Fluorescent, Plasmonic, and Radiotherapeutic Properties of the 177Lu-Dendrimer-AuNP-Folate-Bombesin Nanoprobe Located Inside Cancer Cells.

The integration of fluorescence and plasmonic properties into one molecule is of importance in developing multifunctional imaging and therapy nanoprobes. The aim of this research was to evaluate the fluorescent properties and the plasmonic-photothermal, therapeutic, and radiotherapeutic potential of 177Lu-dendrimer conjugated to folate and bombesin with gold nanoparticles in the dendritic cavity (177Lu-DenAuNP-folate-bombesin) when it is internalized in T47D breast cancer cells. The intense near-Infrared (NIR) fluorescence emitted at 825 nm from the conjugate inside cells corroborated the usefulness of DenAuNP-folate-bombesin for optical imaging. After laser irradiation, the presence of the nanosystem in cells caused a significant increase in the temperature of the medium (46.8°C, compared to 39.1°C without DenAuNP-folate-bombesin, P < 0.05), resulting in a significant decrease in cell viability (down to 16.51% ± 1.52%) due to the 177Lu-DenAuNP-folate-bombesin plasmonic properties. After treatment with 177Lu-DenAuNP-folate-bombesin, the T47D cell viability decreased 90% because of the radiation-absorbed dose (63.16 ± 4.20 Gy) delivered inside the cells. The 177Lu-DenAuNP-folate-bombesin nanoprobe internalized in cancer cells exhibited properties suitable for optical imaging, plasmonic-photothermal therapy, and targeted radiotherapy.

Synthesis and evaluation of Lys¹(α,γ-Folate)Lys³(¹77Lu-DOTA)-Bombesin(1-14) as a potential theranostic radiopharmaceutical for breast cancer.

The aim of this work was to synthesize Lys(1)(α,γ-Folate)-Lys(3)((177)Lu-DOTA)-Bombesin (1-14) ((177)Lu-Folate-BN), as well as to assess its potential for molecular imaging and targeted radiotherapy of breast tumors expressing folate receptors (FR) and gastrin-releasing peptide receptors (GRPR). Radiation absorbed doses of (177)Lu-Folate-BN (74 MBq, i.v.) estimated in athymic mice with T47D-induced breast tumors (positive to FR and GRPR), showed tumor doses of 23.9±2.1 Gy. T47D-tumors were clearly visible (Micro-SPECT/CT images). (177)Lu-Folate-BN demonstrated properties suitable as a theranostic radiopharmaceutical.

Improved radiopharmaceutical based on 99mTc-Bombesin-folate for breast tumour imaging.

BACKGROUND: Clinical studies in women using technetium-99m (Tc)-Bombesin have shown successful radionuclide imaging of breast tumours overexpressing gastrin-releasing peptide receptors (GRPRs). Recent studies have demonstrated that most breast tumours overexpress folate receptors (FRα). AIM: The aim of this work was to synthesize the Lys(α,γ-Folate)-Lys(Tc-EDDA/HYNIC)-Bombesin (1-14) conjugate (Tc-Bombesin-Folate), as well as to assess the in-vitro and in-vivo potential of the radiopharmaceutical to target FRα and GRPR. METHODS: LysLys(HYNIC)-Bombesin (1-14) was conjugated to folic acid and the product was purified by size-exclusion high-performance liquid chromatography. Ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were used for chemical characterization. Tc labelling was performed using ethylenediamine-N,N'-diacetic acid/tricine as coligands. In-vitro binding studies were carried out in T47D breast cancer cells (positive for FRα and GRPR). Biodistribution studies and micro-single-photon emission computed tomography/computed tomography imaging were carried out on athymic mice with T47D-induced tumours. RESULTS: High-performance liquid chromatography analyses indicated that the radioconjugate was obtained with high radiochemical purity (96±2.1%). In-vitro and in-vivo results showed significant uptake of the radiopharmaceutical in T47D cells and tumours (5.43% ID/g), which was significantly inhibited by preincubation with cold folic acid or cold Bombesin. CONCLUSION: The Tc-Bombesin-folate heterobivalent radiopharmaceutical significantly enhances in-vivo tumour uptake because of the concomitant interaction with FRα and GRPR.

Two Novel Nanosized Radiolabeled Analogues of Somatostatin for Neuroendocrine Tumor Imaging.

