Organometallic Tc-technetium(I)- and Re-rhenium(I)-folate derivatives for potential use in nuclear medicine

The folate receptor (FR) is a high affinity membrane protein which is overexpressed on a wide variety of tumor cells, but highly restricted in normal tissues. Therefore folate derivatives labeled with short living isotopes such as ^99mTc (γ, t_1/2 = 6 h) or ¹⁸⁸Re (β⁻, t_1/2 = 17 h) could be used for tumor diagnosis and therapy. In this respect there is a great interest to develop organometallic technetium(I) and rhenium(I) modified folate radiopharmaceuticals. For this purpose folic acid was functionalized with a tridentate picolylamine monoacetic acid chelating system. The chelating system was selectively coupled via an aminohexane spacer to the γ- or α-carboxyl group of the glutamate moiety of folic acid to obtain the corresponding γ- or α-folate derivative or - if directly attached to pteroic acid - the pteroate derivative. The derivatives were reacted with the precursor [M(OH₂)₃(CO)₃]⁺ (M = ^99mTc, Re) to form uniform organometallic folate complexes under mild reaction conditions. All compounds were chemically characterized by means of NMR, MS, IR and HPLC. The determination of the IC₅₀-values for the PAMA-γ-folate derivative (100 nM) and the corresponding organometallic rhenium complex (110 nM) proved retained receptor binding properties. The radiolabeling with [^99mTc(OH₂)₃(CO)₃]⁺ was achieved in excellent yield (>95%) at low ligand concentration (10⁻⁴ M). The cell binding (>45% of total activity) and internalization (>15% of total activity) of all ^99mTc-complexes was very high and specificity for the FR was proved by their complete displacement with excess folic acid. The ^99mTc-complexes were positively tested for their plasma stability and for the absence of binding to plasma proteins.     

Radiosynthesis and in vitro evaluation of 99mTc(CO)3-labeled folic acid derivative

The over-expression of folate receptors in variety of neoplastic tissues makes radiolabeled folate conjugates potential agents for imaging and therapy of such cancers. With the aim of preparing an imaging agent for targeting folate receptors, folic acid has been conjugated with homocysteine for complexation with [^99mTc(CO)₃(H₂O)₃]⁺ core. The radiolabeled complex of the homocysteine-folate could be obtained in >95% radiochemical yield as observed by HPLC. Stability of complex in saline was studied and challenge studies with histidine and cysteine revealed kinetic stability of the complex. Lipophilicity of the radiolabeled complex (log P) was found to be 0.45. In vitro uptake of ^99mTc(CO)₃-labeled folic acid derivative was studied in KB cells and inhibition studies were carried out using ³H-folic acid and cold homocysteine–folate conjugate. The in vitro studies indicated loss of binding affinity of the derivative towards folate receptors.     

Effects on peptide binding affinity for TNFα by PEGylation and conjugation to hyaluronic acid

Conjugation of cytokine-neutralizing monoclonal antibodies (mAb) to hyaluronic acid (HA) having M_w of 1.6 MDa was previously shown to be an effective strategy for localized delivery to sites of inflammation. Despite the disparity in size of the mAb and HA, the mAb–HA conjugate was found bind tumor necrosis factor-α (TNFα) as strongly as the non-conjugated antibody, suggesting conjugation to this charged polysaccharide can provide an alternative to poly(ethylene glycol) (PEG) conjugation, which has been shown to reduce binding interactions for many proteins. To explore conjugation chemistries more systematically, we report a study on a model peptide inhibitor of tumor necrosis factor-α to investigate the effects of site-specific conjugation to HA and PEG. We compared the binding affinities of a variety of WP9QY peptide–polymer conjugates for TNFα in order to examine the effects of PEG molecular weight as well as the effects of PEG versus functionalized hyaluronic acid (HA) conjugation. The results indicate that the binding affinity of the PEG conjugates decreases in comparing PEG with mass 2 k, 10 k, and 30 k, which was attributed to PEG shrouding of the peptide, while conjugation to a 66 kDa HA chain preserved peptide binding affinity. We attribute this difference to the increased solubility of HA compared to PEG, potentially due to the carboxylic acid functional groups. In addition, the results demonstrate that conjugation to HA via a short PEG linker significantly enhances the association rate k_on, which may reflect an increased peptide accessibility. By balancing both the advantages associated with the PEG conjugates and with the HA conjugates, the HA–PEG2k–WP9QY conjugate was able to improve the binding affinity of the peptide for TNFα by a factor of two. Optimization of polymer chemistry could be used to improve delivery of protein therapeutics for localized and systemic administration.     

