Jung Dong Kim

Droplet-born air blowing: Novel dissolving microneedle fabrication

The microneedle-mediated drug delivery system has been developed to provide painless self-administration of drugs in a patient-friendly manner. Current dissolving microneedle fabrication methods, however, require harsh conditions for biological drugs and also have problems standardizing the drug dose. Here, we suggested the droplet-born air blowing (DAB) method, which provides gentle (4-25 °C) and fast (≤10min) microneedle fabrication conditions without drug loss. The amount of drug in the microneedle can be controlled by the pressure and time of droplet dispenser and the air blowing shapes this droplet to the microneedle, providing a force sufficient to penetrate skin. Also, the introduction of a base structure of two layered DAB-microneedle could provide complete drug delivery without wasting of drug. The DAB-based insulin loaded microneedle shows similar bioavailability (96.6±2.4%) and down regulation of glucose level compared with subcutaneous injection. We anticipate that DAB described herein will be suitable to design dissolving microneedles for use in biological drug delivery to patients.

A high-capacity, hybrid electro-microneedle for in-situ cutaneous gene transfer

Cutaneous gene transfer is limited by biological barriers such as skin and cellular membranes; complex approaches are required to overcome these biological barriers, simultaneously. Non-integrated systems that separate cutaneous permeation from intracellular transfection have been used to overcome skin and cellular barriers, respectively, however, do not provide sufficient doses of the gene to local tissue, resulting in inefficient gene transfer in-situ. Although integrated systems for cutaneous gene transfer are available, their safety has been questioned and it is difficult to transfer sufficient amounts of genes due to cumbersome sterilization procedures and the small size of the reservoir. Here, we demonstrate stepwise-aligned cutaneous permeation, cutaneous release, and intracellular transfection using a hybrid electro-microneedle (HEM), which designed as a monolithic hybrid assembly of a dissolving microneedle and an electrode, anomalously. Furthermore, as proof-of-principle, we use the HEM for in-situ cutaneous transfer of p2CMVmIL-12 to successfully treat B16F10 subcutaneous tumors in a mouse model. The HEM described herein holds great promise for cutaneous gene therapy of cancers and for vaccines.

Protein Immobilization Without Purification via Dip‐Pen Nanolithography

Because these coupling reactions require tailored purified pro-teins, purification of the target protein was required. Difficultiesassociated with obtaining a diversity of purified proteins, atime-consuming and labor-intensive process, have no doubtlimited the number of studies using functional protein arrays.

Diagnosis of tuberculous meningitis by detection of immunoglobulin G antibodies to purified protein derivative and lipoarabinomannan antigen in cerebrospinal fluid

Enzyme-linked immunosorbent assay of immunoglobulin G (IgG) activity in cerebrospinal fluid (CSF) and sera was conducted prospectively in 27 patients with tuberculous meningitis (TBM) by using purified protein derivative (PPD) and lipoarabinomannan (LAM) antigens, from January 1989 to August 1990. 29 patients with aseptic meningitis and 49 patients with non-inflammatory neurological illnesses served as controls. All patients had a computed tomography (CT) scan of the head before a lumbar puncture. The IgG antibodies to the antigens were significantly elevated in TBM, and the reactivity was more frequently positive in the CSF than in the sera, suggesting a local synthesis of IgG in the central nervous system (CNS). The sensitivity and the specificity for the diagnosis of TBM were 59.2% and 93.9% for PPD antigen, and 85.2% and 95.9% for LAM antigen, respectively. Assay of IgG reactivity to LAM antigen was clinically very useful for the early diagnosis of TBM and was superior to PPD for detecting the serological evidence of TBM.

Quantitative analysis of human serum leptin using a nanoarray protein chip based on single-molecule sandwich immunoassay.