The somatostatin receptors (SR), which are overexpressed in a majority of neuroendocrine tumors, are targets for radiopeptide-based imaging using for example the 99mTc-Tyr3-Octreotide peptide. Dendrimers are hyperbranched polymeric structures. The nanoscopic size and near-monodisperse nature properties give polyamidoamine (PAMAM) dendrimers an edge over linear polymers in the context of drug delivery. Gold nanoparticles (AuNPs) conjugated to peptides produces stable multimeric systems with target-specific molecular recognition. The aim of this research was to prepare two nanosized multimeric systems for neuroendocrine tumor imaging, 99mTc-PAMAM-Tyr3-Octreotide and 99mTc-AuNP-Tyr-Octreotide, and to compare their in vitro uptake in SR-positive AR42J cancer cells as well as their biodistribution profile in athymic mice bearing AR42J tumors. [Tyr3, Lys(Boc)5]-Octreotide was conjugated to the carboxylate groups of the PAMAM dendrimer (G3.5) with further Boc deprotection using TFA. 99mTc labeling was carried out by a direct method. 99mTc-Tyr3-Octreotide was conjugated to AuNPs (20 nm) by spontaneous reaction with the thiol group of cysteine. Radiochemical purity (RP) was determined by size-exclusion HPLC and ITLC-SG analyses. In vitro binding studies were carried out in AR42J cancer cells. Biodistribution studies were accomplished in athymic mice with AR42J-induced tumors with blocked and unblocked receptors. Elemental analysis demonstrated that 26 Tyr3-Octreotide molecules were successfully conjugated to one molecule of PAMAM. RP for both nanosized conjugates was > 94% and showed recognition for SR in AR42J cells. The tissue distribution of radioactivity 2 h after 99mTc-PAMAM-Tyr3-Octreotide administration in mice showed specific tumor uptake (4.12 ± 0.57% of injected dose/g) and high accumulation in the pancreas (15.08 ± 3.11% of injected dose/g) which expresses SR. No significant difference in the tumor uptake was found between 99mTc-PAMAM-Tyr3-Octreotide and 99mTc-AuNP-Tyr3-Octreotide. However, the dendrimer-peptide conjugate showed a significant renal excretion. Both radiopharmaceuticals demonstrated properties suitable for use as target-specific agents for molecular imaging of tumors that overexpressed SR.

Kit for preparation of multimeric receptor-specific 99mTc-radiopharmaceuticals based on gold nanoparticles.

BACKGROUND: Multivalency is a design principle by which organized arrays amplify the strength of a binding process, such as the binding of multimeric peptides to specific receptors located on cell surfaces. The conjugation of peptides to gold nanoparticles (AuNPs) produces biocompatible and stable multimeric systems with target-specific molecular recognition. AIM: The aim of this research was to develop a kit for technetium-99m (99mTc) labelling of AuNPs that are conjugated to Lys³-bombesin, cyclo[Arg-Gly-Asp-D-Phe-Lys-(Cys)] or thiol-mannose to produce receptor-specific multimeric systems. METHODS: A freeze-dried kit formulation for the instant preparation of 99mTc-ethylenediamine-N,N'-diacetic acid (EDDA)/hydrazinonicotinyl (HYNIC)-Tyr³-octreotide (99mTc-EDDA/HYNIC-TOC) (vial 1) and a second vial containing 1.5 ml of AuNP solution (1 nM, 20 nm diameter, surface area=1260 nm², 37,000 surface Au atoms, 1.05 × 10 particles) plus 10 µl of Lys³-bombesin, cyclo[Arg-Gly-Asp-D-Phe-Lys-(Cys)] or mannose (50 µM, approximately 285 molecules per AuNP) (vial 2) were prepared. Multimeric radiopharmaceuticals were prepared by adding 1 ml of 0.2 mol/l phosphate buffer, pH 7.0, and 1 ml of 99mTcO4⁻ (4 GBq) to vial 1, and the mixture was incubated at 92°C for 20 min in a dry block heater. A total of 100 µl (200 MBq) of 99mTc-EDDA/HYNIC-TOC solution (122 HYNIC-TOC molecules per AuNP) was added to vial 2. No further purification was carried out. Radiochemical purity was determined by instant thin-layer chromatography-silica gel/2-butanone (Rf values for the radiolabelled AuNP and 99mTcO4⁻ were 0.0 and 1.0, respectively), ultrafiltration, size-exclusion high-pressure liquid chromatography and a PD-10 column. The conjugates were characterized by ultraviolet-visible, far-infrared and X-ray photoelectron spectroscopy. In-vitro binding studies were carried out in ανß3 receptor-positive C6 glioma cancer cells, gastrin-releasing peptide receptor-positive PC3 cancer cells or mannose receptor-positive rat liver cells. Biodistribution studies were carried out in athymic mice with induced tumours (PC-3 or C6 cancer cells) or in Wistar rats (99mTc-AuNP-mannose for sentinel lymph node detection). Images were obtained using a micro-single-photon emission computed tomography/computed tomography system. RESULTS: Radiochemical purity was 96 ± 2% for all of the multimeric radiopharmaceuticals. Far-infrared showed a characteristic band at 279 ± 1 cm⁻¹, which was assigned to the Au-S bond. ultraviolet-visible and X-ray photoelectron spectroscopy also indicated that the AuNPs were functionalized with peptides or mannose. Radiopharmaceuticals showed specific recognition for receptors expressed in cancer cells or rat liver cells. Micro-single-photon emission computed tomography/computed tomography images showed clear tumour uptake and lymph node accumulation. The kit (i.e. vial 1 and vial 2) demonstrated excellent stability during storage at 4°C for 6 months. CONCLUSION: Multimeric systems of 99mTc-AuNP-peptide/mannose prepared from kits exhibited properties suitable for use as target-specific agents for molecular imaging of tumours and sentinel lymph node detection.

