Radiolabeling Method Research Articles

Optimized method for fluorine-18 radiolabeling of Affibody molecules using RESCA

BackgroundIn recent years, the interest in Al[18F]F as a labeling agent for Positron Emission Tomography (PET) radiotracers has risen, as it allows for fast and efficient fluorine-18 labeling by harnessing chelation chemistry. The introduction of Restrained Complexing Agent (RESCA) as a chelator has also shown that chelator-based radiolabeling reactions can be performed in mild conditions, making the radiolabeling process attractively more facile than most conventional radiofluorination methods. The aim of the study was to establish optimized conditions for Al[18F]F labeling of Affibody molecules using RESCA as a complexing agent, using Z09591 and Z0185, two Affibody proteins targeting PDGFRβ and TNFα, respectively, as model compounds.ResultsThe Al[18F]F labeling of RESCA-conjugated Z09591 was tested at different temperatures (rt to 60 °C) and with varying reaction times (12 to 60 min), and optimal conditions were then implemented on RESCA-Z0185. The optimized synthesis method was: 1.5–2.5 GBq of cyclotron produced fluorine-18 were trapped on a QMA cartridge and eluted with saline solution to react with 12 nmol of AlCl3 and form Al[18F]F. The respective RESCA-conjugated Affibody molecule (14 nmol) in NaOAc solution was added to the Al[18F]F solution and left to react at 60 °C for 12 min. The mixture was purified on a NAP5 size exclusion column and then analyzed by HPLC. The entire process took approximately 35 min, was highly reproducible, indicating the efficiency and reliability of the method. The labeled compounds demonstrated retained biological function for their respective targets after purification.ConclusionsWe present a general and optimized method for Al[18F]F labeling of RESCA-conjugated Affibody molecules, which can be widely applied to this class of peptide-based imaging agents. Moreover, radiochemical yields were improved when the labeling was conducted at 37 °C or above. In vitro and in vivo assessment of the respective tracers was promising, showing retained binding capacity as well as moderate defluorination, which is usually regarded as a potential downside for RESCA-conjugated tracers.Graphical abstract

Quantitative Biodistribution of OMVs Using SPECT/CT Imaging with HYNIC-Duramycin Radiolabeling.

Introduction: Bacterial outer membrane vesicles (OMVs) are emerging as important players in the host-microbiome interaction, while also proving to be a promising platform for vaccine development and targeted drug delivery. The available methods for measuring their biodistribution, however, are limited. We aimed to establish a high-efficiency radiolabeling method for the treatment of OMVs. Methods: 99mTc-HYNIC-duramycin was incubated with OMVs isolated from E. coli BL21(DE3) ΔnlpI ΔlpxM. Radiolabeling efficiency (RLE) and radiochemical purity (RCP) were measured with size-exclusion high-performance liquid chromatography. The biodistribution was quantitatively measured in mice using SPECT/CT imaging. Results: RLE was 81.84 ± 2.03% for undiluted OMV suspension and 56.17 ± 2.29% for 100× dilution. Postlabeling purification with a spin-desalting column results in 100% radioactivity in the OMV fraction according to HPLC, indicating 100% RCP of the final product. The biodistribution was found to be in line with previous data reported in the literature using other OMV tracking attempts. Conclusions: Our findings illustrate that using HYNIC-duramycin for labeling of the OMVs enhances efficiency and is easily implementable for in vivo imaging studies, significantly improving upon earlier methods.

