Jong-Sang Park

Intranasal Delivery of HMGB1 siRNA Confers Target Gene Knockdown and Robust Neuroprotection in the Postischemic Brain

Noninvasive intranasal drug administration has been noted to allow direct delivery of drugs to the brain. In the present study, the therapeutic efficacy of intranasal small interfering RNA (siRNA) delivery was investigated in the postischemic rat brain. Fluorescein isothiocyanate (FITC)-labeled control siRNA was delivered intranasally in normal adult rats using e-PAM-R, a biodegradable PAMAM dendrimer, as gene carrier. Florescence-tagged siRNA was found in the cytoplasm and processes of neurons and of glial cells in many brain regions, including the hypothalamus, amygdala, cerebral cortex, and striatum, in 1 hour after infusion, and the FITC-fluorescence was continuously detected for at least 12 hours. When siRNA for high mobility group box 1 (HMGB1), which functions as an endogenous danger molecule and aggravates inflammation, was delivered intranasally, the target gene was significantly depleted in many brain regions, including the prefrontal cortex and striatum. More importantly, intranasal delivery of HMGB1 siRNA markedly suppressed infarct volume in the postischemic rat brain (maximal reduction to 42.8 ± 5.6% at 48 hours after 60 minutes middle cerebral artery occlusion (MCAO)) and this protective effect was manifested by recoveries from neurological and behavioral deficits. These results indicate that the intranasal delivery of HMGB1 siRNA offers an efficient means of gene knockdown-mediated therapy in the ischemic brain.

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.

Effective healing of diabetic skin wounds by using nonviral gene therapy based on minicircle vascular endothelial growth factor DNA and a cationic dendrimer.

The development of an efficient method to improve the wound healing process is urgently required for diabetic patients suffering a threat of limb amputations. Various growth factors have been proposed for treatment; however, more research still has to be carried out to maintain their curative effect. In the present study, we describe a simple nonviral gene therapy method for improving wound healing.

Arginine modified PAMAM dendrimer for interferon beta gene delivery to malignant glioma.

A xenograft brain tumor model was established by the subcutaneous injection of U87MG cells into nude mice to investigate the efficacy of a non-viral vector, arginine-modified polyamidoamine dendrimer (PAMAM-R), in delivering a therapeutic gene, human interferon beta (IFN-β). We used 4,6-diamidino-2-phenylindole staining, the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, and the caspase-3 activity assay to determine the induction of apoptosis upon transfection with the PAMAM-R/IFN-β gene polyplex in vitro. The polyplex was injected into xenograft brain tumors. Mice treated with PAMAM-R/pORF-IFN-β exhibited a significantly smaller tumor size than control mice and PAMAM-R/pORF treated mice. Hematoxylin/eosin staining and immunohistochemistry with the endothelial growth factor receptor antibody also revealed inhibition of tumor growth. Furthermore, reverse transcription polymerase chain reaction and the TUNEL assay also verified the expression of IFN-β and induction of apoptosis in vivo. These results indicate that the PAMAM-R/pORF-IFN-β polyplex is an effective therapeutic candidate for glioblastoma multiforme due to its selective induction of apoptosis in tumor cells.

Recombinant soluble neprilysin reduces amyloid-beta accumulation and improves memory impairment in Alzheimer's disease mice.

Accumulation of amyloid-β (Aβ) is thought to be a central pathology in the brain of patients with Alzheimers disease (AD). Neprilysin (NEP), a plasma membrane glycoprotein of the neutral zinc metalloendopeptidase family, is known as a major Aβ-degrading enzyme in the brain. The level of NEP is reduced in the brains of patients with AD; therefore, NEP is under intense investigation as a potential therapeutic source for degradation of deposited Aβ in AD. Previous studies have utilized viral vectors expressing NEP for reduction of Aβ deposition in the brain. However, viral vectors have disadvantages regarding difficulty in control of insert size, expression desired (short- or long-term), and target cell type. Here, in order to overcome these disadvantages, we produced recombinant soluble NEP from insect cells using an NEP expression vector, which was administered by intracerebral injection into AD mice, resulting in significantly reduced accumulation of Aβ. In addition, AD mice treated with NEP showed improved behavioral performance on the water maze test. These data support a role of recombinant soluble NEP in improving memory impairment by regulation of Aβ deposition and suggest the possibility that approaches using protein therapy might have potential for development of alternative therapies for treatment of AD.

Ion pairs of risedronate for transdermal delivery and enhanced permeation rate on hairless mouse skin

Ion-paired solutions of risedronate (RIS) with L-arginine (ARG), L-lysine (LYS), and diethylenetriamine (DETA) were tested in vitro for their potential to enhance the penetration of RIS across the skin of hairless mouse. The xylene solubilities of RIS paired with ARG, LYS, and DETA in molar ratios of 1:2, 1:2, and 1:1 were 8.9%, 12.0%, and 2.1%, respectively, in comparison with the solubility in deionized water, but non-ion-paired RIS was not detected in xylene. In vitro permeation tests were performed on the skin of hairless mice, and the results indicated that ion-paired RIS could penetrate mice skin about 36 times more effectively than RIS alone. The cumulative amount of ion paired RIS after 24 h resulted in 475.18±94.19 μg/cm(2) and 511.21±106.52 μg/cm(2) at molar ratio of 1:2 and 1:1. The cumulative amount of RIS alone was as low as 14.13±5.49 μg/cm(2) in 24h. The hairless mice showed no skin irritation after a single administration of RIS alone and ion-paired RIS (1:2 molar ratio with ARG, and 1:1 molar ratio with DETA). In this study, we found that RIS can be delivered transdermally, and the ion-paired system in an aqueous solution showed an enhanced flux through the skin barrier.

