Effects of Bile Salt Sodium Glycodeoxycholate on the Self-Assembly of PEO-PPO-PEO Triblock Copolymer P123 in Aqueous Solution.

A comprehensive experimental study on the interaction between the PEO-PPO-PEO block copolymer P123 (EO20PO68EO20) and the anionic bile salt sodium glycodeoxycholate (NaGDC) in water has been performed. The work was aimed at investigating the suitability of using P123 as bile salt sequestrant beside the fundamental aspects of PEO-PPO-PEO block copolymer-bile salt interactions. Various experimental techniques including dynamic and static light scattering, small-angle X-ray scattering, and differential scanning calorimetry (DSC) were employed in combination with electrophoretic mobility measurements. The system was investigated at a constant P123 concentration of 1.74 mM and with varying bile salt concentrations up to approximately 250 mM NaGDC (or a molar ratio n(NaGDC)/n(P123) = 144). In the mixed P123-NaGDC solutions, the endothermic process related to the self-assembly of P123 was observed to gradually decrease in enthalpy and shift to higher temperatures upon progressive addition of NaGDC. To explain this effect, the formation of NaGDC micelles carrying partly dehydrated P123 unimers was proposed and translated into a stoichiometric model, which was able to fit the experimental DSC data. In the mixtures at low molar ratios, NaGDC monomers associated with the P123 micelle forming a charged "P123 micelle-NaGDC" complex with a dehydrated PPO core. These complexes disintegrated upon increasing NaGDC concentration to form small "NaGDC-P123" complexes visualized as bile salt micelles including one or a few P123 copolymer chains.

Interactions in Aqueous Mixtures of Cationic Hydroxyethyl Cellulose and Different Anionic Bile Salts.

It is known that the reduction of blood cholesterol can be accomplished through foods containing a large number of dietary fibers; this process is partially related to the binding of bile salt to fibers. To gain new insights into the interactions between dietary fibers and bile salts, this study investigates the interactions between cationic hydroxyethyl cellulose (catHEC) and sodium deoxycholate (NaDC) or sodium cholate (NaC), which have a similar structure. Turbidity measurements reveal strong interactions between catHEC and NaDC, and under some conditions, macroscopic phase separation occurs. In contrast, the interactions with NaC are weak. At a catHEC concentration of 2 wt %, incipient phase separation is approached at concentrations of NaC and NaDC of 32.5 and 19.3 mM, respectively. The rheological results show strong interactions and a prominent viscosification effect for the catHEC/NaDC system but only moderate interactions for the catHEC/NaC system. Both cryogenic transmission electron microscopy and small-angle X-ray scattering results display fundamental structural differences between the two systems, which may explain the stronger interactions in the presence of NaDC. The surmise is that the extended structures formed in the presence of NaDC can easily form connections and entanglements in the network.

Poloxamer/sodium cholate co-formulation for micellar encapsulation of doxorubicin with high efficiency for intracellular delivery: An in-vitro bioavailability study.

HYPOTHESIS: Doxorubicin hydrochloride (DX) is widely used as a chemotherapeutic agent, though its severe side-effects limit its clinical use. A way to overcome these limitations is to increase DX latency through encapsulation in suitable carriers. However, DX has a high solubility in water, hindering encapsulation. The formulation of DX with sodium cholate (NaC) will reduce aqueous solubility through charge neutralization and hydrophobic interactions thus facilitating DX encapsulation into poloxamer (F127) micelles, increasing drug latency. EXPERIMENTS: DX/NaC/PEO-PPO-PEO triblock copolymer (F127) formulations with high DX content (DX-PMs) have been prepared and characterized by scattering techniques, transmission electron microscopy and fluorescence spectroscopy. Cell proliferation has been evaluated after DX-PMs uptake in three cell lines (A549, Hela, 4T1). Cell uptake of DX has been studied by means of confocal laser scanning microscopy and flow cytometry. FINDINGS: DX-PMs formulations result in small and stable pluronic micelles, with the drug located in the apolar core of the polymeric micelles. Cell proliferation assays show a delayed cell toxicity for the encapsulated DX compared with the free drug. Data show a good correlation between cytotoxic response and slow DX delivery to nuclei. DX-PMs offer the means to restrict DX delivery to the cell interior in a highly stable and biocompatible formulation, suitable for cancer therapy.

