Cell Uptake Studies Research Articles

Improved antitumor efficacy and reduced toxicity of docetaxel using anacardic acid functionalized stealth liposomes.

Potential toxicity due to nonspecific distribution is one of the major challenges with currently available chemotherapeutics. In the present report we have developed Docetaxel (DTX) loaded Anacardic acid (AA) functionalized liposomes (DTX-AA-PEG-Liposomes) to have the advantage of selective distribution to cancer cells due to recognition and enhanced uptake by VEGF receptors. AA dual conjugate (AA-PEG-AA) was synthesized by using carbodiimide chemistry and further used to formulate the AA functionalized DTX loaded liposomes by using film hydration method. Extensive optimization of different process variables resulted in the formation of liposomes with particle size 126.4 ± 6.2 nm and PDI 0.239 ± 0.03. The freeze dried DTX-AA-PEG-Liposomes demonstrated sustained release for up to 24 h and excellent stability at accelerated storage stability conditions. Qualitative cell uptake studies demonstrated remarkably higher cellular uptake of Coumarin-6 (C-6) loaded liposomes, while quantitative determination revealed 2.64 and 2.88-fold higher uptake of DTX-AA-PEG-Liposomes in comparison with free DTX. Cell culture studies in MCF-7 to determine cellular uptake mechanism demonstrated clathrin and caveolae mediated internalization of liposomes, independent of Organic Anion Transporting Polypeptides (OATPs) transporters. Moreover, developed liposomes demonstrated relatively higher cell inhibition and apoptosis in MCF-7 cells as compared to free DTX. Furthermore, in vivo pharmacokinetics demonstrated 3.7 and 4.5-fold increase in AUC and t1/2 value of DTX-AA-PEG-Liposomes as compared to Taxotere®, respectively. Moreover, DTX-AA-PEG-Liposomes demonstrated significant reduction in tumor volume and toxicity in comparison with marketed formulation (Taxotere®), confirming enhanced efficacy and safety of the developed formulation.

Multimerization results in formation of re-bindable metabolites: A proof of concept study with FSC-based minigastrin imaging probes targeting CCK2R expression.

Positron emission tomography (PET) with radiolabelled peptide-based tracers has attracted great interest in oncology over the past decades. The success of imaging is closely related to sufficient uptake of the radiotracer in malignant tissue and for this sufficient biological half-life, particularly in the bloodstream, is mandatory. Fast enzymatic degradation during circulation leading to insufficient imaging abilities of peptide-based radioligands remains a major issue. The design of multimeric constructs, bearing multiple targeting moieties, has been widely applied to improve target interaction. This concept may also be applied to prolong the biological half-life of peptide-based radiopharmaceuticals as enzymatic degradation can result in formation of metabolites still capable to interact with the target binding site. In this study we aimed to identify such metabolites and therefore we utilized the siderophore-based bifunctional chelator fusarinine C (FSC) for the design of novel mono- and multimeric constructs, bearing minigastrin (MG) analogues as targeting moieties to address cholecystokinin-2 receptors (CCK2R) which are overexpressed in a variety of cancerous diseases and are well known for fast enzymatic degradation, particularly for truncated des-(Glu)5-MG members, such as MG11. FSC-based imaging probes were radiolabelled with gallium-68 and characterized in vitro (logD, protein binding, affinity and cell-uptake studies, stability and metabolite studies, as well as generation of corresponding metabolites by artificial enzymatic degradation) and in vivo (biodistribution in A431-CCK2R/A431-mock tumour xenografted BALB/c nude mice and stability in blood of living BALB/c mice and analysis of corresponding organ homogenates and urine to identify degradation products). In summary, multimerization was accompanied by partial improvement towards targeting abilities. Identified metabolites formed by artificial enzymatic cleavage of trimeric FSC-MG conjugates in vitro contained intact binding sequences for the receptor. Furthermore, the 68Ga-labelled trimers exhibiting increasing uptake of radioligand in tumour tissue over time and improved in vivo stability in blood samples of living animals of the trimers compared to corresponding mono- and dimers, strongly supporting our hypothesis.

99mTc labelled complexes with secnidazole xanthate: Synthesis and evaluation as potential radiotracers to target tumor hypoxia

In this study, the commercially available secnidazole was successfully converted to secnidazole xanthate (SNXT), in which the xanthate group can act as a bifunctional chelator to coordinate with 99mTc. 99mTc-nitrido complex of SNXT(99mTcN-SNXT) and 99mTc-oxo complex of SNXT(99mTcO-SNXT) were prepared with high radiochemical purity. Both of the complexes were found to be stable in vitro and to exhibit similar hydrophilicity. In addition, comparative in vitro cell uptake studies under anoxic and normoxic conditions demonstrated that both agents were preferentially taken up by hypoxic cells. Biodistribution studies in mice bearing S180 tumor showed 99mTcO-SNXT exhibited a higher tumor uptake and tumor-to-muscle ratio than 99mTcN-SNXT. Furthermore, in SPECT imaging study, 99mTcO-SNXT exhibited a clear accumulation in tumor at 2 h post-injection, suggesting its potential to be a novel hypoxia imaging agent.

