Diethylenetriaminepentaacetic Acid Dianhydride Research Articles

Evaluation of adsorption performance and quantum chemical modeling of pesticides removal using Cell-MG hybrid adsorbent

Magnetite (MG) modified cellulose membrane (Cell-MG), obtained by reaction of 3-aminosilane and subsequently with diethylenetriaminepentaacetic acid dianhydride functionalized waste Cell fibers (Cell-NH2 and Cell-DTPA, respectively), and amino-modified diatomite was used for Azoxystrobin and Iprodione removal from water. Cell-MG membrane was structurally and morphologically characterized using FT-IR and FE-SEM techniques. The influences of operational parameters, i.e. pH, contact time, temperature, and the mass of adsorbent on adsorption and kinetics were studied in a batch system. The calculated capacities of 35.32 and 30.16 mg g-1 for Azoxystrobin and Iprodione, respectively, were obtained from non-linear Langmuir model fitting. Weber-Morris model fitting indicates the main contribution of intra-particle diffusion to overall mass transport resistance. Thermodynamic data indicate spontaneous and endothermic adsorption. The reusability of adsorbent and results from wastewater purification showed that Cell-MG could be used as general-purpose adsorbent. The adsorbent/adsorbate surface interaction was considered from the results obtained using density functional theory (DFT) and calculation of molecular electrostatic potential (MEP). Thus, a better understanding of the relation between the adsorption performances and contribution of non-specific and specific interactions to adsorption performances and design of novel adsorbent with improved properties was deduced

Batch and column adsorption of cations, oxyanions and dyes on a magnetite modified cellulose-based membrane

An optimized method is presented to make magnetite (MG) modified cellulose membrane (Cell-MG) from 3-aminopropyltriethoxysilane and diethylenetriaminepentaacetic acid dianhydride functionalized waste cell fibers; (Cell-NH2 and Cell-DTPA), and amino-modified diatomite. Functionalized Cell-NH2, Cell-DTPA fibers, and diatomite were structurally and morphologically characterized using FT-IR, Raman, and FE-SEM analysis. Amino and carboxyl group content was determined via standard volumetric methods. Response surface method was applied to rationalize the number of experiments related to Cell-MG synthesis and heavy metal ions column adsorption experiments. The effects of pH, contact time, temperature, and initial concentration of pollutants on adsorption and kinetics were studied in a batch, while initial concentration and flow rate were studied in a flow system. The calculated capacities of 88.2, 100.7, 95.8 and 78.2 mg g−1 for Ni2+, Pb2+, Cr(VI) and As(V) ions, respectively, were obtained from Langmuir model fitting. Intra-particle diffusion as a rate-limiting step was evaluated from pseudo-second-order and Weber–Morris model fitting. Thermodynamic parameters indicated spontaneous and low endothermic processes. The results from reusability study, wastewater purification and fixed-bed column study proved the high applicability of Cell-MG. Additionally, high removal capacity of four dyes together with density functional theory and molecular interaction fields, help in the establishment of relation between the adsorption performances and contribution of non-specific and specific interactions at adsorbate/adsorbent interface.

DTPAA-Gd Functionalized Ultrasmall Au15NCs Nanohybrids for Multimodal Imaging.

IntroductionMultimodal imaging agent has the potential to overcome the shortage and incorporate the advantages of different imaging tools for extremely sensitive diagnosis. To achieve multimodal imaging, combining multiple contrast agents into a special nanostructure has become a main strategy; However, the combination of all of these functions into one nanoplatform usually requires a complicated synthetic procedure that results in heterogeneous nanostructure.MethodsIn this study, we develop ultrasmall gold nanoclusters with 15 gold atoms (Au15NCs) functionalized with diethylenetriamine-pentaacetic acid dianhydride (DTPAA-Gd) as an optimized multimodal imaging agent to enhance imaging ability.ResultsThe Au15NCs-DTPAA-Gd nanohybrids possess the ultra-small size and are capable of enhancing the contrast in near-infrared fluorescence (NIRF), magnetic resonance (MR) and X-ray computed tomography (CT) imaging. Meanwhile, the integrated DTPAA-Gd component not only endow the nanohybrids to produce higher T1 relaxivity (r1 = 21.4 mM−1 s−1) than Omnipaque (r1 = 3.973 mM−1s−1) but also further enhance X-ray attenuation property of Au15NCs. Importantly, the fluorescence intensity of Au15NCs-DTPAA-Gd did not decrease compared with Au15NCs. Ultimately, in vivo imaging experiments have demonstrated that Au15NCs-DTPAA-Gd nanohybrids can be quickly eliminated from the body through the urinary system and has great potential for anatomical imaging.ConclusionThese data manifest Au15NCs-DTPAA-Gd present great potential as a multimodal contrast agent for disease diagnosis, especially for early accurate detection of tumors.

