Synthesis and In Vitro Comparison of DOTA,NODAGA and 15-5 Macrocycles as Chelators for the 64Cu-Labelling of Immunoconjugates

The development of ⁶⁴Cu-based immuno-PET radiotracers requires the use of copper-specific bifunctional chelators (BFCs) that contain functional groups allowing both convenient bioconjugation and stable copper complexes to limit in vivo bioreduction, transmetallation and/or transchelation. The excellent in vivo kinetic inertness of the pentaazamacrocyclic [⁶⁴Cu]Cu-15-5 complex prompted us to investigate its potential for the ⁶⁴Cu-labelling of monoclonal antibodies (mAbs), compared with the well-known NODAGA and DOTA chelators. To this end, three NODAGA, DOTA and 15-5-derived BFCs, containing a pendant azadibenzocyclooctyne moiety, were synthesised and a robust methodology was determined to form covalent bonds between them and azide-functionalised trastuzumab, an anti-HER2 mAb, using strain-promoted azide-alkyne cycloaddition. Unlike the DOTA derivative, the NODAGA- and 15-5-mAb conjugates were radiolabelled with ⁶⁴Cu, obtaining excellent radiochemical yields, under mild conditions. Although all the radioimmunoconjugates showed excellent stability in PBS or mouse serum, [⁶⁴Cu]Cu-15-5- and [⁶⁴Cu]Cu-NODAGA-trastuzumab presented higher resistance to transchelation when challenged by EDTA. Finally, the immunoreactive fraction of the radioimmunoconjugates (88–94%) was determined in HER-2 positive BT474 human breast cancer cells, confirming that the bioconjugation and radiolabelling processes implemented had no significant impact on antigen recognition.     

Rapid Perturbation of Free‐Energy Landscapes: From In Vitro to In Vivo

Biological systems are often studied under the most “physiological” conditions possible. However, purposeful perturbation of biological systems can provide much information about their dynamics, robustness, and function. Such perturbations are particularly easy to apply at the interface of molecular biophysics and cellular biology, at which complex and highly regulated networks emerge from the behavior of individual biomolecules. Due to the size of diffusion coefficients and the length scale of biomolecules, the fastest timescales at this interface extend to below a microsecond. Thus perturbations must be induced and detected rapidly. We focus on examples of proteins and RNAs interacting with themselves (folding) or one another (binding, signaling). Beginning with general principles that have been learned from simple models and perturbation experiments in vitro, we progress to more complex environments that mimic aspects of the living cell, and finally rapid perturbation experiments in living cells. On the experimental side we highlight in particular two classes of rapid perturbation methods (nanoseconds to seconds) that have been traditionally employed in biophysical chemistry, but that will become increasingly important in cell biology and in vivo: fast relaxation techniques and phase‐sensitive modulation techniques. These techniques are now increasingly married with imaging to produce a spatiotemporal map of biomolecular stability, dynamics and, in the near future, interaction networks inside cells. Many important chemical processes occur on biologically fast timescales, and yet have important ramifications for slower biological networks.     

Toward Libraries of Biotinylated Chondroitin Sulfate Analogues: From Synthesis to In Vivo Studies

Chondroitin sulfate‐E (CS‐E) oligosaccharidic analogues (di to hexa) were prepared from lactose. In these compounds, the 2‐acetamido group was replaced by a hydroxyl group. This modification speeded up the synthesis, and large oligosaccharides were constructed in a few steps from a lactose‐originated block. The protecting groups used were as follows; Fmoc for hydroxyl groups to be glycosylated, allyl group for anomeric position protection, and trichoroacetimidate leaving groups were used to prepare up to octasaccharides. We took advantage of the presence of allyl group to develop a click biotinylation, through its transformation into a 3‐azido‐2‐hydroxyl propyl group in two steps (epoxidation and sodium azide epoxide opening). The biotinylating agent was a water‐soluble propargylated and biotinylated triethylene glycol (PEG). By using surface plasmon resonance (SPR), it was shown that the di‐, tetra‐, and hexasaccharides display a binding affinity and selectivity toward HSF/GSF and CXCL12 similar to that of CS‐E. A parallel study confirmed their mimicry of natural compounds, based on the hexasaccharide interaction with Otx2, a homeodomain protein involved in brain maturation, thus validating our simplification approach to synthesize bioactive GAG.     

