Systematic study of liposomes composition towards efficient delivery of plasmid DNA as potential application of dermal fibroblasts targeting.

The use of non-viral DNA vectors to topically treat skin diseases has demonstrated a high potential. However, vectors applied on the skin face extracellular barriers including the stratum corneum and intracellular barriers such as the endosomal escape and the nuclear targeting of the plasmid DNA. The aim of this study was to develop a formulation suitable for dermal application and effective for delivering plasmid DNA into cells. Different formulations were prepared using different cationic lipids (DOTAP, DC-Chol, DOTMA) and co-lipids (DOPE, DSPE). Lipoplexes were produced by complexing liposomes with plasmid DNA at different pDNA/CL (w/w) ratios. Our results showed that appropriate pDNA/CL ratios allowing total complexation of plasmid DNA differed depending on the structure of the lipid used. The transfection rates showed that (i) higher rates were obtained with DOTMA lipoplexes, (ii) DC-Chol lipoplexes provided a transfection twice as important as DOTAP lipoplexes and (iii) when DSPE was added, the cytotoxicity decreased while transfection rates were similar. We found that formulations composed of DC-Chol:DOPE:DSPE or DOTMA:DOPE were appropriate to complex plasmid DNA and to transfect human primary dermal fibroblasts with efficacy and limited cytotoxicity. Therefore, these formulations are highly promising in the context of gene therapy to treat skin diseases.

In vitro skin penetration enhancement techniques: A combined approach of ethosomes and microneedles.

Dermal administration of different macromolecules, such as nucleic acids, remains a real challenge because of the difficulty of crossing the main skin barrier, the stratum corneum (SC). To overcome this barrier, the use of deformable lipid-based nanovectors were developed to increase topical penetration through the SC and to promote the intercellular delivery of drugs. The purpose of this study is to compare the skin penetration of different liposome formulations according to their composition. In vitro and ex vivo experiments using Franz diffusion cells were performed to highlight the effect of (i) lipid charge, (ii) edge activators (EA) and (iii) ethanol on the diffusion properties of nanovectors. We showed that all formulations were not able to cross the SC. However, on a tape stripped skin, we showed that cationic formulations containing an EA and ethanol improved the skin penetration. The use of microneedles was considered to bypass the SC. We have shown that sodium cholate and ethanol were necessary to ensure an appropriate diffusion of liposomes into the dermis when applied by means of microneedles. This could be a promising approach to further deliver efficiently macromolecules such as genes into the skin.

Development of a quantitative approach using surface-enhanced Raman chemical imaging: first step for the determination of an impurity in a pharmaceutical model.

This publication reports, for the first time, the development of a quantitative approach using surface-enhanced Raman chemical imaging (SER-CI). A pharmaceutical model presented as tablets based on paracetamol, which is the most sold drug around the world, was used to develop this approach. 4-Aminophenol is the main impurity of paracetamol and is actively researched in pharmaceutical formulations because of its toxicity. As its concentration is generally very low (<0.1%, w/w), conventional Raman chemical imaging cannot be used. In this context, a SER-CI method was developed to quantify 4-aminophenol assessing a limit of quantification below its limit of specification of 1000 ppm. Citrate-reduced silver nanoparticles were used as SERS substrate and these nanoparticles were functionalized using 1-butanethiol. Different ways to cover the tablets surface by butanethiol-functionalized silver nanoparticles were tested and a homogeneity study of the silver nanoparticles covering was realized. This homogeneity study was performed in order to choose the best way to cover the surface of tablets by silver colloid. Afterwards, the optimization of the SER-CI approach was necessary and different spectral intensity normalizations were tested. Finally, a quantitative approach using SER-CI was developed enabling to quantify 4-aminophenol from 0.025% to 0.2% in paracetamol tablets. This quantitative approach was tested on two different series of tablets using different batches of silver nanoparticles.

Determination of 4-aminophenol in a pharmaceutical formulation using surface enhanced Raman scattering: from development to method validation.

