Preparation and Characterization of Collagen–Chitosan–Chondroitin Sulfate Composite Membranes

Collagen (Col)–chitosan (Chi) membrane was modified by a hot dehydrogenation cross-linking method. Carbodiimide was added for further crossing modification. Chondroitin sulfate (CS) was added so that Col–Chi sulfate composite membranes were prepared. The structure of the composite membranes was characterized by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, and its mechanical properties, degradation, and cytotoxicity were characterized. The composite membrane was applied to a full-thickness skin injury in animal experiments performed in rabbits. Strong interactions and good compatibility among Col, Chi, and CS in the composite membrane were present. The good mechanical properties, biocompatibility, digestion resistance, and wound healing promotion of the composite membrane make it a potential wound dressing or skin scaffold for tissue engineering.     

Preparation and Characterization of Oligonucleotides of D- and L-2’ Deoxyuridine

Abstract

The synthesis of oligonucleotides of 2'deoxyuridine containing both the natural D-2'deoxyribose and the unnatural L-2'deoxyribose is described. Units up to the 18-mer have been made via a modified triester procedure and characterized by HPLC.     

Preparation and Physicochemical Characterization of Amoxicillin β-cyclodextrin Complexes

Amoxicillin (AMOX), a penicillin A, belongs to the β-lactam family It is usually the drug of choice within the class because it is better absorbed, following oral administration, than other β-lactam antibiotics. Its β-lactamase degradation might be prevented by using a molecular [AMOX:β-CD] complex. The aim of this work was to prepare complexes using two methods and then characterize interactions between AMOX and the native β-CD. The extent of complexation in solution has been evaluated by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and 2D rotating-frame Overhauser enhancement spectroscopy (2D ROESY). Mass changes (TG), calorimetric effects (DSC), and mass spectrometry (MS) were determined on the same sample under identical conditions using the Skimmer coupling system. Skimmer and infrared spectroscopy (FT-IR) were used to characterize the solid state of the binary system. Complexation of AMOX with β-CD was proven by FT-IR, NMR, DSC, and HPLC. The 2D ROESY spectra did not show any dipolar proton interaction of the AMOX with cyclodextrin. The 1:1 stoichiometry of the complex was obtained by HPLC. The stability constant for AMOX with β-CD was determined to be 1,878 M⁻¹. In the [AMOX:β-CD] complex, the phenyl group is included inside the β-CD, and the ionized carboxyl group on the penam ring forms hydrogen bonds with the secondary hydroxyl groups of another β-CD to keep the complex stable. Preparation methods allowed exactly the same complex.     

Preparation, Characterization and Evaluation of Marsupsin–Phospholipid Complex

The aim of this research was to formulate Marsupsin–phospholipid complex (M–P Complex) in attempt to increase the bioavailability of marsupsin and to characterize this new formulation along with its evaluation. Marsupsin–phospholipid complex was formulated by mechanical dispersion method. In this new formulation, complex formation was confirmed by carrying out transmission electron microscopy (TEM), IR, ¹H-NMR and RP-HPLC analysis. TEM showed M–P Complex diameter range of 0.05–0.5 μm. The entrapment efficiency of M–P Complex was found to be 44%. In vitro release study revealed its first order release profile. Mean blood serum concentration vs time curve of marsupsin was of first order after oral administration of M–P Complex in albino rabbits which clearly showed remarkably increased bioavailability of M–P Complex than standardized marsupsin. The average value of C _max and T _max of M–P Complex were found to be 3.02 mg/ml and 10.2 h, respectively. Hence the findings demonstrate that complexing marsupsin with phospholipids results in better oral bioavailability and improved biological response than free form of standardized marsupsin.     

Preparation and Physicochemical Properties of Catechin/β-cyclodextrin Inclusion Complex Nanoparticles

Nano-size catechin/β-cyclodextrin inclusion complex (CA/β-CD IC) with 1:1 molar ratio was obtained by cooling precipitation at 4 °C. Physicochemical properties of the CA/β-CD IC nanoparticles were characterized. Results of dynamic light scattering and SEM observation showed that CA/β-CD IC molecules underwent a process of assembling and shaping nano-size particles. In the range of 10–14 mM, the higher the concentration of β-CD aqueous solution, the faster the CA/β-CD IC nanoparticles form and the larger the size of the nanoparticles (195.2–438.6 nm). The total recovery, inclusion ratio and loading capacity of the CA/β-CD IC nanoparticles were determined. Results of FT-IR and DSC indicated that stability of CA was enhanced after it was embedded into β-CD cavity. XRD results showed that the strongest three diffraction peaks (located at 2θ = 10.6°, 12.4° and 19.6°) of the CA/β-CD IC nanoparticles was different from that (located at 2θ = 6.6°, 11.7° and 17.7°) of micro-size CA/β-CD IC and the nanoparticles obtained from higher concentration solution possessed higher crystallinity.