Facile synthesis of fluorescent Au@SiO2 nanocomposites for application in cellular imaging

A novel fluorescent Au@SiO₂ nanocomposite, with average size of ca. 30 nm in the diameter, was prepared via a simple microemulsion method. Additionally, transmission electron microscopy (TEM), UV-Vis absorption spectra, Fourier transform infrared (FT-IR) spectra and fluorescence spectra were used to characterize this nanocomposite. This newly synthesized, silica-wrapped, gold nanocluster has the following advantages: good water solubility, exceptional biocompatibility, favorable surface properties and excellent fluorescence properties. Because of these advantages, a Au@SiO₂ nanocomposite is exceptionally suitable for biological applications. In this study, cellular imaging, as a form of biological application, has been fully investigated, and it was discovered, after covalent conjugation of folic acid (FA), that the nanocomposite effectively recognized over expressed folic acid receptors (FARs) on the HeLa cell's surface. Therefore, this fluorescent Au@SiO₂ nanocomposite could be used as a new fluorescent probe for selective biological imaging.     

Synthesis and screening of bifunctional radiolabelled carborane-carbohydrate derivatives

A new bifunctional carborane ligand was prepared as a platform for the development of targeted molecular radioimaging and therapy agents. The carborane derivative was synthesized bearing a glucose substituent to increase the water solubility of the ligand and a benzoic acid group as a site for linking to amine containing targeting vectors. A convenient method to conjugate the ligand and the non-glycosylated analogue to amino groups was developed using simple active esters which were combined with a model amine generating two new N,N-diethyl(aminoethyl) benzamide derivatives. These were labelled with ¹²⁵I in good yield and the log P values measured for [¹²⁵I]-15 (log P = 0.82 ± 0.04) and [¹²⁵I]-16 (log P = 1.53 ± 0.01). The benzamides were also evaluated for their capacity to bind to B16F10 melanoma cells where the hydrophilic compound showed low binding while [¹²⁵I]-16 showed modest uptake (30.7 ± 2.2%) after 24 h.     

Synthesis,Characterization, and Biological Evaluation of a Dual-Action Ligand Targeting αvβ3 Integrin and VEGF Receptors

A dual-action ligand targeting both integrin α_Vβ₃ and vascular endothelial growth factor receptors (VEGFRs), was synthesized via conjugation of a cyclic peptidomimetic α_Vβ₃ Arg-Gly-Asp (RGD) ligand with a decapentapeptide. The latter was obtained from a known VEGFR antagonist by acetylation at the Lys13 side chain. Functionalization of the precursor ligands was carried out in solution and in the solid phase, affording two fragments: an alkyne VEGFR ligand and the azide integrin α_Vβ₃ ligand, which were conjugated by click chemistry. Circular dichroism studies confirmed that both the RGD and VEGFR ligand portions of the dual-action compound substantially adopt the biologically active conformation. In vitro binding assays on isolated integrin α_Vβ₃ and VEGFR-1 showed that the dual-action conjugate retains a good level of affinity for both its target receptors, although with one order of magnitude (10/20 times) decrease in potency. The dual-action ligand strongly inhibited the VEGF-induced morphogenesis in Human Umbilical Vein Endothelial Cells (HUVECs). Remarkably, its efficiency in preventing the formation of new blood vessels was similar to that of the original individual ligands, despite the worse affinity towards integrin α_Vβ₃ and VEGFR-1.     

