RadioLabeling of the Mutant form of Anti-PlGF Nanobody by 99mTc-Tricarbonyl.

Background: Molecular imaging is a highly effective method for diagnosing cancer and evaluating treatment. A molecular tracer often consists of two segments: A targeting segment, which can be antibodies, antibody fragments, or VHH (nanobody), and a detection segment, such as radioisotopes. The small size of VHH allows for excellent tissue penetration and fast clearance, resulting in minimal nonspecific background, which makes them appealing for use as imaging agents. 99m-technetium (99mTc), one of the well-known radioisotopes, is particularly useful in routine clinical imaging. Objectives: This study aims to construct 99mTc-anti-placenta growth factor (PlGF) nanobody and assess its radiochemical purity (RCP). Methods: The mutant form of anti-PlGF nanobody was expressed in E. coli TG1 and purified using Ni-NTA column affinity chromatography. The purified nanobodies were confirmed by SDS-PAGE and western blotting. A 99mTc-tricarbonyl solution was added to phosphate-buffered saline (PBS) containing the mutant nanobody for labeling, and the mixture was purified using a PD-10 column. Results: The RCP of 99mTc-tricarbonyl is > 98%. After the addition of radioisotopes to the mixture of nanobodies, purity reached 70% in 2 hours and remained constant during incubation. After purifying the labeled nanobody, activity was measured, and the highest amount of labeled nanobodies was collected in the second part. The stability of the labeled nanobody in PBS and in competition with histidine for 4 hours was checked by thin-layer chromatography (TLC). Conclusions: The findings of this study reveal that the RCP of the labeled nanobody was above 95% after 4 hours, indicating the labeled antibody's stability. These results are promising and could be utilized in future in vitro and in vivo studies.

Recent Clinical Implications of FAPI: Imaging and Therapy.

ABSTRACT: The fibroblast activation protein (FAP) is a biomarker that is selectively overexpressed on cancer-associated fibroblasts (CAFs) in various types of tumoral tissues and some nonmalignant diseases, including fibrosis, arthritis, cardiovascular, and metabolic diseases. FAP plays a critical role in tumor microenvironment through facilitating proliferation, invasion, angiogenesis, immunosuppression, and drug resistance. Recent studies reveal that FAP might be regarded as a promising target for cancer diagnosis and treatment. FAP-targeted imaging modalities, especially PET, have shown high sensitivity and specificity in detecting FAP-expressing tumors. FAP-targeted imaging can potentially enhance tumor detection, staging, and monitoring of treatment response, and facilitate the development of personalized treatment strategies. This study provides a comprehensive view of FAP and its function in the pathophysiology of cancer and nonmalignant diseases. It also will discuss the characteristics of radiolabeled FAP inhibitors, particularly those based on small molecules, their recent clinical implications in imaging and therapy, and the associated clinical challenges with them. In addition, we present the results of imaging and biodistribution radiotracer 68Ga-FAPI-46 in patients with nonmalignant diseases, including interstitial lung disease, primary biliary cirrhosis, and myocardial infarction, who were referred to our department. Our results show that cardiac FAP-targeted imaging can provide a novel potential biomarker for managing left ventricle remodeling. Moreover, this study has been organized and presented in a manner that offers a comprehensive overview of the current status and prospects of FAPI inhibitors in the diagnosis and treatment of diseases.

Self-assembled peptide/polymer hybrid nanoplatform for cancer immunostimulating therapies.

Integrating peptide epitopes in self-assembling materials is a successful strategy to obtain nanovaccines with high antigen density and improved efficacy. In this study, self-assembling peptides containing MAGE-A3/PADRE epitopes were designed to generate functional therapeutic nanovaccines. To achieve higher stability, peptide/polymer hybrid nanoparticles were formulated by controlled self-assembly of the engineered peptides. The nanoparticles showed good biocompatibility to both human red blood- and dendritic cells. Incubation of the nanoparticles with immature dendritic cells triggered immune effects that ultimately activated CD8 + cells. The antigen-specific and IgG antibody responses of healthy C57BL/6 mice vaccinated with the nanoparticles were analyzed. The in vivo results indicate a specific response to the nanovaccines, mainly mediated through a cellular pathway. This research indicates that the immunogenicity of peptide epitope vaccines can be effectively enhanced by developing self-assembled peptide-polymer hybrid nanostructures.