     

Preparation and Characterization of Stable pH-Sensitive Vesicles Composed of α-Tocopherol Hemisuccinate

The current study aims to develop a stable pH-sensitive drug delivery system. First, cleavable polyethylene glycol-α-tocopherol hemisuccinate (PEG-THS) was synthesized. Conventional pH-sensitive vesicles composed of the Tris salt of α-tocopherol hemisuccinate (THST) were then prepared using the detergent removal technique. The vesicles had a mean particle size of (163.8 ± 5.5) nm and a zeta potential of −74.5 ± 6.4 mV. The THST vesicles were then modified using PEG-THS or uncleavable PEG-cholesterol (PEG-CHOL) (THST/PEG-lipids, 100:6 molar ratio). The mean vesicle particle size and absolute zeta potential decreased with increasing PEG-THS proportion. When the pH was decreased, the vesicle particle size and calcein release rate increased. The THST vesicles were initially Ca²⁺-unstable but exhibited significantly improved stability after modification with PEG-THS, especially at PEG-lipid ratios above 6%. Incubation in an acid serum increased the calcein release rate of conventional THST vesicles to 45 ± 1.98% at 10 min. However, the release rate of the PEG-CHOL vesicles remained low. The calcein release rate of PEG-THS vesicles was between those of conventional and PEG-CHOL-V. Therefore, PEG-THS can protect vesicles in serum and reconstitute their pH sensitivity in acidic conditions. Cleavable PEG-THS can be used in stable pH-sensitive preparations without loss of pH sensitivity. Free calcein and conventional vesicles eliminated from the plasma soon after injection, as well as the half-life (t_1/2) and area under the curve of PEG-THS-V encapsulating calcein, were dramatically increased. This phenomenon indicates that the use of PEG-lipid derivatives has gained a favorably long circulation effect in mice.Key words: cleavage, long circulation, PEG-α-tocopherol hemisuccinate, pH-sensitive, vesicles     

Preparation and Enzymatic Hydrolysis of Maltosyl-α-cyclodextrin

Maltosyl-α-cyclodextrin (6-α-maltosylcyclomaltohexaose, M-CD) was prepared from maltose and α-cyclodextrin by the reverse action of Bacillus pullulanase, and the action of α-amylases on this dextrin was examined. Among α-amylases tested, Thermoactinomyces vulgaris α-amylase (TVA) and Taka-amylase A (TAA) were found to attack the M-CD. Their action pattern on M-CD was studied. These α-amylases cleaved, first the cyclodextrin ring of M-CD, and the branched octasaccharides formed were immediately degraded to form glucose, branched tetraose, or pentaose, though the action pattern was different for TVA and TAA. In addition, TAA also split M-CD into glucose and glucosyl-α-cyclodextrin. Fission products at various stages of the reaction were separated and analyzed by paper chromatography and high performance liquid chromatography, their structures were analyzed, and the degradation pattern of M-CD was found.     

Preparation,Characterization and Reactivity of Diastereomeric Sulfoxides of 8,2′-S-Anhydroadenosine

Abstract

The oxidation of 8,2′-S-anhydroadenosine (1a) has been investigated. The major product from the oxidation of 1a using 1-chlorobenzotriazole was the R-sulfoxide. The oxidation of 3′,5′-di-O-acetyl-8,2′-S-anhydroadenosine (1b) gave predominately the S-sulfoxide. These sulfoxides were found to be very succeptible to nucleophilic attack at C-8.     