Structural characterization of nonionic mixed micelles formed by C12EO6 surfactant and P123 triblock copolymer.

A structural characterization of mixed micelles formed in aqueous solution by the PEO-PPO-PEO triblock copolymer P123 and the nonionic surfactant C(12)EO(6) was carried out using various techniques, including ultralow shear viscosimetry, depolarized dynamic light scattering (VH-DLS), depolarized static light scattering (VH-SLS), and small-angle X-ray scattering (SAXS). The sphere-to-rod transition of the mixed micelles was studied in a diluted regime (P123 concentrations ranging from 0.5 to 10 wt %) at C(12)EO(6)/P123 molar ratios of 2.2, 3.2, 6.0, and 11 as well as for the pure C(12)EO(6). The data from VH-SLS and viscosimetry displayed a sharp increase in the intensity and viscosity, respectively, at the sphere-to-rod transition, and the results from the two methods were in accordance. In both techniques, an increased transition temperature with increasing content of C(12)EO(6) (in the molar ratio regime from 2.2 to 11) was observed. SAXS was used as the main technique, and a thorough structural characterization was performed, where indirect Fourier transformation (IFT) and generalized indirect Fourier transformation (GIFT) were employed in the analysis procedure of the SAXS data. The p(r) functions obtained from the IFT (employed at low P123 concentrations, i.e., 1.0 and 2.0 wt %) and GIFT (employed above 2.0 wt %) analyses revealed increased inhomogeneities in the mixed micelles when the molar ratio was increased. This suggested that the C(12)EO(6) organized themselves at the interface between the PPO core and the PEO corona of the P123 micelles, with the C(12) alkyl chain stretching into the hydrophobic core and the EO(6) part residing in the hydrophilic corona. The structure factor parameters obtained with GIFT for a molar ratio of 2.2 at a P123 concentration of 5.0 wt % showed radius values smaller than what was estimated from the p(r) functions. This was explained by an interpenetration of the PEO chains from one mixed micelle into a neighboring one. VH-DLS was performed on the mixed micelles at a temperature 3 degrees C above the transition temperature and at a molar ratio of 2.2. From the analyzed data, the average length L of the rods was estimated to be 102 nm.

A fluorescence study of the loading and time stability of doxorubicin in sodium cholate/PEO-PPO-PEO triblock copolymer mixed micelles.

HYPOTHESIS: Doxorubicin hydrochloride (DX) is one of the most powerful anticancer agents though its clinical use is impaired by severe undesired side effects. DX encapsulation in nanocarrier systems has been introduced as a mean to reduce its toxicity. Micelles of the nonionic triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) (PEO-PPO-PEO), are very promising carrier systems. The positive charge of DX confines the drug to the hydrophilic corona region of the micelles. The use of mixed micelles of PEO-PPO-PEO copolymers and a negatively charged bile salt should favour the solubilization of DX in the apolar core region of the micelles. EXPERIMENTS: We studied the DX uptake in the micellar systems formed by sodium cholate (NaC) and the PEO100PPO65PEO100 (F127) copolymer, prepared with different mole ratios (MR = nNaC/nF127) in the range 0 ÷ 1. The systems were characterized by small angle X-ray scattering (SAXS) and dynamic light scattering (DLS); DX encapsulation was followed by steady-state and time-resolved fluorescence spectroscopy. FINDINGS: The successful solubilization of DX in the host micellar systems did not affect their structure, as evidenced by both SAXS and DLS data. In the presence of NaC, DX experiences a more apolar environment as indicated by its characteristic fluorescent behaviour. The almost complete uptake of the drug occurred shortly after the sample preparation; however, time resolved fluorescence revealed a slow partition of DX between corona and core regions of the micelles. DX degradation in the mixed micellar systems was markedly reduced relative to aqueous DX solutions.

