Hye Yeong Nam

Biodegradable PAMAM ester for enhanced transfection efficiency with low cytotoxicity

We synthesized biodegradable polycationic PAMAM (polyamidoamine) esters (e-PAM-R, e-PAM-K) that contain arginines or lysines at the peripheral ends of PAMAM-OH dendrimer through ester bond linkages. The PAMAM esters were readily degradable under physiological conditions (pH 7.4, 37 degrees C), with more than 50% of the grafted amino acids hydrolyzed within 5h. However, polyplexes were very stable and were hardly degraded in the endosomal pH range. Moreover, these amino-acid-modified polymers showed excellent buffering capacities between pH 5.1 and 7.4, facilitating endosomal escape of polyplexes. While the lysine-grafted PAMAM ester did not display significant improvement in transfection efficiency, the arginine-conjugated PAMAM ester-mediated transfection of a luciferase gene showed better transfection efficiency than the branched 25 kDa PEI (polyethylenimine) and PAM-R (peptide bond), and lower cytotoxicity, especially with primary cells such as HUVECs (human umbilical vein endothelial cells) and SMCs (primary rat aorta vascular smooth muscle cells). Furthermore, after DNA release, free e-PAM-R degraded completely into nontoxic PAMAM-OH and arginines by hydrolysis, which resulted in lower cytotoxicity in contrast to the poorly degradable arginine-modified PAMAM with amide bonds. These findings demonstrated that the arginine-grafted biodegradable PAMAM dendrimer, e-PAM-R, is a potential candidate as a safe and efficient gene delivery carrier for gene therapy.

Neuroprotection by biodegradable PAMAM ester (e-PAM-R)-mediated HMGB1 siRNA delivery in primary cortical cultures and in the postischemic brain

Although RNA interference (RNAi)-mediated gene silencing provides a powerful strategy for modulating specific gene functions, difficulties associated with siRNA delivery have impeded the development of efficient therapeutic applications. In particular, the efficacy of siRNA delivery into neurons has been limited by extremely low transfection efficiencies. e-PAM-R is a biodegradable arginine ester of PAMAM dendrimer, which is readily degradable under physiological conditions (pH 7.4, 37 degrees C). In the present study, we investigated the efficiency of siRNA delivery by e-PAM-R in primary cortical cultures and in rat brain. e-PAM-R/siRNA complexes showed high transfection efficiencies and low cytotoxicities in primary cortical cultures. Localization of fluorescence-tagged siRNA revealed that siRNA was delivered not only into the nucleus and cytoplasm, but also along the processes of the neuron. e-PAM-R/siRNA complex-mediated target gene reduction was observed in over 40% of cells and it was persistent for over 48 h. The potential use of e-PAM-R was demonstrated by gene knockdown after transfecting High mobility group box-1 (HMGB1, a novel cytokine-like molecule) siRNA into H(2)O(2)- or NMDA-treated primary cortical cultures. In these cells, HMGB1 siRNA delivery successfully reduced both basal and H(2)O(2)- or NMDA-induced HMGB1 levels, and as a result of that, neuronal cell death was significantly suppressed in both cases. Furthermore, we showed that e-PAM-R successfully delivered HMGB1 siRNA into the rat brain, wherein HMGB1 expression was depleted in over 40% of neurons and astrocytes of the normal brain. Moreover, e-PAM-R-mediated HMGB1 siRNA delivery notably reduced infarct volume in the postischemic rat brain, which is generated by occluding the middle cerebral artery for 60 min. These results indicate that e-PAM-R, a novel biodegradable nonviral gene carrier, offers an efficient means of transfecting siRNA into primary neuronal cells and in the brain and of performing siRNA-mediated gene knockdown.

Evaluation of generations 2, 3 and 4 arginine modified PAMAM dendrimers for gene delivery

It is a matter of concern to develop and design synthetic non-viral gene carriers with high transfection efficiency and low cytotoxicity in gene therapy. Recently, various arginine conjugated dendrimers showed better performance in transfection and greater viability than polyethyleneimine (PEI). In this study, we synthesized and investigated e-PAM-R G2, 3 and 4 which are biodegradable polyamidoamine (PAMAM) dendrimers modified with arginine and compared that with PAMAM-R series containing amide bonds for gene carriers. For plasmid DNA delivery, the transfection efficiency of e-PAM-R G4 was higher than G3 and G2 and similar to PAMAM-R G4 with favorable cell viability. Moreover, they indicated significantly higher suppression of TEL/AML1 protein, maybe due to rapid olidonucleotide (ODNs) release through biodegradability of e-PAM-R. These results suggest that biodegradable and non-toxic e-PAM-R may be useful carriers for the gene including plasmid DNA, antisense ODNs and si-RNA.

A new biodegradable crosslinked polyethylene oxide sulfide (PEOS) hydrogel for controlled drug release

We developed a polyethylene glycol (PEG)-based biodegradable hydrogel through disulfide crosslinking of polyethylene oxide sulfide (PEOS). The crosslinking rate was highly dependent on temperature, and incubation at about 40-50 degrees C was required for efficient crosslinking. The crosslinked PEOS hydrogel showed glutathione-dependent dissolution and corresponding controlled release of a model drug-fluorescein isothiocyanate (FITC)-labeled dextran-because the disulfide bond, the main linker, is selectively degraded in response to the high concentration of glutathione. The temperature-sensitive crosslinking and the hydrogel formation have the potential for use as an injectable biogel precursor, which was confirmed by in situ gel formation in mice.