Preparation and Solid-State Characterization of Inclusion Complexes Formed Between Miconazole and Methyl-β-Cyclodextrin

The aim of this study is to confirm the formation of inclusion complexes between miconazole (MCZ) and two derivatives of beta-cyclodextrin, methyl-beta-cyclodextrin (MβCD) and 2-hydroxypropyl-beta-cyclodextrin (HPβCD) in aqueous solution by phase solubility studies. Inclusion complexes with MβCD in the solid state were then prepared by different methods, i.e., kneading, coevaporation (COE), spray-drying (SD), and lyophilization (LPh). The physicochemical properties of these complexes were subsequently studied by means of differential scanning calorimetry, Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction techniques. Phase solubility diagrams with MβCD and HPβCD were classified as A_P type, indicating the formation of 1:1 and 1:2 stoichiometric inclusion complexes. The apparent stability constants (K_S) calculated from the phase solubility diagram were 145.69 M⁻¹ (K _1:1) and 11.11 M⁻¹ (K _1:2) for MβCD and 126.94 M⁻¹ (K _1:1) and 2.20 M⁻¹ (K _1:2) for HPβCD. The method of preparation of the inclusion complexes in the solid state was shown to greatly affect the properties of the formed complex. Hence, the LPh, SD, and COE methods produce true inclusion complexes between MCZ and MβCD. In contrast, crystalline drug was still clearly detectable in the kneaded (KN) product.     

Preparation and Characterization of Gas-filled Liposomes: Can They Improve Oil Recovery?

Although well known for delivering various pharmaceutical agents, liposomes can be prepared to entrap gas rather than aqueous media and have the potential to be used as pressure probes in magnetic resonance imaging (MRI). Using these gas-filled liposomes (GFL) as tracers, MRI imaging of pressure regions of a fluid flowing through a porous medium could be established. This knowledge can be exploited to enhance recovery of oil from the porous rock regions within oil fields. In the preliminary studies, we have optimized the lipid composition of GFL prepared using a simple homogenization technique and investigated key physico-chemical characteristics (size and the physical stability) and their efficacy as pressure probes. In contrast to the liposomes possessing an aqueous core which are prepared at temperatures above their phase transition temperature (T_c), homogenization of the phospholipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocoline (DSPC) in aqueous medium below their T_c was found to be crucial in formation of stable GFL. DSPC based preparations yielded a GFL volume of more than five times compared to their DPPC counter part. Although the initial vesicle sizes of both DSPC and DPPC based GFL were about 10 μm, after 7 days storage at 25°C, the vesicle sizes of both formulations significantly (p < 0.05) increased to 28.3 ± 0.3 μm and 12.3 ± 1.0 μm, respectively. When the DPPC preparation was supplemented with cholesterol at a 1:0.5 or 1:1 molar ratio, significantly (p < 0.05) larger vesicles were formed (12–13 μm), however, compared to DPPC only vesicles, both cholesterol supplemented formulations displayed enhanced stability on storage indicating a stabilizing effect of cholesterol on these gas-filled vesicles. In order to induce surface charge on the GFL, DPPC and cholesterol (1: 0.5 molar ratio) liposomes were supplemented with a cationic surfactant, stearylamine, at a molar ratio of 0.25 or 0.125. Interestingly, the ζ potential values remained around neutrality at both stearylamine ratios suggesting the cationic surfactant was not incorporated within the bilayers of the GFL. Microscopic analysis of GFL confirmed the presence of spherical structures with a size distribution between 1–8 μm. This study has identified that DSPC based GFL in aqueous medium dispersed in 2% w/v methyl cellulose although yielded higher vesicle sizes over time were most stable under high pressures exerted in MRI.     

Purification and Characterization of l-Leucine-α-ketoglutarate Transaminase from Acetobader suboxydans

l-Leucine-α-ketoglutarate (α-KGA) transaminase from Acetobacter suboxydans was purified to the state of homogeneity by the criteria of ultracentrifugation and electrophoresis on a cellulose acetate membrane. The molecular weight was about 80,000 and one mole of pyridoxal 5′-phosphate was bound per mole of enzyme as a coenzyme. The enzyme exhibited absorption maxima at 280, 337 and 414 nm.

The branched-chain amino acids and α-KGA were specific as amino donors and an acceptor. l-Leucine-α-KGA transaminase is suggested to correspond to the enzyme so-called Transaminase B.     

Interferon-Tau Attenuates Uptake of Nanoparticles and Secretion of Interleukin-1β in Macrophages

Background

Type I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1β, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1β secretion in human THP-1 macrophages.

Methods and Results

IFNT dose-dependently inhibited IL-1β secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1β and mature IL-1β. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1β mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1β secretion was restored by anti-IL-10 neutralizing antibody.

