The Design of Polymer‐based Nanocarriers for Effective Transdermal Delivery

This study reports a facile and practical means to non‐invasively deliver biologically active ingredients through the skin using polymer‐based nanocarriers. For this, polymer nanocapsules were fabricated with different surface charges as well as glass transition temperatures and we observed their ability to deliver the encapsulated active ingredient, coenzyme Q10, through the skin layer. Direct imaging of a probe molecule, Nile Red, and a matrix polymer labeled with fluorescence moiety, Lucifer Yellow, allowed us to demonstrate that the probe molecule readily permeates into the deep skin, while the matrix polymer stays in the stratum corneum layer due to electrostatic interactions. Quantitative characterization of the penetrating amount of coenzyme Q10 using the Frantz cell method proved that, to achieve improved delivery efficiency, the nanocapsule should have a low glass transition temperature as well as positive surface charges.

     

Development of positively charged colloidal drug carriers: Chitosan-coated polyester nanocapsules and submicron-emulsions

 Positively charged colloidal drug carriers have shown interesting properties with respect to the negatively charged systems: they have improved stability in the presence of biological cations and their interaction with negatively charged biological membranes is facilitated. In the present work, a new approach in order to provide a positive charge to colloidal systems, i.e., poly-ɛ-caprolactone (PECL) nanocapsules and submicron emulsions, is presented. This is based on the coating of the colloidal droplets with the cationic polysaccharide chitosan (CS). An experimental factorial design 3³ was used to investigate the influence of several factors (CS viscosity, PECL concentration and lecithin concentration) on the physicochemical properties of the systems. All the formulations displayed a particle size in the nanometer range (200–500 nm) and a high positive surface charge (from +30 up to +60 mV). The statistical analysis of these data (surface response methodology) indicated that both size and surface charge of the nanocapsules and submicron emulsions, were significantly affected by all factors under investigation, the CS viscosity being the most relevant factor. The CS coating of the nanocapsules was found to be efficient in preventing their destabilization in the presence of Ca²⁺. Furthermore, the presence of CS permitted the adequate dispersion of the nano-capsules upon freeze-drying. Finally, using diazepam as model drug, it was observed that the encapsulation efficiency was, in all cases, higher than 90% irrespective of the presence of CS in the preparation. As expected, the diazepam release rate from the nanocapsules and submicron emulsions occurred rapidly and it was slightly slowed down due to the CS coating. These results clearly demonstrated that coating nano-capsules and submicron emulsion with CS increases their potential use as drug delivery systems. Received: 17 May 1996 Accepted: 15 August 1996     

Preparation of (2′-5′)-oligonucleotides based on 6-<Emphasis Type="Italic">N</Emphasis>-benzylaminopurineriboside using the <Emphasis Type="Italic">Spicaria violacea</Emphasis> fungal enzyme complex

A method for chemico-enzymatic synthesis of (2′-5′)-oligonucleotides with 6-N-benzylaminopurineriboside as the nucleoside units was proposed. The method consisted of enzymatic hydrolysis of the oligonucleotides with mixed (2′-5′)-(3′-5′)-phosphodiesterbonds that were prepared by polymerization of 6-N-benzyladenosine-2′(3′)-monophosphate by using (3′-5′)-specific nuclease and phosphatase contained in the filtrate of culture medium of the mycelial fungus Spicaria violacea. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 64–68, January–February, 2009.     

Drug permeation through skin from matrix-type drug delivery systems

Nitroglycerin permeation through hairless mouse skin from various matrix-type transdermal delivery systems has been explained fairly well on the basis of a two-layer skin model. The diffusivity and solubility of the drug in the stratum corneum and in the viable skin have been calculated. The diffusivity in the stratum corneum was found to be affected by the type of delivery system, while that in the viable skin was hardly influenced by the delivery system. The loading dose, diffusivity and solubility of the drug in the delivery system were found to be the important design parameters for skin-control transdermal drug delivery and, therefore, the transdermal delivery system has to be optimized by controlling these three parameters to achieve a maximum rate of skin permeation with minimum drug loading dose.     

