Ursolic and oleanolic acid derivatives with cholinesterase inhibiting potential.

Triterpenoids are in the focus of scientific interest, and they were evaluated for many pharmacological applications among them their ability to act as inhibitors of cholinesterases. These inhibitors are still of interest as drugs that improve the life quality of patients suffering from age-related dementia illnesses especially of Alzheimer's disease. Herein, we prepared several derivatives of ursolic and oleanolic acid and screened them in Ellman's assays for their ability to inhibit acetylcholinesterase and/or butyrylcholinesterase, and for each of the active compounds the type of inhibition was determined. As a result, several compounds were shown as good inhibitors for acetylcholinesterase and butyrylcholinesterase even in a micromolar range. An ursolic acid derived hydroxyl-propinyl derivative 10 was a competitive inhibitor for butyrylcholinesterase with an inhibition constant of Ki = 4.29 µM, and therefore being twice as active as gold standard galantamine hydrobromide. The best inhibitor for acetylcholinesterase, however, was 2-methyl-3-oxo-methyl-ursoloate (18), acting as a mixed-type inhibitor showing Ki = 1.72 µM and Ki' = 1.28 µM, respectively.

Applications of Metals for Bone Regeneration.

The regeneration of bone tissue is the main purpose of most therapies in dental medicine. For bone regeneration, calcium phosphate (CaP)-based substitute materials based on natural (allo- and xenografts) and synthetic origins (alloplastic materials) are applied for guiding the regeneration processes. The optimal bone substitute has to act as a substrate for bone ingrowth into a defect, as well as resorb in the time frame needed for complete regeneration up to the condition of restitution ad integrum. In this context, the modes of action of CaP-based substitute materials have been frequently investigated, where it has been shown that such materials strongly influence regenerative processes such as osteoblast growth or differentiation and also osteoclastic resorption due to different physicochemical properties of the materials. However, the material characteristics needed for the required ratio between new bone tissue formation and material degradation has not been found, until now. The addition of different substances such as collagen or growth factors and also of different cell types has already been tested but did not allow for sufficient or prompt application. Moreover, metals or metal ions are used differently as a basis or as supplement for different materials in the field of bone regeneration. Moreover, it has already been shown that different metal ions are integral components of bone tissue, playing functional roles in the physiological cellular environment as well as in the course of bone healing. The present review focuses on frequently used metals as integral parts of materials designed for bone regeneration, with the aim to provide an overview of currently existing knowledge about the effects of metals in the field of bone regeneration.

Stimuli-Responsive Multilayers Based on Thiolated Polysaccharides That Affect Fibroblast Cell Adhesion.

Control of the biomaterial properties through stimuli-responsive polymeric platforms has become an essential technique in recent biomedical applications. A multilayer system of thiolated chitosan (t-Chi) and thiolated chondroitin sulfate (t-CS), consisting of five double layers ([t-Chi/t-CS]5), was fabricated here by applying a layer-by-layer coating strategy. To represent a novel class of chemically tunable nanostructures, the ability to cross-link pendant thiol groups was tested by a rise from pH 4 during layer formation to pH 9.3 and a more powerful chemical stimulus by using chloramine-T (ChT). Following both treatments, the resulting multilayers showed stimuli-dependent behavior, as demonstrated by their content of free thiols, wettability, surface charge, elastic modulus, roughness, topography, thickness, and binding of fibronectin. Studies with human dermal fibroblasts further demonstrated the favorable potential of the ChT-responsive multilayers as a cell-adhesive surface compared to pH-induced cross-linking. Because the [t-Chi/t-CS]5 multilayer system is responsive to stimuli such as the pH and redox environment, multilayer systems with disulfide bond formation may help to tailor their interaction with cells, film degradation, and controlled release of bioactive substances like growth factors in a stimuli-responsive manner useful in future wound healing and tissue engineering applications.

Medical application of glycosaminoglycans: a review.

