Effect of Nanoparticle Surface on the HPLC Elution Profile of Liposomal Nanoparticles.

PURPOSE: Nanoparticles have been used in diverse areas, and even broader applications are expected in the future. Since surface modification can influence the configuration and toxicity of nanoparticles, a rapid screening method is important to ensure nanoparticle quality. METHODS: We examined the effect of the nanoparticle surface morphology on the HPLC elution profile using two types of 100-nm liposomal nanoparticles (AmBisome(Ⓡ) and DOXIL(Ⓡ)). RESULTS: These 100-nm-sized nanoparticles eluted before the holdup time (about 4 min), even when a column packed with particles with a relatively large pore size (30 nm) was used. The elution time of the nanoparticles increased with pegylation of the nanoparticles and protein adsorption to the nanoparticles; however, the nanoparticles still eluted before the holdup time. CONCLUSIONS: The results of this study indicate that HPLC is a suitable tool for rapid evaluation of the surface of liposomal nanoparticles.

Rapid evaluation of the quantity of drugs encapsulated within nanoparticles by high-performance liquid chromatography in a monolithic silica column.

Drug-containing nanoparticles, the foundation of nanomedicine, provide promise for the safe and effective delivery of drugs to their targets. In this study, we developed a simple method to determine the relative quantities of nanoparticle-encapsulated drugs by HPLC using a commercially available monolithic silica column. Amphotericin B- and irinotecan-containing nanoparticles produced nearly simultaneous elution peaks (~7 min), suggesting that elution was largely driven by hydrodynamic effects and was relatively unaffected by differences in the encapsulated drug. A good correlation was observed between the intensity of the nanoparticle peak and the relative quantity of encapsulated drug. We used our method to characterize the effects of drug quantity and nanoparticle size on drug encapsulation rates within the nanoparticles. Encapsulation increased with increasing quantities of the drug in the preparation solution. This effect was greater for irinotecan than for amphotericin B. Although absolute encapsulation also increased with increasing nanoparticle size, encapsulation efficiency decreased. Thus, the monolith column is suitable for evaluating nanomedicine quality and may be used to evaluate many kinds of nanomaterials. Graphical Abstract Evaluation method of quantity of drug encapsulated within nanoparticles was developed. The method can be applicable for a rapid quality assurance of nanomedicine.

Comparison of the migration behavior of nanoparticles based on polyethylene glycol and silica using micellar electrokinetic chromatography.

Nanoparticles, spherical particles with diameters less than 100 nm, are promising theranostic devices for noninvasive diagnosis and therapy. In this study, nanoparticles composed of polyethylene glycol and silica were prepared, and their migration behavior was examined using capillary electrophoresis. The effects of the sodium dodecyl sulfate concentration in the electrolyte, the nanoparticle size, and the encapsulated molecule on the migration were examined. The addition of sodium dodecyl sulfate into the electrolyte had a significant effect on the electrophoretic mobility of polyethylene glycol nanoparticles, but a small effect on that of silica nanoparticles. As for the size effect, the mobility became a little faster for smaller nanoparticle sizes for both polyethylene glycol and silica nanoparticles. The encapsulated molecule affected the mobility of the nanoparticles through interactions between the encapsulated molecules and sodium dodecyl sulfate. We propose that the large effect of sodium dodecyl sulfate on the migration of the polyethylene glycol nanoparticles was due to the large spaces within the nanoparticles. These results indicate that nanoparticle migration is mainly determined by the nanoparticle components.

A computer simulation of the networked structure of a hydrogel prepared from a tetra-armed star pre-polymer.

We used a coarse-grained (CG) molecular dynamics model with potentials convertible to actual units to simulate the polymerization of a gel of a tetra-armed poly(ethylene glycol) derivative (MW ≈ 6000) under aqueous conditions and analysed its three-dimensional network structure. The radius of gyration of individual pre-polymers after gelation was slightly increased compared with that of the single pre-polymer before gelation, and its distribution was broad, attributable to inter- and intra-molecular bonds. The largest pores in the unit cell were about 3.5-3.9 nm. The existence of large pores seems to explain the protein encapsulation capability of and protein leakage from the gel indicating that the CG simulation, which maintains information about potentials in actual units, is an effective tool for investigating gel properties that are difficult to measure in real experiments.

Simultaneous analysis of nanoparticles and small molecules by high-performance liquid chromatography using a silica monolithic column.

A high-performance liquid chromatography method using a commercially available silica monolithic column for the simultaneous analysis of nanoparticles and small molecules was developed. The method uses the micrometer-sized flow-through pores and nanometer-sized mesopores of the monolithic column for separation: first, size separation of nanoparticles was performed by the micrometer-sized pores using the hydrodynamic mode, and then small molecules were separated by the nanometer-sized pores using the normal-phase mode. The method was used to evaluate and compare three existing methods for purifying nanoparticles and to analyse nanoparticle stability. The bimodal structure of the monolithic column is promising for the simultaneous separation of nanoparticles and small molecules.

