International Journal of Pharmaceutics (v.196, #2)

A study of microemulsions with retinol and its esters. Physical properties of w/o and o/w microemulsions containing Tween 60, Tween 80, Epicurone 135 (soy bean lecithin) as surfactants, n-butanol, triacetin, propylene glycol as cosurfactants were examined. The drug-containing systems were characterised in regard to their ophthalmic parameters. Physiologically well-tolerated and physically stable multiple-components were developed. The concentrations of surfactants and cosurfactants which are necessary to form stable systems were evaluated. The values of the following parameters — refractive index, viscosity, pH value, osmotic tension, obtained in the study, proved suitable for the purpose and the preparations were physiologically tolerated, the use of microemulsions as potential drug delivery systems for ocular administration has been discussed. The influence of retinol and its esters on the physical parameters the preparation was investigated. Microemulsion stored at 20°C up to 6 months showed no significant physical changes.
Keywords: Microemulsion; Retinol; Lecithin; Physical parameters; Mixture design;

Immune-stimulating complexes (ISCOMs) are stable colloidal complexes of the adjuvant Quil A, cholesterol and phospholipid, which are effective carriers for subunit vaccines. The techniques currently available for the preparation of ISCOMs from the constituent components are rather complex and are based on either centrifugation or dialysis. This note reports a new simple procedure for the preparation of ISCOM matrices based on hydration of a cholesterol/phospholipid film with an aqueous solution of Quil A. It is demonstrated that ISCOM matrices do not form in the absence of phospholipid when prepared by this method. Further, the ratio by weight of phospholipid to either cholesterol or Quil A must be greater than that required for preparation by either dialysis or centrifugation. Photon correlation spectroscopy, negative stain transmission electron microscopy and centrifugation through a sucrose gradient demonstrate that ISCOM matrices can be prepared from cholesterol/lipid films by hydration with an aqueous solution of Quil A when the ratio of phospholipid:cholesterol:Quil A by weight is 6:1:4, respectively. Lower ratios of phospholipid:cholesterol reduce the efficiency of ISCOM formation while higher ratios produce systems containing a mixture of ISCOMs together with liposomes.
Keywords: ISCOM; Immune-stimulating complex; Quil A; Phospholipid; Vaccine; Adjuvant;

Effects of alcohols and diols on the phase behaviour of quaternary systems by R.G Alany; T Rades; S Agatonovic-Kustrin; N.M Davies; I.G Tucker (141-145).
The aim of the current study was to investigate the effect of different co-surfactants on the phase behaviour of the pseudoternary system water:ethyl oleate:nonionic surfactant blend (sorbitan monolaurate/polyoxyethylene 20 sorbitan mono-oleate). Four aliphatic alcohols (1-propanol, 1-butanol, 1-hexanol and 1-octanol) and four 1,2-alkanediols (1,2-propanediol, 1,2-pentanediol, 1,2-hexanediol and 1,2-octanediol) were used. The co-surfactant-free system forms two different colloidal structures, a water-in-oil microemulsion (w/o ME) and lamellar liquid crystals (LC) and two coarse dispersions, water-in-oil (w/o EM) and oil-in-water (o/w EM) emulsions. Microemulsion region area (%ME), liquid crystalline region area (%LC), amount of amphiphile blend required to produce a balanced microemulsion (%AMPH) and amount of water solubilised (%W) were used as assessment criteria to evaluate the co-surfactants. Seven calculated physico-chemical descriptors were used to represent the different co-surfactants. 1-butanol, 1,2-hexanediol and 1,2-octanediol produced balanced MEs capable of solubilising a high percentage of both oil and water. A similarity was observed between the descriptors attributed to 1-butanol and 1,2-hexanediol. The requirements of a co-surfactant molecule to produce a balanced microemulsion were: HLB value 7.0–8.0, a carbon backbone of 4–6 atoms, percentage carbon of 60–65%, percentage oxygen of 20–30%, log  P value 0.2–0.9 and log 1/S (S: aqueous solubility) close to zero.
Keywords: Microemulsions; Cosurfactants; Alcohols; Diols; Phase diagrams;

