Joon Sig Choi

Characterization of a Targeted Gene Carrier, Lactose- Polyethylene Glycol-Grafted Poly- L-Lysine, and Its Complex with Plasmid DNA

The physicochemical properties and gene transfer ability of lactose-polyethylene glycol-grafted poly-L-lysine (Lac-PEG-PLL) were investigated. A dye displacement assay showed that plasmid DNA self-assembled with Lac-PEG-PLL, and condensation began at a <1:1 charge ratio of plasmid DNA to polymer. In atomic force microscopy, spontaneously assembled Lac-PEG-PLL/DNA complexes revealed a compact structure, with a size of about 100-200 nm. Circular dichroism spectra of Lac-PEG-PLL/DNA complexes revealed that the secondary structure of DNA was altered by complex formation and was similar to that of the poly-L-lysine/DNA complex. Lac-PEG-PLL was shown to protect DNA against nuclease action in a DNase I protection assay. The cytotoxicity test demonstrated that the complex composed of plasmid DNA and Lac-PEG-PLL had little influence on the viability of HepG2 cells, especially in comparison with that of poly-L-lysine/DNA complexes. In conclusion, our copolymer, Lac-PEG-PLI, formed complexes with plasmid DNA (on average, 150 nm), gave little cytotoxicity, and showed increased efficiency of gene transfer into hepatoma cells in vitro. Lactose-polyethylene glycol was grafted to poly-L-lysine to be used as a gene carrier for hepatoma cell targeting and to improve the solubility of the polyplexes. The average size of the carrier/DNA complexes was about 150 nm. The complexes also proved to have high resistance against nuclease attack and little cytotoxicity. The polymer also delivered plasmid DNA efficiently into a HepG2 cell line. Lac-PEG-PLL was more efficient than Lipofectin or galactose-PEG-PLL in transfection efficiency.

Synthesis of PAMAM Dendrimer Derivatives with Enhanced Buffering Capacity and Remarkable Gene Transfection Efficiency

In this study, we introduced histidine residues into l-arginine grafted PAMAM G4 dendrimers to enhance proton buffering capacity and evaluated the physicochemical characteristics and transfection efficacies in vitro. The results showed that the synthesized PAMAM G4 derivatives effectively delivered pDNA inside cells and the transfection level improved considerably as the number of histidine residues increased. Grafting histidine residues into the established polymer vector PAMAM G4-arginine improved their proton buffering capacity. The cytotoxicity of PAMAM G4 derivatives was tested and it was confirmed that they displayed relatively lower cytotoxicity compared to PEI25KD in various cell lines. Also, confocal microscopy results revealed that PAMAM G4 derivatives effectively delivered pDNA into cells, particularly into the nucleus. These PAMAM dendrimer derivatives conjugated with histidines and arginines may provide a promising polymeric gene carrier system.

DNA delivery to the mitochondria sites using mitochondrial leader peptide conjugated polyethylenimine.

Some genetic diseases are associated with the defects of the mitochondrial genome. Direct DNA delivery to the mitochondrial matrix has been suggested as an approach for mitochondrial gene therapy for these diseases. We hypothesized that a mitochondrial leader peptide (LP) conjugated polyethylenimine (PEI) could deliver DNA to the mitochondrial sites. PEI-LP was synthesized by the conjugation of LP to PEI using disulfide bond. The complex formation of PEI-LP with DNA was confirmed by a gel retardation assay. In this study, DNA was completely retarded at a 0.4/1 PEI-LP/DNA weight ratio. In vitro delivery tests into isolated mitochondria or living cells were performed with rhodamin-labeled DNA and PEI-LP. In vitro cell-free delivery assay with isolated mitochondria showed that PEI-LP/DNA complexes were localized at mitochondria sites. Furthermore, the PEL-LP/DNA complexes were localized at the mitochondrial sites in living cells. However, a control carrier, PEI, did not show this effect. In addition, MTT assay showed that PEI-LP showed lower cytotoxicity than PEI. These results suggest that PEI-LP can deliver DNA to the mitochondrial sites and may be useful for the development of mitochondrial gene therapy.

Combinational therapy of ischemic brain stroke by delivery of heme oxygenase-1 gene and dexamethasone.

