Jeanne Leblond

pH-Responsive molecular tweezers.

Molecular tweezers are dynamic devices that are able to switch from one conformation to another upon stimulation by an external trigger. In this work, we report a new water-soluble macromolecular carrier bearing a pH-responsive molecular tweezer, whose affinity for a substrate depends on the external pH. The conformational change of the switching unit was evidenced by (1)H NMR spectroscopy, and fluorescence studies conducted in aqueous media demonstrated the ability of the carrier to bind to substrates in a pH-dependent fashion.

Reduction of Advanced-Glycation End Products Levels and Inhibition of RAGE Signaling Decreases Rat Vascular Calcification Induced by Diabetes

Advanced-glycation end products (AGEs) were recently implicated in vascular calcification, through a process mediated by RAGE (receptor for AGEs). Although a correlation between AGEs levels and vascular calcification was established, there is no evidence that reducing in vivo AGEs deposition or inhibiting AGEs-RAGE signaling pathways can decrease medial calcification. We evaluated the impact of inhibiting AGEs formation by pyridoxamine or elimination of AGEs by alagebrium on diabetic medial calcification. We also evaluated if the inhibition of AGEs-RAGE signaling pathways can prevent calcification. Rats were fed a high fat diet during 2 months before receiving a low dose of streptozotocin. Then, calcification was induced with warfarin. Pyridoxamine was administered at the beginning of warfarin treatment while alagebrium was administered 3 weeks after the beginning of warfarin treatment. Results demonstrate that AGEs inhibitors prevent the time-dependent accumulation of AGEs in femoral arteries of diabetic rats. This effect was accompanied by a reduced diabetes-accelerated calcification. Ex vivo experiments showed that N-methylpyridinium, an agonist of RAGE, induced calcification of diabetic femoral arteries, a process inhibited by antioxidants and different inhibitors of signaling pathways associated to RAGE activation. The physiological importance of oxidative stress was demonstrated by the reduction of femoral artery calcification in diabetic rats treated with apocynin, an inhibitor of reactive oxygen species production. We demonstrated that AGE inhibitors prevent or limit medial calcification. We also showed that diabetes-accelerated calcification is prevented by antioxidants. Thus, inhibiting the association of AGE-RAGE or the downstream signaling reduced medial calcification in diabetes.

New pharmaceutical applications for macromolecular binders

Macromolecular binders consist of polymers, dendrimers, and oligomers with binding properties for endogenous or exogenous substrates. This field, at the frontier of host/guest chemistry and pharmacology, has met a renewed interest in the past decade due to the clinical success of several sequestrants, like sevelamer hydrochloride (Renagel®) or sugammadex (Bridion®). In many instances, multivalent binding by the macromolecular drugs can modify the properties of the substrate, and may prevent it from reaching its site of action and/or trigger a biological response. From small (e.g., ions) to larger substrates (e.g., bacteria and cells), this review presents the state-of-the-art of macromolecular binders and provides detailed illustrative examples of recent developments bearing much promise for future pharmaceutical applications.

Comparative gene transfer between cationic and thiourea lipoplexes

We have previously developed lipopolythiourea lipids as neutral DNA condensing agents for systemic gene delivery. Optimization of the lipopolythiourea structure led to efficient transfecting agents. To further evaluate these lipids, we investigated the internalization process of the thiourea lipoplexes and their intracellular mechanism of transfection versus that of cationic lipoplexes.

Switchable Lipids: Conformational Change for Fast pH-Triggered Cytoplasmic Delivery.

We report the use of switchable lipids to improve the endosomal escape and cytosolic delivery of cell-impermeable compounds. The system is based on a conformational reorganization of the lipid structure upon acidification, as demonstrated by NMR spectroscopic studies. When incorporated in a liposome formulation, the switchable lipids triggered bilayer destabilization through fusion even in the presence of poly(ethylene glycol). We observed 88 % release of sulforhodamine B in 15 min at pH 5, and the liposome formulations demonstrated high stability at pH 7.4 for several months. By using sulforhodamine B as a model of a highly polar drug, we demonstrated fast cytosolic delivery mediated by endosomal escape in HeLa cells, and no toxicity.

Lipopolythiourea/DNA interaction: A biophysical study

Lipopolythioureas (LPT) are original non cationic systems representing an alternative to cationic lipids. Their high transfection efficiency prompted us to investigate further their biophysical properties, and in particular how thiourea lipids interact with DNA. The interaction of lipopolythiourea with DNA was investigated by fluorescence correlation microscopy (FCS). Influence of the lipid length and nature of the thiourea head on the thiourea/DNA interaction were studied. FCS revealed a strong interaction between lipopolythiourea and DNA, occurring at 1 equivalent of a thiourea lipid by a DNA phosphate group, and leading to a condensed plasmid state. From previous in vitro experiments, we could conclude that the lipid leading to the more condensed state of DNA was also the more efficient to transfect cells.

