Analytical Gel Permeation Research Articles

Eudragit® L100 Post-Modification via Ugi Multicomponent Reaction

This work investigated the post-modification of commercial poly(methyl mechacrylate-coacrylic acid) using the versatile multicomponent Ugi reaction. This reaction introduces diverse functionalities to tailor the polymer properties effectively. The commercial copolymer which is known for biocompatibility and drug delivery application was subjected to the Ugi reaction, altering its chemical structure. The structure of the modified polymer was checked by Fourier transform infrared spectroscopic and nuclear magnetic resonance and thermal characterizations, confirming successful Ugi component integration. Additionally, gel permeation chromatography assessed molecular weight changes. Results revealed enhanced thermal stability of the modified polymers compared to unmodified polymer, potentially advantageous in high-temperature applications. In summary, the study demonstrates efficient poly(methyl mechacrylate-co-acrylic acid) post-modification through a multicomponent Ugi reaction, yielding tailored polymers. Comprehensive spectroscopic, thermal, and gel permeation analyses shed light on structural changes and improved stability, pivotal for innovative applications like drug delivery.

Long-term monitoring of the biomass and production of lipids by Nitzschia palea for biodiesel production

Pennate diatom Nitzschia palea can be cultured in outdoor vertical-bed photobioreactors to produce biodiesel. To assess the production of biomass and lipids, non-axenic cultures of Nitzschia palea were grown outdoors, and the growth of these cultures was measured biweekly. During the annual cycle of algal culture, the culture temperature ranged from 17.3 °C to 33.5 °C, the dry weight biomass ranged from 0.11 g l−1 to 0.25 g l−1, light energy] ranged from 1.94 Wm−2 to 3.9 Wm−2 and intracellular lipid content ranged from 7.1% to 11.4% of biomass weight after drying at 60 °C. Gas chromatography/mass spectroscopy (GC/MS) analysis of n-hexane extracts showed that the intracellular lipids were primarily C14:0 myristic acid (9.01%), C15:0 pentadecyclic acid (8.26%) and two types of C16:0, palmitic acid (41.13%) and palmitoleic acid (29.25%). Gel permeation analysis showed that carboxylic acids comprised 28.9% of lipids, 16.3% of monoglycerides, 27.3% of diglycerides and 24.3% of triglycerides. Alcoholysis of lipids resulted in the conversion of about 93.9% of fatty acids to equivalent fatty acid methyl esters (FAME) or biodiesel, which, on basis of wt%, consisted primarily of C15:0 methyl myristate (8.3%), C16:0 methyl pentadecanoate] (7.2%), C17:1methyl palmitoleate (28.7%) and methyl palimtate](39.8%).

Tunable molecular weights of poly(triphenylamine‐2,2′‐bithiophene) and their effects on photovoltaic performance as sensitizers for dye‐sensitized solar cells

ABSTRACTDespite the huge progress achieved over the past decade, the relationship between the molecular weights of dyes and the performance of dye‐sensitized solar cells (DSSCs) remains unclear. In this article, we report on the fine control of the number‐average molecular weight (Mn) of poly(triphenylamine‐2,2′‐bithiophene) (PPAT) dyes with cyanoacrylic acid moieties as acceptors. We found a correlation between the Mn and photovoltaic performance of these polymers when they were used for DSSC applications. In this study, three samples (PPAT‐01, PPAT‐02, and PPAT‐03) with different Mn values (Mns = 1700, 2800, and 3500 g/mol) were prepared through the control of the polymerization time and characterized by analytical gel permeation chromatography and NMR. Under the same experimental conditions, the overall cell efficiency of the oligomer dyes showed a nonmonotonic tendency with increasing molecular weight. The power‐conversion efficiencies were 2.81% for PPAT‐01, 4.72% for PPAT‐02, and 1.88% for PPAT‐03. UV absorption measurements proved that PPAT‐03 formed aggregation, whereas PPAT‐01 and PPAT‐02 were in the monolayer state adsorbed on TiO2. The larger aggregation decreased charge transfer; thus, poor photoelectric conversion performance was observed. Furthermore, a higher molecular weight reduced the amount of PPAT‐03 adsorbed on TiO2, and this had a crucial effect on the performance of the cells because of the reduced photocurrent. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 44182.

