Preparation and radiolabeling of human serum albumin (HSA)-coated magnetite nanoparticles for magnetically targeted therapy

In this paper, we describe the preparation of human serum albumin-coated magnetic particles of about 200 nm in diameter with narrow size distribution radiolabeled with ¹⁸⁸Re for the purpose of magnetically targeted therapy. The optimum radiolabeling conditions are: SnCl₂·2H₂O 8 mg/ml, citric acid 20 mg/ml, vitamin C 8 mg/ml, labeling volume 500 μl and a reaction time of 3 h. The stability of the radiolabeled particles is suitable for in vivo study.     

Microfluidic radiolabeling of biomolecules with PET radiometals

IntroductionA robust, versatile and compact microreactor has been designed, fabricated and tested for the labeling of bifunctional chelate conjugated biomolecules (BFC-BM) with PET radiometals.MethodsThe developed microreactor was used to radiolabel a chelate, either 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) that had been conjugated to cyclo(Arg-Gly-Asp-DPhe-Lys) peptide, with both ⁶⁴Cu and ⁶⁸Ga respectively. The microreactor radiolabeling conditions were optimized by varying temperature, concentration and residence time.ResultsDirect comparisons between the microreactor approach and conventional methods showed improved labeling yields and increased reproducibility with the microreactor under identical labeling conditions, due to enhanced mass and heat transfer at the microscale. More importantly, over 90% radiolabeling yields (incorporation of radiometal) were achieved with a 1:1 stoichiometry of bifunctional chelate biomolecule conjugate (BFC-BM) to radiometal in the microreactor, which potentially obviates extensive chromatographic purification that is typically required to remove the large excess of unlabeled biomolecule in radioligands prepared using conventional methods. Moreover, higher yields for radiolabeling of DOTA-functionalized BSA protein (Bovine Serum Albumin) were observed with ⁶⁴Cu/⁶⁸Ga using the microreactor, which demonstrates the ability to label both small and large molecules.ConclusionsA robust, reliable, compact microreactor capable of chelating radiometals with common chelates has been developed and validated. Based on our radiolabeling results, the reported microfluidic approach overall outperforms conventional radiosynthetic methods, and is a promising technology for the radiometal labeling of commonly utilized BFC-BM in aqueous solutions.     

Iodination and radiolabeling of alpha-allocryptopine with iodine-125.

A procedure for labeling of alpha-allocryptopine with 125I is reported in this article. Alpha-allocryptopine is first iodinated with largely excessive iodine monochloride, and then radiolabeled by isotopic exchange with Na125I in a dichloromethane-water two-phase system. The radiochemical purity and yield of the labeled product are typically as high as 94% and 72%, respectively. Further chromatographic purification gives a highly pure product (radiochemical purity > 99%). The distribution of 125I-labeled alpha-allocryptopine in mice at 2 h after injection shows that the uptake of radioiodine in thyroid is negligibly low.     

Recent developments in the radiolabeling of antibodies with iodine, indium, and technetium

The use of radiolabeled antibodies for tumor detection and therapy has provided some striking successes despite unfavorable tumor-to-normal tissue radioactivity ratios due, in part, to the accumulation of the label in normal tissues. One approach, which has been and is still under consideration to reduce unwanted background levels, is the improvement of ways in which radiolabels are attached to antibody, especially with the goal of increasing in vivo stability. Although these improvements have occurred throughout the history of this field, important developments have recently been reported in the labeling of antibodies with three radiolabels, namely radioiodine, 111In, and 99mTc. Thus, antibodies may now be labeled with radioisotopes of iodine in ways that minimize the extent of in vivo dehalogenation leading to thyroid, stomach, and gut radioactivity uptake. Newer and stronger chelates for 111In have been developed in the hope that their use would result in lower radioactivity levels in the liver. Finally, newer methods, both direct and indirect, for the attachment of 99mTc to antibodies have been developed and are now being clinically tested. Although these developments have taken place only recently and the in vivo behavior of labels attached in these ways have not yet been fully characterized, it is possible to make tentative conclusions regarding their impact. Thus, the use of stably radioiodinated antibodies appears to have resulted in modest improvements in patient images. In contrast, the use of stable chelates for labeling antibodies with 111In may have had no appreciable effect on liver radioactivity levels. The use of antibodies radiolabeled with 99mTc, especially via the newer direct labeling methods, are providing superior images in patients with low radioactivity levels in organs such as liver. However, it must still be established whether the short physical half-life of 99mTc lowers sensitivities and specificities of detection relative to other labels.     

Direct radiolabeling of monoclonal antibodies with rhenium-188 for radioimmunotherapy of solid tumors--a review of radiolabeling characteristics, quality control and in vitro stability studies.

