[Targeting for drug delivery and imaging: an overview of the state of the art in cancer]. / Vectorisation à visée thérapeutique ou diagnostique : une synthèse de l'état de l'art dans le domaine du cancer.

The concept of drug targeting has resulted in a considerable variety of tools within nanotechnology, whose potentials in the special field of cancer are very interesting. There is a strong link between the structure and the surface of the vector on the one hand, and the biological target thus accessible on the other hand, as illustrated a number of typical examples. Parallel developments were made in the fields of therapy and diagnosis, stage by stage. The challenges now live in the combination of approaches widely reported under the terminology of "theranostics".

PEGylation and preliminary biocompatibility evaluation of magnetite-silica nanocomposites obtained by high energy ball milling.

High energy ball milling (HEBM) has been used for the first time to prepare PEGylated magnetite-silica (Fe(3)O(4)-SiO(2)) nanocomposites intended to be used for biological purposes. Surface amine groups were introduced by a silanization reaction involving 3-aminopropyl triethoxysilane (APTS) followed by PEGylation to yield long-term stable and stealth nanocomposites of 200nm in diameter. The efficient coverage by PEG chains was shown by isothermal titration calorimetry (ITC) where PEGylated nanocomposites did not interact with BSA compared to non-PEGylated counterparts which led to a significant change in enthalpy. By cell viability (MTT) assays and cell morphology investigations, it was evidenced that PEGylated Fe(3)O(4)-SiO(2) nanocomposites did not provide any appreciable cytotoxicity on J774 macrophage and MCF-7 breast cancer cell lines. Furthermore, noticeable internalization was evidenced by J774 cells with PEGylated Fe(3)O(4)-SiO(2) nanocomposites in contrast to MCF-7 cells, in good agreement with the respective tendency of each cell line for endocytosis.

Folic acid-targeted mesoporous silica nanoparticles for two-photon fluorescence.

New nanotools for the imaging of cancer cells have been synthesized. Two-photon dye-doped 3-aminopropyltriethoxysilane-grafted mesoporous silica nanoparticles (MSN) have been grafted with folic acid (FA) functionalized PEG groups. Amine-PEG groups were first reacted with an activated ester derivative of FA. A mixture of FA- and hydroxyl-PEG has then been reacted with the amino groups at the surface of the particles. Cell culture experiments performed with MCF7 and HeLa cancer cells demonstrated that these functionalized MSN showed a low cytotoxicity even after a 24 hours incubation time at high concentrations. These modified MSN are promising for applications in the field of two-photon imaging and their potentiality for photodynamic therapy is currently being investigated.

[Mesoporous silica nanoparticles for two-photon fluorescence]. / Nanoparticules de silice mésoporeuse optimisées pour la fluorescence à deux photons.

Mesoporous silica nanoparticles have unique properties: a specific large surface or a narrow casting of the sizes of pores. The perspectives of use are the creation of new tools for the premature diagnosis. For these potential biological applications, the harmlessness of these nanoparticles must be established.

Squalenoyl nanomedicine of gemcitabine is more potent after oral administration in leukemia-bearing rats: study of mechanisms.

In an earlier report, we demonstrated the superior anticancer efficacy of orally administered squalenoyl gemcitabine (SQdFdC) nanomedicine over its parent drug gemcitabine on rats bearing RNK-16 large granular lymphocytic (LGL) leukemia. In the present communication, we investigated the mechanisms behind this observation both at the cell and tissue level. The mechanisms were investigated by performing cytotoxicity, cell uptake, and biodistribution experiments. In the presence of cytidine deaminase, SQdFdC nanoassemblies resisted deamination and exerted significant anticancer activity in vitro against RNK-16 LGL leukemia cells, whereas the cytotoxicity of free gemcitabine decreased by approximately 83-fold, indicating its degradation due to deamination. Additionally, the SQdFdC showed considerably higher intracellular accumulation and retention compared with gemcitabine (P<0.05). Unlike gemcitabine, the cellular access to SQdFdC was not influenced by nucleoside transporters. When administered orally to rats, unlike H-gemcitabine, the H-SQdFdC absorbed slowly, but exhibited an improved pharmacokinetics and tissue distribution profile, particularly in the lymphoid organs (the major organs of metastasis). The resistance to deamination, followed by the improved pharmacokinetic and tissue distribution, and greater accumulation and retention at the level of cancer cells, are the key factors for the superiority of SQdFdC nanoassemblies over free gemcitabine against RNK-16 LGL leukemia in rats.

Squalenoylation favorably modifies the in vivo pharmacokinetics and biodistribution of gemcitabine in mice.

