Octa-arginine-modified pegylated liposomal doxorubicin: an effective treatment strategy for non-small cell lung cancer

Abstract

The present study aims to evaluate the efficacy of octa-arginine (R8)-modified pegylated liposomal doxorubicin (R8-PLD) for the treatment of non-small cell lung cancer, for which the primary treatment modality currently consists of surgery and radiotherapy. Cell-penetrating peptide R8 modification of Doxorubicin-(Dox)-loaded liposomes was performed by post-insertion of an R8-conjugated amphiphilic PEG-PE copolymer (R8-PEG-DOPE) into the liposomal lipid bilayer. In vitro analysis with the non-small cell lung cancer cell line, A549 confirmed the efficient cellular accumulation of Dox, delivered by R8-PLD compared to PLD. It led to the early initiation of apoptosis and a 9-fold higher level of the apoptotic regulator, caspase 3/7 (9.24±0.34) compared to PLD (1.07±0.19) at Dox concentration of 100 μg/mL. The treatment of A549 monolayers with R8-PLD increased the level of cell death marker lactate dehydrogenase (LDH) secretion (1.2±0.1 for PLD and 2.3±0.1 for R8-PLD at Dox concentration of 100 μg/mL) confirming higher cytotoxicity of R8-PLD than PLD, which was ineffective under the same treatment regimen (cell viability 90±6% in PLD vs. 45±2% in R8-PLD after 24h). R8-PLD had significantly higher penetration into the hypoxic A549 tumor spheroids compared to PLD. R8-PLD induced greater level of apoptosis to A549 tumor xenograft and dramatic inhibition of tumor volume and tumor weight reduction. The R8-PLD treated tumor lysate had a elevated caspase 3/7 expression than with R8-PLD treatment. This suggested system improved the delivery efficiency of Dox in selected model of cancer which supports the potential usefulness of R8-PLD in cancer treatment, lung cancer in particular.

Figure 1

Comparison of cellular uptake of R8-modified and unmodified PEGylated liposomal doxorubicin by flow cytometry. The A549 cells were incubated with PLD and R8-PLD at 6 µg/mL of Dox for 1 and 4 h, after which the FACS analysis was performed. The cell-associated Dox fluorescence was measured. (A) Representative dot plot obtained from FACS analysis showing the cell population labeled with Dox after incubation with PLD or R8-PLD at 1 h and 4 h time points. (B) Comparison of the geometric mean of fluorescence of the cells treated with PLD or R8-PLD (left panel) and the representative histogram plot obtained from histogram statistics of the FACS analysis (right panel). The data are mean ±SD, averaged from three separate experiments. The significance of difference between the mean was analyzed by Student's t-test, *** indicates p<0.001.

Figure 2

Assessment of cellular internalization by confocal microscopy. (A) Confocal laser scanning micrographs of A549 cells obtained from z-experiment. A549 cells were incubated with PLD or R8-PLD for 90 min, were taken at a fixed XY plane along a consecutive Z-axes (Z-5, 7, 9, 11 from total 16 slices). The nuclei were stained with Hoechst 33342. The lower panel represents merged pictures of Dox and Hoechst 33342 fluorescence. Scale bar: 20 µm. (B) Assessment of nuclear localization of Doxorubicin delivered by PLD and R8-PLD. The merged (nuclei stained and PLD/R8-PLD treated) pictures of the center z-slices were analyzed with Image J software and the colocalization coefficients were obtained. The data are mean ± SD, averaged from 3 images of the same treatment. *, p<0.05, paired Student's t-test.

Figure 3

Accumulation of PLD and R8-PLD in A549 spheroids by confocal laser scanning microscopy. (A) Selected Z-stacked micrographs of A549 spheroids, taken at consecutive Z-axes were shown. (B) Graphical representation of mean intensity of the Doxorubicin signal of the center slice (100 µm inside from top) of PLD and R8-PLD dosed spheroids quantified with Image J software. The cells were treated with PLD and R8-PLD at 100 µM of Dox for 1 and 4 h. Images were selected out of 16 Z-slices. Scale bar: 200 µm. The data in figure B are mean ± SD, averaged from 9 different regions of interest from the images of two spheroids. ***, p<0.001, paired Student's t-test.

Figure 4

Comparison of up-regulation of an early apoptotic marker, phosphatidyl serine, upon treatment with PLD and R8-PLD by flow cytometry. The A549 cells were treated with PLD or R8-PLD at 30 µg/mL of Dox for 4 h, then incubated for 18 h in complete media. The cells were treated with Annexin V-Alexa Fluor 488 conjugate, the ligand of phosphatidyl serine. The phosphatidyl serine receptor translocates from cytoplasm to the outer cell surface on the initiation of apoptosis. The labeling of cells with Annexin V-Alexa Fluor 488 conjugate was measured by FACS analysis. (A) Representative dot plot showing the cell population with or without Annexin V labeling. (B) Geometric mean of fluorescence of PLD/R8-PLD dosed cells treated with or without Annexin V Alexa Fluor 488 conjugate, and (C) Representative histogram plot of cells treated with PLD or R8-PLD followed by Annexin V-Alexa 488. Data in (B) are mean ± SD, averaged from three separate experiments. ** indicates p<0.01, analyzed by the paired Student's t-test.

Figure 5

Assessment of Dox-induced cell death of A549 cells treated with PLD and R8-PLD. Cells were incubated with PLD or R8-PLD at a Dox concentration of 0 to 100 µg/mL for 4 h followed by the incubation period of 24 and 48 h before cell viability was measured. The data are mean ± SD, averaged from three separate experiments. ***, p<0.001, paired Student's t-test.

Figure 6

The effect of PLD and R8-PLD on Dox-induced LDH release (A) and caspase 3/7 activity (B) in A549 cells. The cells were treated with PLD or R8-PLD at varied Dox concentration for 4 h, media removed and cells incubated for 48 h in complete media before analyzing the media for LDH release and the cell lysate for caspase 3/7 activity. Each bar represents mean ± SD of n=3 from 3 separate experiments. The significance of differences between the means was analyzed by the paired Student's t-test. ***indicates p<0.001.

Figure 7

Assessment of the in vivo therapeutic efficacy of R8-modified PLD compared to unmodified PLD i.v. in A549-tumor xenograft. (A) Tumor volume and weight (arrows indicate treatment). (B) Apoptosis analysis. Apoptotic cells were detected in frozen tumor sections, determined by TUNEL assay and visualized by fluorescence microscopy. The upper panel shows the sections stained with Hoechst 33342 and the right panel shows the TUNEL staining. Magnifications-20X objectives. (C) Measurement of caspase 3/7 level in A549 tumor. The tumors isolated from the treatment groups of PBS, PLD or R8-PLD were homogenized, centrifuged and a tumor lysate equivalent to 25 µg of proteins was analyzed using the Apo-ONE Homogeneous Caspase 3/7 Assay kit. ** and ***, P<0.01 and 0.001, unpaired Student's t test. *** indicates p<0.001 compared to PBS.