In vivo EPR extracellular pH-metry in tumors using a triphosphonated trityl radical.

PURPOSE: The ability to assess the extracellular pH (pHe) is an important issue in oncology, because extracellular acidification is associated with tumor aggressiveness and resistance to cytotoxic therapies. In this study, a stable triphosphonated triarylmethyl (TPTAM) radical was qualified as a pHe electron paramagnetic resonance (EPR) molecular reporter. METHODS: Calibration of hyperfine splitting as a function of pH was performed using a 1.2-GHz EPR spectrometer. Gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) was used as an extracellular paramagnetic broadening agent to assess the localization of TPTAM when incubated with cells. In vivo EPR pH-metry was performed in MDA, SiHa, and TLT tumor models and in muscle. Bicarbonate therapy was used to modulate the tumor pHe. EPR measurements were compared with microelectrode readouts. RESULTS: The hyperfine splitting of TPTAM was strongly pH-dependent around the pKa of the probe (pKa = 6.99). Experiments with Gd-DTPA demonstrated that TPTAM remained in the extracellular compartment. pHe was found to be more acidic in the MDA, SiHa, and TLT tumor models compared with muscle. Treatment of animals by bicarbonate induced an increase in pHe in tumors: similar variations in pHe were found when using in vivo EPR or invasive microelectrodes measurements. CONCLUSION: This study demonstrates the potential usefulness of TPTAM for monitoring pHe in tumors. Magn Reson Med 77:2438-2443, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Delivery of siRNA targeting tumor metabolism using non-covalent PEGylated chitosan nanoparticles: Identification of an optimal combination of ligand structure, linker and grafting method.

PEGylated chitosan-based nanoparticles offer attractive platforms for siRNA cocktail delivery into tumors. Still, therapeutic efficacy requires us to select a rational combination of siRNAs and an efficient tumor delivery after systemic administration. Here, we showed that non-covalent PEGylation of chitosan-based nanoparticles loaded with siRNA targeting two key transporters of energy fuels for cancer cells, namely the lactate transporter MCT1 and the glutamine transporter ASCT2, could lead to significant antitumor effects. As a ligand, we tested variations of the prototypical RGD peptidomimetic (RGDp). A higher siRNA delivery was obtained with naphthyridine-containing RGDp randomly conjugated on the PEG chain by clip photochemistry and the use of a lipophilic linker than when using traditional chain-end grafting and RGDp with a hydrophilic linker. The antiproliferative effects resulting from ASCT2 and MCT1 silencing were validated separately in vitro in conditions mimicking specific metabolic profiles of cancer cells and in vivo upon concomitant delivery. The combination of those siRNA and the selected components of targeted RGDp nanoparticles led to a dramatic tumor growth inhibition upon peri-tumoral but also systemic administration in mice. Altogether these data emphasize the convenience of using non-covalent PEGylated chitosan particles to produce sheddable stealth protection compatible with an efficient siRNA delivery in tumors.

Intracellular siRNA delivery dynamics of integrin-targeted, PEGylated chitosan-poly(ethylene imine) hybrid nanoparticles: A mechanistic insight.

Integrin-targeted nanoparticles are promising for the delivery of small interfering RNA (siRNA) to tumor cells or tumor endothelium in cancer therapy aiming at silencing genes essential for tumor growth. However, during the process of optimizing and realizing their full potential, it is pertinent to gain a basic mechanistic understanding of the bottlenecks existing for nanoparticle-mediated intracellular delivery. We designed αvß3 integrin-targeted nanoparticles by coupling arginine-glycine-aspartate (RGD) or RGD peptidomimetic (RGDp) ligands to the surface of poly(ethylene glycol) (PEG) grafted chitosan-poly(ethylene imine) hybrid nanoparticles. The amount of intracellular siRNA delivered by αvß3-targeted versus non-targeted nanoparticles was quantified in the human non-small cell lung carcinoma cell line H1299 expressing enhanced green fluorescent protein (EGFP) using a stem-loop reverse transcription quantitative polymerase chain reaction (RT-qPCR) approach. Data demonstrated that the internalization of αvß3-targeted nanoparticles was highly dependent on the surface concentration of the ligand. Above a certain threshold concentration, the use of targeted nanoparticles provided a two-fold increase in the number of siRNA copies/cell, subsequently resulting in as much as 90% silencing of EGFP at well-tolerated carrier concentrations. In contrast, non-targeted nanoparticles mediated low levels of gene silencing, despite relatively high intracellular siRNA concentrations, indicating that these nanoparticles might end up in late endosomes or lysosomes without releasing their cargo to the cell cytoplasm. Thus, the silencing efficiency of the chitosan-based nanoparticles is strongly dependent on the uptake and the intracellular trafficking in H1299 EGFP cells, which is critical information towards a more complete understanding of the delivery mechanism that can facilitate the future design of efficient siRNA delivery systems.

