Metal Organic Polyhedra: A Click-and-Clack Approach Toward Targeted Delivery.

Mixed self-assembly of ligands 1 and 2, PXDA (3), and Pd(NO3)2 afforded metal organic polyhedra (MOP 1 - MOP 3) which bear 24 covalently attached CB[7] and cyclooctyne moieties. Post assembly modification (PAM) of MOP 3 by covalent strain promoted alkyne azide click reaction provided MOP 4 R bearing covalently attached functionality (PEG, sulfonate, biotin, c-RGD, fluorescein and cyanine). Orthogonal CB[7] guest mediated non-covalent PAM of MOP 4 R with Ad-FITC afforded MOP 5 RGD Ad-FITC and MOP 5 biotin 0020Ad-FITC. Flow cytometry analysis of the uptake of MOP 5 RGD Ad-FITC toward U87 cells demonstrated improved uptake relative to control MOP lacking c-RGD ligands. These results suggest a broad applicability of orthogonally functionalizable (covalent and non-covalent) MOPs in targeted drug delivery and imaging applications.

Metal-Organic Polyhedron Capped with Cucurbit[8]uril Delivers Doxorubicin to Cancer Cells.

Self-assembly of ligand 1 and Pd(NO3)2 delivers Fujita-type metal-organic polyhedron (MOP) 3 which bears 24 covalently attached methyl viologen units on its external surface, as evidenced by 1H NMR, diffusion-ordered spectroscopy NMR, electrospray mass spectrometry, transmission electron microscopy, and atomic force microscopy measurements. MOP 3 undergoes noncovalent complexation with cucurbit[n]urils to yield MOPs 4-6 with diameter ≈5-6 nm. MOP 5 can be fully loaded with doxorubicin (DOX) prodrug 2 via hetero-ternary complex formation to yield 7. The MOPs exhibit excellent stability toward neutral to slightly acidic pH in 10 mM sodium phosphate buffer, mitigating the concern of disassembly during circulation. The results of MTS assays show that MOP 7 is 10-fold more cytotoxic toward HeLa cells than equimolar quantities of DOX prodrug 2. The enhanced cytotoxicity can be traced to a combination of enhanced cellular uptake of 7 and DOX release as demonstrated by flow cytometry and confocal fluorescence microscopy. The confluence of properties imparted by the polycationic MOP architecture and plug-and-play CB[n] complexation provides a potent new platform for drug delivery application.

Glycoluril-Derived Molecular Clips are Potent and Selective Receptors for Cationic Dyes in Water.

Molecular clip 1 remains monomeric in water and engages in host-guest recognition processes with suitable guests. We report the Ka values for 32 1⋅guest complexes measured by 1 H NMR, UV/Vis, and fluorescence titrations. The cavity of 1 is shaped by aromatic surfaces of negative electrostatic potential and therefore displays high affinity and selectivity for planar and cationic aromatic guests that distinguishes it from CB[n] receptors that prefer aliphatic over aromatic guests. Electrostatic effects play a dominant role in the recognition process whereby ion-dipole interactions may occur between ammonium ions and the C=O groups of 1, between the SO3- groups of 1 and pendant cationic groups on the guest, and within the cavity of 1 by cation-π interactions. Host 1 displays a high affinity toward dicationic guests with large planar aromatic surfaces (e.g. naphthalene diimide NDI+ and perylene diimide PDI+) and cationic dyes derived from acridine (e.g. methylene blue and azure A). The critical importance of cation-π interactions was ascertained by a comparison of analogous neutral and cationic guests (e.g. methylene violet vs. methylene blue; quinoline vs. N-methylquinolinium; acridine vs. N-methylacridinium; neutral red vs. neutral red H+ ) the affinities of which differ by up to 380-fold. We demonstrate that the high affinity of 1 toward methylene blue (Ka =3.92×107 m-1 ; Kd =25 nm) allows for the selective sequestration and destaining of U87 cells stained with methylene blue.

Acyclic Cucurbit[n]uril-Type Molecular Containers: Influence of Linker Length on Their Function as Solubilizing Agents.