Electrically enhanced percutaneous delivery of delta-aminolevulinic acid using electric pulses and a DC potential

Selectivity of photodynamic therapy can be improved with localized photosensitizer delivery, but topical administration is restricted by poor diffusion across the stratum corneum. We used electric pulses to increase transdermal transport of delta-aminolevulinic acid (ALA), a precursor to the photosensitizer protoporphyrin IX (PpIX). ALA-filled electrodes were attached to the surface of excised porcine skin or the dorsal surface of mice. Pulses were administered and, in some in vivo cases, a continuous DC potential (6 V) was concomitantly applied. For in vitro 14C ALA penetration, 10 microm layers parallel to the stratum corneum were assayed by liquid scintillation analysis, and 10 microm cross sections were examined autoradiographically. As the electrical dose (voltage x frequency x pulse width x treatment duration) increased, there was an increase in penetration depth. In vivo delivery was assayed by measuring the fluorescence of PpIX in skin samples. A greater than two-fold enhancement of PpIX production with electroporative delivery was seen versus that obtained with passive delivery. Superimposition of a DC potential resulted in a nearly three-fold enhancement of PpIX production versus passive delivery. Levels were higher than the sum of PpIX detected after pulse-alone and DC-alone delivery. Electroporation and electrophoresis are likely factors in electrically enhanced delivery.     

Development of an efficient transdermal delivery system of small interfering RNA using functional peptides, Tat and AT-1002

Topical use of small interfering RNA (siRNA) as a therapeutic nucleic acid is increasingly studied for the treatment of skin diseases and for the improvement of skin properties. However, naked siRNA transdermal delivery is limited by its low stability in the body and low permeability into target cells. This is due to various skin barriers such as the stratum corneum that has multiple lipid bilayers and epidermal layers that have tight junctions. In this study, we investigate non-invasive transdermal siRNA delivery using two functional peptides: AT1002, which is a tight junction modulator and 6-mer synthetic peptide belonging to a novel class of compounds that reversibly increases paracellular transport of molecules across the epithelial barrier; and Tat, which is a cell-penetrating peptide applicable as a transdermal siRNA delivery enhancer. We examined whether expression of the tight junction protein zonula occludens protein 1 (ZO-1) was detected in mouse skin applied with AT1002. Additionally, siRNA stabilities for RNaseA using Tat and AT1002 were assessed. We also determined the intradermal delivery efficiency of siRNA using functional peptides by confocal laser microscopy of fluorescently labeled siRNA in mouse skin. We found that the Tat analog and AT1002 strongly increased siRNA stability against RNaseA. In addition, ZO-1 disappeared from the skin after treatment with AT1002, yet recovered with time after washing. Finally, we also found that Tat and AT1002 peptides accelerate transdermal siRNA delivery both widely and effectively. Thus, combination of Tat and AT1002 is expected to be a transdermal delivery enhancer of siRNA.     

Dynamic surface tension and dilational viscoelasticity of adsorption layers of alkylated chitosans and surfactant–chitosan complexes

The kinetics of the adsorption at the air-water interface and the processes of the structure formation inside the adsorption layers of hydrophobically modified systems [alkylated chitosans and sodium dodecyl sulfate (SDS)–chitosan (Ch) complexes] have been studied by the tensiometric method based on the axisymmetric rising-bubble-shape analysis as a function of the bulk concentration of polymers and the ageing time of their adsorption layers. The kinetics of the adsorption of chitosan, alkylated chitosans (ChC₃, ChC₈, and ChC₁₂), and surfactant–polyelectrolyte (PE) complexes formed by the chitosan and the polysoaps with oppositely charged anionic surfactant SDS is characterized by an induction time (the so-called lag time), τ_lag, corresponding to the diffusion stage of the formation of adsorption layers. During this time, the decrease in the surface tension (or the increase in the surface pressure π) does not exceed several millinewtons per meter that corresponds to the “gaseous” state of adsorption layers. The postlag stage of the formation of the adsorption layer is characterized by the remarkable rate of increase in the surface pressure π that corresponds to the conformational rearrangement of PEs inside the adsorption layer by increasing the number of hydrophobic groups (adsorbing centres) in contact with the non-polar phase at the interface. It has been found that during the lag time, the adsorption of alkylated chitosans (cationic polysoaps) increases with increasing alkyl chain length, whereas during the postlag time, the adsorption of the ChC₃ is maximal with regard to other polysoaps. It has been confirmed that at equal content of alkyl groups in the system, the surface activity of the SDS–Ch complexes is much higher with regard to that of the polysoaps. The viscoelasticity of adsorption layers of individual PEs and their complexes continuously increases with the ageing time, giving evidence for the interaction between the polymers inside the adsorption layers. It has been found that the rate of increase in the dilational storage module E′ of the adsorption layers of SDS–Ch complexes is much higher than for the polysoaps that correlates with the higher surface activity of the former with regard to the latter. For the mentioned systems, the module E′ is much higher than the loss module E″ that confirms the solid-like properties of their adsorption layers. On the other hand, the adsorption layers of the chitosan are liquid-like, while E′<<E′′.     

