Relationship between intraprostatic tracer deposits and sentinel lymph node mapping in prostate cancer patients.

UNLABELLED: Intraprostatic injection of the hybrid tracer indocyanine green (ICG)-(99m)Tc-nanocolloid enables both preoperative sentinel node (SN) identification and intraoperative visualization of the SN. Relating the fluorescence deposits in embedded prostate tissue specimens to the preoperatively detected SNs also provides the opportunity to study the influence of their placement on lymphatic drainage pattern. METHODS: Nineteen patients with prostate carcinoma scheduled for robot-assisted laparoscopic prostatectomy and lymph node (LN) dissection were included. ICG-(99m)Tc-nanocolloid was injected intraprostatically, guided by ultrasound. SN biopsy was performed using a combination of radioguidance and fluorescence guidance. Tracer distribution was visualized in paraffin-embedded prostate samples using ex vivo fluorescence imaging. This distribution was correlated to the number and location of the SNs identified on preoperative lymphoscintigraphy and SPECT/CT. RESULTS: ICG-(99m)Tc-nanocolloid helped guide surgical excision of the SNs. Ex vivo fluorescence imaging revealed a large variation in the locations of intraprostatic tracer deposits among patients. Tracer deposits in the peripheral zone correlated with a higher number of visualized LNs than deposits in the central zone (on average, 4.7 vs. 2.4 LNs per patient). Furthermore, tracer deposits in the mid gland correlated with a higher number of visualized LNs than deposits near the base or apex of the prostate (on average, 6 vs. 3.5 LNs per patient). CONCLUSION: The hybrid nature of the tracer not only enables surgical guidance but also provides an opportunity to study the correlation between the location of tracer deposits within the prostate and the number and location of preoperatively visualized SNs. These data suggest that the location at which a tracer deposit is placed influences the lymphatic drainage pattern.

Synthesis, pharmacoscintigraphic evaluation and antitumor efficacy of methotrexate-loaded, folate-conjugated, stealth albumin nanoparticles.

UNLABELLED: The present study aims to develop a multifunctional nanoformulation based on technetium-99m labeled, folate conjugated, methotrexate-loaded human serum albumin nanoparticles (HSA NPs) and explore their potential in cancer theragnostics. MATERIALS & METHODS: Methotrexate-loaded HSA NPs were synthesized by a reverse microemulsion technique, followed by chemical crosslinking with glutaraldehyde. These NPs were conjugated with folic acid (FA) through a hydrophilic polyethylene glycol spacer to render them long-circulatory and augment their tumor-specific localization. The therapeutic conjugate was further radiolabeled with a γ-emitter technetium-99m for real-time monitoring of its blood clearance kinetics and biodistribution through the measurement of blood/organ-associated radioactivity and scintigraphic imaging. RESULTS & CONCLUSION: In vitro cell-uptake and cytotoxicity studies corroborated that FA conjugation enabled these NPs to specifically target and kill folate-receptor overexpressing cancer cells via S phase arrest. Blood clearance kinetics and biodistribution studies clearly indicated that circulation time, as well as tumor-specific localization of methotrexate-loaded HSA nanocarriers, could be significantly augmented upon polyethylene glycolylation and conjugation of FA. Finally, we demonstrated that these targeted HSA NPs inhibited tumor growth more effectively, as compared with the nontargeted controls.

Differential receptor targeting of liver cells using 99mTc-neoglycosylated human serum albumins.

Neolactosyl human serum albumin (LSA) targets asialoglycoprotein receptor and shows high liver uptake due to accumulation in hepatocytes. Although neomannosyl human serum albumin (MSA) also shows high liver uptake, it has been reported to be taken up by Kupffer cells and endothelial cells. We compared the biological properties of LSA and MSA. 99mTc-LSA and 99mTc-MSA biodistribution in mice were investigated after intravenous injection. In vivo localization of rhodaminisothiocyanate (RITC)-LSA and fluoresceineisothiocyanate (FITC)-MSA were investigated in mouse liver. Excretion routes of 99mTc-LSA and 99mTc-MSA metabolites were examined. Both 99mTc-LSA and 99mTc-MSA showed high liver uptakes. RITC-LSA was taken up by hepatocytes whereas FITC-MSA was taken up by Kupffer cells and endothelial cells. 99mTc-MSA showed higher spleen and kidney uptakes than 99mTc-LSA. 99mTc-LSA metabolites excreted in urine and feces accounted for 44.4 and 50.0% of 99mTc-LSA injected, respectively, while 99mTc-MSA metabolites accounted for 51.5 and 10.3%, respectively. In conclusion, LSA is specifically taken up by hepatcytes while MSA by Kupffer cells and endothelial cells. After taken up by the liver, LSA is metabolized by the hepatocytes and then excreted through both the hepatobiliary tract and kidney, whereas MSA is metabolized by Kupffer cells and endoghelial cells and then excreted mainly through the kidney.

Preparation of 99mTc-Nanocoll for use in sentinel node localization: Validation of a protocol for supplying in unit-dose syringes.

OBJECTIVES: To determine if 99mTc-Nanocoll is affected by storage in a syringe, passage through an R-Lock or mixing with Patent Blue V dye. METHODS: A Nanocoll kit was reconstituted at 280 MBq/5 ml. Samples of 0.5 ml were drawn into 3 ml Plastipak syringes. After 1 h and 6 h, adsorption of 99mTc on a syringe was measured and the following tests of quality were performed on samples from the kit and a syringe: 99mTc-pertechnetate impurity by thin-layer chromatography, the percentage of 99mTc on particles >100 nm by Nuclepore filtration and particle size by photon correlation spectroscopy. These tests were also applied to samples that had been passed through an R-Lock or mixed with Patent Blue V. Each experiment was performed five times. RESULTS: In all samples, adsorption of 99mTc on syringes was <1%, 99mTc-pertechnetate impurity was <2%, >95% of 99mTc was labelled to particles <100 nm in diameter, the mean particle diameter was 6.6 nm and the particles had a diameter of <12 nm. All tests showed no significant difference (P > 0.05, n = 5) between 99mTc-Nanocoll from the original kit and a syringe at either 1 h or 6 h. Similar results were obtained with samples that had been passed through an R-Lock or mixed with Patent Blue V. CONCLUSIONS: A capped 3 ml Plastipak syringe is a suitable container in which to supply 99mTc-Nanocoll. Neither passage through an R-Lock nor mixing with Patent Blue V affects the quality of 99mTc-Nanocoll.