Recent advances in development and application of derivatization reagents having a benzofurazan structure: a brief overview.

Chemical derivatization is often used to improve the separation efficiency and to enhance the detectability of the target compounds in high-performance liquid chromatography and capillary electrophoresis. The derivatization reagents having a benzofurazan (2,1,3-benzoxadiazole) structure are one of the most often used reagent for this purpose. In this paper, the recent advances in the development and the application of benzofurazan derivatization reagents are reviewed.

Reduction of molecular leaching from a gel matrix for the precisely controlled release of encapsulated molecules by light stimulus.

We have developed a general method for controlling molecular functions using a photodegradable hydrogel; gels containing molecules made from such materials are capable of release and activation by light stimulus. As the elimination of molecular leaching from the gel before irradiation was a barrier to the precise control of molecular function, we optimized the monomer used in gel preparation during this study. The addition of N,N'-methylenebis(acrylamide) (MBAA) inhibited molecular leaching from the gel; the MBAA concentration is a critical factor in controlling the leaching of encapsulated molecules. We succeeded in preparing a gel that halved the leaching of small encapsulated molecules, while the leaching of large molecules, such as albumin (66 kDa) and ferritin (450 kDa), was at negligible levels, or disappeared. The on/off ratios (released amount/leached amount) of albumin and ferritin were 8 and 17, respectively.

Ticlopidine, a cholestatic liver injury-inducible drug, causes dysfunction of bile formation via diminished biliary secretion of phospholipids: involvement of biliary-excreted glutathione-conjugated ticlopidine metabolites.

The antiplatelet drug, ticlopidine (TIC), reportedly causes cholestatic liver injuries. The present study analyzed the effect of TIC on bile formation, revealing that the biliary secretion of phospholipids was significantly decreased in TIC-administered Sprague Dawley (SD) rats. However, the effect of TIC on biliary phospholipids was not observed in SD rats pretreated with diethylaminoethyl diphenylpropylacetate that inhibits cytochrome P450s (P450), or in Eisai hyperbilirubinemic rats (EHBR) lacking functional multidrug resistance-associated protein 2 (MRP2/ABCC2). These results suggest that glutathione-conjugated TIC metabolites (TIC-SGs), which were formed in the liver after P450s-mediated metabolism and were excreted extensively into bile by MRP2, mediated the observed alterations of the bile composition. Administration of TIC caused significant liver injuries in SD rats, with decreased biliary phospholipids, but not in EHBR, consistent with the in vitro observation that phospholipid-bile acid-mixed micelles moderated the cytotoxic effects of bile acids. Further analyses revealed that TIC-SGs did not directly inhibit multidrug resistance 3 P-glycoprotein (MDR3/ABCB4)-mediated phosphatidylcholine efflux in vitro. Because the diminished biliary secretion of phospholipids with TIC administration was restored by taurocholate infusion in SD rats, the decreased biliary concentration of bile acids, due to the stimulation of bile acid-independent bile flow driven by TIC-SGs, might have indirectly attenuated phospholipid secretion. In conclusion, extensive biliary excretion of TIC-SGs decreased the biliary secretion of phospholipids, which might have increased the risk of TIC-induced cholestatic liver injury.

Small mesh size hydrogel for functional photocontrol of encapsulated enzymes and small probe molecules.

Previously, we developed the "protein activation and release from cage by external light" (PARCEL) method for controlling the function of proteins by encapsulating them in a photodegradable hydrogel and subsequently releasing them by ultraviolet (UV) irradiation of the gel. However, controlling small proteins is difficult because small proteins can leak from the gap (ca. 12.4 nm) of the mesh structure of the hydrogel without irradiation. Here, we developed a photodegradable gel with a smaller mesh size (~3.6 nm) and used the new gel to control the function of three small enzymes (trypsin, chymotrypsin, and elastase) and several small nonprotein molecules. The new gel showed reduced leakage of the proteins without irradiation, and tryptic activity increased approximately 78-fold upon irradiation of gel-encapsulated trypsin. The new gel also permitted encapsulation and release of 4',6-diamidino-2-phenylindole (DAPI, molecular weight 277), a small DNA-specific fluorescent probe. After irradiation to the gel-encapsulated DAPI and subsequent addition of DNA, strong fluorescence of the DAPI-DNA complex was observed. Our results indicate that reducing the gel mesh size from 12.4 to 3.6 nm should allow the encapsulation of various proteins and small molecules in an inactive state and their subsequent light-induced release. We expect this method to be useful in preparation of photoactivated biosensors, drug delivery systems, and catalysis.

