Monoolein cubic phase including in situ ionically gelled alginate and its salt-responsive release property

Salt-responsive monoolein (MO) cubic phase was prepared by in situ ionically gelling alginate contained in its water channels. On the TEM micrographs, bilayers, and water channels, characteristic of MO cubic phase were observed, and alginate and CaCl₂ had little effect on the structure. According to the differential scanning calorimetric thermogram, the cubic-to-hexagonal phase transition temperature of the cubic phase containing CaCl₂ solution was 46.8°C and it was much lower than that of the cubic phase containing distilled water, 60.5°C. The transition temperature was not significantly affected by alginate. The phase transition temperatures measured by the calorimetric analysis were in accordance with those determined by polarized optical microscopy. An initial burst release of dye (i.e., amaranth) was observed when the gelled alginate was not contained in the water channel of the cubic phase. A sustained release was obtained with the cubic phase containing the gelled alginate. The release of dye loaded in the cubic phase containing the gelled alginate was significantly promoted when the cubic phase came into contact with PBS (10?mM, pH 7.4), possibly because the multivalent cation (Ca²⁺) bound to alginate chains could be replaced by the monovalent cation (Na⁺).     

Monoolein cubic phase containing disulfide proteinoid and its reduction-responsive release property

Monoolein (MO) cubic phase, which can release its payload in answering to reducing condition, was prepared by including a disulfide proteinoid composed of Asp, DL-leucine (Leu), and dithiodipropionic acid (DTPA) in its water channel. On the TEM micrograph, the cubic phase could accommodate the proteinoid with no change in its bilayer structure. The phase transition temperature of the cubic phase was about 58.7°C, and it was little affected by the proteinoid. The release degree of allura red loaded in the cubic phase was higher at a higher dithiothreitol (DTT) concentration. The proteinoid would be broken down by the reducing agent, facilitating the release.     

Monoolein cubic phase containing azobenzene and its UV/visible light irradiation-dependent release property

Monoolein (MO) cubic phase, whose MO/azobenzene mass ratios were 1:0, 1:0.0025, 1:0.005, and 1:0.025, was prepared by a melt-hydration method. According to the polarized optical micrographs and the differential thermograms, the phase transition temperature of the cubic phase was lower as the content of azobenzene was higher, and it decreased upon the subsequent irradiation of UV light for 1 hour and visible light for 1 hour. The photoirradiation significantly promoted the release of methylene blue (a water-soluble dye) loaded in the cubic phase only when the MO/azobenzene mass ratio was 1:0.025. The photoirradiation could promote the release of Nile red (an oil-soluble dye) even when the MO/azobenzene mass ratio was less than 1:0.025. The higher photo-susceptibility of Nile red release was possibly because the dye would be intercalated in the MO lipid matrix and the photoirradiation could affect the lipid matrix property.     

大茴香醛改性海藻酸钠凝胶的制备及释药性能

在酸催化下使海藻酸钠与对甲氧基苯甲醛(又名大茴香醛)发生缩醛化反应,使其疏水改性,并将其制备成凝胶。利用红外(FTIR)、荧光、透射电镜(TEM)、扫描电镜(SEM)、紫外、热重分析(TGA)对产物进行了表征。结果表明,大茴香醛成功地与海藻酸钠发生了反应。该凝胶可以作为药物载体对牛血清白蛋白进行包埋释放,结果发现,改性后的产物其载药率和缓释性能比未改性的海藻酸钠有了一定的提高。     

Chemical characterization of recombinant human epidermal growth factor

Human epidermal growth factor (EGF) produced from yeast and purified by ion-exchange and gel filtration chromatography was analysed by reversed-phase liquid chromatography (RP-LC) and showed two main peaks (EGF-1 and EGF-2) with different relative yields from batch to batch and similar biological activities (radioreceptor analysis). Each component isolated by RP-LC was reduced, carboxymethylated and digested with endopeptidase Lys-C and endopeptidase Glu-C separately. Each digest was analysed by fast atom bombardment mass spectrometry (FAB-MS) without further purification. It was verified that both EGF-1 and EGF-2 have the same amino acid sequence of human EGF except at the C-terminal end. EGF-2 lacks the C-terminal arginine and EGF-1 lacks the C-terminal leucine- arginine residues. The location of the SS bonds was also verified by FAB-MS of the peptide mixture obtained by 2% acetic acid hydrolysis. The fermentation and purification processes were followed by RP-LC and FAB-MS and allowed the amount of EGF-1 to be reduced to 10–15%, but intact EGF was never detected.     

