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.

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.

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.

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.

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.

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.

Tissue factor and tumor: clinical and laboratory aspects.

This review summarizes data demonstrating the role of TF in tumor development, metastasis and angiogenesis. TF is a transmembrane protein that is expressed constitutively in some kinds of extravascular cells and transiently in intravascular cells after stimulation with cytokines and growth factors. Originally TF was considered to have a function in the initiation of coagulation. In the last years it became evident that TF plays a role in physiological and pathological processes outside the hemostasis. Up-regulation of TF expression appears to be characteristic of tumor tissue. In a variety of human tumors it was shown by immunohistochemistry, that TF can be expressed in malignant cells as well as in tumor-infiltrating macrophages or endothelial cells. Such abnormal TF expression contributes to the angiogenic process by a shift in the balance between endogenous proangiogenic and antiangiogenic factors. Observations of a significant correlation between elevated TF expression with increased microvessel density and VEGF expression underline the TF involvement in tumor angiogenesis. Furthermore, TF expression influences also metastasis. The effect of TF on metastasis may result from its angiogenic effect, but also from the production of growth factors or adhesion proteins.

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.

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.

A potent inhibitor of prothrombin gene expression as a result of standardized target site selection and design of antisense oligonucleotides.

The development of antisense oligonucleotides (AS-ODN) always had the limitation that because of complex mRNA secondary structures, not every designed AS-ODN inhibited the expression of its target. There have been many investigations to overcome this problem in the last few years. This produced a great deal of theoretical and empirical findings about characteristics of effective AS-ODNs in respect to their target regions but no standardized selection procedure of AS-ODN target regions within a given mRNA or standardized design of AS-ODNs against a specific target region. We present here a standardized method based on secondary structure prediction for target site selection and AS-ODN design, followed by validation of the antisense effect caused by our predicted AS-ODNs in cell culture. The combination of theoretical design and experimental selection procedure led to an AS-ODN that efficiently and specifically reduces prothrombin mRNA and antigen.

Antisense-mediated hTERT inhibition specifically reduces the growth of human bladder cancer cells.

PURPOSE: The expression of human telomerase reverse transcriptase (hTERT) is associated with cellular aging and tumorigenesis. It was found in nearly all cancer types but not in most normal, somatic cells. The aim of this study was to investigate whether hTERT inhibition by antisense oligodeoxynucleotides (AS-ODN) can act as an efficient strategy to specifically impair the growth of bladder cancer (BCa) cells in vitro. EXPERIMENTAL DESIGN: Twenty-three AS-ODNs were designed complementary to five putative single-stranded target sites using a computer-aided secondary structure prediction of hTERT mRNA. The BCa cell lines were transfected once or several times with AS-ODNs, and the influences on cell growth, hTERT mRNA, and hTERT protein levels, as well as on telomerase activity, were examined. RESULTS: An immediate and continuous reduction of cell viability (up to a complete cell loss) was achieved by treatment with 5 of 23 tested AS-ODNs in EJ28 cells. Additionally, significant inhibition of proliferation (doubling time, clonogenic survival), as well as an induction of G(1) arrest were observed. The specificity of the growth-inhibitory action of the five efficient AS-ODNs was confirmed by diminished hTERT transcript amount (< or =88%) and reduced hTERT protein content in EJ28 cells. As a consequence, the telomerase activity was inhibited by anti-hTERT treatment < or =60%. Inhibition of viability was shown for an additional three tested BCa cell lines but not for primary fibroblasts after treatment with the five most effective AS-ODNs supporting an antitumor action of these constructs. CONCLUSION: Specific hTERT inhibition causes remarkable short- and long-term effects on the growth of BCa cells and represents a promising new treatment option of solid tumors. We propose that this alternative treatment could be applied in terms of an instillation therapy.

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.

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.

Antisense-mediated VEGF suppression in bladder and breast cancer cells.