Functionalization of the PEG Corona of Nanoparticles by Clip Photochemistry in Water: Application to the Grafting of RGD Ligands on PEGylated USPIO Imaging Agent.

The fast development of nanomedicines requires more and more reliable chemical tools in order to accurately design materials and control the surface properties of the nano-objects used in biomedical applications. In this study we describe a smooth and simple photografting technique, i.e., the clip photochemistry, that allows the introduction of molecules of interest in inert polymers or on stealth nanoparticles directly in aqueous solution. First we developed the methodology on polyethylene glycol (PEG) and looked for critical parameters of the process (irradiation times, concentrations, washings) by using several molecular probes and adapted analytical techniques ((19)F qNMR, EA, LSC). We found that the clip photochemistry in water is a robust and efficient method to functionalize PEG. Second we applied it on PEGylated USPIO (USPIO-PEG) magnetic resonance imaging agent and succeeded in introducing RGD peptide and homemade peptidomimetics on their PEG segments. The magnetic abilities of the conjugated nanoparticles were unchanged by the derivatization process as evidenced by their relaxometric properties and their NMRD profile. When tested on Jurkat lymphocyte T Cells, which express αvß3 integrins, the USPIO conjugated with RGD ligands leads to an increase of the transverse relaxation rate (R2) by a factor 10 to 14 as compared to USPIO-PEG. Consequently, it makes them good candidates for targeted imaging technology in cancer therapy.

Self-assembling doxorubicin-tocopherol succinate prodrug as a new drug delivery system: synthesis, characterization, and in vitro and in vivo anticancer activity.

Self-assembled prodrugs forming nanoaggregates are a promising approach to enhance the antitumor efficacy and to reduce the toxicity of anticancer drugs. To achieve this goal, doxorubicin was chemically conjugated to d-α-tocopherol succinate through an amide bond to form N-doxorubicin-α-d-tocopherol succinate (N-DOX-TOS). The prodrug self-assembled in water into 250 nm nanostructures when stabilized with d-α-tocopherol poly(ethylene glycol) 2000 succinate. Cryo-TEM analysis revealed the formation of nanoparticles with a highly ordered lamellar inner structure. NMR spectra of the N-DOX-TOS nanoparticles indicated that N-DOX-TOS is located in the core of the nanoparticles while PEG chains and part of the tocopherol are in the corona. High drug loading (34% w/w) and low in vitro drug release were achieved. In vitro biological assessment showed significant anticancer activity and temperature-dependent cellular uptake of N-DOX-TOS nanoparticles. In vivo, these nanoparticles showed a greater antitumor efficacy than free DOX. N-DOX-TOS nanoparticles might have the potential to improve DOX-based chemotherapy.

(S)-1-(Pent-4'-enoyl)-4-(hydroxymethyl)-azetidin-2-one derivatives as inhibitors of human fatty acid amide hydrolase (hFAAH): synthesis, biological evaluation and molecular modelling.