A calorimetry and light scattering study of the formation and shape transition of mixed micelles of EO20PO68EO20 triblock copolymer (P123) and nonionic surfactant (C12EO6).

The interaction between the nonionic surfactant C12EO6 and the poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer EO20PO68EO20 (P123) has been investigated by means of isothermal titration and differential scanning calorimetry (DSC) as well as static and dynamic light scattering (SLS and DLS). P123 self-assembles in water into spherical micelles at ambient temperatures. At raised temperatures, the DSC data revealed a sphere-to-rod transition of the P123 micelles around 60 degrees C. C12EO6 interacts strongly with P123 micelles in aqueous solution to give mixed micelles with a critical micelle concentration (cmc) well below the cmc for pure C12EO6. The presence of C12EO6 also lowers the critical micelle temperature of P123 so aggregation starts at significantly lower temperatures. A new phenomenon was observed in the P123-C12EO6 system, namely, a well-defined sphere-to-rod transition of the mixed micelles. A visual phase study of mixtures containing 1.00 wt % P123 showed that in a narrow concentration range of C12EO6 both the sphere-to-rod transition and the liquid-liquid phase separation temperature are strongly depressed compared to the pure P123-water system. The hydrodynamic radius of spherical mixed micelles at a C12EO6/P123 molar ratio of 2.2 was estimated from DLS to be 9.1 nm, whereas it is 24.1 nm for the rodlike micelles. Furthermore, the hydrodynamic length of the rods at a molar ratio of 2.2 is in the range of 100 nm. The retarded kinetics of the shape transition was detected in titration calorimetric experiments at 40 degrees C and further studied by using time-resolved DLS and SLS. The rate of growth, which was slow (>2000 s), was found to increase with the total concentration.

Tryptophan-tagged cutinase studied by steady state fluorescence for understanding of tag interactions in aqueous two-phase systems.

Genetic engineering has been used to construct fusion proteins of Fusarium solani pisi cutinase and tryptophan-based tags, expressed in Saccharomyces cerevisiae, to increase the partitioning in aqueous two-phase systems. The separation systems were composed of thermoseparating polymers (random copolymers of ethylene oxide and propylene oxide, EOPO) and detergents (C(12)EO(n)). In this study, the fluorescence behaviour of the peptide-tagged protein, free peptide tag and tryptophan was investigated. The tryptophan-tagged proteins, cutinase-(WP)(4) and cutinase-TGGSGG-(WP)(4), showed emission spectra similar to the free peptides and tryptophan, indicating solvent exposure of the tag. The influence of polymers and detergents on the fluorescence of tagged proteins was examined. When peptides and tagged proteins were exposed to polymer, a slight blue shift of the emission maximum was observed. Larger blue shifts of the emission maximum were observed when C(12)EO(n) detergents were utilised. The results correlate with aqueous two-phase partitioning where addition of C(12)EO(n) detergents results in more extreme partitioning compared to systems containing only polymers. Dynamic light scattering (DLS) measurements of the EOPO copolymers were carried out, showing that the polymers did not aggregate at concentrations used in aqueous two-phase systems. Quenching of fluorescence with iodide for both proteins and peptide tags was studied. Plots according to the Stern-Volmer equation resulted in a linear fit, indicating exposed tryptophan residues for both free peptides and fusion proteins. The quenching constants were similar for both tagged protein and free peptide tag. The fluorescence results indicated that the tryptophan residues in the tag were exposed to the solvent and could interact with detergents and polymers in the two-phase systems.

Cubosome formulations stabilized by a dansyl-conjugated block copolymer for possible nanomedicine applications.

