CEACAM6 Promotes Lung Metastasis via Enhancing Proliferation, Migration and Suppressing Apoptosis of Prostate Cancer Cells.

BACKGROUND/AIM: Metastatic prostate cancer (mPCa) results in high morbidity and mortality. Visceral metastases in particular are associated with a shortened survival. Our aim was to unravel the molecular mechanisms that underly pulmonary spread in mPCa. MATERIALS AND METHODS: We performed a comprehensive transcriptomic analysis of PCa lung metastases, followed by functional validation of candidate genes. Digital gene expression analysis utilizing the NanoString technology was performed on mRNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue from PCa lung metastases. The gene expression data from primary PCa and PCa lung metastases were compared, and several publicly available bioinformatic analysis tools were used to annotate and validate the data. RESULTS: In PCa lung metastases, 234 genes were considerably up-regulated, and 78 genes were significantly down-regulated when compared to primary PCa. Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) was identified as suitable candidate gene for further functional validation. CEACAM6 as a cell adhesion molecule has been implicated in promoting metastatic disease in several solid tumors, such as colorectal or gastric cancer. We showed that siRNA knockdown of CEACAM6 in PC-3 and LNCaP cells resulted in decreased cell viability and migration as well as enhanced apoptosis. Comprehensive transcriptomic analyses identified several genes of interest that might promote metastatic spread to the lung. CONCLUSION: Functional validation revealed that CEACAM6 might play an important role in fostering metastatic spread to the lung of PCa patients via enhancing proliferation, migration and suppressing apoptosis in PC-3 and LNCaP cells. CEACAM6 might pose an attractive therapeutic target to prevent metastatic disease.

Influence of PEGDA Molecular Weight and Concentration on the In Vitro Release of the Model Protein BSA-FITC from Photo Crosslinked Systems.

Novel 3D printing techniques enable the development of medical devices with drug delivery systems that are tailored to the patient in terms of scaffold shape and the desired pharmaceutically active substance release. Gentle curing methods such as photopolymerization are also relevant for the incorporation of potent and sensitive drugs including proteins. However, retaining the pharmaceutical functions of proteins remains challenging due to the possible crosslinking between the functional groups of proteins, and the used photopolymers such as acrylates. In this work, the in vitro release of the model protein drug, albumin-fluorescein isothiocyanate conjugate (BSA-FITC) from differently composed, photopolymerized poly(ethylene) glycol diacrylate (PEGDA), an often employed, nontoxic, easily curable resin, was investigated. Different PEGDA concentrations in water (20, 30, and 40 wt %) and their different molecular masses (4000, 10,000, and 20,000 g/mol) were used to prepare a protein carrier with photopolymerization and molding. The viscosity measurements of photomonomer solutions revealed exponentially increasing values with increasing PEGDA concentration and molecular mass. Polymerized samples showed increasing medium uptake with an increasing molecular mass and decreasing uptake with increasing PEGDA content. Therefore, the modification of the inner network resulted in the most swollen samples (20 wt %) also releasing the highest amount of incorporated BSA-FITC for all PEGDA molecular masses.

Dual Drug Delivery in Cochlear Implants: In Vivo Study of Dexamethasone Combined with Diclofenac or Immunophilin Inhibitor MM284 in Guinea Pigs.

Cochlear implants are well established to treat severe hearing impairments. Despite many different approaches to reduce the formation of connective tissue after electrode insertion and to keep electrical impedances low, results are not yet satisfying. Therefore, the aim of the current study was to combine the incorporation of 5% dexamethasone in the silicone body of the electrode array with an additional polymeric coating releasing diclofenac or the immunophilin inhibitor MM284, some anti-inflammatory substances not yet tested in the inner ear. Guinea pigs were implanted for four weeks and hearing thresholds were determined before implantation and after the observation time. Impedances were monitored over time and, finally, connective tissue and the survival of spiral ganglion neurons (SGNs) were quantified. Impedances increased in all groups to a similar extent but this increase was delayed in the groups with an additional release of diclofenac or MM284. Using Poly-L-lactide (PLLA)-coated electrodes, the damage caused during insertion was much higher than without the coating. Only in these groups, connective tissue could extend to the apex of the cochlea. Despite this, numbers of SGNs were only reduced in PLLA and PLLA plus diclofenac groups. Even though the polymeric coating was not flexible enough, MM284 seems to especially have potential for further evaluation in connection with cochlear implantation.

Novel dalbavancin-PLLA implant coating prevents hematogenous Staphylococcus aureus infection in a minimally invasive mouse tail vein model.

