Lipid-based particle engineering via spray-drying for targeted delivery of antibiotics to the lung.

Pulmonary delivery of antibiotics for the treatment of tuberculosis provides several benefits compared to conventional oral and parenteral administration. API-loaded particles delivered directly to alveolar macrophages, where Mycobacterium tuberculosis resides, can reduce the required dose and decrease the severe side effects of conventional treatment. In this work, lipid-microparticles loaded with rifampicin were engineered via spray-drying to be administered as a carrier-free dry powder for inhalation. Although, it is well-known that spray-drying of lipid-based excipients is strongly limited, a completely lipid-based formulation using diglycerol full ester of behenic acid was produced. The solid state of the lipid, providing high melting temperature, absence of polymorphism and monophasic crystallization, led to high yield of spray-dried particles (83%). Inhalable particles of mass median aerodynamic diameter of 2.36 µm, median geometric size of 2.05 µm, and negative surface (-50.03 mV) were engineered. Such attributes were defined for deep lung deposition and targeted delivery of antibiotics to alveolar macrophages. Superior aerodynamic performance as carrier-free DPI was associated to a high fine particle fraction of 79.5 %. No in vitro cytotoxic effects were found after exposing epithelial cell lines and alveolar macrophages. In vitro uptake of particles into alveolar macrophages indicated the efficiency of their targeted delivery. The use of highly processable and safe lipid-based excipients for particle engineering via spray-drying can extend the availability of materials for functionalized applications for pulmonary delivery.

Mechanically promoted lipid-based filaments via composition tuning for extrusion-based 3D-printing.

Lipid excipients are favorable materials in pharmaceutical formulations owing to their natural, biodegradable, low-toxic and solubility/permeability enhancing properties. The application of these materials with advanced manufacturing platforms, particularly filament-based 3D-printing, is attractive for personalized manufacturing of thermolabile drugs. However, the filament's weak mechanical properties limit their full potential. In this study, highly flexible filaments were extruded using PG6-C16P, a lipid-based excipient belonging to the group of polyglycerol esters of fatty acids (PGFAs), based on tuning the ratio between its major and minor composition fractions. Increasing the percentage of the minor fractions in the system was found to enhance the relevant mechanical filament properties by 50-fold, guaranteeing a flawless 3D-printability. Applying a novel liquid feeding approach further improved the mechanical filament properties at lower percentage of minor fractions, whilst circumventing the issues associated with the standard extrusion approach such as low throughput. Upon drug incorporation, the filaments retained high mechanical properties with a controlled drug release pattern. This work demonstrates PG6-C16 P as an advanced lipid-based material and a competitive printing excipient that can empower filament-based 3D-printing.

Filament-based 3D-printing of placebo dosage forms using brittle lipid-based excipients.

In order to expand the limited portfolio of available polymer-based excipients for fabricating three-dimensional (3D) printed pharmaceutical products, Lipid-based excipients (LBEs) have yet to be thoroughly investigated. The technical obstacle of LBEs application is, however their crystalline nature that renders them very brittle and challenging for processing via 3D-printing. In this work, we evaluated the functionality of LBEs for filament-based 3D-printing of oral dosage forms. Polyglycerol partial ester of palmitic acid and polyethylene glycols monostearate were selected as LBEs, based on their chemical structure, possessing polar groups for providing hydrogen-bonding sites. A fundamental understanding of structure-function relationship was built to screen the critical material attributes relevant for both extrusion and 3D-printing processes. The thermal behavior of lipids, including the degree of their supercooling, was the critical attribute for their processing. The extrudability of materials was improved through different feeding approaches, including the common powder feeding and a devised liquid feeding setup. Liquid feeding was found to be more efficient, allowing the production of filaments with high flexibility and improved printability. Filaments with superior performance were produced using polyglycerol ester of palmitic acid. In-house designed modifications of the utilized 3D-printer were essential for a flawless processing of the filaments.

Lipid-microparticles for pulmonary delivery of active pharmaceutical ingredients: Impact of lipid crystallization on spray-drying processability.