Analysis of the Overall Structure of the Multi-Domain Amyloid Precursor Protein (APP)

The amyloid precursor protein (APP) and its processing by the α-, β- and γ-secretases is widely believed to play a central role during the development of Alzheimer´s disease. The three-dimensional structure of the entire protein, its physiologic function and the regulation of its proteolytic processing remain, however, largely unclear to date. To gain a deeper understanding of the structure of APP that underlies all of its functions, we first cloned and recombinantly expressed different constructs in E. coli. Using limited proteolysis followed by mass spectrometry and Edman degradation as well as analytical gel permeation chromatography coupled static light scattering, we experimentally analyzed the structural domain boundaries and determined that the large ectodomain of APP consists of exactly two rigidly folded domains – the E1-domain (Leu18-Ala190) and the E2-domain (Ser295-Asp500). Both, the acidic domain (AcD) connecting E1 and E2 as well as the juxtamembrane region (JMR) connecting E2 to the single transmembrane helix are highly flexible and extended. We identified in-between the E1-domain and the AcD an additional domain of conservation and partial flexibility that we termed extension domain (ED, Glu191-Glu227). Using Bio-layer interferometry, pull-down assays and analytical gel filtration experiments we demonstrated that the E1-domain does not tightly interact with the E2-domain, both in the presence and in the absence of heparin. APP hence forms an extended molecule that is flexibly tethered to the membrane. Its multi-domain architecture enables together with the many known functionalities the concomitant performance of several, independent functions, which might be regulated by cellular, compartment specific pH-changes.

Preparation and physicochemical properties of digested collagen fragments with varying molecular weights

The porcine dermal collagen molecules were successfully digested by clostridium histolyticun collagenases (CHC) and then ultra-filtrated using varying grades of ultra-filtration membranes. Gel permeation analyses revealed that collagen fragments with varying molecular weights were successfully segregated using ultra-filtration membranes with varying grades of pore sizes. Fourier transform infra-red analyses suggest that digested collagen fragments and digested collagen fragments prepared after ultra-filtration still preserve certain percentages of triple helix structures of collagen molecules, although the percentages of preserved triple helix structures present in digested and ultra-filtrated collagen fragments reduce significantly as their Mw values reduce. Thermal and denaturation temperature analysis suggest that denaturation temperature and thermal degradation temperature values of digested collagen fragments and ultra-filtrated collagen fragments decrease significantly as their Mw values reduce. The absorbed/desorbed amounts of digested and ultra-filtrated collagen fragments in PA6/PP flocking specimens are significantly higher than those of the original collagens, and increase significantly as their Mw values reduce. Possible reasons accounting for the above degradation, ultra-filtration physicochemical, absorbing and desorbing properties of original, digested and ultra-filtrated digested collagen molecules are reported.

Functional and Metabolic Effects of Adaptive Glycerol Kinase (GLPK) Mutants in Escherichia coli

Herein we measure the effect of four adaptive non-synonymous mutations to the glycerol kinase (glpK) gene on catalytic function and regulation, to identify changes that correlate to increased fitness in glycerol media. The mutations significantly reduce affinity for the allosteric inhibitor fructose-1,6-bisphosphate (FBP) and formation of the tetramer, which are structurally related, in a manner that correlates inversely with imparted fitness during growth on glycerol, which strongly suggests that these enzymatic parameters drive growth improvement. Counterintuitively, the glpK mutations also increase glycerol-induced auto-catabolite repression that reduces glpK transcription in a manner that correlates to fitness. This suggests that increased specific GlpK activity is attenuated by negative feedback on glpK expression via catabolite repression, possibly to prevent methylglyoxal toxicity. We additionally report that glpK mutations were fixed in 47 of 50 independent glycerol-adapted lineages. By far the most frequently mutated locus (nucleotide 218) was mutated in 20 lineages, strongly suggesting this position has an elevated mutation rate. This study demonstrates that fitness correlations can be used to interrogate adaptive processes at the protein level and to identify the regulatory constraints underlying selection and improved growth.