188Re is one of the radioisotopes expected to emerge as useful for therapy. Development of new radiopharmaceuticals based on 188Re depends on the radiolabeling methods used, which would give stable complexes having predefined radiochemical properties and in vitro and in vivo stability. This paper has attempted to provide a perspective of 188Re-labeled monoclonal antibodies, their radiolabeling characteristics, methods for quality control of radioimmunoconjugates and in vitro stability for radioimmunotherapy of solid tumors. The direct method of 188Re radiolabeling of antibodies by reductive attachment of 188Re in which free sulfhydryl groups have been generated by reduction of the intramolecular S-S disulfide bonds has been shown to be a promising approach in particular. Moreover, excellent methods have been developed to test the radionuclide, radiochemical purity and stability of 188Re-radioimmunoconjugates using high performance liquid chromatography (HPLC) and paper chromatography.     

The radiolabeling of proteins by the [18F]AlF method

A new ([(18)F]AlF)(2+)-binding ligand that contains 1,4,7-triazacyclononane-1,4-diacetate (NODA) attached to a methyl phenylacetic acid group (MPA) was conjugated to N-(2-aminoethyl)maleimide (EM) to form NODA-MPAEM. The NODA-MPAEM was labeled with ([(18)F]AlF)(2+) at 105°C in 49-82% yield and conjugated at room temperature to an antibody Fab' fragment in 69-80% yield (total time ～50min) and with retention of immunoreactivity. These data indicate that the rapid and simple [(18)F]AlF-labeling method can be easily adapted for preparing heat-sensitive compounds with (18)F quickly and in high yields.     

A facile no-carrier-added radioiodination procedure suitable for radiolabeling kits

A simple, straightforward radioiodination procedure has been developed that is ideal for use in the construction of radioiodination kits. Isolation of no-carrier-added radioiodinated products from the ionic precursors is readily achieved using simple Sep-Pak filtrations.     

Catabolism of neurotensins. Implications for the design of radiolabeling strategies of peptides.

A major impact in diagnosis and treatment of cancer with peptide based radiopharmaceuticals is expected. Among others neurotensin is considered to be a promising candidate. However, most neurotensin analogues, which bind to the neurotensin receptor have a too short biological half live due to catabolism. Therefore, stabilized fragments have been prepared and labeled with the newly developed [Tc(CO)3](+)-moiety. A single histidine or a (N alpha-His)-Ac group coupled to the N-terminus of the neurotensin fragments were used as a bidentate or a tridentate ligand respectively, which coordinate the metal carbonyl efficiently. Affinity and binding studies of the 99mTc(I) radiolabeled neurotensin fragments revealed a behavior influenced by catabolism and properties of the metal complex.     

Isotype switch variant anti-idiotype monoclonal antibodies: comparative radiolabeling and in vitro binding

The influence of antibody isotype on radiolabeling efficacy and immunoreactivity has been difficult to systematically examine as antibodies of different isotypes generally vary in both constant and variable regions. The recent ability to isolate class or isotype switch monoclonal antibodies that have identical binding regions but different constant regions allows for such a comparison to be undertaken. Two isotype switch variant families of murine anti-idiotypic monoclonal antibodies were studied for radioiodination efficacy and immunoreactivity following labeling. In one of the families (S3H5), the IgG2a isotype switch variant derived from an IgG2b parent had nearly 70% greater iodine incorporation and over 50% greater immunoreactivity than the IgG2b parent. In the other family (S5A8), the IgG2a isotype switch variant had virtually identical efficacy of iodine incorporation and binding to antigen after labeling as did its IgG2b parent. Differences in relative heavy and light chain iodine incorporation were seen among isotype switch variants and their parents regardless of alterations in quantitative iodine incorporation or immunoreactivity. We conclude that in certain instances, cloning of an isotype switch variant antibody can result in an antibody offspring that has superior radiolabeling characteristics to its parent antibody. This isotype switching approach may find utility in converting highly-specific, but difficult to label antibodies, to more useful agents.     

Synthesis, radiolabeling, biodistribution and fluorescent imaging of histidine-coupled hematoporphyrin

IntroductionHematoporphyrin (Hp) and hematoporphyrin derivatives (HpDs) have been widely used as photosensitizers in photodynamic therapy (PDT). Radiolabeling of HpDs is helpful for preclinical and clinical studies of PDT.MethodsThe histidine-coupled hematoporphyrin (His-Hp) was synthesized and radiolabeled with [^99mTc(CO)₃(H₂O)₃]⁺. Biodistribution of the radioligand and fluorescent imaging of His-Hp in mice bearing S180 tumor were investigated.Results[^99mTc(CO)₃]⁺-labeled His-Hp was electrically neutral, hydrophilic and stable. The biodistribution of the radioligand in S180 tumor-bearing mice was similar with that of nonlabeled HpD in the literature. The uptake of His-Hp in tumors and livers was confirmed by fluorescent imaging.ConclusionsThe complex [^99mTc(CO)₃]⁺–His-Hp might be suitable for in vivo dose evaluation of HpD in PDT.     

