Chi-Hsien Liu

Terpene microemulsions for transdermal curcumin delivery: Effects of terpenes and cosurfactants

Microemulsion systems composed of terpenes, polysorbate 80, cosurfactants, and water were investigated as transdermal delivery vehicles for curcumin. Pseudoternary phase diagrams of three terpenes (limonene, 1,8-cineole, and α-terpineol) at a constant surfactant/cosurfactant ratio (1:1) were constructed to illustrate their phase behaviors. Limonene combined with cosurfactants like ethanol, isopropanol, and propylene glycol were employed as microemulsion ingredients to study their potential for transdermal curcumin delivery. The transdermal delivery efficacy and skin retention of curcumin were evaluated using neonate pig skin mounted on a Franz diffusion cell. The curcumin permeation rates in the limonene microemulsion studied were 30- and 44-fold higher than those of 1,8-cineole and α-terpineol microemulsions, respectively. Significant effects on the skin permeation rates were observed from microemulsions containing different limonene/water contents. Histological examination of treated skin was performed to investigate the change of skin morphologies. Characteristics such as droplet size, conductivity, interfacial tension, and viscosity were analyzed to understand the physicochemical properties of the transdermal microemulsions. In conclusion, microemulsions loaded with curcumin were successfully optimized for transdermal delivery after screening various terpenes, cosurfactants, and limonene/water ratios. These results indicate that the limonene microemulsion system is a promising tool for the percutaneous delivery of curcumin.

Cationic nanoemulsions as non-viral vectors for plasmid DNA delivery.

Non-viral gene carriers have been extensively investigated as alternatives to viral vectors for therapeutic gene delivery. Many cationic lipid carriers including liposomes, emulsions, and solid lipid nanoparticles are used to transfer plasmid DNA. Stable nanoemulsions were prepared and modified by conjugating fatty acids with cationic amino acids including lysine, arginine, and histidine with the help of carbodiimide. Concentrations of crosslinker and amino acids were optimized to obtain the maximal surface potential. The zeta potential and size distribution of the cationic nanoemulsions were measured using photon correlation spectroscopy. The morphology of nanoemulsion-DNA complexes was examined by transmission electron microscopy. The transfection efficiencies and cytotoxicity of three cationic nanoemulsions were evaluated using 3T3 fibroblast cells. The maximal zeta potentials of lysine-, arginine-, and histidine-modified nanoemulsions were 50, 43, and 7 mV, respectively. The transfection efficiencies of amino acid-modified nanoemulsions were in the order of lysine > arginine > histidine. Low cytotoxicities of these three amino acid-modified nanoemulsions were observed. A facile and inexpensive in situ modification for producing cationic nanoemulsions was developed. The results show the potential of amino acid-modified cationic nanoemulsions as non-viral vectors for gene delivery.

Characterization and formulation optimization of solid lipid nanoparticles in vitamin K1 delivery

Background: Solid lipid nanoparticle (SLN) systems have been applied to various drugs and delivery routes. Vitamin K1 is an important cofactor for maintaining hemostasis and preventing hemorrhage. Method: Vitamin K1-loaded SLNs are systematically being developed by optimizing triglycerides and lipophilic and hydrophilic surfactants based on the size and stability of the resulting SLNs. Concentrations of the surfactants, Myverol and Pluronic, were optimized by a central composite design and response surface methodology. Vitamin K1 (phylloquinone) was used as a lipophilic drug in the SLN system to evaluate the potential for oral delivery. Results: Vitamin K1-loaded SLNs had a mean size of 125 nm and a zeta potential of −23 mV as measured by photon correlation spectroscopy. The prepared SLNs were examined by differential scanning calorimetry and transmission electron microscopy and found to have an imperfect crystalline lattice and a spherical morphology. Effects of ultrasonication duration and drug load on the particle size and entrapment efficiency of the SLNs were also evaluated. Conclusion: More than 85% of the vitamin K1 was entrapped in SLNs when the payload was <5%. The vitamin K1 in SLNs was stable for a 54-h duration in simulated gastric and intestinal fluids. The particle size and vitamin K1 entrapped in the SLN were stable after 4 months of storage at 25°C. The results demonstrated that SLNs prepared herein can potentially be exploited as carriers for the oral delivery of vitamin K1.

Novel lutein loaded lipid nanoparticles on porcine corneal distribution.