Preparation of Tc-99m-macroaggregated albumin from recombinant human albumin for lung perfusion imaging.

Human serum albumin (HSA) extracted from pooled blood taken from human donors is used in the production of (99m)Tc-labelled macroaggregated albumin (MAA) for lung perfusion imaging. However, concerns for the safety of blood-derived products due to potential contamination by infective agents (e.g. new variant CJD), make alternative production methods necessary. Recombinant DNA technology is a promising method of albumin production avoiding problems associated with human-derived HSA. This paper presents results comparing MAA prepared from recombinant human albumin (rHA, Recombumin) (rMAA) with in-house produced HSA MAA (hMAA) and commercially available MAA (cMAA). (99m)Tc-MAA was prepared using previously published production methods by heating a mixture of albumin and stannous chloride in acetate buffer (pH 5.4) at 70 degrees C for 20 min. Parameters investigated include aggregate size, radiolabelling efficiency, radiochemical and aggregate stability at 4 degrees C and in vitro (in whole human blood) at 37 degrees C and biodistribution studies. Results showed that rMAA could be produced with similar morphology, labelling efficiency and stability to hMAA and cMAA. Our findings confirm that rHA shows significant potential as a direct replacement for HSA in commercially available MAA.

Comparative evaluation of 99Tcm-Hynic-HSA and 99Tcm-MAG3-HSA as possible blood pool agents.

Two strategies have been used to increase the 99Tcm binding strength of human serum albumin (HSA) and thus enhance its blood retention. In a first approach, HSA was derivatized with a varying number of hydrazino nicotinyl (Hynic) side-chains using N-hydroxysuccinimidyl hydrazino nicotinate. Labelling of this albumin derivative with 99Tcm resulted in labelling yields of 90-95%. On the other hand, a 99Tcm-MAG3-HSA conjugate was prepared using the preformed chelate approach. In this way, non-specific binding of 99Tcm to HSA could be excluded. The in vitro stability of both 99Tcm-HSA derivatives was evaluated by cysteine challenge experiments and revealed a much higher stability for 99Tcm-Hynic-HSA than for 99Tcm-MAG3-HSA. The biological behaviour of the preparations was evaluated in mice and a rabbit using 125I-HSA as an internal biological standard. The blood retention of 99Tcm-MAG3-HSA decreased more rapidly than that of 125I-HSA in both animal species, whereas 99Tcm-Hynic-HSA seemed to provide a quasi-perfect 99Tcm-labelled analogue for 125I-HSA and 99Tcm-red blood cells (99Tcm-RBCs). In addition, the blood retention of 99Tcm-Hynic-HSA appeared to be similar to that of 99Tcm-RBCs in a volunteer. These results clearly indicate the superiority of 99Tcm-Hynic-HSA over 99Tcm-MAG3-HSA as a possible blood pool agent.

Technetium-99m mercaptoalbumin as a potential substitute or technetium-99m labelled red blood cells.

Technetium-99m labelled red blood cells (99mTc-RBCs) are far superior to 99mTc-labelled human serum albumin (99mTc-HSA) for radionuclide ventriculography, but their labelling is more complex, time consuming and risk bearing (in vitro labelling) or suffers from interference by some medications (in vivo labelling). We have now modified HSA by the introduction of mercapto groups with the purpose of preparing stable and practical 99mTc-mercaptoalbumin with long retention in the vascular system, that could replace 99mTc-RBCs. HSA was incubated with N-succinimidyl S-acetylthioacetate (SATA) or N-succinimidyl 2,3-di(S-acetylthio) propionate (SATP) to introduce a chain containing one or two protected sulfhydryl groups on some of the lysine amino groups. After purification by size-exclusion chromatography (SEC), the mercapto groups were deprotected by incubation at alkaline pH or by treatment with hydroxylamine. The reaction products were used with or without SEC purification for direct or exchange labelling experiments with 99mTc at neutral pH. SEC-HPLC was used to determine labelling yields and to isolate pure 99mTc-mercaptoalbumin. Stable 99mTc-mercaptoalbumin complexes could be formed in 90%-95% yield after coupling albumin with SATA or SATP in all molar ratios used followed by deacetylation in one of the mentioned conditions. The most favourable results were obtained after reaction of SATA or SATP with HSA in a 25:1 ratio and deprotection with NH2OH. The stability of the resulting 99mTc-mercaptoacetyl-albumin (99mTc-MA-HSA) and 99mTc-dimercaptopropionyl-albumin (99mTc-DMP-HSA) and their retention in vivo in plasma of mice and rabbits are clearly higher than that of conventional 99mTc-HSA preparations. 99mTc-DMP-HSA approaches the behaviour of 125I-HSA quite well in both animal species. A preliminary study with 99mTc-DMP-HSA in a volunteer showed a retention in the vascular compartment almost identical to that of 99mTc-RBCs and clearly higher than that of a common 99mTc-HSA preparation. The results indicate that these 99mTc-mercaptoalbumins and especially 99mTc-DMP-HSA are very promising as a practical alternative to 99mTc-RBCs.