The possibility of preparing uniform nanoparticles consisting of proteins such as gelatin followed by covalent linkage of avidin was investigated. Gelatin nanoparticles were prepared by two step desolvation. Functional groups at the surface of the particulate system were quantified with site-specific reagents. The surface of the nanoparticles was thiolated and avidin was covalently attached to the nanoparticles via a bifunctional spacer at high levels. Biotinylated peptide nucleic acid (PNA) was effectively complexed by the avidin-conjugated nanoparticles. Avidin-conjugated protein nanoparticles should prove as potential carrier system for biotinylated drug derivatives in antisense therapy.
Keywords: Nanoparticles; Gelatin; Covalent linkage; Avidin; Peptide nucleic acid (PNA);

Liposomes are belonging to the modern kinds of drugs. They are facilitating the secure and well-targeted transport of substances inside the organism, and are gaining increasing importance in the pharmacological treatment of tumors and in gene-therapeutic strategies. Multilamellar liposomes have advantages to one-layer liposomes: the multiplicity of coats increases the effect of a reservoir and makes extremely prolonged releases of drugs possible. This study describes the aseptical manufacturing of different kinds of multilamellar liposomes. It has been shown that it is possible to produce liposome-suspensions with different shares of multilamellar liposomes in the scale of litres under aseptical circumstances using a newly constructed multi-stage-pressure-filtration-device.
Keywords: Liposomes; Multiple layer vesicles (MLV); Aseptical production;

The major problem of many newly developed pharmaceutical drugs is their poor solubility in water and simultaneously in organic media. To solve these problems formulation as nanosuspensions is an attractive alternative. During the drug development process screening for an optimal formulation by homogenisation is essential. Time and cost effective production in an initial phase of R&D can be conducted on lab scale by using the Micron Lab 40 in its discontinuous version. In this report reproducibility of small scale production parameters (particle size, size distribution, content of microparticles) was exemplary studied for the drug RMKP22.
Keywords: Drugs; Nanoparticles; Polydispersity index;

Poorly soluble drugs are often a challenging problem in drug formulation, especially when the drug is not soluble in either aqueous media or organic solvents. Attempts to overcome the solubility problem are, e.g. solubilisation with mixed micelles or forming a complex using cyclodextrines, but these approaches are of limited success. Another problem with new high potential drug is that these drugs often show bioavailability problems. One tried to improve the in vivo performance of poorly soluble drugs by reducing the particles size of the drug thus leading to an increased surface area and an increased dissolution velocity (Müller et al., 1994, 1999). Some of these problems occurred with tarazepide and therefore it was tried to create a formulation with this drug as nanosuspension which is suitable for intravenous administration.
Keywords: Nanosuspension; Tarazepide; Long-term stability;

Solid lipid nanoparticles as drug carriers for topical glucocorticoids by C.Santos Maia; W Mehnert; M Schäfer-Korting (165-167).
Recent investigations both in vitro and in human subjects proved the benefit/risk ratio of prednicarbate (PC) to exceed those of halogenated topical glucocorticoids about 2-fold. To obtain a further highly desired increase by drug targeting to viable epidermis, PC was incorporated into solid lipid nanoparticles (SLN). Keratinocyte and fibroblast monolayer cultures, reconstructed epidermis and excised human skin served to evaluate SLN toxicity and PC absorption. Well-tolerated preparations (e.g. cellular viability 94.5% following 18 h incubation of reconstructed epidermis) were obtained. PC penetration into human skin increased by 30% as compared to PC cream, permeation of reconstructed epidermis increased even 3-fold. The present study shows the great potential of SLN to improve drug absorption by the skin.
Keywords: Prednicarbate; Benefit-risk ratio; Solid lipid nanoparticles; Dermal absorption; Drug targeting;