Topical delivery has the advantages including being user friendly and cost effective. Development of topical delivery carriers for lutein is becoming an important issue for the ocular drug delivery. Quantification of the partition coefficient of drug in the ocular tissue is the first step for the evaluation of delivery efficacy. The objectives of this study were to evaluate the effects of lipid nanoparticles and cyclodextrin (CD) on the corneal lutein accumulation and to measure the partition coefficients in the porcine cornea. Lipid nanoparticles combined with 2% HPβCD could enhance lutein accumulation up to 209.2 ± 18 (μg/g) which is 4.9-fold higher than that of the nanoparticles. CD combined nanoparticles have 68% of drug loading efficiency and lower cytotoxicity in the bovine cornea cells. From the confocal images, this improvement is due to the increased partitioning of lutein to the corneal epithelium by CD in the lipid nanoparticles. The novel lipid nanoparticles could not only improve the stability and entrapment efficacy of lutein but also enhance the lutein accumulation and partition in the cornea. Additionally the corneal accumulation of lutein was further enhanced by increasing the lutein payload in the vehicles.

Acridine orange coated magnetic nanoparticles for nucleus labeling and DNA adsorption.

The magnetic-fluorescent nanoparticles are the integration of fluorophores and magnetic nanoparticles (MNP), which are superior to traditional single-modal nanoparticles. Here, we develop magnetic nanoparticles functionalized by acridine orange (ACO) for labeling nucleus and separating DNA. The ACO, a cell-permeant nucleic acid binding dye, is conjugated with amine on magnetic nanoparticles by glutaraldehyde-mediated coupling and characterized by TEM and FT-IR. Fluorescence spectroscopy, INCell analyzer, and confocal microscopy analyses confirmed the fluorescent property of ACO modified MNP. Furthermore, the modified magnetic nanoparticles showed strong intracellular fluorescence when incubated with 293T cells for a short period of time. The adsorption capacities measured at various concentrations showed enhanced adsorption capacities for double or single stranded DNA when compared to amine MNP conjugated with glutaraldehyde. The cell viability tests of the composite nanoparticles on 293T cells showed low cytotoxicity indicating the safeness of the nanoparticles. The modified magnetic nanoparticles pave a versatile platform for biological applications such as cell labeling and DNA adsorption.

In Vitro Scleral Lutein Distribution by Cyclodextrin Containing Nanoemulsions

Lutein is a macular pigment that contributes to maintaining eye health. The development of lutein-laden nanocarriers for ocular delivery would have the advantages of user friendliness and cost-effectiveness. Nano-scaled vehicles such as cyclodextrin (CD) and nanoemulsion could overcome the barriers caused by the scleral structure. This study focused on the development of hybrid nanocarriers containing nanoemulsion and CD for scleral lutein accumulation. In the presence of the nanoemulsion, CD forms such as βCD and hydroxyethyl (HE) βCD increased the partition of lutein into the porcine sclera. A combination of nanoemulsion and 2% HEβCD enhanced lutein accumulation to 119±6 µg g(-1) h(-1), which was 9.2-fold higher than that with lutein suspension alone. We explored the dose effect of CD in nanoemulsion on scleral lutein and found that the scleral accumulation of lutein was enhanced by increasing the CD content. The novel nanoemulsion had 95% drug-loading efficiency and low cytotoxicity in retinal cells. The CD-modified nanoemulsion not only improved the stability and entrapment efficacy of lutein in the aqueous system but also enhanced scleral lutein accumulation. An increase in the partition coefficient of lutein in porcine sclera when using the CD-modified nanoemulsion was also confirmed.

Adsorption of Nattokinase by Amino Acid-Conjugated Magnetic Nanoadsorbents

Purified nattokinase has the potential to be developed as functional food additives and drugs to prevent or treat several diseases. Arginine modified magnetic nanoparticles to isolate nattokinase from Natto broth were developed in this study. Functional nanoparticles were prepared by covalently conjugating cationic ligands onto the surface via a carbonyldiimidazole coupling method. The arginine-modified magnetic nanoparticles demonstrated the 2.4-fold adsorption of nattokinase compared with the lysine modified nanoparticles. The behavior of the adsorption and desorption for nattokinase onto surface-modified magnetic nanoadsorbents from the natto solution was systematically investigated. The results of transmission electron microscopy indicated that surface modification on nanoparticles did not affect size distribution of magnetic nanoadsorbents. The adsorption kinetics was fitted by the pseudo-first-order and pseudo-second-order equations. It was found that the pseudo-first-order rate equation was the best one for characterizing the adsorption kinetics. In this study the purification factor, the maximum adsorption capacity, and recovery yield were 8.0, 3.79 U/mg nanoparticles, and 15%, respectively. Overall, our results demonstrated that the arginine-coated magnetic nanoadsorbents could efficiently adsorb nattokinase from the Natto broth.

