Aggregation of dextran hydrophobically modified by sterically-hindered phenols in aqueous solutions: Aggregates vs. single molecules

The hydrodynamic properties of dextrans hydrophobically modified by sterically-hindered phenols in aqueous solution were characterized by a combination of Cryogenic Transmission Electron Microscopy (Cryo-TEM), fluorescent spectroscopy, and Dynamic Light Scattering (DLS). All solutions investigated contained aggregates the structure, shape, and critical aggregation concentration of which are influenced by the amount of the inserted phenol groups in the polymer matrix. We found that increasing of temperature enhances aggregation. It was established that the critical aggregation concentration could be well approximated by a logarithmic function of the substitution degree of the glycoside units. The results suggest that the proportion of aggregates is nevertheless negligible in comparison with the proportion of single molecules.     

Molecular properties of conjugates formed by synthetic hydrophilic polymers and sterically hindered phenols

Water-soluble conjugates are prepared via the chemical modification of poly(vinyl alcohol) and poly(ethylene glycol) by antioxidants taken from the family of sterically hindered phenols. The effects of the degree of substitution of conjugates on the dimensions of molecules and their aggregates are studied by viscometry and light scattering in dilute solutions. It is shown that an increase in the amount of antioxidant groups incorporated into the poly(vinyl alcohol) chain leads to a decrease in the dimensions of single conjugate molecules owing to attraction of hydrophobic groups. Poly(ethylene glycol) molecules carrying end groups of sterically hindered phenols form micellar aggregates that are absent in the solution of the initial polymer. The mean number of molecules involved in such an aggregate is 83. It is found that the presence of hydrophobic end groups in poly(ethylene glycol) molecules causes a sharp reduction in the lower critical solution temperature of a solution relative to that of the initial polymer.     

High-performance size-exclusion chromatographic procedure for the determination of fluoresceinyl isothiocyanate dextrans of various molecular masses in biological media

A high-performance size-exclusion chromatographic procedure, using Nucleosil Diol, for the quantitative analysis of fluoresceinyl isothiocyanate dextrans of various molecular masses (10,000-150,000) in biological media was developed. The influence of the molecular mass and the degree of substitution of the conjugates on the chromatographic behaviour are discussed. In addition to quantitation, the molecular mass of the conjugates with degree of substitution below 1.6 could be estimated from the chromatograms. Linear standard calibration curves were obtained at concentrations down to 0.050 micrograms ml-1 in rabbit plasma and urine and homogenates of rabbit liver, lymph node and muscle when the derivative (degree of substitution 0.85) was monitored by fluorescence detection (lambda ex = 493 nm, lambda em = 520 mm). The fluoresceinyl isothiocyanate dextrans were found to be stable for more than three days at 37 degrees in all biological media under investigation. A pH-rate profile for the alkaline hydrolysis of fluoresceinyl isothiocyanate dextrans was constructed. The applicability of the method to pharmacokinetic studies was demonstrated by recording the plasma concentration-time profile of a fluoresceinyl isothiocyanate dextran T-70 conjugate following intravenous injection to a rabbit. In relation to future pharmacokinetic investigations on dextran conjugates, the results reported indicate that labelling of the parent dextran with fluoresceinyl isothiocyanate and monitoring of the fluoresceinyl isothiocyanate dextran conjugate throughout the organism using the described method is a promising development.     

Preparation and characterization of self-assembled nanoparticles of heparin-deoxycholic acid conjugates

Various deoxycholic acid (DOCA) bearing heparin (HD) amphiphilic conjugates with different degrees of substitution (DS) with DOCA were synthesized using heparin as a hydrophilic segment and DOCA as a hydrophobic segment. Structural characteristics of these HD conjugates were investigated using 1H NMR, dynamic light scattering, zeta potential, transmission electron microscopy (TEM), and fluorescence spectroscopy. HD conjugates provided monodispersed self-aggregates in water, with mean diameters decreasing with increasing DOCA DS in the range of 120-200 nm. HD aggregates were covered with negatively charged heparin shells, exhibiting xi potentials near -56 mV. The critical aggregation concentration (cac) of the HD (0.02-0.003 mg/mL) depended upon DOCA DS. TEM images demonstrated that the shape of the self-aggregates was spherical. Partition equilibrium constants, Kv, for pyrene in the self-aggregates in water indicated that increasing DS enhanced the hydrophobicity of the self-aggregate inner core. The mean aggregation number of DOCA per hydrophobic microdomain, estimated by the fluorescence quenching methods using cetylpyridinium chloride, indicated that five to nine of HD chains comprised a hydrophobic domain in the conjugates.     