A surfactant-based, regularly arrayed nanostructure gel matrix for migration of small molecules.

The preparation of nanometer-scale pores, or nanopores, has become easy because of the progress in nanotechnology. Surfactants are promising materials for the preparation of nanostructures containing nanopores, because surfactants form many different phase structures, including cubic, micellar, and lamellar structures. We prepared a gel matrix with a cubic structure from a commercially available surfactant, polyoxyethylene(50) lauryl ether (C12EO50, Adekatol LA-50). This gel matrix had regularly arrayed nanopores between the packed spherical micelles. We used the gel to separate biomolecules by means of slab gel electrophoresis. The gel was applicable to migration of amino acids and peptides; however, larger molecules, such as proteins and single-walled carbon nanotubes, did not migrate through the gel. We concluded that the pore size was too small for the penetration of large molecules, and that only small molecules could penetrate the gel matrix. The migration mechanism of small molecules was similar to that observed in conventional gel electrophoresis. We concluded that the gel matrix prepared from surfactant is a promising matrix for migration and purification of small molecules. We also expect that the gel can be used as a nanoscale filter to trap large molecules, allowing only small molecules to pass.

Itraconazole-induced cholestasis: involvement of the inhibition of bile canalicular phospholipid translocator MDR3/ABCB4.

Biliary secretion of bile acids and phospholipids, both of which are essential components of biliary micelles, are mediated by the bile salt export pump (BSEP/ABCB11) and multidrug resistance 3 P-glycoprotein (MDR3/ABCB4), respectively, and their genetic dysfunction leads to the acquisition of severe cholestatic diseases. In the present study, we found two patients with itraconazole (ITZ)-induced cholestatic liver injury with markedly high serum ITZ concentrations. To characterize the effect of ITZ on bile formation in vivo, biliary bile acids and phospholipids were analyzed in ITZ-treated rats, and it was revealed that biliary phospholipids, rather than bile acids, were drastically reduced in the presence of clinically relevant concentrations of ITZ. Moreover, by using MDR3-expressing LLC-PK1 cells, we found that MDR3-mediated efflux of [¹4C]phosphatidylcholine was significantly reduced by ITZ. In contrast, BSEP-mediated transport of [³H]taurocholate was not significantly affected by ITZ, which is consistent with our in vivo observations. In conclusion, this study suggests the involvement of the inhibition of MDR3-mediated biliary phospholipids secretion in ITZ-induced cholestasis. Our approach may be useful for analyzing mechanisms of drug-induced cholestasis and evaluating the cholestatic potential of clinically used drugs and drug candidates.

Diagnosis of glutaric aciduria type 1 by measuring 3-hydroxyglutaric acid in dried urine spots by liquid chromatography tandem mass spectrometry.

Accumulation of glutaric acid (GA) and 3-hydroxyglutaric acid (3HGA) in body fluids is the biochemical hallmark of type 1 glutaric aciduria (GA1), a disorder characterized by acute striatal degeneration and a subsequent dystonia. To date, methods for quantification of 3HGA are mainly based on stable isotope dilution gas chromatography mass spectrometry (GC-MS) and require extensive sample preparation. Here we describe a simple liquid chromatography tandem MS (LC-MS/MS) method to quantify this important metabolite in dried urine spots (DUS). This method is based on derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). Derivatization was adopted to improve the chromatographic and mass spectrometric properties of the studied analytes. Derivatization was performed directly on a 3.2-mm disc of DUS as a sample without extraction. Sample mixture was heated at 60°C for 45 min, and 5 µl of the reaction solution was analyzed by LC-MS/MS. Reference ranges obtained were in excellent agreement with the literature. The method was applied retrospectively for the analysis of DUS samples from established low- and high-excreter GA1 patients as well as controls (n = 100). Comparison of results obtained versus those obtained by GC-MS was satisfactory (n = 14). In populations with a high risk of GA1, this approach will be useful as a primary screening method for high- or low-excreter variants. In these populations, however, DUS analysis should not be implemented before completing a parallel comparative study with the standard screening method (i.e., molecular testing). In addition, follow-up DUS GA and 3HGA testing of babies with elevated dried blood spot C5DC acylcarnitines will be useful as a first-tier diagnostic test, thus reducing the number of cases requiring enzymatic and molecular analyses to establish or refute the diagnosis of GA1.

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry.

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.

Compounds having thiourea moiety as derivatization reagents in liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS): synthesis of derivatization reagents for carboxylic acids.