Copper Sulfide Nanoparticles with Phospholipid‐PEG Coating for In Vivo Near‐Infrared Photothermal Cancer Therapy

In this work, small sizes of hydrophobic copper sulfide nanoparticles (CuS NPs, ～3.8 nm in diameter) have been successfully prepared from the reaction of copper chloride with sodium diethyldithiocarbamate (SDEDTC) inside a heated oleylamine solution. These CuS NPs displayed strong absorption in the 700–1100 nm near‐infrared (NIR) region. By coating CuS NPs with DSPE‐PEG2000 on the surface, the as‐synthesized CuS@DSPE‐PEG NPs exhibited good water solubility, significant stability and biocompatibility, as well as excellent photothermal conversion effects upon exposure to an 808 nm laser. After intravenous administration to mice, the CuS@DSPE‐PEG NPs were found to passively target to the tumor site, and tumor tissues could be ablated efficiency under laser irradiation. In addition, CuS@DSPE‐PEG NPs do not show significant toxicity by histological and blood chemistry analysis, and can be effectively excreted via metabolism. Our results indicated that CuS@DSPE‐PEG NPs can act as an ideal photothermal agent for cancer photothermal therapy.     

Immobilization of the Gas Signaling Molecule H2S by Radioisotopes: Detection,Quantification, and In Vivo Imaging

Hydrogen sulfide (H₂S) has multifunctional roles as a gas signaling molecule in living systems. However, the efficient detection and imaging of H₂S in live animals is very challenging. Herein, we report the first radioisotope‐based immobilization technique for the detection, quantification, and in vivo imaging of endogenous H₂S. Macrocyclic ⁶⁴Cu complexes that instantly reacted with gaseous H₂S to form insoluble ⁶⁴CuS in a highly sensitive and selective manner were prepared. The H₂S concentration in biological samples was measured by a thin‐layer radiochromatography method. When ⁶⁴Cu–cyclen was injected into mice, an elevated H₂S concentration in the inflamed paw was clearly visualized and quantified by Cerenkov luminescence and positron emission tomography (PET) imaging. PET imaging was also able to pinpoint increased H₂S levels in a millimeter‐sized infarcted lesion of the rat heart.     

Immobilization of the Gas Signaling Molecule H2S by Radioisotopes: Detection,Quantification, and In Vivo Imaging

Hydrogen sulfide (H₂S) has multifunctional roles as a gas signaling molecule in living systems. However, the efficient detection and imaging of H₂S in live animals is very challenging. Herein, we report the first radioisotope‐based immobilization technique for the detection, quantification, and in vivo imaging of endogenous H₂S. Macrocyclic ⁶⁴Cu complexes that instantly reacted with gaseous H₂S to form insoluble ⁶⁴CuS in a highly sensitive and selective manner were prepared. The H₂S concentration in biological samples was measured by a thin‐layer radiochromatography method. When ⁶⁴Cu–cyclen was injected into mice, an elevated H₂S concentration in the inflamed paw was clearly visualized and quantified by Cerenkov luminescence and positron emission tomography (PET) imaging. PET imaging was also able to pinpoint increased H₂S levels in a millimeter‐sized infarcted lesion of the rat heart.     