Gemcitabine (2',2'-difluorodeoxyribofuranosylcytosine; dFdC) is an anticancer nucleoside analog active against wide variety of solid tumors. However, this compound is rapidly inactivated by enzymatic deamination and can also induce drug resistance. To overcome the above drawbacks, we recently designed a new squalenoyl nanomedicine of dFdC [4-N-trisnorsqualenoyl-gemcitabine (SQdFdC)] by covalently coupling gemcitabine with the 1,1',2-trisnorsqualenic acid; the resultant nanomedicine displayed impressively greater anticancer activity compared with the parent drug in an experimental murine model. In the present study, we report that SQdFdC nanoassemblies triggered controlled and prolonged release of dFdC and displayed considerably greater t(1/2) (approximately 3.9-fold), mean residence time (approximately 7.5-fold) compared with the dFdC administered as a free drug in mice. It was also observed that the linkage of gemcitabine to the 1,1',2-trisnorsqualenic acid noticeably delayed the metabolism of dFdC into its inactive difluorodeoxyuridine (dFdU) metabolite, compared with dFdC. Additionally, the elimination of SQdFdC nanoassemblies was considerably lower compared with free dFdC, as indicated by lower radioactivity found in urine and kidneys, in accordance with the plasmatic concentrations of dFdU. SQdFdC nanoassemblies also underwent considerably higher distribution to the organs of the reticuloendothelial system, such as spleen and liver (p < 0.05), both after single- or multiple-dose administration schedule. Herein, this paper brings comprehensive pharmacokinetic and biodistribution insights that may explain the previously observed greater efficacy of SQdFdC nanoassemblies against experimental leukemia.

Preclinical toxicology (subacute and acute) and efficacy of a new squalenoyl gemcitabine anticancer nanomedicine.

This study investigates 1) the anticancer efficacy of a new squalenoyl prodrug of gemcitabine (SQgem) in nanoassembly form compared with gemcitabine at equitoxic doses and 2) the subacute and acute preclinical toxicity of these compounds. The toxicity studies revealed that SQgem nanoassemblies, like gemcitabine, were toxic, and they led to dose-dependent mortality after daily i.v. injections for 1 week, irrespective of the route of administration. However, a 4- to 5-day spaced dosing schedule (injections on day 0, 4, 8, and 13) was proved to be safer in terms of weight loss and hematological and other toxicity. Using this spaced dosing schedule, SQgem nanoassemblies exhibited impressive anticancer activity in mice bearing L1210 leukemia because this treatment led to 75% long-term survivors. In contrast, at equitoxic doses, neither free gemcitabine nor cytarabine led to longterm survivors and all the mice of these groups died of the disease. Further toxicity studies performed at lethal doses by blood and serum analysis and organ weight determinations revealed that the hematological toxicity was the dose-limiting toxicity in both SQgem nanoassemblies and gemcitabine, whereas probable gastrointestinal toxicity was also associated with free gemcitabine. The SQgem nanoassemblies did not display hepatotoxicity, which is one of the clinically encountered toxicities of gemcitabine. To summarize, these preclinical studies demonstrated that the toxicological profile of new squalenoyl gemcitabine nanomedicine was not distinct from that of the parent gemcitabine, whereas it was much more potent than gemcitabine at equitoxic doses and cytarabine at clinically relevant doses. These data support the candidature of SQgem for clinical trials.

A new nanomedicine of gemcitabine displays enhanced anticancer activity in sensitive and resistant leukemia types.

Gemcitabine is an anticancer nucleoside analogue active against various solid tumors. However, it possesses important drawbacks like a poor biological half-life and the induction of resistance. With the objective of overcoming the above drawbacks, we designed a new nanomedicine of gemcitabine and studied its anticancer efficacy against leukemia at preclinic. Gemcitabine has been covalently coupled with 1,1',2-tris-nor-squalenic acid to obtain the new anticancer nanomedicine 4-(N)-Tris-nor-squalenoyl-gemcitabine (SQdFdC NA). The SQdFdC NA exhibited, in comparison to gemcitabine, 3.26- and 3.22-folds higher cytotoxicity respectively, in murine resistant leukemia L1210 10K cells and in human leukemia resistant cell line CEM/ARAC8C. Following intravenous treatment of murine aggressive metastatic leukemia L1210 wt bearing mice, the SQdFdC NA caused significant increase in survival time compared to gemcitabine and also led to long-term survivals, which was not the case after gemcitabine treatment. This was attributed to significantly higher deposition of SQdFdC NA in spleen and liver (P<0.05), the major metastatic organs. In comparison to gemcitabine, SQdFdC NA displayed greater ability to induce S-phase arrest of the cancer cells followed by increased apoptotic induction. Interestingly, like gemcitabine, SQdFdC NA didn't induce appreciable differences in blood parameters even at doses higher than those used for anticancer evaluation. The preclinical data obtained in vitro and in vivo with SQdFdC NA demonstrate that this nanomedicine represents a new therapeutic system for the effective treatment of leukemia.

