Tailoring stimuli-responsive delivery system driven by metal–ligand coordination bonding

Figures & data

Figure 1 Particle size and PDI in culture media as a function of time: Z₁/CMCS₁ NPs (A), Z₁/CMCS₁-TA/Cu ²⁺1 NPs (B) and Z₁/CMCS₁-TA/Ca²⁺ NPs (C).

Notes: Control referred to the freshly prepared NPs without 10-fold diluting in culture media. Data displayed as mean ± SD (n=3). The Z₁/CMCS₁ NPs were prepared at a zein:CMCS ratio of 1:1 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺or Ca²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; NPs, nanoparticles; PDI, polydispersity index; TA, tannic acid; SD, standard deviation; Z₁, zein₁.

Figure 2 TEM image and size distribution of Z₁/CMCS₁ NPs (A) and (B), Z₁/CMCS₁-TA/Cu₁²⁺ NPs (C) and (D) and Z₁/CMCS₁-TA/Ca²⁺ NPs (E) and (F). TEM image of Z₁/CMCS₁-TA/Cu₂²⁺ NPs (G) and Z₁/CMCS -TA/Cu₃²⁺ NPs (H). Digital photographs of the NPs are shown in I. 1 to 5 in I refer to Z₁/CMCS₁ NPs, Z₁/CMCS₁-TA/Cu₁²⁺ NPs, Z₁/CMCS₁-TA/Cu₂²⁺ NPs, Z₁/CMCS₁-TA/Cu₃²⁺ NPs and Z₁/CMCS₁-TA/Ca²⁺ NPs, respectively. The Z₁/CMCS₁ NPs were prepared at a zein:CMCS ratio of 1:1 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺), 0.48 mM (Cu₂²⁺) and 0.72 mM (Cu₃²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; NPs, nanoparticles; TA, tannic acid; TEM, transmission electron microscope; SD, standard deviation; Z₁, zein₁.

Figure 3 TEM image and size distribution of DOX-loaded Z₁/CMCS₁-TA/Cu₁²⁺ NPs (A–C) and Z₁/CMCS₁-TA/Ca²⁺ (D–F). The Z₁/CMCS₁ NPs were prepared at zein:CMCS ratio of 1:1 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; DOX, doxorubicin hydrochloride; NPs, nanoparticles; TA, tannic acid; TEM, transmission electron microscope; Z₁, zein₁.

Figure 4 In vitro release profiles of DOX-loaded Z₁/CMCS₁ NPs (A), Z₂/CMCS₁-TA/Cu₁²⁺ NPs (B), Z₂/CMCS₁-TA/Cu₂²⁺ NPs (C), Z₂/CMCS₁-TA/Cu₃²⁺ NPs (D), Z₁/CMCS₁-TA/Cu₁²⁺ NPs (E), Z₁/CMCS₁-TA/Cu₂²⁺ NPs (F) and Z₁/CMCS₁-TA/Cu₃²⁺ NPs (G) in 0.01 M buffer under different pH conditions.

Notes: The Z₂/CMCS₁-TA/metal NPs were prepared at a zein:CMCS ratio of 2:1 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺), 0.48 mM (Cu₂²⁺) and 0.72 mM (Cu₃²⁺). The Z₁/CMCS₁-TA/metal NPs were prepared at a zein:CMCS ratio of 1:1 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺), 0.48 mM (Cu₂²⁺) and 0.72 mM (Cu₃²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; DOX, doxorubicin hydrochloride; NPs, nanoparticles; TA, tannic acid; Z₁, zein₁; Z₂, zein₂.

Figure 5 In vitro release profiles of DOX-loaded Z₁/CMCS₂-TA/Cu₁²⁺ NPs (A), Z₂/CMCS₁-TA/Ca²⁺ NPs (B), Z₁/CMCS₁-TA/Ca²⁺ (C) and Z₁/CMCS₂-TA/Ca²⁺ (D) in 0.01 M buffer under different pH conditions.

Abbreviations: CMCS, carboxymethyl chitosan; DOX, doxorubicin hydrochloride; NPs, nanoparticles; TA, tannic acid; Z₁, zein₁; Z₂, zein₂.

Figure 6 In vitro cytotoxicity of the blank NPs (A) and DOX-loaded NPs (B) against HepG2 cells incubated for 24 h.

Notes: In all panels, the indicated concentrations are DOX doses. It should be noted that for evaluating DOX-loaded NPs, equal concentrations of blank NPs were employed to eliminate the effect of vehicles in MTT assay. Data displayed as mean ± SD (n=6). **P<0.01 versus DOX-NPs group. The morphology of the cells after incubating for 24 h with NPs (D), NPs-TA/Cu₁²⁺ (E) and NPs-TA/Ca²⁺ (F) or DOX-NPs (G), DOX-NPs-TA/Cu₁²⁺ (H) and DOX-NPs-TA/Ca²⁺ (I) was evaluated by using optical microscopy with a 10× objective. (C) was the control. The scale bars represent 100 μm.

