Influence of oil content on physicochemical properties and skin distribution of Nile red-loaded NLC.

The aims of this study were to investigate the effect of the oil content on the physicochemical properties of NLC and to elucidate the potential of NLC for skin targeting. The obtained results showed that an increase in the oil content did not affect the mean particle size of NLC but impacted on the zeta potential. The inner structure of NLC was influenced by the increasing proportion of oil towards the less ordered structure as confirmed by differential scanning calorimetry (DSC) and X-ray diffraction (XRD), particularly for the higher medium chain triglycerides (MCT) loading. The data from proton nuclear magnetic resonance (1H NMR) revealed that cetyl palmitate nanoparticles did not completely recrystallize after cooling down to room temperature. 1H NMR and DSC results indicate that MCT molecules were restricted in the NLC as compared to the nanoemulsions (NE). Nile red distribution and penetration into skin from NLC were pronounced as compared to NE and dependent on the MCT loading. The deep penetration and high amount of Nile red were related to the occlusion factor. Moreover, the epidermal targeting was achieved by NLC applications, particularly those containing 5% MCT (NLC-5) depending on the amount of MCT loading.

Encapsulation of ascorbyl palmitate in nanostructured lipid carriers (NLC)--effects of formulation parameters on physicochemical stability.

Enhancement of the chemical stability of ascorbyl palmitate (AP) after incorporation into nanostructured lipid carriers (NLC) has been reported. However, the formulation parameters of AP-loaded NLC have not been completely investigated. Moreover, the long-term chemical stability of AP in any colloidal systems has not been yet achieved. Therefore, in this study the formulation parameters affecting the stability of AP after incorporation into NLC were evaluated including types of lipids, types of surfactants, storage conditions, i.e. temperature and nitrogen gas flushing, the effects of drug loading as well as types of antioxidants. After storage for 90 days, the mean particle size analyzed by photon correlation spectroscopy (PCS) was lower than 350 nm. The zeta potential measured by the Zetasizer IV was higher than -30 mV in all developed AP-loaded NLC formulations which varied according to the types of lipid and surfactant. Concerning the chemical stability of AP, it was found that AP-loaded NLC prepared and stored in non-degassing conditions, a higher percentage of AP loading in NLC, lower storage temperature (4 degrees C), addition of antioxidants as well as selection of suitable surfactants and solid lipids improved the chemical stability of AP. Moreover, an improvement of long-term chemical stability of AP was achieved by addition of antioxidants with nitrogen gas flushing as compared to those without antioxidant. The percentage of drug remaining at both 4 degrees C and room temperature (25 degrees C) was higher than 85% during 90 days of storage.

Cytotoxicity studies of Dynasan 114 solid lipid nanoparticles (SLN) on RAW 264.7 macrophages-impact of phagocytosis on viability and cytokine production.

Solid lipid nanoparticles (SLN) based on Dynasan 114 (D114) were tested using RAW 264.7 cells. The influence of different surfactants on the cytotoxicity of this type of SLN was examined, expressed as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability and the production of cytokines such as interleukin 6 (IL-6), IL-12 and tumour necrosis factor-alpha (TNF-alpha). Results were compared with previously obtained data when peritoneal mouse macrophages were used. SLN produced with stabilizers/surfactants such as poloxamer 188, sodium cholate, Lipoid S75, Tween 80, Poloxamine 908 and sodium dodecylsulfate were shown to be nontoxic towards RAW 264.7 cells. Cytokine production was reduced and stimulation, expressed in elevated cytokine levels, could not be found. Using cetylpyridinium chloride (CPC) as stabilizing surfactant, SLN became cytotoxic in a concentration-dependent manner. Not only were the viabilities reduced but also cytokine production. Cytotoxic effects of CPC stabilized SLN could be antagonized using cytochalasin B to block phagocytosis. D114-SLN produced with pharmaceutically accepted surfactants for intravenous injection (poloxamer 188, Lipoid S75, sodium cholate, Tween 80) were very well tolerated by the cells. Even sodium dodecylsulfate-stabilized D114-SLN did not exert toxic effects. Comparison of the RAW 264.7 data with previously obtained data from toxicity studies of D114-SLN towards peritoneal mouse macrophages showed similar results. This offers the possibility of using the RAW 264.7 cell line for cytotoxicity studies of colloidal drug carrier systems, rather than using laboratory animals as source of macrophages for these kinds of studies.

