99m Technetium-HMPAO-labeled platelet scan in practice: Preparation, quality control, and biodistribution studies

Abstract There is no biodistribution or imaging data on 99mtechnetium (Tc)-hexamethyl propylamine oxime (HMPAO)-labeled platelets in the literature. The current study aimed to present updated information about the clinical procedures for preparation and use of labeled platelets. Following two-step centrifugation at 1500 and 2500 rpm, the platelets were extracted from whole blood into platelet-rich plasma (PRP) above the buffy coat and then from PRP into a platelet pellet at the bottom of the tube. The 99mTc-HMPAO-labeled platelets were inspected for purity, viability, release of 99mTc from platelets, and sterility. Also, microscopic examination and thin layer chromatography (TLC) were performed. Biodistribution was assessed following necropsy in BALB/c mice and through imaging of New Zealand rabbits. The separation ratio was estimated at 98%, and radiochemical purity was measured to be 80%. The labeling efficiency was above 30% in more than half of the assays (range: 17-43%). The release of 99mTc from platelets was 9% per hour at 37ºC. After 24 hours, stability was estimated at 54% in the human serum. The target organs of mice included the spleen and liver. In rabbits, the imaging results indicated liver as the target organ. Thyroid uptake was negligible up to 90 minutes. Based on the findings, extraction of platelets and labeling them with 99mTc-HMPAO is a feasible and safe approach in routine practice.

Radiolabeling of Preformed Niosomes with [99mTc]: In Vitro Stability, Biodistribution, and In Vivo Performance.

Nanocarriers radiolabeled with [99mTc] can be used for diagnostic imaging and radionuclide therapy, as well as tracking their pharmacokinetic and biodistribution characteristics. Due to the advantages of niosomes as an ideal drug delivery system, in this study, the radiolabeling procedure of niosomes by [99mTc]-HMPAO complexes was investigated and optimized. Glutathione (GSH)-loaded niosomes were prepared using a thin-film hydration method. To label the niosomes with [99mTc], the preformed GSH-loaded niosomes were incubated with the [99mTc]-HMPAO complex and were characterized for particle size, size distribution, zeta potential, morphology, and radiolabeling efficiency (RE). The effects of GSH concentration, incubation time, incubation temperature, and niosomal composition on RE were investigated. The biodistribution profile and in vivo SPECT/CT imaging of the niosomes and free [99mTc]-HMPAO were also studied. Based on the results, all vesicles had nano-sized structure (160-235 nm) and negative surface charge. Among the different experimental conditions that were tested, including various incubation times, incubation temperatures, and GSH concentrations, the optimum condition that resulted in a RE of 92% was 200-mM GSH and 15-min incubation at 40°C. The in vitro release study in plasma showed that about 20% of radioactivity was released after 24 h, indicating an acceptable radiolabeling stability in plasma. The biodistribution of niosomes was clearly different from the free radiolabel. Niosomes carrying radionuclide were successfully used for tracking the in vivo disposition of these carriers and SPECT/CT imaging in rats. Furthermore, biodistribution studies in tumor-bearing mice revealed higher tumor accumulation of the niosomal formulation as compared with [99mTc]-HMPAO.

Effect of coating thickness of iron oxide nanoparticles on their relaxivity in the MRI.

OBJECTIVES: Iron oxide nanoparticles have found prevalent applications in various fields including drug delivery, cell separation and as contrast agents. Super paramagnetic iron oxide (SPIO) nanoparticles allow researchers and clinicians to enhance the tissue contrast of an area of interest by increasing the relaxation rate of water. In this study, we evaluate the dependency of hydrodynamic size of iron oxide nanoparticles coated with Polyethylene glycol (PEG) on their relativities with 3 Tesla clinical MRI. MATERIALS AND METHODS: We used three groups of nanoparticles with nominal sizes 20, 50 and 100 nm with a core size of 8.86 nm, 8.69 nm and 10.4 nm that they were covered with PEG 300 and 600 Da. A clinical magnetic resonance scanner determines the T1 and T2 relaxation times for various concentrations of PEG-coated nanoparticles. RESULTS: The size measurement by photon correlation spectroscopy showed the hydrodynamic sizes of MNPs with nominal 20, 50 and 100 nm with 70, 82 and 116 nm for particles with PEG 600 coating and 74, 93 and 100 nm for particles with PEG 300 coating, respectively. We foud that the relaxivity decreased with increasing overall particle size (via coating thickness). Magnetic resonance imaging showed that by increasing the size of the nanoparticles, r2/r1 increases linearly. CONCLUSION: According to the data obtained from this study it can be concluded that increments in coating thickness have more influence on relaxivities compared to the changes in core size of magnetic nanoparticles.