Abbreviations: DOX, doxorubicin hydrochloride; MTT, methylthiazolyldiphenyltetrazolium bromide; NPs, nanoparticles; TA, tannic acid; SD, standard deviation.

Figure 7 The qualitative (A–C) and quantitative (D) study of cellular uptake.

Notes: CLSM images of intracellular uptake of DOX-NPs (A), DOX-NPs-TA/Cu₁²⁺ NPs (B) and DOX-NPs-TA/Ca²⁺ NPs (C) by HepG2 cells. The scale bars represent 20 μm. Flow cytometry results of HepG2 cells as a function of time treated with DOX-loaded NPs (D). Data displayed as mean ± SD (n=3). **P<0.01 versus the DOX-NPs group.

Abbreviations: CLSM, confocal laser scanning microscopy; DOX, doxorubicin hydrochloride; NPs, nanoparticles; TA, tannic acid; SD, standard deviation.

Scheme 1 Illustration of the synthesis and structures of DOX-loaded zein/CMCS NPs coated by metal–TA films and the proposed model for pH-dependent drug release in tumor cells.

Abbreviations: CMCS, carboxymethyl chitosan; DOX, doxorubicin hydrochloride; NPs, nanoparticles; TA, tannic acid.

Figure S1 Fourier transform infrared spectroscopy (FTIR) spectra of different samples.

Notes: NPs-Cu₁²⁺, NPs-Cu₂²⁺, NPs-Cu₃²⁺, NPs-Ca²⁺: metal–TA-coated zein/CMCS nanoparticles prepared at a zein:CMCS ratio of 1:1 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺), 0.48 mM (Cu₂²⁺) and 0.72 mM (Cu₃²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; NPs, nanoparticles; TA, tannic acid.

Figure S2 XPS survey spectra of Z₁/CMCS₁-TA/Cu₁²⁺ (A) and Z₁/CMCS₁-TA/Ca²⁺ (B).

Notes: The Z₁/CMCS₁ NPs were prepared at zein:CMCS ratio of 1:1 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; TA, tannic acid; XPS, X-ray photoelectron spectroscopy; Z₁, zein₁.

Table S1 Characterization of NPs (pH 7.4)

Samples	Size, nm	PDI	Zeta potential, mV
Z2/CMCS1	100.3±2.2	0.14±0.02	−24.3±0.7
Z2/CMCS1-TA/Cu1^2+	108.3±3.5	0.12±0.02	−32.7±2.2
Z2/CMCS1-TA/Cu2^2+	116.1±2.2	0.16±0.01	−31.0±1.5
Z2/CMCS1-TA/Cu3^2+	122.2±3.3	0.25±0.02	−32.1±1.6
Z2/CMCS1-TA/Ca^2+	129.4±2.3	0.20±0.01	−25.4±0.3

Notes: The results displayed as mean ± standard deviation (n=3). The NPs were prepared at a zein:CMCS ratio of 2:1 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺), 0.48 mM (Cu₂²⁺) and 0.72 mM (Cu₃²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; NPs, nanoparticles; PDI, polydispersity index; TA, tannic acid; Z₂, zein₂.

Table S2 Characterization of NPs (pH 7.4)

Samples	Size, nm	PDI	Zeta potential, mV
Z1/CMCS1	82.1±1.6	0.15±0.01	−24.6±1.9
Z1/CMCS1-TA/Cu1^2+	96.6±2.7	0.19±0.01	−31.9±5.1
Z1/CMCS1-TA/Cu2^2+	114.5 ±1.3	0.17±0.02	−32.2±1.2
Z1/CMCS1-TA/Cu3^2+	118.3±2.8	0.26±0.01	−30.0±2.0
Z1/CMCS1-TA/Ca^2+	90.1±0.4	0.21±0.01	−27.9±2.4

Notes: The results displayed as mean ± standard deviation (n=3). The NPs were prepared at a zein:CMCS ratio of 1:1 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺), 0.48 mM (Cu₂²⁺) and 0.72 mM (Cu₃²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; NPs, nanoparticles; PDI, polydispersity index; TA, tannic acid; Z₁, zein₁.

Table S3 Characterization of NPs (pH 7.4)

Samples	Size, nm	PDI	Zeta potential, mV
Z1/CMCS2	103.3±0.6	0.17±0.01	−27.8±1.8
Z1/CMCS2-TA/Cu1^2+	113.3±1.9	0.25±0.01	−32.0±1.4
Z1/CMCS2-TA/Cu^2+	89.3±1.1	0.17±0.01	−29.4±0.6

Notes: The results displayed as mean ± standard deviation (n=3). The NPs were prepared at a zein:CMCS ratio of 1:2 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; NPs, nanoparticles; PDI, polydispersity index; TA, tannic acid; Z₁, zein₁.