Dextran as antioxidant's activity carrier

Two series of conjugates of dextran and antioxidants from the class of sterically hindered phenols were prepared. The conjugates were characterized by the substitution degree of glycoside units, solubility in different solvents, intrinsic viscosity. The investigation of radical scavenging activity (RSA) of conjugates was carried out in their reactions with two free radicals — 2,2-diphenyl-1-picrylhydrazyl (DPPH) and sodium salt of 2,2-diphenyl-1-picrylhydrazyl sulfonic acid (DPPH-salt). The usage of water soluble DPPH-salt enabled to estimate the RSA of the conjugates in water. It was shown that the rate constants of interaction of the DPPH-salt and the conjugates were 10–30 times higher than this value for low-molecular analogue of phenoxan. High RSA of the conjugates in water can be explained by large solvation shell formed due to high content of hydroxy groups in dextran.     

Self-aggregated nanoparticles from methoxy poly(ethylene glycol)-modified chitosan: synthesis; characterization; aggregation and methotrexate release in vitro

Methoxy poly(ethylene glycol)-grafted-chitosan (mPEG-g-CS) conjugates were synthesized by formaldehyde linking method and characterized by Fourier transform infrared (FT-IR) and proton nuclear magnetic resonance (¹H-NMR). The degree of substitution (DS) of methoxy poly (ethylene glycol) (mPEG) in the mPEG-g-CS molecules determined by ¹H-NMR ranged from 19% to 42%. The critical aggregation concentration (CAC) was determined by fluorescence spectroscopy using pyrene as fluorescence probe and its value was 0.07 mg/mL in water. mPEG-g-CS formed monodisperse self-aggregated nanoparticles with a roughly spherical shape and a mean diameter of 261.9 nm were prepared by the dialysis method. mPEG-g-CS self-aggregated nanoparticles were used as carriers of poorly water-soluble anticancer drug methotrexate (MTX). MTX was physically entrapped inside mPEG-g-CS self-aggregated nanoparticles by dialysis method and the characteristics of MTX-loaded mPEG-g-CS self-aggregated nanoparticles were analyzed using dynamic laser light scattering (DLLS), transmission electron microscopy (TEM). Moreover, in vitro release behavior of MTX was also investigated and the results showed that MTX was continuously released more than 50% in 48 h.     

In situ detection of birefringent mesoscopic H and J aggregates of thiacarbocyanine dye in solution

Polarized-light microscopy, fluorescence microscopy, atomic force microscopy as well as absorption and fluorescence spectroscopy were used to characterize mesoscopic structures of both supramolecular H and J aggregates of 3,3'-disulfopropyl-5,5'-dichloro-9-methyl thiacarbocyanine dye in aqueous solution. Polarized-light microscopy visualizes in situ the mesoscopic morphology of the H and J aggregates and distinguishes between them by their own colors. The H aggregate having a fibrous structure showed negative birefringence, namely, the refractive index along the fiber short axis was higher than that of the long axis, so that pi-electron chromophores of the dye molecule are likely to orient along the short axis of the elongated fibers. The degree of birefringence of the H aggregate fiber was approximately -0.3. Investigations on the concentration dependence of the absorption spectra showed that the amount of J aggregates increased at the expense of a decrease in the amount of H aggregates. With respect to the J aggregates, a small dot morphology was observed at a relatively low dye concentration of 3.0 mM. With an increase of the dye concentration up to 10 mM, the morphology changed into mesoscopic fibers. In contrast, fluorescence microscopy for the fibrous J aggregates reveals that the constituent molecules are approximately aligned along the long axis of the fibers.     