Development of matrix patches for transdermal delivery of a highly lipophilic antiestrogen.

The aim of this study was to develop matrix-type transdermal systems (TDSs) containing the highly lipophilic (log P = 5.82) antiestrogen (AE) and the permeation enhancers propylene glycol and lauric acid. For that purpose, permeation of AE from various adhesive matrices through excised skin of hairless mice was evaluated. It was found that pretreatment of the skin with permeation enhancers raised the transdermal flux of subsequently applied antiestrogen. Highest steady-state transdermal fluxes (1.1 microg cm(-2) h(-1)) were obtained from Gelva, polyacrylate adhesive, followed by 0.55 microg cm(-2) h(-1) from Oppanol polyisobutylene, 0.31 microg cm(-2) h(-1) from BIO-PSA silicone, and 0.12 microg cm(-2) h(-1) from Sekisui polyacrylate matrices. In order to develop TDS with high content of fluid permeation enhancer propylene glycol, two different strategies were investigated. One strategy was the addition of hydroxypropyl cellulose (HPC) as thickening agent to Gelva matrices. This allowed for propylene glycol loading levels of up to 30%, resulting in transdermal AE fluxes of 0.09 microg cm(-2) h(-1). On the other hand, a fleece-laminated backing foil was loaded with the described permeation enhancer formulation and laminated with polyacrylate adhesive layer, resulting in transdermal AE fluxes of 0.06 microg cm(-2) h(-1). However, application of these TDSs on skin pretreated with permeation enhancers raised the fluxes to 2.6 microg cm(-2) h(-1) from Gelva/HPC and 0.46 microg cm(-2) h(-1) from fleece/Sekisui.

Lipid-drug-conjugate (LDC) nanoparticles as novel carrier system for the hydrophilic antitrypanosomal drug diminazenediaceturate.

The objective of the present study was to incorporate the hydrophilic drug diminazenediaceturate at a high loading into lipid nanoparticles by creating nanoparticles from lipid-drug conjugates (LDC). IR and DSC data showed that the antitrypanosomal drug diminazene is able to react with fatty acids to form water-insoluble salts like diminazenedistearate and -dioleate. The salts could be transformed into nanoparticles using high-pressure homogenization technique, established for solid lipid nanoparticles (SLN). By using polysorbate 80 as surfactant, physically stable LDC nanoparticle dispersions of both salts could be obtained. The mean PCS diameters and polydispersity indices were 364 nm and 0.233 for diminazenedistearate and 442 nm and 0.268 for diminazenedioleate, respectively. Due to the composition of the LDC bulk materials, nanoparticles with a high drug load of 33% (w/w) were obtained even for this highly water-soluble drug diminazenediaceturate. The new carrier system of LDC nanoparticles overcomes one limitation of SLN, i.e. the limited loading capacity for hydrophilic drugs. Transforming water-soluble hydrophilic drugs into LDC and formation of nanoparticles allows prolonged drug release and targeting to specific sites by i.v. injection. These results provide a first basis of using LDC-polysorbate 80 nanoparticles for brain delivery of diminazene to treat second stage human African trypanosomiasis (HAT).

Stable biocompatible adjuvants--a new type of adjuvant based on solid lipid nanoparticles: a study on cytotoxicity, compatibility and efficacy in chicken.