Table S4 Element composition and content on the surface of Z₁/CMCS₁-TA/Cu₁²⁺ NPs and Z₁/CMCS₁-TA/Ca²⁺ NPs

Sample	C	O	N	S	Cu	Ca
Z1/CMCS1-TA/Cu1^2+	64.33	25.45	8.85	1.14	0.23	–
Z1/CMCS1-TA/Cu^2+	62.85	27.04	8.54	1.09	–	0.48

Note: ‘–’ indicates element not detected.

Abbreviations: CMCS, carboxymethyl chitosan; NPs, nanoparticles; TA, tannic acid; Z₁, zein₁.

Table S5 Characterization of DOX-loaded NPs (pH 7.4)

Samples	Size, nm	PDI	Zeta potential, mV	Encapsulation efficiency, %
DOX-Z1/CMCS1	135.2±1.2	0.18±0.01	−24.5±0.7	91.0±0.9
DOX-Z1/CMCS1-TA/Cu1^2+	140.6±1.6	0.23±0.01	−32.7±0.3	96.1±1.1
DOX-Z1/C MCS1-TA/Cu2^2+	144.3±2.0	0.21±0.03	−33.2±0.4	95.9±0.8
DOX-Z1/CMCS1-TA/Cu3^2+	160.1±4.3	0.27±0.02	−32.2±0.4	96.4±0.7
DOX-Z1/CMCS1-TA/Ca^2+	137.6±2.1	0.21±0.02	−28.1±0.8	93.4±1.3

Notes: The results displayed as mean ± standard deviation (n=3). The NPs were prepared at a zein:CMCS ratio of 1:1 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺), 0.48 mM (Cu₂²⁺) and 0.72 mM (Cu₃²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; DOX, doxorubicin hydrochloride; NPs, nanoparticles; TA, tannic acid; Z₁, zein₁.

Table S6 Characterization of DOX-loaded NPs (pH 7.4)

Samples	Size, nm	PDI	Zeta potential, mV
DOX-Z1/CMCS2	130.8±1.6	0.17±0.02	−27.3±0.9
DOX-Z1/CMCS2-TA/Cu1^2+	136.7±1.8	0.26±0.01	−32.3±0.7
DOX-Z1/C MCS2-TA/Cu^2+	128.1±1.1	0.22±0.01	−32.0±0.8

Notes: The results displayed as mean ± standard deviation (n=3). The NPs were prepared at a zein:CMCS ratio of 1:2 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; DOX, doxorubicin hydrochloride; NPs, nanoparticles; TA, tannic acid; Z₁, zein₁.

Table S7 Characterization of DOX-loaded NPs (pH 7.4)

Samples	Size, nm	PDI	Zeta potential, mV
DOX-Z2/CMCS1	156.4±4.9	0.15±0.01	−23.0±1.7
DOX-Z2/CMCS1-TA/Cu1^2+	156.8±3.5	0.23±0.02	−32.9±0.6
DOX-Z2/C MCS1-TA/Cu2^2+	165.5±3.6	0.26±0.01	−37.7±1.4
DOX-Z2/CMCS1-TA/Cu3^2+	181.7±3.8	0.26±0.01	−37.4±1.3
DOX-Z2/CMCS1-TA/Ca^2+	172.4±3.7	0.20±0.01	−27.6±0.7

Notes: The results displayed as mean ± standard deviation (n=3). The NPs were prepared at a zein:CMCS ratio of 2:1 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺), 0.48 mM (Cu₂²⁺) and 0.72 mM (Cu₃²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; DOX, doxorubicin hydrochloride; NPs, nanoparticles; TA, tannic acid; Z₂, zein₂.

Table S8 IC₅₀ values for DOX-loaded NPs against HepG2 cells

Samples	Cell	IC50 (µg/mL)
DOX-Z1/CMCS1	HepG2	2.36±0.18
DOX-Z1/CMCS1-TA/Cu1^2+	HepG2	2.91±0.12*
DOX-Z1/C MCS1-TA/Cu^2+	HepG2	2.67±0.28

Notes: The results displayed as mean ± standard deviation (n=3).

* P<0.05 versus DOX-Z₁/CMCS₁ group. The NPs were prepared at zein:CMCS ratio of 1:1 w/w with the final metal ion concentration of 0.24 mM (Cu₁²⁺ or Ca²⁺).

Abbreviations: CMCS, carboxymethyl chitosan; DOX, doxorubicin hydrochloride; IC₅₀, half maximal inhibitory concentration; NPs, nanoparticles; TA, tannic acid; Z₁, zein₁.