A radioactive CRISPR interference system using 89Zr-labeled LbCas12a.

Recently, CRISPR proteins have been recognized as promising candidates for drug development. However, there is still a lack of substances with the appropriate sensitivity and stability for targeted drug delivery systems. 89Zr is a radioactive isotope that emits positrons, allowing real-time in vivo tracking with proven safety. In this study, we confirmed that labeling with 89Zr did not compromise the functionality of CRISPR proteins during in vivo behavioral imaging. Furthermore, we demonstrated the therapeutic efficacy of the CRISPR interference system in a mouse model of liver fibrosis, highlighting the theragnostic potential of isotope-labeled CRISPR proteins. The findings of this research could contribute to various aspects of ongoing clinical studies exploring the in vivo applications of CRISPR proteins.

Theranostics through Utilizing Cherenkov Radiation of Radioisotope Zr-89 with a Nanocomposite Combination of TiO2 and MnO2.

Cherenkov radiation (CR) derived from the decay of diagnostic and therapeutic radionuclides is currently being studied by the scientific community to determine if these emissions can be harnessed for cancer detection and therapy. While Cherenkov luminescence imaging (CLI) has been studied in the preclinical and clinical settings, Cherenkov radiation-induced cancer therapy (CRICT) is a relatively new area of research that harnesses the emitted photons to kill cancer cells through free radical generation and DNA damage. Nanoparticles seem well suited for developing a theranostic platform that would allow researchers to visualize therapy delivery and also generate the reactive oxygen species necessary to kill cancer cells. Herein, we report the preparation of an 89Zr-TiO2-MnO2 nanocomposite that incorporates transferrin onto the nanoparticle surface to enhance cancer cell growth inhibition. The incorporation of the positron emission tomography (PET) radioisotope 89Zr (half-life: 3.3 days) allowed for the detection of the nanoparticle using PET and for the creation of Cherenkov emissions that interacted with the nanoparticle surface to generate free radicals for therapy delivery. After preparation, these systems were observed to be stable in various media and provided excellent tumor growth control after being intratumorally injected into mice bearing CT-26 tumors. These results demonstrate that a therapeutically efficient CRICT platform can be generated using commercially available and affordable materials.

Theragnostic 64Cu/67Cu Radioisotopes Production With RFT-30 Cyclotron.

64Cu and 67Cu are theragnostic pair radionuclides with promising application in the nuclear medicine. 64Cu is PET nuclide for the non-invasive diagnosis and 67Cu is beta emitter for therapy of various cancers. This study discusses optimization efforts in the production of these radioactive coppers carried out with 30 MeV cyclotron. Optimized conditions include target preparation, chemical separation, and quality control. The production routes of 64Cu and 67Cu were studied based on the nuclear reactions of 64Ni(p,n)64Cu and 70Zn(p,α)67Cu. The produced 64Cu and 67Cu have >99.9% of the radionuclidic purity. The yield at the end of bombardment (EOB) of 64Cu and 67Cu is 28.5 MBq/µAh and 67Cu is 0.58 MBq/µAh, respectively.

Chitosan-TiO2 composite: A potential 68Ge/68Ga generator column material.

A durable and ready to use 68Ge-68Ga generator column material is required for its routine use in radiopharmaceutical procedures. The present work comprises preliminary studies for development and evaluation of chitosan-TiO2 based microsphere (C-TOM) composite towards its competence as a column material. The batch uptake studies showed higher distribution coefficients for 68Ge vis-à-vis 68Ga in the complete concentration range of HCl examined (0.01-1 mol.L-1). Furthermore, C-TOM showed enduring physical and chemical stability in 0.01 mol.L-1 HCl with persistent 68Ga elution profiles (>95%) and negligible 68Ge breakthrough (2 × 10-4%) for the preliminary evaluation period of ∼2 months. Overall, the studies indicated that, 68Ga with high radionuclidic purity (≥99.99%) can be eluted routinely in a small volume (∼1.5 mL) of 0.01 mol.L-1 HCl proving its potentials as a novel solid phase extractant for 68Ge/68Ge generator system.

Red Blood Cell Membrane Bioengineered Zr-89 Labelled Hollow Mesoporous Silica Nanosphere for Overcoming Phagocytosis.