Tripolyphosphate-responsive release property of monoolein cubic phase containing sodium dodecyl sulfate and oligo chitosan

Tripolyphosphate (TPP)-responsive MO cubic phase was prepared by immobilizing oligo chitosan in the water channel through its electrostatic attraction with sodium dodecyl sulfate (SDS). The phase transition temperature (PTT) increased with increasing the content of SDS. The PTT of cubic phase whose SDS content was 0%, 0.21%, 0.42%, 0.84%, and 1.68%, determined by polarized microscopy, was about 69.5°C, 72°C, 75°C, 80.5°C, and 95°C, respectively. The PTT did not markedly deviate from that determined by differential scanning calorimetry. The release degree for 72 h of dye (i.e., amaranth and methylene blue) was dependent on the pH value of release medium (pH 3.0 and pH 7.0). Moreover, the release degree significantly increased when the TPP concentration in the release medium increased to 0.4% (w/v). Oligo chitosan was electrostatically complexed with TPP and the complexation took place extensively at the oligo chitosan/TTP mass ratio of 1:0.125 and 1:0.25 and at the oligo chitosan concentration of 1.6% (w/v), evidenced by optical spectroscopy and scanning electron microscopy. It was thought that the complexation was responsible for the TPP concentration-dependent release.     

Effect of tris(hydroxymethyl) aminomethane on the phase behavior of poly(ethylene imine)/cinnamic acid conjugate and the release property of cubic phase containing the conjugate

The upper critical solution temperature (UCST) of poly(ethylene imine)/cinnamic acid (PEI/CA) conjugate decreased as the tris(hydroxymethyl) aminomethane (THMAM) concentration increased. On the optical micrographs of PEI/CA mixture solutions at 25°C, the microspheres were found when the THMAM concentration was 0?mM and 100?mM, but hardly found at 200?mM. Monoolein (MO) cubic phase containing PEI, CA, and THMAM exhibited a bilayer structure on the TEM micrograph. The release degree of methylene blue loaded in the cubic phase was not strongly dependent on THMAM concentration at 25°C and 37°C, but strongly dependent on the concentration at 50°C.     

The controlled release behavior and pH‐ and thermo‐sensitivity of alginate/poly(vinyl alcohol) blended hydrogels

Poly(vinyl alcohol) (PVA) was blended with sodium alginate (Alg) in various ratios and crosslinked with calcium chloride and made into hydrogel membranes. The dependence of the swelling behavior of these Alg‐Ca/PVA hydrogels on pH was investigated. The temperature‐dependent swelling behavior of the semi‐interpenetrating network (semi‐IPN) hydrogels was examined at temperatures from 2 to 45°C and the enthalpy of mixing (ΔH_mix) was determined at various temperatures. The molecular structure of the hydrogels was studied by infrared spectroscopy and their water structure in the semi‐IPN hydrogels was measured by differential scanning calorimetry (DSC). The influence of Ca²⁺ content on the network structure of Alg‐Ca/PVA hydrogels was investigated in terms of the compressive elastic modulus, effective crosslinking density, and the polymer–solvent interaction parameter based on the Flory theory. The loading of alizarin red S (ARS) followed the Langmuir isotherm mechanism and the release kinetics of ARS from the Alg‐Ca/PVA hydrogels followed the Fickian diffusion mechanism. Copyright © 2008 John Wiley & Sons, Ltd.     

Hydrophobically modified alginate for extended release of pharmaceuticals

Hydrophobically modified alginate hydrogels have great potential in drug delivery as they are biologically compatible and cost efficient. While previous works have shown successful protein, and hydrophobic and hydrophilic drug delivery, little information regarding the relationship between crosslinker density and drug release rate is known. This paper investigates the impact of crosslinker density and hydrophobic degree of substitution within modified alginate gels and solutions on the release kinetics using model hydrophobic drug, sulindac. Near zero‐order release was obtained for an extended period of 5 days. Drug release rates decreased as the crosslinker density within both modified alginate hydrogels and solutions increased. Release data fit well to a simplified Fickian relationship, suggesting that the release mechanism is diffusion‐limited. These release characteristics also correlate with bulk rheological measurements, indicating a strong interrelationship between the mechanical properties and the drug release characteristics of the hydrogels.     

