Jee-Heon Jeong

The effects of 8-arm-PEG-catechol/heparin shielding system and immunosuppressive drug, FK506 on the survival of intraportally allotransplanted islets.

This study proposed a double-layer shielding method of using 8-arm-PEG-catechol (PEG(8)) and N-hydroxysuccinimidyl-linked unfractionated heparin (UFH-NHS) for the prevention of instant blood-mediated inflammatory reaction (IBMIR) and immune reactions against transplanted pancreatic islets. The surface of islet was evenly covered by PEG(8) and UFH-NHS. Both viability and functionality of islets were evaluated in vitro, and the anti-coagulation effect of conjugated heparin on the islet surface was also evaluated. The inhibition effects of PEG(8)/UFH double-layer shielding system on immune reactions and IBMIR induced by transplanted islets were evaluated in an allograft model. When pancreatic islets of Sprague-Dawley (SD) rats were transplanted in the liver of F344 rats, the mean survival time (MST) of PEG(8)/UFH double-layer shielded islets (6.8 ± 1.6 days) was statistically increased, compared to that of unmodified islets (3.6 ± 1.1 days). Furthermore, when 0.5 mg/kg of FK506 was daily administered, the MST of double-layer shielded islet (15.0 ± 2.1 days) was increased by two-fold, compared to that of unmodified islets treated with the same dose of FK506 (8.0 ± 2.4 days). Therefore, a newly developed strategy of combining the PEG(8)/UFH double-layer shielding system with FK506 would certainly be effective for preventing immune activation and IBMIR against allotransplanted islets.

Functional enhancement of beta cells in transplanted pancreatic islets by secretion signal peptide-linked exendin-4 gene transduction.

This study assessed whether the newly designed exendin-4 (Ex-4) gene with highly releasable characteristics could enhance the beta cell function, thereby attenuating the essential islet mass required to cure diabetes. We constructed a lentivirus system encoding for a highly releasable secretion signal peptide, the peptide linked Ex-4 (SP-Ex-4) gene. After the transduction of lentivirus encoding for SP-Ex-4 (LV-SP-Ex-4) gene into the islets, the therapeutic effects of Ex-4 secreted were evaluated by conducting glucose-stimulated insulin secretion and cytokine- or hypoxia-induced apoptosis. Additionally, the effect of reduced islet numbers for transplantation was evaluated via in vivo models. The transduction of LV-SP-Ex-4 gene did not affect the viability of islets. In diabetic animal models, 50 islets expressing Ex-4 were transplanted to cure the diabetic nude mice, whereas at least 150 untransduced islets had to be transplanted to cure the diabetic nude mice. When the transduced islets were transplanted into diabetic immunocompetent mice, the survival rate of the mice was 18.0±4.9 days; however, when the untransduced islets were transplanted, they were rejected within 10.0±0.6 days. Therefore, the highly releasable Ex-4 could enhance the beta cell function with slightly enhanced viability of transplanted islets, presenting as a potential technology for overcoming islet shortage.

Molecularly engineered islet cell clusters for diabetes mellitus treatment.

Pancreatic islet transplantation is a promising method for curing diabetes mellitus. We proposed in this study a molecularly engineered islet cell clusters (ICCs) that could overcome problems posed by islet transplantation circumstances and hosts immune reactions. A gene containing highly releasable exendin-4, an insulinotropic protein, was delivered into single islet cells to enhance glucose sensitivity; thereafter, the cells were reaggregated into small size ICCs. Then the surface of ICCs was modified with biocompatible poly(ethylene glycol)-lipid (PEG) (C18) for preventing immune reactions. The regimen of ICCs with low doses of anti-CD154 mAb and tacrolimus could effectively maintain the normal glucose level in diabetic mice. This molecularly engineered PEG-Sp-Ex-4 ICC regimen prevented cell death in transplantation site, partly through improving the regulation of glucose metabolism and by preventing hypoxia- and immune response-induced apoptosis. Application of this remedy is also potentially far-reaching; one would be to help overcome islet supply shortage due to the limited availability of pancreas donors and reduce the immunosuppressant regimens to eliminate their adverse effects.