Colloidal stability of ultrasmall superparamagnetic iron oxide (USPIO) particles with different coatings.

Ultrasmall superparamagnetic iron oxide (USPIO) particles are efficient contrast agents used in vivo to enhance relaxation differences between healthy and pathological tissues. Detailed understanding of their physicochemical properties in suspension is necessary to guarantee the quality and safety of biological USPIO particles application. The ferrofluids stability against aggregation and gravitational settling affects their biodistribution and consequently the resulting contrast. In this study, the stability of iron oxide particles was investigated by dynamic light scattering (DLS) at different NaCl concentrations in order to monitor the evolution of the hydrodynamic radius of the particles with time. The results were interpreted using the classical DLVO theory of colloidal stability. The electrophoretic mobility and the models generally used to convert it to zeta potential were discussed and related to the stability results.

Physicochemical characterization of ultrasmall superparamagnetic iron oxide particles (USPIO) for biomedical application as MRI contrast agents.

Ultrasmall superparamagnetic iron oxide (USPIO) particles are maghemite or magnetite nanoparticles currently used as contrast agent in magnetic resonance imaging. The coatings surrounding the USPIO inorganic core play a major role in both the in vitro stability and, over all, USPIO's in vivo fate. Different physicochemical properties such as final size, surface charge and coating density are key factors in this respect. Up to now no precise structure--activity relationship has been described to predict entirely the USPIOs stability, as well as their pharmacokinetics and their safety. This review is focused on both the classical and the latest available techniques allowing a better insight in the magnetic core structure and the organic surface of these particles. Concurrently, this work clearly shows the difficulty to obtain a complete physicochemical characterization of USPIOs particles owing to their small dimensions, reaching the analytical resolution limits of many commercial instruments. An extended characterization is therefore necessary to improve the understanding of the properties of USPIOs when dispersed in an aqueous environment and to set the specifications and limits for their conception.

Squalenoyl nanomedicines as potential therapeutics.

Nucleoside analogues display significant anticancer or antiviral activity by interfering with DNA synthesis. However, there are some serious restrictions to their use, including their rapid metabolism and the induction of resistance. We have discovered that the linkage of nucleoside analogues to squalene leads to amphiphilic molecules that self-organize in water as nanoassemblies of 100-300 nm, irrespective of the nucleoside analogue used. The squalenoyl gemcitabine exhibited superior anticancer activity in vitro in human cancer cells and gemcitabine-resistant murine leukemia cells, and in vivo in experimental leukemia both after intravenous and oral administration. The squalenoylation of other antiretroviral nucleosides also led to more potent drugs when tested in primary cultures of HIV-infected lymphocytes. Thus, the squalenoylation is an original technology platform for generating more potent anticancer and antiviral nanomedicines.

Structural characterization of ultrasmall superparamagnetic iron oxide (USPIO) particles in aqueous suspension by energy dispersive X-ray diffraction (EDXD).

Ultrasmall superparamagnetic iron oxide (USPIO) particles were structurally characterized in situ in an aqueous dilute suspension by energy dispersive X-ray diffraction (EDXD) and ex situ as powders obtained by lyophilization of the suspension by angular dispersive X-ray diffraction (ADXD) at 20 degrees C. Structural parameters obtained by the Rietveld method on ADXD data were used as starting parameters for modeling the structure of the particles in suspension. Although each particle is a single crystal, as evidenced by conventional X-ray diffraction, our results indicate that the structural order, specific to a crystal, does not extend to the entire volume of the particle. In fact, each individual particle, averagely, has a crystalline structural extension ca. 4.0 nm smaller than the apparent dimensions obtained by both ADXD and TEM (ca. 8.0 nm).

Folate-conjugated iron oxide nanoparticles for solid tumor targeting as potential specific magnetic hyperthermia mediators: synthesis, physicochemical characterization, and in vitro experiments.

