Systematic evaluation of oligodeoxynucleotide binding and hybridization to modified multi-walled carbon nanotubes.

BACKGROUND: In addition to conventional chemotherapeutics, nucleic acid-based therapeutics like antisense oligodeoxynucleotides (AS-ODN) represent a novel approach for the treatment of bladder cancer (BCa). An efficient delivery of AS-ODN to the urothelium and then into cancer cells might be achieved by the local application of multi-walled carbon nanotubes (MWCNT). In the present study, pristine MWCNT and MWCNT functionalized with hydrophilic moieties were synthesized and then investigated regarding their physicochemical characteristics, dispersibility, biocompatibility, cellular uptake and mucoadhesive properties. Finally, their binding capacity for AS-ODN via hybridization to carrier strand oligodeoxynucleotides (CS-ODN), which were either non-covalently adsorbed or covalently bound to the different MWCNT types, was evaluated. RESULTS: Pristine MWCNT were successfully functionalized with hydrophilic moieties (MWCNT-OH, -COOH, -NH2, -SH), which led to an improved dispersibility and an enhanced dispersion stability. A viability assay revealed that MWCNT-OH, MWCNT-NH2 and MWCNT-SH were most biocompatible. All MWCNT were internalized by BCa cells, whereupon the highest uptake was observed for MWCNT-OH with 40% of the cells showing an engulfment. Furthermore, all types of MWCNT could adhere to the urothelium of explanted mouse bladders, but the amount of the covered urothelial area was with 2-7% rather low. As indicated by fluorescence measurements, it was possible to attach CS-ODN by adsorption and covalent binding to functionalized MWCNT. Adsorption of CS-ODN to pristine MWCNT, MWCNT-COOH and MWCNT-NH2 as well as covalent coupling to MWCNT-NH2 and MWCNT-SH resulted in the best binding capacity and stability. Subsequently, therapeutic AS-ODN could be hybridized to and reversibly released from the CS-ODN coupled via both strategies to the functionalized MWCNT. The release of AS-ODN at experimental conditions (80 °C, buffer) was most effective from CS-ODN adsorbed to MWCNT-OH and MWCNT-NH2 as well as from CS-ODN covalently attached to MWCNT-COOH, MWCNT-NH2 and MWCNT-SH. Furthermore, we could exemplarily demonstrate that AS-ODN could be released following hybridization to CS-ODN adsorbed to MWCNT-OH at physiological settings (37 °C, urine). CONCLUSIONS: In conclusion, functionalized MWCNT might be used as nanotransporters in antisense therapy for the local treatment of BCa.

Bone Morphogenetic Protein-2 Hybridized with Nano-Anchored Oligonucleotides on Titanium Implants Enhances Osteogenic Differentiation In Vivo.

PURPOSE: Previous in vitro studies have shown that DNA oligonucleotides (ODN) can be successfully used as anchor strands for the binding and retarded release of biologically active recombinant human bone morphogenetic protein 2 (rhBMP-2). The aim of the present study was to test the hypothesis that rhBMP-2 bound to the surface of titanium implants through hybridization with nano-anchored ODN strands is biologically active and can enhance the induction of osteogenic markers in peri-implant bone in vivo. MATERIALS AND METHODS: Custom-made, surface acid-etched (SAE) titanium discs and implants were coated with ODN anchor strands and subsequently hybridized with complementary ODN strands conjugated to rhBMP-2 (AS_CS_BMP-2). Discs/implants with SAE surface, ODN-coated surface (AS), and ODN-coated surface with nonconjugated rhBMP-2 (AS_BMP-2) served as controls. Release of rhBMP-2 from the coated discs was evaluated in vitro using enzyme-linked immunosorbent assay (ELISA), and bone-specific activity was assessed through pNPP turnover by induced alkaline phosphatase (AP) up to a period of 56 days. In vivo expression of bone-specific markers was analyzed after bilateral placement of coated implants into the tibiae of 36 Wistar rats (72 tibiae total). Immunostaining for AP and runt-related transcription factor 2 (Runx2) was carried out after 1, 4, and 13 weeks. RESULTS: Release from the AS_CS_ BMP-2-coated titanium surfaces was significantly retarded compared to surfaces loaded with AS_BMP-2. The in vitro biologic activity of the released rhBMP-2 conjugates measured by AP induction was equivalent to released nonconjugated rhBMP-2. Immunostaining revealed a significant increase in the in vivo induction of AP around AS_CS_BMP-2 implants compared to the controls after 1 and 4 weeks. CONCLUSION: Titanium AS_CS_BMP-2 implants can significantly enhance osteogenic differentiation in vivo in peri-implant bone in early periods of osseointegration.