Evaluation of diffusive gradients in thin-films using a Diphonix resin for monitoring dissolved uranium in natural waters

Commercially available Diphonix^® resin (TrisKem International) was evaluated as a receiving phase for use with the diffusive gradients in thin-films (DGT) passive sampler for measuring uranium. This resin has a high partition coefficient for actinides and is used in the nuclear industry. Other resins used as receiving phases with DGT for measuring uranium have been prone to saturation and significant chemical interferences. The performance of the device was evaluated in the laboratory and in field trials. In laboratory experiments uptake of uranium (all 100% efficiency) by the resin was unaffected by varying pH (4–9), ionic strength (0.01–1.00 M, as NaNO₃) and varying aqueous concentrations of Ca²⁺ (100–500 mg L⁻¹) and HCO₃⁻ (100–500 mg L⁻¹). Due to the high partition coefficient of Diphonex^®, several elution techniques for uranium were evaluated. The optimal eluent mixture was 1 M NaOH/1 M H₂O₂, eluting 90% of the uranium from the resin. Uptake of uranium was linear (R² = 0.99) over time (5 days) in laboratory experiments using artificial freshwater showing no saturation effects of the resin. In field deployments (River Lambourn, UK) the devices quantitatively accumulated uranium for up to 7 days. In both studies uptake of uranium matched that theoretically predicted for the DGT. Similar experiments in seawater did not follow the DGT theoretical uptake and the Diphonix^® appeared to be capacity limited and also affected by matrix interferences. Isotopes of uranium (U²³⁵/U²³⁸) were measured in both environments with a precision and accuracy of 1.6–2.2% and 1.2–1.4%, respectively. This initial study shows the potential of using Diphonix^®-DGT for monitoring of uranium in the aquatic environment.     

Thiol-functionalized shell crosslinked knedel-like (SCK) nanoparticles: a versatile entry for their conjugation with biomacromolecules

Shell crosslinked knedel-like (SCK) nanoparticles were prepared having thiol-terminated poly(ethylene glycol) (PEG) chains extending throughout their shell layers and were then conjugated with bovine serum albumin (BSA) as a model biomacromolecule. The SCKs originated from amphiphilic block copolymers of acrylic acid and styrene, PAA₆₆-b-PS₇₁, pre-functionalized with ca. five mono-tert-Boc-protected diamino PEG₃₂ per polymer chain, which then had undergone deprotection and amidation with N-succinimidyl-S-acetylthiohexanionate to introduce an acetyl-protected thiol chain terminus on the end of each PEG graft. Assembly of these amphiphilic graft block copolymers into micelles, by transitioning from N,N-dimethylformamide to water, was followed by amidation-based crosslinking throughout the shell layer, with the introduction of 2,2′-(ethylenedioxy)-bis(ethylamine) and 1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide methiodide, to afford SCKs bearing the acetyl-protected thiol groups. Deprotection in aqueous buffer solution by reaction with hydroxylamine hydrochloride gave the SCKs presenting a nominal number of ca. 750 thiols per nanoparticle. The solution was assayed by Ellman's method resulting in a concentration of 55±6 μM [HS], theoretical of concentration 58 μM [HS], after which the coupling with BSA was performed immediately. Tetramethylrhodamine-labeled, maleimido-functionalized BSA was allowed to react with the thiol-functionalized SCKs at stoichiometries of ca. 10, 20, and 30 BSAs/SCK, after which UV-vis spectroscopy and Bradford's assay determined a coupling efficiency of >50-60%. The SCK particle diameters were measured by TEM to be 16 nm and 20 nm and their hydrodynamic diameters were measured by dynamic light scattering to be 20 nm and 30 nm, before and after BSA conjugation, respectively.     

<Superscript>99m</Superscript>Tc(CO)<Subscript>3</Subscript>–AOPA colchicine conjugate as a potential tumor imaging agent

This work reports the synthesis, radiolabeling and preliminary biodistribution results in tumor-bearing mice of the ^99mTc(CO)₃–AOPA colchicine conjugate. The novel ligand was successfully synthesized by conjugation of N-(acetyloxy)-2-picolylamino (AOPA) to deacetylcolchicine via a short carbonyl-methylene linker. Radiolabeling was performed in high yield with [^99mTc(CO)₃]⁺ core. ^99mTc(CO)₃–AOPA colchicine conjugate was hydrophilic and was stable at room temperature. Biodistribution studies in tumor-bearing mice showed that ^99mTc(CO)₃–AOPA colchicine conjugate accumulated in the tumor with good uptake and retention. However, its clearance from normal organs was not so fast, resulting in poor T/NT ratios. Further modification on the linker or/and ^99mTc-chelate to improve the tumor targeting efficacy and in vivo kinetic profiles is currently in progress.     

