Effective Cryopreservation of a Bioluminescent Auxotrophic Escherichia coli-Based Amino Acid Array to Enable Long-Term Ready-to-Use Applications.

Amino acid arrays comprising bioluminescent amino acid auxotrophic Escherichia coli are effective systems to quantitatively determine multiple amino acids. However, there is a need to develop a method for convenient long-term preservation of the array to enable its practical applications. Here, we reported a potential strategy to efficiently maintain cell viability within the portable array. The method involves immobilization of cells within agarose gel supplemented with an appropriate cryoprotectant in individual wells of a 96-well plate, followed by storage under freezing conditions. Six cryoprotectants, namely dimethyl sulfoxide, glycerol, ethylene glycol, polyethylene glycol, sucrose, and trehalose, were tested in the methionine (Met) auxotroph-based array. Carbohydrate-type cryoprotectants (glycerol, sucrose, and trehalose) efficiently preserved the linearity of determination of Met concentration. In particular, the array with 5% trehalose exhibited the best performance. The Met array with 5% trehalose could determine Met concentration with high linearity (R2 value = approximately 0.99) even after storage at -20 °C for up to 3 months. The clinical utilities of the Met and Leu array, preserved at -20 °C for 3 months, were also verified by successfully quantifying Met and Leu in spiked blood serum samples for the diagnosis of the corresponding metabolic diseases. This long-term preservation protocol enables the development of a ready-to-use bioluminescent E. coli-based amino acid array to quantify multiple amino acids and can replace the currently used laborious analytical methods.

Poly(γ-Glutamic Acid)/Chitosan Hydrogel Nanoparticles For Effective Preservation And Delivery Of Fermented Herbal Extract For Enlarging Hair Bulb And Enhancing Hair Growth.

INTRODUCTION: Hair growth-promoting herbal extract mixtures (4HGF) exhibits significant anti-inflammatory activities relevant to promoting hair growth; however, its efficacy in patients with hair loss has been limited majorly due to its low penetration ability into hair follicles. Herein, we prepared hydrogels via dropwise addition of poly(γ-glutamic acid) (PGA) solution containing 4HGF into chitosan (CS) solution, resulting in quick formation of ~400 nm-sized hydrogel particles through electrostatic interaction-derived ionic gelation with over 50% encapsulation efficiency of 4HGF (PGA-4HGF). METHODS: The size and morphology of PGA-4HGF were characterized by TEM, SEM, and dynamic light scattering analyses. Encapsulation efficiency and loading capacity of 4HGF within PGA-4HGF, as well as in vitro release profiles were determined by simply measuring the characteristic absorbance of 4HGF. Penetrating efficiency of PGA-4HGF was evaluated by tracking the respective fluorescence through model porcine skin with confocal laser microscope system. By treating PGA-4HGF on telogenic mice and dermal papilla cells (DPCs), we evaluated the size of hair bulbs in mice, as well as morphological changes in DPCs. RESULTS: Negligible and sustained release of entrapped 4HGF from the hydrogel nanoparticles were observed under acidic and physiological pH conditions, respectively, which is quite advantageous to control their release and prolong their hair growth-promoting effect. The hydrogel nanoparticles were penetrable through the porcine skin after incubation with or without shaking. After treating telogenic mice and DPCs with PGA-4HGF, we detected enlargement of hair bulbs and remarkable shape changes, respectively, thereby showing its potential in induction of hair growth. CONCLUSION: These results suggest that the hydrogel nanoparticle formulation developed in this study can be employed as a potential approach for the preservation of hair growth-promoting compounds, their delivery of into hair follicles, and enhancing hair growth.

Hair Growth Promoting Effect of 4HGF Encapsulated with PGA Nanoparticles (PGA-4HGF) by ß-Catenin Activation and Its Related Cell Cycle Molecules.

Poly-γ-glutamic acid (γ-PGA)-based nanoparticles draw remarkable attention as drug delivery agents due to their controlled release characteristics, low toxicity, and biocompatibility. 4HGF is an herbal mixture of Phellinus linteus grown on germinated brown rice, Cordyceps militaris grown on germinated soybeans, Polygonum multiflorum, Ficus carica, and Cocos nucifera oil. Here, we encapsulated 4HGF within PGA-based hydrogel nanoparticles, prepared by simple ionic gelation with chitosan, to facilitate its penetration into hair follicles (HFs). In this study, we report the hair promoting activity of 4HGF encapsulated with PGA nanoparticles (PGA-4HGF) and their mechanism, compared to 4HGF alone. The average size of spherical nanoparticles was ~400 nm in diameter. Continuous release of PGA-4HGF was observed in a simulated physiological condition. As expected, PGA-4HGF treatment increased hair length, induced earlier anagen initiation, and elongated the duration of the anagen phase in C57BL/6N mice, compared with free 4HGF treatment. PGA-4HGF significantly increased dermal papilla cell proliferation and induced cell cycle progression. PGA-4HGF also significantly increased the total amount of ß-catenin protein expression, a stimulator of the anagen phase, through induction of cyclinD1 and CDK4 protein levels, compared to free 4HGF treatment. Our findings underscore the potential of PGA nanocapsules to efficiently deliver 4HGF into HFs, hence promoting hair-growth. Therefore, PGA-4HGF nanoparticles may be promising therapeutic agents for hair growth disorders.

Pediococcus pentosaceus-Fermented Cordyceps militaris Inhibits Inflammatory Reactions and Alleviates Contact Dermatitis.

Cordyceps militaris is a medicinal mushroom used to treat immune-related diseases in East Asia. We investigated the anti-inflammatory effect of the extract of C. militaris grown on germinated Rhynchosia nulubilis (GRC) fermented with Pediococcus pentosaceus ON89A isolated from onion (GRC-ON89A) in vivo as well as in vitro. The anti-inflammatory effect of GRC-ON89A was investigated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The total polyphenol content (TPC) and total flavonoid content (TFC) in the GRC-ON89A ethanol extract were significantly increased compared to that in GRC. GRC-ON89A hexane fraction (GRC-ON89A-Hex) inhibited the release of nitric oxide (NO) compared to that of the LPS-treated control without cytotoxicity in LPS-stimulated RAW 264.7 macrophages. GRC-ON89A-Hex decreased the inducible NO synthase (iNOS), cyclooxygenase 2 (COX2), and tumor necrosis factor (TNF)-α mRNA expression in LPS-stimulated RAW 264.7 macrophages. In addition, pre-treatment with GRC-ON89A-Hex significantly inhibited LPS-stimulated phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB. To induce allergic contact dermatitis (ACD), 1-fluoro-2, 4-dinitrofluorobenzene (DNFB) was applied to the surface of the right ears of C57BL/6N mice. GRC-ON89A reduced the ear swelling and thickness in DNFB-induced ACD mice. This study demonstrates the potential usefulness of GRC-ON89A as an anti-inflammatory dietary supplement or drug.