Progressive slowdown/prevention of cellular senescence by CD9-targeted delivery of rapamycin using lactose-wrapped calcium carbonate nanoparticles

Cellular senescence, a state of irreversible growth arrest and altered cell function, causes aging-related diseases. Hence, treatment modalities that could target aging cells would provide a robust therapeutic avenue. Herein, for the first time, we utilized CD9 receptors (overexpressed in senescent cells) for nanoparticle targeting in addition to the inherent β-galactosidase activity. In our study, CD9 monoclonal antibody-conjugated lactose-wrapped calcium carbonate nanoparticles loaded with rapamycin (CD9-Lac/CaCO3/Rapa) were prepared for targeted rapamycin delivery to senescent cells. The nanoparticles exhibited an appropriate particle size (~130 nm) with high drug-loading capacity (~20%). In vitro drug release was enhanced in the presence of β-galactosidase suggesting potential cargo drug delivery to the senescent cells. Furthermore, CD9-Lac/CaCO3/Rapa exhibited high uptake and anti-senescence effects (reduced β-galactosidase and p53/p21/CD9/cyclin D1 expression, reduced population doubling time, enhanced cell proliferation and migration, and prevention of cell cycle arrest) in old human dermal fibroblasts. Importantly, CD9-Lac/CaCO3/Rapa significantly improved the proliferation capability of old cells as suggested by BrdU staining along with significant reductions in senescence-associated secretory phenotypes (IL-6 and IL-1β) (P < 0.05). Altogether, our findings suggest the potential applicability of CD9-Lac/CaCO3/Rapa in targeted treatment of senescence.

Xenotransplantation of exendin-4 gene transduced pancreatic islets using multi-component (alginate, poly-L-lysine, and polyethylene glycol) microcapsules for the treatment of type 1 diabetes mellitus.

This study proposed that microencapsulation of exendin-4 gene transduced islets using alginate, poly-L-lysine, and polyethylene glycol could lead to increased viability and functionality of islets in a rat to mouse xenograft model. The stability of the microcapsules was determined using an osmotic pressure test and a rotational stress test. Exendin-4 gene was transduced into pancreatic islets using lenti-viral vectors and the transduced islets were encapsulated using multi-component microcapsules mentioned above. Both viability and functionality of microencapsulated islets were evaluated in both in vitro and in vivo xenograft model. The viabilities of the unmodified islets (control) and the exendin-4 transduced islets (test) on 14th day were 18.6 ± 11.1 and 49.2 ± 13.4%, respectively (p < 0.05). The stimulation index of the control and the test groups was 2.3 ± 1.7 and 3.0 ± 1.6, respectively. The mean survival times (MST) of the control and the test groups were 20.2 ± 8.0 and 35.2 ± 10.0 days, respectively (p < 0.05). Significant differences in MST suggested that transduction of exendin-4 gene had a great potential to increase the function of encapsulated islets. In conclusion, exendin-4 gene transduced islets encapsulated by poly(ethylene glycol) conjugated alginate/PLL microcapsules significantly improved both viability and functionality of encapsulated islets.

Effects of surface camouflaged islet transplantation on pathophysiological progression in a db/db type 2 diabetic mouse model

To investigate the inhibition effects of pancreatic islet transplantation on the progression of obese type 2 diabetes, we analyzed the effects of surface camouflaged islet transplantation on delaying the disease progression in a db/db diabetic mouse model. Surface camouflaged islets using 6-arm-PEG-catechol were transplanted in db/db diabetic mice. The fat accumulation and toxicity in the liver, the expansion of islets in the pancreas, and the size change of abdominal adipocyte were analyzed. In addition, the blood glucose control, insulin levels and immunohistochemical staining of recovered tissues were analyzed after transplantation. Then co-administration of anti-CD154 monoclonal antibody and Tacrolimus (IT group) deterred the pathophysiological progression of obese type 2 diabetes. At day 3 of transplantation, the serum insulin concentration of IT group was increased compared to the db/db diabetic mice group. The immunohistochemical studies demonstrated that the mass of 6-arm-PEG-catechol grafted islet was preserved in the transplantation site for 14 days. Surface modification using 6-arm-PEG-catechol effectively inhibited the immune cell infiltration and activation of host immune cells when immunosuppressive drug was given to the db/db type 2 diabetes mice. Therefore, 6-arm-PEG-catechol grafted islets effectively restored the insulin secretion in islet recipients and prevented the disease progression in type 2 diabetes.

Potential differentiation ability of gingiva originated human mesenchymal stem cell in the presence of tacrolimus.