Monoclonal antibody conjugated magnetic nanoparticles could target MUC-1-positive cells in vitro but not in vivo.

MUC1 antigen is recognized as a high-molecular-weight glycoprotein that is unexpectedly over-expressed in human breast and other carcinomas. In contrast, C595 a monoclonal antibody (mAb) against the protein core of the human urinary epithelial machine, is commonly expressed in breast carcinomas. The aim of this study was to conjugate ultra-small super paramagnetic iron oxide nanoparticles (USPIO) with C595 mAb, in order to detect in vivo MUC1 expression. A dual contrast agent (the C595 antibody-conjugated USPIO labeled with 99mTc) was prepared for targeted imaging and therapy of anti-MUC1-expressing cancers. The C595 antibody-conjugated USPIO had good stability and reactivity in the presence of blood plasma at 37 °C. No significant differences were observed in immunoreactivity results between conjugated and nonconjugated nanoparticles. The T1 and T2 measurements show >79 and 29% increments (for 0.02 mg/ml iron concentrations) in T1 and T2 values for USPIO-C595 in comparison with USPIO, respectively. The nanoprobes showed the interesting targeting capability of finding the MUC1-positive cell line in vitro. However, we found disappointing in vivo results (i.e. very low accumulation of nanoprobes in the targeted site while >80% of the injected dose per gram was taken up by the liver and spleen), not only due to the coverage of targeting site by protein corona but also because of absorption of opsonin-based proteins at the surface of nanoprobes.

Preparation, Biological Evaluation and Dosimetry Studies of 175Yb-Bis-Phosphonates for Palliative Treatment of Bone Pain.

OBJECTIVE: Optimized production and quality control of ytterbium-175 (Yb-175) labeled pamidronate and alendronate complexes as efficient agents for bone pain palliation has been presented. METHODS: Yb-175 labeled pamidronate and alendronate (175Yb-PMD and 175Yb-ALN) complexes were prepared successfully at optimized conditions with acceptable radiochemical purity, stability and significant hydroxyapatite absorption. The biodistribution of complexes were evaluated up to 48 h, which demonstrated significant bone uptake ratios for 175Yb-PAM at all-time intervals. It was also detected that 175Yb-PAM mostly washed out and excreted through the kidneys. RESULTS: The performance of 175Yb-PAM in an animal model was better or comparable to other 175Yb-bone seeking complexes previously reported. CONCLUSION: Based on calculations, the total body dose for 175Yb-ALN is 40% higher as compared to 175Yb-PAM (especially kidneys) indicating that 175Yb-PAM is probably a safer agent than 175Yb-ALN.

Synthesis and receptor binding studies of novel 4,4-disubstituted arylalkyl/arylalkylsulfonyl piperazine and piperidine-based derivatives as a new class of σ1 ligands.

This study presents the synthesis and biological evaluation of a new series of arylalkyl/arylalkylsulfonyl piperazine and piperidine-based derivatives as sigma receptor ligands. It was found that a number of halogen substituted sulfonamides display relatively high and low affinities to σ1 and σ2 receptors, respectively. The σ1 affinities and subtype selectivities of four piperidine derivatives were also found to be generally comparable to those of piperazine analogues. Compared to σ1-Rs compounds with n = 0 and 2, those with n = 1 proved to have optimal length of carbon chain by exhibiting higher affinities. Within this series, the 4-benzyl-1-(3-iodobenzylsulfonyl)piperidine sigma ligand was identified with 96-fold σ1/σ2 selectivity ratio (Kiσ1 = 0.96 ± 0.05 nM and Kiσ2 = 91.8 ± 8.1 nM).