Tracing proliferating potential of lung tumours by [11C]Nicotinamide

Many tumours rely on aerobic glycolysis, known as the Warburg effect, to satisfy energy needs and sustain rapid proliferation [1]. High demands of glucose in tumours can be measured by [18F[fluorodeoxyglucose, [18F]FDG, positron emission tomography (PET). Nicotinamide adenine dinucleotide (NAD) is a coenzyme involved in redox reactions in many metabolic pathways, including glycolysis. To maintain the high rates of glycolysis and proliferation, tumours need increased NAD+ levels and use nicotinamide for the upregulated NAD+ salvage pathway that is the main source of NAD+ in proliferative cancer cells [2]. Herein, we show an improved synthesis method of [11C]Nicotinamide, [11C]NAM, and studied the uptake of [11C]NAM and [18F]FDG to measure proliferating capacity in a lung cancer model. [Carboxyl-11C]nicotinamide was synthesized by a one-pot radiolabeling method [3] on the SYNTHRA synthesizer (Synthra GmbH, Germany) (see Figure 1A). We used a KrasG12D/+ genetically engineered mouse model of lung cancer and evaluated the uptake of [11C]nicotinamide and [18F]FDG with dual-tracer sequential imaging protocol (see Figure 1B) using a NanoScan PET/MRI scanner (Mediso Medical Imaging Systems, Hungary). We obtained [carboxyl-11C]nicotinamide in a radiochemical yield of 15 ± 5 %, volumic activity of 50 ± 10 MBq/ml, and radiochemical purity was > 99 %. We optimized the imaging protocol and found the uptake of [11C]nicotinamide co-localized with the uptake of [18F]FDG in the KrasG12D/+ model of lung cancer as shown in Figure 1C suggesting the higher NAM uptake was associated with high NAD+ production in highly proliferating tumours. We demonstrated that metabolic PET/MRI imaging with [11C]NAM can complement [18F]FDG PET and be useful for probing proliferative potential in lung cancer. This method could be translated and applied in clinical practice to enable visualization of NAD+ metabolism in lung tumours and targeted therapeutically. Please click on the 'PDF' for the full abstract!

A Novel Bifunctional Chelating Agent for Tyrosine-Specific Radiolabeling of Peptides and Proteins.

Site-specific radiolabeling is utilized for the development of antibody- or peptide-based radiotheranostic agents. Although tyrosine can be exploited as one of the target residues for site-specific radiolabeling of peptides and proteins, a tyrosine-specific radiolabeling method has not been established. In this study, we newly designed and synthesized a novel bifunctional chelating agent, TBD-DO3A, consisting of a triazabutadiene (TBD) scaffold and metal chelator, 1,4,7,10-tetraazacyclododecane 1,4,7-triacetic acid (DO3A). Conjugation of TBD-DO3A with Ac-Tyr-NHMe followed by 111In-labeling afforded [111In]In-Tyr-DO3A, which showed high-level stability in mouse plasma. Then, we selected the tyrosine-containing cyclic peptide c(RGDyK) as a model ligand and synthesized [111In]In-RYD. [111/natIn]In-RYD showed in vitro binding properties for integrin αvβ3 equivalent to those of [111/natIn]In-RKD, a lysine residue-labeled control compound. In in vivo biodistribution and SPECT/CT imaging studies using U87MG/PC-3 tumor-bearing mice, [111In]In-RYD and [111In]In-RKD were selectively accumulated and facilitated U87MG tumor visualization at 24 h postinjection. These results indicate that TBD-DO3A has fundamental properties as a bifunctional chelator for tyrosine-specific radiolabeling of peptides and proteins.

"One Method to Label Them All": A Single Fully Automated Protocol for GMP-Compliant 68Ga Radiolabeling of PSMA-11, Transposable to PSMA-I&T and PSMA-617.

Prostate-specific membrane antigen (PSMA) is an ideal target for molecular imaging and targeted radionuclide therapy in prostate cancer. Consequently, various PSMA ligands were developed. Some of these molecules are functionalized with a chelator that can host radiometals, such as 68Ga for PET imaging. The 68Ga radiolabeling step benefits from process automation, making it more robust and reducing radiation exposure. To design a single automated radiolabeling protocol for the GMP-compliant preparation of [68Ga]Ga-PSMA-11, transposable to the production of [68Ga]Ga-PSMA-617 and [68Ga]Ga-PSMA-I&T. A GAIA® synthesis module and a GALLIAD® generator were used. Radio-TLC and radio-HPLC methods were validated for radiochemical purity (RCP) determination. Three [68Ga]Ga-PSMA-11 validation batches were produced and thoroughly tested for appearance and pH, radionuclide identity and purity, RCP, stability, residual solvent and sterility. Minimal modifications were made to the reagents and disposables for optimal application to other PSMA ligands. [68Ga]Ga-PSMA-11 for clinical application was produced in 27 min. The 3 validation batches met the quality criteria expected by the European Pharmacopoeia to allow routine production. For optimal transposition to PSMA-617, the solid phase extraction cartridge was changed to improve purification of the radiolabeled product. For application to PSMA-I&T, the buffer solution initially used was replaced by HEPES 2.7 M to achieve good radiochemical yields. Residual HEPES content was checked in the final product and was below the Ph. Eur. threshold. A single automated radiolabeling method on the GAIA® module was developed and implemented for 68Ga radiolabeling of 3 PSMA ligands, with slight adjustments for each molecule.