Y3+ embedded in polymeric nanoparticles: Morphology, dimension and stability of composite colloidal system

Radiolabeled nanoparticles are promising tools in cancer diagnosis and therapy. Moreover, yttrium-90 (90Y) is a good candidate as suitable β− emitting radioisotope for a new approach to radio-guided surgery (RGS) proposed by some researchers of our group. In this work, we developed new composite nanoparticles, based on polymethylmethacrylate (PMMA), and poly(methylmethacrylate-co-acrylic acid), P(MMA/AA), embedded with yttrium ion (89Y3+), as a first step for the development of new 90Y3+ based nanocomposites. The composite nanoparticles were synthesised by emulsion polymerization technique, in the presence of potassium persulfate (KPS) as radical initiator, toluene as cosolvent, diethylenetriamine penta acetic acid dianhydride (DTPA) as chelating agent, using different MMA/89Y3+ molar ratios. The yttrium doped polymeric nanoparticles were characterized by means of FTIR spectroscopy, SEM-EDX, AFM, and DLS measurements. The influence of experimental parameters, such as cosolvent and reaction time, on morphology and dimension of composite nanoparticles was investigated, and monodispersed nanospheres with diameters from 20nm to 400nm were obtained. The composite material was purified and studied by means of DLS to confirm the colloid stability in water solution at different temperatures (25°C and 37°C) up to 3 weeks.

Safety and efficacy of self-assembling bubble carriers stabilized with sodium dodecyl sulfate for oral delivery of therapeutic proteins

Sodium dodecyl sulfate (SDS) is generally regarded as a potent permeability enhancer in oral formulations; however, one concern related to the use of any permeation enhancer is its possible absorption of unwanted toxins during the period of epithelial permeability enhancement. In this work, the safety and efficacy of an SDS-containing bubble carrier system that is developed from an orally administered enteric-coated capsule are evaluated. The bubble carriers comprise diethylene triamine pentaacetic acid (DTPA) dianhydride, sodium bicarbonate (SBC), SDS, and insulin. Upon exposure to the intestinal fluid, DTPA dianhydride hydrolyzes to yield acids, and SBC rapidly reacts with these acids to generate CO2, producing bubble carriers, each containing a self-assembling water film. The hydrophilic insulin is entrapped in the self-assembled water film, which is stabilized by SDS. The SDS in the bubble carrier system can act as a dissolution enhancer in the dispersion of insulin molecules, as a surfactant that stabilizes the bubble carriers, as a protease inhibitor that protects the protein drug, and as a permeation enhancer that augments its oral bioavailability. Hence, a significant increase in the plasma insulin level and an excellent blood glucose-lowering response in diabetic rats are effectively achieved. Moreover, the enhancement of epithelial permeation by this SDS-containing formulation does not promote the absorption of intestinal endotoxins. The above facts indicate that the bubble carrier system that is stabilized by SDS can be used as a safe and potent carrier in the oral delivery of therapeutic proteins.

Functionalized Metal Chelates Based on Diethylenetriaminetetraacetic Acids for Chemical Modification of Proteins and Small Biomolecules