Evaluating the effect of ultrasmall superparamagnetic iron oxide nanoparticles for a long-term magnetic cell labeling.

In order to evaluate the long-term viability, the iron content stability, and the labeling efficiency of mammalian cells using magnetic cell labeling; dextran-coated ultrasmall superparamagnetic iron oxide (USPIOs) nanoparticles with plain surfaces having a hydrodynamic size of 25 nm were used for this study. Tests were carried out in four groups each containing 5 flasks of 5.5 × 10(6) AD-293 embryonic kidney cells. The cell lines were incubated for 24 h using four different iron concentrations with and without protamine sulfate (Pro), washed with phosphate-buffered saline (PBS) and centrifuged three times to remove the unbounded USPIOs. Cell viability was also verified using USPIOs. There were no significant differences in the cell viability between the control group of cells and those groups with iron uptake at the specified iron concentrations. The average iron uptake ratio compared to that of the control group was (114 ± 1). The magnetic resonance images (MRI) at post-labeling day 1 and day 21 showed (75 ± 4)% and (22 ± 5)% signal decrements compared to that of the control, respectively. The Perl's Prussian blue test showed that 98% of the cells were labeled, and the iron concentration within the media did not affect the cell iron uptake. Magnetic cellular labeling with the USPIO-Pro complex had no short or medium term (3 weeks) toxic effects on AD-293 embryonic kidney cells.

Comparison of (99m)Tc-labeled PR81 and its F(ab')2 fragments as radioimmunoscintigraphy agents for breast cancer imaging.

OBJECTIVE: We digested anti-MUC1 monoclonal antibody PR81 to produce F(ab')2 fragments. A comparison was performed between the two radiolabeled PR81 and F(ab')2 fragments for breast tumor imaging in a mouse model. METHODS: The optimum conditions for pepsin digestion of PR81 were investigated in terms of enzymes: antibody ratio, digestion time duration and preserved immunoreactivity of the produced fragments. The F(ab')2 fragments were labeled with Technetium-99m using HYNIC as a chelator and tricine as a co-ligand. The immunoreactivity of the complexes was assessed by radioimmunoassay using MCF7 cells. Biodistribution and imaging studies were performed in female BALB/c mice with breast tumor xenograft at 4, 8 and 24 h post-administration. The PR81 was labeled with technetium-99m in the same way for comparison. RESULTS: The optimum time duration for PR81 digestion was found to be 28 h at an enzyme:antibody weight ratio of 1:20 that resulted in 95.2 ± 4.7% purity. The labeling of intact PR81 and its F(ab')2 fragments were 87.6 ± 4.2 and 76.1 ± 3.3% after 1 h, respectively (p value <0.05). The percentage of immunoreactivity of F(ab')2 fragments and intact PR81 were 75.4 ± 2.1% and 85.7 ± 2.9%, respectively (p value <0.05). The biodistribution and imaging studies demonstrated localization of the fragments at 4 h post-administration with high sensitivity and specificity. CONCLUSION: The results showed that F(ab')2 fragment of PR81 is more suitable than intact PR81 for safer and more rapid detection of human breast cancer.

177Lu labeling of Herceptin and preclinical validation as a new radiopharmaceutical for radioimmunotherapy of breast cancer.

INTRODUCTION: In the present study, Herceptin was labeled with lutetium-177 via DOTA, and the necessary preclinical quality control tests (in vitro and in vivo) were performed to evaluate its use as a radioimmunotherapy agent. MATERIAL AND METHODS: Herceptin was conjugated to DOTA as a chelator in three different conjugation buffers (ammonium acetate, carbonate and HEPES buffer); each of the resulting conjugates was compared with respect to in vitro characteristics such as number of chelates per antibody, incorporated activity, immunoreactivity and in vitro stability in PBS buffer and blood serum. The biodistribution study and gamma camera imaging were performed in mice bearing breast tumors. To assess the therapeutic effects of (177)Lu-Herceptin, cytotoxicity was investigated for 7 days in a SKBr3 breast cancer cell line. RESULTS: Carbonate buffer was the best conjugation buffer (number of chelates per antibody: 6; incorporated activity: 81%; immunoreactivity: 87%; buffer stability: 86%; serum stability: 81%, after 4 days). The efficient tumor uptake observed in the biodistribution studies was consistent with the gamma camera image results. At a concentration of 4 µg ml(-1), (177)Lu-Herceptin (surviving cells: 5 ± 0.6% of the total cells) of the total cells corresponded to an approximately eightfold increase in cytotoxicity in comparison to unmodified Herceptin (surviving cells: 43 ± 3.9%). CONCLUSION: The new complex described herein could be considered for further evaluation in animals and potentially in humans as a radiopharmaceutical for use in the radioimmunotherapy of breast cancer. These results may be important for patients who cannot tolerate the therapeutic dosage of Herceptin currently used because of heart problems.

