In Vivo Imaging of [60]Fullerene-Based Molecular Spherical Nucleic Acids by Positron Emission Tomography.

18F-Labeled [60]fullerene-based molecular spherical nucleic acids (MSNAs), consisting of a human epidermal growth factor receptor 2 (HER2) mRNA antisense oligonucleotide sequence with a native phosphodiester and phosphorothioate backbone, were synthesized, site-specifically labeled with a positron emitting fluorine-18 and intravenously administrated via tail vein to HER2 expressing HCC1954 tumor-bearing mice. The biodistribution of the MSNAs was monitored in vivo by positron emission tomography/computed tomography (PET/CT) imaging. MSNA with a native phosphodiester backbone (MSNA-PO) was prone to rapid nuclease-mediated degradation, whereas the corresponding phosphorothioate analogue (MSNA-PS) with improved enzymatic stability showed an interesting biodistribution profile in vivo. One hour after the injection, majority of the radioactivity was observed in spleen and liver but also in blood with an average tumor-to-muscle ratio of 2. The prolonged radioactivity in blood circulation may open possibilities to the targeted delivery of the MSNAs.

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [89Zr]Zr-DFO-PEG5-Tz.

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-of-concept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new 89Zr-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels-Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t1/2 = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [89Zr]Zr-DFO-PEG5-Tz ([89Zr]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 ± 0.2 vs 17.1 ± 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for 89Zr-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.

Development of [18F]AmBF3 Tetrazine for Radiolabeling of Peptides: Preclinical Evaluation and PET Imaging of [18F]AmBF3-PEG7-Tyr3-Octreotide in an AR42J Pancreatic Carcinoma Model.

Radiolabeled peptides have emerged as highly specific agents for targeting receptors expressed in tumors for therapeutic and diagnostic purposes. Peptides developed for positron emission tomography (PET) are typically radiolabeled using prosthetic groups or bifunctional chelators for fast "kit-like" incorporation of the radionuclide into the structure. A novel [18F]alkylammoniomethyltrifluoroborate ([18F]AmBF3) tetrazine (Tz), [18F]AmBF3-Tz, was developed for the [18F]fluorination of trans-cyclooctene (TCO)-modified biomolecules using Tyr3-octreotides (TOCs) as model peptides. [18F]AmBF3-Tz (Am = 15.4 ± 9.2 GBq/µmol, n = 14) was evaluated in healthy mice by ex vivo biodistribution and PET/computed tomography (CT), where the radiolabel in the prosthetic group was found stable in vivo, indicated by the low bone uptake in tibia (0.4 ± 0.1% ID/g, t = 270 min). TCO-TOCs tailored with polyethylene glycol (PEG) linkers were radiolabeled with [18F]AmBF3-Tz, forming two new tracers, [18F]AmBF3-PEG4-TOC (Am = 2.8 ± 1.8 GBq/µmol, n = 3) and [18F]AmBF3-PEG7-TOC (Am of 6.0 ± 3.4 GBq/µmol, n = 13), which were evaluated by cell uptake studies and ex vivo biodistribution in subcutaneous AR42J rat pancreatic carcinoma tumor-bearing nude mice. The tracer demonstrating superior behavior ex vivo, the [18F]AmBF3-PEG7-TOC, was further evaluated with PET/CT, where the tracer provided clear tumor visualization (SUVbaseline = 1.01 ± 0.07, vs SUVblocked = 0.76 ± 0.04) at 25 min post injection. The novel AmBF3-Tz demonstrated that it offers potential as a prosthetic group for rapid radiolabeling of biomolecules in mild conditions using bioorthogonal chemistry.

Development of 68Ga-Labeled Hepatitis E Virus Nanoparticles for Targeted Drug Delivery and Diagnostics with PET.

Targeted delivery of diagnostics and therapeutics offers essential advantages over nontargeted systemic delivery. These include the reduction of toxicity, the ability to reach sites beyond biological barriers, and the delivery of higher cargo concentrations to diseased sites. Virus-like particles (VLPs) can efficiently be used for targeted delivery purposes. VLPs are derived from the coat proteins of viral capsids. They are self-assembled, biodegradable, and homogeneously distributed. In this study, hepatitis E virus (HEV) VLP derivatives, hepatitis E virus nanoparticles (HEVNPs), were radiolabeled with gallium-68, and consequently, the biodistribution of the labeled [68Ga]Ga-DOTA-HEVNPs was studied in mice. The results indicated that [68Ga]Ga-DOTA-HEVNPs can be considered as promising theranostic nanocarriers, especially for hepatocyte-targeting therapies.

