Biodistribution and preliminary toxicity studies of nanoparticles made of Biotransesterified ß-cyclodextrins and PEGylated phospholipids.

BACKGROUND: The modification of ß-cyclodextrins (ßCDs) by grafting alkyl chains on the primary and/or secondary face yields derivatives (ßCD-C10) able to self-organize under nanoprecipitating conditions into nanoparticles (ßCD-C10-NP) potentially useful for drug delivery. The co-nanoprecipitation of ßCD-C10 with polyethylene glycol (PEG) chains yields PEGylated NPs (ßCD-C10-PEG-NP) with potentially improved stealthiness. The objectives of the present study were to characterize the in vivo biodistribution of ßCD-C10-PEG-NP with PEG chain length of 2000 and 5000Da using nuclear imaging, and to preliminarily evaluate the in vivo acute and extended acute toxicity of the most suitable system. RESEARCH DESIGN AND METHODS: The in vivo and ex vivo biodistribution features of naked and decorated nanoparticles were investigated over time following intravenous injection of 125I-radiolabeled nanoparticles to mice. The potential toxicity of PEGylated ßCD-C10 nanosuspensions was evaluated in a preliminary in vivo toxicity study involving blood assays and tissue histology following repeated intraperitoneal injections of nanoparticles to healthy mice. RESULTS: The results indicated that ßCD-C10-PEG5000-NP presented increased stealthiness with decreased in vivo elimination and increased blood kinetics without inducing blood, kidney, spleen, and liver acute and extended acute toxicity. CONCLUSIONS: ßCD-C10-PEG5000-NPs are stealth and safe systems with potential for drug delivery.

Biodistribution of intravenously administered amphiphilic beta-cyclodextrin nanospheres.

Amphiphilic beta-cyclodextrin (betaCDa) nanospheres (mean diameter 90-110 nm) prepared by the solvent displacement method were developed as a colloidal drug delivery system. In order to survey the fate of these nanoparticles, the amphiphilic beta-cyclodextrin was first iodinated by a two-step procedure involving iodination of the primary face followed by an acylation of the secondary face. After radiolabeling of this derivative with (125)I, nanospheres made of betaCDa/betaCDa (125)I were formulated. After a single intravenous injection of labeled nanoparticles in mice, the organ distribution was analyzed from 10 min to 6 days. A rapid clearance of (125)I-labeled betaCDa nanospheres from the blood circulation to the mononuclear phagocyte system was visualized by non-invasive planar imaging study. Radioactivity measurements in organs showed that the nanospheres mainly concentrated in the liver and the spleen where 28 and 24% of the radioactivity per gram of organ was, respectively, found 10 min after injection. At the opposite, the blood activity was low at that time and become negligible thereafter. Finally, the fact that no particular sign of toxicity is observed in injected animals should be emphasized since it is the first report on intravenous administration of betaCDa nanoparticles.

Miscellaneous nanoaggregates made of beta-CD esters synthesised by an enzymatic pathway.

Various beta-cyclodextrin (beta-CD) fatty esters with different chain lengths (C4-C14) were synthesised by transesterification of beta-cyclodextrin by vinyl fatty ester using thermolysin in DMSO. For each cyclodextrin derivatives, two batches of synthesis were realized. The ability of these derivatives to form nano-organized systems was investigated through the solvent displacement technique. During the formulation step, the effects of the initial concentration of beta-CD fatty esters in the organic phase and that of the final volume of the aqueous non-solvent phase were studied. Except for the beta-CD C4 ester, the transesterified beta-CD derivatives led to measurable nanoparticles. Cryo-electron microscopy images showed a significant morphological variability. Spherical, rod-like or more irregularly-shaped nano-objects were observed with either matricial or lamellar structures. A statistical analysis by a two-way ANOVA was computed for each class of beta-cyclodextrin esters in order to determine the effects of batch and formulation on the final size of nanoparticles.

Enhanced degradation of fluorene in soil slurry by Absidia cylindrospora and maltosyl-cyclodextrin.

This study investigates the fungal biodegradation of fluorene, a polycyclic aromatic hydrocarbon, in liquid medium and soil slurry. Fungal strains and cyclodextrins were used in order to degrade fluorene and optimize fluorene bioavailability and degradation in soil slurries. After a procedure of selection in solid and liquid media, maltosyl-cyclodextrin, a branched cyclodextrin was chosen. 47 fungal strains isolated from a contaminated site were tested for biodegradation. Results showed the greater efficiency of "adapted" fungi isolated from contaminated soil vs reference strains belonging to the collection of the laboratory. These assays allowed us to select the most efficient strain, Absidia cylindrospora, which was used in a bioaugmentation process. Bioaugmentation tests were performed in an artificially contaminated non-sterile soil. In the presence of A. cylindrospora, more than 90% of the fluorene was degraded within 288 h, while 576 h were necessary in the absence of fungal bioremediation. It also appeared that biodegradation was enhanced by amendment with previously selected maltosyl-cyclodextrin. The results of this study indicate that A. cylindrospora and maltosyl-cyclodextrin could be used successfully in fluorene bioremediation systems.