Biomimetic nanoparticles (NPs) have been actively studied for their biological compatibility due to its distinguished abilities viz. long-term circulation, low toxicity, ease for surface modification, and its ability to avoid phagocytosis of NPs by macrophages. Coating the NPs with a variety of cell membranes bearing the immune control proteins increases drug efficacy while complementing the intrinsic advantages of the NPs. In this study, efforts were made to introduce oxophilic radiometal 89Zr with hollow mesoporous silica nanospheres (HMSNs) having abundant silanol groups and were bioengineered with red blood cell membrane (Rm) having cluster of differentiation 47 (CD47) protein to evaluate its long-term in vivo behavior. We were successful in demonstrating the increased in vivo stability of synthesized Rm-camouflaged, 89Zr-labelled HMSNs with the markedly reduced 89Zr release. Rm camouflaged 89Zr-HMSNs effectively accumulated in the tumor by avoiding phagocytosis of macrophages. In addition, re-injecting the Rm isolated using the blood of the same animal helped to overcome the immune barrier. This novel strategy can be applied extensively to identify the long-term in vivo behavior of nano-drugs while enhancing their biocompatibility.

Development of 68Ga-SCN-DOTA-Capsaicin as an Imaging Agent Targeting Apoptosis and Cell Cycle Arrest in Breast Cancer.

68Ga-labeled capsaicin using a DOTA (1,4,7,10-tetraazocyclododecane-N,N',N″,N'″-tetraacetic acid) derivative [68Ga-SCN-Benzyl(Bn)-DOTA-capsaicin] was studied for the diagnosis of breast cancers, such as MCF-7 and SK-BR-3. The standard compound, 69Ga-SCN-Bn-DOTA-capsaicin, was also prepared and characterized by spectroscopic analysis. The binding affinity of 68Ga-SCN-Bn-DOTA-capsaicin was evaluated by using breast cancer cell lines (MCF-7, SK-BR-3) and colon cancer cell (CT-26); the biodistribution was carried out by using MCF-7-bearing nude mice, after which the positron emission tomography (PET) images were obtained at different time intervals (15-120 minutes). 68Ga-SCN-Bn-DOTA-capsaicin showed a cellular uptake of 0.93% Injected Dose (ID) after 30 minutes of incubation, whereas 68Ga-SCN-Bn-DOTA showed a lower uptake of 0.25% ID. The tumor-to-blood ID/g% ratios increased and were found to be 0.49, 0.22, and 0.77 for 15, 30, and 60 minutes, respectively. The small-animal PET study showed that the uptake of 68Ga-SCN-Bn-DOTA-capsaicin was higher in the tumor regions even at 30 minutes after injection. These results suggest that 68Ga-SCN-Bn-DOTA-capsaicin is a potential targeting agent for PET imaging of MCF-7.

Acid resistant zirconium phosphate for the long term application of 68Ge/68Ga generator system.

The 68Ge/68Ga generator system is an excellent source for producing ready-to-use Ga-68 in clinical Positron Emission Tomography (PET) applications. The column adsorbent is the key component for the 68Ge/68Ga generator system. Therefore, several studies have been conducted to identify column materials with a stable and superior elution yield in an acidic eluent (0.1 N HCl solution). In this study, four different zirconium phosphates were synthesized with a particle size of 200-800nm, pore-size of 55∼190Šand surface area of 0.72-268m2g-1. Synthesized and studied amorphous zirconium phosphate (ZrP-1) exhibited excellent acid resistant properties for the 0.1 N HCl eluent and a large surface area of 268m2g-1. Amorphous ZrP-1 showed a good Ga-68 elution yield of 74% in 0.1 N HCl eluent accompanying extraordinary low breakthrough of Ge-68 (0.007%).

Discovery of boronic acid-based fluorescent probes targeting amyloid-beta plaques in Alzheimer's disease.