A Theranostic Cellulose Nanocrystal-Based Drug Delivery System with Enhanced Retention in Pulmonary Metastasis of Melanoma.

Metastatic melanoma can be difficult to detect until at the advanced state that decreases the survival rate of patients. Several FDA-approved BRAF inhibitors have been used for treatment of metastatic melanoma, but overall therapeutic efficacy has been limited. Lutetium-177 (177 Lu) enables simultaneous tracking of tracer accumulation with single-photon emission computed tomography and radiotherapy. Therefore, the codelivery of 177 Lu alongside chemotherapeutic agents using nanoparticles (NPs) might improve the therapeutic outcome in metastatic melanoma. Cellulose nanocrystals (CNC NPs) can particularly deliver payloads to lung capillaries in vivo. Herein, 177 Lu-labeled CNC NPs loaded with vemurafenib ([177 Lu]Lu-CNC-V NPs) is developed and the therapeutic effect in BRAF V600E mutation-harboring YUMM1.G1 murine model of lung metastatic melanoma is investigated. The [177 Lu]Lu-CNC-V NPs demonstrate favorable radiolabel stability, drug release profile, cellular uptake, and cell growth inhibition in vitro. In vivo biodistribution reveals significant retention of the [177 Lu]Lu-CNC-V NPs in the lung, liver, and spleen. Ultimately, the median survival time of animals is doubly increased after treatment with [177 Lu]Lu-CNC-V NPs compared to control groups. The enhanced therapeutic efficacy of [177 Lu]Lu-CNC-V NPs in the lung metastatic melanoma animal model provides convincing evidence for the potential of clinical translation for theranostic CNC NP-based drug delivery systems after intravenous administration.

Exploring the Biochemical Foundations of a Successful GLUT1-Targeting Strategy to BNCT: Chemical Synthesis and In Vitro Evaluation of the Entire Positional Isomer Library of ortho-Carboranylmethyl-Bearing Glucoconjugates.

Boron neutron capture therapy (BNCT) is a noninvasive binary therapeutic modality applicable to the treatment of cancers. While BNCT offers a tumor-targeting selectivity that is difficult to match by other means, the last obstacles preventing the full harness of this potential come in the form of the suboptimal boron delivery strategies presently used in the clinics. To address these challenges, we have developed delivery agents that target the glucose transporter GLUT1. Here, we present the chemical synthesis of a number of ortho-carboranylmethyl-substituted glucoconjugates and the biological assessment of all positional isomers. Altogether, the study provides protocols for the synthesis and structural characterization of such glucoconjugates and insights into their essential properties, for example, cytotoxicity, GLUT1-affinity, metabolism, and boron delivery capacity. In addition to solidifying the biochemical foundations of a successful GLUT1-targeting approach to BNCT, we identify the most promising modification sites in d-glucose, which are critical in order to further develop this strategy toward clinical use.

Radiolabeling of Theranostic Nanosystems.

In the recent years, progress in nanotechnology has significantly contributed to the development of novel pharmaceutical formulations to overcome the drawbacks of conventional treatments and improve the therapeutic outcome in many diseases, especially cancer. Nanoparticle vectors have demonstrated the potential to concomitantly deliver diagnostic and therapeutic payloads to diseased tissue. Due to their special physical and chemical properties, the characteristics and function of nanoparticles are tunable based on biological molecular targets and specific desired features (e.g., surface chemistry and diagnostic radioisotope labeling). Within the past decade, several theranostic nanoparticles have been developed as a multifunctional nanosystems which combine the diagnostic and therapeutic functionalities into a single drug delivery platform. Theranostic nanosystems can provide useful information on a real-time systemic distribution of the developed nanosystem and simultaneously transport the therapeutic payload. In general, the diagnostic functionality of theranostic nanoparticles can be achieved through labeling gamma-emitted radioactive isotopes on the surface of nanoparticles which facilitates noninvasive detection using nuclear molecular imaging techniques, such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT), meanwhile, the therapeutic effect arises from the potent drug released from the nanoparticle. Moreover, some radioisotopes can concurrently emit both gamma radiation and high-energy particles (e.g., alpha, beta, and Auger electrons), prompting the use either alone for radiotheranostics or synergistically with chemotherapy. This chapter provides an overview of the fundamentals of radiochemistry and relevant radiolabeling strategies for theranostic nanosystem development as well as the methods for the preclinical evaluation of radiolabeled nanoparticles. Furthermore, preclinical case studies of recently developed theranostic nanosystems will be highlighted.