Topically administered Risedronate shows powerful anti-osteoporosis effect in ovariectomized mouse model

We investigated the therapeutic effect of topical Risedronate (RIS) on a mouse model of estrogen-deficient osteoporosis. Fourteen-week-old female mice were ovariectomized and assigned to 4 groups: SHAM-operated (SHAM), OVX mice treated with vehicle (OVX-V), OVX mice treated with 0.2% RIS (OVX-0.2% RIS), and OVX-mice treated with 0.02% RIS (OVX-0.02% RIS). Topical samples containing RIS were prepared in 10% (w/w) polyethylene glycol (PEG, MW 400) and 80 μg of sample was spread on the mices mid-backs every 3 days for 5 weeks. Micro-CT analysis of femora demonstrated that OVX-0.2% RIS exhibited a 29% greater bone mineral density and 24% greater bone volume fraction than that of OVX-V group. Investigation of the trabecular bone in OVX-0.2% RIS revealed a 24% higher bone volume (BV/TV), 51% higher trabecular number (Tb.N), and 40% lower trabecular separation (Tb.Sp) compared to OVX-V mice. Additionally, bone phenotypes of tibiae were further confirmed by histological analysis. OVX-0.2% RIS group exhibited a 494% greater BV/TV, 464% less Tb.Sp, 81% greater active osteoclast surface (Oc.S/BS) and 26% less osteoclast number (N.Oc/BS) than that of OVX-V group. Collectively, these results indicated that topical delivery of RIS has powerful pharmaceutical effects on the prevention of osteoporosis and bone turnover.

Nonviral gene therapy in vivo with PAM-RG4/apoptin as a potential brain tumor therapeutic

Background Glioma is still one of the most complicated forms of brain tumor to remove completely due to its location and the lack of an efficient means to specifically eliminate tumor cells. For these reasons, this study has examined the effectiveness of a nonviral gene therapy approach utilizing a tumor-selective killer gene on a brain tumor xenograft model. Methods and results The therapeutic apoptin gene was recombined into the JDK plasmid and delivered into human brain tumor cells (U87MG) by using a polyamidoamine dendrimer with an arginine surface (PAM-RG4). Studies in vitro showed that the PAM-RG4/apoptin plasmid polyplex exhibited a particularly high transfection activity of .40%. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, 4′,6-Diamidino-2-phenylindole (DAPI) TUNEL assay, DAPI staining, and caspase-3 activity assay verified that the tumor cells had undergone apoptosis induced by apoptin. For in vivo studies, the polyplex was injected into tumors, which were induced by injecting U87MG cells intradermally into nude mice. Based on hematoxylin and eosin staining, epidermal growth factor receptor immunohistochemistry results and tumor volume measurement results, tumor growth was effectively inhibited and no specific edema, irritation, or other harm to the skin was observed after polyplex injection. The in vivo expression of apoptin and the induction of apoptosis were verified by reverse-transcription polymerase chain reaction analysis, TUNEL assay, and DAPI staining. Conclusion The PAM-RG4/apoptin gene polyplex is a strong candidate for brain tumor therapeutics because of the synergistic effect of the carrier’s high transfection efficiency (35%–40%) in glioma cells and the selective apoptosis-inducing activity of apoptin in tumor cells.

Suppression of neointimal hyperplasia by sirolimus-eluting expanded polytetrafluoroethylene (ePTFE) haemodialysis grafts in comparison with paclitaxel-coated grafts

BACKGROUNDnHaemodialysis vascular access dysfunction caused by aggressive venous neointimal hyperplasia is a major problem for haemodialysis patients with synthetic arteriovenous (AV) grafts. Several different strategies to prevent venous stenosis by inhibiting smooth muscle cell proliferation and migration using local delivery of potent antiproliferative agents are currently under investigation. We performed this study to evaluate the efficacy of sirolimus-eluting vascular grafts in preventing stenosis and to compare the effectiveness of sirolimus-coated grafts with that of paclitaxel-coated vascular grafts that we characterized in a previous study.nnnMETHODSnAV grafts were implanted laterally between the common carotid artery and external jugular vein of 14 female Landrace pigs. Three types of grafts were implanted: grafts coated with 1.08 μg/mm(2) sirolimus (low dose, n = 6), grafts coated with 2.41 μg/mm(2) sirolimus (high dose, n = 2) and uncoated control grafts (n = 6). Animals were sacrificed 6 weeks after surgery. Cross-sections of the venous anastomoses were analysed to determine the percentage of luminal stenosis in each group, and immunohistochemistry was performed to identify the cellular phenotypes of the neointimal hyperplasia and tissues adjacent to the implanted grafts.nnnRESULTSnCompared with the control group, neointimal hyperplasia in the venous anastomoses of the groups implanted with sirolimus-coated vascular grafts was significantly suppressed without infection. The mean ± standard error values for the percentage of luminal stenosis were 75.7 ± 12.7% in the control group and 22.2 ± 1.41% in the low-dose sirolimus-coated group. Myofibroblasts and fibroblasts were the major cell types found in the neointimal hyperplasia.nnnCONCLUSIONSnNeointimal hyperplasia was effectively suppressed by sirolimus-eluting grafts. However, the inhibitory effects of sirolimus-eluting grafts were weaker than those observed for paclitaxel-coated grafts in our previous study.

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

OBJECTIVESnPaclitaxel 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.nnnMETHODSnA 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.nnnRESULTSnNo 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.nnnCONCLUSIONSnPaclitaxel 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.