A series of lipophilic ester derivatives (2a-g) of (S)-1-(pent-4'-enoyl)-4-(hydroxymethyl)-azetidin-2-one has been synthesised in three steps from (S)-4-(benzyloxycarbonyl)-azetidin-2-one and evaluated as novel, reversible, ß-lactamic inhibitors of endocannabinoid-degrading enzymes (human fatty acid amide hydrolase (hFAAH) and monoacylglycerol lipase (hMAGL)). The compounds showed IC50 values in the micromolar range and selectivity for hFAAH versus hMAGL. The unexpected 1000-fold decrease in activity of 2a comparatively to the known regioisomeric structure 1a (i.e. lipophilic chains placed on N1 and C3 positions of the ß-lactam core) could be explained on the basis of docking studies into a revisited model of hFAAH active site, considering one or two water molecules in interaction with the catalytic triad.

Macrocycle-embedded ß-lactams as novel inhibitors of the Penicillin Binding Protein PBP2a from MRSA.

Assuming that bicyclic ß-lactams endowed with high conformational adaptability should more easily form acyl-enzyme complexes with PBP2a than the traditional antibiotics, we have prepared a series of bis-2-oxo-azetidinyl macrocycles as potential inhibitors. The compounds are formally "head-head" (HH) cyclodimers of 1-(ω-alkenoyl)-3-(S)-(ω'-alkenoylamino)-2-azetidinones, with various lengths of the alkene chains, obtained by two successive metathesis reactions using the Grubbs catalyst. All compounds behave as acylating inhibitors of PBP2a and one ß-lactam (5c), embedded into the largest ring (32 atoms), features an activity close to that of Ceftobiprole. Conformational analyses, theoretical reactivity models and docking experiments in PBP2a cavity allow to propose a novel pharmacophore, i.e. the 3-(S)-acylamino-1-acyl-2-azetidinone ring, with the syn-conformation of the imide function, associated to a flexible macrocycle favoring the opening of the active site.

Polyethylene terephthalate membrane grafted with peptidomimetics: endothelial cell compatibility and retention under shear stress.

The present work aimed to treat a polyethylene terephthalate (PET) surface to make the biomaterial more 'attractive' in terms of attachment and shear stress response to endothelial cells with a view to possible applications in vascular grafting. A surface wet-chemistry protocol was applied to graft track-etched PET membranes with RGD peptidomimetics based on the tyrosine template and active at the nano-level vs. isolated human αvß3 receptor, which was monitored by X-ray photoelectron spectroscopy, contact angle measurement and atomic force microscopy for characterization. A primary culture of human saphenous vein endothelial cells was used before and after sterilization of the membranes (heat treatment or γ-ray irradiation) to test the benefit of grafting. The optimal surface concentrations of grafted molecules were around 50 pmol/cm². Compared to GRGDS, the peptidomimetics promoted cell attachment with similar or slightly better performances. Endothelialized grafted supports were further exposed to 2 h of shear stress mimicking arterial conditions. Cells were lost on non-grafted PET whereas cells on grafted polymers sterilized by γ-ray irradiation withstood forces with no significant difference in focal contacts. At the mRNA level, cells on functionalized PET were able to respond to shear stress with NFkB upregulation. Thus, grafting of peptidomimetics as ligands of the αvß3 integrin could be a relevant strategy to improve the adhesion of human endothelial cells and to obtain an efficient endothelialized PET for the surgery of small-diameter vascular prostheses.

Targeted nanoparticles with novel non-peptidic ligands for oral delivery.

Orally administered targeted nanoparticles have a large number of potential biomedical applications and display several putative advantages for oral drug delivery, such as the protection of fragile drugs or modification of drug pharmacokinetics. These advantages notwithstanding, oral drug delivery by nanoparticles remains challenging. The optimization of particle size and surface properties and targeting by ligand grafting have been shown to enhance nanoparticle transport across the intestinal epithelium. Here, different grafting strategies for non-peptidic ligands, e.g., peptidomimetics, lectin mimetics, sugars and vitamins, that are stable in the gastrointestinal tract are discussed. We demonstrate that the grafting of these non-peptidic ligands allows nanoparticles to be targeted to M cells, enterocytes, immune cells or L cells. We show that these grafted nanoparticles could be promising vehicles for oral vaccination by targeting M cells or for the delivery of therapeutic proteins. We suggest that targeting L cells could be useful for the treatment of type 2 diabetes or obesity.

