An efficient Trojan delivery of tetrandrine by poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) (PVP-b-PCL) nanoparticles shows enhanced apoptotic induction of lung cancer cells and inhibition of its migration and invasion

Figures & data

Figure 1 Schematic illustration of synthesis of tetrandrine-loaded PVP-b-PCL nanoparticles.

Abbreviations: PVP-b-PCL, poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone); Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles.

Table 1 Characterization of Tet-NPs with different drug feeding

Tet feeding (copolymer based w/w)	Size (nm)a/PDI	Zeta potential (mV)	DLC (%)	EE (%)
0	125.3±7.3/0.13	−3.8±0.5	N/A	N/A
10%	115.5±6.9/0.14	−4.5±0.4	9.3±2.1	91.2±5.1
20%	120.2±8.2/0.13	−5.6±1.1	15.2±3.5	89.3±7.3
40%	111.4±9.2/0.14	−6.2±1.2	27.4±4.2	83.2±5.9

a Note: Mean diameter was measured by dynamic light scattering.

Abbreviations: Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly (ε-caprolactone) nanoparticles; PDI, polydispersity index; DLC, drug loading content; EE, encapsulation efficiency; Tet, tetrandrine.

Figure 2 Size distribution of Tet-NPs detected by DLS.

Abbreviations: Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles; DLS, dynamic light scattering.

Figure 3 Release curves of Tet-NPs at different feeding ratios.

Abbreviations: Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly (ε-caprolactone) nanoparticles; NPs, nanoparticles; h, hours; wt, weight; Tet, tetrandrine.

Figure 4 Fluorescent microscope images of A549 cells incubated with coumarin-6-loaded Tet-NPs.

Notes: (A) Cells incubated with coumarin-6 loaded Tet-NPs at 37°C. (B) Cells incubated with coumarin-6 loaded Tet-NPs at 4°C. (C) Cells incubated with both coumarin-6 loaded Tet-NPs and NaN₃ at 37°C. Magnification =400X. (D) The cellular uptake efficiency of coumarin-6 loaded Tet-NPs detected by HPLC. **Represents P<0.01 versus control.

Abbreviations: Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles; NaN₃, sodium azide; HPLC, high-performance liquid chromatography.

Figure 5 AO staining of A549 cells by fluorescent microscopy exposed to different agents including E-NPs, Tet and Tet-NPs.

Abbreviations: AO, acridine orange; E-NPs, empty nanoparticles; Tet, tetrandrine; Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles; Con, control.

Figure 6 Apoptosis rates induced by a series of doses of Tet or Tet-NPs by FACS.

Abbreviations: Tet, tetrandrine; Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles; FACS, fluorescence activated cell sorting; Con, control; FITC, fluorescein isothiocyanate; E-NPs, empty nanoparticles.

Figure 7 The expression of anti-apoptotic protein Bcl-2 and Bcl-xL in A549 cells exposed to an equivalent dose of Tet or Tet-NPs detected by Western blot analysis.

Abbreviations: Con, control; E-NPs, empty nanoparticles; Tet, tetrandrine; Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles.

Figure 8 Semi-quantification of the gel image, normalized to β-actin control.

Notes: **Represents P<0.01 versus control; ^#represents P<0.05 versus the equivalent dose of Tet (n=3).

Abbreviations: Con, control; E-NPs, empty nanoparticles; Tet, tetrandrine; Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles.

Figure 9 Wound healing ability of A549 cells exposed to equivalent doses of Tet or Tet-NPs. Phase micrograph of A549 cells at various time points after monolayer wounding.

Abbreviations: Con, control; E-NPs, empty nanoparticles; Tet, tetrandrine; Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles; h, hours.

Figure 10 Quantification of cell migration using the monolayer wound scratching assay.

Notes: **Represents P<0.01 versus the equivalent dose of Tet. Each data point represents the mean ± SD from three independent experiments.

Abbreviations: Con, control; E-NPs, empty nanoparticles; Tet, tetrandrine; Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles; h, hours; SD, standard deviation.

Figure 11 Cell invasive ability of A549 cells exposed to equivalent doses of Tet or Tet-NPs.

Notes: A549 cells were treated with Tet or Tet-NPs at equivalent doses of 0, 1, 2, and 4 μg/mL for 48 hours. Invading cells were fixed with methanol, stained with crystal violet, and photographed under fluorescent microscopy.

Abbreviations: Con, control; E-NPs, empty nanoparticles; Tet, tetrandrine; Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles.

Figure 12 Quantification of cell invasion.

Notes: Each data point represents the mean ± SD from three independent experiments; *represents P<0.05 versus equivalent doses of Tet.

Abbreviations: SD, standard deviation; Tet, tetrandrine; Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles.

Figure 13 The expression of metastatic relative protein MMP-2, MMP-9 and TIMP-3 in A549 cells exposed to an equivalent dose of Tet or Tet-NPs.

Abbreviations: MMP, matrix metalloproteinase; TIMP-3, tissue inhibitor of MMP-3; Con, control; E-NPs, empty nanoparticles; Tet, tetrandrine; Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles.

Figure 14 Semi-quantification of the gel image, normalized to β-actin control.

Notes: **Represents P<0.01 versus control; ^#represents P<0.05 versus the equivalent dose of Tet (n=3).

Abbreviations: MMP, matrix metalloproteinase; TIMP-3, tissue inhibitor of MMP-3; Con, control; E-NPs, empty nanoparticles; Tet, tetrandrine; Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles.

Figure S1 Release curves of lyophilized Tet-NPs in PBS and serum.

Abbreviations: Tet-NPs, tetrandrine-loaded poly(N-vinylpyrrolidone)-block-poly(ε-caprolactone) nanoparticles; PBS, phosphate buffered saline; h, hours.