Metabolism of a Bioorthogonal PET Tracer Candidate [19F/18F]SiFA-Tetrazine in Mouse Liver Microsomes: Biotransformation Pathways and Defluorination Investigated by UHPLC-HRMS.

Organofluorosilicon based 18F-radiolabeling is an efficient method for incorporating fluorine-18 into 18F-radiopharmaceuticals for positron emission tomography (PET) by 19F/18F isotopic exchange (IE). The first PET radiopharmaceutical, 18F-SiFAlin-TATE, radiolabeled with a silicon-based [18F]fluoride acceptor (SiFA), namely, a para-substituted di-tert-butyl[18F]fluorosilylbenzene, has entered clinical trials, and is paving the way for other potential [18F]SiFA-labeled radiopharmaceuticals for diagnostic use. In this study, we report the in vitro metabolism of an oxime-linked SiFA tetrazine (SiFA-Tz), a new PET-radiotracer candidate, recently evaluated for pretargeted PET imaging and macromolecule labeling. Metabolism of SiFA-Tz was studied in mouse liver microsomes (MLM) for elucidating its major biotransformation pathways. Nontargeted screening by ultrahigh performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) was utilized for detection of unknown metabolites. The oxime bond between the SiFA and Tz groups forms two geometric (E/Z) isomers, which underwent the same biotransformations, but unexpectedly with different kinetics. In total, nine proposed metabolites of SiFA-Tz from phase I and II reactions were detected, five of which were defluorinated in MLMs, elucidating the metabolic pathway leading to previously reported defluorination of [18F]SiFA-Tz in vivo. Based on the HRMS studies a biotransformation pathway is proposed: hydroxylation (+O) to tert-butyl group adjacent to the silicon, followed by oxidative defluorination (+OH/-F) cleaving the fluorine off the silicon. Interestingly, eight proposed metabolites of a reduced dihydrotetrazine analogue, SiFA-H2Tz, from phase I and II reactions were additionally detected. To the best of our knowledge, this is the first reported comprehensive investigation of enzyme mediated metabolic pathway of tetrazines and para-substituted di-tert-butylfluorosilylbenzene fluoride acceptors, providing novel structural information on the biotransformation and fragmentation patterns of radiotracers bearing these structural motifs. By investigating the metabolism preceding defluorination, structurally optimized new SiFA compounds can be designed for expanding the portfolio of efficient 19F/18F isotopic exchange labeling probes for PET imaging.

Addressing the Biochemical Foundations of a Glucose-Based "Trojan Horse"-Strategy to Boron Neutron Capture Therapy: From Chemical Synthesis to In Vitro Assessment.

Boron neutron capture therapy (BNCT) for cancer is on the rise worldwide due to recent developments of in-hospital neutron accelerators which are expected to revolutionize patient treatments. There is an urgent need for improved boron delivery agents, and herein we have focused on studying the biochemical foundations upon which a successful GLUT1-targeting strategy to BNCT could be based. By combining synthesis and molecular modeling with affinity and cytotoxicity studies, we unravel the mechanisms behind the considerable potential of appropriately designed glucoconjugates as boron delivery agents for BNCT. In addition to addressing the biochemical premises of the approach in detail, we report on a hit glucoconjugate which displays good cytocompatibility, aqueous solubility, high transporter affinity, and, crucially, an exceptional boron delivery capacity in the in vitro assessment thereby pointing toward the significant potential embedded in this approach.