人血清脂蛋白与胆固醇修饰的葡聚糖的相互作用

采用浊度法和高效液相色谱法研究了人血清低密度脂蛋白（LDL）和极低密度脂蛋白（VLDL）与胆固醇修饰的葡聚糖（CHD）的相互作用。浊度法研究结果表明，CHD与VLDL混合溶液的浊度大于CHD与LDL混俣溶液的浊度。CHD浓度和CHD链上胆固醇含量都对溶液的浊度产生影响。当CHD浓度为0．3mg/mL，CHD链上胆固醇含量为4个／100个糖环时，混合溶液的浊度可达到最大值，表明LDL或VLDL要的程度最大。Ca^2 对CHD与LDL或VLDL的相互作用没有影响。高效液相色谱的研究结果表明，CHD与LDL的结合使得复合物分子量增加；CHD分子量增大，复合物分子量也增加；当葡聚糖上胆固醇含量为4．0时，复合物分子量最大。实验结果表明，CHD能引起LDL或VLDL的自聚集。     

Surfactant-induced amorphous aggregation of tobacco mosaic virus coat protein: a physical methods approach

The interactions of non-ionic surfactant Triton X-100 and the coat protein of tobacco mosaic virus, which is an established model for both ordered and non-ordered protein aggregation, were studied using turbidimetry, differential scanning calorimetry, isothermal titration calorimetry, and dynamic light scattering. It was found that at the critical aggregation concentration (equal to critical micelle concentration) of 138 x 10(-6) M, Triton X-100 induces partial denaturation of tobacco mosaic virus coat protein molecules followed by protein amorphous aggregation. Protein aggregation has profound ionic strength dependence and proceeds due to hydrophobic sticking of surfactant-protein complexes (start aggregates) with initial radii of 46 nm. It has been suggested that the anionic surfactant sodium dodecyl sulfate forms mixed micelles with Triton X-100 and therefore reverses protein amorphous aggregation with release of protein molecules from the amorphous aggregates. A stoichiometric ratio of 5 was found for Triton X-100-sodium dodecyl sulfate interactions.     

Polyamidoamine dendrimer and dextran conjugates: preparation,characterization, and in vitro and in vivo evaluation

Amide and ester conjugates of aceclofenac with polyamidoamine (PAMAM-G0) dendrimer zero generation and dextran (40 kDa) polymeric carrier, respectively, are presented. The prepared conjugates were characterized by UV, TLC, HPLC, IR, and ¹H NMR spectroscopy. The average degrees of substitution of amide and ester conjugates were determined and found to be (12.5 ± 0.24) % and (7.5 ± 0.25) %, respectively. The in vitro hydrolysis studies showed that dextran ester conjugate hydrolyzed faster in a phosphate buffer solution of pH 9.0 as compared to PAMAM dendrimer G0 amide conjugate, and followed the first order kinetics. No amount of the drug was regenerated at pH 1.2 in simulated gastric fluid. The dextran conjugate showed short half-life as compared to the PAMAM dendrimer conjugate. Anti-inflammatory and analgesic activities of the dendrimer conjugate were found to be similar to those of the standard drug. Results of chronic ulceroginic activity showed deep ulceration and high ulcer index for aceclofenac, whereas lower ulcer index was found for the PAMAM dendrimer and dextran (40 kDa) conjugates. Experimental data suggest that PAMAM dendrimer and dextran (40 kDa) can be used as carriers for the sustained delivery of aceclofenac along with a remarkable reduction in gastrointestinal toxicity.     

Aqueous biphase formation by mixtures of dextran and hydrophobically modified dextran

 A series of modified dextrans was prepared by condensation of straight chain saturated C3, C4 and C6 fatty acids and the phase behavior of aqueous solutions of these materials with unmodified dextran was measured as a function of temperature, concentration and degree of substitution. At a constant degree of substitution the tendency towards aqueous biphase formation increased with the length of the hydrophobic substituent, the temperature and the molecular weight. Fluorescence studies of the modified dextrans with pyrene as a probe indicated the presence of hydrophobic micro-domains. Rheological study showed that there was no large-scale association for C3 and C4 substituted dextran, mainly intramolecular association, however some intermolecular association existed for C6 substituted dextran. The results are compared with the behavior of the classical PEG/dextran biphase systems, and mechanisms driving phase separation are discussed. Received: 7 October 1997 Accepted: 20 January 1998     

Cosurfactant and cosolvent effects on surfactant self-assembly in supercritical carbon dioxide