Two acyclic cucurbit[n]uril (CB[n])-type molecular containers that differ in the length of the (CH2 )n linker (M2C2: n=2, M2C4: n=4) between their aromatic sidewalls and sulfonate solubilizing groups were prepared and studied. The inherent solubilities of M2C2 (68 mm) and M2C4 (196 mm) are higher than the analogue with a (CH2 )3 linker (M2, 14 mm) studied previously. (1) H NMR dilution experiments show that M2C2 and M2C4 do not self-associate in water, which enables their use as solubilizing excipients. We used phase solubility diagrams (PSDs) to compare the solubilizing capacities of M2, M2C2, M2C4, hydroxypropyl-ß-cyclodextrin (HP-ß-CD), and sulfobutylether-ß-cyclodextrin (SBE-ß-CD) toward 15 insoluble drugs. We found that M2C2 and M2C4-as gauged by the slope of their PSDs-are less potent solubilizing agents than M2. However, the higher inherent solubility of M2C2 allows higher concentrations of drug to be formulated using M2C2 than with M2 in several cases. The solubilizing ability of M2C2 and SBE-ß-CD were similar in many cases, with Krel values averaging 23 and 12, respectively, relative to HP-ß-CD. In vitro cytotoxicity and in vivo maximum tolerated dose studies document the biocompatibility of M2C2.

Acyclic Cucurbit[n]uril-Type Molecular Container Enables Systemic Delivery of Effective Doses of Albendazole for Treatment of SK-OV-3 Xenograft Tumors.

Approximately, 40-70% of active pharmaceutical ingredients (API) are severely limited by their extremely poor aqueous solubility, and consequently, there is a high demand for excipients that can be used to formulate clinically relevant doses of these drug candidates. Here, proof-of-concept studies demonstrate the potential of our recently discovered acyclic cucurbit[n]uril-type molecular container Motor1 (M1) as a solubilizing agent for insoluble drugs. M1 did not induce significant rates of mutations in various Salmonella typhimurium test strains during the Ames test, suggesting low genotoxicity. M1 also has low risk of causing cardiac toxicity in humans since it did not inhibit the human Ether-à-go-go-Related Gene channel as tested on transfected CHO cell lines via patch clamp analysis. Albendazole (ABZ) is a widely used antihelminthic agent but that has also shown promising efficacy against cancerous cells in vitro. However, due to its low aqueous solubility (2.7 µM) and poor pharmacokinetics, ABZ is clinically limited as an anticancer agent. Here we investigated the potential of M1 as a solubilizing excipient for ABZ formulation. A pharmacokinetic study indicated that ABZ escapes the peritoneal cavity resulting in 78% absolute bioavailability, while its active intermediate metabolite, albendazole sulfoxide, achieved 43% absolute bioavailability. The daily dosing of 681 mg/kg M1 complexed with 3.2 mg/kg of ABZ for 14 days did not result in significant weight loss or pathology in Swiss Webster mice. In vivo efficacy studies using this M1·ABZ inclusion complex showed significant decreases in tumor growth rates and increases in survival of mice bearing SK-OV-3 xenograft tumors. In conclusion, we provide substantial new evidence demonstrating that M1 is a safe and efficient excipient that enables in vivo parenteral delivery of poorly water-soluble APIs.

Orotracheal administration of contrast agents: a new protocol for brain tumor targeting.