Evaluation of Organo [18F]Fluorosilicon Tetrazine as a Prosthetic Group for the Synthesis of PET Radiotracers.

Fluorine-18 is the most widely used positron emission tomography (PET) radionuclide currently in clinical application, due to its optimal nuclear properties. The synthesis of 18F-labeled radiotracers often requires harsh reaction conditions, limiting the use of sensitive bio- and macromolecules as precursors for direct radiolabeling with fluorine-18. We aimed to develop a milder and efficient in vitro and in vivo labeling method for trans-cyclooctene (TCO) functionalized proteins, through the bioorthogonal inverse-electron demand Diels-Alder (IEDDA) reaction with fluorine-18 radiolabeled tetrazine ([18F]SiFA-Tz). Here, we used TCO-modified bovine serum albumin (BSA) as the model protein, and isotopic exchange (IE) (19F/18F) chemistry as the labeling strategy. The radiolabeling of albumin-TCO with [18F]SiFA-Tz ([18F]6), providing [18F]fluoroalbumin ([18F]10) in high radiochemical yield (99.1 ± 0.2%, n = 3) and a molar activity (MA) of 1.1 GBq/µmol, confirmed the applicability of [18F]6 as a quick in vitro fluorination reagent for the TCO functionalized proteins. While the biological evaluation of [18F]6 demonstrated defluorination in vivo, limiting the utility for pretargeted applications, the in vivo stability of the radiotracer was dramatically improved when [18F]6 was used for the radiolabeling of albumin-TCO ([18F]10) in vitro, prior to administration. Due to the detected defluorination in vivo, structural optimization of the prosthetic group for improved stability is needed before further biological studies and application of pretargeted PET imaging.

Intravitreal Pharmacokinetics in Mice: SPECT/CT Imaging and Scaling to Rabbits and Humans.

Preclinical in vivo tests of retinal drug responses are carried out in mice and rats, often after intravitreal injections. However, quantitative pharmacokinetics in the mouse eye is poorly understood. Ocular pharmacokinetics studies are usually done in rabbits. We investigated elimination of three compounds ([99mTc]Tc-pentetate, [111In]In-pentetreotide, [99mTc]Tc-human serum albumin with molecular weights of 510.2 Da, 1506.4 Da, and 66.5 kDa, respectively) from mouse vitreous using imaging with single photon emission computed tomography/computed tomography (SPECT/CT). Increasing molecular weight decreased elimination of the compounds from the mouse eyes. Half-lives of [99mTc]Tc-pentetate, [111In]In-pentetreotide, and [99mTc]Tc-human serum albumin in the mouse eyes were 1.8 ± 0.5 h, 4.3 ± 1.7 h, and 30.0 ± 9.0 h, respectively. These values are 3-12-fold shorter than half-lives of similar compounds in the rabbit vitreous. Dose scaling factors were calculated for mouse-to-rabbit and mouse-to-man translation. They were 27-90 and 38-126, respectively, for intravitreal injections in rabbit and man. We show ocular pharmacokinetic parameters for mice and interspecies scaling factors that may augment ocular drug discovery and development.

Radiolabeled Molecular Imaging Probes for the In Vivo Evaluation of Cellulose Nanocrystals for Biomedical Applications.

Cellulose nanocrystals (CNCs) have remarkable potential to improve the delivery of diagnostic and therapeutic agents to tumors; however, the in vivo studies on CNC biodistribution are still limited. We developed CNC-based imaging probes for the in vitro and in vivo evaluation using two labeling strategies: site-specific hydrazone linkage to the terminal aldehyde of the CNC and nonsite-specific activation using 1,1'-carbonyldiimidazole (CDI). The in vivo behavior of unmodified CNC, DOTA-CNC (ald.), and DOTA-CNC (OH) was investigated in healthy and 4T1 breast cancer mouse models. They displayed good biocompatibility in cell models. Moreover, the biodistribution profile and SPECT/CT imaging confirmed that the accumulation of 111In-labeled DOTA-CNC (ald.) and 111In-DOTA-CNC (OH) was primarily in hepatic, splenic, and pulmonary ducts in accordance with the clearance of nontargeted nanoparticles. The developed CNC imaging probes can be used to obtain information with noninvasive imaging on the behavior in vivo to guide structural optimization for targeted delivery.