A surface enhanced Raman scattering (SERS) method able to quantify 4-aminophenol in a pharmaceutical formulation based on acetaminophen, also called paracetamol, was developed and, for the first time, successfully validated. In this context, silver nanoparticles were synthesized according to the method described by Lee-Meisel and used as SERS substrate. The repeatability of the silver colloid synthesis was tested using different methods to characterize the size and the zeta potential of silver nanoparticles freshly synthesized. To optimize the SERS samples preparation, a design of experiments implicating concentrations of citrate-reduced silver nanoparticles and aggregating agent was performed in order to maximize the Raman signal enhancement. Finally, an approach based on tolerance intervals and accuracy profiles was applied in order to thoroughly validate the method in a range of concentrations comprised from 3 to 15 µg mL(-1) using normalized band intensities. The standard addition method was selected as method calibration. Therefore, measurements were carried out on 4-aminophenol spiked solutions of the pharmaceutical formulation. Despite the well-known stability and reproducibility problems of SERS, the validation was performed using two operators and five batches of nanoparticles, one for each validation day.

Nuclear delivery of a therapeutic peptide by long circulating pH-sensitive liposomes: benefits over classical vesicles.

The purpose of this study is to propose a suitable vector combining increased circulation lifetime and intracellular delivery capacities for a therapeutic peptide. Long circulating classical liposomes [SPC:CHOL:PEG-750-DSPE (47:47:6 molar% ratio)] or pH-sensitive stealth liposomes [DOPE:CHEMS:CHOL:PEG(750)-DSPE (43:21:30:6 molar% ratio)] were used to deliver a therapeutic peptide to its nuclear site of action. The benefit of using stealth pH-sensitive liposomes was investigated and formulations were compared to classical liposomes in terms of size, shape, charge, encapsulation efficiency, stability and, most importantly, in terms of cellular uptake. Confocal microscopy and flow cytometry were used to evaluate the intracellular fate of liposomes themselves and of their hydrophilic encapsulated material. Cellular uptake of peptide-loaded liposomes was also investigated in three cell lines: Hs578t human epithelial cells from breast carcinoma, MDA-MB-231 human breast carcinoma cells and WI-26 human diploid lung fibroblast cells. The difference between formulations in terms of peptide delivery from the endosome to the cytoplasm and even to the nucleus was investigated as a function of time. Characterization studies showed that both formulations possess acceptable size, shape and encapsulation efficiency but cellular uptake studies showed the important benefit of the pH-sensitive formulation over the classical one, in spite of liposome PEGylation. Indeed, stealth pH-sensitive liposomes were able to deliver hydrophilic materials strongly to the cytoplasm. Most importantly, when encapsulated in pH-sensitive stealth liposomes, the peptide was able to reach the nucleus of tumorigenic and non tumorigenic breast cancer cells.

Liposome surface charge influence on skin penetration behaviour.

Vesicular systems have shown their ability to increase dermal and transdermal drug delivery. Their mechanism of drug transport into and through the skin has been investigated but remains a much debated question. Several researchers have outlined that drug penetration can be influenced by modifying the surface charge of liposomes. In the present work we study the influence of particle surface charge on skin penetration. The final purpose is the development of a carrier system which is able to enhance the skin delivery of two model drugs, betamethasone and betamethasone dipropionate. Liposomes were characterised by their size, morphology, zeta potential, encapsulation efficiency and stability. Ex vivo diffusion studies using Franz diffusion cells were performed. Confocal microscopy was performed to visualise the penetration of fluorescently labelled liposomes into the skin. This study showed the potential of negatively charged liposomes to enhance the skin penetration of betamethasone and betamethasone dipropionate.

Skin penetration behaviour of liposomes as a function of their composition.