Determination of 232Th in seawater by ICP-MS after preconcentration and separation using a chelating resin

This article describes an analytical method for the separation, preconcentration and determination of ²³²Th in seawater samples at sub-ng/L levels using a NOBIAS CHELATE PA1 resin and a sector field (SF) inductively coupled plasma mass spectrometer (ICP-MS). The resin showed excellent adsorption of ²³²Th at a low pH of 2.4 ± 0.4 in a relatively small volume (200 mL) of seawater. ²³²Th adsorbed on the resin was easily eluted using 5 mL of 0.8 M HNO₃. An enrichment factor of 40 was achieved for ²³²Th analysis. Ethylenediamine-tetraacetic acid disodium salt dehydrate (EDTA) was used to investigate the effect of ²³²Th-binding organic ligand on the retention of ²³²Th on the chelating resin. Results obtained using acidified samples (pH of 2.4 ± 0.4) showed EDTA had no significant effect on ²³²Th recovery, indicating that at this low pH, ²³²Th was dissociated from the ²³²Th-binding organic ligand and quantitatively retained on the NOBIAS CHELATE PA1 resin. The developed analytical method was characterized by a separation and preconcentration taking approximately 4 h and a low detection limit of 0.0038 ng/L for ²³²Th, and was successfully applied to the determination of ²³²Th in seawater samples collected from coastal areas, Japan.     

Separating Ag, B, Cd, Dy, Eu, and Sm in a Gd matrix using 2-ethylhexyl phosphonic acid mono-2-ethylhexyl ester extraction chromatography for ICP-AES analysis

The separation procedure for Ag, B, Cd, Dy, Eu and Sm as impurities in Gd matrix using ICP-AES technique with an extraction chromatographic column has been developed. The spectral interference of the Gd matrix on the elements was eliminated using a chromatography technique with 2-ethylhexyl phosphonic acid mono-2-ethylhexyl ester (PC-88A) as the mobile phase and XAD-16 resin as the stationary phase. Ag⁺, B₄O₇²⁻, and Cd²⁺ were eluted with 0.1 M HNO₃, while rare earth ions were not. The best eluent for separating Eu and Sm in the Gd matrix was 0.3 M HNO₃. The limit of quantitation for these elements was 0.6-3.0 ng mL⁻¹. The recovery of Ag, B, and Cd was 90-104% using 0.1 M HNO₃ as the eluent, while that of Eu, Gd, and Sm ranged from 100 to 102% with 0.3 M HNO₃. Dy was recovered quantitatively with 4 M HNO₃. The relative standard deviation of the methods for a set of three replicates was between 1.0 and 15.4% for the synthetic and standard Gd solutions. The proposed separation procedure was used to measure Ag, B, Cd, Dy, Eu, and Sm in a standard Gd solution.     

High acidity of polycyano azatriquinanes—theoretical prediction by the DFT calculations

It is shown by the B3LYP/6-311+G(2d,p)//B3LYP/6-31G(d) calculations that the hexacyano derivative of aza-acepentalene is an extremely powerful superacid both in the gas phase and in DMSO as evidenced by the ΔH_acid = 255.1  kcal mol⁻¹ and pK_a (DMSO) = −26.5. Its synthesis is strongly recommended, in particular, since the related conjugate base hexachloro aza-acepentalenide anion was prepared recently.     

Synthesis and preliminary biological evaluation of [<Superscript>99m</Superscript>Tc(CO)<Subscript>3</Subscript>(IDA–PEG<Subscript>3</Subscript>–CB)]<Superscript>−</Superscript> for tumor imaging

This work reports the synthesis, radiolabeling and preliminary biodistribution results in tumor-bearing mice of [^99mTc(CO)₃(IDA–PEG₃–CB)]⁻. The novel chlorambucil derivative was successfully synthesized by conjugation of iminodiacetic acid (IDA) to chlorambucil via a pegylated linker. The ligand could be labeled by [^99mTc(CO)₃]⁺ core in high yield to get [^99mTc(CO)₃(IDA–PEG₃–CB)]⁻, which was very hydrophilic and was stable at room temperature. Biodistribution studies in tumor-bearing mice showed that [^99mTc(CO)₃(IDA–PEG₃–CB)]⁻ accumulated in the tumor with favorable uptake and retention. The good accumulation in tumor tissue with high tumor/muscle ratios warrants further research to improve tumor targeting efficacy and pharmacokinetic profile of radiolabeled chlorambucil derivative by structural modification.     