Application of Lateral and Distance Spacers in an Oligonucleotide Based Immobilization System for Bioactive Molecules onto Titanium Implants.

Immobilization of bioactive molecules (BAMs) on a nanometer scale is of great interest for functionalization of implant and scaffold surfaces in current biomaterials research. A system for immobilization of one or more compounds is described, which is based on nanomechanical fixation of single-stranded nucleic acids into an anodic titanium oxide layer and their subsequent hybridization with BAMs conjugated to the respective complementary strands. This paper focuses on further development and in depth understanding of the immobilization system, as some of the major findings established for common sensor applications for immobilization of single-stranded DNA onto gold surfaces cannot be transferred to the TiO2 surface. The first approach concerning the influence of the internal spacer sequence revealed the best performance for a polyadenine based sequence out of four homologous spacer sequences (A30, T30, C30, and G30). This overall best performance of the A30 spacer is attributed to an increased contour length by nucleotide staggering, which resulted in the best protection of the hybridization sequence from unfavorable interactions with the surface or damaging attacks by reactive oxygen species. The second approach comprises the implementation of a lateral spacer, also based on a homologous sequence of A30. Simultaneous as well as sequential adsorption of anchor strands and spacer strands were performed, and it could be shown that a preadsorption with high density of the spacer was most effective to increase hybridization efficiency.

Functionalization of titanium implants using a modular system for binding and release of VEGF enhances bone-implant contact in a rodent model.

AIMS: To test the immobilization of vascular endothelial growth factor (VEGF165 ) on the surface of titanium implants using DNA oligonucleotide (ODN) anchor strands for the ability to enhance periimplant bone formation. MATERIALS AND METHODS: DNA oligonucleotides were anchored to the surface of sandblasted acid-etched (SAE) titanium screw implants and were hybridized with complementary strands of ODN conjugated to rhVEGF165 . The implants were tested against blank SAE implants and SAE implants with nano-anchored ODN. The implants were inserted into the tibiae of 36 Sprague-Dawley rats. Primary outcome parameters were bone-implant contact (BIC), amount of new bone formation and periimplant bone density (BD). density after 1, 4 and 13 weeks. Unit of analysis has been the individual implant. RESULTS: Implants with rhVEGF165 hybridized to ODN anchor strands exhibited significantly increased average BIC after 1 month compared to blank implants and implants with anchored ODN strands. CONCLUSIONS: It is concluded that rhVEGF165 immobilized on the surface of titanium implants through nano-anchored oligonucleotide strands can accelerate BIC of sandblasted and etched titanium implants to a certain extent. The radius of effect of the growth factor appears to be limited to tissue immediately adjacent to the implant surface.

Characterization of different carbon nanotubes for the development of a mucoadhesive drug delivery system for intravesical treatment of bladder cancer.