Heavy metal contamination of nanosuspensions produced by high-pressure homogenisation by K.P. Krause; O. Kayser; K. Mäder; R. Gust; R.H. Müller (169-172).
High pressure homogenisation is a method for the production of nanosuspensions. In this process crystalline drug particles are pressed with high pressure through a narrow homogenisation gap. Due to the conditions in the gap it seems possible that metal erosion can occur. In this study the heavy metal (Fe) contamination of nanosuspensions produced by high pressure homogenisation was determined. Therefore nanosuspensions were analysed by atom absorption spectroscopy concerning their load of iron which is chosen as reference metal. The results show that the erosion of metal is below 1 ppm and will not cause any toxicological problems.
Keywords: Nanosuspensions; Heavy metal contamination; High pressure homogenisation; Metal erosion;

Microencapsulation of peptides and proteins by Gesine E Hildebrand; Johannes W Tack (173-176).
Microcapsules were prepared by using a double-emulsion technique. A new production method called ‘induced phase separation method’ was applied to encapsulate peptides and proteins. To find the optimal adjuvants a matrix was set up combining the appropriate organic solvents and the suitable surfactants. The polymer was chosen with regard to the required release period. The aqueous drug solution was intensively mixed with the organic polymer solution. An aqueous surfactant solution was slowly added to the O/W emulsion. The obtained W/O/W emulsion is stirred under partial vacuum conditions until the organic solvent was removed. After removing the solvent from the W/O/W emulsion the microcapsules were washed and lyophilized. The morphology of the microparticles (spheres, sponges, capsules, surplus polymer) was checked by microscopy, particle size distributions were measured by laser diffraction.
Keywords: Microencapsulation; Bioerodible polymers; Peptides; Proteins;

Influences of process parameters on nanoparticle preparation performed by a double emulsion pressure homogenization technique by A Lamprecht; N Ubrich; M Hombreiro Pérez; C.-M Lehr; M Hoffman; P Maincent (177-182).
The preparation of nanoparticles (NP) as an improved colloidal carrier system for proteins was investigated. Bovine serum albumin (BSA) was used as model drug. Owing to the high solubility of the protein in water, the double emulsion technique has been chosen as one of the most appropriate method. In order to both reaching submicron size as well as increasing the grade of monodispersity compared to previous preparation techniques, a microfluidizer as homogenization device was used. All experiments were performed using two biodegradable polymers, poly[d,l-lactic-co-glycolic acid] 50/50 (PLGA) and poly[ε-caprolactone] (PCL). The homogenization procedure has been optimized with regard to particle size and monodispersity by studying the influence of the homogenization time as well as the amount of polymer and surfactant in the external aqueous phase. The drug loading has been improved by varying the concentration of the protein in the inner aqueous phase. By increasing the protein concentration in the inner aqueous phase the polydispersity was slightly higher, while the particle size was not influenced significantly. The BSA encapsulation efficiency decreased with higher protein concentration in the inner aqueous phase. All release profiles were characterized by a initial burst effect, a higher release rate was obtained after 4 weeks for PLGA NP (60%) compared with PCL NP (47%).
Keywords: Nanoparticles; Encapsulation; Double emulsion technique; Poly[lactic-co-glycolic acid]; Poly[ε-caprolactone]; BSA;

Influence of high pressure homogenisation equipment on nanodispersions characteristics by S Liedtke; S Wissing; R.H Müller; K Mäder (183-185).
In this study a comparison of the influence of the homogenising equipment supplied by different manufacturers on the quality of the lipid nanodispersions is given. An Avestin EmulsiFlex-B3 (B3) and APV Micron Lab 40 (LAB 40) were used for high pressure homogenisation. Particle size and particle size distribution were chosen as quality parameters. The influence of different process parameters was evaluated. The two homogenisers were compared in their quality of nanoparticles-production by hot and cold homogenisation technique and in processing nanoemulsions. Working with the B3 appeared as useful for preformulation studies and processing of expensive or rare drugs and excipients. This first scaling up within laboratory scale is evaluated and the problems and remarkable aspects working with the B3 are pointed out.
Keywords: Nanoparticles; High pressure homogenisation; SLN; Nanoemulsions;