Spray-drying is an extensively used technology for engineering inhalable particles. Important technical hurdles are however experienced when lipid-based excipients (LBEs) are spray-dried. Stickiness, extensive wall deposition, or simply inability to yield a solid product have been associated to the low melting points of LBEs. In this work, solutions containing polyglycerol esters of behenic acid (PGFA-behenates), or other high melting point LBEs, were spray-dried to produce ibuprofen (IBU)-loaded inhalable lipid-microparticles. Prior to spray-drying, rational boundaries for the outlet temperature of the process were defined using LBE-IBU phase diagrams. Despite spray-drying the solutions at outlet temperatures below the boundaries, process performance and yield among LBEs were entirely different. Lipid crystallization into polymorphs or multi-phases negatively impacted the yield (10-47%), associated to liquid fractions unable to recrystallize at the surrounding gas temperature in the spray-dryer. The highest yields (76-82%), ascribed to PGFA-behenates, resulted from monophasic crystallization and absence of polymorphism. Lipid-microparticles, composed of a PGFA-behenate, were characterized by a volume mean diameter of 6.586 µm, tap density of 0.389 g/cm3 and corrugated surface. Application as carrier-free dry powder for inhalation resulted in high emitted fraction (90.9%), median mass aerodynamic diameter of 3.568 µm, fine particle fraction of 45.6% and modified release in simulated lung fluid.

Novel approach for overcoming the stability challenges of lipid-based excipients. Part 3: Application of polyglycerol esters of fatty acids for the next generation of solid lipid nanoparticles.

Solid lipid nanoparticles (SLN) are an advantageous carrier system for the delivery of lipophilic active pharmaceutical ingredients (APIs). The use of SLN has been limited due to stability issues attributed to the unstable solid state of the lipid matrix. A novel approach for overcoming this problem is the application of polyglycerol esters of fatty acids (PGFAs) as lipid matrices with stable solid state. PG2-C18 full, a PGFA molecule, was used to develop SLN loaded with dexamethasone as a model API. Dexamethasone-loaded SLN were manufactured via melt-emulsification and high pressure homogenization in the dosage form of a lipid nanosuspension. SLN with median particle size of 242.1 ± 12.4 nm, zeta potential of -28.5 ± 7.8 mV, entrapment efficiency of 90.2 ± 0.7% and API released after 24 h of 81.7 ± 0.7%, were produced. Differential Scanning Calorimetry (DSC) and Small and Wide Angle X-Ray Scattering (SWAXS) analysis of the lipid nanosuspension evidenced the crystallization of PG2-C18 full in a monophasic system in α-form and absence of polymorphism and crystallite growth up to 6 months storage at room temperature. This resulted in stable performance of the SLN after storage: absence of particle agglomeration, no API expulsion, and stable release profile. The potential pulmonary administration of SLN was tested by the nebulization capacity of the lipid nanosuspension. Cellular exposures to SLN did not induce cytotoxicity or immune effect on pulmonary cells. The application of PGFAs as safe and stable lipid matrices provide a promising approach for the development of the next generation of SLN.

Novel approach for overcoming the stability challenges of lipid-based excipients. Part 1: Screening of solid-state and physical properties of polyglycerol esters of fatty acids as advanced pharmaceutical excipients.

The major challenge of conventional lipid-based excipients (LBE) for drug delivery is their unstable solid state, affecting the stability of pharmaceutical product. Polyglycerol esters of fatty acids (PGFAs) are oligomeric hydroxyethers of glycerol fully or partially esterified with fatty acids. Tuning the number of polyglycerol moieties, fatty acids chain length and free hydroxyl groups per molecule results in diverse physicochemical properties, e.g. HLB, melting point, and wettability, which makes these molecules attractive candidates as novel LBE for different pharmaceutical applications. In this first part of our studies the solid state of PGFAs and the stability thereof were profiled on molecular, nano, and microstructural level and the resulting properties as LBE. DSC analysis confirmed the single phase system of PGFAs without phase separation. WAXS patterns revealed the absence of polymorphism and the direct crystallization into a stable α-form; without transition to more dense configurations. SAXS patterns exposed the lamellar arrangement. The lamellae stacks were characterized by the crystallite thickness and growth. The nano, microstructure and physicochemical properties of PGFAs remained stable during storage. The stable solid state and the broad functionality of PGFAs offer a novel approach to overcome the challenges faced by conventional LBE for advanced pharmaceutical applications. Examples for such applications are presented in the next parts of this study.

Novel approach for overcoming the stability challenges of lipid-based excipients. Part 2: Application of polyglycerol esters of fatty acids as hot melt coating excipients.