Diastereomerically Pure 6R- and 6S-3'-Aza-2'-18F-Fluoro-5-Methyltetrahydrofolates Show Unprecedentedly High Uptake in Folate Receptor-Positive KB Tumors.

The aim of this study was to develop the radiosyntheses of diastereomerically pure 6R- and 6S-3'-aza-2'-18F-fluoro-5-methyltetrahydrofolate (MTHF) (6R-18F-1 and 6S-18F-1) using the integrated approach and to compare the in vitro and in vivo performance characteristics of both radioligands with the previously reported 3'-aza-2'-18F-fluorofolic acid tracer (18F-2), the oxidized form. Methods: 6R-18F-1, 6S-18F-1, and 18F-2 were radiolabeled with 18F using aromatic nucleophilic substitution reaction. In vitro cell uptake studies and binding affinity assays were performed using folate receptor (FR)-α-expressing KB cells. PET/CT imaging and biodistribution experiments were performed with KB tumor-bearing mice. Results: Reference compounds 6R-1 and 6S-1 were obtained after acidic hydrolysis of the corresponding protected intermediates 6R-3 and 6S-3 in high chemical yields (81%-87%) and chemical purities of more than 95%. 6R-18F-1, 6S-18F-1, and 18F-2 were obtained after a 2-step radiosynthetic procedure in a decay-corrected radiochemical yield of up to 5% and molar radioactivities ranging from 20 to 250 GBq/μmol. In vitro binding affinity studies using FR-α-positive KB cells gave half-maximal inhibitory concentrations of 27.1 ± 3.7 and 23.8 ± 4.0 nM for 6R-1 and 6S-1, respectively, which were higher than for the previously reported 3'-aza-2'-fluorofolic acid 2 (1.4 ± 0.5 nM). Comparably high cell uptake values in FR-α-expressing KB cells were found for all 3 radiofolates. In biodistribution studies, exceptionally high KB tumor uptake value of over 32% injected activity per gram of tissue for both 6R-18F-1 and 6S-18F-1 was observed at 180 min after injection, whereas for 18F-2 only 15% injected activity per gram was found in the KB tumors. Radioactivity uptake in the kidneys, liver, salivary glands, and spleen was substantially different for the 6R- and 6S-diastereoisomers and 18F-2 Excellent KB tumor visualization was found in PET/CT images with 6R-18F-1 and 6S-18F-1, both of which outperformed the corresponding oxidized 18F-2. Conclusion: We have successfully radiolabeled 6R- and 6S-3'-aza-2'-18F-fluoro-5-MTHF with 18F using the integrated approach. Our results suggest that both 6R- and 6S-3'-aza-2'-18F-fluoro-5-MTHF are promising reduced radiofolates for imaging FR-α-expressing cancers.

Abstract 3031: Positron emission tomography imaging of activated T cells by targeting OX40 reveals spatiotemporal immune dynamics and predicts response to in situ tumor vaccination

Abstract Clinical success of cancer immunotherapies has renewed interest in imaging the behavior of immune cells. Due to the spatiotemporally varying signatures of immune response, it has been difficult to monitor and predict patient outcomes using traditional clinical tests. ImmunoPET, defined herein as positron emission tomography utilizing radiolabeled antibodies, has the potential to enable noninvasive, sensitive and longitudinal interrogation of immune cell subset and state. Cell states including activation, anergy, and exhaustion may be more prognostic of disease outcome than the presence of tumor-infiltrating immune cells alone. In particular, T cell activation is thought to be critical to treatment success across many classes of cancer immunotherapy. In this work, we present the first radionuclide imaging of OX40, a novel and specific biomarker of activated antigen-specific T cells. Activation dependent and T cell restricted expression of OX40 was validated in vitro via flow cytometric analysis. Cell uptake studies with radiolabeled 64Cu-DOTA-AbOX40 demonstrated ~11 fold [p < .0001] higher uptake in dyna-bead activated T cells compared to resting. The tracer showed negligible nonspecific uptake in OX40 blocked or OX40-/- T cells and low background levels across a panel of 5 cancer cell lines tested. In vivo, ImmunoPET imaging revealed new insights into response following in situ tumor vaccination with CpG, an adjuvant immunotherapy currently in clinical trials. Balb-C mice bearing dual A20 lymphoma tumors were administered low dose CPG directly in the left tumor (n=7-10), while vehicle control mice received PBS (n=7-10). Early after vaccination, imaging revealed increased OX40 radiotracer uptake in the CPG treated tumor (TT) [~37%; p<0.05] confirmed by immunofluorescent staining. ViSNE, a visualization technique for high-dimensional cytometry data, classified OX40+ single cells in a cluster associated with a nonregulatory, activated CD4 T cell phenotype. CPG treatment led to local expansion of this unique OX40 cell population [~63%; p<0.05]. By late time points, a full systemic response emerged as evidenced by increased Luminex cytokine measurements in the plasma of CPG-treated mice. Unsupervised hierarchical clustering based on radiotracer or cytokine measurements correctly assigned mice into CPG-treated or vehicle cohorts, with few exceptions. More remarkably, a generalized linear regression model indicated early PET signal (mean %ID/g) in the local tumor environment to be highly predictive of response outcomes at late timepoints [r2=0.746]. OX40 ImmunoPET provides a readily translatable approach for monitoring activated T cells with high sensitivity and specificity. In this instance, integration of molecular imaging and computational immunology enabled systems-level interrogation of vaccine response. Citation Format: Aaron T. Mayer, Israt S. Alam, Idit Sagiv-Barfi, Kezheng Wang, Ophir Vermesh, Debra K. Czerwinski, Emily M. Johnson, Michelle L. James, Ronald Levy, Sanjiv S. Gambhir. Positron emission tomography imaging of activated T cells by targeting OX40 reveals spatiotemporal immune dynamics and predicts response to in situ tumor vaccination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3031.