Sortase-Mediated Site-Specific Conjugation to Prepare Fluorine-18-Labeled Nanobodies.

Single-domain antibodies, or nanobodies (Nbs), are promising biomolecules for use in molecular imaging due to their excellent affinity, specificity, and fast clearance from the blood. Given their short blood half-life, pairing Nbs with short-lived imaging radioisotopes is desirable. Because fluorine-18 (18F) is routinely used for clinical imaging, it is an attractive radioisotope for Nbs. We report a novel sortase-based, site-specific 18F-labeling method applied to three nanobodies. Labeled nanobodies were synthesized either by a two-step indirect radiolabeling method in one pot or by a one-step direct labeling method using a sortase-mediated conjugation of either the radiolabeled chelator (H-GGGK((±)-Al[18F]FH3RESCA)-NH2) or the unlabeled chelator (H-GGGK((±)-H3RESCA)-NH2) followed by labeling with Al[18F]F, respectively. The overall radiochemical yields were 15-43% (n = 22, decay-corrected) in 70 min (indirect labeling) and 23-58% (n = 12, decay-corrected) in 50 min (direct labeling). The radiochemical purities of the labeled nanobodies prepared by both methods were >98% with a specific activity of 400-600 Ci/mmol (n = 22) for each of the three Nbs tested and exhibited excellent stability profiles under physiological conditions. This simple, site-specific, reproducible, and generalizable 18F-labeling method to prepare nanobodies (Nb-Al[18F]F-RESCA) or other low molecular weight biomolecules can easily be adopted in various settings for preclinical and clinical studies.

Application of Microfluidic Devices for Automated Two-Step Radiolabeling of Antibodies.

Radioimmunoconjugates (RICs) composed of tumor-targeting monoclonal antibodies and radionuclides have been developed for diagnostic and therapeutic application. A new radiolabeling method using microfluidic devices is expected to facilitate simpler and more rapid synthesis of RICs. In the microfluidic method, microfluidic chips can promote the reaction between reactants by mixing them efficiently, and pumping systems enable automated synthesis. In this study, we synthesized RICs by the pre-labeling method, in which the radiometal is coordinated to the chelator and then the radiolabeled chelator is incorporated into the antibodies, using microfluidic devices for the first time. As a result of examining the reaction parameters including the material of mixing units, reaction temperature, and flow rate, RICs with radiochemical purity (RCP) exceeding 90% were obtained. These high-purity RICs were successfully synthesized without any purification simply by pumping three solutions of a chelating agent, radiometal, and antibody into microfluidic devices. Under the same conditions, the RCP of RICs labeled by conventional methods was below 50%. These findings indicate the utility of microfluidic devices for automatic and rapid synthesis of high-quality RICs.

A third generation PSMA-targeted agent [211At]YF2: Synthesis and in vivo evaluation