Bifunctional reagents based on diethylenetriaminetetraacetic acid containing a bound metal ion and a reactive functional group for the interaction with proteins and low-molecular-weight substances have been synthesized. An Amino-derivative of a complexonate was obtained by acylation of monosubstituted diamine with diethylenetriaminepentaacetic acid dianhydride followed by deprotection ofthe amino group, purification by anion exchange chromatography and chelation of Eu3+. This metal chelate derivative was used for labeling 17α-hydroxyprogesterone 3-(O-carboxymethyl)oxime and horseradish peroxidase. The enzyme modified with the Eu3+ complexonate at the carbohydrate component and with a cortisol derivative at the polypeptide chain was used in a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) as well as in an enzyme immunoassay of the steroid hormone. DELFIA showed that labeled 17α-hydroxyprogesterone retained the affinity for corresponding antibodies. A Eu(3+)-complexonate carboxy-derivative N-succinimide ester was obtained by acylation of the aminochelate with p-phthalic acid di-N-succinimide ester. It was used for modification of amino groups of lysine residues in polypeptide chains of human serum albumin and some immunoglobulins G. Purification of Eu3+ complexonate-protein conjugates by gel-chromatography on a Superose- 12 column allowed to separate the modified proteins from unreacted low molecular weight Eu(3+)-derivatives and to determine a degree of lanthanide inclusion into a protein. The amount of Eu3+ covalently attached to a protein was determined by measuring the fluorescence of a conjugate in the dissociative-enhancement solution. The obtained values correlated well with the results of ICP-MS determination of Eu3+ concentration in a conjugate solution. It was shown that conjugates of monoclonal antibodies obtained by the proposed method possessed the required characteristics of fluorescence intensity, signal-to-noise ratio, sensitivity and specificity in DELFIA medical diagnostic systems.

Self-assembling bubble carriers for oral protein delivery

Successful oral delivery of therapeutic proteins such as insulin can greatly improve the quality of life of patients. This study develops a bubble carrier system by loading diethylene triamine pentaacetic acid (DTPA) dianhydride, a foaming agent (sodium bicarbonate; SBC), a surfactant (sodium dodecyl sulfate; SDS), and a protein drug (insulin) in an enteric-coated gelatin capsule. Following oral administration to diabetic rats, the intestinal fluid that has passed through the gelatin capsule saturates the mixture; concomitantly, DTPA dianhydride produces an acidic environment, while SBC decomposes to form CO2 bubbles at acidic pH. The gas bubbles grow among the surfactant molecules (SDS) owing to the expansion of the generated CO2. The walls of the CO2 bubbles consist of a self-assembled film of water that is in nanoscale and may serve as a colloidal carrier to transport insulin and DTPA. The grown gas bubbles continue to expand until they bump into the wall and burst, releasing their transported insulin, DTPA, and SDS into the mucosal layer. The released DTPA and SDS function as protease inhibitors to protect the insulin molecules as well as absorption enhancers to augment their epithelial permeability and eventual absorption into systemic circulation, exerting their hypoglycemic effects.

HPLC ANALYSIS OF RADIOGALLIUM LABELED PROTEINS USING A TWO-SOLVENT SYSTEM

A two-solvent system was used in high-performance liquid chromatography (HPLC) analysis of radiolabeled pharmaceutically active proteins. The proteins were successively labeled with [67Ga]-gallium chloride separately after conjugation with diethylenetriaminepentaacetic acid dianhydride (ccDTPA). The radiolabelded proteins were purified using size exclusion chromatography and/or solid-phase purification. A two-solvent system consisting sodium acetate/citrate buffer was used for the determination of radiochemical purity (90–98%), specific activity (22–23 TeBq/mM), and labeling efficiency (70–82%) at optimized conditions in each case. The retention times for insulin, β-HCG, gonadotropin, erythropoietin, glucagon, (1-24-corticotrophin, rituximab, oxytocine, and streptokinase were 17.2, 14.8, 18.31, 12.77, 20.0, 21.97, 15.2, and 17.2 min, respectively, while for 67Ga3+ cation and/or 67Ga-DTPA were at the range of 2.70–2.90 min. This is a simple and inexpensive HPLC method for the analysis of radiolabeled peptides and proteins using reverse-phase columns.

Infrared spectroscopic study of the model metal–ligand–antibody systems: What information on the structure and stability of systems can be obtained?

In this study we investigated the possibility of using infrared spectroscopy for the characterization of the ligand (diethylenetriaminepentaacetic acid, DTPA) in various aqueous media (water and different buffers), the antibody (immunoglobulin G, IgG), the metal–ligand complexes (Y–DTPA and Lu–DTPA) and the metal–ligand–antibody conjugates (Y–DTPA–IgG and Lu–DTPA–IgG). The aim was to describe behaviour of individual components, and to find spectral markers of complexes and/or conjugates formation. The reason of the choice of Lu and Y was the potential use of their radionuclides to form radiopharmaceutical species for nuclear medicine. We performed kinetic measurements to describe the stability/instability of individual systems. Multivariate data analysis allowed finding out spectral regions related to different chemical processes. Hydrolysis effect of the diethylenetriaminepentaacetic acid dianhydride (cDTPAA) in water was confirmed in unbuffered aqueous solution. Nevertheless, the study of cDTPAA in various buffered media showed evident suppression of hydrolysis. Thus, appropriate buffers were selected as media for complexation reactions with Y and Lu. Characteristic marker bands of complexation were identified. Human IgG in phosphate buffer and its interaction with the metal–ligand complexes were studied. Observation of new bands, band shifts and intensity changes revealed formation of the metal–ligand–IgG conjugates together with a somewhat changed secondary structure of IgG. We demonstrated the possibility of vibrational spectroscopic monitoring of the preparation steps of the metal–ligand–antibody conjugates as potential radiopharmaceutical products.