In order to increase the effectiveness of therapeutics for bladder carcinoma (BCa) treatment, alternative strategies for intravesical applications are needed. The use of carbon nanotubes (CNTs) as basis for a multifunctional drug transporter is a promising possibility to combine traditional chemotherapeutics with innovative therapeutic agents such as antisense oligodeoxynucleotides or small interfering RNA. In the current study four CNT types varying in length and diameter (CNT-1, CNT-2, CNT-3, CNT-4) were synthesized and then characterized with different spectroscopic techniques. Compared to the pristine CNT-1 and CNT-3, the shortened CNT-2 and CNT-4 exhibited more defects and lower aspect ratios. To analyze their mucoadhesive properties, CNTs were exposed to mouse bladders ex vivo by using Franz diffusion cells. All four tested CNT types were able to adhere to the urothelium with a mean covering area of 5-10%. In vitro studies on UM-UC-3 and EJ28 BCa cells were conducted to evaluate the toxic potential of these CNTs. Viability and cytotoxicity assays revealed that the shortened CNT-2 and CNT-4 induced stronger inhibitory effects on BCa cells than CNT-1 and CNT-3. In conclusion, CNT-1 and CNT-3 showed the most promising properties for further optimization of a multifunctional drug transporter.

A modular peptide-based immobilization system for ZrO2, TiZr and TiO2 surfaces.

The present study describes a novel versatile immobilization system for the modification of implant materials with biologically active molecules (BAMs), e.g. antibiotics or growth factors. Specific adsorbing peptides are used as anchor molecules to immobilize oligodesoxynucleotides (ODNs) on the implant surface (anchor strand, AS). The BAM is conjugated to a complementary ODN strand (CS) which is able to hybridize to the AS on the implant surface to immobilize the BAM. The ODN double strand allows for a controlled release of the BAM adjustable by the ODN sequence and length. The immobilization system was developed and proven on three typical implant materials, namely ZrO2, TiZr and Ti, respectively. The parathyroid hormone (PTH) fragment 1-34 was conjugated to the CS and immobilized on these different implant materials. To investigate the biological activity of the immobilized PTH, alkaline phosphatase was quantified after incubation of the osteoblast precursor cells C2C12 on the modified samples. The results demonstrate the successful immobilization of biologically active PTH (1-34) and the high potential of the established surfaces to achieve an increased osseointegration of variable implants, especially for patients with risk factors.

Investigation of the peptide adsorption on ZrO2, TiZr, and TiO2 surfaces as a method for surface modification.

Specific surface binding peptides offer a versatile and interesting possibility for the development of biocompatible implant materials. Therefore, eight peptide sequences were examined in regard to their adsorption on zirconium oxide (ZrO2), titanium zircon (TiZr), and titanium (c.p. Ti). Surface plasmon resonance (SPR) measurements were performed on Ti coated sensor chips to determine the kinetics of the interactions and kinetic rate constants (kon, koff, KD, and Rmax). We also investigated the interactions which are present in our system. Electrostatic and coordinative interactions were found to play a major role in the adsorption process. Four of the eight examined peptide sequences showed a significant adsorption on all investigated materials. Moreover, the two peptides with the highest adsorption could be quantified (up to 370 pmol/cm(2)). For potential biomaterials applications, we proved the stability of the adsorption of selected peptides in cell culture media, under competition with proteins and at body temperature (37 °C), and their biocompatibility via their effects on the adhesion and proliferation of human mesenchymal stem cells (hMSCs). The results qualify the peptides as anchor peptides for the biofunctionalization of implants.

Improved anchorage of Ti6Al4V orthopaedic bone implants through oligonucleotide mediated immobilization of BMP-2 in osteoporotic rats.