Physicochemical properties and theoretical modeling of actinide complexes with a para-tert-Butylcalix[6]arene bearing phosphinoyl pendants. Extraction capability of the calixarene toward f elements.

The coordination ability of the hexaphosphinoylated p-tert-butylcalix[6]arene B6bL6 toward actinides is established, as well as its good separation ability of the actinide ions UO2 2+ and Th(IV) over trivalent rare earths such as La(III), Eu(III), and Y(III). Spectrophotometric titration of uranyl with B6bL6 in CH 3CN yields log beta 11 = 7.1 and log beta 12 = 12.5 for the 1:1 and 1:2 (UO2 2+/B 6bL6) species, respectively. Actinide complexes with 1:1 and 1:2 (M/L) stoichiometries are isolated and characterized by elemental analysis, IR, and UV-vis. Compounds 1 and 3 fulfill their CN = 8 just with B 6bL (6), while compounds 2 and 4 require coordinated nitrates and/or water molecules. The luminescence spectra of the uranyl complexes and the parameters such as FWMH, vibronic spacing (upsilon sp), and the U-O bond length, as well as the luminescence lifetimes, permit the understanding of the coordination chemistry of these actinide calixarene complexes. Energy transfer from the B6bL6 ligand to the uranyl ion is demonstrated to be relevant in compound 1 with Q abs = 2.0%. The uranyl complex emission reveals a biexponential decay with tau s from 210 to 220 micros and tau L from 490 to 650 micros for compounds 1 and 3, respectively. The liquid-liquid extraction results demonstrate the good extraction capability of B 6bL (6) toward actinides but not for rare earths at room temperature. The extracted species keeps the 1(cation)/1(calixarene) ratio for the UO2 2+, Th 4+, and Eu 3+ ions. A good capacity of B6bL 6 toward Th4+ ions using aqueous phase 2 containing even up to 0.3 M thorium nitrate and an organic phase of 2.47 x 10 (-4) M B6bL6 in chloroform is found. The spectroscopic properties of the isolated uranyl complexes and the extraction studies reveal a uranophilic nature of B6bL6. The molecular modeling results are in good agreement with the experimental findings.

Lys and Arg in UBI: a specific site for a stable Tc-99m complex?

The aim of this study was to help establish if ubiquicidin peptide 29-41 fragment (UBI) contains a specific site for 99mTc labeling by a new direct method under alkaline conditions. Since this peptide does not have cysteine residues, it is possible that neighboring arginine and lysine in the peptide amino acid sequence (Thr-Gly-Arg-Ala-Lys-Arg-Arg-Met-Gln-Tyr-Asn-Arg-Arg) could be a specific coordination site to form a stable 99mTc-UBI complex. Following direct labeling, the in vitro stability of 99mTc-UBI was compared to UBI radiolabeled by one indirect method using HYNIC/tricine and HYNIC/tricine/EDDA. Radiochemical purity of 99mTc-UBI averaged 97% compared to 88% for 99mTc-HYNIC-UBI/tricine and 98% for 99mTc-HYNIC-UBI/tricine/EDDA. Both 99mTc-HYNIC-UBI (tricine or EDDA) and 99mTc-UBI showed stability in human serum and solutions of cysteine. 99mTc-UBI radiochemical purity 24 h after dilution in 0.9% NaCl was greater than 90% at pH 9 and greater than 95% at pH 6.5. Under one set of experimental conditions, in vitro binding to bacteria of 99mTc-UBI was 35% and identical to that of 99mTc-HYNIC-UBI/tricine and 99mTc-HYNIC-UBI/tricine/EDDA at 32% and 31% respectively. The biodistribution of 99mTc-UBI in mice showed a rapid renal clearance. To help identify the site(s) of 99mTc binding following direct labeling, molecular mechanics and quantum-mechanical calculations were performed which showed that the amine groups of Arg(7) and Lys are the most probable site. The calculations show that these groups can form a square pyramid with two water molecules for the Tc cation (dxysp(3)). It will be necessary to isolate and characterize the 99Tc(V)(O)-UBI.(H2O)n complex to confirm these results.