The development of new non-invasive diagnostic and therapeutic approaches is of paramount importance in order to improve the outcome of patients with glioblastoma (GBM). In this work we investigated a completely non-invasive pre-clinical protocol to effectively target and detect brain tumors through the orotracheal route, using ultra-small nanoparticles (USRPs) and MRI. A mouse model of GBM was developed. In vivo MRI acquisitions were performed before and after intravenous or orotracheal administration of the nanoparticles to identify and segment the tumor. The accumulation of the nanoparticles in neoplastic lesions was assessed ex vivo through fluorescence microscopy. Before the administration of contrast agents, MR images allowed the identification of the presence of abnormal brain tissue in 73% of animals. After orotracheal or intravenous administration of USRPs, in all the mice an excellent co-localization of the position of the tumor with MRI and histology was observed. The elimination time of the USRPs from the tumor after the orotracheal administration was approximately 70% longer compared with intravenous injection. MRI and USRPs were shown to be powerful imaging tools able to detect, quantify and longitudinally monitor the development of GBMs. The absence of ionizing radiation and high resolution of MRI, along with the complete non-invasiveness and good reproducibility of the proposed protocol, make this technique potentially translatable to humans. To our knowledge, this is the first time that the advantages of a needle-free orotracheal administration route have been demonstrated for the investigation of the pathomorphological changes due to GBMs.

Alkoxyamines: toward a new family of theranostic agents against cancer.

Theranostics combines therapeutic and diagnostic or drug deposition monitoring abilities of suitable molecules. Here we describe the first steps of building an alkoxyamine-based theranostic agent against cancer. The labile alkoxyamine ALK-1 (t(1/2) = 50 min at 37 °C) cleaves spontaneously to generate (1) a highly reactive free alkyl radical used as therapeutic agents to induce cell damages leading to cell death and (2) a stable nitroxide used as contrast agent for Overhauser-enhanced magnetic resonance imaging (OMRI). The ALK-1 toxicity was studied extensively in vitro on the glioblastoma cell line U87-MG. Cell viability appeared to be dependent on ALK-1 concentration and on the time of the observation following alkoxyamine treatment. For instance, the LC50 at 72 h was 250 µM. Data showed that cell toxicity was specifically due to the in situ released alkyl radical. This radical induced oxidative stress, mitochondrial changes, and ultimately the U87 cell apoptosis. The nitroxide production, during the alkoxyamine homolysis, was monitored by OMRI, showing a progressive MRI signal enhancement to 6-fold concomitant to the ALK-1 homolysis. In conclusion, we have demonstrated for the first time that the alkoxyamines are promising molecules to build theranostic tools against solid tumors.

Overhauser-enhanced MRI of elastase activity from in vitro human neutrophil degranulation.

BACKGROUND: Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research. METHODOLOGY/PRINCIPAL FINDINGS: We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI) contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4) cells per milliliter, well under the physiological neutrophils concentrations. CONCLUSIONS/SIGNIFICANCE: These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis) by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

Cellular density effect on RGD ligand internalization in glioblastoma for MRI application.

Cellular density is a parameter measured for glioma grade and invasiveness diagnosis. The characterization of the cellular density can be performed, non invasively, by magnetic resonance imaging (MRI), since, this technique displays a good resolution. Nevertheless MRI sensitivity is critical. Development of smart contrast agents appears useful to increase MRI signal to noise ratio (SNR). Tumor invasiveness is correlated with high expression of integrins that can be targeted by RGD motif. In this study, MRI contrast agents or fluorescent probes linked to RGD-peptides were used, in a glioma model, to assess the relation between RGD uptake/signal improvement/cell density and consequently tumor invasiveness. Experiments were performed in vitro with U87-MG glioma cells. Flow cytometry and microscopy experiments with RGD and iRGD-alexa488 demonstrated that cell internalization was dependent on cell density. The internalization involved a clathrin-dependent endocytosis. Cytoskeleton and particularly the microtubules were concerned. Actin filaments played a minor role. The internalization was also dependent on the glycolysis and the oxidative phosphorylations. The cellular density modulated the importance of the endocytosis pathways and of the metabolism but not the cytoskeleton contribution. The internalization of the RGD-peptide associated to gadolinium chelate increased the SNR of U87 cells. Moreover, following the cell density augmentation, the SNR increased with a low amplitude but a trend was clearly determined. In conclusion, RGD-peptide internalization appeared, in vitro, as a marker of cellular density. In perspective, the combination of these peptides with contrast agents associated to more sensitive MRI techniques could improve the MRI signal allowing the characterization of cellular density for tumor diagnosis.