A pyrene- and phosphonate-containing fluorescent probe as guest molecule in a host polymer matrix.

New host-guest materials have been prepared by incorporation of a home-made organic probe displaying a pyrene motif and a phosphonate function into a regular amphiphilic copolymer. Using powder X-Ray diffraction, photoluminescence and FT-IR spectroscopy, we have been able to study the non-covalent interactions between the host matrix and the guest molecule in the solid state. Interestingly, we have shown that the matrix directs the guest spatial localization and alters its properties. Thanks to the comparison of pyrene vs. N-pyrenylmaleimide derivatives, the influence of the chemical nature of the guest molecules on the non-covalent interactions with the host have been studied. In addition, using polyethylene glycol as a reference host, we have been able to evidence a true matrix effect within our new insertion materials. The phosphonated guest molecule appears to be a novel probe targeting the hydrophilic domain of the host copolymer.

An unprecedented reversible mode of action of ß-lactams for the inhibition of human fatty acid amide hydrolase (hFAAH).

A series of compound was prepared to clarify the reversible mechanism of ß-lactamic hFAAH inhibitors on the one hand, and to modulate some of their physicochemical parameters on the other hand. In particular, two compounds (4b and 4e) were designed to display a potential good leaving group on the crucial carbonyl with a view to possibly acylating the active serine of the hFAAH catalytic triad. Reversibility studies showed that these two compounds retain the reversible mode of inhibition, suggesting a noncovalent interaction between our ß-lactams and hFAAH. Finally, pharmacological evaluations of bioisosteres of the lead compound (4a, IC(50) = 5.3 nM) revealed that log P values and PSA could be optimized without altering the FAAH inhibition (IC(50) values from 3.65 nM to 70.9 nM).

Surface modification of polypropylene nonwovens with LDV peptidomimetics and their application in the leukodepletion of blood products.

For clinical indications and according to European standards, a depletion of the leukocytes from whole blood has to be realized before transfusion to a patient. In this study, the surface of a layer of meltblown oxygen-plasma treated polypropylene nonwoven (O(2)-PP), making part of the composition of leukodepletion filters, was modified with bioactive molecules to enhance the adhesion of leukocytes. These synthetic small molecules (called peptidomimetics) mimic the "Leu-Asp-Val" (LDV) tripeptide sequence recognized by the α(4)ß(1) integrin, a receptor expressed on leukocytes. Their activity, as ligands of the α(4)ß(1) integrin, was confirmed through cell adhesion assays. The peptidomimetics were covalently immobilized on O(2)-PP nonwoven via activation of the hydroxyl- and carboxyl-functions displayed on the polymer surface with trifluoro-triazine reagent, or were simply deposited on the O(2)-PP surface. The treated materials were characterized by X-ray photoelectron spectroscopy, wettability, and morphological analyses, before and after steam sterilization. Experimental filters composed of 10 layers of O(2)-PP nonwovens and a last layer modified with the peptidomimetics were evaluated, using whole blood filtration assays, for their leukodepletion efficiency, the recovery of red cells and platelets and the waste of blood, with an objective to design new filters fulfilling practical and medical criteria.

Vibrational circular dichroism versus optical rotation dispersion and electronic circular dichroism for diastereomers: the stereochemistry of 3-(1'-hydroxyethyl)-1-(3'-phenylpropanoyl)-azetidin-2-one.

The absolute configuration of a relatively large and conformationally flexible chiral compound, 3-(1'-hydroxyethyl)-1-(3'-phenylpropanoyl)-azetidin-2-one, is determined using Vibrational Circular Dichroism (VCD) spectroscopy, Optical Rotation Dispersion (ORD) and Electronic Circular Dichroism (ECD). To that end a state of the art experimental VCD spectrum is compared to a theoretical spectrum and the absolute configuration is assigned. ORD and ECD are also used in the assignment to investigate the complementarity of the three techniques. VCD spectroscopy is found to have important advantages over ORD and ECD for diastereomers. The concept of robust modes is applied to this conformationally flexible molecule, showing that its use is limited for such large and flexible molecules.

