(Poly-cyanoacrylate) nanomedicines for cancer and beyond: Lessons learned.

This « Magnum Opus ¼ emphasizes that serendipity is a corner stone in research. The paths of discovery and innovation often result from the interdisciplinarity of scientific areas that are a priori disconnected from each other. In the 1970s, fundamental discoveries in cell biology led to unexpected advances in galenic pharmacy with the emergence of nanotechnologies for the intracellular delivery of non diffusing molecules. As well, fluorescein-loaded polyacrylamide nanocapsules were shown to deliver this fluorescent agent precisely into cellular lysosomes which represented a seminal observation. However, due to the lack of biodegradability of this carrier polymer, this approach was still far from therapeutic application. The use of cyanoacrylates as surgical glue inspired us to use this material in the design of the first biodegradable nanoparticles for human use. Capable of transporting compounds with anti-tumor activity, these polyalkylcyanoacrylate nanoparticles demonstrated the unexpected property of overcoming multi-drug resistance. This discovery led to the development of a nanomedicine that has completed phase III clinical trials for the treatment of resistant hepatocarcinoma. Going beyond the state-of-the art, a step ahead in the nanomedicine field was the drug « squalenoylation ¼ technology, which represents a shift from the « physical ¼ to the « chemical ¼ encapsulation paradigm. The bioconjugation of anticancer and other drugs to squalene, a natural and biocompatible lipid, enabled a dramatic increase in drug payload, and eliminated the so-called « burst release ¼ of drug: Two major drawbacks commonly associated with drug nanoencapsulation. The drug « squalenoylation ¼ approach resulted in a generic nanomedicine platform with broad pharmacological applications.

Vitamin C-squalene bioconjugate promotes epidermal thickening and collagen production in human skin.

Vitamin C (Vit C) benefits to human skin physiology notably by stimulating the biosynthesis of collagen. The main cutaneous collagens are types I and III, which are less synthesized with aging. Vit C is one of the main promotors of collagen formation but it poorly bypasses the epidermis stratum corneum barrier. To address this challenge, we developed a lipophilic version of Vit C for improving skin diffusion and delivery. Vit C was covalently conjugated to squalene (SQ), a natural lipid of the skin, forming a novel Vit C-SQ derivative suitable for cream formulation. Its biological activity was investigated on human whole skin explants in an ex vivo model, through histology and protein and gene expression analyses. Results were compared to Vit C coupled to the reference lipophilic compound palmitic acid, (Vit C-Palmitate). It was observed that Vit C-SQ significantly increased epidermal thickness and preferentially favored collagen III production in human skin after application for 10 days. It also promoted glycosaminoglycans production in a higher extent comparatively to Vit C-Palmitate and free Vit C. Microdissection of the explants to separate dermis and epidermis allowed to measure higher transcriptional effects either in epidermis or in dermis. Among the formulations studied, the strongest effects were observed with Vit C-SQ.

Squalenoyl-gemcitabine/edelfosine nanoassemblies: Anticancer activity in pediatric cancer cells and pharmacokinetic profile in mice.

Despite the great advances accomplished in the treatment of pediatric cancers, recurrences and metastases still exacerbate prognosis in some aggressive solid tumors such as neuroblastoma and osteosarcoma. In view of the poor efficacy and toxicity of current chemotherapeutic treatments, we propose a single multitherapeutic nanotechnology-based strategy by co-assembling in the same nanodevice two amphiphilic antitumor agents: squalenoyl-gemcitabine and edelfosine. Homogeneous batches of nanoassemblies were easily formulated by the nanoprecipitation method. Their anticancer activity was tested in pediatric cancer cell lines and pharmacokinetic studies were performed in mice. In vitro assays revealed a synergistic effect when gemcitabine was co-administered with edelfosine. Squalenoyl-gemcitabine/edelfosine nanoassemblies were found to be capable of intracellular translocation in patient-derived metastatic pediatric osteosarcoma cells and showed a better antitumor profile than squalenoyl-gemcitabine nanoassemblies alone. The intravenous administration of this combinatorial nanomedicine in mice exhibited a controlled release behavior of gemcitabine and diminished edelfosine plasma peak concentrations. These findings make it a suitable pre-clinical candidate for childhood cancer therapy.

A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine.

