Unraveling the effects of low protein-phenol binding affinity on the structural properties of beta-lactoglobulin.

Non-covalent interactions of phenolics with proteins cannot always be readily identified, often leading to contradictory results described in the literature. This results in uncertainties as to what extent phenolics can be added to protein solutions (for example for bioactivity studies) without affecting the protein structure. Here, we clarify which tea phenolics (epigallocatechin gallate (EGCG), epicatechin and gallic acid) interact with the whey protein ß-lactoglobulin by combining various state-of-the-art-methods. STD-NMR revealed that all rings of EGCG can interact with native ß-lactoglobulin, indicating multidentate binding, as confirmed by the small angle X-ray scattering experiments. For epicatechin, unspecific interactions were found only at higher protein:epicatechin molar ratios and only with 1H NMR shift perturbation and FTIR. For gallic acid, none of the methods found evidence for an interaction with ß-lactoglobulin. Thus, gallic acid and epicatechin can be added to native BLG, for example as antioxidants without causing modification within wide concentration ranges.

The Impact of Redox, Hydrolysis and Dehydration Chemistry on the Structural and Magnetic Properties of Magnetoferritin Prepared in Variable Thermal Conditions.

Ferritin, a spherically shaped protein complex, is responsible for iron storage in bacteria, plants, animals, and humans. Various ferritin iron core compositions in organisms are associated with specific living requirements, health state, and different biochemical roles of ferritin isomers. Magnetoferritin, a synthetic ferritin derivative, serves as an artificial model system of unusual iron phase structures found in humans. We present the results of a complex structural study of magnetoferritins prepared by controlled in vitro synthesis. Using various complementary methods, it was observed that manipulation of the synthesis technology can improve the physicochemical parameters of the system, which is useful in applications. Thus, a higher synthesis temperature leads to an increase in magnetization due to the formation of the magnetite phase. An increase in the iron loading factor has a more pronounced impact on the protein shell structure in comparison with the pH of the aqueous medium. On the other hand, a higher loading factor at physiological temperature enhances the formation of an amorphous phase instead of magnetite crystallization. It was confirmed that the iron-overloading effect alone (observed during pathological events) cannot contribute to the formation of magnetite.

pH Responsiveness of Hexosomes and Cubosomes for Combined Delivery of Brucea javanica Oil and Doxorubicin.

We report pH-responsive liquid crystalline lipid nanoparticles, which are dual-loaded by Brucea javanica oil (BJO) and doxorubicin hydrochloride (DOX) and display a pH-induced inverted hexagonal (pH = 7.4) to cubic (pH = 6.8) to emulsified microemulsion (pH = 5.3) phase transition with a therapeutic application in cancer inhibition. BJO is a traditional herbal medicine that strongly inhibits the proliferation and metastasis of various cancers. Doxorubicin is an antitumor drug, which prevents DNA replication and hampers protein synthesis through intercalation between the base pairs of the DNA helices. Its dose-dependent cardiotoxicity imposes the need for safe delivery carriers. Here, pH-induced changes in the structural and interfacial properties of designed multicomponent drug delivery (monoolein-oleic acid-BJO-DOX) systems are determined by synchrotron small-angle X-ray scattering and the Langmuir film balance technique. The nanocarrier assemblies display good physical stability in the studied pH range and adequate particle sizes and ζ-potentials. Their interaction with model lipid membrane interfaces is enhanced under acidic pH conditions, which mimic the microenvironment around tumor cells. In vitro cytotoxicity and apoptosis studies with BJO-DOX dual-loaded pH-switchable liquid crystalline nanoparticles are performed on the human breast cancer Michigan Cancer Foundation-7 (MCF-7) cell line and MCF-7 cells with doxorubicin resistance (MCF-7/DOX), respectively. The obtained pH-sensitive nanomedicines exhibit enhanced antitumor efficacy. The performed preliminary studies suggest a potential reversal of the resistance of the MCF-7/DOX cells to DOX. These results highlight the necessity for further understanding the link between the established pH-dependent drug release profiles of the nanocarriers and the role of their pH-switchable inverted hexagonal, bicontinuous cubic, and emulsified microemulsion inner organizations for therapeutic outcomes.

Folate receptor targeted bufalin/ß-cyclodextrin supramolecular inclusion complex for enhanced solubility and anti-tumor efficiency of bufalin.