The characteristic molecular composition of the different glycosaminoglycans (GAGs) is related to their role as structural components and regulators of a multitude of functions of proteins, cells and tissues in the human body. Therefore, it is not surprising that GAGs are widely used as coating materials for implants, components of 3D-constructs such as tissue engineering scaffolds and hydrogels, but also as diagnostic devices such as biosensors and in controlled release applications. Beside a physisorption or encapsulation of GAGs, these applications often require their chemical modification to allow a stable covalent attachment on surfaces or cross-linking reactions with other molecules. Then, the preservation of the functionality of GAGs under maintenance of their biocompatibility is a challenging task and must be addressed in accordance with the designated field of application. Here, we will give a brief overview on structure and biological functions of GAGs, different methods of their activation and immobilization, the recent progress in GAG-related biomaterials development, as well as some examples of their application in the field of tissue engineering and regenerative medicine. Copyright © 2017 John Wiley & Sons, Ltd.

Effect of thiolated glycosaminoglycans on the behavior of breast cancer cells: toward the development of in vitro models of cancer.

PURPOSE: The influence of extracellular matrix components like glycosaminoglycans (GAG) or adhesive proteins on the migration of cancer cells and the progression of tumorigenesis remains a challenging task. Therefore, this study aims to give insight into the interaction of cancer cells exhibiting different metastatic potential (MDA-MB-231, MDA-MB-468) with surface immobilized GAG interacting with serum proteins like fibronectin. METHODS: Model substrata were covalently coated with different thiolated GAG (hyaluronan (HA), chondroitin sulfate (CS), heparin (Hep)) and investigated for the adsorption of fibronectin (FN) with surface plasmon resonance. Then, adhesion of breast cancer cells in the presence of and without serum proteins was studied. Further, the outgrow behavior of confluent cancer cells was examined with the help of cell migration chambers and single-cell migration with time-lapse microscopy. RESULTS: FN adsorption revealed that the Hep-coated surfaces were able to adsorb significantly more protein than CS and HA. Generally, initial adhesion of breast cancer cells on GAG-coated substrata was inhibited for HA- and CS-coated substrata in the presence of serum proteins for both cell lines in comparison to serum-free conditions. The cell size was also significantly decreased by the influence of serum proteins. The outgrow studies clearly confirmed the different migration speed of both cancer cells while single-cell migration was particularly enhanced on HA-coated surfaces. CONCLUSIONS: The results reveal that adsorption of serum proteins (e.g. albumin) possess an inhibiting effect on the adhesion of breast cancer cells and that single-cell migration is enhanced for both breast cancer cell lines on HA.

Synthesis of thiolated polysaccharides for formation of polyelectrolyte multilayers with improved cellular adhesion.

Intrinsic cross-linking is not only useful for increasing stability, but also for tailoring mechanical properties of polyelectrolyte multilayers (PEM) on implants and tissue engineering scaffolds. Here, a novel route for synthesizing thiolated chitosan (t-Chi) based on the application of 3,3'-dithiodipropionic acid was applied, while thiolated chondroitin sulfate (t-CS) was conjugated by 3,3'-dithiobis (propanoic hydrazide). Both products were subsequently reduced to obtain the free thiols. The thiol content, structural changes and degree of substitution were studied by UV-vis, FTIR, Raman and 1H NMR spectroscopy, respectively. Chi and CS can be used for PEM formation with the layer-by-layer method, due to the cationic nature of Chi at pH values below 5.0 and the anionic character of CS. Comparative studies on the formation of native Chi/CS versus t-Chi/t-CS PEM with surface plasmon resonance and ellipsometry revealed higher layer mass. We also found that the PEM composed of t-Chi/t-CS had superior cell adhesion properties for human keratinocytes in comparison to the native PEM.

Effect of Immobilized Thiolated Glycosaminoglycans on Fibronectin Adsorption and Behavior of Fibroblasts.

Glycosaminoglycans (GAGs) chondroitin sulfate, heparin, hyaluronan, and sulfated hyaluronan are lower and higher thiolated to enable a one-step covalent modification of gold or vinyl-terminated surfaces. Measurements of water contact angle and zeta potentials reveal that sulfated GAG-modified surfaces are more wettable and possess a negative surface potential. Additionally, higher thiolated GAGs (tGAGs) exhibit increased wettability and higher surface roughness. Fibronectin (FN) adsorption increases with sulfation degree of tGAGs. The tGAG-functionalized surfaces with higher degree of sulfation promote fibroblast adhesion most under serum-free conditions. The preadsorption of FN allows for more cell adhesion on tGAG surfaces. Metabolic activity measurements show that cell growth is enhanced for tGAGs up to a certain thiolation degree. Overall, thiolation of GAGs does not hamper their bioactivity toward proteins and cells, which make them highly interesting for biomimetic surface modification of implants and tissue engineering scaffolds.