The aim of the present study was to test the biocompatibility and functionality of orthopaedic bone implants with immobilized oligonucleotides serving as anchor stands for rhBMP-2 and rhVEGF-A conjugated with complementary oligonucleotides in an osteoporotic rat model. Al2O3-blasted acid etched Ti6Al4V implants, carrying oligonucleotide anchor strands and hybridized with rhBMP-2 or rhVEGF-A through complementary 31-mer oligonucleotide stands were inserted into the proximal tibia of ovariectomized rats. At the time of surgery (15 weeks after ovariectomy) microCT analysis showed significantly lower bone mineral density compared to non-ovariectomized animals. Bone-implant contact (BIC) and pullout-force were not negatively affected by non-hybridized anchor strands. Twelve weeks after surgery, a significantly higher pullout force was found for BMP-2 hybridized to the anchor strands compared to non-hybridized anchor strands or native samples, and on histomorphometric analysis BIC was highest in the BMP group. Thus, we could show the biocompatibility and in vivo functionality of this modular, self-organizing system for immobilization and subsequent release of BMP-2 in vivo.

Functionalized carbon nanotubes as transporters for antisense oligodeoxynucleotides.

The use of DNA-based therapeutics requires efficient delivery systems to transport the DNA to their place of action within the cell. To accomplish this, we investigated multiwalled carbon nanotubes (pristine MWCNT, p-MWCNT) functionalized with hydroxyl groups via 1,3-dipolar cycloaddition. In this way, we have obtained MWCNT-f-OH with improved stability in aqueous dispersions which is an advantageous property for their use in cellular environments. Afterwards, a carrier strand oligodeoxynucleotide (CS-ODN) was adsorbed to MWCNT-f-OH followed by hybridization with a therapeutic antisense oligodeoxynucleotide (AS-ODN). The amount of adsorbed CS-ODN, as well as the complementary AS-ODN and a non-complementary oligodeoxynucleotide (NS-ODN) as reference, was directly measured by radionuclide labeling of ODNs. We show that subsequent release of AS-ODNs and NS-ODNs was possible for MWCNT-f-OH above the melting temperature of AS-ODNs at 80 °C and under physiological conditions at different pH values at 37 °C. We also show a very low influence of p-MWCNT and MWCNT-f-OH on the cell viability of the bladder carcinoma (BCa) cell line EJ28 and that both MWCNT types were internalized by EJ28. Therefore, MWCNT-f-OH represents a promising carrier able to transport and release AS-ODNs inside cells.

Peptide linkers for the immobilization of bioactive molecules on biphasic calcium phosphate via a modular immobilization system.

Herein we describe the use of peptide linkers to establish a nucleic acid-based immobilization system based on biphasic calcium phosphates (BCP), with which different molecules can be immobilized at the same time in defined ratios. It consists of single-stranded oligonucleotides, anchor strands (AS) which are immobilized to the surface and conjugates of complementary strands (CS) and bioactive molecules that bind to the AS via Watson-Crick base pairing. AS immobilization can be achieved on calcium phosphate ceramics using conjugates of AS and peptides that bind specifically to the ceramic. We successfully immobilized three different peptide sequences on BCP. Among them, we identified Stath (DpSpS EEK FLR RIG RFG, phosphoserine) as the most suitable one and further immobilized Stath-AS conjugates on BCP. This immobilized AS was able to hybridize with CS. Unspecific adsorption of oligonucleotides on the BCP surface was negligible. The stability of the system was proven by short term desorption experiments. The amounts of immobilized peptides, oligonucleotides and peptide-AS conjugates were determined by an enzymatic assay using biotin-streptavidin interactions, and were found to reach surface densities that are of therapeutic relevance (0.03 pmol cm(-2)).

Effect of oligonucleotide mediated immobilization of bone morphogenic proteins on titanium surfaces.