IntroductionTargeted α-particle therapy agents have shown promising responses in patients who have developed resistance to β−-particle emitting radionuclides, albeit off-target toxicity remains a concern. Astatine-211 emits only one α-particle per decay and may alleviate the toxicity from α-emitting daughter radionuclides. Previously, we developed the low-molecular-weight PSMA-targeted agent [211At]L3-Lu that showed suitable therapeutic efficacy and was well tolerated in mice. Although [211At]L3-Lu had good characteristics, we now have evaluated a closely related analogue, [211At]YF2, to determine the better molecule for clinical translation. MethodsThe tin precursors and unlabeled iodo standards for [211At]YF2 and [211At]L3-Lu each were synthesized and a new one-step labeling method was developed to produce [211At]YF2 and [211At]L3-Lu from the respective tin precursor. RCY and RCP were determined using RP-HPLC. Cell uptake, internalization and in vitro cell-killing (MTT) assays were performed on PSMA+ PC-3 PIP cells in parallel experiments to compare [211At]YF2 and [211At]L3-Lu directly. A paired-label biodistribution study was performed in athymic mice with subcutaneous PSMA-positive PC-3 PIP xenografts as a head-to-head comparison of [131I]YF2 and [125I]L3-Lu. The tissue distribution of [211At]YF2 and [211At]L3-Lu were determined individually in the same animal model. ResultsThe syntheses of tin precursors and unlabeled iodo standards were accomplished in reasonable yields. A streamlined and scalable radiolabeling method (1 h total synthesis time) was developed for the radiosynthesis of both [211At]YF2 and [211At]L3-Lu with 86 ± 7 % (n = 10) and 87 ± 5 % (n = 7) RCY, respectively, and > 95 % RCP for both. The maximum activity of [211At]YF2 produced to date was 666 MBq. An alternative method that did not involve HPLC purification was developed that provided similar RCY and RCP. Significantly higher cell uptake, internalization and cytotoxicity was seen for [211At]YF2 compared with [211At]L3-Lu. Significantly higher uptake and longer retention in tumor was seen for [131I]YF2 than for co-administered [125I]L3-Lu, while considerably higher renal uptake was seen for [131I]YF2. The biodistribution of [211At]YF2 was consistent with that of [131I]YF2. Conclusion[211At]YF2 exhibited higher cellular uptake, internalization and cytotoxicity than [211At]L3-Lu on PSMA-positive PC3 PIP cells. Likewise, higher uptake and longer retention in tumor was seen for [211At]YF2. Experiments to evaluate the dosimetry and therapeutic efficacy of [211At]YF2 are under way.

Approaches to Reducing Normal Tissue Radiation from Radiolabeled Antibodies.

Radiolabeled antibodies are powerful tools for both imaging and therapy in the field of nuclear medicine. Radiolabeling methods that do not release radionuclides from parent antibodies are essential for radiolabeling antibodies, and practical radiolabeling protocols that provide high in vivo stability have been established for many radionuclides, with a few exceptions. However, several limitations remain, including undesirable side effects on the biodistribution profiles of antibodies. This review summarizes the numerous efforts made to tackle this problem and the recent advances, mainly in preclinical studies. These include pretargeting approaches, engineered antibody fragments and constructs, the secondary injection of clearing agents, and the insertion of metabolizable linkages. Finally, we discuss the potential of these approaches and their prospects for further clinical application.

Preparation of a Zirconium-89 Labeled Clickable DOTA Complex and Its Antibody Conjugate.

Desferrioxamine B (DFO) is the clinical standard chelator for preparing zirconium-89 labeled antibodies. In the current study, the stabilities of a zirconium-89 labeled panitumumab (PAN; Vectibix®) with three different chelators (DFO, DFO*, and DOTA) were compared. PAN is an anti-HER1/EGFR monoclonal antibody approved by the FDA for the treatment of HER1-expressing colorectal cancers and was used as the model antibody for this study. DFO/DFO* conjugates of PAN were directly radiolabeled with zirconium-89 at room temperature to produce [89Zr]Zr-DFO/DFO*-PAN conjugates following a well-established procedure. A zirconium-89 labeled DOTA-PAN conjugate was prepared by an indirect radiolabeling method. A cyclooctyne-linked DOTA chelator (BCN-DOTA-GA) was first radiolabeled with zirconium-89 at 90 °C under a two-step basic pH adjustment method followed by conjugation with PAN-tetrazene at 37 °C to produce a labeled conjugate, BCN-[89Zr]Zr-DOTA-GA-PAN. High reproducibility of the radiolabeling was observed via this two-step basic pH adjustment. The overall radiochemical yield was 40-50% (n = 12, decay uncorrected) with a radiochemical purity of >95% in 2 h synthesis time. All three conjugates were stable in whole human serum for up to 7 days at 37 °C. The kinetic inertness of the conjugates was assessed against the EDTA challenge. BCN-[89Zr]Zr-DOTA-GA-PAN exhibited excellent inertness followed by [89Zr]Zr-DFO*-PAN. [89Zr]Zr-DFO-PAN displayed the lowest level of inertness.