Stimulation of In Vivo Antitumor Immunity with Hollow Mesoporous Silica Nanospheres

The use of appropriate adjuvants that support the generation of robust and long‐lasting antitumor immune responses is crucial for tumor immunotherapy owing to the immunosuppressive environment of the growing tumor. However, the most commonly used adjuvant, aluminum hydroxide, is ineffective for generating such immune responses and therefore not suitable for cancer immunotherapy. It is now shown that plain hollow mesoporous silica nanospheres markedly improve the antitumor immunity, the Th1 and Th2 immunity, and the CD4⁺ and CD8⁺ effector memory T cell population in bone marrow in vivo and may thus be used as immunoadjuvants to treat cancer in humans.     

Enzyme‐Mediated Site‐Specific Bioconjugation of Metal Complexes to Proteins: Sortase‐Mediated Coupling of Copper‐64 to a Single‐Chain Antibody

The enzyme‐mediated site‐specific bioconjugation of a radioactive metal complex to a single‐chain antibody using the transpeptidase sortase A is reported. Cage amine sarcophagine ligands that were designed to function as substrates for the sortase A mediated bioconjugation to antibodies were synthesized and enzymatically conjugated to a single‐chain variable fragment. The antibody fragment scFv_anti‐LIBS targets ligand‐induced binding sites (LIBS) on the glycoprotein receptor GPIIb/IIIa, which is present on activated platelets. The immunoconjugates were radiolabeled with the positron‐emitting isotope ⁶⁴Cu. The new radiolabeled conjugates were shown to bind selectively to activated platelets. The diagnostic potential of the most promising conjugate was demonstrated in an in vivo model of carotid artery thrombosis using positron emission tomography. This approach gives homogeneous products through site‐specific enzyme‐mediated conjugation and should be broadly applicable to other metal complexes and proteins.     

In Vivo Anticancer Activity of AZD3965: A Systematic Review

Proliferating cancer cells have high energy demands, which is mainly obtained through glycolysis. The transmembrane trafficking of lactate, a major metabolite produced by glycolytic cancer cells, relies on monocarboxylate transporters (MCTs). MCT1 optimally imports lactate, although it can work bidirectionally, and its activity has been linked to cancer aggressiveness and poor outcomes. AZD3965, a specific MCT1 inhibitor, was tested both in vitro and in vivo, with encouraging results; a phase I clinical trial has already been undertaken. Thus, analysis of the experimental evidence using AZD3965 in different cancer types could give valuable information for its clinical use. This systematic review aimed to assess the in vivo anticancer activity of AZD3965 either alone (monotherapy) or with other interventions (combination therapy). Study search was performed in nine different databases using the keywords “AZD3965 in vivo” as search terms. The results show that AZD3965 successfully decreased tumor growth and promoted intracellular lactate accumulation, which confirmed its effectiveness, especially in combined therapy. These results support the setup of clinical trials, but other important findings, namely AZD3965 enhanced activity when given in combination with other therapies, or MCT4-induced treatment resistance, should be further considered in the clinical trial design to improve therapy response.     

Antilymphoma Effect of Incomptine A: In Vivo,In Silico,and Toxicological Studies

Incomptine A (IA) is a sesquiterpene lactone isolated from Decachaeta incompta that induces apoptosis, reactive oxygen species production, and a differential protein expression on the U-937 (diffuse histiocytic lymphoma) cell line. In this work, the antitumor potential of IA was investigated on Balb/c mice inoculated with U-937 cells and through the brine shrimp lethality (BSL) test. Furthermore, IA was subjected to molecular docking study using as targets proteins associated with processes of cancer as apoptosis, oxidative stress, and glycolytic metabolism. In addition to determining the potential toxicity of IA in human, its acute toxicity was performed in mice. Results reveals that IA showed high antilymphoma activity and BSL with an EC₅₀ of 2.4 mg/kg and LC₅₀ 16.7 µg/mL, respectively. The molecular docking study revealed that IA has strong interaction on all targets used. In the acute oral toxicity, IA had a LD₅₀ of 149 mg/kg. The results showed that the activities of IA including antilymphoma activity, BSL, acute toxicity, and in silico interactions were close to the methotrexate, an anticancer drug used as positive control. These findings suggest that IA may serve as a candidate for the development of a new drug to combat lymphoma.     