The derivatization reagents for carboxylic acids, N-(Pyridin-3-yl)hydrazinecarbothioamide, N-[4-(dimethylamino)phenyl]hydrazinecarbothioamide, 1-(2-aminoethyl)-3-(pyridin-3-yl)thiourea, 1-(2-aminoethyl)-3-[4-(dimethylamino)phenyl]thiourea and 4-(2-aminoethyl)-N-phenylpiperazine-1-carbothioamide were synthesized. These reagents reacted with carboxylic acids at 60°C for 45 min in the presence of the condensation reagents. The generated derivatives were favorably separated on the reversed-phase column and sensitively detected by electrospray ionization tandem mass spectrometry. These reagents enhanced the electrospray ionization response of the analyte and generated a particular product ion efficiently by collision-induced dissociation, and thus they were suitable for MS/MS detection.

Isothiocyanates as derivatization reagents for amines in liquid chromatography/electrospray ionization-tandem mass spectrometry.

The applicability of 3-pyridyl isothiocyanate, p-(dimethylamino)phenyl isothiocyanate and m-nitrophenyl isothiocyanate as the derivatization reagents for amines in high-performance liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) was examined. The generated derivatives of amines with these reagents were favorably separated on the reversed-phase column and detected by ESI-MS/MS. The C-N bond of the generated thiourea structure was efficiently cleaved by collision-induced dissociation and gave the single and intense product ion. Among the three reagents, 3-pyridyl isothiocyanate was the most suitable as the derivatization reagent with regard to the reactivity to amines and the detection sensitivity.

Synthesis of benzofurazan derivatization reagents for short chain carboxylic acids in liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS).

Benzofurazan derivatization reagents, 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminopentylamino)-2,1,3-benzoxadiazole (DAABD-AP) and 4-[2-(N,N-dimethylamino) ethylaminosulfonyl]-7-(2-aminobutylamino)-2,1,3-benzoxadiazole (DAABD-AB), for short-chain carboxylic acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) were synthesized. These reagents reacted with short chain carboxylic acids in the presence of the condensation reagents at 60 degrees C for 60 min. The generated derivatives were separated on the reversed-phase column and detected by ESI-MS/MS with the detection limits of 0.1-0.12 pmol on column. Upon collision-induced dissociation, a single and intense product ion at m/z 151 was observed. These results indicated that DAABD-AP and DAABD-AB are suitable as the derivatization reagents in LC/ESI-MS/MS analysis.

High performance liquid chromatography analysis of 100-nm liposomal nanoparticles using polymer-coated, silica monolithic columns with aqueous mobile phase.

Recently, nanoparticles have garnered considerable attention, and the demand for a rapid and simple method for their analysis has increased accordingly. The bimodal pores (few µm- and few tens nm-sized pores) of monolithic columns were thought to be suitable for the separation of nanoparticles and small molecules; however, the residual silanol groups on the column surface resulted in the strong adsorption of liposomes and hindered their analysis. To overcome this problem, we modified the surface of the silica monolith via a two-step process and developed three silica monolithic columns coated with three different polymers: glycidyl methacrylate (GMA), 2-hydroxyethyl methacrylate (HEMA), and N-vinylpyrrolidone (VP). These were used for the analysis of 100-nm liposomal nanoparticles. Since 15% polymer coating prevented the nanoparticle adsorption, liposomes (AmBisome®) and pegylated liposomes (DOXIL®) were eluted rapidly (within 1min) using these columns, without using organic solvents in the mobile phase. Molecular leaching from the liposomes, as well as protein adsorption to the liposomes (corona formation) could be evaluated using the polymer-coated columns, thus illustrating their utility in the rapid and simple analysis of 100-nm liposomal nanoparticles.

Synthesis and properties of a new fluorescent bicyclic 4-N-carbamoyldeoxycytidine derivative.

[reaction: see text] A bicyclic 4-N-carbamoyldeoxycytidine derivative (1, dC(hpp)) geometrically locked was synthesized as a new fluorescent nucleobase. The hybridization properties of oligodeoxynucleotides containing dC(hpp) were investigated by use of T(m) analysis. It was found that dC(hpp) forms stable base pairs not only with the complementary guanine base, but also with the adenine base. Interestingly, the fluorescence of dC(hpp) was suppressed only when a dC(hpp)-dG base pair was formed.

Surface modification of silica nanoparticles using 4-aryloxy boron dipyrromethene (BODIPY) enhances skin permeation.

4-Aryloxy boron dipyrromethene (BODIPY) modification of the surface of silica nanoparticles (NPs) improved permeability through the membrane of HaCaT skin cells and swine skin tissue. The 35 nm BODIPY-modified NPs penetrated tape-stripped skin and reached the dermis within 1 h. Since these NPs can encapsulate a variety of molecules including macromolecules, they are expected to serve as effective carriers for the delivery of drugs, genes, and other compounds through skin and into cells.

Fluorescence enhancement by hydroperoxides based on a change in the intramolecular charge transfer character of benzofurazan.

Strong fluorescence signals were observed after the reaction of novel reagents with hydroperoxides.