Conclusions

Ruminant IFNT inhibits NLRP3 inflammasome-driven IL-1β secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1β induction. It may be a therapeutic alternative to IFNA and IFNB.     

Isolation and Characterization of 2′-amino-modified RNA Aptamersfor Human TNFα

Human tumor necrosis factor α (hTNFα), a pleiotropic cytokine with activities ranging from host defense mechanisms in infection and injury to severe toxicity in septic shock or other related diseases, is a promising target for drug screening. Using the SELEX (systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotide ligands (aptamers) with high affinities for hTNFα.Aptamers were selected from a starting pool of 40 randomized sequences composed of about 10^15 RNA molecules. Representative aptamers were truncated to the minimal length with high affinity for hTNFα and were further modified by replacement of 2′-OH with 2′-F and 2′-NH2 at all ribopurine positions. These modified RNA aptamers were resistant to nuclease. The specificity of these aptamers for hTNFα was confirmed, and their activity to inhibit the cytotoxicity of hTNFα on mouse L929 cells was determined. Results demonstrated that four 2′-NH2-modified aptamers bound to hTNFα with high affinity and blocked the binding of hTNFα to its receptor, thus protecting the L929 cells from the cytotoxicity of hTNFα. Oligonucleotide aptamers described here are potential therapeutics and diagnostics for hTNFα-related diseases.     

In vivo processing of CXCL12α/SDF-1α after intravenous and subcutaneous administration to mice

CXCL12α has been shown to be selectively processed at the N‐ and C‐termini in blood and plasma in vitro. In order to study the processing in vivo, several versions of CXCL12α were expressed and purified. The protein was administered either iv or sc to mice, and at different time points postadministration plasma was collected and analyzed. To detect modifications of the CXCL12α molecule in crude plasma a SELDI TOF‐MS‐based method was developed. Anti‐CXCL12 antibodies were immobilized on the SELDI chip and CXCL12α binding to the antibodies was detected by SELDI‐TOF‐MS. The protein was found to be processed both at the C‐ and N‐termini. The same processed CXCL12α forms as detected in vitro were found; however, in addition further processing was detected at the N‐terminus, where altogether seven amino acids were removed. At the C‐terminus the lysine was removed as has been seen in vitro, and no further processing was detected. The full‐length CXCL12α disappeared within minutes after administration, whereas the processed forms of the protein were detectable for up to 6–8 h postadministration. The same processed forms appeared after iv and sc administration, only the kinetics was different. When the shortest processed form detected in plasma, 7ΔN1ΔC‐CXCL12α, was administered directly, no further processed forms were detected. Interestingly, a version of CXCL12α containing a N‐terminal methionine was protected against N‐terminal processing in plasma in vitro; however, in vivo no protection was seen, the protein was processed in the same way as full‐length CXCL12α.     

Isolation and Characterization of 2'-amino-modified RNA Aptamers for Human TNFα

Human tumor necrosis factor a (hTNFa), a pleiotropic cytokine with activities ranging from host defense mechanisms in infection and injury to severe toxicity in septic shock or other related diseases, is a promising target for drug screening. Using the SELEX (systematic evolution of ligands by exponential enrichment) process, we isolated oligonucleotide ligands (aptamers) with high affinities for hTNFa. Aptamers were selected from a starting pool of 40 randomized sequences composed of about 1015 RNA molecules. Representative aptamers were truncated to the minimal length with high affinity for hTNFa and were further modified by replacement of 2'-OH with 2'-F and 2'-NH2 at all ribopurine positions. These modified RNA aptamers were resistant to nuclease. The specificity of these aptamers for hTNFa was confirmed, and their activity to inhibit the cytotoxicity of hTNFa on mouse L929 cells was determined. Results demonstrated that four 2'-NH2-modified aptamers bound to hTNFa with high affinity and blocked the     

SD大鼠SDF-1α基因克隆与序列分析

目的克隆SD大鼠SDF-1α基因,并进行生物信息学分析。方法采用RT-PCR扩增SDF-1α基因,对扩增产物进行序列的测定,利用Internet和GenBank数据库对测序结果正确的SDF-1α基因进行生物信息学分析。结果克隆了SDF-1α基因,测序结果正确;生物信息学分析表明,SDF-1α基因开放读码为270bp,编码89个氨基酸;同源性分析表明,与人、猫、狗的SDF-1α基因同源性最大。多种组织的半定量RT-PCR研究表明,该基因在心脏、肝脏、肺、脑、肾脏、脾脏、胰脏均有表达,其中脾脏、胰脏表达较高。结论成功克隆SDF-1α基因,对其功能进行了初步预测,为进一步的功能研究打下基础。     