Intramolecular Friedel-Crafts Acylation of [11C]Isocyanates Enabling the Radiolabeling of [carbonyl-11C]DPQ.

α,β-aromatic lactams are highly abundant in biologically active molecules, yet so far they cannot be radiolabeled with short-lived (t1/2=20.3 min), β+-decaying carbon-11, which has prevented their application as positron emission tomography tracers. Herein, we developed, optimized, and applied a widely applicable, one-pot, quick, robust and automatable radiolabeling method for α,β-aromatic lactams starting from [11C]CO2 using the reagent POCl3⋅AlCl3. This method proceeds via intramolecular Friedel-Crafts acylation of in situ formed [11C]isocyanates and shows a broad substrate scope for the formation of five- and six-membered rings. We implemented our developed labeling method for the radiosynthesis of the potential PARP1 PET tracer [carbonyl-11C]DPQ in a clinical radiotracer production facility following the standards of the European Pharmacopoeia.

18F]-Radiolabelled Nanoplatforms: A Critical Review of Their Intrinsic Characteristics, Radiolabelling Methods, and Purification Techniques.

A wide range of nano-objects is found in many applications of our everyday life. Recognition of their peculiar properties and ease of functionalization has prompted their engineering into multifunctional platforms that are supposed to afford efficient tools for the development of biomedical applications. However, bridging the gap between bench to bedside cannot be expected without a good knowledge of their behaviour in vivo, which can be obtained through non-invasive imaging techniques, such as positron emission tomography (PET). Their radiolabelling with [18F]-fluorine, a technique already well established and widely used routinely for PET imaging, with [18F]-FDG for example, and in preclinical investigation using [18F]-radiolabelled biological macromolecules, has, therefore, been developed. In this context, this review highlights the various nano-objects studied so far, the reasons behind their radiolabelling, and main in vitro and/or in vivo results obtained thereof. Then, the methods developed to introduce the radioelement are presented. Detailed indications on the chemical steps involved are provided, and the stability of the radiolabelling is discussed. Emphasis is then made on the techniques used to purify and analyse the radiolabelled nano-objects, a point that is rarely discussed despite its technical relevance and importance for accurate imaging. The pros and cons of the different methods developed are finally discussed from which future work can develop.

Radiosynthesis, structural identification and in vitro tissue binding study of [18F]FNA-S-ACooP, a novel radiopeptide for targeted PET imaging of fatty acid binding protein 3

BackgroundFatty acid binding protein 3 (FABP3) is a target with clinical relevance and the peptide ligand ACooP has been identified for FABP3 targeting. ACooP is a linear decapeptide containing a free amino and thiol group, which provides opportunities for conjugation. This work is to develop methods for radiolabeling of ACooP with fluorine-18 (18F) for positron emission tomography (PET) applications, and evaluate the binding of the radiolabeled ACooP in human tumor tissue sections with high FABP3 expression.ResultsThe prosthetic compound 6-[18F]fluoronicotinic acid 4-nitrophenyl ester was conveniently prepared with an on-resin 18F-fluorination in 29.9% radiochemical yield and 96.6% radiochemical purity. Interestingly, 6-[18F]fluoronicotinic acid 4-nitrophenyl ester conjugated to ACooP exclusively by S-acylation instead of the expected N-acylation, and the chemical identity of the product [18F]FNA-S-ACooP was confirmed. In the in vitro binding experiments, [18F]FNA-S-ACooP exhibited heterogeneous and high focal binding in malignant tissue sections, where we also observed abundant FABP3 positivity by immunofluorescence staining. Blocking study further confirmed the [18F]FNA-S-ACooP binding specificity.ConclusionsFABP3 targeted ACooP peptide was successfully radiolabeled by S-acylation using 6-[18F]fluoronicotinic acid 4-nitrophenyl ester as the prosthetic compound. The tissue binding and blocking studies together with anti-FABP3 immunostaining confirmed [18F]FNA-S-ACooP binding specificity. Further preclinical studies of [18F]FNA-S-ACooP are warranted.