Immobilized glucose-6-phosphate dehydrogenase as a substrate for solubilized epidermal growth factor receptor tyrosine kinase

Based on our previously reported solution assay protocol, a solid-phase assay for the tyrosine kinase activity of the epidermal growth factor receptor has been developed. Glucose-6-phosphate dehydrogenase, immobilized noncovalently on microtiter plates, was used as the substrate in the solid-phase assay. Phosphorylation of the immobilized substrate takes place in the presence of ATP and a solubilized epidermal growth factor receptor preparation. After washing off the soluble reaction mixture, the phosphotyrosine-containing dehydrogenase produced on the well surface is quantitated by an ELISA method using a polyclonal antiphosphotyrosine antibody, a second antibody conjugated with horseradish peroxidase, and finally theo-phenylenediamine reaction. The absorbance at 492 nm developed in the wells is a measure of the kinase activity of the solubilized receptor preparation. Putative inhibitors of receptor kinase can be conveniently incorporated in this assay system to test for potential inhibitory activity. This assay, being rapid and convenient, is useful in drug screening programs where a high through-put rate is required.     

Photopolymerization of alicyclic methacrylate hydrogels for controlled release

Alicyclic hydroxy methacrylate monomer, o‐hydroxycyclohexyl methacrylate (HCMA), was synthesized and characterized by Fourier transformed infrared spectroscopy (FT‐IR) and proton nuclear magnetic resonance spectroscopy (¹H‐NMR). Photopolymerization kinetics of HCMA was investigated via real‐time infrared spectroscopy (RT‐IR). Polymeric network hydrogels based on hydroxyethyl methacrylate (HEMA) and HCMA were prepared by using the photopolymerization technique. Mechanical strength, swelling characteristic, and controlled release behavior of hydrogels with various feed compositions were studied. Poly(HEMA‐co‐HCMA) hydrogel had higher storage modulus than that of poly(HEMA) hydrogel as investigated by dynamic mechanical analysis (DMA). Acid orange 8 was used as a model drug for the investigation of drug release behavior of copolymeric hydrogels. Results indicated that increase in HCMA ratio in hydrogel composition could reduce the swelling rate and prolong the release time. Scanning electron microscopy (SEM) was also utilized to study the surface morphology of hydrogels, and the results indicated that HCMA content influenced pore diameter on the hydrogel surface. Copyright © 2008 John Wiley & Sons, Ltd.     

Biodegradable polymer/clay systems for highly controlled release of NPK fertilizer

The aim of this work was to obtain biodegradable polymeric systems based on poly(hydroxybutyrate) (PHB) for use in the controlled release of agrochemicals and to analyze the relationship between the properties of polymers and the rates of release of active compounds. Two types of systems were obtained: one using nitrogen, phosphorous, and potassium (NPK) fertilizer directly mixed within the polymer matrix and another with the fertilizer previously incorporated in bentonite (Bent) and mixed with the polymer. The systems were obtained by melt processing and then evaluated by their properties. The release of the active compounds was analyzed by conductometric analysis using an aqueous solution as release medium for 240 hours. The obtained results were correlated with the biodegradation process of PHB. All of the systems presented a significant reduction in the active compounds released to the environment as compared with the direct application of NPK. The PHB/NPK systems showed a release of up to 37% of the compounds, while the PHB/m‐Bent showed greater control, with a release between 4% and 11% after 240 hours. In addition, the properties of the polymer systems presented a direct relationship with the rate of active compounds released. The type of production process, properties, and biodegradability indicate interesting potential of these systems for application in the controlled release of active compounds.     

