Fig 1.
1H-NMR of PAMAM (A) and PAMAM-PEG-MAL (B) in D2O.
Fig 2.
Characterization of PAM-Ap/pDNA NPs.
(A) The size and Zeta potential of the PAM-Ap NPs were measured by DLS. (B)The size distribution of PAM/pDNA and PAM-Ap NPs at N/P = 40. (C) The Zeta potential distribution of PAM/pDNA and PAM-Ap NPs at N/P = 40. (D) Gel retardation electrophoresis of PAM-Ap/pDNA NPs. Band 1: Naked pDNA, bands 2–7: PAM-Ap/pDNA NPs were prepared at N/P ratio of 0.5, 1, 2, 4, 8 and 16, respectively. (E) Serum stability test of pDNA, PAM/pDNA and PAM-Ap/pDNA complexes in 50% FBS conditions at 37°C. Data was shown asmean±SD (n = 3).
Fig 3.
Cellular uptake of YOYO-1 labeled PAM-Ap/pDNA NPs (A) and proportion of YOYO-1 positive cells (B).
The untreated cells were used as control. Data was shown as mean±SD (n = 3). *p < 0.05, significant difference between these groups.
Fig 4.
Representative Confocal microscopy images show the cellular uptake of PAM-Ap/pDNA NPs.
A549 cells treated with YOYO-1 labeled pDNA complexed with PAM and PAM-Ap (N/P ratio: 40) with or without S6 aptamer pretreated. The cells were incubated at 37°C. All scale bars are 20 μm.
Fig 5.
The fluorescent images (A) and quantitative measurements (B) of in vitro transfection of PAM /pMiR-34a and PAM-Ap/pMiR-34a NPs on A549 cells at different N/P ratios.
Data was shown as mean±SD (n = 3). All scale bars are 200 μm. *p < 0.05, significant difference between these groups.
Fig 6.
BCL-2 and p53 mRNA (A) and protein (B) expression in the A549 cells.
Cells were cultured for 48 h after transfected with PAM-Ap/pMiR-NC NPs, PAM/pMiR-34a NPs and PAM-Ap/pMiR-34a NPs for 4 h. Quantitative real-time PCR analysis was adopted to quantify the mRNA expression, and western blotting was used to determine the protein expression. The untreated cells were used as control. Data was shown as mean±SD (n = 3). *p < 0.05, significant difference as compared with PAM/pMiR-34a.
Fig 7.
In vitro cytotoxicity and apoptosis assay of A549 cell, with the untreated cells as control.
(A) In vitro cytotoxicity of PAM-Ap/pMiR-NC, PAM/pMiR-34a and PAM-Ap/pMiR-34a NPs in A549 cells at different time point after transfection. (B) Cellular apoptosis of A549 cells after treatment with PAM-Ap/pMiR-NC, PAM/pMiR-34a and PAM-Ap/pMiR-34a NPs for 48 h. CCK-8 assay was used to evaluate cell viability and flow cytometry was used to determine cell apoptosis with Annexin/PI staining. Data was shown as mean±SD (n = 3). *p < 0.05, significant difference between these groups.
Fig 8.
Migration and invasion assay of A549 cells.
Cells were pre-treated with PAM-Ap/pMiR-NC, PAM/pMiR-34a and PAM-Ap/pMiR-34a NPs. The untreated cells were used as control. (A) Representative microscopy images of migration and quantitative analysis of migratory cells. (B) Representative microscopy images of invasion and quantitative analysis of invasive cells. Data was shown as mean±SD (n = 3). *p < 0.05, significant difference between these groups.