Technology of DTPA and immunoglobulins conjugation and their attachment to 90Y and 177Lu radionuclides

The aim of the study of labeling of ligand–antibody conjugates was to find optimal conditions of preparing of these conjugates and appropriate radioactivity of selected nuclide for applications in nuclear medicine. Conjugation of the γ-immunoglobulin G (human or bovine IgG, polyclonal antibodies) and bifunctional chelating agent, diethylenetriaminepentaacetic acid dianhydride (cDTPAA), was carried out. Various values of the cDTPAA/antibody ratio, the weight concentration of polyclonal or monoclonal antibodies (MEM-97) and buffers were used. Further, the labeling conditions of the DTPA–IgG conjugate by radionuclides 90Y and 177Lu were optimized, and the labeling yield and the conjugation ratio of prepared radionuclide–DTPA–IgG conjugates was determined. Optimal incubation time of the immunoglobulin conjugation was obtained at 30 min from mixing of individual components. The labeling yield of radionuclide–DTPA–antibody conjugate higher than 95% was achieved. Higher values of conjugation ratio of radionuclide–DTPA–antibody conjugate were achieved in 0.1 mol L−1 carbonate buffer, pH 8.5, and the 0.1 mol L−1 carbonate buffer is suitable for studied conjugation systems. This study showed that the labeling yield as well as the conjugation ratio of tested systems depend on the amount of antibody substance, bifunctional chelating agent/antibody molar ratio and pH value of the buffer used.

Porous Polymersomes with Encapsulated Gd‐Labeled Dendrimers as Highly Efficient MRI Contrast Agents

The use of nanovesicles with encapsulated Gd as MR contrast agents has largely been ignored due to the detrimental effects of the slow water exchange rate through the vesicle bilayer on the relaxivity of encapsulated Gd. Here, we describe the facile synthesis of a composite MR contrast platform, consisting of dendrimer conjugates encapsulated in porous polymersomes. These nanoparticles exhibit improved permeability to water flux and a large capacity to store chelated Gd within the aqueous lumen, resulting in enhanced longitudinal relaxivity. The porous polymersomes, ~130 nm in diameter, were produced through the aqueous assembly of the polymers, polyethylene oxide-b-polybutadiene (PBdEO), and polyethylene oxide-b-polycaprolactone (PEOCL). Subsequent hydrolysis of the caprolactone (CL) block resulted in a highly permeable outer membrane. To prevent the leakage of small Gd-chelate through the pores, Gd was conjugated to PAMAM dendrimer via diethylenetriaminepentaacetic acid dianhydride (DTPA dianhydride) prior to encapsulation. As a result of the slower rotational correlation time of Gd-labeled dendrimers, the porous outer membrane of the nanovesicle, and the high Gd payload, these functional nanoparticles were found to exhibit a relaxivity (R1) of 292,109 mM(-1) s(-1) per particle. The polymersomes were also found to exhibit unique pharmacokinetics with a circulation half-life of >3.5 hrs and predominantly renal clearance.

Development of a radiolabeled β-human chorionic gonadotropin

beta-Human chorionic gonadotropin (beta-hCG) was successively labeled with [(67)Ga]-gallium chloride after conjugation with freshly prepared diethylenetriaminepentaacetic acid dianhydride (ccDTPA). After solid phase purification of the radiolabeled hormone, high performance liquid chromatography showed radiochemical purity higher than 95 % under optimized conditions (specific activity = 22-23 TBq mM(-1), labeling efficiency 80 %). Preliminary in vivo studies (ID g(-1), %) in male wild-type rats showed marked gonadal uptake of the tracer after 240 minutes in agreement with the biodistribution studies and reported beta-hCG receptors. Target to blood ratios were 5.1 and 15.2 after 3 and 24 hours, respectively, while target to muscle ratios were 35 and 40 after 3 and 24 hours, respectively.