We present here an innovative, fluorescent, monoolein-based cubosome dispersion. Rather than embedded within the monoolein palisade, the fluorescent imaging agent, namely dansyl, was conjugated to the terminal ethylene oxide moieties of the block copolymer Pluronic F108. We discuss the physicochemical and photophysical properties of this fluorescent Pluronic and of a cubosome formulation stabilized by a mixture of dansyl-conjugated and non-conjugated Pluronic, also including an anticancer drug (quercetin). Furthermore, we performed biocompatibility tests against HeLa cells to assess internalization and cytotoxicity features of this nanoparticles aqueous dispersion. Cryo-TEM, SAXS, and DLS analysis, proved the bicontinuous cubic inner nanostructure and the morphology of this fluorescent cubosome dispersion, while photophysical measurements and biocompatibility results basically validate their potential use for theranostic nanomedicine applications.

Soluble aggregates in aqueous solutions of polyion-surfactant ion complex salts and a nonionic surfactant.

Water-soluble aggregates based on two polyion-surfactant ion "complex salts", consisting of hexadecyltrimethylammonium (C16TA(+)) and polyacrylate (PA(-)) with either 25 or 6000 repeating units, with added nonionic surfactant octaethylene glycol monododecyl ether (C12E8) have been investigated. A previous phase study has shown that added C12E5 or C12E8 can solubilize complex salts in aqueous systems, and that increasing the poly(ethylene oxide) chain length of the nonionic surfactant and/or decreasing the polyion length favors dissolution. In this work we report on dynamic light scattering, NMR diffusometry, small-angle X-ray scattering, and isothermal titration calorimetry measurements performed to characterize the solubilized composite aggregates in dilute aqueous solution in terms of size and stoichiometry. It was found that mixed aggregates of polyacrylate, C16TA(+) ions, and C12E8, with almost constant stoichiometry, coexist with free micelles of C12E8 at all investigated mixing ratios. The length of the polyion only weakly affects the stoichiometry of the mixed aggregates while strongly affecting their size and water solubility.

Flavonoids: precipitation kinetics and interaction with surfactant micelles.

Polyphenolic substances, such as flavonoids, attract considerable interest due to their ability to act as antioxidants. The vast majority of studies published deal with the nutritional, biochemical, or chemical structure aspects of these substances while few reports exist on the physico-chemical properties as well as solution behavior of the substances. In this article we report on the precipitation kinetics of naringenin, quercetin, and rutin in pure water as well as in micellar solutions of Tween 80. The methods used are dynamic and static light scattering in conjunction with cryo transmission electron microscopy. The results show that the substances precipitate rapidly in pure water. In the presence of Tween 80 micelles, however, the flavonoids can become solubilized in the micelles, which can result in solutions which are stable for days. The results suggest that the extent of solubilization is related to the chemical structure of the flavonoids. This study illustrates that precipitation kinetics and the distribution of polyphenolic substances in solutions and dispersions are factors of importance, which should be taken into account when designing investigations and interpreting results.

Interaction between DNA and charged colloids could be hydrophobically driven.

The interaction of DNA with amino-functionalized polystyrene particles has been studied by using a dynamic light scattering (DLS) technique. In 10 mM NaBr solution the particles have a hydrodynamic radius of 76 nm and the DNA macromolecule investigated (double stranded) has a hydrodynamic radius of 107 nm. At very low DNA concentrations, DNA adopts a flat conformation on the particle surface. If the DNA concentration is increased above 0.1 microg/mL, the thickness of the DNA layer increases, suggesting the presence of large loops and tails. Although the particles contain primary amino groups, they have a negative net charge under the conditions used in this work. Thus, the driving force for DNA adsorption is not of electrostatic origin but rather due to a hydrophobic effect. Addition of cationic surfactant to the DNA-precoated amino-functionalized particles induces changes in the adsorbed layer conformation, in agreement with the coadsorption of cationic surfactant.