Angiogenesis plays a key role in tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is one of the major angiogenic factors. In the study we have evaluated the efficiency of antisense oligodeoxynucleotides (AS-ODN) against VEGF selected from computational prediction of VEGF mRNA structure. Twenty-five different AS-ODN in two different tumor cell lines were investigated. Treatment of cell line EJ28 by VEGF723 resulted in a 83.5% suppression of VEGF protein when compared with control-ODN. Three further AS-ODN reduced VEGF protein more than 45% in comparison to control-ODN. This was caused by an antisense-specific downregulation of the VEGF transcript determined by real-time PCR. Furthermore, antisense-mediated inhibition of VEGF was associated by a reduced cell viability. In MCF-7 cells VEGF protein was inhibited more than 45% by two AS-ODN. In conclusion, we found that computational prediction of potential single strand mRNA motifs is a well suitable method to elect effective AS-ODN.

Tissue specific expression and serum levels of human tissue factor in patients with urological cancer.

Human tissue factor (TF) is involved in tumor angiogenesis and metastasis. However, little is known about the distribution of TF in urological cancer. In this study we investigated the TF expression in tumor tissue and autologous non-malignant tissue as well as in serum of patients with renal cell carcinoma (RCC), bladder cancer, and prostate cancer (PCa). To study the distribution of TF in tumor tissue and in the surrounding non-malignant tissue, we measured TF protein by ELISA in tissue specimens obtained intraoperatively from 18 RCC, seven bladder cancer and six PCa patients. Differences in TF expression were found between tumor tissue and nonmalignant tissue for the three tumor types at the protein level (in the order RCC < bladder cancer < PCa). In all but one of the 18 RCC patients (94 %) higher TF protein level was observed in non-malignant tissue as compared to the tumor tissue. In addition, the relative TF mRNA expression analyzed by a quantitative RT-PCR assay in the same RCC tissue sample pairs was higher in 78% of non-malignant tissues in comparison to the tumor tissue specimens. Moreover, using enzyme linked immunosorbent assay the TF protein content was measured in serum samples of 66 patients with bladder cancer, 75 RCC patients and 157 PCa patients, and was compared with the TF serum level of 92 healthy volunteers. Whereas no differences were detected between normal volunteers and patients with PCa or RCC, patients with bladder cancer showed a significantly increased level of serum TF (P=0.0076). However, no causal association between TF levels in serum and TF content in tissue extracts for all three tumor types of urological tumors was found. Our results suggest that TF in non-malignant renal tissues was expressed at a higher level compared to the supposed de novo TF expression in RCC tissue specimens. This indicates a tumor-associated induction of TF expression in the TF-negative RCC progenitor cells. The increased serum TF levels are alike the reported higher urinary TF levels found in bladder cancer patients. The potential clinical relevance of this finding should be further elucidated.

Inhibition of TF gene expression by antisense oligonucleotides in different cancer cell lines.

Human tissue factor (TF) is the initiator of blood coagulation. Beside this function it is involved in tumor angiogenesis and metastasis. In the study we have evaluated the efficiency of antisense oligonucleotides (AS-ODNs) against TF selected from computational prediction of TF mRNA structure. Fourteen different AS-ODNs were tested in three cell lines of different origin with a high TF content. In cell line MCF-7 expression of TF gene was inhibited up to 50% by the AS-ODN AS-7 in comparison to reference. To investigate the dependence of inhibition efficiency on the AS-ODN position inside a potential target motive we designed further AS-ODNs shifted 2-3 nt among AS-7. One AS-ODN was found as more effective than AS-7. In cell line T508 were obtained moderate effects in inhibition of TF gene expression of 30% by AS-4. In J82 cells TF protein was inhibited up to 68% by two AS-ODNs. In conclusion, we compared inhibition of TF gene expression in different cancer cell lines and found that all effective AS-ODNs were located in the translated region of TF mRNA. Suitability of a target region of an AS-ODN is relatively independent on cell line. In contrast, optimal transfection conditions are dependent on cell line.

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.

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.

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.