Drug-Loaded Multifunctional Nanoparticles Targeted to the Endocardial Layer of the Injured Heart Modulate Hypertrophic Signaling.

Ischemic heart disease is the leading cause of death globally. Severe myocardial ischemia results in a massive loss of myocytes and acute myocardial infarction, the endocardium being the most vulnerable region. At present, current therapeutic lines only ameliorate modestly the quality of life of these patients. Here, an engineered nanocarrier is reported for targeted drug delivery into the endocardial layer of the left ventricle for cardiac repair. Biodegradable porous silicon (PSi) nanoparticles are functionalized with atrial natriuretic peptide (ANP), which is known to be expressed predominantly in the endocardium of the failing heart. The ANP-PSi nanoparticles exhibit improved colloidal stability and enhanced cellular interactions with cardiomyocytes and non-myocytes with minimal toxicity. After confirmation of good retention of the radioisotope 111-Indium in relevant physiological buffers over 4 h, in vivo single-photon emission computed tomography (SPECT/CT) imaging and autoradiography demonstrate increased accumulation of ANP-PSi nanoparticles in the ischemic heart, particularly in the endocardial layer of the left ventricle. Moreover, ANP-PSi nanoparticles loaded with a novel cardioprotective small molecule attenuate hypertrophic signaling in the endocardium, demonstrating cardioprotective potential. These results provide unique insights into the development of nanotherapies targeted to the injured region of the myocardium.

Adenoviral Delivery of Tumor Necrosis Factor-α and Interleukin-2 Enables Successful Adoptive Cell Therapy of Immunosuppressive Melanoma.

Adoptive T-cell transfer is a promising treatment approach for metastatic cancer, but efficacy in solid tumors has only been achieved with toxic pre- and postconditioning regimens. Thus, adoptive T-cell therapies would benefit from complementary modalities that enable their full potential without excessive toxicity. We aimed to improve the efficacy and safety of adoptive T-cell transfer by using adenoviral vectors for direct delivery of immunomodulatory murine cytokines into B16.OVA melanoma tumors with concomitant T-cell receptor transgenic OT-I T-cell transfer. Armed adenoviruses expressed high local and low systemic levels of cytokine when injected into B16.OVA tumors, suggesting safety of virus-mediated cytokine delivery. Antitumor efficacy was significantly enhanced with adenoviruses coding for murine interleukin-2 (mIL-2) and tumor necrosis factor-α (mTNFα) when compared with T-cell transfer alone or viruses alone. Further improvement in efficacy was achieved with a triple combination of mIL-2, mTNFα, and OT-I T-cells. Mechanistic studies suggest that mIL-2 has an important role in activating T-cells at the tumor, while mTNFα induces chemokine expression. Furthermore, adenovirus treatments enhanced tumor-infiltration of OT-I T-cells as demonstrated by SPECT/CT imaging of (111)In-labeled cells. Our results suggest the utility of cytokine-coding adenoviruses for improving the efficacy of adoptive T-cell therapies.

Oxidative stress protection by exogenous delivery of rhHsp70 chaperone to the retinal pigment epithelium (RPE), a possible therapeutic strategy against RPE degeneration.

PURPOSE: To measure the cytoprotective effects of rhHsp70 against oxidative stress and study its cellular uptake, intracellular and intraocular distribution in the retinal pigment epithelium. METHODS: Human retinal pigment epithelial cells (ARPE-19) were pre-treated with rhHsp70 for 24 h, 48 h, and 72 h before being exposed to 1.25 mM hydrogen peroxide. Non-treated cells served as control. We analysed interleukin 6 secretion, cell viability, and cytolysis. Uptake and intracellular distribution of fluorescently labelled rhHsp70 were investigated with flow cytometry and confocal microscopy, respectively. Ocular distribution of radioactively labelled rhHsp70 was followed ex vivo in porcine eyes by micro SPECT/CT. RESULTS: After exposure to hydrogen peroxide, IL-6 secretion decreased by 35-39% when ARPE-19 cells were pre-treated with rhHsp70. Cell viability increased by 17-32%, and cell lysis, measured by the release of lactate dehydrogenase, decreased by 6-43%. ARPE-19 cells endocytosed rhHsp70 added to the culture medium and the protein was localized in late endosomes and lysosomes. Following intravitreal injection into isolated porcine eyes, we found 20% rhHsp70 in the RPE. CONCLUSIONS: Recombinant hHsp70 protein offers protection against oxidative stress. RPE cells take up the exogenously delivered rhHsp70 and localize it in late endosomes and lysosomes. This work provides the basis for a therapeutic strategy to target aggregate-associated neurodegeneration in AMD.