Design of a New 99mTc-radiolabeled Cyclo-peptide as Promising Molecular Imaging Agent of CXCR4 Receptor: Molecular Docking, Synthesis, Radiolabeling, and Biological Evaluation.

INTRODUCTION: C-X-C Chemokine receptor type 4 (CXCR4) is often overexpressed or overactivated in different types and stages of cancer disease. Therefore, it is considered a promising target for imaging and early detection of primary tumors and metastasis. In the present research, a new cyclo-peptide radiolabelled with 99mTc, 99mTc-Cyclo [D-Phe-D-Tyr-Lys (HYNIC)- D-Arg-2-Nal-Gly-Lys(iPr)], was designed based on the parental LY251029 peptide, as a potential in vivo imaging agent of CXCR4-expressing tumors. METHODS: The radioligand was successfully prepared using the method of Fmoc solid-phase peptide synthesis and was evaluated in biological assessment. Molecular docking findings revealed high affinity (binding energy of -9.7 kcal/mol) and effective interaction of Cyclo [D-Phe- D-Tyr-Lys (HYNIC)-D-Arg-2-Nal-Gly-Lys(iPr)] in the binding pocket of CXCR4 receptor (PDB code: 3OE0) as well. RESULT: The synthesized peptide and its purity were assessed by both reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectroscopy. High stability (95%, n = 3) in human serum and favorable affinity (Kd = 28.70 ± 13.56 nM and Bmax = 1.896 ± 0.123 fmol/mg protein) in the B16-F10 cell line resulted. Biodistribution evaluation findings and planar image interpretation of mice both showed high affinity and selectivity of the radiotracer to the CXCR4 receptors. CONCLUSION: Therefore, the findings indicate this designed radioligand could be used as a potential SPECT imaging agent in highly proliferated CXCR4 receptor tumors.

The Footprint of Exosomes in the Radiation-Induced Bystander Effects.

Radiation therapy is widely used as the primary treatment option for several cancer types. However, radiation therapy is a nonspecific method and associated with significant challenges such as radioresistance and non-targeted effects. The radiation-induced non-targeted effects on nonirradiated cells nearby are known as bystander effects, while effects far from the ionising radiation-exposed cells are known as abscopal effects. These effects are presented as a consequence of intercellular communications. Therefore, a better understanding of the involved intercellular signals may bring promising new strategies for radiation risk assessment and potential targets for developing novel radiotherapy strategies. Recent studies indicate that radiation-derived extracellular vesicles, particularly exosomes, play a vital role in intercellular communications and may result in radioresistance and non-targeted effects. This review describes exosome biology, intercellular interactions, and response to different environmental stressors and diseases, and focuses on their role as functional mediators in inducing radiation-induced bystander effect (RIBE).

Targeting Caveolin-1 and Claudin-5 with AY9944, Improve Blood-Brain Barrier Permeability; Computational Simulation and Experimental Study.

The current study aimed to determine the protective effect of AY9944 related to Caveolin-1 and Claudin-5 role in lipid raft, which can rescue the blood-brain barrier from enhanced permeability. Therefore, in vivo analyses were performed following ischemia in normal, ischemic, and AY9944-treated animal groups. The results revealed that AY9944 reduced the infarct size, edema, and brain water content. The extravasation of Alb-Alexa 594 and biocytin-TMR was minimum in the AY9944-treated animals. The results showed a significant decrease in the expression level of Caveolin-1 over 8 h and 48 h and a remarkable increase in the level of Claudin-5 over 48 h following ischemia in AY9944-treated animals. Molecular docking simulation demonstrated that AY9944 exerts a possible protective role via attenuating the interaction of the Caveolin-1 and cholesterol in lipid raft. These findings point out that AY9944 plays a protective role in stroke by means of blood-brain barrier preservation. Proper neural function essentially needs a constant homeostatic brain environment which is provided by the blood-brain barrier. Rescuing blood-brain barrier from enhanced permeability via inducing the protective effect of AY9944 related to caveolin-1 and claudin-5 role in lipid raft was the aim of the current study.

Design of peptide-based inhibitor agent against amyloid-ß aggregation: Molecular docking, synthesis and in vitro evaluation.