Reactions of Osmabenzene with Silver/Copper Acetylides: From Metallabenzene to Benzene

The transformations of metallabenzene to substituted benzenes have been achieved by reactions of osmabenzenes with silver/copper acetylides. In this investigation, novel tetraphenylphosphonium salts containing two phosphonium substituents on the same benzene ring are generated.     

Formulation and Optimization of Alogliptin-Loaded Polymeric Nanoparticles: In Vitro to In Vivo Assessment

The nano-drug delivery system has gained greater acceptability for poorly soluble drugs. Alogliptin (ALG) is a FDA-approved oral anti-hyperglycemic drug that inhibits dipeptidyl peptidase-4. The present study is designed to prepare polymeric ALG nanoparticles (NPs) for the management of diabetes. ALG-NPs were prepared using the nanoprecipitation method and further optimized by Box–Behnken experimental design (BBD). The formulation was optimized by varying the independent variables Eudragit RSPO (A), Tween 20 (B), and sonication time (C), and the effects on the hydrodynamic diameter (Y1) and entrapment efficiency (Y2) were evaluated. The optimized ALG-NPs were further evaluated for in vitro release, intestinal permeation, and pharmacokinetic and anti-diabetic activity. The prepared ALG-NPs show a hydrodynamic diameter of between 272.34 nm and 482.87 nm, and an entrapment efficiency of between 64.43 and 95.21%. The in vitro release data of ALG-NPs reveals a prolonged release pattern (84.52 ± 4.1%) in 24 h. The permeation study results show a 2.35-fold higher permeation flux than pure ALG. ALG-NPs exhibit a significantly (p < 0.05) higher pharmacokinetic profile than pure ALG. They also significantly (p < 0.05) reduce the blood sugar levels as compared to pure ALG. The findings of the study support the application of ALG-entrapped Eudragit RSPO nanoparticles as an alternative carrier for the improvement of therapeutic activity.     

Preparation,Characterization, and Pharmacological Investigation of Withaferin-A Loaded Nanosponges for Cancer Therapy; In Vitro,In Vivo and Molecular Docking Studies

The rapidly growing global burden of cancer poses a major challenge to public health and demands a robust approach to access promising anticancer therapeutics. In parallel, nanotechnology approaches with various pharmacological properties offer efficacious clinical outcomes. The use of new artificial variants of nanosponges (NS) as a transporter of chemotherapeutic drugs to target cells has emerged as a very promising tool. Therefore, in this research, ethylcellulose (EC) NS were prepared using the ultrasonication assisted-emulsion solvent evaporation technique. Withaferin-A (WFA), an active ingredient in Withania somnifera, has been implanted into the nanospongic framework with enhanced anticancer properties. Inside the polymeric structure, WFA was efficiently entrapped (85 ± 11%). The drug (WFA) was found to be stable within polymeric nanosponges, as demonstrated by Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) studies. The WFA-NS had a diameter of 117 ± 4 nm and zeta potential of −39.02 ± 5.71 mV with a polydispersity index (PDI) of 0.419 ± 0.073. In addition, scanning electron microscopy (SEM) revealed the porous surface texture of WFA-NS. In vitro anticancer activity (SRB assay) results showed that WFA–NS exhibited almost twice the anticancer efficacy against MCF-7 cells (IC₅₀ = 1.57 ± 0.091 µM), as quantified by flow cytometry and comet tests. Moreover, fluorescence microscopy with DAPI staining and analysis of DNA fragmentation revealed apoptosis as a mechanism of cancer cell death. The anticancer activity of WFA-NS was further determined in vivo and results were compared to cisplatin. The anticancer activity of WFA-NS was further investigated in vivo, and the data were consistent to those obtained with cisplatin. At Day 10, WFA-NS (10 mg/kg) significantly reduced tumour volume to 72 ± 6%, which was comparable to cisplatin (10 mg/kg), which reduced tumour volume to 78 ± 8%. Finally, the outcomes of molecular modeling (in silico) also suggested that WFA established a stable connection with nanosponges, generating persistent hydrophobic contacts (polar and nonpolar) and helping with the attractive delayed-release features of the formulation. Collectively, all the findings support the use of WFA in nanosponges as a prototype for cancer treatment, and opened up new avenues for increasing the efficacy of natural product-derived medications.     