Deformable liposomes have been developed and evaluated as a novel topical and transdermal delivery system. Their mechanism of drug transport into and through the skin has been investigated but remains a much debated question. The present study concerns ex vivo diffusion experiments using pig ear skin in order to explain the penetration mechanism of classical and deformable liposomes. Classical and deformable vesicles containing betamethasone in the aqueous compartment through the use of cyclodextrin inclusion complexes were compared to vesicles encapsulating betamethasone in their lipid bilayer. Deformable liposomes contained sodium deoxycholate as the edge activator. Liposomes were characterised by their diameter, encapsulation efficiency, deformability, stability (in terms of change in diameter) and release of encapsulated drug. Ex vivo diffusion studies using Franz diffusion cells were performed. Confocal microscopy was performed to visualise the penetration of fluorescently labelled liposomes into the skin. This study showed that liposomes do not stay intact when they penetrate the deepest layers of the skin. Betamethasone is released from the vesicles after which free drug molecules can diffuse through the stratum corneum and partition into the viable skin tissue.

ESR technique for noninvasive way to quantify cyclodextrins effect on cell membranes.

A new way to study the action of cyclodextrin was developed to quantify the damage caused on cell membrane and lipid bilayer. The Electron Spin Resonance (ESR) spectroscopy was used to study the action of Randomly methylated-beta-cyclodextrin (Rameb) on living cells (HCT-116). The relative anisotropy observed in ESR spectrum of nitroxide spin probe (5-DSA and cholestane) is directly related to the rotational mobility of the probe, which can be further correlated with the microviscosity. The use of ESR probes clearly shows a close correlation between cholesterol contained in cells and cellular membrane microviscosity. This study also demonstrates the Rameb ability to extract cholesterol and phospholipids in time- and dose-dependent ways. In addition, ESR spectra enabled to establish that cholesterol is extracted from lipid rafts to form stable aggregates. The present work supports that ESR is an easy, reproducible and noninvasive technique to study the effect of cyclodextrins on cell membranes.

Development and validation of a sensitive solid phase extraction/hydrophilic interaction liquid chromatography/mass spectrometry method for the accurate determination of glucosamine in dog plasma.

A sensitive and accurate LC/MS method was developed for the monitoring of glucosamine (GLcN) dog plasmatic concentration. In this scope, relatively low plasmatic concentrations of GLcN were expected, ranging from 50 to 1000 ng/mL. Liquid chromatography coupled to simple quadrupole mass spectrometry detection (LC/MS) was selected bringing the selectivity and the sensitivity needed for this application. Additionally, a solid phase extraction (SPE) step was performed to reduce matrix and ion suppression effects. Due to the ionisable character of the compound of interest, a mixed-mode strong cation exchange (Plexa PCX) disposable extraction cartridge (DEC) was selected. The separation was carried out on a Zorbax SB-CN column (5 microm, 4.6mm i.d. x 250 mm), considering hydrophilic interaction liquid chromatography (HILIC). Indeed, the mobile phase was made of methanol and 5mM ammonium hydrogen carbonate buffer at pH 7.5 (95/5, v/v). The detection was led at m/z ratios of 180.0 and 417.0, for GLcN and IS, respectively. Reliability of the results was demonstrated through the validation of the method using an approach based on the accuracy profile allowing managing the risk associated to the use of these methods in routine analysis: it is thus guaranteed that each future result will fall in the +/-30% acceptance limits with a probability of at least 90%. Successful application of the method to a preliminary pharmacokinetic study illustrated the usefulness of the method for pre-clinical studies.

Comparison of chitosan/siRNA and trimethylchitosan/siRNA complexes behaviour in vitro.

Chitosan and trimethylchitosan (TMC)-siRNA nanoparticles were produced by simple complexation technique or by ionic gelation using tripolyphosphate (TPP). The obtained complexes were characterized in terms of physicochemical properties such as size, zeta potential, complexation efficiency and stability. Furthermore, cytotoxicity, cell uptake and transfection efficiency of polyplexes were evaluated in vitro. Under pH condition of cell culture medium, a strong decrease in siRNA condensation efficiency was observed with chitosan nanoparticles. This characteristic resulted in low transfection efficiencies in HEK293 cell line. Formulation of chitosan polyplexes with TPP led to improvement of polyplexes stability but no significant increase in transfection efficiency was observed compared to simple chitosan complexes. By contrast, TMC complexes did not have pH dependency on siRNA complexation. TMC-siRNA nanoparticles were stable in physiological condition. Accordingly, cellular uptake was increased compared to chitosan polyplexes. However, improvement of transfection efficiency was low regarding to cellular uptake of these complexes. Chitosan and TMC complexes present some characteristics favourable for siRNA delivery, such as ability to integrate siRNA into small discrete particles or low toxicity of the complexes. This study also highlights the importance of complexes stability in physiological environment for siRNA transfection purposes.

