Application of a lipid-coated hollow calcium phosphate nanoparticle in synergistic co-delivery of doxorubicin and paclitaxel for the treatment of human lung cancer A549 cells

Figures & data

Table 1 Characterization of PTX/DOX–LCP with different prescriptions (n=3)

Formulation	Prescription	Particle size (nm)	PDI	Zeta potential (mV)	EE (%)
HCP	–	257.3	0.187	–	–
LCP	A2B2	297.8	0.259	−40.1	–
PTX/DOX–LCP1	A1B1C1	300.1	0.016	−29.6	63.14
PTX/DOX–LCP2	A1B2C3	311.5	0.084	−36.2	56.43
PTX/DOX–LCP3	A1B3C2	308.0	0.100	−16.8	60.61
PTX/DOX–LCP4	A2B1C3	348.7	0.211	−39.8	78.92
PTX/DOX–LCP5	A2B2C2	335.0	0.228	−41.1	80.40
PTX/DOX–LCP6	A2B3C1	313.4	0.062	−40.1	79.95
PTX/DOX–LCP7	A3B1C2	389.1	0.134	−37.3	66.83
PTX/DOX–LCP8	A3B2C1	371.2	0.390	−38.3	78.71
PTX/DOX–LCP9	A3B3C3	473.8	0.048	−42.1	65.41

Notes: A, molar ratio of soya lecithin/cholesterol (A₁, 3:1; A₂, 4:1; A₃, 5:1); B, the concentration of DOX-HCP (B₁, 100 mg; B₂, 200 mg; B₃, 300 mg); C, the concentration of PTX (C₁, 10 mg; C₂, 20 mg; C₃, 30 mg). ‘–’ indicates absence of data.

Abbreviations: HCP, hollow calcium phosphate; LCP, lipid-coated hollow calcium phosphate; DOX, doxorubicin; PTX, paclitaxel; PDI, polydispersity index; EE, encapsulation efficiency.

Figure 1 TEM images of SPS nanospheres (A), HCP (B), and LCP (C). Magnification ×30,000.

Abbreviations: TEM, transmission electron microscopy; SPS, sulfonated polystyrene nanospheres; HCP, hollow calcium phosphate; LCP, lipid-coated hollow calcium phosphate.

Table 2 IC₅₀, CI₅₀, and DRI of different compositions to A549 cells

Compositions	IC50 (ng/mL)	CI50	DRI for DOX	DRI for PTX
DOX	407.38	–	–	–
DOX/PTX =12/1	112.20/9.33	0.61	3.64	3.02
DOX/PTX =6/1	109.65/18.20	0.92	3.72	1.55
DOX/PTX =3/1	69.18/22.91	0.98	5.88	1.23
DOX/PTX =1/3	64.57/194.98	7.08	6.29	0.14
DOX/PTX =1/6	39.81/239.88	8.61	10.20	0.12
PTX	28.18	–	–	–

Note: ‘–’ indicates absence of data.

Abbreviations: DOX, doxorubicin; PTX, paclitaxel; DRI, dose-reduction index; IC₅₀, the inhibitory concentration to produce 50% cell death; CI₅₀, combination index.

Figure 2 (A) Cell viability of LCP at various concentrations and (B) cell inhibition rate of PTX/DOX–LCP and DOX/PTX.

Abbreviations: LCP, lipid-coated hollow calcium phosphate; DOX, doxorubicin; PTX, paclitaxel.

Figure 3 (A) In vitro drug-release curves of PTX/DOX–LCP powders. (B–D) The PXRD patterns, DSC patterns, and FT-IR spectra of PTX, DOX, HCP, LCP, PTX–LCP, DOX–LCP, and the physical mixture (PM) with the same proportion of PTX/DOX–LCP and PTX/DOX–LCP.

Abbreviations: LCP, lipid-coated hollow calcium phosphate; DOX, doxorubicin; PTX, paclitaxel; PXRD, powder X-ray diffractometer; DSC, differential scanning calorimeter; FT-IR, Fourier-transform infrared; HCP, hollow calcium phosphate.

Figure 4 Apoptosis rate images of the control, DOX/PTX, and PTX/DOX–LCP groups.

Abbreviations: LCP, lipid-coated hollow calcium phosphate; DOX, doxorubicin; PTX, paclitaxel.

Figure 5 The flow cytometry images of the control group (A), DOX/PTX group (B), and PTX/DOX–LCP group (C).

Abbreviations: LCP, lipid-coated hollow calcium phosphate; DOX, doxorubicin; PTX, paclitaxel.

Figure 6 The CLSM images of A549 cells treated with FITC-LCP at various time periods (A) and at various concentrations (B). Magnification ×600.

Abbreviations: CLSM, confocal laser scanning microscopy; FITC, fluorescein isothiocyanate; LCP, lipid-coated hollow calcium phosphate.

Figure 7 TEM images of FITC-LCP not taken up (A) and intracellular uptake in A549 cells (B). Magnification ×20,000.

Note: Arrows point at microfilaments.

Abbreviations: FITC, fluorescein isothiocyanate; LCP, lipid-coated hollow calcium phosphate; TEM, transmission electron microscopy.

Figure 8 (A) The tumor volume curves of the saline, DOX/PTX, and PTX/DOX–LCP groups. (B) The images of tumor tissues excised from the tumor-bearing nude mice of the saline, DOX/PTX, and PTX/DOX–LCP groups.

Abbreviations: LCP, lipid-coated hollow calcium phosphate; DOX, doxorubicin; PTX, paclitaxel.

Figure 9 Histological examination of excised tumor tissues of the saline, DOX/PTX, and PTX/DOX–LCP groups under different magnification.

Abbreviations: LCP, lipid-coated hollow calcium phosphate; DOX, doxorubicin; PTX, paclitaxel.

Figure 10 Images of Bax, Bcl-2, and Caspase-3 protein expression levels of the blank, DOX/PTX, and PTX/DOX–LCP groups.

Abbreviations: LCP, lipid-coated hollow calcium phosphate; DOX, doxorubicin; PTX, paclitaxel.