Abstract 2015: Exosomes secreted by highly migratory premalignant lung epithelial cells promote epithelial mesenchymal transition and migration

Abstract Lung cancer is a highly metastatic disease. Contrary to the common belief that metastasis is a late event, recent findings have suggested that micrometastasis may also occur as an early phenomenon. An increasing body of evidence supports a role for epithelial-mesenchymal transition (EMT) in inducing field cancerization. Reports suggest that cancer-derived exosomes can act via intra-cellular communication to induce EMT and migration. However, the role of premalignant cell-derived exosomes in EMT induction, cellular migration and establishment of field cancerization in unknown. Herein, we describe the role of premalignant cell-derived exosomes on early metastatic behavior. Our laboratory has recently discovered a unique highly motile (HM) subpopulation of human bronchial epithelial cells (HBECs), using a novel “constricted migration” selection strategy, with enhanced metastatic potential in vivo. The HBECs used for selection were modified with changes reciprocating premalignancy (p53null, activated Kras-G12D). Comparative RNA-seq datasets illustrate increased expression of key EMT genes in HM-HBECs. This unique subpopulation of HM-HBECs offer a unique model to investigate premalignant cell migration. We used exosomes derived from HM, SM (slow motile) and unselected (UN) HBECs to evaluate EMT induction, migration, and invasion in vitro and in vivo. Exosomes were isolated by ultracentrifugation and characterized for size distribution using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy, and characterized by western blotting with antibodies specific for Alix, Flotillin, CD9 and CD63. Exosome labeling for cell uptake studies was performed using Dil dye entrapment and by transient transfection with CD63-GFP, releasing GFP+ exosomes. 6X108 HBEC cells produced approximately 6 x 108 exosomes with size ranging from 50 to 130 nm. The exosome signature of HM-, SM-, and UN-HBECs were characterized by mass spectroscopy and micro RNA sequencing. Exosomes from HM-HBECs induce UN-HBECs to obtain a HM phenotype with enhanced EMT, faster migration and increased invasive capacity. Western blot and qPCR data suggest a time-dependent activation of an EMT signature. Further in vivo experiments are in progress to evaluate the role of HM-HBEC derived exosomes on metastatic homing, tumor formation and spread of premalignant HBEC cells and tumor cells. Our data suggest that premalignant cell-derived exosomes possess distinct molecular signatures and could potentially mediate early migration, resulting in the creation of a premalignant field. As such, exosomes afford novel targets in premalignancy for early intervention and cancer interception. Citation Format: Manash K. Paul, Suman Dutta, Bharti Bisht, Salehi-Rad Ramin, Paul Pagano, Gal Bitan, John D. Minna, Steven M. Dubinett. Exosomes secreted by highly migratory premalignant lung epithelial cells promote epithelial mesenchymal transition and migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2015.

Abstract LB-271: Antitumor activity of new epidermal growth factor receptor inhibitors in non-small cell lung and breast cancer

Abstract Non-small cell lung cancer (NSCLC) and breast cancer (BrCa) are two of the most common forms of cancer. Since EGFR and HER2 are proven to be key targets in cancer therapy, we have designed and synthesized new molecules, belonging to a distinct chemical category of the existing drugs. Lapatinib was selected as an ideal prototype compound for the design of new analogues with potential action against EGFR and HER2 due to its ability to target and inhibit both receptors with great selectivity and efficiency. Structural analogues of lapatinib were synthesized by replacing the central ring of quinazoline by purine or bioisosteric rings aiming to investigate and potentially ameliorate the biological and pharmacological action. For the preparation of the target molecules, synthetic pathways have been designed and developed, which allow their high yielding preparation. The potency of the various analogues was evaluated in vitro using the following methodologies: MTT cytotoxic assay, Western Blot and ELISA assays to evaluate the ability of the analogues to inhibit EGFR phosphorylation in comparison with the activity of lapatinib and cell uptake studies based on mass spectrometry techniques. From a pool of 12 different analogues, two compounds, an imidazopyridine and an imidazopyrimidine derivative had the highest potencies, which were comparable to lapatinib in the NSCLC cell line A549 and in the BrCa cell line HCC-1954 (using MTT cytotoxic assays average IC50 values of 3 μM & 30 μM, respectively versus 11 μM for lapatinib). The same compounds were also tested in the NSCLC cell line NCI-H1975, which harbours the L858R + T790M mutations responsible for resistance (average IC50 values of 10 μM & 30 μM, respectively versus 9 μM for lapatinib). In addition, by using Western Blot and ELISA assays the two agents were the most potent compared to lapatinib, as they caused 50% inhibition of pEGFR at a 10 μM concentration. We have shown that the imidazopyridine derivative enters the A549 cells at a relatively slower rate in comparison to lapatinib. Presumably the physicochemical properties of the analogue are responsible for its higher accumulation (at 48 h incubation intracellular concentration of 17 ng/100.000 cells versus 10 ng/100.000 cells for lapatinib). Encouraged by the preliminary results two key molecules with similar potency to lapatinib will undergo further evaluation in vivo to determine their pharmacokinetic characteristics using advanced mass spectrometric approaches and their efficacy in clinically relevant mouse models. Citation Format: ARIS DOUKATAS, Efthymios-Spyridon Gavriil, Theodoros Karampelas, Panagiotis Marakos, Nicole Pouli, Constantin Tamvakopoulos. Antitumor activity of new epidermal growth factor receptor inhibitors in non-small cell lung and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-271.