Characterization of an endophytic whorl-forming Streptomyces from Catharanthus roseus stems producing polyene macrolide antibiotic.

An endophytic whorl-forming Streptomyces sp. designated as TS3RO having antifungal activity against a large number of fungal pathogens, including Sclerotinia sclerotiorum, Rhizoctonia solani, Colletotrichum gloeosporioides, Cryphonectria parasitica, Fusarium oxysporum, Pyrenophora tritici-repentis, Epidermophyton floccosum, and Trichophyton rubrum, was isolated from surface-sterilized Catharanthus roseus stems. Preliminary identification showed that Streptomyces cinnamoneus subsp. sparsus was its closest related species. However, strain TS3RO could readily be distinguished from this species using a combination of phenotypic properties, 16S rDNA sequence similarity, and phylogenetic analyses. Thus, the whorl-forming Streptomyces sp. strain TS3RO is likely a new subspecies within the Streptomyces cinnamoneus group. Direct bioautography on a thin-layer chromatography plate with Cladosporium cucumerinum was conducted throughout the purification steps for bioassay-guided isolation of the active antifungal compounds from the crude extract. Structural elucidation of the isolated bioactive compound was obtained via LC-MS spectrometry, UV-visible spectra, and nuclear magnetic resonance data. It revealed that fungichromin, a known methylpentaene macrolide antibiotic, was the main antifungal component of TS3RO strain, as shown by thin-layer chromatography bioautography. This is the first report of an endophytic whorl-forming Streptomyces isolated from the medically important plant Catharanthus roseus.

Targeting of tumor endothelium by RGD-grafted PLGA-nanoparticles.

The destruction of the neovessels in solid tumors can cause the death of tumor cells resulting from the lack of oxygen and nutrients. Peculiarities of the tumor vasculature, however, also position angiogenic endothelial cells as obvious targets to address cytotoxic drugs into the tumor. In particular, the identification of a three-amino acids sequence, arginine-glycine-aspartate (RGD), as a fundamental recognition site for proliferating endothelial attachment to the extracellular matrix leads to the development of tumor-targeting ligands for nanoparticles. The RGD peptide can target the α(v)ß(3) integrin overexpressed by the tumor endothelium, and thereby increases the accumulation of drug-loaded RGD-grafted nanoparticles. RGD-nanoparticles may thus extravasate more efficiently and enter the tumor via the enhanced permeability and retention (EPR) effect. This combination of active and passive processes leads to the penetration of nanoparticles into the tumor tissue, followed by cellular uptake and intracellular delivery of the cytotoxic payload. Since cancer cells may also express α(v)ß(3) integrin, the entrapping of RGD-nanoparticles into the tumor interstitial fluid may yet be facilitated through direct binding to cancer cells. Here, we describe methods used for the preparation of RGD-nanoparticles and for the validation of their potential of tumor endothelium targeting both in vitro and in vivo. We also illustrate how RGD-nanoparticles may be more suited than nontargeted modalities for the tumor delivery of poorly soluble and/or highly cytotoxic drugs, using different mouse tumor xenograft models.

Synthesis of poly(lactide-co-glycolide-co-ε-caprolactone)-graft-mannosylated poly(ethylene oxide) copolymers by combination of "clip" and "click" chemistries.