Bioactive Tryptophan-Based Copper Complex with Auxiliary β-Carboline Spectacle Potential on Human Breast Cancer Cells: In Vitro and In Vivo Studies

Biocompatible tryptophan-derived copper (1) and zinc (2) complexes with norharmane (β-carboline) were designed, synthesized, characterized, and evaluated for the potential anticancer activity in vitro and in vivo. The in vitro cytotoxicity of both complexes 1 and 2 were assessed against two cancerous cells: (human breast cancer) MCF7 and (liver hepatocellular cancer) HepG2 cells with a non-tumorigenic: (human embryonic kidney) HEK293 cells. The results exhibited a potentially decent selectivity of 1 against MCF7 cells with an IC₅₀ value of 7.8 ± 0.4 μM compared to 2 (less active, IC₅₀ ~ 20 μM). Furthermore, we analyzed the level of glutathione, lipid peroxidation, and visualized ROS generation to get an insight into the mechanistic pathway and witnessed oxidative stress. These in vitro results were ascertained by in vivo experiments, which also supported the free radical-mediated oxidative stress. The comet assay confirmed the oxidative stress that leads to DNA damage. The histopathology of the liver also ascertained the low toxicity of 1.     

From Double‐Four‐Ring Germanosilicates to New Zeolites: In Silico Investigation

To date, the majority of zeolites have been prepared by the solvothermal route using organic structure directing agents. Two new zeolites with structural codes PCR and OKO were recently prepared from UTL germanosilicate by removal of the double‐four ring (D4R) connecting the dense two‐dimensional layers [Nature Chem. 2013 , 5, 628]. The corresponding experimental protocol, Assembly–Disassembly–Organization–Reassembly (ADOR), is explored in this contribution with an in silico investigation. The structure and properties of hypothetical zeolites that could be obtained from zeolites with IWW, IWV, IWR, ITR, and ITH topologies using the ADOR protocol are reported based on a computational investigation. A total of 20 new structures are presented together with their characteristics.     

Near‐Infrared‐Emitting BODIPY‐trisDOTA111In as a Monomolecular Multifunctional Imaging Probe: From Synthesis to In Vivo Investigations

A new generation of monomolecular imaging probes (MOMIP) based on a distyryl‐BODIPY (BODIPY=boron‐dipyrromethene) coupled with three DOTA macrocycles has been prepared (DOTA=1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid). The MOMIP presents good fluorescence properties and is very stable in serum. The bimodal probe was conjugated to trastuzumab, and an optical in vivo study showed high accumulation of the imaging agent at the tumor site. ¹¹¹In radiometallation of the bioconjugate was performed in high radiochemical yield, highlighting the potential of this new BODIPY‐chelators derivative as a bimodal imaging probe.     

HPLC with Fluorescence and Photodiode Array Detection for Quantifying Capmatinib in Biological Samples: Application to In Vivo and In Vitro Studies

