Water-Based Synthesis of ß-Sheet-Like Supramolecular Metallohydrogel Organized by Using a Native Ultrashort Peptide Sequence.

Designing supramolecular hydrogels using short peptides is challenging. To control self-assembly, a certain amount of organic solvent is typically added to the system, or the short peptide is modified with a functional group that is hydrophobic, hydrophilic, or highly coordinative. We discovered that l-His-l-Ile-l-Thr (HIT), a very short unmodified "native" tripeptide, selectively responds to Cu2+ ions in pure water to form a transparent supramolecular metallohydrogel. Circular dichroism analysis revealed that Cu2+ ions, but no other metal species, caused HIT to change from a random-coil-like to a ß-sheet-like structure. Other spectroscopic methods were used to characterize the properties of the supramolecular metallohydrogel. These results are expected to facilitate the development of native short peptides as advanced functional biomaterials.

Enhanced water dispersibility and Caco-2 cell monolayer permeability of quercetin by complexation with casein hydrolysate.

Although quercetin (Que) has many beneficial therapeutic effects on the human body, its oral bioavailability is poor because of its low water solubility. Herein, we describe the preparation of casein hydrolysate (Pepcas ) and its use as a dispersant to enhance the water dispersibility of Que resulting in an improvement of its bioavailability. By complexation with Pepcas , the apparent solubility of Que in the complex (Que@Pepcas ) was significantly increased under acidic and neutral conditions. Que@Pepcas was pre-digested in vitro using a simulated digestive juice, and a Caco-2 cell monolayer permeation assay was performed to evaluate the oral bioavailability of Que. The cumulative amount of Que that permeated the monolayer using Que@Pepcas after 8, 16, and 24 h was increased compared with a blank sample (Que@Blank), and the apparent permeation coefficient Papp of Que@Pepcas was approximately 2.6-fold better than that of Que@Blank. PRACTICAL APPLICATION: Pepcas can be used as a high absorption supplement and food additive, and our Pepcas complexation technique should be widely applicable to not only Que but also to other poorly water-soluble substances.

Dominant factors that determine the dissolution state of complexes between poorly water-soluble ingredients and casein hydrolysate.

Casein hydrolysate (Pep) is a dispersant for poorly water-soluble drugs and nutraceutical ingredients. However, two types of complexes may be between Pep and poorly water-soluble molecules: those that are (1) dispersed as hydrocolloids in aqueous media with a particle size of 100-500 nm; and (2) not hydrocolloids, as indicated by permeability of the complex through an ultrafiltration (UF) membrane and the fact that the particle size is ambiguous by dynamic light scattering. This study was conducted to clarify the factors that determine the dissolution state of the complexes between poorly water-soluble ingredients and casein hydrolysate. We classified the dissolution state of the complexes between poorly water-soluble ingredients and Pep by the permeability using a UF membrane. Complexes containing larger and more-hydrophobic molecules are hydrocolloids and do not permeate the UF membrane, whereas complexes containing comparatively smaller and less-hydrophobic molecules do permeate the UF membrane. A complex containing indomethacin, which has borderline solubility properties, does not permeate the UF membrane at pH values in which the indomethacin carboxylic acid group is protonated, but it is permeable at pH values in which its carboxylic acid group is deprotonated. Furthermore, we determined the stoichiometry and association constant for the complex between a major peptide in Pep and poorly water-soluble resveratrol (Res) based on fluorescence quenching. We calculated the stoichiometry of Res and PepY to be 2:3 and the association constant to be 2.4 × 107 M-1.

Enhanced water dispersibility and permeability through a Caco-2 cell monolayer of ß-cryptoxanthin extracted from kumquats by complexation with casein.

ß-Cryptoxanthin (BCX) possesses potential therapeutic and health benefits. However, BCX absorption is low because of its poor aqueous solubility. In this study, a complex between BCX and casein (Cas) was prepared to improve the water dispersibility and bioavailability of BCX. BCX was recovered quantitatively from freeze-dried kumquat powder through solid-liquid extraction and saponification. The complexation significantly improved the apparent solubility of BCX under acidic and neutral conditions. A cell membrane permeation test using a Caco-2 cell monolayer was performed to evaluate the bioavailability of the BCX-Cas complex. This complex and a blank sample were digested in vitro and added to the apical side of the Caco-2 cell membrane. The quantity of BCX that permeated using the BCX-Cas complex after 24 h was 22.7 times greater than that of the blank. Thus, complexation of BCX with Cas improved dramatically the bioavailability of BCX from a kumquat extract.

Poly(amine-co-ester) nanoparticles for effective Nogo-B knockdown in the liver.

Degradable poly(amine-co-ester) (PACE) terpolymers hold tremendous promise for siRNA delivery because these materials can be formulated into delivery vehicles with highly efficient siRNA encapsulation, providing effective knockdown with low toxicity. Here, we demonstrate that PACE nanoparticles (NPs) provide substantial protein knockdown in human embryonic kidney cells (HEK293) and hard-to-transfect primary human umbilical vein endothelial cells (HUVECs). After intravenous administration, NPs of solid PACE (sPACE)-synthesized with high monomer content of a hydrophobic lactone-accumulated in the liver and, to a lesser extent, in other tissues. Within the liver, a substantial fraction of sPACE NPs were phagocytosed by liver macrophages, while a smaller fraction of NPs accumulated in hepatic stellate cells and liver sinusoidal endothelial cells, suggesting that sPACE NPs could deliver siRNA to diverse cell populations within the liver. To test this hypothesis, we loaded sPACE NPs with siRNA designed to knockdown Nogo-B, a protein that has been implicated in the progression of alcoholic liver disease and liver fibrosis. These sPACE:siRNA NPs produced up to 60% Nogo-B protein suppression in the liver after systemic administration. We demonstrate that sPACE NPs can effectively deliver siRNA therapeutics to the liver to mediate protein knockdown in vivo.