Infective/bacterial endocarditis is a rare but life-threatening disease with a hospital mortality rate of 22.7% and a 1-year mortality rate of 40%. Therefore, continued research efforts to develop efficient anti-infective implant materials are of the utmost importance. Equally important is the development of test systems that allow the performance of new materials to be comprehensively evaluated. In this study, a novel antibacterial coating based on dalbavancin was tested in comparison to rifampicin/minocycline, and the suitability of a recently developed mouse tail vein model for testing the implant coatings was validated. Small polymeric stent grafts coated with a poly-L-lactic acid (PLLA) layer and incorporated antibiotics were colonized with Staphylococcus (S.) aureus before implantation into the tail vein of mice. The main assessment criteria were the hematogenous spread of the bacteria and the local tissue reaction to the contaminated implant. For this purpose, colony-forming units (CFU) in the blood, spleen and kidneys were determined. Tail cross sections were prepared for histological analysis, and plasma cytokine levels and expression values of inflammation-associated genes were examined. Both antibiotic coatings performed excellently, preventing the onset of infection. The present study expands the range of available methods for testing the anti-infectivity of cardiovascular implants, and the spectrum of agents for effective surface coating.

A PLLA Coating Does Not Affect the Insertion Pressure or Frictional Behavior of a CI Electrode Array at Higher Insertion Speeds.

To prevent endocochlear insertion trauma, the development of drug delivery coatings in the field of CI electrodes has become an increasing focus of research. However, so far, the effect of a polymer coating of PLLA on the mechanical properties, such as the insertion pressure and friction of an electrode array, has not been investigated. In this study, the insertion pressure of a PLLA-coated, 31.5-mm long standard electrode array was examined during placement in a linear cochlear model. Additionally, the friction coefficients between a PLLA-coated electrode array and a tissue simulating the endocochlear lining were acquired. All data were obtained at different insertion speeds (0.1, 0.5, 1.0, 1.5, and 2.0 mm/s) and compared with those of an uncoated electrode array. It was shown that both the maximum insertion pressure generated in the linear model and the friction coefficient of the PLLA-coated electrode did not depend on the insertion speed. At higher insertion speeds above 1.0 mm/s, the insertion pressure (1.268 ± 0.032 mmHg) and the friction coefficient (0.40 ± 0.15) of the coated electrode array were similar to those of an uncoated array (1.252 ± 0.034 mmHg and 0.36 ± 0.15). The present study reveals that a PLLA coating on cochlear electrode arrays has a negligible effect on the electrode array insertion pressure and the friction when higher insertion speeds are used compared with an uncoated electrode array. Therefore, PLLA is a suitable material to be used as a coating for CI electrode arrays and can be considered for a potential drug delivery system.

Accelerated Endothelialization of Nanofibrous Scaffolds for Biomimetic Cardiovascular Implants.

Nanofiber nonwovens are highly promising to serve as biomimetic scaffolds for pioneering cardiac implants such as drug-eluting stent systems or heart valve prosthetics. For successful implant integration, rapid and homogeneous endothelialization is of utmost importance as it forms a hemocompatible surface. This study aims at physicochemical and biological evaluation of various electrospun polymer scaffolds, made of FDA approved medical-grade plastics. Human endothelial cells (EA.hy926) were examined for cell attachment, morphology, viability, as well as actin and PECAM 1 expression. The appraisal of the untreated poly-L-lactide (PLLA L210), poly-ε-caprolactone (PCL) and polyamide-6 (PA-6) nonwovens shows that the hydrophilicity (water contact angle > 80°) and surface free energy (<60 mN/m) is mostly insufficient for rapid cell colonization. Therefore, modification of the surface tension of nonpolar polymer scaffolds by plasma energy was initiated, leading to more than 60% increased wettability and improved colonization. Additionally, NH3-plasma surface functionalization resulted in a more physiological localization of cell−cell contact markers, promoting endothelialization on all polymeric surfaces, while fiber diameter remained unaltered. Our data indicates that hydrophobic nonwovens are often insufficient to mimic the native extracellular matrix but also that they can be easily adapted by targeted post-processing steps such as plasma treatment. The results achieved increase the understanding of cell−implant interactions of nanostructured polymer-based biomaterial surfaces in blood contact while also advocating for plasma technology to increase the surface energy of nonpolar biostable, as well as biodegradable polymer scaffolds. Thus, we highlight the potential of plasma-activated electrospun polymer scaffolds for the development of advanced cardiac implants.

PLLA Coating of Active Implants for Dual Drug Release.

