We describe a facile method to prepare water-compatible molecularly imprinted polymer nanogels (MIP NGs) as synthetic antibodies against target glycans. Three different phenylboronic acid (PBA) derivatives were explored as monomers for the synthesis of MIP NGs targeting either α2,6- or α2,3-sialyllactose, taken as oversimplified models of cancer-related sT and sTn antigens. Starting from commercially available 3-acrylamidophenylboronic acid, also its 2-substituted isomer and the 5-acrylamido-2-hydroxymethyl cyclic PBA monoester derivative were initially evaluated by NMR studies. Then, a small library of MIP NGs imprinted with the α2,6-linked template was synthesized and tested by mobility shift Affinity Capillary Electrophoresis (msACE) to rapidly assess an affinity ranking. Finally, the best monomer o-acrylamido PBA was selected for the synthesis of polymers targeting both sialyllactoses. The resulting MIP NGs display an affinity constant ≈ 106 M-1 and selectivity towards imprinted glycans. This general procedure could be applied to any non-modified carbohydrate template possessing a reducing end.
Poly (glycerol sebacate) is a widely studied elastomeric copolymer obtained from the polycondensation of two bioresorbable monomers, glycerol and sebacic acid. Due to its biocompatibility and the possibility to tailor its biodegradability rate and mechanical properties, PGS has gained lots of interest in the last two decades, especially in the soft tissue engineering field. Different synthetic approaches have been proposed, ranging from classic thermal polyesterification and curing to microwave-assisted organic synthesis, UV crosslinking and enzymatic catalysis. Each technique, characterized by its advantages and disadvantages, can be tailored by controlling the crosslinking density, which depends on specific synthetic parameters. In this work, classic and alternative synthetic methods, as well as characterisation and tailoring techniques, are critically reviewed with the aim to provide a valuable tool for the reproducible and customized production of PGS for tissue engineering applications.
In this work, an innovative and accurate affinity capillary electrophoresis (ACE) method was set up to monitor the complexation of aqueous MIP nanogels (NGs) with model cancer-related antigens. Using α2,6'- and α2,3'-sialyllactose as oversimplified cancer biomarker-mimicking templates, NGs were synthesized and characterized in terms of size, polydispersity, and overall charge. A stability study was also carried out in order to select the best storage conditions and to ensure product quality. After optimization of capillary electrophoresis conditions, injection of MIP NGs resulted in a single, sharp, and efficient peak. The mobility shift approach was applied to quantitatively estimate binding affinity, in this case resulting in an association constant of K ≈ 106 M-1. The optimized polymers further displayed a pronounced discrimination between the two sialylated sugars. The newly developed ACE protocol has the potential to become a very effective method for nonconstrained affinity screening of NG in solution, especially during the NG development phase and/or for a final accurate quantitation of the observed binding.
Metastasis is the main cause of anti-cancer therapy failure, leading to unfavorable prognosis for patients. The true challenge to increase cancer patient life expectancy by making cancer a chronic disease with periodic but manageable relapses relies on the development of efficient therapeutic strategies specifically directed against key targets in the metastatic process. Traditional chemotherapy with classical alkylating agents, microtubule inhibitors, and antimetabolites has demonstrated its limited efficacy against metastatic cells due to their capacity to select chemo-resistant cell populations that undergo epithelial-to-mesenchymal transition (EMT), thus promoting the colonization of distant sites that, in turn, sustain the initial metastatic process. This scenario has prompted efforts aimed at discovering a wide variety of small molecules and biologics as potential anti-metastatic drugs directed against more specific targets known to be involved in the various stages of metastasis. In this short review, we give an overview of the most recent advances related to important families of antimetastatic small molecules: intracellular tyrosine kinase inhibitors, cyclin-dependent kinase inhibitors, KRAS inhibitors, and integrin antagonists. Although the majority of these small molecules are not yet approved and not available in the drug market, any information related to their stage of development could represent a precious and valuable tool to identify new targets in the endless fight against metastasis.
Conjugation via disuccinimidyl homobifunctional linkers is reported in the literature as a convenient approach for the synthesis of glycoconjugate vaccines. However, the high tendency for hydrolysis of disuccinimidyl linkers hampers their extensive purification, which unavoidably results in side-reactions and non-pure glycoconjugates. In this paper, conjugation of 3-aminopropyl saccharides via disuccinimidyl glutarate (DSG) was exploited for the synthesis of glycoconjugates. A model protein, ribonuclease A (RNase A), was first considered to set up the conjugation strategy with mono- to tri- mannose saccharides. Through a detailed characterization of synthetized glycoconjugates, purification protocols and conjugation conditions have been revised and optimized with a dual aim: ensure high sugar-loading and avoid the presence of side reaction products. An alternative purification approach based on hydrophilic interaction liquid chromatography (HILIC) allowed the formation of glutaric acid conjugates to be avoided, and a design of experiment (DoE) approach led to optimal glycan loading. Once its suitability was proven, the developed conjugation strategy was applied to the chemical glycosylation of two recombinant antigens, native Ag85B and its variant Ag85B-dm, that are candidate carriers for the development of a novel antitubercular vaccine. Pure glycoconjugates (≥99.5%) were obtained. Altogether, the results suggest that, with an adequate protocol, conjugation via disuccinimidyl linkers can be a valuable approach to produce high sugar-loaded and well-defined glycovaccines.
In the last few years, nanomaterials based on fullerene have begun to be considered promising tools in the development of efficient adjuvant/delivery systems for vaccination, thanks to their several advantages such as biocompatibility, size, and easy preparation and modification. In this work we reported the chemoenzymatic synthesis of natural polymannan analogues (di- and tri-mannan oligosaccharides characterized by α1,6man and/or α1,2man motifs) endowed with an anomeric propargyl group. These sugar derivatives were submitted to 1,3 Huisgen dipolar cycloaddition with a malondiamide-based chain equipped with two azido terminal groups. The obtained sugar-modified malondiamide derivatives were used to functionalize the surface of Buckminster fullerene (C60) in a highly controlled fashion, and yields (11-41%) higher than those so far reported by employing analogue linkers. The same strategy has been exploited to obtain C60 endowed with natural and unnatural amino acid derivatives. Finally, the first double functionalization of fullerene with both sugar- and amino acid-modified malondiamide chains was successfully performed, paving the way to the possible derivatization of fullerenes with immunogenic sugars and more complex antigenic peptides.
Fullerenes , Amino Acids , Fullerenes/chemistry , Organic Chemicals , Peptides , Sugars