A Simple Method for Preparation of 18α-Glycyrrhetinic Acid and Its Derivatives

Treatment of 18-glycyrrhizic acid with a methanolic solution of HCl resulted in 1 : 1 mixture of methyl esters of 18- and 18-glycyrrhetinic acids. Benzoylation of the mixture led to methyl esters of 3-benzoyl-18-glycyrrhetinic acid and 3-benzoyl-18-glycyrrhetinic acid, which were separated by chromatography on silica gel. 18-Glycyrrhetinic acid was prepared by alkaline hydrolysis of methyl 3-benzoyl-18-glycyrrhetinate and was further used for the syntheses of 3-keto-18-glycyrrhetinic acid and methyl esters of 18-glycyrrhetinic acid and 3-keto-18-glycyrrhetinic acid.     

E-Selectin Mediated Adhesion and Migration of Endothelial Colony Forming Cells Is Enhanced by SDF-1α/CXCR4

Objective

Endothelial-colony forming cells (ECFCs) can be readily expanded from human umbilical cord blood and can facilitate repair of endothelial injury. E-selectin and SDF-1α are produced following endothelial injury and can regulate endothelial progenitor homing. Mechanisms of vascular repair specific to the mode of injury have not been well described in homogenous cell populations such as ECFCs and are needed for development of more effective vascular repair strategies.

Methods and Results

Lipopolysaccharide (LPS)-induced endotoxic injury to mature human umbilical vein endothelial cells (HUVEC) was compared with hypoxic and radiation injury. E-selectin expression in HUVEC cells is markedly increased (208-fold) following LPS-induced injury and facilitates increased ECFC adhesion and migration function in vitro. SDF-1α expression remains unchanged in LPS-treated HUVEC cells but increases more than 2 fold in fibroblasts undergoing similar endotoxic injury. SDF-1α induces expression of E-selectin ligands on ECFCs and facilitates greater E-selectin-mediated adhesion and migration of ECFCs in a CXCR4-dependent manner. Induction of E-selectin expression in HUVECs following hypoxic or radiation injury is negligible, however, while SDF-1α is increased markedly following hypoxia, highlighting injury-specific synergism between mediators of vascular repair.

Conclusion

E-selectin mediates adhesion and migration of ECFCs following endotoxic endothelial injury. SDF-1α augments E-selectin mediated ECFC adhesion and migration in a CXCR4-dependent manner.     

6-Mercaptopurine and Daunorubicin Double Drug Liposomes—Preparation,Drug-Drug Interaction and Characterization

This article addresses and investigates the dual incorporation of daunorubicin (DR) and 6-mercaptopurine (6-MP) in liposomes for better chemotherapy. These drugs are potential candidates for interaction due to the quinone (H acceptor) and hydroxyl (H donor) groups on DR and 6-MP, respectively. Interactions between the two drugs in solution were monitored by UV/Vis and fluorescence spectroscopy. Interaction between the two drugs inside the liposomes was evaluated by HPLC (for 6-MP) and by fluorescence spectroscopy (for daunorubicin) after phospholipase-mediated liposome lysis. Our results provide evidence for the lack of interaction between the two drugs in solution and in liposomes. The entrapment efficiencies of 6-MP in the neutral Phosphatidyl choline (PC):Cholesterol (Chol):: 2:1 and anionic PC:Chol:Cardiolipin (CL) :: 4:5:1 single and double drug liposomes were found to be 0.4% and 1.5% (on average), respectively. The entrapment efficiencies of DR in the neutral and anionic double drug liposomes were found to be 55% and 31%, respectively. The corresponding entrapment of daunorubicin in the single drug liposomes was found to be 62% on average. Our thin layer chromatography (TLC) and transmission electron microscopy (TEM) results suggest stability of lipid and liposomes, thus pointing plausible existence of double drug liposomes. Cytotoxicity experiments were performed by using both single drug and double drug liposomes. By comparing the results of phase contrast and fluorescence microscopy, it was observed that the double drug liposomes were internalized in the jurkat and Hut78 (highly resistant cell line) leukemia cells as viewed by the fluorescence of daunorubicin. The cytotoxicity was dose dependent and had shown a synergistic effect when double drug liposome was used.