Cochlear implants, like other active implants, rely on precise and effective electrical stimulation of the target tissue but become encapsulated by different amounts of fibrous tissue. The current study aimed at the development of a dual drug release from a PLLA coating and from the bulk material to address short-term and long-lasting release of anti-inflammatory drugs. Inner-ear cytocompatibility of drugs was studied in vitro. A PLLA coating (containing diclofenac) of medical-grade silicone (containing 5% dexamethasone) was developed and release profiles were determined. The influence of different coating thicknesses (2.5, 5 and 10 µm) and loadings (10% and 20% diclofenac) on impedances of electrical contacts were measured with and without pulsatile electrical stimulation. Diclofenac can be applied to the inner ear at concentrations of or below 4 × 10-5 mol/L. Release of dexamethasone from the silicone is diminished by surface coating but not blocked. Addition of 20% diclofenac enhances the dexamethasone release again. All PLLA coatings serve as insulator. This can be overcome by using removable masking on the contacts during the coating process. Dual drug release with different kinetics can be realized by adding drug-loaded coatings to drug-loaded silicone arrays without compromising electrical stimulation.

Evaluation of a Murine Model for Testing Antimicrobial Implant Materials in the Blood Circulation System.

Medical device-related infections are becoming a steadily increasing challenge for the health care system regarding the difficulties in the clinical treatment. In particular, cardiovascular implant infections, catheter-related infections, as well as infective endocarditis are associated with high morbidity and mortality risks for the patients. Antimicrobial materials may help to prevent medical device-associated infections and supplement the currently available therapies. In this study, we present an easy-to-handle and simplified in vivo model to test antimicrobial materials in the bloodstream of mice. The model system is composed of the implantation of a bacteria-laden micro-stent scaffold into the murine tail vein. Our model enables the simulation of catheter-related infections as well as the development of infective endocarditis specific pathologies in combination with material testing. Furthermore, this in vivo model can cover two phases of the biofilm formation, including both the local tissue response to the bacterial biofilm and the systemic inflammatory response against circulating bacteria in the bloodstream that detached from a mature biofilm.

The Role of Biodegradable Poly-(L-lactide)-Based Polymers in Blood Cell Activation and Platelet-Monocyte Interaction.

The main purpose of new stent technologies is to overcome unfavorable material-related incompatibilities by producing bio- and hemo-compatible polymers with anti-inflammatory and anti-thrombogenic properties. In this context, wettability is an important surface property, which has a major impact on the biological response of blood cells. However, the influence of local hemodynamic changes also influences blood cell activation. Therefore, we investigated biodegradable polymers with different wettability to identify possible aspects for a better prediction of blood compatibility. We applied shear rates of 100 s-1 and 1500 s-1 and assessed platelet and monocyte activation as well as the formation of CD62P+ monocyte-bound platelets via flow cytometry. Aggregation of circulating platelets induced by collagen was assessed by light transmission aggregometry. Via live cell imaging, leukocytes were tracked on biomaterial surfaces to assess their average velocity. Monocyte adhesion on biomaterials was determined by fluorescence microscopy. In response to low shear rates of 100 s-1, activation of circulating platelets and monocytes as well as the formation of CD62P+ monocyte-bound platelets corresponded to the wettability of the underlying material with the most favorable conditions on more hydrophilic surfaces. Under high shear rates, however, blood compatibility cannot only be predicted by the concept of wettability. We assume that the mechanisms of blood cell-polymer interactions do not allow for a rule-of-thumb prediction of the blood compatibility of a material, which makes extensive in vitro testing mandatory.

Smart releasing electrospun nanofibers-poly: L.lactide fibers as dual drug delivery system for biomedical application.

An ongoing challenge in drug delivery systems for a variety of medical applications, including cardiovascular diseases, is the delivery of multiple drugs to address numerous phases of a treatment or healing process. Therefore, an extended dual drug delivery system (DDDS) based on our previously reported cardiac DDDS was generated. Here we use the polymer poly(L-lactide) (PLLA) as drug carrier with the cytostatic drug Paclitaxel (PTX) and the endothelial cell proliferation enhancing growth factor, human vascular endothelial growth factor (VEGF), to overcome typical in-stent restenosis complications. We succeeded in using one solution to generate two separate DDDS via spray coating (film) and electrospinning (nonwoven) with the same content of PTX and the same post processing for VEGF immobilisation. Both processes are suitable as coating techniques for implants. The contact angle analysis revealed differences between films and nonwovens. Whereas, the morphological analysis demonstrated nearly no changes occurred after immobilisation of both drugs. Glass transition temperatures (Tg ) and degree of crystallinity (χ) show only minor changes. The amount of immobilised VEGF on nonwovens was over 300% higher compared to the films. Also, the nonwovens revealed a much faster and over three times higher PTX release over 70 d compared to the films. The almost equal physical properties of nonwovens and films allow the comparison of both DDDS independently of their fabrication process. Both films and nonwovens have significantly increased in vitro cell viability for human umbilical vein endothelial cells (EA.hy926) with dual loaded PTX and VEGF compared to PTX-only loaded samples.