A boronic acid-based fluorescent probe was developed for diagnosis of amyloid-ß (Aß) plaques from Alzheimer's disease (AD). Probe 4c, which included boronic acid as a functional group, exhibited a significant increase (64.37-fold, FAß/F0) in fluorescence intensity as a response to Aß aggregates, with a blue shift (105nm) in the maximum emission wavelength. We found that boronic acid as a functional group improved the binding affinity (KD value=0.79±0.05µM for 4c) for Aß aggregates and confirmed that 4c selectively stained Aß plaques in brain sections from APP/PS1 mice. Ex vivo fluorescence imaging using mice (normal and APP/PS1) also revealed that 4c was able to penetrate the blood-brain barrier (BBB) and to stain Aß plaques in the brain. From these results, we believe that 4c will be useful as a fluorescent probe in preclinical research related to AD. Furthermore, we believe that our results with boronic acid also provide valuable information for the development of a probe for Aß plaques.

Development of fluorescent probes that bind and stain amyloid plaques in Alzheimer's disease.

ß-amyloid (Aß) plaques in the brain are composed of Aß40 and Aß42 peptides, and are the defining pathological feature of Alzheimer's disease (AD). Fluorescent probes that can detect Aß plaques have gained increasing interest as potential tools for in vitro and in vivo monitoring of the progression of AD. In this study, chalcone-mimic fluorescent probe 5 was designed and prepared. Probe 5 exhibited an approximately 50-fold increase in emission intensity after mixing with Aß42 aggregates, a high affinity for Aß42 aggregates (K D = 1.59 µM), and reasonable lipophilicity (log P value = 2.55). Probe 5 also exhibited specific staining of Aß plaques in the transgenic mice (APP/PS1) brain sections. Ex vivo fluorescence imaging of the brain from normal and TG mice revealed that probe 5 was able to penetrate the BBB and stain the Aß plaques. These results suggest that chalcone-mimic probe 5 possessed the requirements of a fluorescent probe for Aß plaques and may be useful in AD research.

In-house development of an optimized synthetic module for routine [11C]acetate production.

[11C]Acetate, a radiotracer for PET imaging, is a promising radiopharmaceutical for overcoming the limitation of 2-deoxy-2-[18F]fluoro-D-glucose in a number of cancers. Here, the optimized automatic synthesis of [11C]acetate using an in-house-developed module under different conditions has been reported for routine production. [11C]CO2 was produced in a 16.4 MeV PETtrace cyclotron, and methyl magnesium chloride was used for synthesis. For product purification, ion-exchange solid-phase extraction cartridges were used, connected in series. High-performance liquid chromatography and gas chromatography were used to measure radiochemical and chemical purity. The Limulus amebocyte lysate test and the fluid thioglycollate medium test were performed for quality control of [11C]acetate. The total reaction time of [11C]acetate was within 15 min, and the overall decay-corrected radiochemical yield was 84.33±8.85%. Radiochemical purity was greater than 98% when evaluated on an analytical high-performance liquid chromatography system. No endotoxins or anaerobic bacteria were seen on quality control checks. Optimized production of [11C]acetate was achieved by the in-house module. Radiochemical and biological properties of the [11C]acetate produced were appropriate for clinical PET study.

Synthesis and inhibitory effects of some novel 1,3-diarylprop-2-en-1-one analogues in Foxp3 expression: a novel class of anti-cancer candidates.

A series of novel analogues of 1,3-diarylprop-2-en-1-one (3a-m) were synthesized and evaluated for their inhibitory activity of FOX P3 gene expression and apoptosis in CD4(+)T cells that had been isolated from the spleen of 8 to 10 weeks old mice. The structure-activity relationship (SAR) of the R1 and R2 modification was studied to identify a candidate with the maximum potency. Of these compounds, 3d showed the highest inhibitory activity of FOX P3 gene expression and apoptosis (66.5 % inhibition at 10 µM). To the best of the authors' knowledge this is the first report of 1,3- diarylprop-2-en-1-ones as regulators of FOX P3 gene expression.

Micropatterning of cells on electron-irradiated poly(dimethylsiloxane) surface.

In this study, a facile route to fabricate micropatterns of cells is presented on the basis of electron irradiation of poly(dimethylsiloxane) (PDMS). PDMS films were irradiated with electron beams through a pattern mask with micrometer-sized grids. After irradiation, the changes in the chemical composition, morphology, and wettability of the PDMS surface were investigated by using an X-ray photoelectron spectrometer, an atomic force microscope, and a contact anglometer. The results of the surface analysis revealed that the hydrophobic PDMS surface was changed into a hydrophilic one by the electron irradiation. Furthermore, on the basis of cell culturing on the selectively-irradiated PDMS, cells such as NIH3T3 and L929 were selectively adhered to and proliferated on the irradiated regions of the PDMS surface, resulting in the micropatterns of the cells on the PDMS surface.