New folate-conjugated superparamagnetic maghemite nanoparticles have been synthesized for the intracellular hyperthermia treatment of solid tumors. These ultradispersed nanosystems have been characterized for their physicochemical properties and tumor cell targeting ability, facilitated by surface modification with folic acid. Preliminary experiments of nanoparticles heating under the influence of an alternating magnetic field at 108 kHz have been also performed. The nanoparticle size, surface charge, and colloidal stability have been assessed in various conditions of ionic strength and pH. The ability of these folate "decorated" maghemite nanoparticles to recognize the folate receptor has been investigated both by surface plasmon resonance and in folate receptor expressing cell lines, using radiolabeled folic acid in competitive binding experiments. The specificity of nanoparticle cellular uptake has been further investigated by transmission electron microscopy after incubation of these nanoparticles in the presence of three cell lines with differing folate receptor expression levels. Qualitative and quantitative determinations of both folate nanoparticles and nontargeted control nanoparticles demonstrated a specific cell internalization of the folate superparamagnetic nanoparticles.

Metallic colloid nanotechnology, applications in diagnosis and therapeutics.

In recent years the fields of medicine and biology assist to an ever-growing innovation related to the development of nanotechnologies. In the pharmaceutical domain, for example, liposomes, polymer based micro and nanoparticles have been subjects of intense research and development during the last three decades. In this scenario metallic particles, which use was already suggested in the first half of the '80, are now experiencing a real renaissance. In the field of diagnosis, magnetic resonance imaging is one of the first and up to now the most developed application of metallic particles. But beside this application, a very new generation of biosensors based on the optical properties of colloidal gold and fluorescent nanocrystals, called quantum dots seems to be ready to be implemented in diagnosis and medical imaging. Concerning therapeutic applications, the potentialities of metal nanoparticles to help fulfilling the need of time and space controlled release of drugs has been intuited for a long time. Nowadays, magnetically guided carriers or thermal responsive matrices, in which drug release is triggered by the heating of metal nanoparticles, are effective examples of their application in drug delivery, while more recently efforts to develop metallic nanoobjects to be used as vectors of nucleic acids for vaccination and transfection have been multiplied. In the future, one of the most interesting challenges is certainly the use of metallic nanoparticles for an innovating, effective and selective physical treatment of solid tumors via targeted intracellular hyperthermia.

Phospholipid hydrolysis in a pharmaceutical emulsion assessed by physicochemical parameters and a new analytical method.

The aim of this work was to develop a simple high-performance liquid chromatography (HPLC) technique with evaporative light scattering detection (ELSD) for the separation and quantification of the major phospholipid (PL) and lysophospholipid (LPL) classes contained in a pharmaceutical phospholipid-based emulsion. In the established method, phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyeline (SM), lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) were separated with a PVA-Sil stationary phase and a binary gradient from pure chloroform to methanol:water (94:6 v/v) at 3.4%/min. The ELSD detection was enhanced using 0.1% triethylamine and formic acid in each gradient mobile phases. Factors such as stationary phase and ELSD drift tube temperature were optimized, concluding in optimal temperatures of 25 degrees C for separation and 50 degrees C for evaporation. This HPLC-ELSD method was then applied to a PL-emulsion exposed to autoclaving and accelerated thermal conditions at 50 degrees C. Hydrolysis of PC and PE followed first-order kinetics, representing only 45% of the total lipid mass after 3 months. The chemical stability was correlated to commonly measured formulation physical and physico-chemical parameters such as droplet size, emulsion pH and zeta-potential.

Increase of doxorubicin sensitivity by doxorubicin-loading into nanoparticles for hepatocellular carcinoma cells in vitro and in vivo.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is known to be chemoresistant to anticancer drugs due to the multidrug resistant (MDR) transporters expression. Here, we compared in vitro and in vivo the anti-tumor efficacy of doxorubicin-loaded polyisohexylcyanoacrylate nanoparticles (PIHCA-Dox) versus free doxorubicin (Dox). These nanoparticles are known to overcome the MDR phenotype. METHODS: We first determined in vitro the 50% inhibition concentration (IC(50)) of these drugs on different human hepatoma cell lines. Secondly, the efficacy of the drugs in vivo was determined on the X/myc transgenic murine model of HCC by histological counting of apoptotic tumorous hepatocytes and by TUNEL labeling. We characterized by semi-quantitative RT-PCR the MDR-related gene (mdr1, mdr3, mrp1) expression pattern in this model. RESULTS: In vitro, IC(50) was reduced with PIHCA-Dox versus Dox for Huh7 (1.7-fold reduction; P<0.001), HepaRG (4.5-fold reduction; P<0.01), HepG2 (1.5-fold reduction; P<0.001), and HepG2.2.15 (1.5-fold reduction; P=0.059). In vivo, HCC in transgenic mice overexpressed the mdr1 and mdr3 genes and the antitumor drugs efficacy was greatly enhanced after injection of PIHCA-Dox (9.0+/-5.0%; n=15) versus Dox (4.6+/-3.3%; n=13; P=0.01) for apoptotic bodies count. CONCLUSIONS: These promising data showing a higher anti-tumor efficacy on HCC of PIHCA-Dox versus Dox, warrant further studies in both animals and humans.

