Su-Eon Jin

Accelerated wound healing by smad3 antisense oligonucleotides-impregnated chitosan/alginate polyelectrolyte complex

Smad3 mediates the intracellular signaling of TGF-beta1 superfamily and plays a critical role in the cellular proliferation, differentiation and elaboration of matrix pivotal to cutaneous wound healing. Smad3 antisense oligonucleotides (ASOs) impregnated polyelectrolyte complex (PEC) containing chitosan and sodium alginate was prepared for accelerated wound healing. Physicochemical properties of PEC were characterized by zeta potential, scanning electron microscopy and bioadhesive test. Full-thickness, excisional wounds were made on the dorsum of C57BL6 mice. Then, smad3 ASOs-PEC, PEC alone, smad3 ASOs and gauze dressing were applied to determine concentration of TGF-beta1 and collagen in tissues and observe the wound contraction and histology of tissues. Zeta potentials and bioadhesive strengths of ASOs-PEC were increased as the chitosan ratio in PEC. In smad3 ASOs-PEC, the healing process suggested by wound closure and histological observation was faster than other groups because collagen contents increased and level of TGF-beta1 decreased. These results demonstrate that the smad3 ASOs-PEC composed of chitosan and sodium alginate could be applied for accelerated wound healing.

Retained topical delivery of 5-aminolevulinic acid using cationic ultradeformable liposomes for photodynamic therapy

5-Aminolevulinic acid (5-ALA), inducing photodynamic protoporphyrin (PpIX), is a hydrophilic molecule, resulting in leashing the capacity to cross tissue barriers like stratum corneum (SC) of skin. Here, we aimed to develop 5-ALA loaded ultradeformable liposomes (UDL) with different surface charges, and to investigate their physicochemical characteristics and capability for the skin penetration and retention of 5-ALA for topical photodynamic therapy (PDT). The effects of surface charges of UDL on in vitro permeation of 5-ALA and in vivo accumulation of 5-ALA-induced PpIX in viable skin were determined and then compared with conventional neutral liposomes (nLiposome). All UDL showed smaller particle size and better deformability than nLiposome. However, entrapment efficiency of 5-ALA was similar to each vesicle. Among vesicles, the cationic UDL (cUDL) demonstrated higher stability and permeability, and could deliver 5-ALA into deep skin tissue by topical application. Moreover, the 5-ALA loaded in cUDL was long retained, and induced more amount of PpIX in viable skin than those in other UDL and nLiposome. Considering that the conversion of 5-ALA into PpIX occurs preferentially in epidermis, these results suggested that topical delivery of 5-ALA loaded in cUDL could be an interesting proposal to optimize PDT related to 5-ALA.

Subconjunctivally injected, liposome-encapsulated streptokinase enhances the absorption rate of subconjunctival hemorrhages in rabbits

Liposome-encapsulated streptokinase (SK) was prepared with distearoyl-phosphatidyl-ethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG(2000)). In vitro release assay demonstrated over 81% of SK was released from liposomes at 48 h, and the effect of its subconjunctival injection on the absorption rate of induced subconjunctival hemorrhage (SH) in rabbits was evaluated. After 8h of SH induction, eyes were randomly assigned to one of four subconjunctival injection groups (10 eyes each): group A: the free form of SK (1000 IU/mL); group B: liposome-encapsulated SK (1000 IU/mL); group C: 0.1 mL of liposomes; and group D: no injection. SHs were photographed at 8, 24, 48, 72, and 120 h after SH induction and their sizes were compared. Size decrease of the SH was faster in groups A and B than in groups C and D. Group B displayed significantly different absorption rates than group A at 24 and 48 h and with groups C and D at 24, 48, and 72 h, with the shortest mean elapsed time among all groups. The ocular absorption of SK was lower after the injection of the liposome-encapsulated SK than the free form. These results demonstrated that subconjunctival injection of liposome-encapsulated SK enhances the rate of SH absorption, especially in the early phases.

Charge-mediated topical delivery of plasmid DNA with cationic lipid nanoparticles to the skin

Cationic lipid nanoparticles (cLNs) were modified to develop a gene delivery system for topical use via a dermal route. The cLNs were formulated using high pressure homogenization method and were composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), Tween 20, and tricaprin as a solid core (1:1:1:1.67, w/w). The prepared cLNs were nanoscale-sized (<100 nm) and were highly positive (51 mV). The cLN/DNA complexes demonstrated enhanced transfection potential in the cells at the optimal ratio without cytotoxic effects. To evaluate its efficacy in topical application, in vitro skin transfer of the cLN/DNA complexes was monitored using the measurement of the surface zeta potential of hairless mouse skin and validated using confocal microscopy of the sectioned skin. The in vivo delivery of plasmid DNA with the cLN formulation was examined using the relative expression levels of mRNA after non-invasive application with the cLN/DNA complexes on hair-removed dorsal skin of mice. The cLNs successfully transferred plasmid DNA to the skin, which was facilitated by the charge-mediated interaction between the cLN/DNA complexes and the skin. These results suggest the promising potential of cLNs as a topical gene delivery system for gene vaccine delivery and cutaneous gene therapy in preclinical and clinical applications.