Synthesis, structures and anticancer potentials of platinum(II) saccharinate complexes of tertiary phosphines with phenyl and cyclohexyl groups targeting mitochondria and DNA

A series of new Pt(II) saccharinate complexes containing PR3 ligands (PPh3, PPh2Cy, PPhCy2 and PCy3) with progressive phenyl (Ph) replacement by cyclohexyl (Cy) were synthesized and structurally characterized by IR, NMR, ESI-MS and X-ray diffraction. The anticancer activity of the complexes was tested against human breast (MCF-7), lung (A549), colon (HCT116), and prostate (DU145) cancer cell lines as well as against normal bronchial epithelial (BEAS-2B) cells. Trans-configured complexes 1, 3 and 5 emerged as potential anticancer drug candidates. The mechanism of action of the potent complexes was then investigated in detail. The three complexes interacted with DNA by groove binding and with HSA via hydrophobic IIA subdomain. Furthermore, the complexes cleaved plasmid DNA efficiently. Cellular uptake studies in MCF-7 cells showed that the biologically active complexes were mainly localized in cytoplasm. The cytotoxic activity was a function of the lipophilicity and cellular accumulation of the complexes. As determined by M30, Annexin V and Caspase 3/7 activity assays, the complexes induced apoptosis in MCF-7 and HCT116 cells. Mechanistic studies showed that the potent complexes cause excessive generation of reactive oxygen species (ROS) and display a dual action, concurrently targeting both mitochondria and genomic DNA.

Antibody with Infinite Affinity for In Vivo Tracking of Genetically Engineered Lymphocytes.

There remains an urgent need for the noninvasive tracking of transfused chimeric antigen receptor (CAR) T cells to determine their biodistribution, viability, expansion, and antitumor functionality. DOTA antibody reporter 1 (DAbR1) comprises a single-chain fragment of the antilanthanoid-DOTA antibody 2D12.5/G54C fused to the human CD4-transmembrane domain and binds irreversibly to lanthanoid (S)-2-(4-acrylamidobenzyl)-DOTA (AABD). The aim of this study was to investigate whether DAbR1 can be expressed on lymphocytes and used as a reporter gene as well as a suicide gene for therapy of immune-related adverse effects. Methods: DAbR1 was subcloned together with green fluorescent protein into an SFG-retroviral vector and used to transduce CD3/CD28-activated primary human T cells and second-generation 1928z (CAR) T cells. Cell surface expression of DAbR1 was confirmed by cell uptake studies with radiolabeled AABD. In addition, the feasibility of imaging of DAbR1-positive T cells in vivo after intravenous injection of 86Y/177Lu-AABD was studied and radiation doses determined. Results: A panel of DAbR1-expressing T cells and CAR T cells exhibited greater than 8-fold increased uptake of 86Y-AABD in vitro when compared with nontransduced cells. Imaging studies showed 86Y-AABD was retained by DAbR1-positive T cells while it continuously cleared from normal tissues, allowing for in vivo tracking of intravenously administered CAR T cells. Normal-organ dose estimates were favorable for repeated PET/CT studies. Selective T cell ablation in vivo with 177Lu-AABD seems feasible for clustered T-cell populations. Conclusion: We have demonstrated for the first time that T cells can be modified with DAbR1, enabling their in vivo tracking via PET and SPECT. The favorable biodistribution and high image contrast observed warrant further studies of this new reporter gene.

Co-delivery of 5-Fluorouracil and Curcumin Nanohybrid Formulations for Improved Chemotherapy Against Oral Squamous Cell Carcinoma.