Dual-Labeled Trastuzumab-Based Imaging Agent for the Detection of Human Epidermal Growth Factor Receptor 2 Overexpression in Breast Cancer

Overexpression of the human epidermal growth factor receptor (HER) family has been implicated in cancer because of its participation in signaling pathways regulating cellular proliferation, differentiation, motility, and survival. In this work, we exploited the extracellular binding property of trastuzumab, a clinically therapeutic monoclonal antibody to the second member of the HER family (HER2), to design a diagnostic imaging agent, ((111)In-DTPA)(n)-trastuzumab-(IRDye 800CW)(m), that is dual labeled with (111)In, a gamma-emitter, and a near-infrared (NIR) fluorescent dye, IRDye 800CW, to detect HER2 overexpression in breast cancer cells. The stoichiometric ratios "n" and "m" refer to the number of diethylenetriaminepentaacetic acid dianhydride (DTPA) and IRDye 800CW molecules bound per trastuzumab molecule, respectively. Fluorescence microscopy and confocal microscopy were used to determine the molecular specificity of (DTPA)(n)-trastuzumab-(IRDye800)(m) in vitro in SKBr3 (HER2-positive) and MDA-MB-231 (HER2-negative) breast cancer cells. SKBr3 cells were incubated with (DTPA)(n)-trastuzumab-(IRDye800)(m) or IRDye800CW or pretreated with trastuzumab or human IgG followed by (DTPA)(n)-trastuzumab-(IRDye800)(m) and examined under a fluorescence microscope. For in vivo characterization, athymic nude mice bearing HER2-overexpressing SKBr3-luc subcutaneous xenografts were injected intravenously with ((111)In-DTPA)(n)-trastuzumab-(IRDye800)(m) and imaged with SPECT and NIR fluorescence imaging at 48 h. Tumor-bearing mice were also injected intravenously with trastuzumab 24 h before administration of ((111)In-DTPA)(n)-trastuzumab-(IRDye800)(m). Nonspecific uptake in the SKBr3-luc tumors was analyzed by injecting the mice with IRDye 800CW and ((111)In-DTPA)(p)-IgG-(IRDye800)(q), where "p" and "q" are the stoichiometric ratios of DTPA and IRDye 800CW bound per IgG antibody, respectively. (DTPA)(n)-trastuzumab-(IRDye800)(m) showed significantly greater binding to SKBr3 cells than to MDA-MB-231 cells. Confocal imaging revealed that this binding occurred predominantly around the cell membrane. Competitive binding studies with excess trastuzumab before incubation with (DTPA)(n)-trastuzumab-(IRDye800)(m) abolished this binding affinity, but pretreatment with nonspecific IgG did not alter binding. In vivo nuclear and optical imaging of SKBr3-luc xenografts injected with ((111)In-DTPA)(n)-trastuzumab-(IRDye800)(m) revealed significantly more uptake in the tumor region than in the contralateral muscle region. The tumor-to-muscle ratio decreased in mice pretreated with trastuzumab and in mice injected with IRDye 800CW and ((111)In-DTPA)(p)-IgG-(IRDye800)(q). Ex vivo imaging of dissected organs confirmed these results. Finally, coregistration of histologic hematoxylin-eosin stains with autoradiography signals from tumor and muscle tissue slices indicated that ((111)In-DTPA)(n)-trastuzumab-(IRDye800)(m) bound only in tumor tissue and not to muscle. Dual-labeled ((111)In-DTPA)(n)-trastuzumab-(IRDye800)(m) may be an effective diagnostic biomarker capable of tracking HER2 overexpression in breast cancer patients.

The determination of the rate of conjugation immunoglobuline with bifunctional chelator

The work was performed under the GACR project: „Technology of preparation of radionuclides and their labelled compounds for nuclear medicine and pharmacy with the use of the reactor LVR-15“ reg. no. 104/03/0499. Imaging of cell’s antigens with the use of labelled immunoglobulines allows imaging of specific receptors on cell membrane and specific tumours. It is necessary to carry out the labelling of the immunoglobulines with radionuclides of suitable physical properties, which form cations (e.g., 111In, 90Y, 177Lu) that form very strong chelates of sufficiently high stability constant preventing the dissociation of complexes or the radionuclide under “ in-vivo” conditions. The immunoglobuline must be conjugated with the bifunctional chelator (BCH), which contains both chelating unit and reactive group for binding to the immunoglobuline. In our laboratory we have conjugated human IgG and monoclonal antibody CD20 with diethylenetriamine pentaacetic acid dianhydride (cDTPAA). Radionuclides 90Y and 177Lu prepared on the LVR-15 reactor in NRI Rez were used for labelling. After conjugation and labelling the yields in relation to the amount of isotopic carrier have been determined.