Traditional analysis of the dissolution of lipophilic compounds from aqueous phase into oil is often hampered by the necessity to separate the receiver oil compartment from the aqueous phase. In order to avoid possible artefacts associated with additional separation methods, a procedure was developed to selectively detect the entry of a compound into the oil phase of a oil/water dispersion. When a combination of a primary and secondary scintillator was predissolved in the oil, and solid 14C-tetrahydrolipstatin was added, increasing signals from the same container were measured upon prolonged incubation. The data are consistent with the hypothesis that 14C-THL that has dissolved in the oil phase is essentially responsible for the measured signal. The obtained dissolution profiles of 14C-THL into oil match with parallel experiments using classical procedures.
Keywords: Fat; Dissolution; Proximity; Scintillation;

The investigated drug delivery system is a solid-reversed-micellar-solution (SRMS). The composition of this solution is 70% Witepsol W35 and 30% (w/w) lecithin. 1% (w/w) metoclopramide-HCl (MCP) was solubilizied in the SRMS. After melting and on contact with water or any physicological aqueous media the SRMS exihibits an application induced transformation into a semisolid system of liquid crystalline microstructure. The structure of the liquid crystal has been identified by polarized light microscopy as a lamellar mesophase. Due to a low coefficient of diffusion in this mesophase a controlled release of the drug may be possible. The release profiles of the in vitro experiments have shown zero order kinetics and a sustained release of the SRMS-suppositories (SRMS-supp.) in comparison with commercial suppositories (Gastrosil-supp.). To examine bioavailability an in vivo study with rabbits was carried out. Five SRMS-supp. (10 mg MCP) and five Gastrosil-supp. (10 mg MCP) were tested in a parallel-group study. These experiments have shown a five times longer mean residence time (parameter of sustained release) in comparison with Gastrosil-supp. In vitro and in vivo studies have shown that rectal application of SRMS-supp. provides an appropriate route for controlled release of MCP via application induced transformation into liquid crystals.
Keywords: Solid-reversed-micellar-solution; Suppositories; Metoclopramide-HCl;

The objective of the present study was to characterise and optimise the desolvation process of human serum albumin (HSA) for the preparation of nanoparticles. Following the desolvation of the protein, the resulting nanoparticles were stabilised by the addition of varying amounts of glutaraldehyde. The particle size and the number of available amino groups on the surface of the nanoparticles were determined. The results indicated that the particle size depended mainly on the amount of desolvating agent added, but not on the amount of cross-linker. Increasing volumes of glutaraldehyde reduced the number of amino groups on the surface of HSA nanoparticles.
Keywords: Nanoparticles; Human serum albumin (HSA); Surface characterisation; Desolvation procedure; Amino group determination;

Crystallographic investigation of cetylpalmitate solid lipid nanoparticles by G. Lukowski; J. Kasbohm; P. Pflegel; A. Illing; H. Wulff (201-205).
Solid lipid nanoparticles (SLN) as alternative intravenous colloidal drug carriers were produced by high pressure homogenisation of the melted lipid cetylpalmitate. The crystallographic properties of the used cetylpalmitate SLN were characterised by small angle X-ray scattering (SAXS) and X-ray diffraction (XRD). It was found that the SLN are available exclusively in a crystalline form. Different cetylpalmitate formulations showed all the same patterns and a uniform crystal lattice was obtained. A partial indexing of the signals of the cetylpalmitate was carried out and the unit cell of the cetylpalmitate was estimated by the Miller indices. A preferred orientation in 00l-direction was observed. This can be explained by an impressive lamellar lattice structure of the cetylpalmitate. The results were compared with the crystal data of cetylpalmitate known from literature. There was no correlation with the monoclinic structure known so far. This could indicate that the SLN consist of crystallites of another modification of the cetylpalmitate.
Keywords: Cetylpalmitate; Solid lipid nanoparticles; Crystallisation; Small angle X-ray scattering;

Solubilization of timolol maleate in reversed micellar systems by Stefan Mackeben; Christel C Müller-Goymann (207-210).
Small angle X-ray scattering (SAXS) and photon correlation spectroscopy (PCS) are two methods to measure particle sizes in the order of 10 nm magnitude, which can be used to characterize reversed micellar systems, in this case reverse micelles consisting of lecithin and isopropyl myristate (IPM). In this study these micelles are loaded with different concentrations of the amphiphilic anti-glaucoma drug timolol maleate (TM). The PCS results are consistent with those yielded by SAXS, showing a decrease of particle size with higher TM concentration. In addition, SAXS is capable to give information about the particle shape. This kind of evaluation yields an ellipsoidal shape for micelles with low drug loads, which transform into nearly spherical micelles at higher drug concentrations.
Keywords: SAXS; PCS; Reverse micelles; Timolol (maleate);