The chemotherapeutics agent, 5-fluorouracil (5-FU), and curcumin (Cur), a natural antioxidant, has a wide pharmacological window to treat oral carcinoma; however, both drugs have limited bioavailability. This research study designs to develop a nanoemulsions (NEs) formulation by combining 5-FU and Cur to improve anticancer activity against oral cavity squamous cell carcinoma (OSCC) cells from the diversified origin for in vitro analysis, SCC090 (human tongue) and SCC152 (human hypo-pharynx). NEs formulated through homogenization, applying high-energy ultrasonication technique. The prepared 5-FUNE/Cur-NE/5-FU-Cur-NE were characterized and optimized by different in vitro assays to evaluate release system and treatment of OSCC cells to monitor cellular acceptability, such as in vitro anticancer activity by MTT assay, cell uptake studies and protein expression associated apoptotic study. 5-FUNE/Cur-NE/5-FU-Cur-NE successfully formulated and show mean-value of the particle size (150-200nm), surface charge (-25.70 to -37.91mV), and PDI (0.194). In vitro release of 5-FUNE/Cur-NE/5-FU-Cur-NEs was monitored over a course of 04days, where acidic pH shows higher release as compared to alkaline pH, along with acceptable stability data. Cytotoxicity study has shown higher-dose-dependent anticancer effect with a reduced IC50 value of NEs as compared to BLNE. Cellular uptake study of 5-FUNE/Cur-NE/5-FU-Cur-NEs upgraded many folds, comparatively BLNE and show potential cell arrest. Additionally, the cell protein (Blc2, Bax, P53, and P21) expression was revised and raised cell apoptosis. The combinational loaded, 5-FU and Cur in nanoformulation system have proven their potency to deliver improved anticancer activity, against oral cancer.

Glyceryl ester surfactants: Promising excipients to enhance the cell permeating properties of SEDDS

AimThe aim of the study is the evaluation of the impact of glyceryl ester surfactants on cell permeating properties of SEDDS (self-emulsifying drug delivery systems). MethodsSEDDS containing the glyceryl ester surfactants polyglyceryl-3-stearate (TGlysurf9), polyglyceryl-5-oleate (TGlysurf11.5) and glyceryl stearate citrate (TGlysurf12) were prepared and characterized regarding droplet size and zeta potential. Toxicity studies were performed on Caco-2 cells using resazuring assay. The formulations were loaded with fluorescein diacetate (FDA) and curcumin, and cell uptake studies on Caco-2 cells were performed. Cell uptake was visualized via real time live confocal microscopy. Cell permeability of the SEDDS was tested and trans-epithelial electrical resistance (TEER) measurements were performed. Furthermore, the anti-proliferative and anti-migration activity of curcumin loaded in the SEEDS was investigated. ResultsThe developed SEDDS (0.05% m/v) showed no cytotoxicity on Caco-2 cells after 3 h of incubation. Glyceryl esters-SEDDS showed a significant higher FDA and curcumin cell uptake than SEDDS without glyceryl ester surfactants (p < 0.05). TGlysurf9-SEDDS showed thereby the most pronounced permeation enhancing properties. TEER remained constant during the permeation study. Curcumin loaded in TGlysurf9-SEDDS exhibited 1.9-fold higher anti-proliferative effect than curcumin loaded in SEDDS without glyceryl ester surfactants. Furthermore, curcumin loaded in glyceryl ester-SEDDS inhibited Caco-2 cells migration to a higher extent than unloaded curcumin and curcumin loaded in SEDDS without the glyceryl ester surfactants. ConclusionsGlyceryl ester surfactants and in particular polyglyceryl-3-stearate might be a promising excipient for the formulation of SEDDS exhibiting enhanced cellular uptake and permeation enhancing properties.

Radiosynthesis and evaluation of novel 99mTc(CO)3-labelled thymidine dithiocarbamate derivatives for tumor imaging with SPECT

A series of novel thymidine dithiocarbamate derivatives (DTC-TdR) were successfully synthesized and then radiolabelled using [99mTc(CO)3]+ core with high yields. The chemical characterizations of 99mTc(CO)3-labelled dithiocarbamate derivatives have been carried out by preparing their corresponding rhenium complexes. The radiotracers were stable in vitro, and the partition coefficient results indicated that they were lipophilic. The cell uptake studies showed the uptakes of these 99mTc(CO)3-labelled thymidine derivatives were mediated by nucleoside transporters. Biodistribution of the complexes in mice bearing tumor showed that they had high tumor uptake and good tumor/muscle ratio. A clear SPECT imaging of the tumor location was obtained in mice bearing S180 tumor with one of radiotracers, suggesting they would be potential tumor imaging agents.

Impact of structural alterations on the radiopharmacological profile of 18F-labeled pyrimidines as cyclooxygenase-2 (COX-2) imaging agents