The aim of the present study was to test the hypothesis that oligonucleotides can be used for anchorage and slow release of osteogenic growth factors such as BMP to enhance the osteogenic activity of a titanium implant surface. Strands of 60-mer non-coding DNA oligonucleotides (ODN) were bound to an acid-etched sandblasted cp Ti-surface by nanomechanical fixation using anodic polarization. RhBMP2 that had been conjugated to complementary strands of DNA oligonucleotides was then bound to the anchored ODN strands by hybridization. Binding studies showed a higher binding capacity compared to non-conjugated BMP2. Long term release experiments demonstrated a continuous release from all surfaces that was lowest for the conjugated BMP2 bound to the ODN anchor strands. Proliferation of human bone marrow stroma cells (hBMSC) was significantly increased on these surfaces. Immunofluorescence showed that hBMSC grown on surfaces coated with specifically bound conjugated BMP2 developed significantly higher numbers of focal adhesion points and exhibited significantly higher levels of transcription of osteogenic markers alkaline phosphatase and osteopontin at early intervals. Biological activity (induction of alkaline phosphatase) of conjugated BMP2 released from the surface was comparable to released non-conjugated BMP2, indicating that conjugation did not negatively affect the activity of the released molecules. In conclusion the present study has shown that BMP2 conjugated to ODN strands and hybridized to complementary ODN strands anchored to a titanium surface has led to slow growth factor release and can enhance the osteogenic activity of the titanium surface.

Biological nano-functionalization of titanium-based biomaterial surfaces: a flexible toolbox.

Surface functionalization with bioactive molecules (BAMs) on a nanometre scale is a main field in current biomaterial research. The immobilization of a vast number of substances and molecules, ranging from inorganic calcium phosphate phases up to peptides and proteins, has been investigated throughout recent decades. However, in vitro and in vivo results are heterogeneous. This may be at least partially attributed to the limits of the applied immobilization methods. Therefore, this paper highlights, in the first part, advantages and limits of the currently applied methods for the biological nano-functionalization of titanium-based biomaterial surfaces. The second part describes a new immobilization system recently developed in our groups. It uses the nanomechanical fixation of at least partially single-stranded nucleic acids (NAs) into an anodic titanium oxide layer as an immobilization principle and their hybridization ability for the functionalization of the surface with BAMs conjugated to the respective complementary NA strands.

Oligonucleotide-RGD peptide conjugates for surface modification of titanium implants and improvement of osteoblast adhesion.

A new concept for modular biosurface engineering of titanium implants based on the self-assembly of complementary oligonucleotides was biochemically investigated and optimized. This study describes the synthesis and characterization (RP-HPLC and Sakaguchi assay) of oligodeoxyribonucleotide (ODN) conjugates of the hexapeptide GRGDSP containing the RGD sequence as the recognition motif for cellular adhesion receptors (integrins). The peptide was chosen exemplarily as a model molecule, because it is a simple but potent bioactive molecule and relatively well investigated. The conjugation products must fulfill two main requirements: (I) the ability to hybridize and (II) the preservation of biological activity of the RGD peptide for the enhancement of osteoblast adhesion. In the following text, the term "hybridization" is generally used for Watson-Crick base pairing. The ability of the conjugates to hybridize to surface-immobilized complementary ODN was verified by competitive hybridization with radiolabeled ((32)P) complementary strands and by hybridization experiments using a quartz crystal microbalance (QCM). Surface hybridization was further characterized using different adsorption isotherms (e.g., Freundlich and Frumkin types), since the type of isotherm and the derived thermodynamic parameters may reveal characteristic differences between ODN and conjugates thereof. Biological activity of the conjugates was examined in vitro with osteoblasts. The cells were either cultured directly on the ODN-GRGDSP modified titanium implants or used for competition adhesion studies with dissolved ODN-GRGDSP conjugates. All results support the successful establishment of the new surface modification system. Hybridization of RGD peptide-modified nucleic acids to ODN-modified titanium implant materials is thus a promising method for osteoblast attachment in a modular and self-organizing system on implant surfaces.

Immobilization of oligonucleotides on titanium based materials by partial incorporation in anodic oxide layers.