Influence of different parameters on reconstitution of lyophilized SLN by E Zimmermann; R.H Müller; K Mäder (211-213).
Drug-loaded solid lipid nanoparticles (SLN) suitable for parenteral administration were freeze-dried. The lipid matrix Imwitor 900 (concentration, 2.5%) was stabilized with Lipoid E 80 and sodium glycocholate. The influence of different parameters of lyophilization like the protective effect of cryoprotectants, freezing velocity, and thermal treatment was investigated. The results of this study demonstrate that, by optimizing critical process parameters, i.v.-injectable SLN-dispersions can be freeze-dried, preserving their small particle size.
Keywords: Solid lipid nanoparticles; Lyophilization; Colloidal drug carrier;

In this study, the hydrolytic degradation of phosphatidylcholine in aqueous liposome-dispersions and the stability of the anti cancer drug carboplatin enclosed in the liposomes were investigated in dependence on liposome composition and storage conditions. Cholesterol containing liposomes show a high stability of the phosphatidylcholine and the encapsulated carboplatin during six months storage in refrigerator. The hydrolytic degradation of phosphatidylcholine is strongly increased by addition of the antioxidant ascorbyl palmitate, but despite the partial hydrolysis the advantages of the lipid membrane are retained — no degradation of the drug and no changes in the particle size were detected during six months storage in refrigerator, in contrast to storage at room temperature.
Keywords: Phosphatidylcholine liposomes; Lipids; HPLC; Particle size; Stability;

Comparison of wax and glyceride solid lipid nanoparticles (SLN®) by Volkhard Jenning; Sven Gohla (219-222).
The present study compares solid lipid nanoparticles (SLN) formulated with either wax or glyceride bulk material. While most published data deal with glyceride SLN, little knowledge is reported on wax carriers. The two types were compared with respect to drug encapsulation efficacy, particle size distribution after production and storage, and crystal packing. The inclusion of retinol as a model drug was investigated. Retinol is chemically unstable in water and rather stable in lipid phases. Thus, rapid degradation of retinol indicates rapid drug expulsion from the carrier. Good stability indicates an effective drug encapsulation in the lipid phase of the nanoparticles. Particle size distribution was measured by laser diffractometry. Subcell packing and assignment of polymorphic forms was investigated by WAXS measurements. Glyceride SLN showed good drug encapsulation, while physical stability was poor. In contrast, wax SLN possessed good physical stability but lacked sufficient drug encapsulation in the solidified state. These differences were attributed in part to different crystal packing. Less ordered crystal lattices favour successful drug inclusion, as in the case of glyceryl monosterate and glyceryl behenate SLN. The highly ordered crystal packing of wax SLN comprised of beeswax or cetyl palmitate, for instance, leads to drug expulsion, but also to superior physical stability.
Keywords: Solid lipid nanoparticles; Glyceride; Wax; Drug encapsulation; WAXS;

Confocal laser scanning microscopy (CLSM) was employed in order to characterize microcapsules. Microcapsules were prepared by complex coacervation: gelatin and arabic gum were labelled with fluorescent markers. In the capsule wall a homogeneous distribution for both gelatin and arabic gum throughout the capsule wall was depicted. By the use of CLSM and a computational image analysis the quantification of the labelled polymer in the wall material was possible. Adding fluorescently labelled casein as a macromolecular model compound to the coacervation process, a gradiental distribution in the wall material was observed with highest concentration of casein at the oil–wall interface. The influence of casein concentration on its deposition behaviour in the capsule wall was imaged successfully and thereafter quantified by computational image analysis.
Keywords: Confocal laser scanning microscopy (CLSM); Microcapsules; Microencapsulation; Complex coacervation; Polymer distribution;