The impact of alcohol additives on the self-assembly of surfactants in supercritical carbon dioxide is investigated using lattice Monte Carlo simulations. We observe that all studied (model) alcohols reduce the critical micelle concentration. The reduction is stronger the longer the hydrocarbon chain of the alcohol, and the higher the alcohol concentration. Short-chain alcohols are found to concentrate in the surfactant layer of the aggregates, replacing surfactant molecules and leading to a strong decrease of the aggregation number and a large increase of the number of aggregates. On the other hand, only a small number of alcohol molecules with longer chain length are found in the aggregates, leading to a slight increase in the aggregation number. However, structural properties such as size and density profiles of aggregates at the same aggregation number are not influenced markedly. Consequently, short-chain alcohols act as cosurfactants, directly influencing the properties of the aggregates, while alcohols with longer hydrocarbon chains work as cosolvents, altering the properties of the solvent. However, the transition between both extremes is gradual.     

萘和丹酰基修饰的扇形树枝状分子聚集体的能量转移行为

分别对1-3代聚(酰胺-胺)(PAMAM)结构的dendron分子的外端基和focal point进行了修饰,得到了外端基为萘(给体)色团、焦点(focal point)为丹酰(受体)色团的树枝状化合物Dan-ABπ-Nap(n=2,4,8).利用荧光光谱测定了不同浓度下所得一系列树枝状分子在水中的荧光强度,并计算了它...     

Nystatin—dextran conjugates: Synthesis and characterization

The coupling of nystatin (Nys), a water-insoluble antifungal agent, to dextran via an imine or amine bond was systematically investigated. Dextran was first oxidized to dialdehyde dextran using potassium periodate, purified from the oxidizing agent, and reacted with Nys to form the Schiff base. The Schiff base was reduced to the amine using borohydride. All reactions took place in water. The purification of the oxidized dextran from the oxidizing agent was essential to prevent oxidative degradation of Nys at the coupling step. The effects on the coupling yield of the following factors: dextran molecular weight, degree of oxidation (aldehyde content), Nys to dextran ratio, temperature, and reaction pH were studied. A 95% coupling yield was obtained at the optimized coupling conditions: pH 8.9 ± 0.1, 50% degree of oxidation, and initial ratio of Nys to dialdehyde dextran 1:2.5. In all experiments, dextran was decreased in molecular weight during the oxidation step. Both imine and amine forms of Nys-dextran conjugates were soluble in water and exhibited improved stability in aqueous solutions as compared to the unbound drug. The conjugates showed comparable minimum inhibitory concentration (MIC) values against Candida albicans and Cryptococcus neoformans. The conjugates were about 25 times less toxic than free Nys after a single injection in mice. © 1996 John Wiley & Sons, Inc.     

Aggregation of antifreeze protein and impact on antifreeze activity

Antifreeze protein type III aggregates once the concentration exceeds a critical value, the so-called critical aggregation concentration (CAC). It was found for the first time that the aggregation of antifreeze protein exerts a direct impact on the antifreeze efficiency. It follows from our measurements that the AFP III above CAC will enhance the antifreeze activity because of the increase of the kink kinetics barrier of surface integration. This is attributed to the optimal packing of AFP III molecules on the surface of the ice nucleus as well as ice crystals above CAC. This study will extend our understanding of the antifreeze mechanism of antifreeze protein monomers as well as antifreeze aggregates on ice nucleation and shed light on the selection of antifreeze agents.     

The aggregation behavior between anionic carboxymethylchitosan and cetyltrimethylammonium bromide: MesoDyn simulation and experiments

The aggregation behavior between carboxymethylchitosan (CMCHS) and cetyltrimethylammonium bromide (CTAB) is investigated by MesoDyn simulation and experimental techniques, for increasing CTAB concentrations. Mixed CMCHS/CTAB bulk aggregates are formed in the solution. Simulation results give the morphologies of aggregates clearly and illustrate the two stages for the formation of aggregates: the first stage is CTAB molecules aggregating on the CMCHS chain and the second stage is the equilibrium stage. A viscosity maximum and a hydrodynamic radius minimum at a certain CTAB concentration reveal the bridging structure of the polymer chains by the micelles. Transmission electron microscopy (TEM) images give the bridging structure clearly. At higher surfactant concentrations, light scattering and TEM show the existence of larger structures, whose size increases with CTAB concentration. According to the simulation and experimental results, the process of aggregate formation and aggregation mechanism are analyzed. Initially CMCHS and CTAB form network structure due to the bridge action of CTAB micelles, while the network structure disappears gradually and is replaced by ellipsoidal CMCHS/CTAB aggregate structure with CTAB concentration increasing.     

