Liposomes as Gene Delivery Vectors for Human Placental Cells.

Nanomedicine as a therapeutic approach for pregnancy-related diseases could offer improved treatments for the mother while avoiding side effects for the fetus. In this study, we evaluated the potential of liposomes as carriers for small interfering RNAs to placental cells. Three neutral formulations carrying rhodamine-labelled siRNAs were evaluated on an in vitro model, i.e., human primary villous cytotrophoblasts. siRNA internalization rate from lipoplexes were compared to the one in the presence of the lipofectamine reagent and assessed by confocal microscopy. Results showed cellular internalization of nucleic acid with all three formulations, based on two cationic lipids, either DMAPAP or CSL-3. Moreover, incubation with DMAPAP+AA provided a rate of labelled cells as high as with lipofectamine (53 ± 15% and 44 ± 12%, respectively) while being more biocompatible. The proportion of cells which internalized siRNA were similar when using DMAPAP/DDSTU (16 ± 5%) and CSL-3 (22 ± 5%). This work highlights that liposomes could be a promising approach for gene therapy dedicated to pregnant patients.

Bile Acid-Based Drug Delivery Systems for Enhanced Doxorubicin Encapsulation: Comparing Hydrophobic and Ionic Interactions in Drug Loading and Release.

Doxorubicin (Dox) is a drug of choice in the design of drug delivery systems directed toward breast cancers, but is often limited by loading and control over its release from polymer micelles. Bile acid-based block copolymers present certain advantages over traditional polymer-based systems for drug delivery purposes, since they can enable a higher drug loading via the formation of a reservoir through their aggregation process. In this study, hydrophobic and electrostatic interactions are compared for their influence on Dox loading inside cholic acid based block copolymers. Poly(allyl glycidyl ether) (PAGE) and poly(ethylene glycol) (PEG) were grafted from the cholic acid (CA) core yielding a star-shaped block copolymer with 4 arms (CA-(PAGE- b-PEG)4) and then loaded with Dox via a nanoprecipitation technique. A high Dox loading of 14 wt % was achieved via electrostatic as opposed to hydrophobic interactions with or without oleic acid as a cosurfactant. The electrostatic interactions confer a pH responsiveness to the system. 50% of the loaded Dox was released at pH 5 in comparison to 12% at pH 7.4. The nanoparticles with Dox loaded via hydrophobic interactions did not show such a pH responsiveness. The systems with Dox loaded via electrostatic interactions showed the lowest IC50 and highest cellular internalization, indicating the pre-eminence of this interaction in Dox loading. The blank formulations are biocompatible and did not show cytotoxicity up to 0.17 mg/mL. The new functionalized star block copolymers based on cholic acid show great potential as drug delivery carriers.

Aptamer-based liposomes improve specific drug loading and release.

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 15equivalents 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 aptamer's 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.

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.

Econazole imprinted textiles with antifungal activity.

In this work, we propose pharmaceutical textiles imprinted with lipid microparticles of Econazole nitrate (ECN) as a mean to improve patient compliance while maintaining drug activity. Lipid microparticles were prepared and characterized by laser diffraction (3.5±0.1 µm). Using an optimized screen-printing method, microparticles were deposited on textiles, as observed by scanning electron microscopy. The drug content of textiles (97±3 µg/cm(2)) was reproducible and stable up to 4 months storage at 25 °C/65% Relative Humidity. Imprinted textiles exhibited a thermosensitive behavior, as witnessed by a fusion temperature of 34.8 °C, which enabled a larger drug release at 32 °C (temperature of the skin) than at room temperature. In vitro antifungal activity of ECN textiles was compared to commercial 1% (wt/wt) ECN cream Pevaryl®. ECN textiles maintained their antifungal activity against a broad range of Candida species as well as major dermatophyte species. In vivo, ECN textiles also preserved the antifungal efficacy of ECN on cutaneous candidiasis infection in mice. Ex vivo percutaneous absorption studies demonstrated that ECN released from pharmaceutical textiles concentrated more in the upper skin layers, where the fungal infections develop, as compared to dermal absorption of Pevaryl®. Overall, these results showed that this technology is promising to develop pharmaceutical garments textiles for the treatment of superficial fungal infections.

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.

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.

Molecular tweezers: concepts and applications.

Taken to the molecular level, the concept of "tweezers" opens a rich and fascinating field at the convergence of molecular recognition, biomimetic chemistry and nanomachines. Composed of a spacer bridging two interaction sites, the behaviour of molecular tweezers is strongly influenced by the flexibility of their spacer. Operating through an "induced-fit" recognition mechanism, flexible molecular tweezers select the conformation(s) most appropriate for substrate binding. Their adaptability allows them to be used in a variety of binding modes and they have found applications in chirality signalling. Rigid spacers, on the contrary, display a limited number of binding states, which lead to selective and strong substrate binding following a "lock and key" model. Exquisite selectivity may be expressed with substrates as varied as C(60) , nanotubes and natural cofactors, and applications to molecular electronics and enzyme inhibition are emerging. At the crossroad between flexible and rigid spacers, stimulus-responsive molecular tweezers controlled by ionic, redox or light triggers belong to the realm of molecular machines, and, applied to molecular tweezing, open doors to the selective binding, transport and release of their cargo. Applications to controlled drug delivery are already appearing. The past 30 years have seen the birth of molecular tweezers; the next many years to come will surely see them blooming in exciting applications.