The application of hot melt coating (HMC) as an economic and solvent-free technology is restricted in pharmaceutical development, due to the instable solid-state of HMC excipients resulting in drug release instability. We have previously introduced polyglycerol esters of fatty acids (PGFAs) with stable solid-state (Part 1). In this work we showed a novel application of PGFAs as HMC excipients with stable performance. Three PGFA compounds with a HLB range of 5.1-6.2 were selected for developing immediate-release formulations. The HMC properties were investigated. The viscosity of molten lipids at 100 °C was suitable for atomizing. The DSC data showed the absence of low solidification fractions, thus reduced risk of agglomeration during the coating process. The driving force for crystallization of selected compounds was lower and the heat flow exotherms were broader compared to conventional HMC formulations, indicating a lower energy barrier for nucleation and lower crystallization rate. Lower spray rates and a process temperature close to solidification temperature were desired to provide homogeneous coating. DSC and X-ray diffraction data revealed stable solid state during 6 months storage at 40 °C. API release was directly proportional to HLB and indirectly proportional to crystalline network density and was stable during investigated 3 months. Cytotoxicity was assessed by dehydrogenase activity and no in vitro cytotoxic effect was observed.

In vitro toxicity screening of polyglycerol esters of fatty acids as excipients for pulmonary formulations.

One of the main problems for the development of pulmonary formulations is the low availability of approved excipients. Polyglycerol esters of fatty acids (PGFA) are promising molecules for acting as excipient for formulation development and drug delivery to the lung. However, their biocompatibility in the deep lung has not been studied so far. Main exposed cells include alveolar epithelial cells and alveolar macrophages. Due to the poor water-solubility of PGFAs, the exposure of alveolar macrophages is expected to be much higher than that of epithelial cells. In this study, two PGFAs and their mixture were tested regarding cytotoxicity to epithelial cells and cytotoxicity and functional impairment of macrophages. Cytotoxicity was assessed by dehydrogenase activity and lactate dehydrogenase release. Lysosome function, phospholipid accumulation, phagocytosis, nitric oxide production, and cytokine release were used to evaluate macrophage function. Cytotoxicity was increased with the increased polarity of PGFA molecules. At concentrations above 1 mg/ml accumulation in lysosomes, impairment of phagocytosis, secretion of nitric oxide, and increased release of cytokines were noted. The investigated PGFAs in concentrations up to 1 mg/ml can be considered as uncritical and are promising for advanced pulmonary delivery of high powder doses and drug targeting to alveolar macrophages.

Monitoring of a Hot Melt Coating Process via a Novel Multipoint Near-Infrared Spectrometer.

The aim of the present work was to develop a PAT strategy for the supervision of hot melt coating processes. Optical fibers were placed at various positions in the process chamber of a fluid bed device. Experiments were performed to determine the most suitable position for in-line process monitoring, taking into account such requirements as a good signal to noise ratio, the mitigation of dead zones, the ability to monitor the product over the entire process, and reproducibility. The experimental evidence suggested that the position at medium fluid bed height, looking towards the center, i.e., normal to particle movement, proved to be the most reliable position. In this study, the advantages of multipoint monitoring are shown, and an in-line-implementation was created. This enabled the real-time supervision of the process, including the fast detection of inhomogeneities and disturbances in the process chamber, and the compensation of sensor malfunction. In addition, a model for estimating the particle size distribution via NIR was successfully created. This ensures that the quality of the product and the endpoint of the coating process can be determined correctly.

Advanced stable lipid-based formulations for a patient-centric product design.

Multiparticulate dosage forms are a recent strategy to meet the special needs of children, elderly people and patients suffering from dysphagia. Our study presents a novel and cost-efficient approach for the manufacturing of a taste-masked multiparticulate system with a stable immediate release profile by applying lipid-based excipients in a solvent-free hot melt coating process. The thermosensitive N-acetylcysteine (N-ac) was used as model drug and hot-melt coated with a mixture of tripalmitin and polysorbate 65. A predictive in vitro method for the evaluation of the taste masking efficiency was developed based on the deprotonation of the carboxyl group of N-ac and the decline of pH, responsible for the unpleasant sour taste of the compound. The method was confirmed using in vivo studies. Differential scanning calorimetry and X-ray scattering experiments revealed polymorphic transformation and its dependency on transformation time, temperature and emulsifier concentration. During the process, the coating was transformed almost completely into the stable ß-polymorph, leading to an unaltered dissolution profile during storage. A statistical design was conducted that revealed the critical process parameters affecting the taste masking efficiency and drug release. This study shows the successful application of solvent-free hot-melt coating in the development of a taste-masked and stable formulation.