Among anticancer nanomedicines, squalenoyl nanocomposites have obtained encouraging outcomes in a great variety of tumors. The prodrug squalenoyl-gemcitabine has been chosen in this study to construct a novel multidrug nanosystem in combination with edelfosine, an alkyl-lysophopholipid with proven anticancer activity. Given their amphiphilic nature, it was hypothesized that both anticancer compounds, with complementary molecular targets, could lead to the formation of a new multitherapy nanomedicine. Nanoassemblies were formulated by the nanoprecipitation method and characterized by dynamic light scattering, transmission electron microscopy and X-ray photoelectron spectroscopy. Because free edelfosine is highly hemolytic, hemolysis experiments were performed using human blood erythrocytes and nanoassemblies efficacy was evaluated in a patient-derived metastatic pediatric osteosarcoma cell line. It was observed that these molecules spontaneously self-assembled as stable and monodisperse nanoassemblies of 51 ±â€¯1 nm in a surfactant/polymer free-aqueous suspension. Compared to squalenoyl-gemcitabine nanoassemblies, the combination of squalenoyl-gemcitabine with edelfosine resulted in smaller particle size and a new supramolecular conformation, with higher stability and drug content, and ameliorated antitumor profile.

Towards improved HIV-microbicide activity through the co-encapsulation of NRTI drugs in biocompatible metal organic framework nanocarriers.

The efficacy of the routinely used anti-HIV (Human Immunodeficiency Virus) therapy based on nucleoside reverse transcriptase inhibitors (NRTIs) is limited by the poor cellular uptake of the active triphosphorylated metabolites and the low efficiency of intracellular phosphorylation of their prodrugs. Nanoparticles of iron(iii) polycarboxylate Metal-Organic Frameworks (nanoMOFs) are promising drug nanocarriers. In this study, two active triphosphorylated NRTIs, azidothymidine triphosphate (AZT-Tp) and lamivudine triphosphate (3TC-Tp), were successfully co-encapsulated into the biocompatible mesoporous iron(iii) trimesate MIL-100(Fe) nanoMOF in order to improve anti-HIV therapies. The drug loaded nanoMOFs could be stored for up to 2-months and reconstituted after freeze drying, retaining similar physicochemical properties. Their antiretroviral activity was evidenced in vitro on monocyte-derived macrophages experimentally infected with HIV, making these co-encapsulated nanosystems excellent HIV-microbicide candidates.

In vivo behavior of MIL-100 nanoparticles at early times after intravenous administration.

Metal-organic frameworks have shown interesting features for biomedical applications, such as drug delivery and imaging agents. The benchmarked mesoporous iron(III) trimesate MIL-100 MOF nanocarrier combines progressive release of high drug cargoes with absence of visible in vivo toxicity. Although in a previous study pharmacokinetics and biodistribution of MIL-100 nanoparticles were evaluated in the long term (from 24h to 1 month), the crucial times for drug targeting and delivery applications are shorter (up to 24h). Thus, this work aims to study the blood circulating profile and organ accumulation of MIL-100 nanocarrier at early times after administration. For this purpose, after intravenous administration to rats, both constitutive components of MIL-100 (trimesate and iron) were quantified by high performance liquid chromatography and a spectrophotometric method, respectively. The pharmacokinetic profile suggested that the nanoparticles act as a depot in the blood stream during the first hours before being cleared. Accumulation took mainly place in the liver and, in some extent, in the spleen. Nevertheless, histological studies demonstrated the absence of morphological alterations due to the presence of the particles in these organs. Liver function was however slightly altered as reflected by the increased plasma aspartate aminotransferase concentrations. Finally trimesate was progressively eliminated in urine.

Antineoplastic busulfan encapsulated in a metal organic framework nanocarrier: first in vivo results.

Nanoparticles of a mesoporous iron(iii) trimesate MIL-100 nanocarrier encapsulating high amounts of the challenging antineoplastic busulfan were administered to rats and compared with the commercial Busilvex®. Large differences in serum concentration of both busulfan and trimesate revealed the great impact of drug encapsulation both on the drug and on nanoparticle pharmacokinetics during the first 24 h of administration.

An aquaporin 4 antisense oligonucleotide loaded, brain targeted nanoparticulate system design.