Preparation, characterization, and controlled release of novel nanoparticles based on MMA/beta-CD copolymers

A series of random copolymers with different beta-cyclodextrin contents were synthesized by radical copolymerization of MMA with a monovinyl beta-CD monomer. The copolymers were characterized with IR spectroscopy, elemental analysis, DSC, and TGA. Based on these copolymers, their nanoparticles were prepared by using DMF, water, and acetone as solvents. Aqueous dispersions of the nanoparticles were further obtained by dialysis against water. Zetasizer Nano-ZS dynamic light scattering and transmission electron microscopy were employed to characterize the nanoparticles. Using camptothecin as a model drug molecule, the encapsulation efficiency and release behavior of the nanoparticles were investigated.     

Imaging of epidermal growth factor receptor on single breast cancer cells using surface-enhanced Raman spectroscopy

Epidermal growth factor receptor (EGFR) is widely used as a biomarker for pathological grading and therapeutic targeting of human cancers. This study investigates expression, spatial distribution as well as the endocytosis of EGFR in single breast cancer cells using surface-enhanced Raman spectroscopy (SERS). By incubating anti-EGFR antibody conjugated SERS nanoprobes with an EGFR-over-expressing cancer cell line, A431, EGFR localization was measured over time and found to be located primarily at the cell surface. To further validate the constructed SERS probes, we applied this SERS probes to detect the EGFR expression on breast cancer cells (MDA-MB-435, MDA-MB-231) and their counterpart cell lines in which EGFR expression was down-regulated by breast cancer metastasis suppressor 1 (BRMS1). The results showed that SERS method not only confirms immunoblot data measuring EGFR levels, but also adds new insights regarding EGFR localization and internalization in living cells which is impossible in immunoblot method. Thus, SERS provides a powerful new tool to measure biomarkers in living cancer cells.     

Conversion of a murine monoclonal antibody A13 targeting epidermal growth factor receptor to a human monoclonal antibody by guided selection

Epidermal growth factor receptor (EGFR) is an attractive target for tumor therapy because it is overexpressed in the majority of solid tumors and the increase in receptor expression levels has been linked with a poor clinical prognosis. Also it is well established that blocking the interaction of EGFR and the growth factors could lead to the arrest of tumor growth and possibly result in tumor cell death. A13 is a murine monoclonal antibody (mAb) that specifically binds to various sets of EGFR-expressing tumor cells and inhibits EGF-induced EGFR phosphorylation. We isolated human immunoglobulin genes by guided selection based on the mAb A13. Four different human single chain Fvs (scFvs) were isolated from from hybrid scFv libraries containing a human VH repertoire with the VL of mAb A13 and a human VL repertoire with the VH of mAb A13. All the 4 scFvs bound to EGFR-expressing A431 cells. One scFv (SC414) with the highest affinity was converted to IgG1 (ER414). The ER414 exhibited ～17 fold lower affinity compared to the A13 mAb. In addition the ER414 inhibited an EGF-induced tyrosine phosphorylation of EGFR with much lower efficacy compared to the A13 mAb and Cetuximab (Merck KgaA, Germany). We identified that the epitope of A13 mAb is retained in ER414. This approach will provide an efficient way of converting a murine mAb to a human mAb.     

Molecular dynamics simulation reveals structural and thermodynamic features of kinase activation by cancer mutations within the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a major target for drugs in treating lung carcinoma as it promotes cell growth and tumor progression. Structural studies have demonstrated that EGFR exists in an equilibrium between catalytically active and inactive forms, and dramatic conformational transitions occur during its activation. It is known that EGFR mutations promote such conformational changes that affect its activation and drug efficacy. The most common point mutation in lung cancer patients is a leucine to arginine substitution at amino acid 834 (L834R). In a recent article, we have studied changes in drug binding affinities due to cancer mutations of EGFR using ensemble molecular dynamics (MD) simulations. Here, we address an enhanced activation mechanism thought to be associated with this mutation. Using extended timescale MD simulations, the structural and energetic properties are studied for both active and inactive conformations of EGFR. The thermodynamic stabilities of these two conformations are characterized by free energy landscapes estimated from molecular mechanics/Poisson-Boltzmann solvent area calculations. Our study reveals that the L834R mutation introduces conformational changes in both states, adjusting the relative stabilities of active and inactive conformations and hence the activation of the EGFR kinase.     