Combinational therapies using genes and drugs are promising therapeutic strategies for various diseases. In this research, a co-delivery carrier of dexamethasone and plasmid DNA (pDNA) was developed by conjugation of dexamethasone to polyethylenimine (2 kDa, PEI2k) for combinational therapy of ischemic brain. Dynamic light scattering, atomic force microscopy and flow cytometry studies showed that the pDNA/dexamethasone-conjugated PEI2k (PEI2k-Dexa) complex was 150 nm in size and was taken up by cells more easily than PEI2k-Dexa only. The tumor necrosis factor-α (TNF-α) level was decreased more efficiently by pDNA/PEI2k-Dexa complex than dexamethasone only in hypoxia activated Raw 264.7 macrophage cells, suggesting that pDNA/PEI2k-Dexa complex increased the delivery efficiency and therapeutic effect of dexamethasone. In in vitro transfection assay, PEI2k-Dexa had higher transfection efficiency than PEI2k and lipofectamine. However, the simple mixture of PEI2k and dexamethasone did not show this effect, suggesting that the conjugation of dexamethasone to polyethylenimine increased DNA delivery efficiency of PEI2k. To evaluate the effects of combinational therapy in vivo, pDNA/PEI2k-Dexa complex was applied to a transient focal ischemia animal model. At 24 h after the injection, mean infarction volume and the TNF-α level were reduced more efficiently in the pDNA/PEI2k-Dexa injection group, compared with the control, pDNA/PEI2k, or dexamethasone injection group. The infarction volume and inflammatory cytokines were further decreased by delivery of pSV-HO-1 using PEI2k-Dexa. Magnetic resonance imaging and microPET studies confirmed the therapeutic effect of pSV-HO-1/PEI2k-Dexa complex at 10 days after the injection. Therefore, pSV-HO-1/PEI2k-Dexa complexes may be useful in combinational therapy for ischemic diseases such as stroke.

Synthesis and characterization of dexamethasone‐conjugated linear polyethylenimine as a gene carrier

Linear polyethylenimine (25 kDa, LPEI25k) has been shown to be an effective non‐viral gene carrier with higher transfection and lower toxicity than branched polyethylenimine (BPEI) of comparable molecular weight. In this study, dexamethasone was conjugated to LPEI25k to improve the efficiency of gene delivery. Dexamethasone is a synthetic glucocorticoid receptor ligand. Dexamethasone‐conjugated LPEI25k (LPEI–Dexa) was evaluated as a gene carrier in various cells. Gel retardation assays showed that LPEI–Dexa completely retarded plasmid DNA (pDNA) at a 0.75:1 weight ratio (LPEI/pDNA). LPEI–Dexa had the highest transfection efficiency at a 2:1 weight ratio (LPEI–Dexa/DNA). At this ratio, the size of the LPEI–Dexa/pDNA complex was approximately 125 nm and the zeta potential was 35 mV. LPEI–Dexa had higher transfection efficiency than LPEI and Lipofectamine 2000. In addition, the cytotoxicity of LPEI–Dexa was much lower than that of BPEI (25 kDa, BPEI25k). In conclusion, LPEI–Dexa has a high transfection efficiency and low toxicity and can therefore be used for non‐viral gene delivery. J. Cell. Biochem. 110: 743–751, 2010.

Dexamethasone conjugation to polyamidoamine dendrimers G1 and G2 for enhanced transfection efficiency with an anti-inflammatory effect

Polyamidoamine (PAM) dendrimers with low generation such as PAM generation 1 (PAMG1) and PAM generation 2 (PAMG2) have been widely used as a gene carrier due to low toxicity, albeit their low transfection efficiency. In this study, dexamethasone was conjugated to PAMG1 and PAMG2 in order to increase the transfection efficiency. In a gel retardation assay, the dexamethasone conjugated PAMG1 and PAMG2 (PAMG1-Dexa and PAMG2-Dexa) retarded plasmid DNA (pDNA) completely at 5:1 and 3:1 weight ratios (polymer:pDNA), respectively. In transfection assays, PAMG1-Dexa and PAMG2-Dexa had the highest transfection efficiency at 20:1 and 10:1 weight ratios, respectively. In addition, PAMG1-Dexa and PAMG2-Dexa had higher transfection efficiencies than PAMG1, PAMG2, PEI25k, and lipofectamine. In a MTT assay, PAMG1-Dexa and PAMG2-Dexa were less cytotoxic than lipofectamine. In addition, PAMG1-Dexa and PAMG2-Dexa decreased the TNF-α level more efficiently than dexamethasone only in the lipopolysaccharide (LPS)-induced Raw264.7 cells. Therefore, PAMG1-Dexa and PAMG2-Dexa may prove to be useful as gene delivery carriers with an anti-inflammatory effect.

Characterization of basic amino acids-conjugated PAMAM dendrimers as gene carriers for human adipose-derived mesenchymal stem cells

Since mesenchymal stem cells (MSCs) can self-renew and differentiate into multiple cell types, the delivery of genes to this type of cell can be an important tool in the emerging field of tissue regeneration and engineering. However, development of more efficient and safe nonviral vectors for gene delivery to stem cells in particular still remains a great challenge. In this study, we describe a group of nonviral gene delivery vectors, conjugated PAMAM derivatives (PAMAM-H-R, PAMAM-H-K, and PAMAM-H-O), displaying affinity toward human adipose-derived mesenchymal stem cells (AD-MSCs). Transfection efficiency using pDNA encoding for luciferase (Luc) and enhanced green fluorescent protein (EGFP), and cytotoxicity assays were performed in human AD-MSCs. The results show that transfection efficiencies of conjugated PAMAM derivatives are improved significantly compared to native PAMAM dendrimer, and that among PAMAM derivatives, cytotoxicity of PAMAM-H-K and PAMAM-H-O were very low. Also, treatment of human AD-MSCs to polyplex formation in conjugated PAMAM derivatives, their cellular uptake and localization were analyzed by flow cytometry and confocal microscopy.

