Functionalization of deproteinized bovine bone with a coating-incorporated depot of BMP-2 renders the material efficiently osteoinductive and suppresses foreign-body reactivity

The repair of critical-sized bony defects remains a challenge in the fields of implantology, maxillofacial surgery and orthopaedics. As an alternative bone-defect filler to autologous bone grafts, deproteinized bovine bone (DBB) is highly osteoconductive and clinically now widely used. However, this product suffers from the disadvantage of not being intrinsically osteoinductive. In the present study, this property was conferred by coating DBB with a layer of calcium phosphate into which bone morphogenetic protein 2 (BMP-2) was incorporated. Granules of DBB bearing a coating-incorporated depot of BMP-2--together with the appropriate controls (DBB bearing a coating but no BMP-2; uncoated DBB bearing adsorbed BMP-2; uncoated DBB bearing no BMP-2)--were implanted subcutaneously in rats. Five weeks later, the implants were withdrawn for a histomorphometric analysis of the volume densities of (i) bone, (ii) bone marrow, (iii) foreign-body giant cells and (iv) fibrous capsular tissue. Parameters (i) and (ii) were highest, whilst parameters (iii) and (iv) were lowest in association with DBB bearing a coating-incorporated depot of BMP-2. Hence, this mode of functionalization not only confers DBB with the property of osteoinductivity but also improves its biocompatibility--thus dually enhancing its clinical potential in the repair of bony defects.

Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries

Click chemistry is a powerful technology for the functionalization of therapeutic proteins with effector moieties, because of its potential for bio-orthogonal, regio-selective, and high-yielding conjugation under mild conditions. Designed Ankyrin Repeat Proteins (DARPins), a novel class of highly stable binding proteins, are particularly well suited for the introduction of clickable methionine surrogates such as azidohomoalanine (Aha) or homopropargylglycine (Hpg), since the DARPin scaffold can be made methionine-free by an M34L mutation in the N-cap which fully maintains the biophysical properties of the protein. A single N-terminal azidohomoalanine, replacing the initiator Met, is incorporated in high yield, and allows preparation of "clickable" DARPins at about 30 mg per liter E. coli culture, fully retaining stability, specificity, and affinity. For a second modification, we introduced a cysteine at the C-terminus. Such DARPins could be conveniently site-specifically linked to two moieties, polyethylene glycol (PEG) to the N-terminus and the fluorophore Alexa488 to the C-terminus. We present a DARPin selected against the epithelial cell adhesion molecule (EpCAM) with excellent properties for tumor targeting as an example. We used these doubly modified molecules to measure binding kinetics on tumor cells and found that PEGylation has no effect on dissociation rate, but slightly decreases the association rate and the maximal number of cell-bound DARPins, fully consistent with our previous model of PEG action obtained in vitro. Our data demonstrate the benefit of click chemistry for site-specific modification of binding proteins like DARPins to conveniently add several functional moieties simultaneously for various biomedical applications.

Biomimetic Coatings and Their Biological Functionalization

The biomimetic coating technique can be used to deposit layers of calcium phosphate (CaP) on medical devices to improve their osteoconductivity and osseointegration.The inorganic layer generated is akin to mineralized bone matrix and can be degraded as such. The biomimetic coating technique involves the nucleation and growth of bone-like crystals on a pretreated substrate by immersing it in a supersaturated solution of CaP under physiological conditions of temperature (37°C) and pH (7.4). The method, originally developed by Kokubo and his co-workers in 1990, has since undergone improvement and refinement by several groups of investigators. Biomimetic coatings are valuable in that they can serve as a vehicle for the slow, sustained release of osteogenic agents at the site of implantation. This attribute is rendered possible by the near-physiological conditions under which these coatings are prepared, which permits the incorporation of bioactive agents into the inorganic crystal latticework rather than their superficial adsorption on preformed layers. In addition, the biomimetic coating technique can be applied to implants of an organic as well as of a metallic nature and to those with irregular surface geometries, which is not possible using conventional methodologies.

Synthesis of bicyclo-DNA nucleosides with additional functionalization in the carbocyclic ring

Two novel bicyclo nucleoside isomers carrying the base thymine in the furanose ring and an ester substituent in the carbocyclic ring were synthesized from a common bicyclic sugar precursor via a cyclopropanation/fragmentation pathway in nine steps. The relative configuration of the ester substituent in both isomers as well as the anomeric configuration in one nucleoside was determined by 1H-NMR difference NOE spectroscopy.

Functionalization of β-caryophyllene generates novel polypharmacology in the endocannabinoid system

The widespread dietary plant sesquiterpene hydrocarbon β-caryophyllene (1) is a CB2 cannabinoid receptor-specific agonist showing anti-inflammatory and analgesic effects in vivo. Structural insights into the pharmacophore of this hydrocarbon, which lacks functional groups other than double bonds, are missing. A structure-activity study provided evidence for the existence of a well-defined sesquiterpene hydrocarbon binding site in CB2 receptors, highlighting its exquisite sensitivity to modifications of the strained endocyclic double bond of 1. While most changes on this element were detrimental for activity, ring-opening cross metathesis of 1 with ethyl acrylate followed by amide functionalization generated a series of new monocyclic amides (11a, 11b, 11c) that not only retained the CB2 receptor functional agonism of 1 but also reversibly inhibited fatty acid amide hydrolase (FAAH), the major endocannabinoid degrading enzyme, without affecting monoacylglycerol lipase (MAGL) and α,β hydrolases 6 and 12. Intriguingly, further modification of this monocyclic scaffold generated the FAAH- and endocannabinoid substrate-specific cyclooxygenase-2 (COX-2) dual inhibitors 11e and 11f, which are probes with a novel pharmacological profile. Our study shows that by removing the conformational constraints induced by the medium-sized ring and by introducing functional groups in the sesquiterpene hydrocarbon 1, a new scaffold with pronounced polypharmacological features within the endocannabinoid system could be generated. The structural and functional repertoire of cannabimimetics and their yet poorly understood intrinsic promiscuity may be exploited to generate novel probes and ultimately more effective drugs.

Functionalization of PAMAM dendrimers with nitronyl nitroxide radicals as models for the outer-sphere relaxation in dentritic potential MRI contrast agents

PAMAM dendrimers functionalized with nitronyl nitroxide radicals were characterized. Quantitative determination of substitution with radicals was performed using EPR and electrochemical methods. The study of the 1H NMR relaxation of the surrounding water showed how the outer-sphere contribution to the relaxivity may be limited by the presence of the dendrimer core.