Incorporation of a Michael acceptor enhances the antitumor activity of triterpenoic acids.

Finding and developing drugs for the treatment of cancer has been challenging scientists for many decades, and using compounds of natural origin represents one of several strategies. Triterpenoic acids are a very promising class of secondary metabolites being able to induce apoptosis while their cytotoxicity is low. Therefore, derivatizations have to be conducted to improve cytotoxicity while retaining their ability to induce programmed cell death. The incorporation of a Michael acceptor into molecules resulted very often in drugs of improved cytotoxicity. Thus, in this study we synthesized and evaluated several Michael acceptor substituted compounds derived from glycyrrhetinic, ursolic, oleanolic and platanic acid. The influence of the presence of such a functional group onto the cytotoxicity was investigated in colorimetric sulforhodamine B assays employing several human cancer cell lines. EC50 values in the single-digit micromolar range were measured. Thus, the incorporation of a Michael acceptor unit into triterpenoic acids enhances the cytotoxicity of these compounds significantly.

Both hyaluronan and collagen type II keep proteoglycan 4 (lubricin) at the cartilage surface in a condition that provides low friction during boundary lubrication.

Wear resistant and ultralow friction in synovial joints is the outcome of a sophisticated synergy between the major macromolecules of the synovial fluid, e.g., hyaluronan (HA) and proteoglycan 4 (PRG4), with collagen type II fibrils and other non-collagenous macromolecules of the cartilage superficial zone (SZ). This study aimed at better understanding the mechanism of PRG4 localization at the cartilage surface. We show direct interactions between surface bound HA and freely floating PRG4 using the quartz crystal microbalance with dissipation (QCM-D). Freely floating PRG4 was also shown to bind with surface bound collagen type II fibrils. Albumin, the most abundant protein of the synovial fluid, effectively blocked the adsorption of PRG4 with HA, through interaction with C and N terminals on PRG4, but not that of PRG4 with collagen type II fibrils. The above results indicate that collagen type II fibrils strongly contribute in keeping PRG4 in the SZ during cartilage articulation in situ. Furthermore, PRG4 molecules adsorbed very well on mimicked SZ of absorbed HA molecules with entangled collagen type II fibrils and albumin was not able to block this interaction. In this last condition PRG4 adsorption resulted in a coefficient of friction (COF) of the same order of magnitude as the COF of natural cartilage, measured with an atomic force microscope in lateral mode.

Synthesis of thiolated glycosaminoglycans and grafting to solid surfaces.

Glycosaminoglycans (GAGs) with varying degree of sulfation were chemically modified to obtain thiolated analogues (tGAGs) for subsequent surface grafting on vinyl-terminated self-assembled monolayers. Thiolation was achieved by the use of the disulfide containing crosslinker 3,3'-dithiobis(propanoic hydrazide) and subsequent reduction of the disulfide with dithiothreitol. Two different molar ratios of the crosslinker were used for conjugation. The tGAGs were characterized by (1)H-NMR, Raman and flow-field-flow-fractionation (A4F) to determine the chemical composition, structure and molecular weight of the products. Ellman's reagent was used to quantify the thiol concentration of tGAGs. The tGAGs were immobilized onto vinyl-terminated glass and silicon via thiol-ene reaction. This was achieved by homogeneous immobilization from solution as well as with microcontact printing and exposure to UV light. The results of water contact angle measurement (WCA), ellipsometry and confocal laser scanning microscopy (CLSM) demonstrated that the resulting surface coverage was dependent on the degree of thiolation of GAGs.

Novel mineralized heparin-gelatin nanoparticles for potential application in tissue engineering of bone.

