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.

Recombinant virus like particles as drug delivery system.

The drug delivery system described here is based on a virus like particle consisting of the recombinant expressed major capsid protein of Polyomavirus, VP1. Polyoma, a murine virus belonging to the Papovaviridae, forms a non-enveloped icosahedral capsid. These capsids are organized as a double shell composed of three different proteins: VP1,VP2 and VP3. The outer shell of the vision is composed of 360 VP1 molecules arranged as 72 pentamers. These capsids have a diameter of about 50 nm. The VP1 protein acts as a major ligand for certain membrane receptors during virus infection. Furthermore, the N-terminus of the VP1 protein contains a DNA-binding domain and a nuclear localization sequence. The recombinant production of the VP1 protein offers a save way to obtain a highly purified, non pathogenic pharmaceutical excipient. Combining these aspects, VP1 proteins provide a targeting as well as a drug binding site when used as a save drug carrier for gene therapy. Current applications are also including oligonucleotides as well as small molecules as well as vaccines.

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.

Amyloid beta-induced changes in nitric oxide production and mitochondrial activity lead to apoptosis.

Increasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of Alzheimer's disease. Thus, we investigated the effects of acute and chronic exposure to increasing concentrations of amyloid beta (Abeta) on mitochondrial function and nitric oxide (NO) production in vitro and in vivo. Our data demonstrate that PC12 cells and human embryonic kidney cells bearing the Swedish double mutation in the amyloid precursor protein gene (APPsw), exhibiting substantial Abeta levels, have increased NO levels and reduced ATP levels. The inhibition of intracellular Abeta production by a functional gamma-secretase inhibitor normalizes NO and ATP levels, indicating a direct involvement of Abeta in these processes. Extracellular treatment of PC12 cells with comparable Abeta concentrations only leads to weak changes, demonstrating the important role of intracellular Abeta. In 3-month-old APP transgenic (tg) mice, which exhibit no plaques but already detectable Abeta levels in the brain, reduced ATP levels can also be observed showing the in vivo relevance of our findings. Moreover, we could demonstrate that APP is present in the mitochondria of APPsw PC12 cells. This presence might be directly involved in the impairment of cytochrome c oxidase activity and depletion of ATP levels in APPsw PC12 cells. In addition, APPsw human embryonic kidney cells, which produce 20-fold increased Abeta levels compared with APPsw PC12 cells, and APP tg mice already show a significantly decreased mitochondrial membrane potential under basal conditions. We suggest a hypothetical sequence of pathogenic steps linking mutant APP expression and amyloid production with enhanced NO production and mitochondrial dysfunction finally leading to cell death.

New protamine quantification method in microtiter plates using o-phthaldialdehyde/N-acetyl-L-cysteine reagent.

Protamine is a well-known excipient in pharmaceutics. It represents a peptide consisting of exclusive aliphatic amino acids, hence it cannot be quantified by UV-spectroscopy (lambdamax 280 nm). A new and sensitive quantification method based on the derivatisation of protamine with ortho-phthaldialdehyde (OPA) in the presents of 2-mercaptoethanol (ME) or N-acetyl-L-cysteine (NAC) in basic aqueous solution using 96-well microtiter plates are introduced in this report. The resulting isoindol derivatives reveal a fluorescence excitation (maximum lambdaex 345 nm) and emission (maximum lambdaem 450 nm) spectra. Derivatives of OPA/NAC reagent were found to be useful for protamine quantification in pharmaceutical nanoparticle preparation containing DNA. A sufficient stability of the isoindol derivatives was shown. It was possible to determine protamine free base, protamine sulphate and protamine chloride with limits of detection less than 1.1 microg/ml.

Physicochemical characterisation of cationic polybutylcyanoacrylate-nanoparticles by fluorescence correlation spectroscopy.

The aim of this study was to compare different physical and chemical methods with fluorescence correlation spectroscopy (FCS) in order to characterise cationic acrylate nanoparticles (NP), which can deliver oligonucleotides (ON) into mammalian cells. These positively charged nanoparticles were prepared from diethylaminoethyl dextran (DEAE-dextran) and poly(n-butyl-2-cyanoacrylate) (PBCA). NP consists of PBCA oligochains with an average size of PBCA 9 mer and were formed by entrapping DEAE-dextran and dextran 70,000 in high amounts into the particle matrix. The oligochain length of PBCA was investigated by mass-spectroscopy (MALDI TOF). The molecular weight of a particle with d = 108 nm was estimated to be approximately 3.6 x 10(8) Da. The mean size of the nanoparticles were in a range of dh = 130-140 nm, as determined independently by FCS and dynamic light scattering. Atomic force microscopy and scanning electron microscopy images confirm this size range. Furthermore, the particle mass of the PBCA-NP was estimated by FCS measurements. For this approach two new methods for fluorescence labelling of cationic particles were developed. Fluorescent labelled dextran 70,000 was entrapped into the particle matrix; in addition, the derivatisation of hydroxyl groups of the NP was achieved with 5-([4,6-dichlorotriazin-2-yl]amino) fluorescein (DTAF). ON can be localised in a complex with the NP by dual-colour fluorescence cross correlation spectroscopy measurements. The zetapotential of the unloaded NP was positively charged with about +39 mV and decreased down to -40 mV on addition of excess ON. After centrifugation quantification of the ON loading onto the particles by strong anion exchange high performance liquid chromatography (SAX HPLC) and FCS showed that approximately 20 microg ON per 100 g NP was adsorbed. The FCS measurements of the ON adsorption in situ was found to be much higher with approximately 95 microg ON per 100 g NP.

Rapid screening method for antisense oligonucleotides against human growth factor receptor p185(erbB-2).

The aim of this study was the development of an indirect cell proliferation assay as screening tool for antisense oligonucleotides. Unmodified and phosphorothioate-modified oligonucleotides with different amounts of sulfur in the DNA backbone were examined for biologic activity. The human growth factor receptor p185(erbB-2) was chosen as cellular target. High-level expression of this protein can be related to an early event in tumor development and cell proliferation. We correlated the expression of p185(erbB-2) with the cell proliferation of BT-474. Additionally a control cell line (MCF-7) with very low p185(erbB-2) expression was cultivated. Antisense oligonucleotides were transfected as a liposome formulation (Lipofectin), GIBCO-BRL, Eggenstein, Germany). Cell count was correlated with a total protein quantification assay (BCA method). Stability against nuclease digestion was determined with a DNase I assay. Sequence-specific antisense effects on the p185(erbB-2) protein level were determined by Western blot. An antisense phosphorothioate oligonucleotide was identified to inhibit the cell proliferation in comparison to a random control and a negative control oligonucleotide sequence. The comparison of fully thioated, partly thioated, and unmodified oligonucleotides verified the correlation between the enzymatic stability and the biologic activity of the different modifications. Using the unstable oligonucleotides, more treatments were necessary to achieve an antiproliferative effect. In our study, the indirect proliferation assay was found to be a reliable and potent tool for an antisense oligonucleotide screening by targeting the p185(erbB-2) protein.