Synthesis of polyethylene glycol-oligo (glutamic acid) conjugated with polyethylenimine-dexamethasone for gene delivery applications.

In this study, polyethylenimine-dexamethasone (PEI-Dexa) was conjugated to polyethylene glycol-oligo (glutamic acid) derivatives (PEG-Glu), and the ability of this conjugated derivative's gene transfection efficiency was investigated. Dexamethasone is the potent ligand of the glucocorticoid receptor which facilitates the transfer into nucleus, and it is known to enlarge the nuclear pore complexes. PEG facilitates the formation of polyplexes with improved solubility, reduced aggregation, lower cytotoxicity, and possibly decreases opsonization with serum proteins in the bloodstream. In gel retardation assays, PEG-Glu8-PEI-Dexa/pDNA and PEG-Glu10-PEI-Dexa/pDNA were completely retarded at or above polymer: pDNA weight ratios of 14 and 12, respectively. The physicochemical characteristics were studied by measuring the average size distribution and zeta-potential values of the complexes. In vitro transfection assays showed that PEG-Glu-PEI-Dexa/pDNA complexes displayed higher gene delivery efficiency than the PEI 2 kDa/pDNA complexes. In addition, PEG-Glu-PEI-Dexa was less toxic than PEI 25 kDa. These results indicate that PEG-Glu-PEI-Dexa has the potential to be used as an efficient gene carrier for nonviral gene therapy.

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.

Preparation of dexamethasone-based cationic liposome and its application to gene delivery in vitro.

In this study, dexamethasone was conjugated to PAMAM dendrimer (generation 0) and its gene transfection efficiency was investigated. To make a liposomal solution for gene delivery, DOPE was used as a fusogenic helper lipid. In gel retardation assay, PAMAM-dexamethasone conjugate (PAM-Dex)/DOPE liposome/DNA complex was completely retarded at 8:1 N/P (nitrogen/phosphate) ratio. The physicochemical characteristics are studied by measuring the average size distribution and zeta-potential values of the complexes. In vitro transfection assay showed that the PAM-Dex/DOPE liposome/DNA complex displayed higher gene delivery efficiency compared to PAMAM/DNA complex. In addition, PAM-Dex/DOPE liposome showed the lowest toxicity compared to PAMAM, PEI 25 kD and Lipofectamine. These results indicate that PAM-Dex/DOPE liposome has a potential to be used as an efficient gene carrier for gene therapy.

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.

Synthesis of poly(ethylene glycol)-polydiacetylene conjugates and their micellar and chromic characteristics.

In this report, novel polymer-lipid conjugates were synthesized and their unique micellar and chromic properties were studied. The conjugates were synthesized by the liquid-phase peptide synthesis method using methoxy(polyethylene glycol)-amine (mPEG-NH2, MW2000) as a supporting material. One and two 10,12-pentacosadiynoic acid (PCDA) groups were conjugated to mPEG-NH2 to prepare mPEG-PCDA and mPEG-PCDA2, respectively. The polymer conjugates could form nanometer-sized micelles in aqueous media and be further polymerized under the exposure of UV 254 nm due to the UV sensitive nature of PCDA. It was observed that mPEG-PCDA2 micelle showed very distinctive photochromism and thermochromism in response to UV or heat, whereas mPEG-PCDA did not display any chromic properties. Moreover, a distinctive chromic property change was observed by adding alpha-cyclodextrin as a model biological molecule to the micelle solution. Due to their unique properties such as high water-solubility, cross-linkable micelle formation with a nano-scaled size, and stimuli-responsive chromic nature, the polymer-lipid conjugates would be useful for various biomedical applications, in particular as a nano-carrier for drug delivery and biosensor.

Preparation of cationic polydiacetylene nanovesicles for in vitro gene delivery.

Here, we report novel cationic polydiacetylene-containing nanovesicle system and its application to gene delivery in vitro. The nanovesicle was constructed using a cationic diacetylene monomer lipid (DADMDPA-bis-PCDA) which contains a quaternary ammonium head group. The cationic character of this monomer leads to the electrostatic interaction with genetic materials such as plasmid DNA forming complexes and the diacetylene groups on its hydrophobic moiety provides the further polymerization functionality upon UV irradiation forming polydiacetylene-linkages within the nanovesicle. In the present study, the characteristics of nanovesicle/DNA interaction, and the transfection efficiency and cytotoxicity for the human embryonic kidney cells were characterized and compared between the nonpolymerized and polymerized nanovesicles.

Enhanced splicing correction effect by an oligo-aspartic acid-PNA conjugate and cationic carrier complexes.

Peptide nucleic acids (PNAs) are synthetic structural analogues of DNA and RNA. They recognize specific cellular nucleic acid sequences and form stable complexes with complementary DNA or RNA. Here, we designed an oligo-aspartic acid-PNA conjugate and showed its enhanced delivery into cells with high gene correction efficiency using conventional cationic carriers, such as polyethylenimine (PEI) and Lipofectamine 2000. The negatively charged oligo-aspartic acid-PNA (Asp(n)-PNA) formed complexes with PEI and Lipofectamine, and the resulting Asp(n)-PNA/PEI and Asp(n)-PNA/Lipofectamine complexes were introduced into cells. We observed significantly enhanced cellular uptake of Asp(n)-PNA by cationic carriers and detected an active splicing correction effect even at nanomolar concentrations. We found that the splicing correction efficiency of the complex depended on the kind of the cationic carriers and on the number of repeating aspartic acid units. By enhancing the cellular uptake efficiency of PNAs, these results may provide a novel platform technology of PNAs as bioactive substances for their biological and therapeutic applications.