We report a method for the quantitative analysis of human serum leptin, which is a protein hormone associated with obesity, using a nanoarray protein chip based on a single-molecule sandwich immunoassay. The nanoarray patterning of a biotin-probe with a spot diameter of 150 nm on a self-assembled monolayer functionalized by MPTMS on a glass substrate was successfully accomplished using atomic force microscopy (AFM)-based dip-pen nanolithography (DPN). Unlabeled leptin protein molecules in human serum were detected based on the sandwich fluorescence immunoassay by total internal reflection fluorescence microscopy (TIRFM). The linear regression equation for leptin in the range of 100 zM-400 aM was determined to be y=456.35 x+80,382 (R=0.9901). The accuracy and sensitivity of the chip assay were clinically validated by comparing the leptin level in adult serum obtained by this method with those measured using the enzyme-linked immunosorbent assay (ELISA) performed with the same leptin standards and serum samples. In contrast to conventional ELISA techniques, the proposed chip methodology exhibited the advantages of ultra-sensitivity, a smaller sample volume and faster analysis time.

An ultra-sensitive nanoarray chip based on single-molecule sandwich immunoassay and TIRFM for protein detection in biologic fluids

This paper describes a single-molecule sandwich immunoassay method that utilizes total internal reflection fluorescence microscopy (TIRFM) at the single-molecule level for nanoarray protein chip applications. Nanoarray patterning of a biotin-probe with a spot diameter of 179 +/- 1 nm was performed successfully on a (3-mercaptopropyl)trimethoxysilane (MPTMS)-coated glass substrate by atomic force microscopy (AFM). The formation of biotin patterns was confirmed directly by observing the heights of bound streptavidin and biotin-antibody on glass substrates using an AFM in contact mode. Target protein molecules (or antigen) at the zepto-molar (zM) concentration level (x 10(-21) M) were detected on MPTMS-coated glass nanoarray protein chips by TIRFM. Finally, cytokine clinical samples (i.e. TNF-alpha and IL-1alpha) as cancer marker protein molecules were applied to nanoarray protein chips, and detection limits were at 600 zM.

The use of biodegradable microneedle patches to increase penetration of topical steroid for prurigo nodularis

BackgroundThe stratum corneum is an almost impermeable barrier. Recently, microneedles have been used to increase drug delivery passing the stratum corneum by incorporating the drug within the microneedle or by coating the surface of the microneedle with the drug.ObjectiveThis study was performed to investigate whether applying a biodegradable microneedle patch after topical steroid application increases penetration of the steroid in vitro, as well as treatment efficacy in patients with prurigo nodularis.Materials & methodsIn vitro penetration of topical steroids after biodegradable microneedle patch application was measured using a 3D skin model. To evaluate the treatment efficacy of the combination of biodegradable microneedle and topical steroids, a split-body clinical study was performed.ResultsPenetration of topical steroid in the in vitro skin model was significantly greater in the microneedle-applied skin. In a split-body clinical study with prurigo nodularis patients, the area and height of skin lesions decreased after four weeks of treatment on both sides, however, the microneedle patch side exhibited a significantly greater decrease in both area and height, compared to the control side. The pruritus visual analogue scale was also significantly lower on the microneedle side.ConclusionWe suggest that simply applying a microneedle patch after topical steroid application could be a useful strategy for treating refractory skin diseases such as prurigo nodularis.

Ultrathin Metal Crystals: Growth on Supported Graphene Surfaces and Applications

Controlled nucleation and growth of metal clusters in metal deposition processes is a long-standing issue for thin-film-based electronic devices. When metal atoms are deposited on solid surfaces, unintended defects sites always lead to a heterogeneous nucleation, resulting in a spatially nonuniform nucleation with irregular growth rates for individual nuclei, resulting in a rough film that requires a thicker film to be deposited to reach the percolation threshold. In the present study, it is shown that substrate-supported graphene promotes the lateral 2D growth of metal atoms on the graphene. Transmission electron microscopy reveals that 2D metallic single crystals are grown epitaxially on supported graphene surfaces while a pristine graphene layer hardly yields any metal nucleation. A surface energy barrier calculation based on density functional theory predicts a suppression of diffusion of metal atoms on electronically perturbed graphene (supported graphene). 2D single Au crystals grown on supported graphene surfaces exhibit unusual near-infrared plasmonic resonance, and the unique 2D growth of metal crystals and self-healing nature of graphene lead to the formation of ultrathin, semitransparent, and biodegradable metallic thin films that could be utilized in various biomedical applications.