Synthesis and structure of the two-dimensional coordination networks [Ln(PDC)(N-HPDC)]∞ (PDC = pyridine-3,4-dicarboxylate,Ln = La,Ce, Pr)

The title compounds, [Ln(PDC)(HPDC)]_∞ (H₂PDC = pyridine-3,4-dicarboxylic acid, Ln = La, Ce, Pr), have been prepared and have been characterised by IR, MS, TGA, X-ray crystallography and powder diffraction. These isostructural complexes display both bridging and chelating carboxylate binding modes including a highly unusual chelation in which both carboxylate substituents bind to the same Pr atom, imparting chirality around the metal despite the non-chiral crystal setting. This system represents the first fully characterised metal complex containing the N-protonated ligand derivative (N-HPDC⁻). A polymorph of the ligand precursor, pyridine-3,4-dicarboxylic acid is also reported.     

Diastereo- and enantioseparation of a N-Boc amino acid with a zwitterionic quinine-based stationary phase: Focus on the stereorecognition mechanism

A chiral chromatography method enabling the simultaneous diastereo- and enantioseparation of N^α-Boc-N⁴-(hydroorotyl)-4-aminophenylalanine [Boc-Aph(Hor)-OH, 1] was optimized with a quinine-based zwitterionic stationary phase. The polar-ionic eluent system consisting of ACN:MeOH:water—49.7:49.7:0.6 (v/v/v) with formic acid (4.0 mM) and diethylamine (2.5 mM), allowed the successful separation of the four acid stereoisomers: α_d,d-/d,l-1 = 1.08; α_d,l-/l,d-1 = 1.08; α_l,d-/l,l-1 = 1.40.     

Fabrication of dissolved O2 metric uric acid biosensor using uricase epoxy resin biocomposite membrane

Uricase purified from 20-day-old leaves of cowpea was immobilized on to epoxy resin membrane with 80% retention of initial activity of free enzyme and a conjugation yield of 0.056 mg/cm². The uricase epoxy resin bioconjugate membrane was mounted over the sensing part of the combined electrode of ‘Aqualytic’ dissolved O₂ (DO) meter to construct a uric acid biosensor. The biosensor measures the depletion of dissolved O₂ during the oxidation of uric acid by immobilized uricase, which is directly proportional to uric acid concentration. The biosensor showed optimum response within 10-12 s at a pH 8.5 and 35 °C. A linear relationship was found between uric acid concentration from 0.025 to 0.1 mM and O₂ (mg/l) consumed. The biosensor was employed for measurement of uric acid in serum. The mean value of uric acid in serum was 4.92 mg/dl in apparently healthy males and 3.11 mg/dl in apparently healthy females. The mean analytic recoveries of added uric acid in reaction mixture (8.9 and 9.8 mg/dl) were 93.6 ± 2.34 and 87.18 ± 3.17% respectively. The within and between batch CVs were <6.5 and <5.0%, respectively. The serum uric acid values obtained by present method and standard enzymic colorimetric method, showed a good correlation (r = 0.996) and regression equation being y = 0.984x + 0.0674. Among the various metabolites tested only, glucose (11%), urea (38%), NaCl (25%) and cholesterol (13%) and ascorbic acid (56%) caused decrease, while, MgSO₄ and CaCl₂ had no effect on immobilized enzyme. The enzyme electrode showed only 32% decrease during its use for 100 times over a period of 60 days at 4 °C.     