Metal-to-ligand charge-transfer sensitisation of near-infrared emitting lanthanides in trimetallic arrays M2Ln (M = Ru, Re or Os; Ln = Nd, Er or Yb)

The new pro-ligand 4-methyl-4'-(carbonylamino(2-(tert-butoxycarbonylamino)ethyl))-2,2'-bipyridyl (L1) has been prepared and used to synthesise the complex fac-Re(I)Cl(CO)3(L1) 1 and the complex salts [M(II)(bipy)2(L1)](PF6)2 (M=RuII 8 or OsII 15). Deprotection with trifluoroacetic acid affords the amine-functionalised derivatives fac-Re(I)Cl(CO)3(L2) 2, [M(II)(bipy)2(L2)](PF6)2 (M=RuII 9 or OsII 16) which react with the dianhydride of diethylenetriamine pentaacetic acid (DTPA) to give the binuclear complex {fac-Re(I)Cl(CO)3}2(L3) 3 and the complex salts [{M(II)(bipy)2}2(L3)](PF6)4 (M = RuII 10 or OsII 17). The latter react with salts Ln(OTf)3 to afford a series of 12 heterotrimetallic compounds that contain a lanthanide (Ln) ion in the DTPA binding site; {fac-Re(I)Cl(CO)3}2(L3)LnIII (Ln=Nd 4, Er 5, Yb 6 or Y 7) and [{M(II)(bipy)2}2(L3)LnIII](PF6)(OTf)3 (M=RuII, Ln=Nd 11, Er 12, Yb 13 or Y 14; M=OsII, Ln=Nd 18, Er 19, Yb 20 or Y 21). All of these trimetallic species display absorption bands ascribed to metal-to-ligand charge-transfer (MLCT) excitations, and luminescence measurements show that these excited states can be used to sensitise near-infrared emission from LnIII (Ln=Nd, Er or Yb) ions. Single crystal X-ray structures of L1 and [RuII(bipy)2(L2H)](H2PO4)3.(CH3)2CO.0.8H2O were obtained, the latter revealing the presence of H2PO4- counter anions, the source of which is presumed to be hydrolysis of PF6- ions.

A derivative of diethylenetriaminepentaacetic acid for europium labeling of proteins.

Preparation of a reagent that will incorporate diethylenetriaminepentaacetic acid (DTPA) into proteins under mild conditions and make a strong europium chelate is described. Aminoacetaldehyde diethyl acetal was reacted with DTPA dianhydride, and mono- and disubstituted products as well as unsubstituted DTPA were separated by gel filtration. The monosubstituted product, after conversion into the corresponding aldehyde by mild acid hydrolysis, is conjugated to protein or other amino-containing compounds via reductive amination at neutral pH. Although the DTPA-Eu-labeled proteins are themselves not fluorescent, a strong fluorescence of europium can be generated easily by the dissociation-enhancement mechanism. A direct measurement of lectin-ligand interaction using Eu-labeled ligand and lectin immobilized on 96-well plate illustrates that the assay utilizing Eu fluorescence is as sensitive as the radioactive assays.

Labeling of monoclonal antibodies with diethylenetriaminepentaacetic acid-appended radioiodinated peptides containing D-amino acids.

