Korean J Physiol Pharmacol.  2021 Sep;25(5):467-478. 10.4196/kjpp.2021.25.5.467.

Apoptin gene delivery by a PAMAM dendrimer modified with a nuclear localization signal peptide as a gene carrier for brain cancer therapy

Affiliations
  • 1Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Smart Marine Therapeutics Center, Inje University, Busan 47392, Korea.
  • 2Division of Applied Medicine, Research Institute for Korea Medicine, School of Korean Medicine, Pusan National University, Busan 50612, Korea.
  • 3Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon 34134, Korea.

Abstract

In this study, we aimed to synthesize PAMAMG3 derivatives (PAMAMG3-KRRR and PAMAMG3-HKRRR), using KRRR peptides as a nuclear localization signal and introduced histidine residues into the KRRR-grafted PAMAMG3 for delivering a therapeutic, carcinoma cell-selective apoptosis gene, apoptin into human primary glioma (GBL-14) cells and human dermal fibroblasts. We examined their cytotoxicity and gene expression using luciferase activity and enhanced green fluorescent protein PAMAMG3 derivatives in both cell lines. We treated cells with PAMAMG3 derivative/apoptin complexes and investigated their intracellular distribution using confocal microscopy. The PAMAMG3-KRRR and PAMAMG3-HKRRR dendrimers were found to escape from endolysosomes into the cytosol. The JC-1 assay, glutathione levels, and Annexin V staining results showed that apoptin triggered cell death in GBL-14 cells. Overall, these findings indicated that the PAMAMG3-HKRRR/apoptin complex is a potential candidate for an effective nonviral gene delivery system for brain tumor therapy in vitro.

Keyword

Cell death; Dendrimers; Gene delivery system; Glioma

Figure

  • Fig. 1 Synthesis of nonviral gene delivery systems. (A) 1H NMR spectra of PAMAMG3, PAMAMG3-KRRR, and PAMAMG3-HKRRR. (B) The synthesis scheme of PAMAM derivates conjugated with nuclear localization signal (NLS) peptides. (C) Scheme 1. Diagram of the endocytic pathway and the formation of PAMAMG3-HKRRR/pJDK-apoptin complex and apoptin gene delivery into the nucleus. Apoptin promotes apoptotic cell death of GBL-14 cells.

  • Fig. 2 PicoGreen assays of PAMAMG3 derivative/apoptin complexes. Complexes were prepared using PAMAMG3, PAMAMG3-KRRR, and PAMAMG3-HKRRR with pJDK or pJDK-apoptin at different weight ratios. The fluorescence intensity was normalized to 100% with pJDK and pJDK-apoptin DNA. Values are expressed as the mean ± SD of three independent experiments (n = 3).

  • Fig. 3 Cytotoxicity assay of the PAMAMG3 derivatives. (A, B) GBL-14 cells and (C, D) human dermal fibroblasts (HDFs) were exposed to various concentrations of PAMAMG3, PAMAMG3-KRRR, and PAMAMG3-HKRRR. Cytotoxicity was assessed by WST-1 assay after 24 h (A, C) and 48 h (B, D). Values are expressed as the mean ± SD of three independent experiments (n = 3). (E, F) GBL-14 cells and (G, H) HDFs were incubated under similar condition as in WST-1 assay. Cytotoxicity was assessed using LDH assay after 24 h (E, G) and 48 h (F, H). Values are expressed as the mean ± SD of three independent experiments (n = 3).

  • Fig. 4 In vitro transfection efficiency and cell viability of the PAMAMG3 derivatives. (A) GBL-14 cells and (C) human dermal fibroblasts (HDFs) were exposed to PAMAMG3, PAMAMG3-KRRR, and PAMAMG3-HKRRR complexes with pJDK/Luci at different weight ratios for 24 h. (B, D) Cytotoxicity of PAMAMG3 derivative/Luci complexes. Viability of the complexes was assessed using the WST-1 assay. Values are expressed as the mean ± SD of three independent experiments (n = 3).