Functional nanosome for enhanced mitochondria-targeted gene delivery and expression

Mitochondria dysfunction plays a role in many human diseases. Therapeutic techniques for these disorders require novel delivery systems that can specifically target and penetrate mitochondria. In this study, we report a novel nanosome composed of dequalinium-DOTAP-DOPE (1,2 dioleoyl-3-trimethylammonium-propane-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) (DQA80s) as a potential mitochondria-targeting delivery vector. The functional DQAsome, DQA80s, showed enhanced transfection efficiency compared to a vector DQAsomes in HeLa cells and dermal fibroblasts. In addition, DQA80s/pDNA complexes exhibited rapid escape from the endosome into the cytosol. We observed the delivery of pDNA to mitochondria in living cells using flow cytometry, confocal microscopy, and TME imaging. More specifically, we confirmed our results by co-localization of hmtZsGreen constructs to mitochondria when delivered via DQAsomes and DQA80s in living cells. The mitochondria-targeting DQAsomes and DQA80s induced mitochondrial dysfunction through depolarization of mitochondrial membrane potential. Our data demonstrate that DQA80s show promise for use as a mitochondria-targeted carrier system for treatment of mitochondria diseases in vivo.

Dipeptide-functionalized polyamidoamine dendrimer-mediated apoptin gene delivery facilitates apoptosis of human primary glioma cells

Glioblastoma multiform (GBM) is the most frequent and aggressive form of brain tumors in adults. However, the development of more efficient and safe nonviral vector gene therapy represents a promising therapeutic approach, using a tumor-specific killer gene, named apoptin. In this study, we describe the efficacy of non-viral gene delivery vectors, the amino acid-conjugated PAMAM derivatives (PAMAM-H-R and PAMAM-H-K) in delivering a therapeutic gene, displaying affinity toward human primary glioma cells (GBL-14 cells) and dermal fibroblasts. We analyzed transfection efficiency, using luciferase (Luci) and a pDNA encoding for enhanced fluorescent protein (EGFP), and cytotoxicity in both cells. The results show that transfection efficiency of PAMAM-H-R improved compared to native PAMAM dendrimer, but cytotoxicity of PAMAM-H-R and PAMAM-H-K were very low. We treated both cells with a polyplex formation of PAMAM-H-R or PAMAM-H-K/apoptin, and analyzed their cellular uptake and localization by flow cytometry and confocal microscopy. Furthermore, we analyzed the endosomal escape effect using TEM images, and found that PAMAM-H-R showed very fast escape from endosome to the cytosol. Caspase 3 activity assay, cell cycle distribution, and JC-1 analysis showed apoptosis induced by apoptin in GBL-14 cells. This indicates that PAMAM-H-R can be a potential nonviral vector gene delivery carrier for brain tumor therapy. The present study demonstrates that PAMAM-H-R/apoptin gene polyplex can be used as an effective therapeutic candidate for GBM due to its selective induction of apoptosis in primary glioma cells as a potential nonviral gene delivery carrier for brain tumor therapy.

Apoptin Gene Delivery by the Functionalized Polyamidoamine Dendrimer Derivatives Induces Cell Death of U87-MG Glioblastoma Cells

Malignant glioma is the most common and aggressive form of primary brain tumor in adults. In this study, we describe the efficacy of nonviral gene delivery carriers, histidine- and arginine- or histidine- and lysine-grafted polyamidoamine (PAMAM) dendrimers (PAMAM-H-R and PAMAM-H-K), in delivering a therapeutic and a tumor-selective killer gene, apoptin, using human glioma cells (U87-MG) and newborn human dermal fibroblast cells. We analyzed transfection efficiency using luciferase and a plasmid DNA encoding for enhanced green fluorescent protein and assessed cell viability in both cells. The results show that transfection efficiency of PAMAM-H-R and PAMAM-H-K was greatly increased compared with that of native PAMAM. Moreover, among PAMAM derivatives, cytotoxicity of PAMAM-H-K was very low. We treated both cells with complexes of PAMAM-H-R or PAMAM-H-K and apoptin and analyzed their cellular uptake by flow cytometry and localization by confocal microscopy. Furthermore, cell cycle distribution, caspase 3 activity assay, and JC-1 analysis showed cell death induced by apoptin in U87-MG cells. The present study demonstrates that a PAMAM-H-R/apoptin complex is an effective gene carrier system in glioma cell culture.