Selective infection of ACPP-pc-Ad-eGFP in MMP-overexpressing cells and control.

<p>HBE(control), A549, MDA-MB-231 and HepG2 cells were seeded into 96-well plates (10⁴ cells per well) and after 24 h were infected with 10⁴ particles per cell of ACPP-pc-Ad-eGFP in DMEM/10% FCS (A549, MDA-MB-231) or RRPMI-1640/10% FCS (HBE, HepG2). The supernatant was removed 4 h after infection and incubated with 200 µl DMEM/10% FCS (A549, MDA-MB-231) or RRPMI-1640/10% FCS (HBE, HepG2) for an additional 48 h before fluorescence was measured. The columns depict the following: i, HBE; ii, A549; iii, MDA-MB-231; and iv, HepG2. Data are the means ± SEM. *P<0.05 compared with the HBE cell.</p

Transduction efficiency of Ad-eGFP, pc-Ad-eGFP and ACPP-pc-Ad-eGFP with A549 cells.

<p>(A) A549 cells were seeded into 96-well plates (10⁴ cells/well) and infected after 24 h incubation with 10⁴ particles per cell of Ad-eGFP, pc-Ad-eGFP and ACPP-pc-Ad-eGFP in DMEM/10% fetal calf serum (FCS). Cellular GFP fluorescence was visualized 48 h post-infection using a Nikon TI-S microscope and photographed with a Nikon camera. (i) Uninfected cells; infection with (ii) Ad-eGFP, (iii) pc-Ad-eGFP, and (iv) ACPP-pc-Ad-eGFP. (B) After A549 cells were infected for 48 h as described above, the medium was removed, the cells lysed with 100 ml Triton X-100 (0.2% in H₂O) and GFP fluorescence was measured (λ_ex 488 nm and λ_em 538 nm) with a Fluoroskan fluorescence plate reader (Multiskan GO, Thermo Scientific). The columns depict the following: (i) uninfected cells, (ii) Ad-eGFP, (iii) pc-Ad-eGFP, and (iv) ACPP-pc-Ad-eGFP. Data are the means ± SEM. *P<0.05 compared with i, ^#P<0.05 compared with iii (C) A549 cells were trypsinized, aliquoted at (2×10⁵ cells)/(2 ml DMEM/10% FCS) and incubated in 6-well plates at 37°C until 90% confluence was reached; subsequently, 10⁹ particles Ad-eGFP, pc-Ad-eGFP or ACPP-pc-Ad-eGFP labeled with PI were added. Cells were trypsinized, centrifuged (2 min, 1500 g) and washed in PBS 48 h later. Association of PI-labeled virus with cells was measured using a Coulter EPICS XL flow cytometer with an argon laser (λ_ex 540 nm and λ_em 625 nm). The fluorescence profile of control cells (black line) or cells infected with virus (red line), pc-virus (yellow line) or ACPP-pc-virus (purple line).</p

ACPP-pc-Ad-eGFP distributed in cytoplasm of A549 cells.

<p>(A) cytoplasmic uptake of ACPP marker FITC (green); (B) cytoplasmic uptake of Ad-eGFP marker PI (red); (C) superimposition of the two dyes (green–red staining) confirms ACPP-mediated transport of pc-Ad-eGFP to the cytoplasm. Scale bar represents 100 px.</p

Fluorescence images of A549 cells incubated with ACPP-pc-Ad-eGFP(PI) and pc-Ad-eGFP(PI) after 20 min, 2 h, and 4 h.

<p>Panels A-C depict the cytoplasmic uptake of ACPP-pc-Ad-eGFP at 20 min (A), 2 h (B), and 4 h (C). Panels d-f depict the cytoplasmic uptake of pc-Ad-eGFP at 20 min (D), 2 h (E), and 4 h (F). Scale bar represents 100 px.</p

FITC fluorescence of ACPP in A549, MDA-MB-231, HepG2 and HBE.

<p>(A-C) A549, MDA-MB-231, HepG2 showed high FITC fluorescence of ACPP SP×200. (D) HBE showed low FITC fluorescence of ACPP SP×200. (E-H) FITC fluorescence of ACPP in A549, MDA-MB-231, HepG2 and HBE treated with Doxycycline SP×200.</p

Cytoplasmic delivery of ACPP-pc-Ad-eGFP and pc-Ad-eGFP in A549 cells.

<p>Inverted fluorescence microscopy image show(A, B) cytoplasmic marker CFSE (green), (C) incubated ACPP-pc-Ad-eGFP(PI) with A549 cells for 4 h at 37°C yielded uptake of the conjugate (red); (D) incubation pc-Ad-eGFP(PI) with A549 cells for 4 h at 37°C yielded little uptake (red); (E) superimposition of the two dyes (A and C) confirms ACPP-mediated nonendocytotic transport to the cytoplasm. (F) superimposition of the two dyes (Band D) exhibited only endocytotic vesicles. Scale bar represents 100 px.</p

Virus neutralization assay of Ad-eGFP or ACPP-pc-Ad-eGFP.

<p>Ad-eGFP or ACPP-pc-Ad-eGFP (10⁸ particles per 100 µl) was incubated with A549 cells in a 96-well plate (10⁴ cells per well; 10⁴ virus particles per cell) in the presence or absence of neutralizing antibodies (NAb). Expression of GFP was measured at 48 h after Triton X-100 lysis and was expressed as a percentage of the fluorescence signal in the absence of human serum. The yellow bar indicates Ad-eGFP, while the black bar denotes ACPP-pc-Ad-eGFP. Data are the means ± SEM. *P<0.05 compared with Ad-eGFP.</p

Protein of MMP-2 and MMP-9 in cell lines HepG2, HBE, A549 and MDA-MB-231 (n = 3).

<p><i>Left</i>, a representative Western blot; <i>Right</i>, densitometric analysis of the representative Western blot, <i>bars</i> represent the relative amounts of MMP-2 and MMP-9. Data are the means ± SEM. *P<0.05 compared with the HBE cell.</p