Pectin from Passion Fruit Fiber and Its Modification by Pectinmethylesterase

Passion fruit fiber pectin gels represent a new alternative pectin source with potential for food and non-food applications on a commercial scale. Pectic polysaccharides were extracted from passion fruit (Passiflora edulis) fiber using citric acid as a clean catalyst and autoclaved for 20 to 60 min at 121℃. The best condition of pectin yield with the highest molecular weight was obtained with 1.0% of citric acid (250 ㎎/g dry passion fruit fiber pectin) for 20 min of autoclaving. Spectroscopic analyses by Fourier transform infrared, enzymatic degradation reactions, and ion-exchange chromatography assays showed that passion fruit pectin extracted for 20 min was homogeneous high methoxylated pectin (70%). Gel permeation analysis confirmed that the pectin extract obtained by autoclaving by 20 min showed higher molecular weights than those autoclaved for 40 and 60 min. Passion fruit pectin extracted for 20 min was enzymatically modified with fungal pectinmethylesterase to create restructured gels. Short autoclave treatment (20 min) with citric acid as extractant resulted in a significant increase of gel strength, improving pectin extraction in terms of functionality. The treatment of solubilized material (pectic polysaccharides) in the presence of insoluble material (cellulose and hemicellulose) with pectinmethylesterase and calcium led to the creation of a stiffer passion fruit fiber pectin gel, while syneresis was not observed.

Controlled precipitation and purification of hemicellulose from DMSO and DMSO/water mixtures by carbon dioxide as anti-solvent

Anti-solvent precipitation of xylans and mannans from dimethylsulfoxide (DMSO) or DMSO/water mixtures, and subsequent drying with supercritical carbon dioxide (scCO 2) were developed into a useful technique for preparing spherical hemicellulose micro-particles. Depending on the type of hemicellulose, water content of DMSO, precipitation pressure and temperature, the particle size can be adjusted within a wide range from less than 0.1 to more than 5 μm. For example, fast super-saturation which can be achieved by applying supercritical conditions results in the formation of very small particles as mass transfer between the solvent DMSO and anti-solvent scCO 2 is reduced to a minimum. Anti-solvent precipitation from aqueous DMSO (e.g., 10% water) allows for processing distinctly larger amounts of hemicelluloses compared to pure DMSO without the necessity of increasing the precipitation pressure. The formation of an additional inert aqueous phase increases the mass transfer resistance, which results in the formation of larger, stable agglomerates. Curiepoint pyrolysis GC/MS, gel permeation chromatography (GPC) and analysis of the monosaccharide composition of both the parent hemicellulosic material and the corresponding precipitates demonstrated that hemicelluloses can be purified from residual lignin by supercritical anti-solvent precipitation with carbon dioxide without altering the structure of the biopolymers.

Novel minor lipase from Rhizopus chinensis during solid-state fermentation: Biochemical characterization and its esterification potential for ester synthesis

Rhizopus chinensis produces two lipases that catalyze ester synthesis when cultured under solid-state fermentation. The Lip2 was purified to homogeneity by ammonium sulphate precipitation, hydrophobic interaction chromatography and gel filtration chromatography. It has an apparent molecular weight of 33 kDa estimated from SDS–PAGE and 32 kDa calculated from analytical gel permeation, with synthetic activity and purification fold of 96.8 U/mg and 138.3, respectively. Maximum hydrolytic activity was obtained at pH 8.0–8.5 and 40 °C using pNPP as substrate. Slight activation of the enzyme was observed when Mn 2+ is present. The enzyme was most active on p-nitrophenyl laurate (C12). The purified lipase exhibited maximum synthetic activity at pH memory of 6.0 and 30 oC. Most of ethyl esters synthesized by lyophilized enzyme achieved good yields (>90%), and caprylic acid served as the best acyl donor. The enzyme presented a particular affinity for ethanol, n-propanol and n-hexanol, with conversion of 92%, 93% and 92%, respectively, after 20 h incubation.