Role of Lipid Blooming and Crystallite Size in the Performance of Highly Soluble Drug-Loaded Microcapsules.

Hot-melt coating is of growing interest, because it does not require solvents, resulting in reduced process times and costs. However, excipients for this technology are mainly triacylglycerides (TAGs) or their derivatives, which exhibit polymorphism, surface disruption, and complex crystallite networks, affecting the release profile of produced microcapsules. In this work, anhydrous citric acid crystals were coated with molten tristearin using conventional inlet air temperatures (microcapsules A) and temperatures above the melting point of α-form (microcapsules B). Additionally, microcapsules A were tempered to achieve polymorphic stability (microcapsules AB). The product yield and coating efficacy were above 90% and 97%, respectively, demonstrating the feasibility and efficacy of the process. Small angle X-ray scattering analysis confirmed that the tristearin shell of microcapsules B is in the ß-form with a larger average crystallite size than microcapsules A and AB. Scanning electron microscopy images revealed a nonbloomed surface of microcapsules B. We showed that blooming does not play a critical role in the drug release, but the apparent diffusion coefficient of drug is dramatically reduced by increasing TAGs crystallite size and resulting tortuosity. This work brings new insights on the micrometric properties of solid lipid dosage forms, being an important step to prevent the overuse of excipients with unknown toxicity.

A quality by design study applied to an industrial pharmaceutical fluid bed granulation.

The pharmaceutical industry is encouraged within Quality by Design (QbD) to apply science-based manufacturing principles to assure quality not only of new but also of existing processes. This paper presents how QbD principles can be applied to an existing industrial pharmaceutical fluid bed granulation (FBG) process. A three-step approach is presented as follows: (1) implementation of Process Analytical Technology (PAT) monitoring tools at the industrial scale process, combined with multivariate data analysis (MVDA) of process and PAT data to increase the process knowledge; (2) execution of scaled-down designed experiments at a pilot scale, with adequate PAT monitoring tools, to investigate the process response to intended changes in Critical Process Parameters (CPPs); and finally (3) the definition of a process Design Space (DS) linking CPPs to Critical to Quality Attributes (CQAs), within which product quality is ensured by design, and after scale-up enabling its use at the industrial process scale. The proposed approach was developed for an existing industrial process. Through enhanced process knowledge established a significant reduction in product CQAs, variability already within quality specifications ranges was achieved by a better choice of CPPs values. The results of such step-wise development and implementation are described.

Combining microwave resonance technology to multivariate data analysis as a novel PAT tool to improve process understanding in fluid bed granulation.

A set of 192 fluid bed granulation batches at industrial scale were in-line monitored using microwave resonance technology (MRT) to determine moisture, temperature and density of the granules. Multivariate data analysis techniques such as multiway partial least squares (PLS), multiway principal component analysis (PCA) and multivariate batch control charts were applied onto collected batch data sets. The combination of all these techniques, along with off-line particle size measurements, led to significantly increased process understanding. A seasonality effect could be put into evidence that impacted further processing through its influence on the final granule size. Moreover, it was demonstrated by means of a PLS that a relation between the particle size and the MRT measurements can be quantitatively defined, highlighting a potential ability of the MRT sensor to predict information about the final granule size. This study has contributed to improve a fluid bed granulation process, and the process knowledge obtained shows that the product quality can be built in process design, following Quality by Design (QbD) and Process Analytical Technology (PAT) principles.

Immunostimulatory properties of CpG-oligonucleotides are enhanced by the use of protamine nanoparticles.

The aim of this paper was to investigate if the immunostimulatory effects of CpG-oligonucleotides (CpG-ODN) can be enhanced by the use of biodegradable protamine nanoparticles (proticles). We analyzed size, surface charge, and morphology of protamine nanoparticles containing CpG-ODN with photon correlation spectroscopy and transmission electron microscopy. Immunostimulatory effects of these nanoparticles on B cells, plasmacytoid dendritic cells (PDC), peripheral blood mononuclear cells, and whole blood were studied. Cytokine production, activation of the cells in terms of upregulation of surface molecules and uptake of nanoparticles were examined. We found that the use of protamine nanoparticles significantly increased (20-fold) CpG-ODN mediated interferon (IFN)-alpha production of PDC. ODN uptake in PDC was only marginally enhanced. CpG-ODN mediated IP-10 production in whole blood was strongly enhanced by the use of nanoparticles. Apart from a slight increase in CpG-ODN-induced interleukin (IL)-6 production in B cells, other parameters like the CpG-mediated activation of B cells and PDC as well as tumor necrosis factor (TNF)-alpha production of PDC remained largely unchanged. The use of control ODN indicated that the protamine nanoparticles themselves have no immunostimulatory properties. These results strongly support the use of particulate delivery systems like biodegradable protamine nanoparticles for the development of CpG-ODN-based therapeutics.