  • Fig. 5 GFP expression by the PAMAMG3 derivatives. (A) GBL-14 cells and (B) human dermal fibroblasts (HDFs) were exposed to pEGFP, PAMAMG3/pEGFP, PAMAMG3-KRRR/pEGFP, and PAMAMG3-HKRRR/pEGFP for 24 h. The cells were harvested, and the cell fluorescence intensity of GFP-positive cells was quantitatively analyzed by flow cytometry. The x-axis shows fluorescence intensity and y-axis shows number of cells.

  • Fig. 6 Uptake and intracellular localization of the PAMAMG3 derivatives. (A) GBL-14 cells and (B) human dermal fibroblasts (HDFs) were treated with PAMAMG3, PAMAMG3-KRRR, and PAMAMG3-HKRRR labeled with Alexa Fluor 546 and pJDK or pJDK-apoptin at the ratio of 4:1 for 24 h. After 24 h post-exposure, the images were obtained by confocal microscopy. Red fluorescence indicated DNA-labeled with Alexa Fluor 546 dye and blue fluorescence indicated nuclei stained with DAPI. (C, D) Each cell was exposed to Alexa Fluor 488-labeled PAMAMG3, PAMAMG3-KRRR, and PAMAMG3-HKRRR with pJDK or pJDK-apoptin at the ratio of 4:1 for 24 h. After 24 h post-exposed, the cells were harvested. The live cells were stained with LysoTracker Red for lysosomes and counterstained with DAPI for nuclei.

  • Fig. 7 Mitochondrial membrane depolarization and ROS levels induced by PAMAMG3 derivative/pJDK-apoptin. (A) GBL-14 cells and (B) human dermal fibroblasts (HDFs) were exposed to PAMAMG3, PAMAMG3-KRRR, and PAMAMG3-HKRRR with pJDK or pJDK-apoptin for 24 h. The cells were analyzed for mitochondrial membrane potential (MMP) using flow cytometry after JC-1 staining. The number in the bottom right quadrant indicates the percentage of MMP loss. (C, D) Each cell was exposed to similar conditions as those in panel (A). After 24 h post-exposure, intracellular ROS levels were assessed using GSH assay as descried in methods. Values are indicted as the mean ± SD of three independent experiments (n = 3). Statistical analyses were conducted using the unpaired two-tailed Students t-test. Asterisks indicate statistically significant values (**p < 0.01, *p < 0.1, and not significant [n.s.] [p > 0.05]).

  • Fig. 8 Cytotoxicity of the PAMAMG3 derivative/apoptin complexes. (A, B) GBL-14 cells and (C, D) human dermal fibroblasts (HDFs) were incubated with PAMAMG3, PAMAMG3-KRRR, and PAMAMG3-HKRRR with pJDK or pJDK-apoptin for 24 h. After 24 h post-exposure, cytotoxicity of each polymer and polyplex was determined using the WST-1 assay. Values are expressed as mean ± SD of three independent experiments (n = 3). Statistical analyses were conducted using the unpaired two-tailed Students t-test. Asterisks indicate statistically significant values (**p < 0.01, *p < 0.1, and not significant [n.s.] [p > 0.05]).

  • Fig. 9 Apoptosis induced the PAMAMG3 derivative/pJDK-apoptin. (A, B) Annexin V staining of PAMAMG3 derivative/pJDK-apoptin using flow cytometry. GBL-14 cells and human dermal fibroblasts (HDFs) were incubated with PAMAMG3, PAMAMG3-KRRR, and PAMAMG3-HKRRR with pJDK and pJDK-apoptin. After 24 h post-exposure, apoptosis levels for each polyplex were assessed by flow cytometry. Representative histograms showing four quadrants (Q1: percentage of necrosis, Q2: percentage of late apoptosis, Q3: percentage of live cells, and Q4: percentage of early apoptosis).


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