Formation of the amyloid beta (Aß) peptide aggregations represents an indispensable role in appearing and progression of Alzheimer disease. ß-sheet breaker peptides can be designed and modified with different amino acids in order to improve biological properties and binding affinity to the amyloid beta peptide. In the present study, three peptide sequences were designed based on the hopeful results of LIAIMA peptide and molecular docking studies were carried out onto the monomer and fibril structure of amyloid beta peptide using AutoDock Vina software. According to the obtained interactions and binding energy from docking, the best-designed peptide (d-GABA-FPLIAIMA) was chosen and synthesized in great yield (%96) via the Fmoc solid-phase peptide synthesis. The synthesis and purity of the resulting peptide were estimated and evaluated by Mass spectroscopy and Reversed-phase high-performance liquid chromatography (RP-HPLC) methods, respectively. Stability studies in plasma and Thioflavin T (ThT) assay were performed in order to measure the binding affinity and in vitro aggregation inhibition of Aß peptide. The d-GABA-FPLIAIMA peptide showed good binding energy and affinity to Aß fibrils, high stability (more than 90%) in human serum, and a reduction of 20% in inhibition of the Aß aggregation growth. Finally, the favorable characteristics of our newly designed peptide make it a promising candidate ß-sheet breaker agent for further in vivo studies.

Preclinical study of a new 177Lu-labeled somatostatin receptor antagonist in HT-29 human colorectal cancer cells.

OBJECTIVES: Somatostatin receptor-positive neuroendocrine tumors have been targeted using various peptide analogs radiolabeled with therapeutic radionuclides for years. The better biomedical properties of radioantagonists as higher tumor uptake make these radioligands more attractive than agonists for somatostatin receptor-targeted radionuclide therapy. In this study, we tried to evaluate the efficiency of Luthetium-177 (177Lu) radiolabeled DOTA-Peptide 2 (177Lu-DOTA-Peptide 2) as a new radioantagonist in HT-29 human colorectal cancer in vitro and in vivo. METHODS: DOTA conjugated antagonistic peptide with the sequence of p-Cl-Phe-Cyclo(D-Cys-L-BzThi-D-Aph-Lys-Thr-Cys)-D-Tyr-NH2 (DOTA-Peptide 2) was labeled with 177Lu. In vitro assays (saturation binding assay and internalization test) and animal biodistribution were performed in human colon adenocarcinoma cells (HT-29) and HT-29 tumor-bearing nude mice. RESULTS: 177Lu-DOTA-Peptide 2 showed high stability in acetate buffer and human plasma (>97%). Antagonistic property of 177Lu-DOTA-Peptide 2 was confirmed by low internalization in HT-29 cells (<5%). The desired dissociation constant (Kd =11.14 nM) and effective tumor uptake (10.89 percentage of injected dose per gram of tumor) showed high binding affinity of 177Lu-DOTA-Peptide 2 to somatostatin receptors. CONCLUSION: 177Lu-DOTA-Peptide 2 demonstrated selective and high binding affinity to somatostatin receptors overexpressed on the surface of HT-29 cancer cells, which could make this radiopeptide suitable for somatostatin receptor-targeted radionuclide therapy.

Synthesis, biological evaluation and preclinical study of a novel 99mTc-peptide: A targeting probe of amyloid-ß plaques as a possible diagnostic agent for Alzheimer's disease.

With respect to the main role of amyloid-ß (Aß) plaques as one of the pathological hallmarks in the brain of Alzheimer's patients, the development of new imaging probes for targeted detection of Aß plaques has attracted considerable interests. In this study, a novel cyclopentadienyl tricarbonyl Technetium-99 m (99mTc) agent with peptide scaffold, 99mTc-Cp-GABA-D-(FPLIAIMA)-NH2, for binding to the Aß plaques was designed and successfully synthesized using the Fmoc solid-phase peptide synthesis method. This radiopeptide revealed a good affinity for Aß42 aggregations (Kd = 20 µM) in binding affinity study and this result was confirmed by binding to Aß plaques in brain sections of human Alzheimer's disease (AD) and rat models using in vitro autoradiography, fluorescent staining, and planar scintigraphy. Biodistribution studies of radiopeptide in AD and normal rats demonstrated a moderate initial brain uptake about 0.38 and 0.35% (ID/g) 2 min post-injection, respectively. Whereas, AD rats showed a notable retention time in the brain (0.23% ID/g at 30 min) in comparison with fast clearance in normal rat brains. Normal rats following treatment with cyclosporine A as a p-glycoprotein inhibitor showed a significant increase in the radiopeptide brain accumulation compared to non-treated ones. There was a good correlation between data gathered from single-photon emission computed tomography/computed tomography (SPECT/CT) imaging and biodistribution studies. Therefore, these findings showed that this novel radiopeptide could be a potential SPECT imaging agent for early detection of Aß plaques in the brain of patients with AD.