立体异构单萜醇油酸酯的制备及促透活性评价

本文以互为立体异构体的香叶醇(GER)、橙花醇(NER)为先导化合物,采用酰氯酯化法合成油酸香叶醇酯(GER-dC18)、油酸橙花醇酯(NER-dC18),并考察GER、NER、GER-dC18、NER-dC18作为促透剂对多奈哌齐(DNP)的促透活性差异.通过体外释放试验、红外光谱法和分子模拟技术初步探究其促透机制,...     

Electrochemical investigations of the reaction mechanism and kinetics between NADH and riboflavin immobilised on amorphous zirconium phosphate

Electrochemical investigations of the reaction mechanism and kinetics between riboflavin immobilised on zirconium phosphate (ZPRib) in carbon paste and NADH showed results yielding reliable information about aspects on the mechanism of the electron transfer reaction between the flavin and NADH. The formal potential (E°′) of the adsorbed riboflavin was −220 mV versus SCE at pH 7.0. A shift about 250 mV towards a more positive potential compared with its value in solution was assigned to the interaction between the basic nitrogen of riboflavin and the acid groups of ZP. The invariance of the E°′ with the pH of the contacting solution and the effect of different buffer constituents were attributed to the protection effect of ZP over the riboflavin. The electrocatalytic oxidation of NADH at the electrode was investigated using cyclic voltammetry and rotating disk electrode methodology using a potential of −50 mV versus SCE. The heterogeneous electron transfer rate constant, k _obs, was 816 M⁻¹ s⁻¹ and the Michaelis-Menten constant, K _M, was 1.8 mM (confirming a charge transfer complex intermediate in the reaction) for an electrode with a riboflavin coverage of 6.8 × 10⁻¹⁰ mol cm⁻². This drastic increase in the reaction rate between NADH and the immobilised riboflavin was assigned to the shift of the E°′. A surprising effect with addition of calcium or magnesium ion to the solution was also observed. The E°′ was shifted to −150 mV versus SCE and the reaction rate for NADH oxidation increased drastically. Received: 22 February 1999 / Accepted: 10 March 1999     

Separation of Cytidine 5′-triphosphate Biosynthesized from Cytidine 5′-monophosphate on Ion-exchange Resin and HPLC Analysis of Cytidine Compounds

Conditions were studied in the biosynthesis of cytidine 5′-triphosphate (CTP) from cytidine 5′-monophosphate (CMP). A 201 × 7 anion ion-exchange resin was applied for the separation of CTP from CMP. Adsorption isotherm and elution conditions (eluant, eluant concentration, flow rate, sample volume loaded) were investigated. At the same time, a new high-performance liquid chromatography on an anion ion-exchange column WAX-1 with UV detector at 260 nm was developed to measure CMP, cytidine 5′-diphosphate (CDP), and CTP. The retention time for CMP, CDP, and CTP are 0.723, 1.448, and 4.432 min, respectively. This new rapid high-performance liquid chromatography (HPLC) method for the analysis of cytidine compounds in biological sample has a wide linear range with high precision and repeatability.     

Dual-labeled visual tracer system for topical drug delivery by nanoparticle-triggered P-glycoprotein silencing

Using nanoparticle-based drug delivery systems as enhancers is a robust strategy for transdermal delivery; however, the mechanisms by which these systems promote transdermal penetration are still unclear. Here, we fabricated a dual-labeled nano drug delivery system that allows discrete visualization of both the drug and the nanoparticle carrier. To comprehensively examine its potential mechanism, we investigated its effects on human epidermal keratinocyte HaCaT cells, including changes in cell membrane potential, intracellular Ca²⁺ concentration, and Ca²⁺-ATPase activity. P-glycoprotein (P-gp) expression in nanoparticle-treated human dermal microvascular endothelial cells was detected by western blotting and immunofluorescence. Furthermore, the transdermal absorption and biodistribution of the dual-labeled nanoparticles were deeply investigated by skin permeability study in vitro and in vivo using fluorescence microscopy and in vivo imaging, respectively. In addition to reducing membrane potential, increasing the intracellular Ca²⁺ concentration, and decreasing Ca²⁺-ATPase activity, our results indicate that the dual-labeled nanoparticles can downregulate P-gp to promote transdermal absorption. Fluorescence and in vivo imaging visually demonstrated that the nanoparticle delivery system penetrated into the dermis through the stratum corneum. All these results indicate that this dual-labeled nano delivery system provides a new method for future in-depth visual explorations of transdermal drug delivery mechanisms.     