ATP-driven Reduction by Dark-operative Protochlorophyllide Oxidoreductase from Chlorobium tepidum Mechanistically Resembles Nitrogenase Catalysis

During chlorophyll and bacteriochlorophyll biosynthesis in gymnosperms, algae, and photosynthetic bacteria, dark-operative protochlorophyllide oxidoreductase (DPOR) reduces ring D of aromatic protochlorophyllide stereospecifically to produce chlorophyllide. We describe the heterologous overproduction of DPOR subunits BchN, BchB, and BchL from Chlorobium tepidum in Escherichia coli allowing their purification to apparent homogeneity. The catalytic activity was found to be 3.15 nmol min(-1) mg(-1) with K(m) values of 6.1 microm for protochlorophyllide, 13.5 microm for ATP, and 52.7 microm for the reductant dithionite. To identify residues important in DPOR function, 21 enzyme variants were generated by site-directed mutagenesis and investigated for their metal content, spectroscopic features, and catalytic activity. Two cysteine residues (Cys(97) and Cys(131)) of homodimeric BchL(2) are found to coordinate an intersubunit [4Fe-4S] cluster, essential for low potential electron transfer to (BchNB)(2) as part of the reduction of the protochlorophyllide substrate. Similarly, Lys(10) and Leu(126) are crucial to ATP-driven electron transfer from BchL(2). The activation energy of DPOR electron transfer is 22.2 kJ mol(-1) indicating a requirement for 4 ATP per catalytic cycle. At the amino acid level, BchL is 33% identical to the nitrogenase subunit NifH allowing a first tentative structural model to be proposed. In (BchNB)(2), we find that four cysteine residues, three from BchN (Cys(21), Cys(46), and Cys(103)) and one from BchB (Cys(94)), coordinate a second inter-subunit [4Fe-4S] cluster required for catalysis. No evidence for any type of molybdenum-containing cofactor was found, indicating that the DPOR subunit BchN clearly differs from the homologous nitrogenase subunit NifD. Based on the available data we propose an enzymatic mechanism of DPOR.

Synthesis of Dendron-Protected Porphyrin Wires and Preparation of a One-Dimensional Assembly of Gold Nanoparticles Chemically Linked to the π-Conjugated Wires

A one-dimensional assembly of gold nanoparticles chemically bonded to pi-conjugated porphyrin polymers was prepared on a chemically modified glass surface and on an undoped naturally oxidized silicon surface by the following methods: pi-conjugated porphyrin polymers were prepared by oxidative coupling of 5,15-diethynyl-10,20-bis-((4-dendron)phenyl) porphyrin (6), and its homologues (larger than 40-mer) were collected by analytical gel permeation chromatography (GPC). The porphyrin polymers (>40-mer) were deposited using the Langmuir-Blodgett (LB) method on substrate surfaces, which were then soaked in a solution of gold nanoparticles (2.7 +/- 0.8 nm) protected with t-dodecanethiol and 4-pyridineethanethiol. The topographical images of the surface observed by tapping mode atomic force microscopy (AFM) showed that the polymers could be dispersed on both substrates, with a height of 2.8 +/- 0.5 nm on the modified glass and 3.1 +/- 0.5 nm on silicon. The height clearly increased after soaking in the gold nanoparticle solution, to 5.3 +/- 0.5 nm on glass and 5.4 +/- 0.7 nm on silicon. The differences in height (2.5 nm on glass and 2.3 nm on silicon) corresponded to the diameter of the gold nanoparticles bonded to the porphyrin polymers. The distance between gold nanoparticles observed in scanning electron microscopic images was ca. 5 nm, indicating that they were bonded at every four or five porphyrin units.