Lipothioureas as Lipids for Gene Transfection: A Review

Non-viral gene therapy requires innovative strategies to achieve higher transfection efficacy. A few years ago, our group proposed bioinspired lipids whose interaction with DNA was not based on ionic interactions, but on hydrogen bonds. We thus developed lipids bearing a thiourea head which allowed an interaction with DNA phosphates through hydrogen bonds. After a proof of concept with a lipid bearing three thiourea functions, a molecular and cellular screening was performed by varying all parts of the lipids: the hydrophobic anchor, the spacer, the linker, and the thiourea head. Two lipothiourea-based structures were identified as highly efficient in vitro transfecting agents. The lipothioureas were shown to reduce non specific interactions with cell membranes and deliver their DNA content intracellularly more efficiently, as compared to cationic lipoplexes. These lipids could deliver siRNA efficiently and allowed specific cell targeting in vitro. In vivo, thiourea lipoplexes presented a longer retention time in the blood and less accumulation in the lungs after an intravenous injection in mice. They also induced luciferase gene expression in muscle and tumor after local administration in mice. Therefore, these novel lipoplexes represent an excellent alternative to cationic lipoplexes as transfecting agents. In this review we will focus on the structure activity studies that permitted the identification of the two most efficient thiourea lipids.

Aptamer-based liposomes improve specific drug loading and release

Abstract Aptamer technology has shown much promise in cancer therapeutics for its targeting abilities. However, its potential to improve drug loading and release from nanocarriers has not been thoroughly explored. In this study, we employed drug‐binding aptamers to actively load drugs into liposomes. We designed a series of DNA aptamer sequences specific to doxorubicin, displaying multiple binding sites and various binding affinities. The binding ability of aptamers was preserved when incorporated into cationic liposomes, binding up to 15 equivalents of doxorubicin per aptamer, therefore drawing the drug into liposomes. Optimization of the charge and drug/aptamer ratios resulted in ≥ 80% encapsulation efficiency of doxorubicin, ten times higher than classical passively‐encapsulating liposomal formulations and similar to a pH‐gradient active loading strategy. In addition, kinetic release profiles and cytotoxicity assay on HeLa cells demonstrated that the release and therapeutic efficacy of liposomal doxorubicin could be controlled by the aptamers structure. Our results suggest that the aptamer exhibiting a specific intermediate affinity is the best suited to achieve high drug loading while maintaining efficient drug release and therapeutic activity. This strategy was successfully applied to tobramycin, a hydrophilic drug suffering from low encapsulation into liposomes, where its loading was improved six‐fold using aptamers. Overall, we demonstrate that aptamers could act, in addition to their targeting properties, as multifunctional excipients for liposomal formulations. Graphical abstract Figure. No Caption available.

Single stranded siRNA complexation through non-electrostatic interactions.

As double stranded, single stranded siRNA (ss-siRNA) has demonstrated gene silencing activity but still requires efficient carriers to reach its cytoplasmic target. To better understand the fundamental aspect driving the complexation of ss-siRNA with nanocarriers, the interactions between surfaces of various compositions across a ss-siRNA solution were investigated using the Surface Forces Apparatus. The results show that ss-siRNA can adsorb onto hydrophilic (positively and negatively charged) as well as on hydrophobic substrates suggesting that the complexation can occur through hydrophobic interactions and hydrogen bonding in addition to electrostatic interactions. Moreover, the binding strength and the conformation of ss-siRNA depend on the nature of the interactions between the ss-siRNA and the surfaces. The binding of ss-siRNA with nanocarriers, such as micelles or liposomes through non-electrostatic interactions was also evidenced by a SYBR® Gold cyanine dye. We evidenced the presence of interactions between the dye and oligonucleotides already complexed to non-cationic nanovectors biasing the quantification of the encapsulation. These results suggest that non-electrostatic interactions could be exploited to complement electrostatic interactions in the design of nanocarriers. In particular, the different highlighted interactions can be used to complex ss-siRNA with uncharged or anionic carriers which are related to lower toxicity compared to cationic carriers.

CHAPTER 16:Designing Polymeric Binders for Pharmaceutical Applications

The properties of polyvalent polymers to form supramolecular complexes with biological substrates offer many attractive therapeutic possibilities. Polymeric binders are macromolecules designed to exert a pharmacological effect by selectively interacting with exogenous or endogenous substrates. They can be employed to prevent the harmful effects of toxins, inhibit virus colonization or even trigger apoptosis of diseased cells. This chapter presents the fundamentals of developing polymeric binders as new drug entities. The basics of finding the right target, establishing structure–activity relationships and measuring efficacy are highlighted, with numerous examples of polymeric binders at different development stages, including commercialization. Orally administered scavengers represent the most advanced examples in clinical use. Their binding in the gastrointestinal tract results in either local or systemic therapeutic effects. Although they are designed to be non-absorbable, their low systemic exposure is not always devoid of side effects. The required approaches to confirm innocuousness of the macromolecules and the challenges encountered during the clinical phases are also presented.