Radiochemistry and In Vivo Imaging of [45Ti]Ti-THP-PSMA.

Titanium-45 (45Ti) is a radionuclide with excellent physical characteristics for use in positron emission tomography (PET) imaging, including a moderate half-life (3.08 h), decay by positron emission (85%), and a low mean positron energy of 0.439 MeV. However, challenges associated with titanium chemistry have led to the underdevelopment of this radionuclide for incorporation into radiopharmaceuticals. Expanding on our recent studies, which showed promising results for the complexation of 45Ti with the tris hydroxypyridinone (THPMe) chelator, the current work aimed to optimize the chemistry and imaging attributes of [45Ti]Ti-THP-PSMA as a new PET radiopharmaceutical. Methods. Radiolabeling of THP-PSMA was optimized with [45Ti]Ti-citrate at varying pHs and masses of the precursor. The stability of the radiolabeled complex was assessed in mouse serum for up to 6 h. The affinity of [45Ti]Ti-THP-PSMA for prostate-specific membrane antigen (PSMA) was assessed using LNCaP (PSMA +) and PC3 (PSMA -) cell lines. In vivo imaging and biodistribution analysis were performed in tumor-bearing xenograft mouse models to confirm the specificity of the tumor uptake. Results. > 95% of radiolabeling was achieved with a high specific activity of 5.6 MBq/nmol under mild conditions. In vitro cell binding studies showed significant binding of the radiolabeled complex with the PSMA-expressing LNCaP cell line (11.9 ± 1.5%/mg protein-bound activity) compared to that with the nonexpressing PC3 cells (1.9 ± 0.4%/mg protein-bound activity). In vivo imaging and biodistribution studies confirmed specific uptake in LNCaP tumors (1.6 ± 0.27% ID/g) compared to that in PC3 tumors (0.39 ± 0.2% ID/g). Conclusion. This study showed a simple one-step radiolabeling method for 45Ti with THP-PSMA under mild conditions (pH 8 and 37 °C). In vitro cell studies showed promise, but in vivo tumor xenograft studies indicated low tumor uptake. Overall, this study shows the need for more chelators for 45Ti for the development of a PET radiopharmaceutical for cancer imaging.

Size-dependent therapeutic efficiency of 223Ra-labeled calcium carbonate carriers for internal radionuclide therapy of breast cancer.

The size of drug carriers strongly affects their biodistribution, tissue penetration, and cellular uptake in vivo. As a result, when such carriers are loaded with therapeutic compounds, their size can influence the treatment outcomes. For internal α-radionuclide therapy, the carrier size is particularly important, because short-range α-emitters should be delivered to tumor volumes at a high dose rate without any side effects, i.e. off-target irradiation and toxicity. In this work, we aim to evaluate and compare the therapeutic efficiency of calcium carbonate (CaCO3) microparticles (MPs, >2 μm) and nanoparticles (NPs, <100 nm) labeled with radium-223 (223Ra) for internal α-radionuclide therapy against 4T1 breast cancer. To do this, we comprehensively study the internalization and penetration efficiency of these MPs and NPs, using 2D and 3D cell cultures. For further therapeutic tests, we develop and modify a chelator-free method for radiolabeling of CaCO3 MPs and NPs with 223Ra, improving their radiolabeling efficiency (>97%) and radiochemical stability (>97%). After intratumoral injection of 223Ra-labeled MPs and NPs, we demonstrate their different therapeutic efficiencies against a 4T1 tumor. In particular, 223Ra-labeled NPs show a tumor inhibition of approximately 85%, which is higher compared to 60% for 223Ra-labeled MPs. As a result, we can conclude that 223Ra-labeled NPs have a more suitable biodistribution within 4T1 tumors compared to 223Ra-labeled MPs. Thus, our study reveals that 223Ra-labeled CaCO3 NPs are highly promising for internal α-radionuclide therapy.

Preparation of 18F-Labeled Tracers Targeting Fibroblast Activation Protein via Sulfur [18F]Fluoride Exchange Reaction.