Oligonucleotide-protamine-albumin nanoparticles: Protamine sulfate causes drastic size reduction.

Nanoparticles prepared by self-assembly from oligonucleotides (ONs), protamine free base, and human serum albumin ("ternary proticles") are spheres of diameters around 200 nm. Substitution of the protamine free base by protamine sulfate leads to proticles of only around 40 nm in diameter with otherwise unchanged properties. The availability of drug delivery systems of very similar composition but grossly different size may be advantageous when dealing with cells which show size-dependent particle uptake. These nanoparticles are promising candidates for ON delivery to cells because of the following reasons: (1) They are stable for several hours in solutions of up to physiological ionic strength; (2) they are efficiently taken up by cells; (3) after cellular uptake, they easily release the ONs even when these are present as phosphorothioates.

Albumin-protamine-oligonucleotide nanoparticles as a new antisense delivery system. Part 1: physicochemical characterization.

In this paper, a ternary system of albumin-protamine-oligonucleotide nanoparticles (AlPrO-NP) recently developed by Vogel et al. [V. Vogel, D. Lochmann, J. Weyermann, G. Mayer, C. Tziatzios, J.A. van den Broek, W. Haase, D. Wouters, U.S. Schubert, J. Kreuter, A. Zimmer, D. Schubert, Oligonucleotide-protamine-albumin nanoparticles: preparation, physical properties and intracellular processing, J. Controlled Rel. (in press)] which could serve as a potential drug delivery system for antisense oligonucleotides. Former studies of binary protamine-oligonucleotide nanoparticles showed two main disadvantages: (i) aggregation of the particles within a few minutes in the presence of salt; (ii) low intracellular dissociation between protamine and oligonucleotide, especially phosphorothioates. To overcome these problems, human serum albumin (HSA) as a non-toxic, biodegradable macromolecule was introduced as protective colloid. The assembly process of AlPrO-NP was investigated by small angle X-ray scattering (SAXS), fluorescence correlation spectroscopy (FCS), photon correlation spectroscopy (PCS) measurements and scanning electron microscopy (SEM). 'Initial complexes' of HSA and protamine sulphate with a mean hydrodynamic diameter (dh) of about 10-14 nm were found. After adding oligonucleotides (unmodified, phosphorothioate DNA and small interfering RNA), nanoparticles (NPs) were assembled in water and in isotonic media with a dh in a range of 230-320 nm for most preparations. The chemical composition of the particles was investigated by high performance liquid chromatography and fluorescence spectrometry. The whole amount of oligonucleotides (30 microg) was entrapped into the particles at a 1:2 mass ratio (oligonucleotide/protamine). Approximately 7-10% (w/w) of the HSA was bound to the particles. The surface charge of the particles ranged from about +12 to -60 mV depending on the protamine concentration and the ionic conditions. The size and the molecular weight of the components, initial complexes and two model NP preparations were calculated from FCS data. These data verified the PCS, SEM and SAXS measurements.

Albumin-protamine-oligonucleotide-nanoparticles as a new antisense delivery system. Part 2: cellular uptake and effect.