Long-term shelf stability of amphiphilic beta-cyclodextrin nanosphere suspensions monitored by dynamic light scattering and cryo-transmission electron microscopy.

Amphiphilic beta-cyclodextrins (betaCDa) were synthesized by statistically grafting hexanoyl carbon chains on the secondary hydroxyl functions of the betaCD glucopyranosyl units. The obtained derivative was used to prepare submicronic colloidal nanosphere suspensions using a nano-precipitation method. The fresh suspensions contained particles with a diameter ranging from 60-100 nm. Taking into account that the physical stability of colloidal systems remains one of the major problems which can restrict their use in pharmaceutical particulate carrier formulations, the long-term stability of the aqueous nano-dispersions was investigated. Two complementary characterization methods, namely dynamic light scattering and cryo-transmission electron microscopy, were used to control the size distribution and morphology of the nanospheres during storage. The zeta potential was measured as well. An unexpected good physical stability of the suspensions after 3 year storage at room temperature was observed. This behaviour appears to be related to the small size and structural organization of the nanoparticles. The mean diameters determined from light scattering experiments are consistent with those measured from electron micrographs. The slight difference between the values obtained by both methods is discussed.

Influence of surfactants on solubilization and fungal degradation of fluorene.

Eighteen fungal strains were tested in toxicity assays with surfactants in order to select surfactants and strains tolerant to surfactants for degradation assays. Two nonionic surfactants were used, an alkylphenol ethoxylate, Triton X-100, a sorbitan ester, Tween 80 and an anionic surfactant, sodium dodecyl sulfate. Solubilization and biodegradation tests were conducted in liquid medium batch; fluorene was quantified by HPLC. Results showed the enhancement of fluorene solubilization by the three surfactants, good tolerance of nonionic surfactants by the fungal strains and the enhancement of the biodegradation of fluorene by Doratomyces stemonitis (46-62%) and Penicillium chrysogenum (28-61%) in the presence of Tween 80 (0.324 mM) after 2 days.

[Effect of cyclodextrins on fungal degradation of fluorene]. / Effet des cyclodextrines sur la dégradation fongique du fluorène.

The purpose of this study was to improve the bioavailability of fluorene (PAH) by the use of complexing agents, cyclodextrins. The biodegradation tests were performed in liquid medium batches; fluorene was quantified by HPLC. Experimental results showed the enhancement of fluorene degradation by Penicillium italicum and Phanerochaete chrysosporium in the presence of branched cyclodextrins.

Phase behavior of fully hydrated DMPC-amphiphilic cyclodextrin systems.

With the aim of exploring relationships between the chemical structure and the physico-chemical properties of amphiphilic beta-cyclodextrin, a reappraisal of the obtaining of pure heptakis (2,3-di-O-hexanoyl)-beta-cyclodextrin (beta-CDC(6)) was undertaken. In this paper the chemical characterization of the newly synthesized beta-CDC(6) and its ability to form mixed structures with dimyristoylphosphatidylcholine (DMPC) are reported. Miscibility of the two amphiphiles is examined: (i) in monolayers formed at the air-water interface by analyzing the surface pressure-area isotherms; and (ii) in fully hydrated mixtures by differential scanning calorimetry (DSC) and X-ray diffraction at small and wide angles. Results demonstrate that the beta-cyclodextrin derivative is partially miscible to the phospholipid: intimate mixing occurs at beta-CDC(6) molar ratios smaller than 7-15 mol%, depending on the dimensional scale considered, while beyond these compositions phase separation is observed. At the air-water interface, the miscibility region of the two compounds shows non-ideal behavior characterized by the non-additivity of the molecular areas in the mixed monolayers. At the three-dimension level, the formation of a beta-CDC(6)/DMPC mixed lamellar phase occurs except at beta-CDC(6) molar ratios close to 5 mol% at which a highly ordered structure is depicted below the solid-to-liquid state transition of the DMPC hydrocarbon chains. At beta-CDC(6) contents higher than 7 mol%, the mixed assemblies coexist with excess amphiphilic cyclodextrin which then forms a separated hexagonal structure.

Cyclodextrins and carrier systems.