Early detection and treatment of cancers can significantly increase patient prognosis and enhance the quality of life of affected patients. The emerging significance of the tumor microenvironment (TME) as a new frontier for cancer diagnosis and therapy may be exploited by radiolabeled tracers for diagnostic imaging techniques such as positron emission tomography (PET). Cancer-associated fibroblasts (CAFs) within the TME are identified by biomarkers such as fibroblast activation protein alpha (FAPα), which are expressed on their surfaces. Targeting FAPα using small-molecule 18F-labeled inhibitors (FAPIs) has recently garnered significant attention for non-invasive tumor visualization using PET. Herein, two potent aryl-fluorosulfate-based FAPIs, 12 and 13, were synthetically prepared, and their inhibition potency was determined using a fluorimetric FAP assay to be IC50 9.63 and 4.17 nM, respectively. Radiofluorination was performed via the sulfur [18F]fluoride exchange ([18F]SuFEx) reaction to furnish [18F]12 and [18F]13 in high activity yields (AY) of 39-56% and molar activities (Am) between 20-55 GBq/µmol. In vitro experiments focused on the stability of the radiolabeled FAPIs after incubation with human serum, liver microsomes and liver cytosol. Preliminary PET studies of the radioligands were performed in healthy mice to investigate the in vivo biodistribution and 18F defluorination rate. Fast pharmacokinetics for the FAP-targeting tracers were retained and considerable bone uptake, caused by either 18F defluorination or radioligand accumulation, was observed. In summary, our findings demonstrate the efficiency of [18F]SuFEx as a radiolabeling method as well as its advantages and limitations with respect to PET tracer development.

P-016 - [18F]AlF and RESCA: a novel method for radiolabeling of Affibody molecules

P-016 - [18F]AlF and RESCA: a novel method for radiolabeling of Affibody molecules

Deoxyfluorination of phenols for chemoselective 18F-labeling of peptides.

The challenge of forming C-18F bonds is often a bottleneck in the development of new 18F-labeled tracer molecules for noninvasive functional imaging studies using positron emission tomography (PET). Nucleophilic aromatic substitution is the most widely employed reaction to functionalize aromatic substrates with the radioactive fluorine-18 but its scope is restricted to arenes containing electron-withdrawing substituents. Furthermore, many protic functional groups are incompatible with basic fluoride anions. Peptide substrates, which are highly desirable targets for PET molecular imaging, are particularly challenging to label with fluorine-18 because they are densely functionalized and sensitive to high temperatures and basic conditions. To expand the utility of nucleophilic aromatic substitution with fluorine-18, we describe two complementary procedures for the radiodeoxyfluorination of bench-stable and easy-to-access phenols that ensure rapid access to densely functionalized electron-rich and electron-poor 18F-aryl fluorides. The first procedure details the synthesis of an 18F-synthon and its subsequent ligation to the cysteine residue of Arg-Gly-Asp-Cys in 10.5 h from commercially available starting materials (189-min radiosynthesis). The second procedure describes the incorporation of commercially available CpRu(Fmoc-tyrosine)OTf into a fully protected peptide Lys-Met-Glu-(CpRu-Tyr)-Leu via solid-phase peptide synthesis and subsequent ruthenium-mediated uronium deoxyfluorination with fluorine-18 followed by deprotection, accomplished within 7 d (116-min radiosynthesis). Both radiolabeling methods are highly chemoselective and have conveniently been automated using commercially available radiosynthesis equipment so that the procedures described can be employed for the synthesis of peptide-based PET probes for in vivo imaging studies according to as low as reasonably achievable (ALARA) principles.

Radiolabeled Human Serum Albumin Nanoparticles Co-Loaded with Methotrexate and Decorated with Trastuzumab for Breast Cancer Diagnosis.