IntroductionNon-invasive imaging of COX-2 in cancer represents a powerful tool for assessing COX-2-mediated effects on chemoprevention and radiosensitization using potent and selective COX-2 inhibitors as an emerging class of anticancer drugs. Careful assessment of the pharmacokinetic profile of radiolabeled COX-2 inhibitors is of crucial importance for the development of suitable radiotracers for COX-2 imaging in vivo. The delicate balance between the selection of typical COX-2 pharmacophores and the resulting physicochemical characteristics of the COX-2 inhibitor represents a formidable challenge for the search of radiolabeled COX-2 imaging agents. Several pyrimidine-based COX-2 inhibitors demonstrated favorable in vitro and in vivo COX-2 imaging properties in various COX-2 expressing cancer cell lines. Here, we describe a comparative radiopharmacological study of three 18F-labeled COX-2 inhibitors based on a pyrimidine scaffold. The objective of this study was to investigate how subtle structural alterations influence the pharmacokinetic profile of lead compound [18F]1a ([18F]Pyricoxib) to afford 18F-labeled pyrimidine-based COX-2 inhibitors with improved COX-2 imaging properties in vivo. MethodsRadiosynthesis of radiotracers was accomplished through reaction with 4-[18F]fluorobenzyl amine on a methyl-sulfone labeling precursor ([18F]1a and [18F]2a) or late-stage radiofluorination using a iodyl-containing labeling precursor ([18F]3a). Radiopharmacological profile of 18F-labeled pyrimidine-based COX-2 inhibitors [18F]1a, [18F]2a and [18F]3a was studied in COX-2-expressing human HCA-7 colorectal cancer cell line, including cellular uptake studies in HCA-7 cells and dynamic PET imaging studies in HCA-7 xenografts. ResultsCellular uptake of radiotracers [18F]2a and [18F]3a in HCA-7 cells was 450% and 300% radioactivity/mg protein, respectively, after 90 min incubation, compared to 600% radioactivity/mg protein for radiotracer [18F]1a. Dynamic PET imaging studies revealed a tumor SUV of 0.53 ([18F]2a) and 0.54 ([18F]3a) after 60 min p.i. with a tumor-to-muscle ratio of ~1. Tumor SUV for [18F]1a (60 min p.i.) was 0.76 and a tumor-to-muscle ratio of ~1.5. Pyricoxib analogues [18F]2a and [18F]3a showed distinct pharmacokinetic profiles in comparison to lead compound [18F]1a with a significantly improved lung clearance pattern.Replacing the 4-[18F]fluorobenzyl amine motif in radiotracer [18F]1a with a 4-[18F]fluorobenzyl alcohol motif in radiotracer [18F]3a resulted in re-routing of the metabolic pathway as demonstrated by a more rapid liver clearance and higher initial kidney uptake and more rapid kidney clearance compared to radiotracers [18F]1a and [18F]2a. Moreover, radiotracer [18F]3a displayed favorable rapid brain uptake and retention. ConclusionThe radiopharmacological profile of three 18F-labeled COX-2 inhibitors based on a pyrimidine scaffold were evaluated in COX-2 expressing human colorectal cancer cell line HCA-7 and HCA-7 xenografts in mice. Despite the overall structural similarity and comparable COX-2 inhibitory potency of all three radiotracers, subtle structural alterations led to significantly different in vitro and in vivo metabolic profiles. Advances in knowledgeAmong all tested pyrimidine-based 18F-labeled COX-2 inhibitors, lead compound [18F]1a remains the most suitable radiotracer for assessing COX-2 expression in vivo. Radiotracer [18F]3a showed significantly improved first pass pulmonary passage in comparison to radiotracer [18F]1a and might represents a promising lead compound for the development of radiotracers for PET imaging of COX-2 in neuroinflammation.

The somatostatin receptor 2 antagonist 64Cu-NODAGA-JR11 outperforms 64Cu-DOTA-TATE in a mouse xenograft model.

Copper-64 is an attractive radionuclide for PET imaging and is frequently used in clinical applications. The aim of this study was to perform a side-by-side comparison of the in vitro and in vivo performance of 64Cu-NODAGA-JR11 (NODAGA = 1,4,7-triazacyclononane,1-glutaric acid,4,7-acetic acid, JR11 = p-Cl-Phe-cyclo(D-Cys-Aph(Hor)-D-Aph(cbm)-Lys-Thr-Cys)D-Tyr-NH2), a somatostatin receptor 2 antagonist, with the clinically used sst2 agonist 64Cu-DOTA-TATE ((TATE = D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Thr-Cys)Thr). In vitro studies demonstrated Kd values of 5.7±0.95 nM (Bmax = 4.1±0.18 nM) for the antagonist 64/natCu-NODAGA-JR11 and 20.1±4.4. nM (Bmax = 0.48±0.18 nM) for the agonist 64/natCu-DOTA-TATE. Cell uptake studies showed the expected differences between agonists and antagonists. Whereas 64Cu-DOTA-TATE (the agonist) showed very effective internalization in the cell culture assay (with 50% internalized at 4 hours post-peptide addition under the given experimental conditions), 64Cu-NODAGA-JR11 (the antagonist) showed little internalization but strong receptor-mediated uptake at the cell membrane. Biodistribution studies of 64Cu-NODAGA-JR11 showed rapid blood clearance and tumor uptake with increasing tumor-to-relevant organ ratios within the first 4 hours and in some cases, 24 hours, respectively. The tumor washout was slow or non-existent in the first 4 hours, whereas the kidney washout was very efficient, leading to high and increasing tumor-to-kidney ratios over time. Specificity of tumor uptake was proven by co-injection of high excess of non-radiolabeled peptide, which led to >80% tumor blocking. 64Cu-DOTA-TATE showed less favorable pharmacokinetics, with the exception of lower kidney uptake. Blood clearance was distinctly slower and persistent higher blood values were found at 24 hours. Uptake in the liver and lung was relatively high and also persistent. The tumor uptake was specific and similar to that of 64Cu-NODAGA-JR11 at 1 h, but release from the tumor was very fast, particularly between 4 and 24 hours. Tumor-to-normal organ ratios were distinctly lower after 1 hour. This is indicative of insufficient in vivo stability. PET studies of 64Cu-NODAGA-JR11 reflected the biodistribution data with nicely delineated tumor and low background. 64Cu-NODAGA-JR11 shows promising pharmacokinetic properties for further translation into the clinic.