Biochemical characterization of the recombinant human Nogo‐A ectodomain

Nogo-A is a physiologically relevant inhibitor of neuronal growth and regeneration in the myelin of the adult human central nervous system and has attracted considerable attention as a molecular target for the treatment of spinal cord injuries. To gain insight into the structural and functional properties of the large extramembrane region that is characteristic for the Nogo-A splice form of this member of the Reticulon family of membrane proteins, we cloned and expressed the region comprising residues 334-966 as a soluble homogeneous protein in the periplasm of Escherichia coli. SDS/PAGE, under nonreducing conditions, and a systematic substitution analysis of all six Cys residues of Nogo-A indicated that this domain forms two structural disulfide bonds among Cys residues 424, 464, 559 and 597, whereas the Cys residues at positions 699 and 912 seem to be dispensable for folding. The occurrence of a hot spot for host cell proteases and a limited proteolysis experiment suggest that the N-terminal region of Nogo-A up to residue 373 is structurally disordered. Although analytical gel permeation chromatography revealed a large apparent molecular size for the recombinant Nogo-A fragment, indicating oligomer formation, data from analytical ultracentrifugation and dynamic light scattering support a stable monomeric quaternary structure. Notably, the CD spectrum is indicative of a high content of random coil, such that Nogo-A exhibits--at least in part--features of a natively unfolded protein. Nevertheless, the protein fragment identified in this study, as well as its biochemical analysis, provide a promising starting point for future investigations to track down globular subdomains and functionally important regions as well as putative receptor-binding sites therein.

Avidin bioconjugate with a thermoresponsive polymer for biological and pharmaceutical applications

A thermoresponsive polymer, N-isopropylacrylamide- co-acrylamide (Mn 6 kDa) with a lower critical solution temperature (LCST) of 37 °C, was activated and conjugated to avidin to yield a derivative with 200 kDa molecular weight. Gel permeation analysis demonstrated that the new bioconjugate possessed an apparent size corresponding to a 220 kDa globular protein. Photon correlation spectroscopy and turbidometric studies showed that the bioconjugate underwent temperature dependent phase transitions. The protein–co-polymer bioconjugate displayed the same onset phase transition temperature (LCST) as the original synthetic co-polymer. Nevertheless, the aggregation profile of the bioconjugate shifted at higher temperature as compared to the original polymer. This indicated that the aggregation behaviour coil-to-globule transition of the co-polymer was modified by anchoring to the protein surface. Circular dichroism analysis showed that the co-polymer conjugation did not alter the protein tertiary structure tertiary the aromatic amino acid environment. The bioconjugate maintained 85 ± 3% of native avidin affinity for biotin and biotin-Mab, and high affinity was maintained after three heating cycles. Pharmacokinetic studies demonstrated that the co-polymer bioconjugation increased the avidin residence time in the bloodstream. The distribution phase of avidin-co-polymer was longer than the native protein by a factor of 20. The co-polymer conjugation decreased by three-fold the distribution extent of avidin and reduced significantly its up-take to the liver.

Nascent pectin formed in Golgi apparatus of pea epicotyls by addition of uronic acids has different properties from nascent pectin at the stage of galactan elongation

Microsomal membranes were prepared from etiolated pea (Pisum sativum L.) epicotyls and used to form nascent [Uronic acid-14C]pectin. The enzyme products were characterized by selective enzymic degradation, gel permeation chromatography and analysis of cellulose binding properties. The product obtained had a molecular weight of around 40 kDa, which was significantly lower than that of nascent [Gal-14C]pectin prepared from the same tissues. It is composed mainly of polygalacturonan and perhaps also rhamnogalacturonan (RG-I). Evidence was obtained for the presence of a protein attached to the nascent [Uronic acid-14C]pectin, but it was unaffected by endoglucanase and did not bind to cellulose. Hence, no xyloglucan appeared to be attached to the nascent [Uronic acid-14C]pectin. A model is proposed in which xyloglucan is attached to nascent pectin after formation of homogalacturonan, but before the pectin leaves the Golgi apparatus.

Isoselectivity Distribution of Isospecific Centers in Supported Titanium‐Based Ziegler‐Natta Catalysts