Aquaporins (AQPs), members of the water-channel protein family, are highly expressed in brain tissue especially in astrocytic end-feet. They are important players for water hemostasis during development of cytotoxic as well as vasogenic edema. Increased expression of AQPs is important in pathophysiology of neurological diseases such as neuroinflammation and ischemia. Unfortunately, there are a few pharmacological inhibitors of AQP4 with several side effects limiting their translation as a drug for use in clinical conditions. Another therapeutic approach is using antisense oligonucleotides (ASOs) to block AQP4 activity. These are short, synthetic, modified nucleic acids that bind RNA to modulate its function. However, they cannot pass the blood brain barrier (BBB). To overcome this obstacle we designed a nanoparticulate system made up of chitosan nanoparticles surface modified with PEG and conjugated with monoclonal anti transferrin receptor-1 antibody via streptavidin-biotin binding. The nanocarrier system could be targeted to the transferrin receptor-1 at the brain endothelial capillaries through monoclonal antibodies. It is hypothesized that the nanoparticles could pass the BBB via receptor mediated transcytosis and reach brain parenchyma. Particle size, zeta potential, loading capacity and release profiles of nanoparticles were investigated. It was observed that all types of chitosau (CS) nanoparticles had positive zeta potential values and nanoparticle particle size distribution varied between 100 and 800 nm. The association efficiency of ASOs into the nanoparticles was between 80-97% and the release profiles of the nanoparticles exhibited an initial burst effect followed by a controlled release. The results showed that the designed chitosan based nanocarriers could be a promising carrier system to transport nucleic acid based drugs to brain parenchyma.

Nanomedicine as a promising approach for the treatment and diagnosis of brain diseases: the example of Alzheimer's disease.

Targeting of the central nervous system (CNS) in order to treat disorders is actually challenging due to the necessity to cross the blood brain barrier (BBB). This review aims to show how nanomedicine can propose new approach for the treatment and the diagnosis of CNS diseases focusing on Alzheimer's disease (AD). AD is a neurodegenerative disorder prevalent in the senile population. It is characterized by severe neuronal loss and proliferation of plaques composed of ß-amyloid peptide (Aß) and Tau protein deposites. An imbalance between production and clearance leading to the aggregation of Aß peptides especially in neurotoxic forms, may be the initiating factor in AD. The absence of an effective therapeutic approach nowadays could be, in part, due to the bad knowledge of AD physiopathology and the lack of early diagnosis. Many drawbacks such as poor bioavailability or limited BBB arising of tested molecules in the current or new therapeutic strategies explain their failure but can be resolved by the use of nanotechnology. Examples of recently published works using nanoparticles for improving diagnosis and therapy of AD are presented. Ideal nanocarriers for this aim must be able to pass through the BBB and to interact with an AD marker as soluble extracellular Aß forms which are known as the most toxic ones. These first results, even if many ones were obtained in vitro, brought to light the potential of nanoparticles for this challenging issue.

Nanoparticles in drug delivery: past, present and future.

This opinion paper relates how nanoparticles were discovered in the seventies and how the development of biodegradable materials and nanoparticle surface functionalization has allowed new treatment strategies. The reasons why only very few nanoparticle-based medicines are on the market or in late clinical trials are discussed and some new approaches are identified. Future challenges in the nanoparticle field are also identified.

Porous metal organic framework nanoparticles to address the challenges related to busulfan encapsulation.

Busulfan is an alkylating agent widely used in chemotherapy, but with severe side effects. Many attempts have been made to entrap busulfan in nanocarriers to avoid liver accumulation and to protect it against rapid degradation in aqueous media. However, poor loadings (≤ 5 wt%) and fast release were generally obtained due to the low affinity of busulfan towards the nanocarriers. Moreover, drug crystallization often occurred during nanoparticle preparation. To circumvent these drawbacks, metal organic framework (MOF) nanoparticles, based on crystalline porous iron (III) carboxylates, have shown an unprecedented loading (up to 25 wt%) of busulfan. This was attributed to the high porosity of nanoMOFs as well as to their hydrophilic-hydrophobic internal microenvironment well adapted to the amphiphilic character of busulfan. NanoMOFs formulations have kept busulfan in molecular form, preventing its crystallization and degradation. Indeed, busulfan was released intact, as proved by the maintenance of its pharmacological activity.

Interaction of an amphiphilic squalenoyl prodrug of gemcitabine with cellular membranes.