Cationic vectors in ocular drug delivery.

Despite extensive research in the field, the major problem in the ocular drug delivery domain still is rapid precorneal drug loss and poor corneal permeability. One of the approaches recently developed is the drug incorporation into cationic submicronic vectors which exploit the negative charges present at the corneal surface for increased residence time and penetration. This review will focus on the formulation of three main representative cationic colloids developed for ophthalmic delivery: liposomes, emulsions and nanoparticles (NP). Parameters such as choice of the vector type and size, nature of the cationic molecule, pH and ionic strength of the external phase and characteristics of the encapsulated drug will be discussed with accent on the relevance of the positive charge.

Specific antitumor targetable beta-cyclodextrin-poly(ethylene glycol)-folic acid drug delivery bioconjugate.

The tumor targeting properties of a new drug carrier synthesized by bioconjugation of folic acid (FA) to beta-cyclodextrins through a poly(ethylene glycol) (PEG) spacer (CD-PEG-FA) were investigated. Surface plasmon resonance demonstrated that CD-PEG-FA specifically interacts with immobilized folate binding protein (FBP) while the naked beta-cyclodextrins do not display any specific interaction. In vitro studies demonstrated that CD-PEG-FA was devoid of cell toxicity. [(3)H]-folic acid/CD-PEG-FA competition binding investigations performed with folate receptor overexpressing human epidermal carcinoma KB cells showed that CD-PEG-FA had about 14 times lower tumor cell binding capacity than free folic acid. The carrier cell trafficking properties were investigated using rhodamine-B as fluorescent probe, which possesses 3000 and 4580 M(-)(1) inclusion constants for CD-PEG-FA and beta-cyclodextrins, respectively. Cell-associated fluorescence measurements showed that CD-PEG-FA does not promote the rhodamine-B uptake into non-folate receptor expressing human lung carcinoma MCF7 cells while 19% higher accumulation in KB cells was found with respect to rhodamine-B loaded beta-cyclodextrins. Confocal laser scanning microscopy indicated the presence of cytosolic red fluorescent spots after 2 h of incubation of KB cells with rhodamine-B included CD-PEG-FA. The fluorescent dye resided primarily in small spots, namely, endosomes and multivesicular bodies. At 1 h after pulsed incubation, wider red fluorescent cellular structures appeared as a fusion of previous structures.

Extensive surface studies help to analyse zeta potential data: the case of cationic emulsions.

The present study is aimed to characterize the electrostatic parameters of oil in water emulsion droplets composed of MCT (medium chain triglycerides), PL (phospholipids) and Poloxamer and containing increasing concentrations of the cationic lipid oleylamine (OA), in Hepes 20 mM pH 7.4. The initial zeta-potential data suggesting saturation of the droplet surface at high OA concentrations were completed by supplementary analysis: the distribution of the oleylamine within the droplet was determined by reacting the amino groups with the hydrophilic TNBS (trinitrobenzenesulfonic acid), the method being initially standardised with vesicles. In addition, surface potential and pH at the droplet surface were monitored by the pH-sensitive fluorophore 4-heptadecyl-7-hydroxycoumarin. Our results demonstrate that almost all the OA is localised and fully ionised at the droplet surface for all concentrations and that the observed plateau in the zeta-potential values obeys the Gouy-Chapman theory of ion condensation. It is also shown that the slipping plane separation as estimated by the Eversole-Boardman equation is higher that the expected values of 0.2 nm as a result of the relative position of the fluorophore and the outer boundary of the lipid interface thickness and the Poloxamer anchored at the interface only plays a minor role.

"Smart" delivery of antisense oligonucleotides by anionic pH-sensitive liposomes.

Antisense oligonucleotides are molecules that are able to inhibit gene expression being, therefore, potentially active for the treatment of viral infections cancer or inflammatory diseases. However, because of their poor stability in biological medium and their weak intracellular penetration, "smart" delivery systems such as anionic pH-sensitive liposomes were designed. Most known liposome formulations contain a specific phospholipid, phosphatidylethanolamine (PE), which undergoes a transition from lamellar to inverted micelles structures at low pH and allow fusion of liposomal and endosomal membranes and by consequence destabilization of the endosomes. Therefore, liposomes made of PE are able to release their contents in response to acidic pH within the endosomal system while remaining stable in plasma thus improving the cytoplasmic delivery of oligonucleotides after endocytosis. This review illustrates the advantages of this approach for the delivery of antisense oligonucleotides.