The rheological behaviour of solid lipid nanoparticle dispersions (SLN) prepared by high pressure homogenization was investigated using a Haake RS-100 rheometer. Four preparations differing in their lipid content and macroscopic consistency were tested by continuous shear rheometry and oscillatory testing. Rheological data from continuous shear measurement reveal plastic flow for systems with low lipid content as well as for systems with high lipid content. By using oscillatory testing more detailed information concerning the structure could be achieved. Rheological measurements of 40% lipid dispersions show viscoelastic properties comparable to the data from standard dermal preparations. Therefore high concentrated lipid dispersions might constitute a promising vehicle for topical administration.
Keywords: Continuous flow rheometry; Rheology; Oscillatory testing; Viscoelasticity; Solid lipid nanoparticle dispersions (SLN);

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the method of choice to investigate protein adsorption of blood proteins (opsonization) onto nanoparticular drug carriers. In general the reproducability of the obtained adsorption patterns is satisfying. However, direct comparison between the amounts of single protein spots from gels obtained in different runs is difficult, because 2D-PAGE is a multistep procedure. A possible solution of the problem is to establish a protein as internal standard. Therefore, selected proteins (Bio-rad) were under investigation. Due to its molecular weight and isoelectric point, soybean trypsin inhibitor (TI) does not interfere with plasma components. Therefore, a method was established to use TI as an internal standard protein to improve comparability between the 2D-PAGE gels obtained in different analytical runs.
Keywords: Two-dimensional electrophoresis; Internal standard; Protein adsorption; Nanoparticles; Drug targeting;

Preserved solid lipid nanoparticles (SLN) at low concentrations do cause neither direct nor indirect cytotoxic effects in peritoneal macrophages by Nadja Schöler; Eike Zimmermann; Ulrike Katzfey; Helmut Hahn; Rainer H Müller; Oliver Liesenfeld (235-239).
In order to investigate the interaction of preserved solid lipid nanoparticles (SLN) with murine peritoneal macrophages (MØ), cytotoxicity and proinflammatory effects of two different solid lipid nanoparticles (SLN) preparations consisting of either compritol (CO) or cetyl palmitate (CP) preserved with thiomersal were analyzed. Concentration-dependent cytotoxic effects were observed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Secretion of interleukin-6 by MØ following incubation with CO and CP SLN did not differ from secretion by untreated cells; proinflammatory cytokines interleukin-12 and tumor-necrosis-factor-α as further indicators of immunomodulatory effects were not detectable. These findings paralleled our previous findings that unpreserved CO and CP SLN did not induce immunomodulatory effects but cytotoxicity at higher concentrations. There were no synergistic cytotoxic effects of preservative and SLN. Thus, preservation of SLN using thiomersal does not appear to cause increased cytotoxicity and immunomodulatory effects following incubation with MØ.
Keywords: Solid lipid nanoparticles (SLN); Peritoneal macrophages; Cytokines; Cytotoxicity;

Influence of spin probe structure on its distribution in SLN dispersions by P Ahlin; J Kristl; M Šentjurc; J Štrancar; S Pečar (241-244).
Solid lipid nanoparticles (SLN) are drug carrier system composed of biodegradable substances, which are solid at room temperature. The physico-chemical properties and structure of the incorporated compounds can affect their partitioning in SLN dispersions. In this work the influence of lipophilicity and structure of different SP on its location in SLN were studied. By electron paramagnetic resonance (EPR) measurements it was found that lipophilic SP distribute between a solid glyceride core and a soft phospholipid layer, with the more polar part (piperidine ring or methylcarboxylic groups) oriented toward the water–lipid interface. The majority of SP is located in the phospholipid layer, but the portion in the solid lipid core increases with SP lipophilicity. The hydrophilic Tempol does not incorporate into SLN.
Keywords: Solid lipid nanoparticles; Lipophilic spin probes; Location; Electron paramagnetic resonance;