This paper describes the immobilization of bioactive molecules on titanium based surfaces through a combination of nano-mechanical fixation of nucleic acid anchor strands (ASs) by partial and regioselective incorporation within an anodic oxide layer and their hybridization with complementary strands (CSs) intended to be conjugated to bioactive molecules. We focus on the interaction between the substrate surface and the anchor strands, the integrity of ASs and their hybridization ability. The observed dependence of adsorption on pH suggests that initial interaction of terminally phosphorylated ASs with the substrate surface is mediated by electrostatic interaction. Using ASs labelled with (32)P at different termini, it could be shown that strand breaks occur, which are attributed (i) to the formation of reactive oxygen species during anodic polarization, (ii) the photocatalytic activity of the titanium oxide and (iii) drying effects. Damage to AS could be considerably reduced if the electrolyte contained 5 mol l(-1) ethanol, light was excluded during the experimental procedure, and the number of drying and rewetting steps was minimized. A total surface density of AS of 4.5 pmol cm(-2) was reached and could be hybridized to CS with an efficiency of up to 100%. A non-complementary strand (NS) bound with less than 0.5% of the amount of CS under similar conditions. Therefore, non-specific binding of CS is considered as negligible.

Efficient suppression of tissue factor synthesis using antisense oligonucleotides selected by an enhanced strategy for evaluation of structural characteristics.

Selection of optimal antisense constructs is still a problem. Among a huge number of antisense oligonucleotides (AS-ONs) only a small piece show inhibitory efficacy. We want to develop an enhanced strategy for specific selection of effective AS-ONs based on prediction of secondary structure of the target messenger RNA (mRNA) and analysis of thermodynamic properties of the mRNA/AS-ON hybrid. Numerous AS-ONs targeted on human tissue factor (TF) mRNA were investigated to evaluate the relevance of different thermodynamic and structural properties on inhibitory efficacy. Cell viability, TF protein and TF mRNA were determined after transfection of bladder cancer cell line J82. Inhibitory efficacy was related to GC content, target region within the TF mRNA and stability of the mRNA/AS-ON hybrid or affinity of the AS-ON to the target mRNA. We found effective AS-ONs targeted on translated region or 3'-untranslated region of TF RNA. We also detected a great correlation between inhibitory efficacy and GC content as well as stability of the mRNA/AS-ON hybrid.

Antisense-mediated inhibition of survivin, hTERT and VEGF in bladder cancer cells in vitro and in vivo.

Since cancer cells are characterised by multiple genetic alterations the single inhibition of one tumour- associated gene might not be sufficient as a therapeutic strategy. We examined the effects of a combined inhibition of survivin, human telomerase reverse transcriptase (hTERT) and vascular endothelial growth factor (VEGF) with antisense oligodeoxynucleotides (AS-ODNs) and small interfering RNAs (siRNAs) in EJ28 and 5637 bladder cancer (BCa) cells. Following verification of the uptake of intraperitoneally applied fluorescence-labelled AS-ODNs and siRNAs in subcutaneous BCa xenografts, the target-directed constructs were tested as single agents in SCID mice bearing subcutaneous EJ28. Simultaneous inhibition of two of the selected transcripts significantly enhanced cell viability reduction compared to the controls consisting of a target directed construct and an appropriate control construct without any homology to the human genome. The uptake of both antisense inhibitor types in the subcutaneous BCa was achieved even without a carrier. In vivo studies with 9 consecutive intraperitoneal injections with 20 mg/kg AS-ODNs or 4.6 mg/kg siRNAs revealed the biocompatibility of both antisense inhibitor types and showed anti-tumoural activity of the AS-ODNs used.

Experimental hypoxia is a potent stimulus for radiotracer uptake in vitro: comparison of different tumor cells and primary endothelial cells.