两亲性壳聚糖衍生物的合成及其自聚集现象

以壳聚糖为主链, 聚乙二醇单甲醚为亲水性链段, 癸二酸为疏水链段, 合成了一系列两亲性壳聚糖衍生物. 通过FTIR, 1H NMR和X射线粉末衍射等手段对壳聚糖衍生物进行了结构表征, 由元素分析方法计算出衍生物的取代度. 采用直接溶解法制备了壳聚糖衍生物的空白胶束, 通过透射电子显微镜(TEM)观察了胶束的形态. 由动态光散射(DLS)测定了胶束的粒径及分布, 并以芘为分子探针, 通过荧光光谱法测定了壳聚糖衍生物的临界聚集浓度(CAC). 研究结果表明, 壳聚糖主链上疏水链段的取代度越大, 其衍生物的临界聚集浓度越低, 相同浓度下的胶束的粒径也越小.     

Characterization and control of the aggregation behavior of cyclodextrins

Photon correlation spectroscopy has been employed for the purpose of characterizing the aggregation behavior of cyclodextrin molecules in aqueous solutions. This optical method is generally intended to study particle size distribution of colloidal particles, associates and macromolecules. Herein we report on some general methodological issues of photon correlation spectroscopy aiming to illustrate aggregated and non-aggregated state of parent cyclodextrins and cyclodextrin derivatives, such as (2-hydroxy)propyl-β-cyclodextrin and tetraamino rhodaminyl (2-hydroxypropyl)-β-cyclodextrin in different aqueous media. Based on particle size analysis data we have demonstrated that the tendency of cyclodextrin molecules to form aggregates may be controlled by temperature and by various additives, e.g. urea, citric acid and polyvinylpyrrolidone. In the case of (2-hydroxypropyl)-β-cyclodextrin the effect of degree of substitution was also studied.     

Synthesis and hierarchical self-assembly of cavity-containing facial amphiphiles

The synthesis and aggregation behavior of cavity-containing facial amphiphiles is described. The molecules consist of a glycoluril-based rigid cavity functionalized with two water-soluble benzoate groups. By specific molecular recognition processes in water, the amphiphilic hosts self-assemble in a hierarchical process to form arrays of molecules. Depending on the counterions, these arrays can be assembled into well-defined aggregates of mesoscopic size. The size and shape of the aggregates can be tuned by variations in the size and substitution pattern of the cavities of the host molecules.     

Influence of the aggregation state in solution on the supramolecular organization of adsorbed type I collagen layers

In the last years, adsorbed collagen was shown to form layers with a supramolecular organization depending on the substrate surface properties and on the preparation procedure. If the concentration of collagen and the duration of adsorption are sufficient, fibrillar collagen structures are formed, corresponding to assemblies of a few molecules. This occurs more readily on hydrophobic compared to hydrophilic surfaces. This study aims at understanding the origin of such fibrillar structures and in particular at determining whether they result from the deposition of fibrils formed in solution or from the building of assemblies at the interface. Therefore, type I collagen solutions with an increasing degree of aggregation were prepared, using the “neutral-start” approach, by ageing pH 5.8 solutions at 37 °C for 15 min, 2 or 7 days. The obtained solutions were used to investigate the influence of collagen aggregation in solution on the supramolecular organization of adsorbed collagen layers, which was characterized by X-ray photoelectron spectroscopy and atomic force microscopy. Polystyrene and plasma-oxidized polystyrene were chosen as substrates for the adsorption. The size and the density of collagen fibrils at the interface decreased upon increasing the degree of aggregation of collagen in solution. This is explained by a competitive adsorption process between monomers and aggregates of the solution, turning at the advantage of the monomers. More aggregated solutions, which are thus depleted in free monomers, behave like less concentrated solutions, i.e. lead to a lower adsorbed amount and less fibril formation at the interface. This study shows that the supramolecular fibrils observed in adsorbed collagen layers, especially on hydrophobic substrates, are not formed in the solution, prior to adsorption, but are built at the interface, through the assembly of free segments of adsorbed molecules.