Design, preparation and biological evaluation of a 177Lu-labeled somatostatin receptor antagonist for targeted therapy of neuroendocrine tumors.

Somatostatin receptor-targeted radionuclide therapy has become an effective treatment in patients with neuroendocrine tumors. Recently, investigations on the development of antagonistic peptides are increasing with possible superior biological properties as opposed to the agonists. Herein, we have reported the development of a new somatostatin receptor peptide ligand labeled with 177Lu to achieve a therapeutic ligand for tumor treatment. The interactions of selected and drown ligands using Avogadro software were docked on somatostatin receptor by Dink algorithm. The best docked peptide-chelator conjugate (DOTA-p-Cl-Phe-Cyclo(d-Cys-l-BzThi-d-Aph-Lys-Thr-Cys)-d-Tyr-NH2) (DOTA-Peptide 2) was synthesized using the Fmoc solid-phase method. DOTA-Peptide 2 was radiolabeled with the 177Lu Trichloride (177LuCl3) solution at 95 °C for 30 min and radiochemical purity (RCP) of 177Lu-DOTA-Peptide 2 solution was monitored by radio-HPLC and radio-TLC procedures. The new radiolabeled peptide was evaluated for stability, receptor binding, internalization, biodistribution and single-photon emission computed tomography (SPECT) imaging using C6 glioma cells and C6 tumor-bearing rats. DOTA-Peptide 2 was obtained with 98% purity and efficiently labeled with 177Lu (RCP > 99%). 177Lu-DOTA-Peptide 2 showed a high value of stability in acetate buffer (91.4% at 312 h) and human plasma (>97% at 24 h). Radioconjugate exhibited low internalization (<5%) and high affinity for somatostatin receptors (Kd = 12.06 nM, Bmax = 0.20 pmol/106 cells) using saturation binding assay. Effective tumor uptake of 7.3% ID/g (percentage of injected dose per gram of tumor) at 4 h post-injection and fast clearance of radiopeptide from blood and other organs led to a high tumor-to-normal organ ratios. SPECT/CT imaging clearly showed the activity localization in tumor. The favorable antagonistic properties of 177Lu-DOTA-Peptide 2 on the somatostatin receptors can make it a suitable candidate for peptide receptor radionuclide therapy (PRRT). In the future study, the therapeutic application of this radiopeptide will be evaluated.

Recent advances in the design and applications of amyloid-ß peptide aggregation inhibitors for Alzheimer's disease therapy.

Alzheimer's disease (AD) is an irreversible neurological disorder that progresses gradually and can cause severe cognitive and behavioral impairments. This disease is currently considered a social and economic incurable issue due to its complicated and multifactorial characteristics. Despite decades of extensive research, we still lack definitive AD diagnostic and effective therapeutic tools. Consequently, one of the most challenging subjects in modern medicine is the need for the development of new strategies for the treatment of AD. A large body of evidence indicates that amyloid-ß (Aß) peptide fibrillation plays a key role in the onset and progression of AD. Recent studies have reported that amyloid hypothesis-based treatments can be developed as a new approach to overcome the limitations and challenges associated with conventional AD therapeutics. In this review, we will provide a comprehensive view of the challenges in AD therapy and pathophysiology. We also discuss currently known compounds that can inhibit amyloid-ß (Aß) aggregation and their potential role in advancing current AD treatments. We have specifically focused on Aß aggregation inhibitors including metal chelators, nanostructures, organic molecules, peptides (or peptide mimics), and antibodies. To date, these molecules have been the subject of numerous in vitro and in vivo assays as well as molecular dynamics simulations to explore their mechanism of action and the fundamental structural groups involved in Aß aggregation. Ultimately, the aim of these studies (and current review) is to achieve a rational design for effective therapeutic agents for AD treatment and diagnostics.