Quantification of Randomly-methylated-beta-cyclodextrin effect on liposome: an ESR study.

In the present work, the effect of Randomly-methylated-beta-cyclodextrin (Rameb) on the microviscosity of dimyristoyl-l-alpha phosphatidylcholine (DMPC) bilayer was investigated using the electron spin resonance (ESR) technique. The ability of Rameb to extract membrane cholesterol was demonstrated. For the first time, the percentage of cholesterol extracted by Rameb from cholesterol doped DMPC bilayer was monitored and quantified throughout a wide Rameb concentration range. The effect of cholesterol on the inner part of the membrane was also investigated using 16-doxyl stearic acid spin label (16-DSA). 16-DSA seems to explore two different membrane domains and report their respective microviscosities. ESR experiments also establish that the presence of 30% of cholesterol in DMPC liposomes suppresses the jump in membrane fluidity at lipids phase-transition temperature (23.9 degrees C).

Development of a new topical system: drug-in-cyclodextrin-in-deformable liposome.

A new delivery system for cutaneous administration combining the advantages of cyclodextrin inclusion complexes and those of deformable liposomes was developed, leading to a new concept: drug-in-cyclodextrin-in-deformable liposomes. Deformable liposomes made of soybean phosphatidylcholine (PC) or dimyristoylphosphatidylcholine (DMPC) and sodium deoxycholate as edge activator were compared to classical non-deformable liposomes. Liposomes were prepared by the film evaporation method. Betamethasone, chosen as the model drug, was encapsulated in the aqueous cavity of liposomes by the use of cyclodextrins. Cyclodextrins allow an increase in the aqueous solubility of betamethasone and thus, the encapsulation efficiency in liposome vesicles. Liposome size, deformability and encapsulation efficiency were calculated. The best results were obtained with deformable liposomes made of PC in comparison with DMPC. The stability of PC vesicles was evaluated by measuring the leakage of encapsulated calcein on the one hand and the leakage of encapsulated betamethasone on the other hand. In vitro diffusion studies were carried out on Franz type diffusion cells through polycarbonate membranes. In comparison with non-deformable liposomes, these new vesicles showed improved encapsulation efficiency, good stability and higher in vitro diffusion percentages of encapsulated drug. They are therefore promising for future use in ex vivo and in vivo experiments.

Study of the relationship between lipid binding properties of cyclodextrins and their effect on the integrity of liposomes.

It is well known that cyclodextrins are able to extract lipids constituting membranes, increasing their fluidity and permeability. This behaviour towards biological membranes is directly linked to the toxicological effects of methylated cyclodextrins. However, confusion is currently made in the literature between the different methylated cyclodextrin derivatives. Moreover, a new methylated cyclodextrin derivative recently occurred in the market, the Crysmeb. We wanted to compare and understand the effect of the most currently used cyclodextrins on a model membrane. We studied the influence of natural cyclodextrins (betaCD and gammaCD), methylated derivatives (2,6-dimethyl-betaCD (Dimeb), 2,3,6-trimethyl-betaCD (Trimeb) and randomly methylated-betaCD (Rameb), as well as the new derivative Crysmeb), hydroxypropylated derivatives (HPbetaCD of different substitution degrees and HPgammaCD) and the sulfobutylated derivative (SBEbetaCD) on the release of a fluorescent marker encapsulated in the inner cavity of liposomes. It was shown that the observed effect on calcein release can be directly related to the affinity of cyclodextrins for both lipid components of liposomes, cholesterol and phosphatidylcholine. From this relationship, we were able to determine, for each cyclodextrin, a theoretical concentration giving rise to 50% or 100% calcein release. This theoretical concentration was confirmed experimentally. We have also showed that cyclodextrins which provoke calcein release also induce large structure modifications of liposomes.