This paper describes two new possibilities of using cyclodextrins to increase water solubility and bioavailability of poorly water-soluble drugs intended for targeting delivery by the oral or the parenteral route. They use either amphiphilic cyclodextrin nanoparticles or polymeric nanoparticles containing cyclodextrins. Amphiphilic skirt-shaped cyclodextrins, resulting from the esterification of primary hydroxyl groups by hydrocarbon chains varying from C6 to C14, are capable of forming spontaneously nanoparticles which have been loaded with a series of steroid drugs. The drug in the amphiphilic cyclodextrin nanoparticles is molecularly dispersed and can be released very rapidly. Poly(isobutylcyanoacrylate) nanoparticles can be loaded with natural or hydroxypropyl cyclodextrins. This technique results in a significant increase in the loading capacity of nanoparticles with a series of steroids and in a very rapid release of the drug. Both methods are described as well as their potential interest for water-insoluble drugs.

Cyclodextrins in targeting. Application to nanoparticles.

For some years cyclodextrins and their hydrophilic derivatives have been described in the literature as solubilizers capable of enhancing the loading capacity of liposomes and microparticles. We present here two new possibilities of using cyclodextrins in the design of colloidal carriers. The first possibility consists in increasing the loading capacity of poly(isobutyl cyanoacrylate) nanospheres prepared by anionic polymerization, by employing hydroxypropyl cyclodextrins. The second possibility consists in the spontaneous formation of either nanocapsules or nanospheres by the nanoprecipitation of amphiphilic cyclodextrin diesters. These two new techniques are very promising because of the great interest presented by nanoparticles for drug administration by the oral or parenteral routes.

Evaluation of the hydrophobic drug loading characteristics in nanoprecipitated amphiphilic cyclodextrin nanospheres.

The aim of this work was to evaluate the loading capacity and the association characteristics of the hydrophobic drug progesterone on amphiphilic cyclodextrin nanospheres prepared by the nanoprecipitation method. The colloidal suspensions were prepared in the presence or absence of two different surfactants, Pluronic F68 and Tween 80. The physicochemical characteristics of the nanospheres were assessed using a nanosizer, zetameter, and transmission electron microscope. The physical state of the drug was verified using differential scanning calorimetry (DSC) and x-ray diffraction (XRD) methods. The in vitro progesterone release was investigated at 37 degrees C after dilution of the suspensions in sink conditions. Nanospheres with a mean diameter from 100 to 300 nm and a low degree of polydispersity were prepared from amphiphilic hexanoyl-gamma-cyclodextrin. The progesterone loading capacity was not affected by the formulation parameters tested. The DSC and XRD studies demonstrated the absence of the crystalline domains of progesterone in loaded nanospheres. The DSC studies also demonstrated the presence of interactions between the drug and carrier. The release of the drug from the carrier was extremely rapid and was governed by a partition phenomenon that depends only on the solubility of the drug in the release medium. From these results, we concluded that with this method, the progesterone is molecularly associated at the surface of the cyclodextrin nanospheres, probably through hydrophobic interactions in specific sites. The release profiles obtained can be of value when an improvement in the bioavailability of the drug is desired.

Physicochemical characterization of different batches of ethylated beta-cyclodextrins.

Different ethylated beta-cyclodextrin (Et-beta-CD) derivatives were obtained by various synthetic routes and their chemical structures and physical properties were elucidated. The aqueous solubility studies were carried out at 25 and 37 degrees C. A gas phase chromatography analysis, with head space extraction, was developed to detect the presence of residual solvents in the dry preparations. Electrospray ionization mass spectrometry allowed the determination of the average degree of substitution and the molecular mass of the Et-beta-CDs. Nuclear magnetic resonance analysis was used to elucidate the relationship between the solubility behavior and substituted positions of ethyl groups on the CD glucopyranose units. Finally, this paper deals with different physico-chemical methods used to fully characterize the different batches of Et-beta-CD to correlate the data obtained with the pharmacotechnical behavior of these cyclodextrins.

Polymorphism of spironolactone.

Single crystals of two polymorphic and four solvated crystalline forms of spironolactone (C24H32O4S) were obtained using different solvents. The morphology, symmetry, and crystallographic parameters were determined for all crystal forms except for the one obtained from methanol. The stability and transformation of each type of crystal were studied by DSC, TGA, and X-ray diffraction analysis. The structural data of three forms allowed the observation of the change of conformation of the molecules of spironolactone in the three different lattices.

High-field nuclear magnetic resonance techniques for the investigation of a beta-cyclodextrin:indomethacin inclusion complex.

The inclusion complex of indomethacin sodium salt in beta-cyclodextrin has been studied by proton NMR at high magnetic field. The continuous variation technique was used to evidence the formation of a soluble 1:1 complex in aqueous solution at physiological pH. The effective association constant was determined by the Benesi-Hildebrand procedure to be 760 M-1 at 303 K. This technique requires NMR measurements in the presence of a very large excess of one of the complex components and, since both beta-cyclodextrin and the sodium salt of indomethacin are sparingly soluble in water, NMR spectrometers operating at very high magnetic fields were used. Besides the effective association constant, the Benesi-Hildebrand approach allows a precise determination of all NMR parameters of the pure inclusion complex which may be used for a complete analysis of the geometry of this complex in solution.