Cyclic peptide-poly(HPMA) nanotubes as drug delivery vectors: In vitro assessment, pharmacokinetics and biodistribution

Size and shape have progressively appeared as some of the key factors influencing the properties of nanosized drug delivery systems. In particular, elongated materials are thought to interact differently with cells and therefore may allow alterations of in vivo fate without changes in chemical composition. A challenge, however, remains the creation of stable self-assembled materials with anisotropic shape for delivery applications that still feature the ability to disassemble, avoiding organ accumulation and facilitating clearance from the system. In this context, we report on cyclic peptide-polymer conjugates that self-assemble into supramolecular nanotubes, as confirmed by SANS and SLS. Their behaviour ex and in vivo was studied: the nanostructures are non-toxic up to a concentration of 0.5 g L−1 and cell uptake studies revealed that the pathway of entry was energy-dependent. Pharmacokinetic studies following intravenous injection of the peptide-polymer conjugates and a control polymer to rats showed that the larger size of the nanotubes formed by the conjugates reduced renal clearance and elongated systemic circulation. Importantly, the ability to slowly disassemble into small units allowed effective clearance of the conjugates and reduced organ accumulation, making these materials interesting candidates in the search for effective drug carriers.

Microfluidic Assisted Nanoprecipitation of PLGA Nanoparticles for Curcumin Delivery to Leukemia Jurkat Cells.

The ability to control particle size and size distribution of nanoparticles for drug delivery is essential because it impacts on the biodistribution and cellular uptake of nanoparticles. We present a novel microfluidic assisted nanoprecipitation strategy that enables synthesis of surfactant-free curcumin encapsulated poly(lactide- co-glycolide) nanoparticles (Cur-PLGA NP) with adjustable particle diameters (30-70 nm) and narrow particle size distribution (polydispersity index less than 0.2). Our Cur-PLGA NP exhibit excellent colloidal stability and inhibit degradation of curcumin. We further demonstrate the potential of our Cur-PLGA NP as a nanotoxic delivery system for curcumin. Cellular viability assay validates a dose-dependent cytotoxicity of Cur-PLGA NP in leukemia Jurkat cells. In contrast, Cur-PLGA NP does not alter the viability of fibroblast NIH3T3 cells, which suggests that the cytotoxicity of Cur-PLGA NP is specific to cell types. Furthermore, there is no detectable effect by PLGA NP to both leukemia Jurkat cells and fibroblast NIH3T3 cells, highlighting the nontoxic nature of our delivery system. Confocal cell uptake studies indicate that PLGA NP do not alter the cell uptake of curcumin. Our microfluidic assisted approach offers a controlled and effective nanobiomaterials synthesis of drug delivery system for curcumin, which can be extended to different capsule materials for a variety of biomedical applications.

Biopebbles: DNA‐Functionalized Core–Shell Silica Nanospheres for Cellular Uptake and Cell Guidance Studies

AbstractThe development of a versatile class of silica nanoparticles for cell studies is reported. The particles contain a fluorescent dye‐encoded core and a single‐stranded DNA oligonucleotide‐displaying shell. They are accessible in arbitrary size and color through robust protocols for Stöber‐based colloidal synthesis and sturdy chemical surface functionalization. Silica particles in the size range of 100 nm to 1.5 µm diameter containing fluorescein, Cy3 oder Cy5 dye‐encoded cores are synthesized and functionalized with DNA oligonucleotides. These silica biopebbles are conveniently traceable by microscopy and have a high affinity to live cells, which makes them ideal for cell uptake studies, as demonstrated for MCF7 and A431 cancer cells. The biopebbles can be utilized as building blocks for the self‐assembled formation of arbitrary surface patterns on glass substrates. With these architectures, the privileged internalization of the biopebbles can be exploited for improved adhesion and guidance of cells because the particles are no longer ingested by adhered cells due to their physical connection with the solid support. It is believed that the biopebble approach will be useful for a variety of applications, fundamental studies in cell biology and tissue engineering.

Trastuzumab- and Fab' fragment-modified curcumin PEG-PLGA nanoparticles: preparation and evaluation in vitro and in vivo.