Automated method for the determination of a new matrix metalloproteinase inhibitor in ovine plasma and serum by coupling of restricted access material for on-line sample clean-up to liquid chromatography.

A fully automated liquid chromatographic method was developed for the determination of Ro 28-2653, a new synthetic inhibitor of matrix metalloproteinases (MMPs), in ovine serum and plasma. The method was based on the coupling of a pre-column packed with restricted access material, namely LiChrospher RP-8 ADS (alkyl diol silica), for sample clean-up to an analytical column containing octyl silica stationary phase. One hundred microl of biological sample, to which 2-propanol was automatically added, were injected onto the ADS pre-column, which was then washed with a washing liquid consisting of a mixture of 25 mM phosphate buffer (pH 7.0) and acetonitrile (90:10; v/v) for 10 min. By rotation of the switching valve, the analyte was then eluted in the back-flush mode with the LC mobile phase composed of a mixture of acetonitrile and 25 mM phosphate buffer (pH 7.0) (57:43; v/v). The UV detection was performed at 395 nm. The main parameters likely to influence the sample preparation technique were investigated. The method was then validated over a concentration range from 17.5 to 1950 ng/ml, the first concentration level corresponding to the lower limit of quantitation. At this concentration level, the mean bias and the R.S.D. value for intermediate precision were -2.4% and 4.2%, respectively.

Cyclodextrins as a potential carrier in drug nebulization.

The inhalation route is widely studied for many drug applications focusing on either local or systemic distributions. One matter of concern is the solubilization of hydrophobic drugs. We have studied the feasibility of using different cyclodextrins (CDs) to elaborate pharmaceutical formulations for the inhalation route and tested the short-term toxicity of such formulations administered by inhalation to C57BL/6 mice. We have shown that HP-beta-CD, gamma-CD, as well as RAMEB aqueous solutions can undergo aerosolization and that the resulting droplet-size ranges are compatible with pulmonary deposition. In vivo, we have demonstrated that short-term exposure to inhaled HP-beta-CD, gamma-CD and RAMEB solutions are non-toxic after assessing bronchoalveolar lavage (BAL), lung and kidney histology, bronchial responsiveness to methacholine and blood urea. The only change noted is a slight increase in lymphocyte count in the BAL after HP-beta-CD and gamma-CD inhalation. We conclude that CDs are useful in significantly enhancing the solubility of apolar drugs with a view to inhalation therapy although an increase in lymphocyte counts in the BAL after CDs inhalations needs further investigations.

Skin compatibility of cyclodextrins and their derivatives: a comparative assessment using a corneoxenometry bioassay.

Few studies have been performed to assess the risk of skin damage by cyclodextrins (CD) and they have yielded contradictory results. The present study was conducted using the corneoxenometry bioassay on human stratum corneum to compare the skin compatibility of CD currently used in pharmaceutical preparations (betaCD, gammaCD, Rameb, Dimeb, Trimeb, HP-betaCD and HP-gammaCD) and that of new amphiphilic CD derivatives, namely, the phospholipidyl-CD (DMPE-Dimeb and DMPE-Trimeb). All the tested CD were well tolerated by the stratum corneum at a concentration of 5%. However, inter-individual reactivity was larger for DMPE-Dimeb, suggesting a more aggressive trend for this compound. Cutaneous Index of Mildness values obtained confirm that Dimeb is able to extract some skin components and shows that DMPE-Dimeb performs similarly.

Systematic characterization of oil-in-water emulsions for formulation design.

Oil-in-water emulsions varying in surfactant concentration and manufacturing process were prepared. About 10 experiments were performed to characterize them. The goal of this research was to find out which tests should systematically be carried out to assess efficiently the stability and the properties of an emulsified preparation. Thus, formulation design requires at least the measurement of the droplet size, the determination of the zeta potential, a TurbiScan analysis, the investigation of the stability under centrifugation and freeze/thaw cycles. If the emulsion contains an active substance, stability under storage at 4 degrees C and microscopic analysis are relevant. Quality control should be improved by measurements of viscosity and pH.