Pharmacokinetics of oral calcitriol in healthy human based on the analysis with an enzyme immunoassay

We investigated the pharmacokinetics of 2 microg calcitriol in eight healthy human after a single oral administration. Plasma calcitriol was analyzed by sensitive and quantitative enzyme immunoassay following intra- and inter-day validation. In subjects, AUC(0-infinity) and AUC(0-24h) were 267 and 246 pg h ml(-1), respectively. Although the mean plasma concentration of calcitriol in human subjects did not return to baseline at 24h post-dosing, the mean ratio of AUC(0-24h) to AUC(0-infinity) was greater than 80%. The C(max) of calcitriol was measured at 3.4h after administration as 50.0 pg ml(-1). From our results, it can offer new opportunities to design and determine individually appropriate therapeutic dosage regimens based on a pharmacokinetic profile and be used for bioequivalence and reevaluation study of generic drug.

Effect of Slow-Releasing All-Trans-Retinoic Acid in Bioabsorbable Polymer on Delayed Adjustable Strabismus Surgery in a Rabbit Model

PURPOSE To determine the usefulness of slow-releasing all-trans-retinoic acid (ATRA) in polytetrafluoroethylene (PTFE)/polylactide-co-glycolide (PLGA) for delayed adjustable strabismus surgery. DESIGN Animal study. METHODS A prospective, masked-observer, controlled study was performed in 25 rabbits. Fifty rabbit eyes were divided randomly into three groups. After a recession of the superior rectus muscle, a PTFE/PLGA laminate containing ATRA, PTFE alone, or balanced salt solution was applied beneath and over the superior rectus muscle in the PTFE/PLGA/all-trans-retinoic acid group (ATRA group), the polytetrafluoroethylene group (PTFE group), and the control group, respectively. Delayed adjustment was performed once on each superior rectus muscle at 3 or 5 weeks after surgery by a masked observer. RESULTS In the control group, adjustment was possible in 2 of 5 eyes at 3 weeks after surgery and impossible in any eye at 5 weeks after surgery. In the PTFE and ATRA groups, adjustment was possible in all 10 eyes at 3 and 5 weeks after surgery. On comparing adjustability, a significant difference was observed between the PTFE group and the control group or between the ATRA group and the control group 5 weeks after surgery (P = .0003 and P = .0003, respectively). A significant difference was observed between the ATRA group and the control group in terms of adhesion between superior rectus muscles and sclerae at 5 weeks after surgery (P = .006). CONCLUSIONS Slow-releasing ATRA in PTFE/PLGA was found to reduce adhesion and to allow delayed adjustment in most eyes for up to 5 weeks after surgery.

Long-term stable cationic solid lipid nanoparticles for the enhanced intracellular delivery of SMAD3 antisense oligonucleotides in activated murine macrophages.

PURPOSE Long-term stable cationic solid lipid nanoparticles (cSLNs) were formulated to transfer SMAD3 antisense oligonucleotides (ASOs) into the cells to enhance the intracellular activity of the ASOs. The SMAD3 ASOs were designed to block the inflammatory processes linked to TGFβ/SMAD3 pathway. METHODS The cSLN formulation was prepared by high-pressure homogenization method composed of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), dioleoylphosphoethanolamine (DOPE), Tween 20, and tricaprin as a solid lipid core (1:1:1:1.67, w/w). The size and the zeta potential of the prepared cSLNs were measured by light scattering. The cSLN/ASO complexes were generated and introduced into the murine macrophage cells. After the treatment of the complexes, the cellular uptake of the complexes was determined by flow cytometry and the intracellular activity of SMAD3 ASOs from the complexes was evaluated by western blotting of SMAD3. In addition, TGFβ1, an upstream molecule of TGFβ/SMAD3 pathway, was monitored by ELISA. RESULTS The nano-scale sized cSLNs were positively charged and physically stable at 4oC during the storage up to 24 months. The uptake efficiency of the cSLN/ASO complexes into macrophage cells was enhanced up to 80% without cytotoxicity. After the treatment of the cSLN/ASO complexes, SMAD3 as well as TGFβ1 was significantly suppressed based on the SMAD3 ASO activity in the macrophage cells. In addition, the cSLN/ASO complexes prevented the morphological change to dendritic shape in the activated macrophage cells. CONCLUSION These results suggest that the cSLNs have a potential to deliver the SMAD3 ASOs to intracellular compartments for the anti-inflammatory effect. The development of this strategy might lead to anti-inflammatory and anti-fibrotic therapies in immunological disorders.