Poly(lactide-co-glycolide) (PLGA) is extensively used in pharmaceutical applications, for example, in targeted drug delivery, because of biocompatibility and degradation rate, which is easily tuned by the copolymer composition. Nevertheless, synthesis of sugar-labeled amphiphilic copolymers with a PLGA backbone is quite a challenge because of high sensitivity to hydrolytic degradation. This Article reports on the synthesis of a new amphiphilic copolymer of PLGA grafted by mannosylated poly(ethylene oxide) (PEO). A novel building block, that is, α-methoxy-ω-alkyne PEO-clip-N-hydroxysuccinimide (NHS) ester, was prepared on purpose by photoreaction of a diazirine containing molecular clip. This PEO block was mannosylated by reaction of the NHS ester groups with an aminated sugar, that is, 2-aminoethyl-α-d-mannopyroside. Then, the alkyne ω-end-group of PEO was involved in a copper alkyne- azide coupling (CuAAC) with the pendent azides of the aliphatic copolyester. The targeted mannose-labeled poly(lactide-co-glycolide-co-ε-caprolactone)-graft-poly(ethylene oxide) copolymer was accordingly formed. Copolymerization of d,l-lactide and glycolide with α-chloro-ε-caprolactone, followed by substitution of chlorides by azides provided the azido-functional PLGA backbone. Finally, micelles of the amphiphilic mannosylated graft copolymer were prepared in water, and their interaction with Concanavalin A (ConA), a glyco-receptor protein, was studied by quartz crystal microbalance. This study concluded to the prospect of using this novel bioconjugate in targeted drug delivery.

A phosphonated triarylmethyl radical as a probe for measurement of pH by EPR.

A new water soluble phosphonated tetrathiatriarylmethyl radical has been synthesized and its application for pH measurement in a physiological range by EPR is reported.

Non-symmetrically substituted phenoxazinones from laccase-mediated oxidative cross-coupling of aminophenols: an experimental and theoretical insight.

Oxidative cross-coupling reactions of substituted o-aminophenols were catalyzed by a commercial laccase to produce non-symmetrically substituted phenoxazinones for the first time. Identification by (1)H-, (13)C- and (31)P-NMR, and by HPLC-PDA and HPLC-MS/MS of exclusively two kinds of substituted phenoxazinones out of four potential heterocyclic frameworks was confirmed by a DFT study. The redox-properties of the substrates, their relative rates of conversion and the rigid docking of selected substrates led to a revisited mechanistic pathway for phenoxazinones biosynthesis. Our suggestions concern both the first formal two-electron oxidation by laccase and the first intermolecular 1,4-conjugated addition which secures the observed regioselectivity.

Inhibitors of the endocannabinoid-degrading enzymes, or how to increase endocannabinoid's activity by preventing their hydrolysis.

Endocannabinoids are lipid transmitters binding and activating the cannabinoid receptors. Both cannabinoid receptors and endocannabinoids, such as 2-arachidonoylglycerol and anandamide, have been shown to control numerous physiological and pathological processes, including in the central nervous system. Thus regulating endocannabinoid levels in-vivo represents an interesting therapeutic perspective in several CNS-related diseases. To date four enzymes - Fatty Acid Amide Hydrolase (FAAH), N-Acylethanolamine-hydrolyzing Acid Amidase (NAAA), Monoacylglycerol Lipase (MAGL), α/ß-Hydrolase Domain 6 (ABHD6) - were shown to control endocannabinoid levels in tissues or in intact cells. While the searches for NAAA and ABHD6 inhibitors are still in their beginning, a growing number of selective and potent inhibitors are now available to inhibit FAAH and MAGL activities. Here, based on the literature and patent literature, we review the compounds of the different chemical families that have been developed to inhibit these enzymes, with a special emphasis on FAAH and MAGL inhibitors.

LDV peptidomimetics equipped with biotinylated spacer-arms: synthesis and biological evaluation on CCRF-CEM cell line.

The tripeptide Leu-Asp-Val (LDV) is known to bind α(4)ß(1) integrin in leukemia cells. Here we have synthesized a LDV peptidomimetic equipped with a biotin-conjugated spacer-arm. Compound 9 acts as an inhibitor of the α(4)ß(1) integrin in an adhesion assay using fluorescently labeled, α(4)ß(1) integrin-expressing leukemia CCRF-CEM cells. Furthermore, when bound to neutravidin-coated plates, compound 9 could capture CCRF-CEM cells. Such biotin-conjugated LDV peptidomimetic may thus represent a novel tool for biotechnological applications using avidin interaction for leukapheresis or leukemia cell targeting.