A Novel Technique for Loading of Paclitaxel-PLGA Nanoparticles onto ePTFE Vascular Grafts

The major cause of hemodialysis vascular access dysfunction (HVAD) is the occurrence of stenosis followed by thrombosis at venous anastomosis sites due to the aggressive development of venous neointimal hyperplasia. Local delivery of antiproliferative drugs may be effective in inhibiting hyperplasia without causing systemic side effects. We have previously demonstrated that paclitaxel‐coated expanded poly(tetrafluoroethylene) (ePTFE) grafts, by a dipping method, could prevent neointimal hyperplasia and stenosis of arteriovenous (AV) hemodialysis grafts, especially at the graft‐venous anastomoses; however, large quntities of initial burst release have remained a problem. To achieve controlled drug release, paclitaxel (Ptx)‐loaded poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles (Ptx‐PLGA‐NPs) were prepared by the emulsion‐solvent evaporation method and then transferred to the luminal surface and inner part of ePTFE vascular grafts through our micro tube pumping and spin penetration techniques. Scanning electron microscope (SEM) images of various stages of Ptx‐PLGA‐NPs unequivocally showed that micro tube pumping followed by spin penetration effectively transferred Ptx‐PLGA‐NPs to the inner part, as well as the luminal surface, of an ePTFE graft. In addition, the in vitro release profiles of paclitaxel demonstrated that this new system achieved controlled drug delivery with a reduced initial burst release. These results suggest that loading of Ptx‐PLGA‐NPs to the luminal surface and the inner part of an ePTFE graft is a promising strategy to ultimately inhibit the development of venous neointimal hyperplasia.

Effects of curcumin for preventing restenosis in a hypercholesterolemic rabbit iliac artery stent model.

To evaluate the efficacy of the curcumin‐coating stent (CCS) on the inhibition of restenosis in a rabbit iliac artery stent model.

Paclitaxel coating of the luminal surface of hemodialysis grafts with effective suppression of neointimal hyperplasia

OBJECTIVES Paclitaxel coating of hemodialysis grafts is effective in suppressing neointimal hyperplasia in the graft and vascular anastomosis sites. However, paclitaxel can have unwanted effects on the surrounding tissues. To reduce such problems, we developed a method to coat the drug only on the luminal surface of the graft, with little loading on the outer surface. METHODS A peristaltic pump and a double-solvent (water and acetone) system were used to achieve an inner coating of paclitaxel. At the ratio of 90% acetone, paclitaxel was homogeneously coated only on the luminal surface of the graft without changing the physical properties. To determine its effect, grafts were implanted between the common carotid artery and the external jugular vein in pigs using uncoated control grafts (n = 6) and low-dose (n = 6, 0.22 μg/mm(2)) and high-dose (n = 6, 0.69 μg/mm(2)) paclitaxel inner-coated grafts. Cross-sections of graft-venous anastomoses were analyzed histomorphometrically 6 weeks after placement to measure the patency rate, percentage of luminal stenosis, and neointimal area. RESULTS No signs of infection or bacterial contamination were observed in the paclitaxel inner-coated groups. Only one of the six control grafts was patent, but all of the paclitaxel-coated grafts were patent, with little neointima. The mean ± standard error values of percentage luminal stenosis were 75.7% ± 12.7% (control), 17.5% ± 3.1% (low dose), and 19.7% ± 3.0% (high dose). The values for the neointimal area (in mm(2)) were 8.77 ± 1.66 (control), 3.53 ± 0.73 (lose dose), and 4.24 ± 0.99 (high dose). Compared with the control group, paclitaxel inner-coated vascular grafts significantly suppressed neointimal hyperplasia (low dose, P = .001; high dose, P = .002). Myofibroblast proliferation and migration into the graft interstices confirmed the firm attachment of the implanted graft to the surrounding tissue. CONCLUSIONS Paclitaxel coating on the inner luminal surface of vascular grafts was effective in suppressing neointimal hyperplasia, with little inhibition of myofibroblast infiltration within the graft wall.

Cycloserine-induced encephalopathy: evidence on brain MRI

Cycloserine is a structural analogue of the amino acid D-alanine commonly used as an anti-tuberculosis antibiotic. The nervous system is a frequent target of its adverse effects, which include drowsiness, dizziness, headache, lethargy, depression, tremor, dysarthria, hyperreflexia, paraesthesia, anxiety, confusion, seizure and coma [1]. However, this neurotoxicity has not been confirmed by neuroimaging studies. We report the recent experience of a case potentially related to the toxicity of cycloserine.

Encephalitis associated with motor polyneuropathy

We describe three patients with acute encephalitis and motor‐dominant polyneuropathy. All three patients were in their middle age and presented with features of encephalopathy with flu like illness. Cerebrospinal fluid (CSF) studies showed lymphocytic pleocytosis with increased protein. Brain magnetic resonance imagings (MRIs) showed symmetric high signal intensities in bilateral mesial temporal lobes and bilateral thalami in all patients. All three patients became quadriplegic and areflexic within two hospital days (HDs) and electrophysiologic studies revealed motor polyneuropathy. Viral or atypical bacterial infection was suspected, but no causative agent could be found. One patient expired on the twentieth hospital day, and the other two patients showed no or little improvement. To our knowledge, this combination of acute encephalitis with motor polyneuropathy has not been reported.