Determination of itopride hydrochloride by high-performance liquid chromatography with Ru(bpy)3 electrogenerated chemiluminescence detection

In this work, a stable electrogenerated chemiluminescence (ECL) detector was developed. The detector was prepared by packing cation-exchanged resin particles in a glass tube, followed by inserting Pt wires (working electrode) in this tube and sealing. The leakage of Ru(bpy)₃²⁺ can be compensated by adding a small amount of Ru(bpy)₃²⁺ into solution phase. Coupled with high-performance liquid chromatography separation, the detector has been used for determination of itopride hydrochloride in human serum. Under the optimal conditions, the ECL intensity has a linear relationship with the concentration of itopride hydrochloride in the range of 1.0 × 10⁻⁸ g mL⁻¹ to 1.0 × 10⁻⁶ g mL⁻¹ and the detection limit was 3 × 10⁻⁹ g mL⁻¹ (S/N = 3). The as-prepared ECL detector displayed good sensitivity and stability.     

Liquid chromatography on an amide stationary phase with post-column derivatization and fluorimetric detection for the determination of streptomycin and dihydrostreptomycin in foods

The separation of streptomycin and its derivative dihydrostreptomycin using ion-pair liquid chromatography is proposed. The method is based on the use of a new stationary phase based on a ligand with amide groups and the endcapping of trimethylsilyl which avoids the appearance of tailed peaks. The isocratic mobile phase consisted of a 6:94 (v/v) acetonitrile/10 mM pentanesulfonic acid (pH 3.3) mixture at a flow-rate of 1 mL min⁻¹ and fluorescence detection involved a post-column derivatization reaction using β-naphthoquinone-4-sulfonate. Linearity, precision, recovery and sensitivity were satisfactory. The procedure was applied to the analysis of the aminoglycoside antibiotics in different types of foods, as honey, milk, egg and liver. Extraction was carried out by acidic hydrolysis to release protein-bound antibiotics. Detection limits in the food samples are 7.5 and 15 μg kg⁻¹ for streptomycin and dihydrostreptomycin, respectively.     

Determination of radioactive strontium in seawater

This paper describes the procedures of isolating strontium and yttrium from seawater that enable the determination of ^89,90Sr. In one procedure, strontium is directly isolated from seawater on the column filled with Sr resin by binding of strontium to the resin from 3 M HNO₃ in a seawater, and successive elution with HNO₃. In others, strontium is precipitated from seawater with (NH₄)₂CO₃, followed by isolation on a Sr column or an anion exchange column. It is shown that strontium precipitation is optimal with concentration of 0.3 M (NH₄)₂CO₃ at pH = 11. In these conditions, 100% Y, 78% Sr, 80% Ca and 50% Mg are precipitated. Strontium is bound on to Sr column from 5 to 8 M HNO₃, separated from other elements by elution with 3 M HNO₃ and 0.05 M HNO₃. Strontium and yttrium are bound on to anion exchange column from alcoholic solutions of nitric acid. The optimum mixture of alcohols for sample binding is a mixture of ethanol and methanol with the volume ratio 1:3. Strontium and yttrium are separated from Mg, Ca, K, and other elements by elution with 0.25 M HNO₃ in the mixture of ethanol and methanol. After the separation, yttrium and strontium are eluted from the column with water or methanol.In the procedure of direct isolation from 1 l of the sample, the average recovery of 50% was obtained. In the remaining two procedures, the strontium recovery was about 60% for the Sr column and 65% for anion exchange column. Recovery of yttrium is about 70% for the anion exchange column. It turned out that the procedure with the Sr resin (direct isolation and isolation after precipitation) is simpler and faster in the phase of the isolation on the column in comparison with the procedure with the anion exchanger. The procedure with the anion exchanger, however, enables the simultaneous isolation of yttrium and strontium and rapid determination of ^89,90Sr. These procedures were tested by determination of ^89,90Sr on liquid scintillation counter and Cherenkov counting in 5 M HNO₃. Obtained results showed that activity of 50 mBq l⁻¹ of ^89,90Sr and higher can be simultaneously determined.     

Synthesis and properties of a new SASRIN resin derivative: SASRIN-2-pyridylthiocarbonate (SASRIN-TOPCAT) resin

A new SASRIN resin derivative, SASRIN-TOPCAT resin, was synthesized by the reaction of SASRIN resin with 2-thiopyridyl chloroformate. The new resin can be used for the loading of alcohols and thiols under neutral conditions, and the release of alcohol from the resin is achieved by the treatment of 1% TFA in CH₂Cl₂ for 15-60 min. Compared to the other reported resins, SASRIN-TOPCAT resin is more suitable for the loading of alcohols in solid-phase organic synthesis.