A new type of adjuvant was tested for its ability to initiate antibody production in chickens, and its cellular and tissue compatibility were assessed. The stable biocompatible adjuvants tested are based on surface-modified solid lipid nanoparticles (SLNs), made from paraffin or biodegradable glycerides, and are simply admixed to the antigens before administration. The tissue-damaging potency of four formulations of the new adjuvants (H1, H2, H3 and H4) were first tested in vitro by using human foreskin fibroblasts and RAW 264.7 macrophages. The adjuvants were well tolerated by both cell types. Immunisation studies in chickens were performed by using a Mycoplasma bovis antigen and mouse immunoglobulin G (IgG). The resulting antibodies were non-invasively extracted from egg yolk. The use of the various adjuvant formulations resulted in a significant production of specific antibodies after the first and second booster immunisations. Freund's complete adjuvant (FCA), considered until now to be the "gold standard" among the adjuvants, revealed the highest antibody titre against mouse IgG. SLNs with a particle size of more than 100 nm exhibited a clear adjuvant activity, whereas SLNs with a particle size below 100 nm, in various concentrations, revealed a lower adjuvant activity. Immunisation of chickens with the mouse IgG alone, dissolved in phosphate-buffered saline, resulted in a slow antibody titre development. At the end of the experiment, the chickens were examined for vaccination-associated tissue damage. In contrast to FCA, the SLN formulations caused only minor tissue irritation at the injection sites. In conclusion, SLNs seem to be a promising alternative to FCA for antibody production in chickens, and potentially in other animals.

In-vitro release and transdermal fluxes of a highly lipophilic drug and of enhancers from matrix TDS.

Transdermal systems (TDS) are a well-known application form for small, moderately lipophilic molecules. The aim of this study was to investigate the possibility of applying a highly lipophilic drug, the antiestrogen AE (log P=5.82) transdermally by polyacrylate-based matrix TDS. For this purpose, two effects of both drug and enhancer concentration in TDS were investigated: in-vitro release and transdermal permeation of drug and enhancers. In the TDS investigated, in-vitro release as well as in-vitro permeation of AE through excised skin of hairless mice was found to be independent of concentrations of both drug and enhancers. The steady-state fluxes observed were low (about 50-100 ng cm(-2) h(-1)). But skin pretreatment with permeation enhancers resulted in a markedly enhanced permeability (1400 ng cm(-2) h(-1)). Therefore, the permeation of this highly lipophilic drug seems to be limited by the stratum corneum barrier function. In contrast, the transdermal permeation of the enhancers was dependent on the TDS composition. Increase in enhancer content resulted in a higher permeation of enhancers, whereas skin pretreatment did not. In conclusion, it was shown that the highly lipophilic antiestrogen can be administered transdermally by pretreating the skin with the fluid permeation enhancer combination propylene glycol-lauric acid (9+1) and then applying a matrix TDS.

Production and characterization of a budesonide nanosuspension for pulmonary administration.

PURPOSE: This study describes the production of a budesonide nanosuspension by high-pressure homogenization for pulmonary delivery from 40 mL up to 300 mL. The aim was to obtain a nanosuspension that can be nebulized and is also long-term stable. METHODS: The nanosuspension was produced by high-pressure homogenization. Particle size analysis was performed by laser diffraction and photon correlation spectroscopy. For further particle characterization, zeta potential was determined. To investigate the aerosolization properties, the nanosuspension was nebulized and afterward analyzed on particle size. RESULTS: It was possible to obtain a long-term stable budesonide nanosuspension. Mean particle size of this nanosuspension was about 500-600 nm, analyzed by photon correlation spectroscopy. Analysis by laser diffraction showed that the diameters 95% and 99% were below 3 microm. Budesonide nanosuspension showed a long-term stability; no aggregates and particle growth occurred over the examined period of 1 year. The PCS diameter before and after aerosolization did not change, and the LD diameters increased negligibly, showing the suitability for pulmonary delivery. The scale-up from 40 mL up to 300 mL was performed successfully. CONCLUSIONS: High-pressure homogenization is a production method to obtain nanosuspensions with budesonide for pulmonary applica-