AbstractSummary: This article describes the quantitative aspects of the isoselectivity distribution of isospecific centers in five supported titanium‐based Ziegler–Natta catalyst systems containing different internal and external organic donors. Highly crystalline poly(propylene) fractions of the polymers (insoluble in o‐xylene at 95 °C) were analyzed by analytical Tref and gel permeation chromatography (GPC) methods. Separation of the Tref curves into the peaks of sterically uniform components (each represented by the Lorentz function) showed that all these “isotactic” poly(propylene) fractions are nonuniform materials. Each highly crystalline fraction contains several distinct components that differ in stereoregularity. The [mmmm] values for different components (which have approximately the same isotacticity in different polymers) range from as high as 0.986–0.993 to much lower values, in the 0.935–0.955 range. The number of Tref components and their relative contents are substantially different for the crystalline fractions of the polymers produced with different catalyst systems; these parameters depend on the nature of the internal and the external electron donors in the catalysts. GPC analysis of the crystalline poly(propylene) fractions showed that each consists of four or five Flory components (the components produced by kinetically uniform active centers) with widely different average molecular weights, from ≈1 × 104 to ≈1.3 × 106. In general, the probabilities of chain transfer reactions for different active centers decrease with the increase in their isospecificity.Resolution of the Tref curve of the highly crystalline fraction of poly(propylene) prepared with catalyst system 3.magnified imageResolution of the Tref curve of the highly crystalline fraction of poly(propylene) prepared with catalyst system 3.

New Coplanar (ABA)n-Type Donor−Acceptor π-Conjugated Copolymers Constituted of Alkylthiophene (Unit A) and Pyridazine (Unit B): Synthesis Using Hexamethylditin, Self-Organized Solid Structure, and Optical and Electrochemical Properties of the Copolymers

A new series of π-conjugated (ABA)n-type polymers constructed of electron-donating thiophene or 3-alkylthiophene (as the unit A) and electron-accepting pyridazine (as the unit B) were synthesized via palladium-catalyzed dehalogenative polycondensation of Br−ABA−Br using hexamethylditin as the condensing reagent. The obtained polymers were characterized by NMR, IR, and elemental analysis. The polymers with alkyl side chains were soluble in organic solvents, and gave number-average molecular weights ranging from 3.2 × 104 to 16 × 104 in gel-permeation analysis. The polymers exhibited strong green photoluminescence (PL) with the peak in the range of 510−520 nm and quantum yields of 51−61% in chloroform. The PL intensity was sensitive toward acids. In films, the UV−vis and PL peaks shifted to a longer wavelength by about 30−40 nm. Powder X-ray diffraction data suggested that the polymer formed a highly ordered π-stacked assembly assisted by the side chain crystallization and dipole−dipole interaction. Cyclic ...

Synthesis and self-assembly of novel porphyrin molecular wires

Sub-micrometer long butadiyne-linked porphyrin wires were synthesized by oxidative coupling of diethynylporphyrin. The porphyrin wires were analyzed by analytical gel permeation chromatography, absorption spectroscopy and matrix-assisted laser desorption/ionization time of flight mass spectroscopy. Observations of the wire were performed by atomic force microscopy. Self-assembled structures of the wires were observed on highly oriented pyrolytic graphite. Self-assembling features of the porphyrin wires depended on the length of the porphyrin wires and the concentration of the depositing solution.

Chromatographic analysis and sequencing approach of heparin oligosaccharides using cetyltrimethylammonium dynamically coated stationary phases

C 18 and C 8 bonded stationary phases dynamically coated with cetyltrimethylammonium (CTA) and strong anion exchange (SAX) were developed to obtain separations of oligosaccharide mixtures resulting from chemical or enzymatic depolymerization of heparin. With this method, the retention of sulfated oligosaccharides is directly adjustable depending on the amount of CTA adsorbed into the column. Oligosaccharides containing up to 20 sulfates were separated with a resolving power superior to that of conventional SAX analysis. The stability of the column coating enables hundreds of injections. Using ammonium methane sulfonate aqueous solutions as ultraviolet transparent mobile phases, it was possible to set up double detection, including selective detection of acetylated oligosaccharides. Analytical gel permeation chromatography was directly coupled to CTA-SAX, obtaining a two-dimensional profile of analyzed oligosaccharidic mixtures. A sequencing method of heparin oligosaccharides using partial depolymerization by heparinases according to their size and sulfation pattern and digest analysis by CTA-SAX was developed. A direct application of this method to the analysis of oligosaccharide mixtures obtained by complete digestion of heparins by heparinases I, II, and III was done. It allowed a reliable quantification of heparin building blocks. We also focused our attention on di- and tetrasaccharidic species containing the 3-O-sulfated glucosamines taken as markers of the active sites for antithrombin III. The method was also applied to more complex mixtures resulting from porcine heparin partially depolymerized with heparinase I. The specificity of the reaction was studied up to decasaccharidic fractions.