In vivo evaluation of porous silicon and porous silicon solid lipid nanocomposites for passive targeting and imaging.

The use of nanoparticle carriers for the sustained release of cytotoxic drugs in cancer therapy can result in fewer adverse effects and can thus be of great benefit for the patient. Recently, a novel nanocomposite, prepared by the encapsulation of THCPSi nanoparticles within solid lipids (SLN), was developed and characterized as a promising drug delivery carrier in vitro. The present study describes the in vivo evaluation of unmodified THCPSi nanoparticles and THCPSi-solid lipid nanocomposites (THCPSi-SLNCs) as potential drug delivery carriers for cancer therapy by using (18)F radiolabeling for the detection of the particle biodistribution in mice. Passive tumor targeting of (18)F-THCPSis and (18)F-THCPSi-SLNCs by the enhanced permeation and retention effect was investigated in a murine breast cancer model. Encapsulation of THCPSi nanoparticles with solid lipids improved their accumulation in tumors at a 7 week time point (tumor-to-liver ratio 0.10 ± 0.08 and 0.24 ± 0.09% for (18)F-THCPSis and (18)F-THCPSi-SLNCs, respectively).

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.

BACKGROUND: Fatty 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. RESULTS: The 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. CONCLUSIONS: FABP3 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.

Radiolabelling of peptides with tetrazine ligation based on the inverse electron-demand Diels-Alder reaction: rapid, catalyst-free and mild conversion of 1,4-dihydropyridazines to pyridazines.

Click chemistry reactions, such as the tetrazine ligation, based on the inverse-electron demand Diels-Alder (IEDDA), are chemoselective cycloaddition reactions widely used for chemical modifications and synthesis of biomolecule-based radiopharmaceuticals for positron emission tomography (PET). The reactions have potential also for pretargeted PET imaging. When used as a bioconjugation method in production of biomolecule-based radiopharmaceuticals, IEDDA-based tetrazine ligation has one significant drawback, namely the formation of a mixture comprising reduced metastable dihydropyridazines (DHPs) and oxidized cycloadducts. Conversion of the reduced DHPs to stable pyridazines requires oxidation, which is typically achieved by using oxidants or by photo-irradiated air-oxidation, both methods requiring added reagents or reaction times of several hours, not compatible with short-lived radionuclides. Here we report a mild, rapid, and catalyst-free conversion of the DHPs to pyridazines. In this study, a model peptide Tyr3-octreotide (TOC) was modified with polyethylene glycol (PEG) linkers and with trans-cyclooctenes (TCOs) for rapid IEDDA-mediated radiolabeling. Fluorine-18-labelled alkylammoniomethyltrifluoroborate ([18F]AmBF3) tetrazines were conjugated to the TCO-TOC analogs at room temperature for rapid synthesis of PET imaging agent candidates. The formed DHPs were successfully converted to the oxidized form, after heating the radiolabelled bioconjugates in aqueous solution (≥95% water) at 60 °C for a minimum of 10 minutes in the presence of air, resulting in one-pot back-to-back IEDDA reaction and DHP conversion. The water content of the reaction mixture was to be found critical for the coversion. Our finding offers a straightforward method for conversion of the metastable DHPs from the IEDDA-based tetrazine ligation to stable, oxidized pyridazines. The method is especially suitable for applications requiring rapid conversion.

Biological evaluation of integrin α3ß1-targeted 68Ga-labeled HEVNPs in HCT 116 colorectal tumor-bearing mice.