Enzyme-catalyzed synthesis of nucleoside triphosphates from nucleoside monophosphates

Syntheses of adenosine 5′-triphosphate (ATP) from adenosine 5′-monophosphate (AMP) and ribavirin 5′-triphosphate (RTP) from ribavirin 5′-monophosphate (RMP) (1) were performed using enzymes as catalysts. Synthesis of ATP is based on acetyl phosphate as the phosphate donor, and acetate kinase (Bacillus stearothermophilus, EC 2.7.2.1), adenylate kinase (porcine muscle, EC 2.7.4.3), and inorganic pyrophosphatase (yeast, EC 2.6.1.1) as the catalysts. Three reactions on a 150-mmol scale provided ATP as its barium salt in 82% yield and 67% purity. Synthesis of RTP used phosphoenol pyruvate (PEP) as the phosphate donor, and pyruvate kinase (rabbit muscle, EC 2.7.1.40) and adenylate kinase (rabbit muscle) as the catalysts. A gram-scale reaction provided RTP as its barium salt in 93% yield and 97% purity. This work demonstrates the utility of the autoxidationresistant acetate kinase fromB. stearothermophilus, the value of pyrophosphatase in controlling the level of pyrophosphate in the reactions and the ability of adenylate kinase to accept at least one substrate other than a derivative of adenosine.     

Chelate Adsorption for Trace Voltammetric Determination of Xanthosine 5′-Monophosphate and Xanthosine 5′-Diphosphate

Square-wave cathodic adsorptive stripping voltammetry based on adsorptive accumulation is a very sensitive technique for the trace determination of xanthosine 5′-monophosphate (5′-XMP) and xanthosine 5′-diphosphate (5′-XDP). The determination is based on the strong interaction of the adsorption of xanthosine phosphate compounds on a mercury electrode surface, forming Hg(II)-xanthate. The cathodic reduction of the accumulated Hg(II)-xanthate complex provides the basis for direct stripping measurements of the investigated biological compounds at subnanomolar concentration levels. Moreover, controlled adsorptive accumulation of the Cu(II) complex of xanthosine phosphate is also reported to assay trace amounts of xanthosine phosphate. The height of the sharp chelate peak of adsorbed Cu(II)-xanthate, coupled with the flat baseline, facilitates measurements at nanomolar and submicromolar concentration levels. The adsorption and the redox behaviour of the investigated complexes are indicated by cyclic voltammetry. Experimental and instrumental parameters for the quantitative determination were optimized. Statistical analysis of the calibration curve data is also included.     

Interaction of Antiseptic Compounds with Intercellular Lipids of Stratum Corneum

The interaction between three kinds of antiseptic compounds and components of intercellular lipids in the stratum corneum was characterized in terms of thermodynamics at pH 7.5 and 25°C, and the different mechanisms used to penetrate the stratum corneum were clarified. Anionic surfactants such as benzalkonium chloride and benzethonium chloride mainly bound to cholesterol (CH) and cholesterol sulfate with high affinity (10⁵–10⁶ M⁻¹) to extract endogenous CH from the stratum corneum and penetrated through the intercellular route. Chlorhexidine gluconate also bound to CH and accumulated in the stratum corneum without removing endogenous CH. An amphoteric surfactant of dodecyldiaminoethylglycine hydrochloride seemed to be incorporated into the lipid bilayer and bound to ceramide with its polar end close to the lipid polar heads by hydrophobic interaction. This revised version was published online in August 2006 with corrections to the Cover Date.     

扫描电子显微镜在鹌鹑蛋壳表征中的应用

利用扫描电子显微镜对鹌鹑蛋蛋壳的横截面、内外表面进行观察,蛋壳由内向外分别是壳膜、乳突层、海绵层、垂直晶体层和角质层. 外表面分布有深褐色、浅黄色的不规则颜色斑点,深褐色区域比浅黄色区域的角质层厚,其差异来自于角质层. 对深褐色区域与浅黄色区域进行外表面能谱分析,深褐色区域的C、O和N元素含量相对高于浅黄色区域,浅黄色区域的Mg、P、S和Ca元素含量相对高于深褐色区域.     