IntroductionNanoparticles (NPs) modified with bio-ligands represent a promising strategy for active targeted drug delivery to tumour. However, many targeted ligands, such as trastuzumab (TMAB), have high molecular weight, limiting their application for targeting. In this study, we prepared Fab’ (antigen-binding fragments cut from TMAB)-modified NPs (Fab′-NPs) with curcumin (Cur) as a model drug for more effective targeting of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu), which is overexpressed on breast cancer cells.Material and methodsThe release kinetics was conducted by dialysis bags. The ability to kill HER2-overexpressing BT-474 cells of Fab′-Cur-NPs compared with TMAB-Cur-NPs was conducted by cytotoxicity experiments. Qualitative and quantitative cell uptake studies using coumarin-6 (fluorescent probe)-loaded NPs were performed by fluorescence microscopy and flow cytometry. Pharmacokinetics and biodistribution experiments in vivo were assessed by liquid chromatography–tandem mass spectrometry (LC-MS/MS).ResultsThe release kinetics showed that both Fab′-Cur-NPs and TMAB-Cur-NPs provided continuous, slow release of curcumin for 72 h, with no significant difference. In vitro cytotoxicity experiments showed that Fab′-Cur-NPs manifested prominent ability to kill HER2-overexpressing BT-474 cells compared with TMAB-Cur-NPs. Qualitative and quantitative cell uptake studies indicated that the accumulation of Fab′-NPs was greater than that of TMAB-NPs in BT-474 (HER2+) cells; However, there was no significant difference in MDA-MB-231 (HER2−) cells. Pharmacokinetics and biodistribution experiments in vivo demonstrated that the half-life (t1/2) and area under the blood concentration-time curve (AUC0-t) of Fab′-Cur-NPs increased 5.30-fold and 1.76-fold relative to those of TMAB-Cur-NPs, respectively. Furthermore, the tumor accumulation of Fab′-Cur-NPs was higher than that of TMAB-Cur-NPs.ConclusionFab′ fragment has greater capacity than the intact antibody to achieve tumor targeting through NP-based delivery.

Dextran-coated superparamagnetic iron oxide nanoparticles for magnetic resonance imaging: evaluation of size-dependent imaging properties, storage stability and safety.

BackgroundRising criticism of currently available contrast agents for magnetic resonance imaging, either due to their side effects or limited possibilities in terms of functional imaging, evoked the need for safer and more versatile agents. We previously demonstrated the suitability of novel dextran-coated superparamagnetic iron oxide nanoparticles (SPIONDex) for biomedical applications in terms of safety and biocompatibility.MethodsIn the present study, we investigated the size-dependent cross-linking process of these particles as well as the size dependency of their imaging properties. For the latter purpose, we adopted a simple and easy-to-perform experiment to estimate the relaxivity of the particles. Furthermore, we performed an extensive analysis of the particles’ storage stability under different temperature conditions, showing their superb stability and the lack of any signs of agglomeration or sedimentation during a 12 week period.ResultsIndependent of their size, SPIONDex displayed no irritation potential in a chick chorioallantoic membrane assay. Cell uptake studies of ultra-small (30 nm) SPIONDex confirmed their internalization by macrophages, but not by non-phagocytic cells. Additionally, complement activation-related pseudoallergy (CARPA) experiments in pigs treated with ultra-small SPIONDex indicated the absence of hypersensitivity reactions.ConclusionThese results emphasize the exceptional safety of SPIONDex, setting them apart from the existing SPION-based contrast agents and making them a very promising candidate for further clinical development.

Impact of Novel Antidepressants on Cardiac 123I-Metaiodobenzylguanidine Uptake: Experimental Studies on SK-N-SH Cells and Healthy Rabbits

123I-metaiodobenzylguanidine (123I-MIBG) has independent prognostic value for risk stratification among heart failure patients, but the use of concomitant medication should not affect its quantitative information. We evaluated whether the 4 classes of antidepressants currently most prescribed as first-line treatment for major depressive disorder (MDD) have the potential to alter 123I-MIBG imaging results. Methods: The inhibition effect of desipramine, escitalopram, venlafaxine, and bupropion on 131I-MIBG uptake was assessed by in vitro uptake assays using human neuroblastoma SK-N-SH cells. The half-maximal inhibitory concentration of tracer uptake was determined from dose-response curves. To evaluate the effect of intravenous pretreatment with desipramine (1.5 mg/kg) and escitalopram (2.5 or 15 mg/kg) on 123I-MIBG cardiac uptake, in vivo planar 123I-MIBG scanning of healthy New Zealand White rabbits was performed. Results: The half-maximal inhibitory concentrations of desipramine, escitalopram, venlafaxine, and bupropion on 131I-MIBG cellular uptake were 11.9 nM, 7.5 μM, 4.92 μM, and 12.9 μM, respectively. At the maximum serum concentration (as derived by previous clinical trials), the inhibition rates of 131I-MIBG uptake were 90.6% for desipramine, 25.5% for venlafaxine, 11.7% for bupropion, and 0.72% for escitalopram. A low inhibition rate for escitalopram in the cell uptake study triggered investigation of an in vivo rabbit model: with a dosage considerably higher than used in clinical practice, the noninhibitory effect of escitalopram was confirmed. Furthermore, pretreatment with desipramine markedly reduced cardiac 123I-MIBG uptake. Conclusion: In the present in vitro binding assay and in vivo rabbit study, the selective serotonin reuptake inhibitor escitalopram had no major impact on neuronal cardiac 123I-MIBG uptake within therapeutic dose ranges, whereas other types of first-line antidepressants for MDD treatment led to a significant decrease. These preliminary results warrant further confirmatory clinical trials regarding the reliability of cardiac 123I-MIBG imaging, in particular, if the patient's neuropsychiatric status would not tolerate withdrawal of a potentially norepinephrine-interfering antidepressant.