Radiolabeling of trastuzumab with 177Lu via DOTA, a new radiopharmaceutical for radioimmunotherapy of breast cancer.

AIM: Trastuzumab is a monoclonal antibody that is used in treating breast cancer. We labeled this monoclonal antibody with lutetium-177 and performed in vitro quality control tests as a first step in the production of a new radiopharmaceutical. MATERIAL AND METHODS: Trastuzumab was labeled with lutetium-177 using DOTA as chelator. Radiochemical purity and stability in buffer and human blood serum were determined using thin layer chromatography. Immunoreactivity and toxicity of the complex were tested on MCF7 breast cancer cell line. RESULTS: The radiochemical purity of the complex was 96+/-0.9%. The stabilities in phosphate buffer and in human blood serum at 96 h postpreparation were 93+/-1.2% and 85+/-3.5%, respectively. The immunoreactivity of the complex was 89+/-1.4%. At a concentration of 1 nM, the complex killed 70+/-3% of MCF7 cells. At 1.9 nM, 90+/-5% of the cells were killed. CONCLUSIONS: The results showed that the new complex could be considered for further evaluation in animals and possibly in humans as a new radiopharmaceutical for use in radioimmunotherapy against breast cancer.

99mTc-HMPAO-labeled liposomes: an investigation into the effects of some formulation factors on labeling efficiency and in vitro stability.

Technetium-99m complex of hexamethyl-propylene-amineoxime (HMPAO) is used as an efficient agent to label liposomes. For this, 99mTc-HMPAO is incubated with preformed liposomes that contain glutathione (GSH). Effect of GSH and lipid concentration on labeling efficiency, as well as the effect of lipid composition on in vitro stability of labeled liposomes, was investigated in the present study. d,l-HMPAO was synthesized and kits including d,l-HMPAO and SnCl2.2H2O were optimized at 0.5 mg HMPAO, 5.0 microg SnCl2.2H2O and pH 7, and lyophilized. DSPC/CHOL (molar ratio 2:1) liposomes encapsulating GSH were labeled with 99mTc-HMPAO prepared kits. Increase of GSH concentration in hydration buffer from 5 to 200 mM during liposome preparation resulted in a broad labeling efficiency of liposomes ranging from 4.16% to 69.81%. An initial approximate concentration of 100 mM GSH in the hydration buffer seems to be appropriate for a good labeling efficiency. At the optimum concentration of GSH, change of the total initial lipid concentration from 10 to 70 mM did not produce a remarkable difference in labeling efficiency. Study of the effect of lipid composition on the stability of liposomes showed that all three kinds of labeled liposomes composed of DSPC/CHOL, DPPC/CHOL and DMPC/CHOL (molar ratio 2:1) had good in vitro stability in human plasma at 37 degrees C for 48 h; however, employing DSPC resulted in the most stable ones.

Synthesis of carbon-14 analogue of 1,5 diaryl-5-[14C]-1,2,3-triazoles.

Two 1,2,3-triazole anticonvulsants, 1-(4-methylsulfone-phenyl)-5-(4-methyl-phenyl)-1,2,3-triazole and 1-(4-methylsulfone-phenyl)-5-phenyl-1,2,3-triazole, both labeled with carbon-14 in the 5-position were prepared from para-tolunitrile-[cyano-14C] and benzonitrile-[cyano-14C], respectively.