The optimal use of radioiodinated internalizing monoclonal antibodies (mAbs) for radioimmunotherapy necessitates the development of practical methods for increasing the level of retention of 131I in the tumor. Lysosomally trapped ("residualizing") iodine radiolabels that have been previously designed are based mostly on carbohydrate-tyramine adducts, but these methods have drawbacks of low overall yields and/or high levels of mAb aggregation. We have developed a method using thiol-reactive diethylenetriaminepentaacetic acid (DTPA)-peptide adducts wherein the peptides are assembled with one or more D-amino acids, including D-tyrosine. Two such substrates, R-Gly-D-Tyr-D-Lys[1-(p-thiocarbonylaminobenzyl)DTPA], referred to as IMP-R1, and [R-D-Ala-D-Tyr-D-Tyr-D-Lys]2(CA-DTPA), referred to as IMP-R2, wherein R is 4-(N-maleimidomethyl)cyclohexane-1-carbonyl, were synthesized by preparing functional group-protected peptides on a solid phase, selectively derivatizing the lysine side chain with 1-(p-isothiocyanatobenzyl)DTPA or DTPA dianhydride (CA-DTPA), deprotecting other functional groups, and finally derivatizing the peptide's N-terminus so it contained a maleimide group. Radioiodinations of the peptides followed by conjugations to disulfide-reduced mAbs, carried out as a one-vial procedure, resulted in 32-89% overall yields, at specific activities of 1.8-11. 1 mCi/mg, with less than 2% aggregation. Two internalizing mAbs, LL2 (anti-CD 22 B-cell lymphoma mAb) and RS7 (an anti-adenocarcinoma mAb which targets EGP-1 antigen), labeled with this procedure exhibited a 2-3-fold better cellular retention in Ramos and Calu-3 tumor cell lines, in vitro, respectively, compared to the same mAbs radioiodinated with the chloramine-T method. The rationale for the new approach, syntheses, radiochemistry and in vitro data are presented.

Biodistribution and Imaging of Polyethyleneimine-A Gene Delivery Agent

Polyethyleneimine (PEI) has been described as a potentially effective agent for gene delivery. To track the delivery of this gene vector, the biodistribution and imaging of PEI labeled with 111Indium (111In) was studied in Fischer 344 rats. PEI was conjugated with diethylenetriamine pentaacetic acid dianhydride (DTPA), dialyzed, and chelated with 111In. Breast adenocarcinoma 13762 tumor cells were inoculated into the thighs of the rats. The first group of rats (n = 3) were injected intravenously with 300 mu g of the Indiumlabeled DTPA-polyethlyeimine (111In-DTPA-PEI) (50 mu Ci per rat) or 111In-DTPA. These animals were imaged with a gamma camera with a medium energy parallel hole collimator at 5 min, 2 hr, and 24 hr postinjection. The percentage of uptake in tumor (region of interest) was quantitated by a computer image analyzer and expressed as a percentage of injected dose (%ID) per pixel. To further characterize the tissue distribution of 111In-DTPA-PEI (300 mu g, 10 mu Ci per rat) and 111In-DTPA (10 mu Ci per rat), a second group of animals (n = 18) bearing breast tumors were studied with tissue uptake quantified at 2 hr, 24 hr, and 48 hr using a gamma counter. In addition, autoradiography was used to further characterize the distribution of the labeled polymer in two rats at 2 and 24 hr. From these studies, PEI was found to be rapidly cleared, primarily through the kidneys of the rats. In addition, the distribution of 111In-DTPA-PEI was found to be significantly different from 111In-DTPA with a higher tumor-to-blood ratio. These studies show that radio-labeled PEI may have potential as a gamma scintigraphy imaging agent and in tracking the delivery of genetic material.

Time-resolved fluoroimmunoassay.

A method is reported to set up a standard competitive TR-FIA. A simple and inexpensive way to prepare reagents and carry out operations is presented as well, with the aim to make it possible to perform a very sensitive analytical procedure in a personalized way within a nondedicated biochemistry laboratory. This protocol is general and can be easily modified with consideration to the analytical target. Once the antibody is available, both its labeling with diethylenetriaminepentaacetic acid dianhydride and Eu3+, and the setting-up of the assay with measurement of europium ion time-resolved fluorescence in a home-made enhancement solution become feasible.

Synthesis and Relaxivity of Polyamide Paramagnetic Metal Complexes for Magnetic Resonance Imaging

A new class of paramagnetic macromolecular magnetic resonance imaging contrast agents has been developed. Eight new polyamide ligands were synthesized by copolymerization of ethylenediaminetetraacetic acid dianhydride or diethylenetriaminepentaacetic acid dianhydride and diamine monomers. Their gadolinium(III), manganese(II) and iron(III) complexes were also synthesized. All polyamide ligands and metal complexes were characterized by 1H nuclear magnetic resonance, infrared spectra and elemental analyses. Relaxivity studies showed that the polyamide paramagnetic metal complexes had obviously higher relaxation effectiveness as compared to corresponding simple monomeric paramagnetic metal complexes.