Bufalin (BF), a traditional Chinese medicine, exhibited inhibitory activities against a broad spectrum of tumor cells. The present study elaborates that bufalin was successfully encapsulated into the cavity of ß-cyclodextrin (ß-CD), which was determined by Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance spectroscopy (1H NMR), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The best reaction mole ratio of BF/ß-CD was 1:5. The solubilities of bufalin in water and phosphate buffer solution (pH=7.4) were increased up to 24 and 34 times after encapsulated into the cavity of ß-CD respectively. The inclusion efficiency (IE) and drug loading (DL) of bufalin in the inclusion complex were (94.22±0.85)% and (14.11±0.20)%, respectively. Then ß-CD conjugated with folic acid (FA) were further prepared and employed to improve the anti-tumor efficacy of inclusion complex. The in vitro dissolution and solubility study showed better values of inclusion complex and FA targeted inclusion complex than that of pure BF. Cytotoxicity experiments by using HCT116 cell line revealed that the antitumor efficiency of bufalin were enhanced more than two folds in the presence of ß-CD and folate conjugated ß-CD (FA-PEI-ß-CD), which demonstrated the potential application of ß-CD (FA-PEI-ß-CD) as delivery vehicles of bufalin for antitumor therapy.

Self-assembled stable sponge-type nanocarries for Brucea javanica oil delivery.

Sponge-type nanocarriers (spongosomes) are produced upon dispersion of a liquid crystalline sponge phase formed by self-assembly of an amphiphilic lipid in excess aqueous phase. The inner organization of the spongosomes is built-up by randomly ordered bicontinuous lipid membranes and their surfaces are stabilized by alginate chains providing stealth properties and colloidal stability. The present study elaborates spongosomes for improved encapsulation of Brucea javanica oil (BJO), a traditional Chinese medicine that may strongly inhibit proliferation and metastasis of various cancers. The inner structural organization and the morphology characteristics of BJO-loaded nanocarriers at varying quantities of BJO were determined by cryogenic transmission electron microscopy (Cryo-TEM), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). Additionally, the drug loading and drug release profiles for BJO-loaded spongosome systems also were determined. We found that the sponge-type liquid crystalline lipid membrane organization provides encapsulation efficiency rate of BJO as high as 90%. In vitro cytotoxicity and apoptosis study of BJO spongosome nanoparticles with A549 cells demonstrated enhanced anti-tumor efficiency. These results suggest potential clinical applications of the obtained safe spongosome formulations.

Baicalin loaded in folate-PEG modified liposomes for enhanced stability and tumor targeting.

Bioavailability of baicalin (BAI), an example of traditional Chinese medicine, has been modified by loading into liposome. Several liposome systems of different composition i.e., lipid/cholesterol (L), long-circulating stealth liposome (L-PEG) and folate receptor (FR)-targeted liposome (L-FA) have been used as the drug carrier for BAI. The obtained liposomes were around 80 nm in diameter with proper zeta potentials about -25 mV and sufficient physical stability in 3 months. The entrapment efficiency and loading efficiency of BAI in the liposomes were 41.0-46.4% and 8.8-10.0%, respectively. The morphology details of BAI lipsosome systems i.e., formation of small unilamellar vesicles, have been determined by cryogenic transmission electron microscopy (cryo-TEM) and small angle X-ray scattering (SAXS). In vitro cytotoxicity of BAI liposomes against HeLa cells was evaluated by MTT assay. BAI loaded FR-targeted liposomes showed higher cytotoxicity and cellular uptake compared with non-targeted liposomes. The results suggested that L-FA-BAI could enhance anti-tumor efficiency and should be an effective FR-targeted carrier system for BAI delivery.

Elucidation of density profile of self-assembled sitosterol + oryzanol tubules with small-angle neutron scattering.

Small-angle neutron scattering (SANS) experiments have been performed on self-assembled tubules of sitosterol and oryzanol in triglyceride oils to investigate details of their structure. Alternative organic phases (deuterated and non-deuterated decane, limonene, castor oil and eugenol) were used to both vary the contrast with respect to the tubules and investigate the influence of solvent chemistry. The tubules were found to be composed of an inner and an outer shell containing the androsterol group of sitosterol or oryzanol and the ferulic acid moieties in the oryzanol molecule, respectively. While the inner shell has previously been detected in SAXS experiments, the outer shell was not discernible due to similar scattering length density with respect to the surrounding solvent for X-rays. By performing contrast variation SANS experiments, both for the solvent and structurant, a far more detailed description of the self-assembled system is obtainable. A model is introduced to fit the SANS data; we find that the dimensions of the inner shell agree quantitatively with the analysis performed in earlier SAXS data (radius of 39.4 +/- 5.6 angstroms for core and inner shell together, wall thickness of 15.1 +/- 5.5 angstroms). However, the newly revealed outer shell was found to be thinner than the inner shell (wall thickness 8.0 +/- 6.5 angstroms). The changes in the scattering patterns may be explained in terms of the contrast between the structurant and the organic phase and does not require any subtle indirect effects caused by the presence of water, other than water promoting the formation of sitosterol monohydrate in emulsions with aqueous phases with high water activity.