We have designed an amphiphilic prodrug of the anticancer agent gemcitabine (dFdC), by covalent coupling to squalene. This bioconjugate, which self-assembled into nanoparticles (NPs) in water, was previously found to display an impressive anticancer activity both in vitro and in vivo. The present study aims to investigate the impact of SQdFdC nanoparticles on cellular membranes. MTT assays showed that, in the nanomolar range, squalenoyl gemcitabine (SQdFdC) was slightly less active than dFdC on a panel of human cancer cell lines, in vitro. However, above 10 µmol L(-1) SQdFdC was considerably more cytotoxic than dFdC. Contrarily to its parent drug, SQdFdC also induced cell lysis in a few hours, as evidenced by LDH release assays. Erythrocytes were used as an experimental model insensitive to the antimetabolic activity of dFdC to further investigate the putative membrane-related cytotoxic activity of SQdFdC. The bioconjugate also induced hemolysis in a time- and dose-dependent fashion, unlike squalene or dFdC, which clearly proved that SQdFdC could permeabilize cellular membranes. Structural X-ray diffraction and calorimetry studies were conducted in order to elucidate the mechanism accounting for these observations. They confirmed that SQdFdC could be transferred from NPs to phospholipid bilayers and that the insertion of the prodrug within model membranes resulted in the formation of nonlamellar structures, which are known to promote membrane leakage. As a whole, our results suggested that due to its amphiphilic nature, the cell uptake of SQdFdC resulted in its insertion into cellular membranes, which could lead to the formation of nonlamellar structures and to membrane permeation. Whether this mechanism could be the source of toxicity in vivo, however, remains to be established, since preclinical studies have clearly proven that squalenoyl gemcitabine displayed a good toxicity profile.

Prodrug-based intracellular delivery of anticancer agents.

There are numerous anticancer agents based on a prodrug approach. However, no attempt has been made to review the ample available literature with a specific focus on the altered cell uptake pathways enabled by the conjugation and on the intracellular drug-release mechanisms. This article focuses on the cellular interactions of a broad selection of parenterally administered anticancer prodrugs based on synthetic polymers, proteins or lipids. The report also aims to highlight the prodrug design issues, which are key points to obtain an efficient intracellular drug delivery. The chemical basis of these molecular concepts is put into perspective with the uptake and intracellular activation mechanisms, the in vitro and in vivo proofs of concepts and the clinical results. Several active targeting strategies and stimuli-responsive architectures are discussed throughout the article.

Transmembrane diffusion of gemcitabine by a nanoparticulate squalenoyl prodrug: an original drug delivery pathway.

We have designed an amphiphilic prodrug of gemcitabine (dFdC) by its covalent coupling to a derivative of squalene, a natural lipid. The resulting bioconjugate self-assembled spontaneously in water as nanoparticles that displayed a promising in vivo anticancer activity. The aim of the present study was to provide further insight into the in vitro subcellular localization and on the metabolization pathway of the prodrug. Cells treated with radiolabelled squalenoyl gemcitabine (SQdFdC) were studied by differential detergent permeation, and microautography coupled to fluorescent immunolabeling and confocal microscopy. This revealed that the bioconjugate accumulated within cellular membranes, especially in those of the endoplasmic reticulum. Radio-chromatography analysis proved that SQdFdC delivered dFdC directly in the cell cytoplasm. Mass spectrometry studies confirmed that gemcitabine was then either converted into its biologically active triphosphate metabolite or exported from the cells through membrane transporters. To our knowledge, this is the first description of such an intracellular drug delivery pathway. In vitro cytotoxicity assays revealed that SQdFdC was more active than dFdC on a transporter-deficient human resistant leukemia model, which was explained by the subcellular distribution of the drugs and their metabolites. The squalenoylation drug delivery strategy might, therefore, dramatically improve the efficacy of gemcitabine on transporter-deficient resistant cancer in the clinical context.

Cyclodextrins for drug delivery.

Cyclodextrins (CDs) are macrocyclic oligosaccharides composed of α(1,4)-linked glucopyranose subunits. These molecules possess a cage-like supramolecular structure, comparable with the structures of crown ethers, cryptands, spherands, cyclophanes, or calixarenes. However, it took 50 years to establish the molecular structure of CDs. Owing to their capability to form inclusion complexes with a variety of guest molecules, CDs are considered as the most important supramolecular host family among all supramolecular structures mentioned above. They can form complexes with various types of molecules including inorganic, organic, or organometallic that can be radical, cationic, anionic, or neutral molecules. This phenomenon bears the name "molecular recognition," while the selectivity in the formation of complexes with enantiomeric species as guests is called "chiral recognition." In addition, the properties of the molecules forming the complexes with CDs can be modified significantly. As such, a large number of scientists have attempted to elaborate and evaluate various CD derivatives that are able to complex a variety of drugs, enhancing by this way their in vivo solubility and activity. Moreover, a large number of publications describe CD uses in other fields such as foods, textile, cosmetics, or agriculture. This review reports on the recent developments of CDs in drug delivery using various routes of administration.

Freeze-drying of squalenoylated nucleoside analogue nanoparticles.