Development of highly water-dispersible complexes between coenzyme Q10 and protein hydrolysates.

The water-solubility of coenzyme Q10 (CoQ10) is extremely low because of its hydrophobicity, which results in low bioavailability. In this study, peptide mixtures derived from different proteins (casein, albumin, gelatin, lysozyme, zein and hemoglobin) were prepared and used as dispersants ofCoQ10. Most peptide mixtures, except for that derived from lysozyme, enhanced the water-dispersibility of CoQ10 by forming complexes with CoQ10. In particular, the apparent solubility of CoQ10complexed with the casein hydrolysate (Pepcas) was the highest. Pepcas was fractionated by ammonium sulfate precipitation and ultrafiltration to find the most effective peptides for enhancing water-dispersibility of CoQ10. The peptide fraction that had a relatively hydrophobic nature and peptides of higher-than-average molecular weights (PepcasA) was found to be the most effective. Results from differential thermal analysis and powder X-ray diffraction revealed that the complex between CoQ10 and PepcasA in the solid state was amorphous. In solution, the complex is a hydrocolloid with particle sizes of 154-279 nm.

Improvements in the water dispersibility of paclitaxel by complexing with synthetic peptides derived from ß-casein.

Recently, digestive peptides prepared as a casein hydrolysate have been found to be an effective dispersant for the poorly water-soluble drug paclitaxel (Ptx). A major hydrophobic peptide in the digested peptides was identified as YQEPVLGPVRGPFPIIV (PepY) by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry with the "LIFT" technique. In the present study, three peptides PepY, VVVPPFLQPEVMGVSKV (PepV), and KFQSEEQQQTEDELQDK (PepK) were chemically synthesized by Fmoc solid-phase synthesis to compare their function as dispersants for Ptx. PepV and PepK are the most hydrophobic and hydrophilic peptides, respectively, in the sequence of ß-casein, which are the same length as PepY (PepY, PepV, and PepK are abbreviated as Peps). The complex between Ptx and Peps (Ptx-Peps) was prepared by mixing an ethanol solution of Ptx and an aqueous solution of Peps, followed by lyophilization. The complex with PepV, which is estimated to be the most hydrophobic of the peptides, had the greatest ability to improve the water dispersibility of Ptx. The water dispersibility of the complexes between Ptx and PepY and PepV increased as the amount of the peptides increased, whereas PepK was not effective in enhancing the dispersibility of Ptx. Furthermore, a peptide mixture obtained from a casein hydrolysate [Pep (fraction A)] was more effective for the enhancement of Ptx dispersibility than the single peptide PepY. These results suggests that a variety of peptides in the casein hydrolysate contribute toward complexation with Ptx.

Enhancing the water dispersibility of paclitaxel by complexation with hydrophobic peptides.

The complex between paclitaxel (Ptx) and a peptide mixture (Pep) was prepared to enhance of the water-dispersibility of Ptx. Pep was prepared by enzymatic hydrolysis of casein, followed by fractionation using ammonium sulfate precipitation and ultrafiltration. The Ptx and Pep complex (Ptx-Pep) was prepared by mixing an ethanol solution of Ptx and an aqueous solution of Pep followed by lyophilization. The water dispersibility test of Ptx-Pep prepared using different fractions of Pep demonstrated that a fraction (Pep-A), containing relatively hydrophobic peptides with high molecular weights, was effective in enhancing the water dispersibility of Ptx. The sequences of the major peptides in Pep-A were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry with "LIFT" technique. The water dispersibility of the complex between Ptx and Pep-A (Ptx-Pep-A) was independent of pH, even though it is positively or negatively charged under strongly acidic and neutral conditions. As the particle size of Ptx-Pep-A in aqueous media was 147-215 nm, Ptx-Pep-A was present as a hydrocolloidal material in aqueous media.

Enhancement of water solubility of indomethacin by complexation with protein hydrolysate.

Complex formation between indomethacin (Indo) and casein hydrolysate was developed as a novel technique for enhancing the water solubility of Indo. The complex (Indo-Pep) was prepared by mixing an ethanol solution of Indo and an aqueous solution of peptide mixture, followed by lyophilization. The water solubility of Indo-Pep under weakly acidic and neutral conditions is much higher than that of Indo alone. The water solubility of Indo increased with increasing quantity of peptide. Characterization of Indo-Pep using scanning electron microscopy, differential scanning calorimetry, and X-ray diffraction showed that Indo was incorporated in Indo-Pep in an amorphous state. The fluorescence quenching of Indo-Pep also suggested complexation between Indo and the peptides. An aqueous solution of Indo-Pep was fractioned by centrifugation followed by filtration using membrane filters and ultrafilters. Analysis of the fractions by dynamic light scattering and ultraviolet-visible spectroscopy showed that Indo-Pep consisted of small particles and was not a hydrocolloidal material.