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.

Endocytosis, intracellular transport, and exocytosis of lanthanide-doped upconverting nanoparticles in single living cells.

Lanthanide-doped upconverting nanoparticles (UCNPs) have recently attracted enormous attention in the field of biological imaging owing to their unique optical properties (near-infrared excitation followed by photoluminescence in the visible spectral range). For biological applications, it is critical to understand the interaction between these nanoparticles and biological systems at the cellular level. In this paper, using epi-fluorescence microscopy with 980-nm excitation, a full intracellular pathway composed of endocytosis, active transport, and exocytosis was clearly visualized for PEG-phospholipid-coated UCNPs in single HeLa cells, which was experimentally feasible mostly thanks to the excellent photostability and low cytotoxicity thereof. Each step in the pathway was characterized and identified by various chemical inhibition studies and spectroscopic measurements.

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.

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.

Dexamethasone-conjugated polyethylenimine as an efficient gene carrier with an anti-apoptotic effect to cardiomyocytes.

BACKGROUND: Dexamethasone is a potent glucocorticoid with anti-inflammatory effects. Dexamethasone can protect ischemic cardiomyocytes from apoptosis. To apply the anti-apoptotic effect of dexamethasone to ischemic disease gene therapy, dexamethasone-conjugated polyethylenimine (PEI-Dexa) was synthesized and evaluated as an anti-apoptotic gene carrier. METHODS: PEI-Dexa was synthesized with low molecular weight polyethylenimine (PEI2K, 2 kDa). The transfection efficiency and cytotoxicity of PEI-Dexa were evaluated by luciferase assay and the MTT assay. To evaluate the anti-apoptotic effect, PEI-Dexa/DNA complex was transfected into cells and the cells were treated with H(2)O(2). Cell viability and apoptosis level were measured by the MTT assay and caspase-3 assay, respectively. RESULTS: A transfection assay into H9C2 rat cardiomyocytes showed that PEI-Dexa had the highest transfection efficiency at an 8 : 1 weight ratio (PEI-Dexa/DNA). At this ratio, PEI-Dexa had higher transfection efficiency than high molecular polyethylenimine (PEI25K, 25 kDa) and PEI2K. In addition, the cytotoxicity of PEI-Dexa was lower than that of PEI25K. To evaluate the anti-apoptotic effect, PEI-Dexa/pSV-Luc or PEI2K/pSV-Luc was transfected into H9C2 cells and the cells were treated with H(2)O(2). PEI-Dexa was found to reduce caspase-3 activity and increase cell viability compared to PEI2K. Heme oxygenase-1 (HO-1) can protect ischemic cardiomyocytes from apoptosis. Therefore, pSV-HO-1 was cloned and transfected into H9C2 cells using PEI-Dexa. The cells transfected with PEI-Dexa/pSV-HO-1 complex had lower caspase-3 activity and higher viability than the cells transfected with PEI-Dexa/pSV-Luc complex after the H(2)O(2) treatment. CONCLUSIONS: PEI-Dexa is an efficient gene carrier with an anti-apoptotic effect and may be useful for anti-apoptotic gene therapy in combination with pSV-HO-1.

Preparation of orthogonally functionalized surface using micromolding in capillaries technique for the control of cellular adhesion.

This study presents a simple method for the fabrication of an orthogonal surface that can be applied for cell patterning without the need to immobilize specific adhesive peptides, proteins, or extracellular matrix (ECM) for cell attachment. Micromolding in capillaries (MIMIC) produced two distinctive regions. One region contained poly(ethylene glycol)-poly(D,L-lactide) diblock copolymer (PEG-PLA) designed to provide a biological barrier to the nonspecific binding of proteins and fibroblast cells. The other region was coated with polyelectrolyte (PEL) to promote the adhesion of biomolecules including proteins and cells. Resistance to the adsorption of proteins increased with the length of PEG and PLA chains because the longer PEG chain increased the PEG layer thickness and the longer PLA chain induced stronger interaction with the PEL surface. The PEG5k-PLA2.5k (20mg/ml) was the most efficient candidate for the prevention of protein adhesion among the PEG-PLA copolymers examined. The orthogonal functionality of prepared surfaces having PEL regions and background PEG-PLA regions resulted in rapid patterning of biomolecules. Fluorescein isothiocyanate-tagged bovine serum albumin (FITC-BSA) and fibroblast cells successfully adhered to the exposed PEL surfaces. Although methods for cell patterning generally require an adhesive protein layer on the desired area, these fabricated surfaces without adhesive proteins provide a gentle microenvironment for cells. In addition, our proposed approach could easily control patterns, sizes, and shapes at micron scale.

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.

Patterning of proteins and cells on functionalized surfaces prepared by polyelectrolyte multilayers and micromolding in capillaries.

A method for protein and cell patterning on polyelectrolyte-coated surfaces using simple micromolding in capillaries (MIMIC) is described. MIMIC produced two distinctive regions. One contained polyethylene glycol (PEG) microstructures fabricated using photopolymerization that provided physical, chemical, and biological barriers to the nonspecific binding of proteins, bacteria, and fibroblast cells. The second region was the polyelectrolyte (PEL) coated surface that promoted protein and cell immobilization. The difference in surface functionality between the PEL region and background PEG microstructures resulted in simple patterning of biomolecules. Fluorescein isothiocyanate-tagged bovine serum albumin, E. coli expressing green fluorescence protein (GFP), and fibroblast cells were successfully bound to the exposed PEL surfaces at micron scale. Compared with the simple adsorption of protein, fluorescence intensity was dramatically improved (by about six-fold) on the PEL-modified surfaces. Although animal cell patterning is prerequisite for adhesive protein layer to survive on desired area, the PEL surface without adhesive proteins provides affordable microenvironment for cells. The simple preparation of functionalized surface but universal platform can be applied to various biomolecules such as proteins, bacteria, and cells.