Breast cancer is a leading cause of cancer-related mortality among women worldwide, with millions of new cases diagnosed yearly. Addressing the burden of breast cancer mortality requires a comprehensive approach involving early detection, accurate diagnosis, effective treatment, and equitable access to healthcare services. In this direction, nano-radiopharmaceuticals have shown potential for enhancing breast cancer diagnosis by combining the benefits of nanoparticles and radiopharmaceutical agents. These nanoscale formulations can provide improved imaging capabilities, increased targeting specificity, and enhanced sensitivity for detecting breast cancer lesions. In this study, we developed and evaluated a novel nano-radio radiopharmaceutical, technetium-99m ([99mTc]Tc)-labeled trastuzumab (TRZ)-decorated methotrexate (MTX)-loaded human serum albumin (HSA) nanoparticles ([99mTc]-TRZ-MTX-HSA), for the diagnosis of breast cancer. In this context, HSA and MTX-HSA nanoparticles were prepared. Conjugation of MTX-HSA nanoparticles with TRZ was performed using adsorption and covalent bonding methods. The prepared formulations were evaluated for particle size, PDI value, zeta (ζ) potential, scanning electron microscopy analysis, encapsulation efficiency, and loading capacity and cytotoxicity on MCF-7, 4T1, and MCF-10A cells. Finally, the nanoparticles were radiolabeled with [99mTc]Tc using the direct radiolabeling method, and cellular uptake was performed with the nano-radiopharmaceutical. The results showed the formation of spherical nanoparticles, with a particle size of 224.1 ± 2.46 nm, a PDI value of 0.09 ± 0.07, and a ζ potential value of -16.4 ± 0.53 mV. The encapsulation efficiency of MTX was found to be 32.46 ± 1.12%, and the amount of TRZ was 80.26 ± 1.96%. The labeling with [99mTc]Tc showed a high labeling efficiency (>99%). The cytotoxicity studies showed no effect, and the cellular uptake studies showed 97.54 ± 2.16% uptake in MCF-7 cells at the 120th min and were found to have a 3-fold higher uptake in cancer cells than in healthy cells. In conclusion, [99mTc]Tc-TRZ-MTX-HSA nanoparticles are promising for diagnosing breast cancer and evaluating the response to treatment in breast cancer patients.

Nanotexaphyrins for Cancer Imaging and Therapy

Texaphyrins are expanded porphyrin macrocycles with a 5-coordination pocket that can form stable 1:1 complexes with a large group of metal cations including many lanthanide ions, making them inherently amenable for theranostics (e.g., photodynamic therapy, MRI and radiation therapy). For example, gadolinium-texaphyrin (Gd-Tex, Xcytrin®) developed by the Sessler group was evaluated as a radiosensitizer all the way to Phase III trials. Although Gd-Tex did not gain FDA approval due to its limited radiosensitization efficacy, the fact that Gd-Tex showed good safety profile in phase III clinical trials makes texaphyrins attractive building block to develop new theranostic agents.We recently synthesized a texaphyrin-phospholipid building block via a key 1,2-dinitrophenyl-phospholipid intermediate, along with stable chelation of a library of 18 different metal ions into texaphyrin-lipid without compromising their self-assembly into nanotexaphyrins. To realize nanotexaphyrin’s theranostic properties, we developed a customizable strategy to build a family of nanotexaphyrins with quantitatively mixed and matched metal ions. We also developed a rapid and robust nanotexaphyrin radiolabeling method using a home-made disposable microfluidic system that achieved a high radiochemical yield. The optimized metalated nanotexaphyrin displayed excellent chemical, photo, and colloidal stabilities, and favorable pharmacokinetics and biodistribution profiles, resulting versatile cancer imaging and therapy applications. For example, the PSMA-targeted 111In/Lu-nanotexaphyrin demonstrated a preferential tumor accumulation in PSMA-positive tumor by both NIR fluorescence and SPECT/CT imaging, and a potent PDT effect that successfully inhibited PSMA+ tumor growth. On the radiation therapy front, we developed Gd-nanotexaphyrin to allow high density packing of Gd-Tex in a single nanovesicle to overcome the limited radiosensitization efficacy of monomeric Gd-Tex. As hypoxia is a common cause of radiation resistance, we loaded oxygen-carrying myoglobin into Gd-nanotexaphyrins, which enabled spatiotemporal codelivery of O2 and Gd-Tex into tumors, resulting in efficient relief of tumor hypoxia and significant enhancement of Gd-Tex radiosensitization. The synergistic mechanism between appropriate O2 delivery and enhanced Gd-Tex radiosensitization has been demonstrated. The high versatility and clinical translatability of nanotexaphyrins is a promising platform to develop cancer theranostics.