A new method for radiolabeling of Gelfoam particles with 99mTc

Embolization of the spleen and other organs using Gelfoam has recently gained acceptance as a therapeutic procedure. A simple non-invasive determination of the placement of Gelfoam particles is needed and this requires labeling Gelfoam with radionuclides. We have developed a simple, reliable reproducible, and inexpensive method of labeling Gelfoam with 99mTc using commercial sulfur colloid kits. Optimal reaction conditions include room temperature incubation for 60 min at 12 rpm rotation. In vitro stability indicated no loss of 99mTc activity up to 3 h post labeling. Imaging of intra-arterially placed 99mTc Gelfoam at 1 h and 24 h in animals and humans has consistently produced well-defined images of the radioemboli.     

Lung uptake of technetium-99m microaggregated human albumin in the rat after treatment with microaggregated human albumin or macroaggregated human albumin

Marked accumulation in lung and liver of intravenously-injected ^99mTc-microaggregated human serum albumin (liver imaging agent) was observed in rats pretreated with the subcutaneous injection of microaggregated or macroaggregated human serum albumin, whereas accumulation of ^99mTc-microaggregated human serum albumin was observed only in the liver of rats pretreated with plain human serum albumin or in non-treated control rats. The activity of the intravenously-administered ^99mTc-sulfur colloid was concentrated in the liver and spleen only and not in the lung, of rats previously treated with human serum albumin, microaggregated human serum albumin, or macroaggregated human serum albumin.These observations suggest that the specific accumulation of microaggregated human serum albumin in the lung of rats pretreated with aggregated albumin is due to rapid in vivo clumping of injected particles, possibly due to antigen-antibody reaction.     

Synthesis, radiolabeling, and biodistribution of putative metabolites of iodoazomycin arabinoside

Scintigraphic evaluation of patients with advanced oncological disease showed uptake of radioactivity in the brain following administration of the hypoxic imaging agent 123I-iodoazomycin arabinoside (123I-IAZA). Three proposed metabolites of IAZA--methyl 5-deoxy-5-iodo-D-arabinofuranoside, methyl 2,3-di-O-acetyl-5-deoxy-5-iodo-alpha-D-arabinofuranoside, and 1-(5-deoxy-5-iodo-alpha-D-arabinofuranosyl)-2-aminoimidazole (IAIA)--were synthesized, radiolabeled with 125I, and investigated in normal and tumor-bearing murine models for their contribution to this unusual phenomenon. The three compounds were readily radiolabeled by melt or solvent exchange procedures. Biodistribution data indicated rapid blood clearance, rapid excretion, and little tissue accumulation in the brain. IAIA showed significant tumor to blood ratios at 4 h (4.3:1) and liver to blood ratios at 24 h (30:1).     

Antigen-binding site protection during radiolabeling leads to a higher immunoreactive fraction

It is generally accepted that the immunointegrity of an antibody (Ab) depends on the preservation of its antigen-binding sites. Our goal was to radiolabel an antibody at several iodine:antibody molar ratios under conditions protecting its combining site and to compare its immunoreactive fraction (IRF) and electrophoretic mobility with those of the same antibody radiolabeled without protection. The data indicate that an antibody radiolabeled while its antigen-binding site is occupied by its antigen had the same IRF, regardless of the number of iodine atoms per antibody molecule. On the other hand, even at an I:Ab ratio of 1:1, the IRF of the same antibody radiolabeled without protection was lower than that of a protected one and decreased with increasing I:Ab ratios. In addition, the iodination of these Ab changes their electrophoretic mobility; however, when the Ab is labeled in the protected state, the degree of change is less. The binding of an antibody to its antigen prior to radiolabeling, therefore, enhances its immuno-integrity and prevents major conformational changes as reflected by electrophoresis.     

Preparation and radiolabeling of antimony sulfide nanocolloids with two different particle sizes

Antimony sulfide nanocolloids with two different particle sizes were prepared by a modified traditional method and a new method. Transmission electron microscope (TEM) showed the mean particle sizes to be 10.2±2.3 and 44.7±13.8 nm, respectively. The nanocolloids were then radiolabeled with ^99mTc, or with ¹⁸⁸Re. The labeling yields of ^99mTc-Sb₂S₃ (10.2±2.3 nm and 44.7±13.8 nm)>98% and >96% for ¹⁸⁸Re-Sb₂S₃ (10.2±2.3 and 44.7±13.8 nm). The stability of the labeled products was studied at room temperature and all showed excellent stability except for ¹⁸⁸Re-Sb₂S₃ (10.2±2.3 nm). The mechanism of labeling of nanocolloids with ¹⁸⁸Re is different from that of ^99mTc. The antimony sulfide nanocollides prepared by the new method, after labeling are expected to have diagnostic or therapeutic potential in nuclear medicine.