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.

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.

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.

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.

Rapid and mild purification method for nanoparticles from a dispersed solution using a monolithic silica disk.

A rapid and mild purification method for nanoparticles using the commercially available monolithic silica disk, MonoSpin(®), was developed. The nanoparticles were purified from a dispersed solution by filtration with the aid of centrifugation at 2290×g for 2min. The purification conditions were rapid, mild, and simple compared with those of the conventional purification methods such as ultracentrifugation, dialysis, size exclusion chromatography, and ultrafiltration. The method was shown to be applicable for the purification of various nanoparticles, regardless of their size (from 21 to 100nm), composition material (silica, polyethylene glycol, and pegylated liposome), and encapsulated molecule (rhodamine 110 and doxorubicin). It was shown that this method is applicable to the purification of a wide range of nanoparticles in many different fields.

Spatiotemporal activation of molecules within cells using silica nanoparticles responsive to blue-green light.

The spatiotemporal control of molecular function is important but there are currently few techniques for noninvasively controlling various types of molecules in live cells. Herein we developed nanoparticles with a boron dipyrromethene structure, which are responsive to blue-green visible light. Fluorophores (fluorescein, rhodamine B, and Nile blue A) encapsulated within the nanoparticles were released by irradiation for 3 min with visible light. Nanoparticles were internalised by HeLa cells without the aid of a cell-penetrating peptide, serving as vehicles for the delivery of cargo molecules to the cytoplasm. The release and activation of encapsulated molecules by visible light irradiation demonstrate a novel method for the spatiotemporal control of molecular function that can be used to activate molecules inside the skin that cannot be reached by UV light, which has limited tissue penetration.

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.

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.

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.

Analysis of methylcitrate in dried blood spots by liquid chromatography-tandem mass spectrometry.

Accumulation of propionylcarnitine (C3) in neonatal dried blood spots (DBS) is indicative of inborn errors of propionate metabolism including propionic acidemia (PA), methylmalonic aciduria (MMA), and cobalamin (Cbl) metabolic defects. Concentrations of C3 in affected newborns overlap with healthy individuals rendering this marker neither specific nor sensitive. While a conservative C3 cutoff together with relevant acylcarnitines ratios improve screening sensitivity, existing mass spectrometric methods in newborn screening laboratories are inadequate at improving testing specificity. Therefore, using the original screening DBS, we sought to measure 2-methylcitric acid (MCA), a pathognomonic hallmark of C3 disorders to decrease the false positive rate and improve the positive predictive value of C3 disorders. MCA was derivatized with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). No separate extraction step was required and derivatization was performed directly using a 3.2-mm disc of DBS as a sample (65°C for 45 min). The reaction mixture was analyzed by liquid chromatography tandem mass spectrometry. MCA was well separated and eluted at 2.3 min with a total run time of 7 min. The median and (range) of MCA of 0.06 µmol/L (0-0.63) were in excellent agreement with the literature. The method was applied retrospectively on DBS samples from established patients with PA, MMA, Cbl C, Cbl F, maternal vitamin B12 deficiency (n = 20) and controls (n = 337). Comparison with results obtained by another method was satisfactory (n = 252). This method will be applied as a second tier test for samples which trigger positive PA or MMA results by the primary newborn screening method.

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.

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.

Development of a spatiotemporal method to control molecular function by using silica-based photodegradable nanoparticles.

To effectively and safely use molecules, it is important to be able to control the timing and site of molecule activation. We developed a spatiotemporal method to control molecular function by using silica-based photodegradable nanoparticles that can be prepared under mild conditions. The function of various molecules, such as rhodamine B, Nile blue A, propidium iodide (PI), and rhodamine 110, bis-(N-CBZ-l-arginine amide), dihydrochloride (BZAR), was restricted by wrapping in the network structure of the nanoparticle gel. The encapsulated molecule was released from the gel by the light stimulus and its function was restored. Hence, this technique is applicable to the functional control of various molecules. The PI-encapsulated nanoparticles were internalized by the cells after being conjugated with the cell membrane permeability peptide, octaarginine, and were localized to the cytoplasm. Short-term irradiation (20 s) induced PI release from the nanoparticles and the rapid movement (less than 2 min) of the released PI to the nucleus. These nanoparticles are thus useful tools for the spatiotemporal control of various molecular functions because they permit the quick and transient release of encapsulated molecules after short-term irradiation and can be prepared under mild conditions.

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.

The simple preparation of polyethylene glycol-based soft nanoparticles containing dual imaging probes.

We developed a simple method to prepare PEG-based soft nanoparticles that encapsulate dual imaging probes. Because the probes could be encapsulated by either chemical or physical means, a variety of probe molecules were encapsulated within the nanoparticles simultaneously. The nanoparticles were administrated to mice and the pharmacokinetics of the nanoparticles was analyzed by means of MRI, fluorescence spectroscopy, and transmission electron microscopy. The soft nanoparticles were excreted by the mice rapidly through the urine without collapse of the nanoparticles and without leaking of the probe molecules, and no accumulation of the nanoparticles in the body was observed. The pharmacokinetics of the nanoparticles was not changed by the encapsulated molecules and acute toxicity to mice was negligible. It was expect that these PEG-based soft nanoparticles will be applicable for use as a safe diagnostic agent.

Delivery, stabilization, and spatiotemporal activation of cargo molecules in cells with positively charged nanoparticles.

Positively charged photodegradable nanoparticles that simultaneously encapsulated various compounds including small and large molecules were prepared. The nanoparticles were internalized to the cell by endocytosis and were stable within the cells for at least 4 days. The encapsulated molecules were released into the cytosol using light stimuli.

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.

[Development of advanced educational programs, including research programs, for undergraduate students in National Universities: the facts in 2010].

This article summarizes detailed facts obtained from the questionnaire conducted in 2010 at about 14 National Universities on the topic of "Research programs and advanced educational programs for undergraduate students". The contents of the questionnaire included: (1) Research programs based on the coalition of university and hospital and/or community pharmacy, other Graduate Schools, such as School of Medicine etc., and the University Hospital, (2) Educational systems for the achievement of research programs and their research outcomes, (3) Research programs based on pharmacist practices, (4) Ongoing advanced educational programs for undergraduate students, taking advantage of the coalition with Graduate School, School of Medicine (and Dentistry), and University Hospital. Some of the advanced educational programs outlined in this questionnaire will be carried out by our group in the coming years and the educational benefits together with associated problems shall as well be clarified. This approach will be informative for the development of the leader-oriented pharmacist programs for the college of Pharmacy.

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.

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.