support the findings of this study are included within the article.