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 whoseinteraction with DNA was not based on ionic interactions, but on hydrogen bonds. We thusdeveloped lipids bearing a thiourea head which allowed an interaction with DNAphosphates through hydrogen bonds. After a proof of concept with a lipid bearing threethiourea functions, a molecular and cellular screening was performed by varying all partsof the lipids: the hydrophobic anchor, the spacer, the linker, and the thiourea head. Twolipothiourea-based structures were identified as highly efficient in vitro transfecting agents.The lipothioureas were shown to reduce non specific interactions with cell membranes anddeliver their DNA content intracellularly more efficiently, as compared to cationiclipoplexes. These lipids could deliver siRNA efficiently and allowed specific cell targetingin vitro. In vivo, thiourea lipoplexes presented a longer retention time in the blood and lessaccumulation in the lungs after an intravenous injection in mice. They also inducedluciferase gene expression in muscle and tumor after local administration in mice.Therefore, these novel lipoplexes represent an excellent alternative to cationic lipoplexes astransfecting agents. In this review we will focus on the structure activity studies thatpermitted the identification of the two most efficient thiourea lipids.

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.

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.

Comparative gene transfer between cationic and thiourea lipoplexes.

BACKGROUND: 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. METHODS: The MTT test was used for cytotoxicity assessment. Transfection efficiency was determined by luciferase read-out. Permeation to propidium iodide and enhanced green fluorescent protein was evaluated by flow cytometry. Kinetics of internalization and DNA release were monitored by confocal microscopy with labelled DNA. Endocytosis inhibitors were used to study the mechanisms of lipoplex internalization. RESULTS: Although thiourea/DNA complexes exhibit an almost similar level of transfection compared to that of cationic complexes, the thiourea lipoplexes were shown to be six-fold less internalized. Complexes were able to permeabilize the cytoplasmic membrane to 30 kDa molecules. Finally, DNA was shown to be released in less than 10 min in the cellular cytoplasm versus 30 min for cationic lipoplexes. CONCLUSIONS: Despite a weaker internalization compared to cationic lipids, the thiourea lipoplexes were able to transfect cells at a similar level as a result of its greater ability to destabilize the cytoplasmic membrane and release DNA.

Lipopolythiourea transfecting agents: lysine thiourea derivatives.

Synthetic vectors represent an alternative to recombinant viruses for gene transfer. We have recently explored the transfection potential of a class of noncationic lipids bearing thiourea moieties as DNA-associating headgroups. The encouraging results obtained with lipopolythioureas derived from serinol prompted us to further investigate this family of vectors. In the present study, we considered the transfection properties of a series of derivatives based on a different thiourea polar headgroup bearing a lysine scaffold. The synthesis of these compounds could be readily achieved in three steps with good yields. We found that these lipopolythioureas (LPT) might be considered as alternative systems for gene transfection since their activity reached the same magnitude range as that of cationic vectors in the presence of serum. LPT with 14-carbon length chains appeared to be more efficient as a transfecting agent than the ones with shorter chains. Toxicity studies proved that the hydration film method led to particles well tolerated both by the cells in vitro and by the mice in vivo. The ability to induce gene expression in vivo was demonstrated by intratumoral injection. Finally, biodistribution studies showed that the quantity recovered in blood circulation, 2 h after systemic injection, was improved as compared to that in cationic lipids.

Lipopolythioureas: a new non-cationic system for gene transfer.

A DNA-transfection protocol has been developed that makes use of thiourea non-cationic synthetic lipid, N-[1,3-bis(carbamothioylamino)propan-2-yl]-2-(dialkycarbamoylmethoxy)acetamide. It was found that these new compounds could be formulated without helper lipid and that the N-decanoyl and N-lauryl derivatives transfected B16 cells in the presence of serum with an efficiency at the same level as cationic lipids, under identical conditions. In vivo transfection using intratumoral injection was also investigated. It was found that compounds 18c and 19 showed an efficiency of the same magnitude as naked DNA and cationic lipid.

Design, synthesis, and evaluation of enhanced DNA binding new lipopolythioureas.

Nonviral gene delivery is limited to a large extent by the cationic nature of most of the chemical vector. We have shown that lipopolythioureas interact with DNA. However, lipopolythioureas were not very efficient at transfecting cells, probably due to reduced interaction between the noncationic synthetic lipid and the cell membrane. Here, we report that liposomes made from a new thiourea lipid, DPPC, and a lipid bearing an RGD ligand allowed very efficient entry of the lipopolythioureas into integrin alpha(v)beta(3) expressing cells. In addition, we show that a stable interaction between DNA and lipopolythiourea could be obtain with two thiourea groups. Moreover, the addition of a hydrophilic terminus improves the formulation of these new DNA binding agents.

DNA complexing lipopolythiourea.

We present a neutral lipopolythiourea (DTTU) as a potential DNA-binding agent. Light scattering experiments showed that mixing a lipopolythiourea with dipalmitoylphosphatidylcholine (DPPC/DTTU) led to small particles with sizes ranging from 100 to 150 nm at optimum conditions. Setting a fixed DNA amount, an increasing amount of DTTU/DPPC or DPPC lipids was added. Particle size increased only with DTTU/DPPC, indicating that interaction occurred between the DTTU/DPPC particles and DNA. In the same way, only DTTU/DPPC limited the ethidium bromide accessibility to plasmid DNA. These data suggest that DTTU/DPPC liposomes associate to DNA, which was confirmed by agarose gel experiments. To prove the active part of the DTTU lipid itself in DNA compaction, pegoylated-lipid was used. Cholesterol-PEG(2000) alone was not able to condense DNA. In contrast, DTTU/PEG-cholesterol was able to retain plasmid DNA on an agarose gel. In vivo injection of DTTU/DPPC/complexes was studied. Circulation time increase for noncationic particles as compared to cationic. More obvious was the lack of nonspecific accumulation in the lung, where a gain of 3 to 40 fold was measured.