On‐line LC‐GC method for determination of isomers of nervonic acid in meat‐derived food

AbstractAn on‐line liquid chromatography‐gas chromatography (LC‐GC) method for determination of the cis/trans isomers of nervonic acid in meat‐derived food is described. The technique used a home‐assembled LC‐GC coupled system. A simple LC clean‐up procedure using an analytical gel permeation (5 μm) column and 1% ethyl acetate in hexane as mobile phase has proved to be suitable for the isolation and purification of fatty acid methyl esters from the meat matrix. Use of a silica gel (5 μm) pre‐column improved the separation of cis/trans nervonic acid from cholesterol and the more polar constituents of the extracted fraction. GC analyses were performed using a non‐polar GC column and mass spectrometry (MS) for identification of the analytes and flame ionization detector (FID) for quantification of the compounds studied. Excellent linearity with correlation coefficients higher than 0.999 in the range of 0.05–10 μg mL–1 nervonic acid and a good repeatability with relative standard deviation (RSD) values generally lower than 4% was obtained. The method developed provides a high sensitivity for trace analysis of the cis/trans isomers of nervonic acid and enables complete automation.

Polyethylene Glycol Conjugates of Methotrexate Varying in Their Molecular Weight from MW 750 to MW 40000: Synthesis, Characterization, and Structure−Activity Relationships in Vitro and in Vivo

Poly(ethylene glycol)s (PEGs) are potential drug carriers for improving the therapeutic index of anticancer agents. In this work, the anticancer drug methotrexate (MTX) was activated with N,N'-dicyclohexylcarbodiimide (DCC) and coupled to amino group bearing PEGs of MW 750, 2000, 5000, 10 000, 20,000, and 40,000. First, the activation process of MTX with DCC in the presence and absence of N-hydroxysuccinimide was analyzed through HPLC. Preincubation of methotrexate with DCC alone at 0 degrees C proved to be favorable with respect to the amount of activated species and the formation of byproducts. MTX-PEG conjugates were synthesized according to this procedure, isolated through size-exclusion chromatography, and characterized through analytical HPLC, MALDI-TOF spectrometry, and gel permeation chromatography. In a cell-free assay, all of the drug polymer conjugates inhibited the target enzyme of MTX, dihydrofolate reductase (DHFR), to a similar extent, but were not as active as free MTX. Additionally, incubation of the MTX-PEG40000 conjugate for 6 days at 37 degrees C in phosphate buffered saline (pH 7.4), in cell-conditioned medium, or in human serum revealed no significant release of methotrexate. These results, taken together, indicate that release of MTX from polymer conjugates is not necessary for an effective interaction with the active site of dihydrofolate reductase. Evaluation of the in vitro cytotoxicity of the MTX-PEG conjugates in two adherent and three suspension human tumor cell lines revealed that the IC(50) values of the tested compounds increased with the size of the drug-polymer conjugates. The most effective compound tested in these assays was the free drug MTX itself (IC(50) value ranging from approximately 0.01 to 0.05 microM), while the IC(50) values of the polymer conjugates were higher (IC(50) value for MTX-PEG750, 2000 and 5000: approximately 0.6-3 microM; for MTX-PEG10000 and 20000: approximately 2-7 microM; and for MTX-PEG40000: > 6 microM). Subsequently, MTX-PEG5000, MTX-PEG20000, and MTX-PEG40000 were evaluated in a human mesothelioma MSTO-211H xenograft model, and their antitumor effects were compared with free methotrexate and the albumin conjugate MTX-HSA, a conjugate that is currently in phase II clinical trials. In contrast to the in vitro results, the high molecular weight MTX-PEG conjugates exhibited the highest in vivo antitumor activity: At a dose of 40 and 80 mg/kg MTX-PEG5000 was less active than MTX at its optimal dose of 100 mg/kg; MTX-PEG20000 at a dose of 40 mg/kg showed antitumor efficacy comparable to MTX, but MTX-PEG40000 at a dose of 20 mg/kg was superior to MTX and demonstrated antitumor activity of the same order as MTX-HSA (20 mg/kg).