Cutaneous Drug Delivery System: Characteristics of Drug LoadedDissolving Microneedle Technology

Extended Abstract The microneedle-mediated transdermal delivery system has been developed to provide minimal invasive selfadministration method. Especially, dissolving microneedles, which deliver the target drugs as the drug-loaded microneedle dissolves into the skin, have been spotlighted recently [1]. Droplet-born air blowing (DAB) method has great advantages in stability with precise dose control because DAB provide quick manufacturing process with ambient temperature [2]. This study suggests the novel dissolving microneedle fabrication method, droplet-born air blowing (DAB), which provide gentle temperature and fast manufacturing process with precise dose control. The purpose of this study is to show the characteristics of dissolving microneedles and to confirm with the in vitro and ex vivo models. Microneedle fabricated by DAB method. Briefly, Biodegradable polymer such as HA (hyaluronic acid) was dissolved in water with active ingredients. The polymer and drug mixture were dropped to a patch, and each droplet is shaped to the microneedle. We tested protein and chemical drug. The loaded amount of drug was analyzed by enzyme-linked immunosorbent assay (ELISA) or HPLC/UV system. Skin permeability of microneedle was confirmed by OCT (optical coherence tomography) and delivered amount of drug into the skin was analyzed using Franz diffusion cell (Logan, FDC6T). The dissolution performance of protein and chemical drug was evaluated using dissolution equipment (Hanson, Elite 8 Dissolution Tester). We optimized the DAB process parameters and scaled up without applying any heat. Various ingredients were loaded within microneedles approximately 100% compared to theoretical values independent of microneedle length. In vitro and ex vivo studies using Franz diffusion cell showed excellent delivery efficiency compared to topical solution. In vivo OCT images clearly showed that whole length of microneedles could penetrate into human skin. We developed the appropriate dissolution condition for protein and chemical drugs loaded microneedles. We are studying the pharmacokinetics of small molecule, biomolecule pharmaceutical products in animal models. DAB technology suggest a way to solve the problems of conventional molding method to fabricate dissolving microneedle. Based on the method, we have successfully developed mass production system to manufacture microneedlearrayed patch. We loaded lots of active ingredients with precise dose control, and confirmed the delivery efficiency of labile ingredients such as peptide and anti-oxidants within microneedles. We are investigating the formulations for a biopharmaceutics using this platform technology.

Comparison between Newly Developed and Commercial Inhalant Skin Prick Test Reagents Using In Vivo and In Vitro Methods

Background We developed skin prick test (SPT) reagents for common inhalant allergens that reflected the real exposure in Korea. The study aim was to evaluate diagnostic usefulness and allergen potency of our inhalant SPT reagents in comparison with commercial products. Methods We produced eight common inhalant allergen SPT reagents using total extract (Prolagen): Dermatophagoides farinae, Dermatophagoides pteronyssinus, oak, ragweed, mugwort, Humulus japonicus pollens, as well as cat and dog allergens. We compared the newly developed reagents with three commercially available SPT reagents (Allergopharma, Hollister-Stier, Lofarma). We measured total protein concentrations, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), major allergen concentration, and biological allergen potencies measured by immunoglobulin E (IgE) immunoblotting and ImmunoCAP inhibition test. Results Diagnostic values of these SPT reagents were expressed as positivity rate and concordance rate of the results from ImmunoCAP allergen-specific IgE test in 94 allergic patients. In vitro analysis showed marked differences in protein concentrations, SDS-PAGE features, major allergen concentrations, and biological allergen potencies of four different SPT reagents. In vivo analysis showed that positive rates and concordance rates of Prolagen® SPT reagents were similar compared to the three commercial SPT reagents. Conclusion The newly developed Prolagen® inhalant SPT reagents are not inferior to the commercially available SPT reagents in allergy diagnosis.