Plasmin Treatment Accelerates Vascular Endothelial Growth Factor Clearance from Rabbit Eyes

PURPOSE To investigate the clearance of vascular endothelial growth factor (VEGF) after the induction of posterior vitreous detachment by plasmin and/or SF(6). METHODS The study design included four groups of rabbits: group 1 received an intravitreal injection of plasmin and SF(6) in the right eye, group 2 received an intravitreal injection of plasmin in the right eye, group 3 received an intravitreal injection of SF(6) in the right eye, and group 4 received an intravitreal injection of balanced salt solution in the right eye. Intravitreal injection of human VEGF (50 μL, 10 ng/μL) was performed in study eyes and control eyes 1 month after plasmin and/or SF(6) injection. Serum and vitreous samples were collected on days 1, 3, and 7 after VEGF injection to determine the serum and vitreous concentrations of VEGF. RESULTS One day after VEGF injection, residual human VEGF concentration in the vitreous cavity was significantly lower in the plasmin- and SF(6)-treated eyes (group 1) and the plasmin-treated eyes (group 2) when compared with the control eyes (group 4) (P = 0.047 and 0.027, respectively). Three days after VEGF injection, the residual VEGF concentration in the vitreous cavity was still significantly lower in the plasmin- and SF(6)-treated eyes (group 1) when compared with the control eyes (group 4) (P = 0.025). CONCLUSIONS Eyes treated with plasmin exhibit a more rapid clearance of exogenous VEGF than control eyes. This finding suggests a novel treatment for retinopathies associated with vitreous traction and VEGF elevation.

Synergistic effects of basic fibroblast growth factor and insulin on Chinese hamster ovary cells under serum-free conditions.

Chinese hamster ovary (CHO) cells are commonly used in the biotechnology industry to produce recombinant proteins. The development of serum-free media in order to maintain mammalian cell viability and reduce cell death in the absence of serum is highly desirable. Our aim was to find new growth factors that could maintain viability and enhance the proliferation of CHO cells under serum-free conditions. Macrophage colony-stimulating factor (M-CSF)-secreting CHO cells were used as a model cell line to evaluate the stimulatory effects of different compounds, including two growth factors, on cell proliferation. Flow cytometry and fluorescence microscopy were used to analyze the cell cycle and monitor apoptotic nuclei during serum-free culture. Among the tested compounds, insulin and basic fibroblast growth factor (bFGF), each used alone, prevented CHO cells from dying and helped maintain cell viability, but cells were unable to re-enter the cell cycle and proliferate. Simultaneous supplementation with insulin and bFGF synergistically promoted the growth of CHO cells and recombinant M-CSF synthesis in basal DMEM/F12 medium. The mitogenic effects of bFGF and insulin under serum-free conditions were confirmed in the CHO-K1 cell line. These results provide useful information for the design and development of serum-free media as well as for antiapoptotic studies of CHO cells under serum-free conditions.

Enhancement of photodynamic inactivation against Pseudomonas aeruginosa by a nano-carrier approach

As pathogens steadily develop resistance to widely used antibiotics, new methodologies for their efficient inactivation must be developed. Photodynamic therapy is an upcoming technique that provides an alternative option for treating pathogenic infections. The efficiency of photodynamic therapy has been limited by the use of aqueous mediums for dispersing photosensitising agents. Toluidine Blue O (TBO) was chosen for this study as a cationic photosensitiser to inhibit Gram-negative bacterium Pseudomonas aeruginosa. Enhanced delivery of the photosensitiser was ensured by utilising an essential oil-based microemulsion. The efficiency of photodynamic therapy was further improved by the use of a chemical penetration enhancer to improve permeability of the bacterial outer membrane. TBO accumulation patterns in neonate pig skin were studied using confocal laser scanning microscopy. The physicochemical properties of the TBO loaded microemulsion, including UV-vis absorbance, size distribution and zeta potential, were analysed to understand the enhanced antimicrobial activity. Confocal laser scanning microscopy confirmed the formation of a TBO reservoir in the skin by the TBO-loaded microemulsions. TBO (5 μg/mL) in the vehicles significantly inhibited the growth of P. aeruginosa. All these efforts resulted in inhibition obtained at a drug concentration and light intensity much lower than what is reported by the works of previous investigators.