Oral bioavailability in sheep of albendazole from a suspension and from a solution containing hydroxypropyl-beta-cyclodextrin.

Albendazole (ABZ) is a benzimidazole derivative with a broad spectrum of activity against human and animal helminthe parasites. ABZ has a very poor aqueous solubility. This study shows that hydroxypropyl-beta-cyclodextrin (HP-beta-CD) is able to form inclusion complexes with ABZ and that is able to increase its aqueous solubility. A synergistic effect exists between HP-beta-CD and citric acid. The combination of HP-beta-CD (200 mM) and citric acid (50 mM) allows dissolution of more than 1.5 mg of ABZ per ml. The aim of this study is the in vivo evaluation in sheep of a solution of the inclusion complex of ABZ with HP-beta-CD in comparison with a suspension of the same drug. A significant (P<0.05) increase in the relative bioavailability is obtained with the solution containing the ABZ-HP-beta-CD complex as measured by ABZSO plasma levels. The area under the curve (AUC(0--> proportional, variant )) of the solution is 37% higher than that obtained with the suspension. Likewise the peak plasma concentration (C(max)) is twice that of the solution while the time to reach C(max) (T(max)) is reduced.

IgE-mediated anaphylaxis after first intravenous infusion of cyclosporine.

BACKGROUND: Intravenous administration of cyclosporine, which contains Cremophor EL (a polyethoxylated castor oil; BASF, Berlin, Germany), has occasionally resulted in an anaphylactic reaction. An apparent hypersensitivity reaction (bronchospasm and decrease in blood pressure) had occurred during heart transplantation in a 59-year-old woman after intravenous infusion of cyclosporine. Subsequent oral administration of cyclosporine precipitated no reaction. OBJECTIVE: The purpose of this study was to attempt to ascertain the mechanism responsible for the anaphylactic reaction. METHODS: Hypersensitivity investigations, including total serum IgE and allergen-specific IgE quantifications, skin testing, and basophil activation tests by flow cytometric determination of CD63 upregulation were undertaken in the study patient and in two healthy control subjects who were free of medication. RESULTS: The results of intradermal testing with Cremophor EL were positive after 15 minutes in the study patient only. Both cyclosporine and Cremophor EL induced considerable activation of the basophils from our study patient, with an upregulation of CD63 expression from 1% to 39% and 55%, respectively. In contrast, the expression of CD63 on basophils from the two control subjects remained essentially unchanged. CONCLUSIONS: The negative investigative findings in the control subjects, the patient's clinical manifestations in temporal relationship to the infusion, her positive results on intradermal testing with Cremophor, the basophil activation test results, and her uneventful course after oral administration of cyclosporine strongly support the presence of IgE antibodies to Cremophor EL in our patient.

Molecular modeling study of beta- and gamma-cyclodextrin complexes with miconazole.

Different authors have demonstrated the inclusion of miconazole in cyclodextrins (CD). Miconazole can be included in the CD cavity both in the neutral and in the ionized form. The present study tries to understand which fragment of the miconazole molecule is involved in the inclusion. Austin Model 1 approximate molecular orbital calculations have been performed on several complexes between beta-cyclodextrin (betaCD) or gamma-cyclodextrin (gammaCD) and miconazole in the ionized and the non-ionized forms of the two R and S enantiomers in three different orientations. We observed that betaCD is a good vehicle to transport miconazole which can be very easily released. The complexation energy between miconazole and betaCD is not very high but the entropic factor has a great incidence on the stability of the formed complex. The inclusion of the dichlorobenzene-CH(2)-O- and of the imidazole part of the S isomer gives rise to the most probable complex in acidic conditions (ionized miconazole). Nevertheless, the inclusion should be considered as a dynamic process in which different parts of the molecule could be alternatively included in betaCD. The present work demonstrates the high capability of deformation of betaCD which could easily accommodate several types of ligand. By opposite, the cycle extension in gammaCD leads to a more rigid vehicle with regards to miconazole.