Novel approach for a PTX/VEGF dual drug delivery system in cardiovascular applications-an innovative bulk and surface drug immobilization.

The successive incorporation of several drugs into the polymeric bulk of implants mostly results in loss of considerable quantity of one drug, and/or the loss in quality of the coating and also in changes of drug release time points. A dual drug delivery system (DDDS) based on poly-L-lactide (PLLA) copolymers combining the effective inhibition of smooth muscle cell proliferation while simultaneously promoting re-endothelialization was successfully developed. To overcome possible antagonistic drug interactions and the limitation of the polymeric bulk material as release system for dual drugs, a novel concept which combines the bulk and surface drug immobilization for a DDDS was investigated. The advantage of this DDDS is that the bulk incorporation of fluorescein diacetate (FDAc) (model drug for paclitaxel (PTX)) via spray coating enhanced the subsequent cleavable surface coupling of vascular endothelial growth factor (VEGF) via the crosslinker bissulfosuccinimidyl suberate (BS3). In the presence of the embedded FDAc, the VEGF loading and release are about twice times higher than in absence. Furthermore, the DDDS combines the diffusion drug delivery (FDAc or PTX) and the chemical controlled drug release, VEGF via hydrolysable ester bonds, without loss in quantity and quality of the drug release curves. Additionally, the performed in vitro biocompatibility study showed the bimodal influences of PTX and VEGF on human endothelial EA.hy926 cells. In conclusion, it was possible to show the feasibility to develop a novel DDDS which has a high potential for the medical application due to the possible easy and short modification of a polymer-based PTX delivery system.

Studies on the interaction of polylactid-based planar and nanoparticular biomaterials with serum albumin and fibrinogen.

Knowledge about the interactions of biomaterials with biological matrices is still poor however crucial to future development in terms of how to cope with immunomodulatory adverse effects and to control the integration of implants into tissue or the targeting of nano devices. The appropriate design of a material depends on reliable data about the influence of oligomer or polymer configuration and dispersity as well as surface properties including chemical modifications. This work addressed the interaction of four polylactide (PLA) resomers, a well-established, biodegradable biomaterial, and two physiologically relevant proteins, which are ubiquitous in human serum - albumin (HSA) and fibrinogen (HFG). Bovine serum albumin (BSA) was carried along as reference. The amount and stability of protein binding to plane material was assessed by surface plasmon resonance (SPR), the formation of protein corona around NPs by nanoparticle tracking analysis (NTA). The results demonstrated the particular value of SPR and NTA as techniques for the characterisation and prediction of the interactions of implants or NPs with matrix components. Both techniques are complementary with respect to a deeper understanding of changes in the layer composition with time, which is known as the Vroman effect, and, therefore, are considered of value for affecting the interaction processes by material design.

VEGF-releasing suture material for enhancement of vascularization: development, in vitro and in vivo study.

As it has been demonstrated that bioactive substances can be delivered locally using coated surgical suture materials, the authors developed a vascular endothelial growth factor (VEGF)-releasing suture material that should promote vascularization and potentially wound healing. In this context, the study focused on the characterization of the developed suture material and the verification of its biological activity, as well as establishing a coating process that allows reproducible and stable coating of a commercially available polydioxanone suture material with poly(l-lactide) (PLLA) and 0.1µg and 1.0µg VEGF. The in vitro VEGF release kinetics was studied using a Sandwich ELISA. The biological activity of the released VEGF was investigated in vitro using human umbilical vein endothelial cells. The potential of the VEGF-releasing suture material was also studied in vivo 5days after implantation in the hind limb of Wistar rats, when the histological findings were analyzed. The essential results, enhanced cell viability in vitro as well as significantly increased vascularization in vivo, were achieved using PLLA/1.0µg VEGF-coated suture material. Furthermore, ELISA measurements revealed a high reproducibility of the VEGF release behavior. Based on the results achieved regarding the dose-effect relationship of VEGF, the stability during its processing and the release behavior, it can be predicted that a bioactive suture material would be successful in later in vivo studies. Therefore, this knowledge could be the basis for future studies, where bioactive substances with different modes of action are combined for targeted, overall enhancement of wound healing.