Antisense oligonucleotides have been used as a specific tool to inhibit the expression of disease associated genes for many years. Unfortunately, oligonucleotides are polyanionic macromolecules which have a weak permeability through biological membranes and are rapidly degraded by nucleases. The purpose of this work is to characterise a new drug delivery system developed by [V. Vogel, D.Lochmann, J. Weyermann, G. Mayer, C. Tziatios, J.A. van der Brock, W. Haase, D. Wouters, U.S. Schubert, J. Kreuter, A. Zimmer, D. Schubert, Oligonucleotide-protamine-albumin nanoparticles preparation, physical properties and intracellular processing, J. Controlled Rel. (in press)] which allows an increased cellular uptake and an intracellular dissociation of the oligonucleotides. The new system based on nanoparticles (NPs) consists of human serum albumin, protamine sulphate and antisense-oligonucleotides (AlPrO). We tested these new nanoparticles on mouse fibroblasts which were stably transfected with a N-methyl-D-aspartate (NMDA) receptor (NR). This cell line enabled us to perform in vitro studies of cellular uptake, intracellular dissociation and effect of the antisense-oligonucleotide in a simple excitotoxicity model. We compared our findings with free oligonucleotides and a commercial available liposomal preparation (DOTAP). We found a 12-fold increased cellular uptake of oligonucleotides in comparison to free oligonucleotides while 100% of the cells were transfected. The AlPrO-NPs showed very low cytotoxic side effects during a 24 h application. We saw an antisense effect of about 35% in a functional assay as well as on the protein level (western blot). The results of the cell penetration and the antisense assay demonstrated that AlPrO nanoparticles are promising carriers for oligonucleotide administration.

Oligonucleotide-protamine-albumin nanoparticles: preparation, physical properties, and intracellular distribution.

Oligodesoxynucleotides (ODNs) or the corresponding phosphorothioates (PTOs) spontaneously form spherical nanoparticles ("proticles") with protamine in aqueous solutions. The proticles can cross cellular membranes and release the ODNs within the cells. Thus, they represent a potential drug delivery system. The major disadvantages of this system are a lack of stability in salt solutions and its inability to also release PTOs. The present study shows, using PTOs and protamine free base, that these shortcomings can be eliminated by the addition of human serum albumin (HSA) as a third component to the starting mixture. The "ternary" proticles thus obtained contain maximally a few percent of the HSA that was originally present. Nevertheless, they differ from the previously studied "binary" proticles: (1) They are stable in salt solutions for at least several hours. (2) They show a high cellular uptake into murine fibroblasts, and they readily release the PTOs after uptake. The ternary proticles therefore represent a considerable improvement over binary proticles for use as drug delivery systems.

A practical note on the use of cytotoxicity assays.

In this study, four cytotoxicity detection assays and four cytotoxic mechanisms were compared in one cellular system. Cellular responses and their effects were characterized. The assays used are based on different modes of detection like LDH release, MTT metabolism, neutral red uptake and the ATP content of treated cells. As cytotoxic mechanisms were used the model agents triton X-100, chloroquine and sodium azide (which are common in cell culture) as well as an ion channel (NMDA) mediated excitotoxicity cell death (which is specific for the cell line used). We found major differences in the calculated EC(50)-values for the cytotoxic effect of choroquine (0.1 up to 200 mM) and for sodium azide (4 up to 1300 mM) depending on the assay used. Therefore, it is important to choose a suitable cytotoxicity assay depending on the supposed cell death mechanism. As this study compares the strengths and weaknesses of the most common assays, it can help to find the appropriate one.

Comparison of antisense oligonucleotide drug delivery systems.

Antisense oligonucleotides (AS-ONs) are specific drugs to inhibit gene expression at the transcriptional level. They possess a poor bioavailability and can be degraded by nucleases very rapidly. Therefore, a strong need for the development of oligonucleotide drug delivery systems exists. In the present study, two commercially available liposomes (DOTAP, lipofectin), one artificial virus capsoid (polyoma VP1), two cationic acrylate nanoparticles and two protamine-based nanoparticle preparations (proticles) were compared. Physical parameters of all carrier systems including z-average size, size distribution and surface charge regarding were determined. Cellular uptake was measured by a microplate fluorescence quantification method and, in addition, was visualized in mouse fibroblasts by confocal laser scan microscopy (CLSM). A comparison of cytotoxicity of the different drug delivery systems was performed in vitro using a MTT assay. Mouse fibroblasts which were stable transfected with the cDNA of a N-methyl-D-aspartate (NMDA) receptor also served as functional antisense oligonucleotide test system based on excitotoxicity (cell death). In addition, the efficiency of our oligonucleotide delivery systems was compared on the level of protein expression by Western blotting. Concluding the results, an increased uptake of the ON was found (2-18-fold) for all delivery systems compared to the free ON. Protamine-based nanoparticles showed a very low cytotoxicity in contradiction to all other carrier systems. Lipofectin could be identified as the most potent delivery system in terms of antisense effect, followed by protamine nanoparticles and DOTAP. Sequence-specific antisense effects up to 80% were observed in the functional cell death assay. The highest reduction of NMDA expression was obtained from liposomal preparations with approximately 60% analyzed by Western blot.