Capmatinib, a recently approved tyrosine kinase inhibitor, is used for the treatment of non-small cell lung cancer. We describe two new HPLC methods for capmatinib quantification in vivo and in vitro. HPLC with a fluorescence detection method was used to quantify capmatinib in plasma for the first time. The method was successfully applied in a pharmacokinetic study following a 10 mg/kg oral dose of capmatinib given to rats. The chromatographic separation was performed using a Eurospher II 100-3 C18H (50 × 4 mm, 3 µm) column and a mobile phase containing 10 mM of ammonium acetate buffer (pH 5.5): acetonitrile (70:30, v/v), at a flow rate of 2.0 mL min⁻¹. The study also describes the use of HPLC-PDA for the first time for the determination of capmatinib in human liver microsomes and describes its application to study its metabolic stability in vitro. Our results were in agreement with those reported using LC-MS/MS, demonstrating the reliability of the method. The study utilized a Gemini-NX C18 column and a mobile phase containing methanol: 20 mM ammonium formate buffer pH 3.5 (53:47, v/v), delivered at a flow rate of 1.1 mL min⁻¹. These methods are suitable for supporting pharmacokinetic studies, particularly in bioanalytical labs lacking LC-MS/MS capabilities.     

Lipid-Based Nanoparticle Formulation of Diallyl Trisulfide Chemosensitizes the Growth Inhibitory Activity of Doxorubicin in Colorectal Cancer Model: A Novel In Vitro,In Vivo and In Silico Analysis

Garlic’s main bioactive organosulfur component, diallyl trisulfide (DATS), has been widely investigated in cancer models. However, DATS is not suitable for clinical use due to its low solubility. The current study seeks to improve DATS bioavailability and assess its chemopreventive and chemosensitizing properties in an AOM-induced colorectal cancer model. The polyethylene glycol coated Distearoylphosphatidylcholine/Cholesterol (DSPC/Chol) comprising DATS-loaded DATSL and doxorubicin (DOXO)-encapsulated DOXL liposomes was prepared and characterized. The changes in the sensitivity of DATS and DOXO by DATSL and DOXL were evaluated in RKO and HT-29 colon cancer cells. The synergistic effect of DATSL and DOXL was studied by cell proliferation assay in the combinations of IC₁₀, IC₂₅, and IC₃₅ of DATSL with the IC₁₀ of DOXL. AOM, DATSL, and DOXL were administered to different groups of mice for a period of 21 weeks. The data exhibited ~93% and ~46% entrapment efficiency of DATSL and DOXL, respectively. The size of sham liposomes was 110.5 nm, whereas DATSL and DOXL were 135.5 nm and 169 nm, respectively. DATSL and DOXL exhibited significant sensitivity in the cell proliferation experiment, lowering their IC₅₀ doses by more than 8- and 14-fold, respectively. However, the DATSL IC₁₀, IC₂₅, and IC₃₅ showed escalating chemosensitivity, and treated the cells in combination with DOXL IC₁₀. Analysis of histopathological, cancer marker enzymes, and antioxidant enzymes revealed that the high dose of DATSL pretreatment and DOXL chemotherapy is highly effective in inhibiting AOM-induced colon cancer promotion. The combination of DATSL and DOXL indicated promise as a colorectal cancer treatment in this study. Intermolecular interactions of DATS and DOXO against numerous cancer targets by molecular docking indicated MMP-9 as the most favourable target for DATS exhibiting binding energy of −4.6 kcal/mol. So far, this is the first research to demonstrate the chemopreventive as well as chemosensitizing potential of DATSL in an animal model of colorectal cancer.     

Insulin-Loaded SLN Prepared with the Emulsion Dilution Technique: In Vivo Tracking of Nanoparticles after Oral Administration to Rats

Insulin-loaded solid lipid nanoparticles (SLN) were prepared according to a solvent dilution method from O/W emulsions using isovaleric acid as organic phase. Insulin was derivatized with fluorescein isothyocianate (FITC) obtaining a fluorescent marker to be used in in vivo experiments. FITC-insulin and native insulin–loaded SLN were quite similar with regard to their mean sizes and encapsulation efficiency. SLN intestinal uptake was then investigated administering FITC-insulin loaded SLN on healthy male Wistar rats. Significant drug accumulation within intestinal lymphatic system was recovered, but the immune system seems to play an important role in SLN degradation: further studies are necessary to improve the results on blood glucose level.