Chemical activation and changes in surface morphology of poly(ε-caprolactone) modulate VEGF responsiveness of human endothelial cells.

The high degree of clinical routine in percutaneous transluminal coronary angioplasty (PTCA) with and without stenting has not changed the fact that a large number of coronary heart disease patients are still affected by post-operative complications such as restenosis and thrombosis. Because re-endothelialization is the crucial aspect of wound healing after cardiovascular implant surgery, there is a need for modern biomaterials to aid endothelial cells in their adhesion and functional recovery post-stenting. This study systematically examines the potential of numerous chemical polymer modifications with regard to endothelialization. Poly(ε-caprolactone) (PCL) and its chemically activated forms are investigated in detail, as well as the impact of polymer surface morphology and precoating with matrix protein. Human umbilical vein endothelial cells (HUVECs) are used to characterize endothelial cell responses in terms of in vitro viability and adhesion. As a potential component in drug eluting implants, VEGF is applied as stimulus to boost endothelial cell proliferation on the polymer. In conclusion, plasma chemical activation of PCL combined with VEGF stimulation best enhances in vitro endothelialization. Examining the impact of morphological, chemical and biological modifications of PCL, this study makes an important new contribution towards the existing body of work on polymer endothelialization.

Impact of the cellular prion protein on amyloid-ß and 3PO-tau processing.

Previous studies indicate an important role for the cellular prion protein (PrP(C)) in the development of Alzheimer's disease (AD) pathology. In the present study, we analyzed the involvement of PrP(C) in different pathological mechanisms underlying AD: the processing of the amyloid-ß protein precursor (AßPP) and its interaction with AßPP, tau, and different phosphorylated forms of the tau protein (p-tau). The effect of PrP(C) on tau expression was investigated in various cellular compartments using a HEK293 cell model expressing a tau mutant (3PO-tau) or wild type (WT)-tau. We could show that PrP(C) reduces AßPP cleavage, leading to decreased levels of Aß40 and sAßPP without changing the protein expression of AßPP, ß-secretase, or γ-secretase. Tau and its phosphorylated forms were identified as interactions partners for PrP(C), raising the question as to whether PrP(C) might also be involved in tau pathology. Overexpression of PrP(C) in PRNP and 3PO-tau transfected cells resulted in a reduction of 3PO-tau and p-tau as well as a decrease of 3PO-tau-related toxicity. In addition, we used the transgenic PrP(C) knockout (Prnp0/0) mouse line to study the dynamics of tau phosphorylation, an important pathological hallmark in the pathogenesis of AD in vivo. There, an effect of PrP(C) on tau expression could be observed under oxidative stress conditions but not during aging. In summary, we provide further evidence for interactions of PrP(C) with proteins that are known to be the key players in AD pathogenesis. We identified tau and its phosphorylated forms as potential PrP-interactors and report a novel protective function of PrP(C) in AD-like tau pathology.

Surface functionalization of poly(ε-caprolactone) improves its biocompatibility as scaffold material for bioartificial vessel prostheses.

Within this study, chemically modified polymer surfaces were to be developed, which should enhance the subsequent immobilization of various bioactive substances. To improve the hemocompatibility and endothelialization of poly(ε-caprolactone) (PCL) intended as scaffold material for bioartificial vessel prostheses, terminal amino groups via ammonia (NH3) plasma, oxygen (O2) plasma/aminopropyltriethoxysilane (APTES), and 4,4'-methylenebis(phenyl isocyanate) (MDI)/water were provided. Then, immobilization of the anti-inflammatory and antithrombogenic model drug acetylsalicylic acid (ASA) and vascular endothelial growth factor (VEGF) were performed by employing N,N-disuccinimidyl carbonate (DSC) as crosslinker. Contact angle and fluorescence measurements, X-ray photoelectron spectroscopy and infrared spectroscopy confirmed the surface modification. Here the highest functionalization was observed for the O2 plasma/APTES modification. Furthermore, biocompatibility studies demonstrated that the surface reactions have no negative influence, neither on the viability of L929 mouse fibroblasts, nor on primary or secondary hemostasis. Release studies showed that the immobilization of ASA and VEGF on the modified PCL surface via DSC is greatly improved compared to the adsorption-only reference. The advantage of DSC is that it immobilizes both bioactive substances via non-hydrolyzable and/or hydrolyzable covalent bonding. The highest ASA loading and cumulative release was detected using NH3 plasma-activated PCL samples. For VEGF, the O2 plasma/APTES-modified PCL samples were most efficient with regard to loading and cumulative release. In conclusion, both modifications are promising methods to optimize PCL as scaffold material for bioartificial vessel prostheses.