Nanoparticles with decreasing surface hydrophobicities: influence on plasma protein adsorption by A Gessner; R Waicz; A Lieske; B.-R Paulke; K Mäder; R.H Müller (245-249).
The rapid uptake of i.v. injected nanoparticles by cells of the mononuclear phagocytic system (MPS) is a major obstacle for a long blood circulation time and a drug targeting to sites other than the MPS. The adsorption of proteins on the particles surface after i.v. administration depends on their surface characteristics and is regarded as key factor for the in vivo organ distribution. The objective of this study is to investigate changes in the plasma protein adsorption patterns in the course of surface hydrophobicity variation. Latex particles with decreasing surface hydrophobicity were synthesized as model colloidal carriers. Physicochemical characterization had been performed and considerable differences in the protein adsorption patterns on the particles could be detected by using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Correlations between physicochemical characteristics and the protein adsorption patterns have been found and are discussed.
Keywords: Nanoparticles; Surface hydrophobicity; Protein adsorption; Two-dimensional electrophoresis; Drug targeting;

Nuclear gene targeting using negatively charged liposomes by C Welz; W Neuhuber; H Schreier; R Repp; W Rascher; A Fahr (251-252).
Oligonucleotides are a very useful tool to control gene activity. Oligos work by complementary base-pairing with target sequences either in the nucleus or in the cytosol (Zelphati, O., Szoka, F.C., Jr., 1996. Liposomes as a carrier for intracellular delivery of antisense oligonucleotides: a real or magic bullet? J. Contr. Rel. 41, 99–119). In a new approach using chimeric oligonucleotides (Yoon, K., Cole Strauss, A., Kmiec, E.B., 1996. Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA–DNA oligonucleotide. Proc. Natl. Acad. Sci. USA 93, 2071–2076) conversion of single base mutations with help of intranuclear repair mechanisms maybe an advantageous method to cure genetic diseases which are based on single point mutations. These chimeric oligonucleotides are constructed in a way that they form an intramolecular double strand of DNA and modified RNA-bases. We used a fluorescent labelled pure 68-mer DNA-analogue of a chimeric oligonucleotides to follow the intracellular fate of these kind of genetic material. The oligos were complexed with protamine sulfate and coated with three different liposomal formulations. The AVE™-3 formulation shows enhanced properties compared to a classical neutral and negatively charged formulation. Nuclear localisation of oligos could only be observed with the AVE™-3 formulation. Furthermore only the negatively charged liposome formulations interact with the protamine-complexed oligonucleotides.
Keywords: Nuclear gene targeting; Liposomes; Negatively charged;

Keywords: Aphidicolin; Nanosuspension; Leishmania; Drug targeting; Antiprotozoal;

Silica nanoparticles modified with aminosilanes as carriers for plasmid DNA by Carsten Kneuer; Mohammad Sameti; Eleonore G Haltner; Thomas Schiestel; Hermann Schirra; Helmut Schmidt; Claus-Michael Lehr (257-261).
We synthesised silica nanoparticles (SiNP) with covalently linked cationic surface modifications and demonstrated their ability to electrostatically bind, condense and protect plasmid DNA. These particles might be utilised as DNA carriers for gene delivery. All nanoparticles were sized between 10 and 100 nm and displayed surface charge potentials from +7 to +31mV at pH 7.4. They were produced by modification of commercially available (IPAST) or in-house synthesised silica particles with either N-(2-aminoethyl)-3-aminopropyltrimethoxysilane or N-(6-aminohexyl)-3-aminopropyltrimethoxysilane. All particles formed complexes with pCMVbeta plasmid DNA as evidenced by ratio dependend retardation of DNA in the agarosegel and co-sedimentation of soluble DNA with nanoparticles. High salt and alkaline pH did inhibit complex formation. Absorption onto the particles also decreased the hydrodynamic dimensions of plasmid DNA as shown by photon correlation spectroscopy. Complexes formed in water at a w/w ratio of Si26H:DNA (pCMVbeta) of 300 were smallest with a mean hydrodynamic diameter of 83 nm. For effective condensation a w/w ratio of Si26H:DNA of 30 was sufficient. Further, the absorbed DNA was protected from enzymatic degradation by DNase I.
Keywords: Silica; Nanoparticle; DNA carrier; Gene delivery;

Index (263-265).