The aim of the present study is to evaluate the potential differentiation ability of gingiva originated human mesenchymal stem cell in the presence of tacrolimus. Tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres were prepared using electrospraying technique. In vitro release study of tacrolimus-loaded poly(lactic-co-glycolic acid) microspheres was performed in phosphate-buffered saline (pH 7.4). Gingiva-derived stem cells were isolated and incubated with tacrolimus or tacrolimus-loaded microspheres. Release study of the microspheres revealed prolonged release profiles of tacrolimus without any significant initial burst release. The microsphere itself did not affect the morphology of the mesenchymal stem cells, and cell morphology was retained after incubation with microspheres loaded with tacrolimus at 1 μg/mL to 10 μg/mL. Cultures grown in the presence of microspheres loaded with tacrolimus at 1 μg/mL showed the highest mineralization. Alkaline phosphatase activity increased with an increase in incubation time. The highest expression of pSmad1/5 was achieved in the group receiving tacrolimus 0.1 μg/mL every third day, and the highest expression of osteocalcin was achieved in the group receiving 1 μg/mL every third day. Biodegradable poly(lactic-co-glycolic acid)-based microspheres loaded with tacrolimus promoted mineralization. Microspheres loaded with tacrolimus may be applied for increased osteoblastic differentiation.

Size Controlled Heparin Fragment-Deoxycholic Acid Conjugate Showed Anticancer Property by Inhibiting VEGF165.

Heparin is a highly sulfated, long, and linear polysaccharide, which can inhibit tumor growth by interacting with growth factors such as bFGF and VEGF. Several researchers have shown the anti-angiogenic effect of heparin and its conjugates in relation to growth factor inhibition. For drug development and inhibition of growth factors using heparin conjugates, the molecular size of heparin may be crucial considering the size of the heparin binding site of growth factors. In this study, we synthesized heparin fragments and deoxycholic acid conjugated heparin fragments (HFD) to search for the optimal size-controlled conjugate that will inhibit the angiogenic effect of VEGF165. We have also shown that the HFDs could have an enhanced therapeutic effect in vitro and in vivo consequent to the molecular size control. HFDs have significant anti-angiogenic effects by blocking the angiogenic activity of VEGF165 depending on its molecular size. Among them, HFD2 was a promising candidate for oral angiogenesis inhibitor. These results suggest that size-controlled synthesis is necessary for heparin-based drug development.

Design, Synthesis, and Therapeutic Evaluation of Poly(acrylic acid)-tetraDOCA Conjugate as a Bile Acid Transporter Inhibitor.

Regulation of cholesterol and bile acid homeostasis has been attracting attention as a pharmaceutical target for the treatment of diseases, such as hypercholesterolaemia and type 2 diabetes. In recent years, small bile acid analogues have been developed for the purpose of apical sodium-dependent bile acid transporter (ASBT) inhibition. Here, we designed a novel hydrophilic ASBT inhibitor using oligomeric bile acid with a high affinity with ASBT. Polyacrylic acid-tetraDOCA conjugates (PATD) have the ability to bind to ASBT in order to induce hypocholesterolemic effects. Both the viability and the functionality of PATD were evaluated in vitro, showing that PATDs were effective in inhibiting the increases of cholesterol in the blood and oil in the liver induced by high fat diet (HFD). The results indicated that the newly developed biomaterials with oligomeric bile acids and a hydrophilic polymer are potent therapeutic agents for hyperlipidemia.

Layer-by-layer building up of heparin and glycol chitosan for rat pancreatic islet xenotransplantation to mouse

AbstractImmunoprotective strategies are being developed to improve the graft survival rate of islets. Heparin, which has been widely used due to its anti-coagulant and anti-inflammatory effects, holds great promise for use in preventing blood coagulation and related immune reactions. In this study, the layer-by-layer modification of islets using heparin and glycol chitosan as an immunoprotective remedy was examined in xenogeneic islet transplantation. The surface coverage of heparin and glycol chitosan was evaluated in vitro. The viability and functionality of layer-by-layer modified islets was evaluated using the CCK-8 assay and glucose-stimulated insulin secretion (GSIS) test, respectively. In addition, the effect of layer-by-layer on immunoprotection for transplanted islets was evaluated in a diabetic mouse model. Layer-by-layer modified islets were evenly covered with heparin and glycol chitosan. The viability of islets was not affected by the modification procedures up to the third layer modification. In addition, unmodified and layer-by-layer modified (three layers) islets transplanted into the recipients maintained a normal level of blood glucose for 9.0±0.6 and 9.0±0.5 days, respectively. However, when the recipients were administrated with FK506 and CTLA4-Ig, the median survival time of the unmodified and layer-by-layer modified islets (three layers) were 17±1.2 and 23.0±1.3 days, respectively. These results demonstrated that the combination of FK506 and CTLA4-Ig along with layer-by-layer modification using heparin and glycol chitosan showed highly improved synergic effects on the inhibition of inflammation and immune reactions.