Combination Therapy of Lung Cancer Using Layer-by-Layer Cisplatin Prodrug and Curcumin Co-Encapsulated Nanomedicine

Figures & data

Figure 1 Synthesis route and ¹H NMR spectrum of PLGA conjugated CDDP prodrug (CDDP-PLGA). CDDP-PLGA was synthesized by amidation of the carboxyl groups of CDDP with the amine groups of PLGA.

Abbreviations: PLGA, poly(D,L-lactic-co-glycolic); CDDP, cisplatin.

Figure 2 Scheme and TEM image of CDDP-PLGA/CUR LBL NPs. CDDP-PLGA/CUR LBL NPs were prepared by a solvent diffusion technique.

Abbreviations: TEM, transmission electronic microscopy; CDDP, cisplatin; PLGA, poly(D,L-lactic-co-glycolic); CUR, curcumin; LBL, layer-by-layer; NPs, lipid-polymer hybrid nanoparticles.

Table 1 Characterization of LBL NPs (Mean ± SD, n=6)

Formulation	CDDP-PLGA/CUR LBL NPs	CDDP/CUR LBL NPs	CDDP LBL NPs	CUR LBL NPs	LBL NPs
Particle size (nm)	179.6 ± 6.7	172.3 ± 5.3	177.9 ± 4.4	176.3 ± 4.2	175.1 ± 3.9
PDI	0.157 ± 0.031	0.143 ± 0.028	0.125 ± 0.021	0.131 ± 0.023	0.116 ± 0.018
Zeta potential (mV)	−29.9 ± 3.2	−28.7 ± 3.6	−31.4 ± 3.5	−30.2 ± 3.1	−27.6 ± 2.5
EE of CDDP (%)	85.6 ± 3.9	89.1 ± 3.6	87.9 ± 3.2	N/A	N/A
EE of CUR (%)	82.1 ± 2.8	81.6 ± 2.7	N/A	80.8 ± 2.3	N/A
LE of CDDP (%)	10.2 ± 0.9	11.3 ± 1.1	10.8 ± 0.8	N/A	N/A
LE of CUR (%)	10.8 ± 1.1	11.8 ± 1.3	N/A	12.1 ± 1.4	N/A

Notes: This table summarized the particle size, zeta potential, EE and LE of LBL NPs.

Figure 3 In vitro drug release profiles of CDDP or CUR from LBL NPs. In vitro release of CDDP or CUR from LBL NPs was conducted by dialysis bag diffusion method. Data presented as mean ± SD, n = 3.

Abbreviations: CDDP, cisplatin; CUR, curcumin; LBL, layer-by-layer; NPs, lipid-polymer hybrid nanoparticles.

Figure 4 The viability of A549 cells (A), NCI-H1299 cells (B), and BEAS-2B cells (C) when incubated with CDDP-PLGA/CUR LBL NPs, CDDP/CUR LBL NPs, CDDP LBL NPs, CUR LBL NPs, LBL NPs, free CDDP/CUR, free CDDP and free CUR in different concentrations for 72 h. In vitro cytotoxicity of LBL NPs on A549 cells was assessed by the MTT assay. Data presented as mean ± SD, n = 6. *means P < 0.05.

Abbreviations: NSCLC, non-small cell lung cancer; CDDP, cisplatin; CUR, curcumin; LBL, layer-by-layer; NPs, lipid-polymer hybrid nanoparticles; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.

Table 2 The Uptake Efficiency of LBL NPs (Mean ± SD, n=6)

Formulation	Uptake Efficiency (%)
	A549 Cells	NCI-H1299 Cells	BEAS-2B Cells
CDDP-PLGA/CUR LBL NPs	71.3 ± 4.3	68.9 ± 4.1	50.3 ± 3.1
CDDP/CUR LBL NPs	72.5 ± 3.8	70.6 ± 3.8	52.7 ± 2.9
CDDP LBL NPs	69.7 ± 3.9	72.1 ± 3.9	51.6 ± 3.3
CUR LBL NPs	70.2 ± 3.5	71.7 ± 4.2	52.5 ± 3.5

Notes: This table summarized the tumor inhibition rates of CDDP-PLGA/CUR LBL NPs, CDDP/CUR LBL NPs, and free CDDP/CUR groups.

Figure 5 Cellular uptake efficiency of LBL NPs presented by the fluorescent images (A) and flow cytometry (B). Cou-6 was used as a fluorescent probe to determine the cellular uptake efficiency of LBL NPs. Scale bar: 20 μm. Data presented as mean ± SD, n = 6.

Abbreviations: LBL, layer-by-layer; NPs, lipid-polymer hybrid nanoparticles.

Table 3 Tumor Inhibition Rates (Mean ± SD, n=6)

Formulation	Tumor Inhibition Rates (%)
CDDP-PLGA/CUR LBL NPs	76.7 ± 3.1
CDDP/CUR LBL NPs	56.4 ± 2.2
CDDP LBL NPs	24.6 ± 1.9
CUR LBL NPs	16.1 ± 1.2
Free CDDP/CUR	23.1 ± 1.5
Free CDDP	17.8 ± 1.1
Free CUR	6.3 ± 0.8

Notes: This table summarized the tumor inhibition rates of CDDP-PLGA/CUR LBL NPs, CDDP/CUR LBL NPs, and free CDDP/CUR groups.

Figure 6 In vivo anti-tumor efficacy of CDDP-PLGA/CUR LBL NPs evaluated on NSCLC bearing mice, tumor growth (A) and body weight (B) were presented. The tumor volumes were calculated using formulation: V (mm³) = 1/2×(length)×(width)². After 21 days of injection, mice were sacrificed by cervical decapitation and the final tumor weight and body weight of mice was recorded. Data presented as mean ± SD, n = 6. * means P < 0.05.

Abbreviations: CDDP, cisplatin; CUR, curcumin; LBL, layer-by-layer; NPs, lipid-polymer hybrid nanoparticles; NSCLC, non-small cell lung cancer.

Figure 7 In vivo tissue CDDP (A) and CUR (B) distribution in tissues at 24 h. Mice were sacrificed at 24 h after intravenous injection. The tumor tissue, lung, heart, kidney, liver, and spleen were harvest, decomposed on heating in nitric acid, evaporated to dryness, and redissolved in acetonitrile and water (50/50, v/v) solution. Data presented as mean ± SD, n = 6. * means P < 0.05.

Abbreviations: NSCLC, non-small cell lung cancer; CDDP, cisplatin; CUR, curcumin.