Confirmatory determination of organochlorine pesticides in surface waters using LC/APCI/tandem mass spectrometry⋄

A confirmatory method for the determination of organochlorine pesticides (OCPs) and their metabolites (endrin, α-endosulfan, β-endosulfan, endosulfan sulfate, heptachlor, heptachlor epoxide, 2,4′-DDD, 4,4′-DDD, 2,4′-DDE 4,4′-DDE, 2,4′-DDT, and 4,4′-DDT) in surface waters using liquid chromatography /APCI/tandem mass spectrometry has been developed. Chromatographic separation was carried out on a ChromSpher 5 Pesticide column using a gradient elution with mobile phase 1mM ammonium acetate-acetonitrile. Endrin, α-endosulfan, β-endosulfan , endosulfan sulfate, heptachlor and heptachlor epoxide were determined in the negative ionization mode, while the rest compounds in positive ionization mode. For the identification of the analytes, two multireaction monitoring transitions were selected per compounds except for the heptachlor which selected ion monitoring was used. The linearity of the optimized method ranges after SPE concentration, from 0.009 to 30.60 μgL⁻¹ with correlation coefficients greater than 0.99. The method recovery values varied from 72 to 119 % for the different fortification levels . The developed method was successfully applied to determine OCPs and their metabolites in surface water samples collected near paddy fields in growing season of rice, at year 2005 in Pathumthani province, Thailand. Endosulfan sulfate was detected in five out of seven samples and three of them could be quantitated in the range of 0.31to 0.50 μgL⁻¹.     

Transport processes in responding lipid membranes: a possible mechanism for the pH gradient in the stratum corneum

The "acidic mantle" of the skin surface has been related to several essential functions of the skin, although the origin of the acidity is still obscure. In this paper, we investigate how different transport processes can influence the local proton concentration inside a membrane consisting of oriented lipid bilayers. This system is chosen as a simple model of the extracellular lipids in the upper layer of the skin, the stratum corneum. We present a theoretical model for diffusional transport over the membrane in the presence of an osmotic gradient and a gradient in CO(2), taking into account the influence of these gradients on the lipid structure and the local electrostatics. We are also discussing the complications in applying the concept of pH to the stratum corneum. From this, we make the following conclusions: (i) The definition of pH in the stratum corneum is ambiguous, and thus, all statements regarding pH should always be related to a clear definition. (ii) A natural definition of pH in the stratum corneum can be proposed which takes into account local heterogeneity, local charges, and the fact that the stratum corneum is not in thermodynamical equilibrium. (iii) Diffusive transport across an oriented bilayer stack in the presence of an osmotic gradient and/or a gradient in CO(2) can give rise to a substantial gradient in pH. (iv) The results from the simplified model can be correlated to experimental observations of pH in the stratum corneum.     

二维层状离子型纳米材料在化妆品领域的应用进展

二维层状离子型纳米材料具有独特的结构和界面性能,使其在透皮吸收、防晒、活性物的靶向传递、构建稳定的纳米微胶囊等功效中显示出极大的优势,在化妆品行业中具有良好的市场前景。 该类化合物根据层板电荷不同可分为阳离子型粘土材料和阴离子型水滑石类材料。 基于近年来国内外对二者的研究报道和实验研究成果,本文综述了二维离子型纳米材料的特点、性能及其在化妆品领域的应用历史和发展现状,并进一步展望了该类材料在化妆品领域中的应用前景。     

Microneedle arrays permit enhanced intradermal delivery of a preformed photosensitizer

Silicon microneedle (MN) arrays were used to puncture excised murine and porcine skin in vitro and transdermal and intradermal delivery of meso-tetra ( N -methyl-4-pyridyl) porphine tetra tosylate (TMP) investigated using topical application of a bioadhesive patch containing 19 mg TMP cm⁻². Animal studies, using nude mice, were then conducted to investigate the in vivo performance of the bioadhesive patch following MN puncture of skin. MN puncture significantly enhanced both intradermal and transdermal delivery of TMP in vitro , though the total amounts of drug delivered (25.22% into porcine skin and 0.07% across murine skin) were still quite small in each case. Notwithstanding this, in vivo experiments showed that MN puncture was capable of permitting a prolonged increase in TMP fluorescence at the site of application. Importantly, fluorescence was negligible at distant sites, meaning systemic delivery of the drug was not sufficient to induce TMP accumulation other than at the application site. In this study we have conclusively demonstrated proof of principle; MN puncture allows true intradermal delivery of a preformed photosensitizer in animal skin models in vitro and in vivo . Importantly, transdermal delivery was much reduced in each case. Increasing MN density would allow increased amounts of photosensitizer to be delivered. However, as MNs create aqueous pores in the stratum corneum, a preformed photosensitizer must possess at least some degree of water solubility in order to permit enhanced intradermal delivery in this way. We believe that use of MN array technology in this way has the potential to significantly improve topical photodynamic therapy of skin tumors.     

Fast electron transfer repair of oxidizing OH adduct of dGMP by chlorogenic acid

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