Nanoparticles (NPs) were prepared from succinylated gelatin (s-GL) cross-linked with aldehyde heparin (a-HEP) and used subsequently as a nano-template for the mineralization of hydroxyapatite (HAP). Gelatin was functionalized with succinyl groups that made it soluble at room temperature. Heparin was oxidized to generate aldehyde groups and then used as a cross-linker that can react with s-GL to form NPs via Schiff's base linkage. The polymer concentrations, feed molar ratios and pH conditions were varied to fabricate NPs suspension. NPs were obtained with a spheroid shape of an average size of 196 nm at pH 2.5 and 202 nm at pH 7.4. These NPs had a positive zeta potential of 7.3 ± 3.0 mV and a narrow distribution with PDI 0.123 at pH 2.5, while they had a negative zeta potential of -2.6 ± 0.3 mV and formed aggregates (PDI 0.257) at pH 7.4. The NPs prepared at pH 2.5 with a mean particle size of 196 nm were further used for mineralization studies. The mineralization process was mediated by solution without calcination at 37 °C. The HAP formed on NPs was analyzed by Fourier transform infrared spectroscopy and X-ray diffraction. HAP coated s-GL/a-HEP NPs developed in this study may be used in future as osteoinductive fillers enhancing the mechanical properties of injectable hydrogel or use as potential multifunctional device for nanotherapeutic approaches.

The chemical and biological potential of C ring modified triterpenoids.

A convenient and elegant route has been developed to separate the natural regioisomers triterpenoids ursolic acid (UA) and oleanolic acid (OA) as well as derivatives thereof. Eleven unknown derivatives of OA were designed, synthesized, and their cytotoxicity was investigated. Further sixteen compounds were prepared to correlate all compounds in a SAR study. It could be shown that C-ring modifications of OA and UA have only a moderate influence onto the cytotoxic activity of the compounds but a significant impact onto the ability to trigger apoptosis in ovarian cancer cells (cell line A2780).

Enantiopure chiral poly(glycerol methacrylate) self-assembled monolayers knock down protein adsorption and cell adhesion.

Chirality plays a fundamental role not only in biological systems, but also in synthetic materials intended for bio-applications. Self-assembled monolayers (SAMs) are prepared on gold surfaces through a "grafting to" method from racemic or enantiopure chiral poly(glycerol methacrylate)s (PGMA(rac), PGMA(R), and PGMA(S)), having a thiol endgroup. Such SAMs constitute a chemically and structurally well-defined model substrate for studying protein adsorption and cell adhesion as a function of the polymer chirality. Surface plasmon resonance measurements reveal that PGMA SAMs greatly reduce the adsorption of bovine serum albumin (BSA) compared to bare gold surfaces. Interestingly, enantiopure SAMs based on PGMA(R) or PGMA(S) show a significantly larger reduction in BSA adsorption than PGMA(rac)-covered surfaces. Studies with the monocytic cell line THP-1 show a similar relationship between enantiopurity of PGMA SAMs and the extent of cell adhesion. Ellipsometry and Raman spectroscopy measurements indicate that SAMs formed by PGMA(rac) have a higher grafting density compared to SAMs of PGMA(R) and PGMA(S). This seems to be due to the ability of PGMA(rac) to form more intermolecular hydrogen bonds among polymer chains compared to the enantiopure PGMAs. Circular dichroism spectroscopy provide evidence that enantiopure polymers adopt a chiral ordered conformation, most likely helical, in aqueous solutions. It is concluded that a higher water content of SAMs formed by enantiopure PGMA(S) and PGMA(R) SAMs arises from the macromolecular chiral conformation adopted by their enantiopure PGMA chains, and it is the decisive reason for the reduced BSA adsorption and cell adhesion as compared to PGMA(rac) SAMs.

Bioactivity of immobilized hyaluronic acid derivatives regarding protein adsorption and cell adhesion.

Hyaluronic acid (HA) was chemically modified either by oxidation to obtain aldehyde-HA (aHA) or 3,3'-dithiobis(propanoic hydrazide) to obtain thiol-HA (tHA) that was covalently immobilized on model substrata such as amino-terminated surfaces or gold. Knowledge about the effect of modification with HA on physicochemical surface properties of these substrata and estimates of the quantities of immobilized HA were obtained by different physical methods such as contact angle measurements, ellipsometry, and atomic force microscopy. The bioactivity of aHA and tHA toward their natural binding partner aggrecan was studied by comparing surface plasmon resonance to native HA; this shows that binding of aggrecan was achieved in a similar way. Dermal human fibroblasts were used as a model cell to study how chemical modification and immobilization of HA impact adhesion and spreading of cells, which also affects cell growth and differentiation. A lower number and spreading of cells were observed on HA-modified surfaces compared to amino- and vinyl-terminated glass and silicon surfaces. Immunofluorescence microscopy also revealed that adhesion of fibroblast plated on HA-modified surfaces was mediated primarily by HA receptor CD44, indicating that bioactivity of HA was not significantly reduced by chemical modification.