Development of coated nifedipine dry elixir as a long acting oral delivery with bioavailability enhancement.

To develop the long acting nifedipine oral delivery with bioavailability enhancement, a nifedipine dry elixir (NDE) containing nifedipine ethanol solution in dextrin shell was prepared using a spray-dryer, and then coated nifedipine dry elixir (CNDE) was prepared by coating NDE with Eudragit acrylic resin. The physical characteristics and bioavailability of NDE and CNDE were evaluated, and then compared to those of nifedipine powder. NDE and CNDE, which were spherical in shape, had about 6.64 and 8.68–8.75 μm of geometric mean diameters, respectively. The amount of nifedipine dissolved from NDE for 60 min increased about 7- and 40-fold compared to nifedipine powder in pH 1.2 simulated gastric fluid and pH 6.8 simulated intestinal fluid, respectively. Nifedipine released from CNDE was retarded in both dissolution media compared with that from NDE. After oral administration of NDE, the Cmax and AUC0→8h of nifedipine in rat increased about 13- and 7-fold, respectively, and the Tmax of nifedipine was reduced significantly compared with those after oral administration of nifedipine powder alone. The AUC0→8h and Tmax of nifedipine in CNDE increased markedly and the Cmax of nifedipine in CNDE was significantly reduced compared to those in NDE. It is concluded that CNDE, which could lower the initial burst-out plasma concentration and maintain the plasma level of nifedipine over a longer period with bioavailability enhancement, might be one of potential alternatives to the marketed long acting oral delivery system for nifedipine.

Isolation and identification of steroidogenic peptides from calf spleen

Since women with climacteric syndrome have significantly lower serum levels of estradiol and other related hormones, hormone replacement therapies (HRT) such as estrogen are needed to lessen symptoms. However, HRT can often cause severe adverse effects that include many cancers and stroke. Therefore, new and novel approaches to relieve climacteric syndrome still need to be developed. The aim of this study was to identify biologically active peptides from calf spleen that are responsible for stimulating biosynthesis of steroid hormone and to explore the potential of isolated peptides as therapeutic agents for menopausal syndrome. The reverse phase HPLC system was used to isolate active compounds from calf spleen extract, a cell culture system was used to screen the activity of stimulating hormone secretion, and Matrix-Assisted Laser Desorption/Ionization (MALDI) mass spectrometry was used for molecular weight determination. In the present study, two calf steroidogenic peptides, CSP-1 (MW; 4.655 kDa) and CSP-2 (MW; 8.331 kDa), were isolated and identified from calf spleen and may be putative climacteric syndrome therapeutic agents.

Validation of an Enzyme-Immunoassay for Calcitriol in Human Plasma and Evaluation of Its Pharmacokinetics after Single-dose in Korean Volunteers

An enzyme immunoassay (EIA) was validated for quantitation of cacitriol in human plasma. Calcitriol was immunoextracted with immunocapsules, which contain monoclonal antibodies to calcitriol linked to solid phase particles in suspension with a vitamin D binding protein inhibitor. Calcitrol was eluated and the eluates were evaporated under a gentle stream of nitrogen gas. The absorbance of analytes was determined using a microplate reader (reference wavelength 650 nm; measurement wavelength 450 nm). The method was specific and sensitive enough to detect as low as 6.5 pmol/L of calcitriol. Linear calibration range was 6.5-491 pmol/L with correlation coefficient greater than 0.99. The overall accuracy was in the range of 83.8 to 111.2% and precision C.V. (%) 0.99 to 8.47%. The recovery was approximately 100% and stability was confirmed during storage and sample preparation. The pharmacokinetic parameters were calculated by baseline subtraction because calcitriol is an endogenous material. Following oral dose of calcitriol, the mean AUC was 1038539 pmol/Lhr and C of 12863.1 pmol/L was reached at 3.501.07 hr. The mean t of calcitriol was 5.132.10 hr. The present EIA method was successfully applied to study bioavailability after oral administration of 2 g of calcitriol in healthy Korean subjects.