Integrins are cell surface receptors involved in multiple functions vital for cellular proliferation. Various tumor cells overexpress αß-integrins, making them ideal biomarkers for diagnostic imaging and tumor-targeted drug delivery. LXY30 is a peptide that can specifically recognize and interact with the integrin α3ß1, a molecule overexpressed in breast, ovarian and colorectal cancer. Hepatitis E virus nanoparticles (HEVNPs) are virus-like particles that have been investigated as drug delivery agents for the targeted delivery of nucleic acids and small proteins. HEVNPs can be a theranostic platform for monitoring and evaluating tumor-targeted therapies if tagged with a suitable diagnostic marker. Herein, we describe the radiolabeling and biological evaluation of integrin α3ß1-targeted HEVNPs. HEVNPs were conjugated with DOTA and radiolabeled with gallium-68 (t1/2 = 67.7 min), a short-lived positron emitter used in positron emission tomography (PET). The synthesized [68Ga]Ga-DOTA-HEVNPs were used to evaluate the efficacy of conjugated LXY30 peptide to improve HEVNPs binding and internalization to integrin α3ß1 expressing human colorectal HCT 116 cells. In vivo tumor accumulation of [68Ga]Ga-DOTA-HEVNP-LXY30 was evaluated in HCT 116 colorectal tumor-bearing mice. [68Ga]Ga-DOTA-HEVNP-LXY30 and non-targeted [68Ga]Ga-DOTA-HEVNP were radiolabeled with radiochemical yields (RCY) of 67.9 ± 3.3% and 73.7 ± 9.8%, respectively. [68Ga]Ga-DOTA-HEVNP-LXY30 exhibited significantly higher internalization in HCT 116 cells than the non-targeted [68Ga]Ga-DOTA-HEVNPs (21.0 ± 0.7% vs. 10.5 ± 0.3% at 3 h, ****P<0.0001). After intravenous administration to mice, accumulation of [68Ga]Ga-DOTA-HEVNP-LXY30 to HCT 116 xenograft tumors was at its highest rate of 0.8 ± 0.4%ID/g at 60 min. [68Ga]Ga-DOTA-HEVNP-LXY30 accumulated mainly in the liver and spleen (39.8 ± 13.0%%ID/g and 24.6 ± 24.1%ID/g, respectively). Despite the low targeting efficiency in vivo, we demonstrated that [68Ga]Ga-DOTA-HEVNP is a promising diagnostic platform for quantitative analysis of HEVNP distribution in vivo. This nanosystem can be utilized in future studies assessing the success of further engineered HEVNP structures with optimized targeting efficiency in vivo.

Tetrazine Glycoconjugate for Pretargeted Positron Emission Tomography Imaging of trans-Cyclooctene-Functionalized Molecular Spherical Nucleic Acids.

Pretargeted concept in positron emission tomography (PET) together with bioorthogonal chemistry is an elegant solution to study processes with slow pharmacokinetics by utilizing radiotracers labeled with short-lived radionuclides. Namely, radiotracers based on tetrazine ligation with trans-cyclooctene (TCO) via the inverse electron demand Diels-Alder (IEDDA) reaction have become a state-of-the-art for the pretargeted PET imaging. For radiolabeling of tetrazine scaffolds, indirect radiofluorination methods are often preferred, as tetrazines are vulnerable to harsh conditions typically necessary for the direct radiofluorination. 18F-Fluoroglycosylation is an indirect radiofluorination method, which allows the introduction of a widely accessible glucose analog 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG) to aminooxy-functionalized precursors via oxime formation. Here, we report the biological evaluation of [18F]FDG-Tz as a tracer for pretargeted PET imaging of TCO-functionalized molecular spherical nucleic acids (MSNA) against human epidermal growth factor receptor 2 (HER2) mRNA. The oxime ether formation between [18F]FDG and tetrazine oxyamine resulted in [18F]FDG-Tz with high radiochemical purity (>99%) and moderate yields (6.5 ± 3.6%, n = 5). Biological evaluation of [18F]FDG-Tz in healthy mice indicated favorable pharmacokinetics with quick blood clearance, urinary excretion as the main elimination route, and the absence of GLUT1 transportation. The successful pretargeted experiments with TCO-functionalized MSNA revealed higher tumor uptake compared to preclicked MSNA in HER2-expressing human breast cancer xenograft-bearing mice.