Biodegradable hyaluronic acid/n-carboxyethyl chitosan/protein ternary complexes as implantable carriers for controlled protein release

An ampholytic N-carboxyethyl chitosan (CEC), with various isoelectric points (IPs), was synthesized by grafting acrylic acid on chitosan utilizing Michael's reaction. Compared to native chitosan, CEC has enhanced water solubility and dramatically accelerated enzymatic degradation; the rate of degradation is proportional to the degree of substitution (DS). The results from turbidimetric titration and fluorescence studies revealed that CEC formed complexes with either hyaluronic acid (HA) or bovine serum albumin (BSA) within a certain pH range. The HA/CEC/BSA ternary complexes could be prepared by colloid titration with quantitative yield and BSA entrapment. The rate of BSA release from the complexes was affected by pH, ionic strength, DS of CEC, and the molecular weight (MW) of HA. The endurance of BSA release from the complexes could be extended up to 20 d by formulating them with high-MW HA and CEC with low DS.BSA release profiles from HA/CEC-2/BSA complexes.     

Chemical synthesis of MnFe2O4/chitosan nanocomposites for controlled release of ofloxacin drug

In this study, we synthesized ofloxacin‐loaded MnFe₂O₄ nanoparticles (NPs) surface modified with chitosan (CS‐MnFe₂O₄) for prolonged antibiotic release in a controlled manner. It was found that the synthesized CS‐MnFe₂O₄ was spherical in shape with an average size of 30–50 nm, low aggregation, and good magnetic responsibility. An in vitro drug loading and release kinetics study reveals that the drug delivery system can take 86% of drug load and can release ofloxacin over a sustained period of 3 days. The release kinetics study reveals that the drug follows zero order kinetics and the mechanism of drug release is diffusion‐controlled type. These results indicated that CS‐MnFe₂O₄ NPs with pH‐sensitive properties can be used as candidates for intestinal targeted drug delivery through oral administration by avoiding the drug release in the highly acidic gastric fluid region of the stomach.     

Development of temperature-responsive semi-IPN hydrogels from PVA-PNVC-PAm for controlled release of anti-cancer agent

A series of semi-interpenetrating polymeric network (semi-IPN) hydrogels were synthesized using poly(vinyl alcohol) (PVA), monomers N-vinylcaprolactam (NVC) acrylamide (Am), and cross-linker bis[2-methacryloyloxy] ethyl phosphate (BMEP). The hydrogels were synthesized by using free-radical polymerization using ammonium persulphate (APS) as an initiator at 60°C. The hydrogels were characterized by various techniques such as Fourier transform infrared (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and scanning electron microscopy (SEM) to confirm the formation, crystallinity, and morphological behavior. The swelling behavior at various temperatures and pH conditions showed that the semi-IPN hydrogels were good candidates for temperature-responsive nature. 5-Flurouracil (FU), a model anticancer drug, was successfully encapsulated and the encapsulation efficiency was found in range of 50–74% for different hydrogels. Further, in-vitro release studies were performed to investigate the release mechanism. The cumulative release studies showed that the developed hydrogels are potentially efficient for the gastrointestinal drug delivery of FU.     

Fabrication of extracellular matrix-coated conductive polypyrrole-poly(l-lactide) fiber-films and their synergistic effect with (nerve growth factor)/(epidermal growth factor) on neurites growth

Non-nerve cell-derived extracellular matrix (ECM) was coated on the aligned porous polypyrrole-poly(l-lactide) (PPy-PLLA) fiber-films with the conductivity of ∼12 mS/m via L929 cells culture and lysing, resulting in ∼10% increase of PC12 cells attachment and ∼26 μm increase of neurites length. The neurite length of ∼149 μm in EGF/NGF group (optimal concentration radio of 12.5/50 (ng/mL)) on aligned and ECM-conjugated fiber-films was significantly larger than ∼94 μm in only NGF group (50 ng/mL), confirming the synergy of EGF, NGF and aligned ECM-conjuaged PPy-PLLA fibers. When differentiated PC12 cells were exerted electrical stimulation (ES) of 100 mV/cm for 4 h/day in 2 day through ECM-PPy-PLLA fiber-films, their neurite length reached to ∼251 μm, significantly larger than ∼149 μm of group without ES, due to the higer expression of related neural proteins in ES group. A simple mechanism was proposed to analyze synergistical effect of ECM, EGF, NGF on axons adhesion and elongation along the aligned ECM-coated fibers under ES condition.