Nucleoside analogues are potent anticancer or antiviral agents that however display some limitations (rapid metabolism, induction of resistance). In order to overcome these drawbacks, we recently proposed new prodrugs, in which nucleoside analogues were covalently coupled to squalene (SQ). The resulting amphiphilic compounds spontaneously formed nanoparticles (NPs) and displayed a promising efficacy both in vitro and in vivo. Since long-term stability is essential for further clinical development we needed to develop a laboratory-scale freeze-drying protocol in order to improve the colloidal stability of those NPs. Squalenoylated gemcitabine (SQdFdC) has been successfully freeze-dried with trehalose (10%, w/w) as a cryoprotectant. Concentrations of SQdFdC up to 4mg/mL after freeze-drying and rehydration have been obtained, which is necessary for in vivo studies. Stability measurements by dynamic light scattering showed that trehalose had a stabilizing effect on SQdFdC NPs, and that freeze-dried SQdFdC NPs could be stored up to four months at room temperature before rehydration, without loss of stability. In vitro cytotoxicity studies on three murine cell lines showed that SQdFdC NPs retained their cytotoxic activity after freeze-drying. We showed that this freeze-drying protocol could also be applied to squalenoylated didanosine (SQddI) and zalcitabine (SQddC). Overall, these results allow for the use of freeze-dried NPs in upcoming preclinical trials of the different squalenoylated compounds developed in our laboratory.

Preparation and evaluation of alpha-phenyl-n-tert-butyl nitrone (PBN)-encapsulated chitosan and PEGylated chitosan nanoparticles.

Alpha-phenyl-n-tert-butyl nitrone (PBN) shows its major effect by scavenging free radicals formed in the ischemia and it has the ability to penetrate through the blood brain barrier easily. The in vivo stability of PBN is very low and when administered systemically, it has a mean plasma half life of about three hours. Therefore, formulations which are able to prolong the plasma residence time of PBN are of major interest, because oxygen radicals are usually continuously formed under pathological conditions. In this study, PBN, a nitrone compound having neuroprotective properties, was encapsulated in chitosan (CS) and chitosan-poly(ethylene glycol) (CS-PEG) nanoparticles for treatment of diseases such as stroke, in which sustained free radical production is reported. The nanoparticles were characterized through particle size determination, zeta potential, encapsulation efficiency, surface morphology determinations and in vitro release studies. The surface morphologies were evaluated by transmission electron microscopy (TEM) and nanoparticles having spherical shapes were characterized. The particle size distribution was between approximately 97 nm and approximately 322 nm; and the zeta potentials varied between approximately 9 mV and approximately 33 mV. Size of the nanoparticle formulations was important for the release of PBN from nanoparticles. The quantitative determination of PBN has been evaluated by a validated analytical HPLC method. The presented chitosan-based nanotechnology opens new perspectives for testing antioxidant activity in vivo.

Paraquat detoxication with multiple emulsions.

In this study, we show that detoxifying W/O/W multiple emulsions, prepared with an appropriate extractant/trapping couple, represent a promising technology for quick and safe poisoning treatments, with application to the highly toxic herbicide Paraquat, responsible of poisonings from low-dose exposure leading to several deaths every year. In vitro tests led to the choice of an appropriate extractant/trapping couple system with significant detoxication performance. In vivo tests showed (i) that rats receiving high doses of Paraquat, then a detoxifying emulsion, presented an increase from 50% to 100% of the MST (median survival time) and (ii) that no mortality was observed during 30 days with rats dosed with emulsions initially loaded with Paraquat at a concentration much higher than the lethal dose, proving the stability and the inocuity of the detoxifying multiple emulsion in the gastrointestinal tract.

Polymer nanocarriers for the delivery of small fragments of nucleic acids: oligonucleotides and siRNA.

The success of the application of new therapeutic methods based on RNA interfering strategies requires the in vivo delivery of active ODN or siRNA down to the intracellular compartment of the target cells. This article aims to review the studies related to the formulation of RNA interfering agents in polymer nanocarriers. It will present the different types of polymer nanocarriers used as well as the biological activity of the resulting ODN and siRNA loaded nanocarriers. As will be explained, the part of the in vitro studies provided useful data about the intracellular delivery of the formulated RNA interfering agents. Investigations performed in vivo have considered animal models of different relevant diseases. Results from these investigations have clearly demonstrated the interest of several polymer nanocarriers tested so far to deliver active RNA interfering effectors in vivo making possible their administration by the intravenous route.