Hypoxia causes upregulation of vascular endothelial growth factor (VEGF) which is a key regulator in tumor angiogenesis and essential for the proliferation of endothelial cells. Endothelial cells have been described to accumulate radiotracers like (18)F-FDG. However, the contribution of radiotracer uptake by endothelial cells to uptake measured in tumors by positron emission tomography (PET) is still unclear. In this study (18)F-FDG and (18)F-FMISO radiotracer uptake in various tumor and primary endothelial cells cultured at hypoxic conditions was investigated. Experimental hypoxia was confirmed by significant upregulation of VEGF mRNA. In comparison to normoxic conditions, cellular uptake of (18)F-FDG was significantly increased at hypoxic conditions in two of the tumor and all endothelial cells, whereas (18)F-FMISO uptake was only enhanced in tumor cell lines HT-29 and MCF-7. Our data showed a marked influence of experimental hypoxia on the metabolism and gene expression of tumor and endothelial cells in vitro. This indicates an important contribution of endothelial cells to (18)F-FDG radiotracer uptake in tumors and for the visualization of tumors by means of PET.

Multiplex PCR to assay the effect of nucleic acid-based inhibitors on prothrombin transcript level.

We compared an antisense-oligodeoxynucleotide and four DNAzymes directed to the prothrombin mRNA for their efficiency to reduce prothrombin transcript level in HepG2 cells. The DNAzymes have different binding arm symmetry and cleavage sites, but are directed to the identical target site of the antisense-oligodeoxynucleotide. The nucleic acid-based inhibitors were transfected into HepG2 cells and prothrombin transcript level was quantified and normalized to the beta-actin transcript level by multiplex PCR. All nucleic acid-based inhibitors reduced prothrombin transcript level and the effect was in almost all cases, strongest 24 h after transfection, but still remarkable up to 68 h after transfection. The antisense-oligodeoxynucleotide was more effective than the DNAzymes tested.

The effect of electrochemical functionalization of Ti-alloy surfaces by aptamer-based capture molecules on cell adhesion.

To improve cell seeding efficiency and cytocompatibility, we designed a new coating material for scaffolds. We used aptamers, highly specific cell binding nucleic acids generated by combinatorial chemistry with an in vitro selection called systematic evolution of exponential enrichment (SELEX). In this study, we functionalized Ti-alloy surfaces to enhance cell adhesion. By coating the material with a cell specific aptamer, working as a capture molecule, we could improve the attachment of cells effectively and avoid the limitations of the currently available materials. Aptamers, immobilized by partial electrochemical entrapment in oxide layers on Ti-alloy surfaces were able to capture cells out of a flowing suspension rapidly. This model proves that surface immobilized aptamers can greatly enhance the attachment of seeded cells. This technology opens new perspectives towards clinical application of stem cell and tissue engineering strategies.

A cellular model system for expression studies of coagulation proteins.

INTRODUCTION: The development of novel antithrombotic agents directly affecting gene expression requires well established, reliable and useful in vitro model systems for initial validation of drug effects. Since most proteins involved in coagulation are synthesized by the liver, the hepatoblastoma cell line Hep G2 is introduced, here, as a model system to test nucleic acid based coagulation inhibitors. METHODS: Hep G2 cells were characterized with respect to prothrombin, tissue factor and factor VIII expression in dependence of cell culture conditions. Reliable enzyme linked immuno sorbent assays as well as viability tests were introduced that allow drug screening procedures with multiple probes in microplate format. Furthermore, a multiplex PCR-procedure has been presented that offers the possibility to simultaneously detect the effects of a selected compound on two coagulation proteins in comparison to a house keeping gene. RESULTS: Hep G2 cells were not affected in viability by cell culture conditions, while proliferation and the expression patterns of some coagulation factors were affected by the adhesion factor collagen. The prothrombin expression characteristics allowed us to choose a specific time point for the transfection of Hep G2 cells with prothrombin specific antisense oligonucleotides. Antisense oligonucleotides inhibited prothrombin expression independent from culture conditions and the effects were detected on protein-and mRNA-level. DISCUSSION: Nucleic acid based agents require cellular in vitro model systems since they affect the process of gene expression and not the gene product. Hep G2 cells are a useful model to study effects of novel nucleic acid based coagulation inhibitors with an antisense mechanism of action on protein and mRNA level.