Synthesis and characterization of a (68)Ga-labeled N-(2-diethylaminoethyl)benzamide derivative as potential PET probe for malignant melanoma.

Radiolabeled benzamides have been reported to be attractive agents for targeting malignant melanoma as they bind melanin and display high accumulation in melanoma cells. Herein, we report the synthesis and bioevaluation of a novel (68)Ga-labeled benzamide as a potential PET agent for malignant melanoma. The novel radiotracer was synthesized in good radiochemical yields (80% decay corrected yield) and high specific radioactivity (10 GBq/µmol). Cellular uptake of (68)Ga-SCN-NOTA-BZA was significantly higher in B16F10 cells (mouse melanoma) treated with L-tyrosine. Biodistribution and micro-PET studies of (68)Ga-SCN-NOTA-BZA in B16F10-bearing mice showed selective uptake into the tumor. The radiotracer was cleared via renal excretion without further metabolism. These results demonstrate that (68)Ga-SCN-NOTA-BZA is a potential PET probe for malignant melanoma.

Synthesis and evaluation of a novel 68Ga-labeled DOTA-benzamide derivative for malignant melanoma imaging.

Malignant melanoma displays a highly aggressive metastasis. Thus, early diagnosis of malignant melanoma is important for patient survival. We designed and synthesized a novel (68)Ga-labeled benzamide derivative that specifically binds to melanoma as demonstrated by its ability to bind to melanin. (68)Ga-SCN-DOTA-PCA was synthesized with a radiochemical yield of ~80% and a radiochemical purity of >97% by analytical HPLC. The in vitro binding of (68)Ga-SCN-DOTA-PCA to melanin and its cellular uptake demonstrated the selective uptake in melanin. In addition, the biodistribution and micro-PET imaging of (68)Ga-SCN-DOTA-PCA in B16F10 tumor models showed the specific accumulation in melanoma. These results suggest that (68)Ga-SCN-DOTA-PCA would be a promising agent for melanoma diagnosis.

Design, synthesis, and anti-influenza viral activities of 1,3-diarylprop-2-en-1-ones: a novel class of neuraminidase inhibitors.

A series of 1,3-diarylprop-2-en-1-one derivatives 3a-v have been synthesized and evaluated for their ability to inhibit neuraminidase (NA). Among the prepared compounds, the less lipophilic derivative 3k showed the most potent in vitro inhibitory activity against NA with an IC(50) value of 1.5 µM.

Fluorescent 2-styrylpyridazin-3(2H)-one derivatives as probes targeting amyloid-beta plaques in Alzheimer's disease.

Amyloid plaques, which are primarily composed of aggregated amyloid-beta (Aß) peptide, are the neuropathological hallmarks of Alzheimer's disease (AD). Fluorescent markers containing 2-styrylpyridazin-3(2H)-ones were developed to detect intracellular aggregated Aß peptides. Nine compounds exhibited a greater than 10-fold increase of in emission spectra before and after mixing with Aß aggregates compared with before mixing. Among these compounds, compound 9n exhibited the highest affinity for Aß aggregates (K(d)=1.84 µM) and selectively stained both aggregated intracellular Aß and Aß plaques in the transgenic AD model mice (APP/PS1). These preliminary results indicate that 2-styrylpyridazin-3(2H)-one derivatives are promising alternative fluorescence imaging agent for the study of AD.

Design and evaluation of the tandem target for a simultaneous production of [11C]CH4 and [18F]-fluoride.

The tandem target for a simultaneous production of [11C]CH4 and [(18)F]-fluoride has been designed and evaluated. A separate recovery system has been applied for a simultaneous collection of [11C]CH4 and [(18)F]-fluoride after a bombardment. The [11C]CH4 target was placed in front, and the [18F]-fluoride target was posted successively. An aluminum grid was employed between the two target cavities to improve the burst pressure of the titanium foil during an irradiation. It was demonstrated that a useful amount of [11C]CH4 and [18F]-fluoride can be produced simultaneously by this newly designed system.