Mara Soares da Silva

Development of 2-(dimethylamino)ethyl methacrylate-based molecular recognition devices for controlled drug delivery using supercritical fluid technology.

This work reports the development of a novel potential body-friendly oral drug delivery system, which consists of a biocompatible molecularly imprinted polymer (MIP), with pH sensitive character and low cross-linking degree (20.2wt%), synthesized and processed in supercritical carbon dioxide. The MIP is synthesized using 2-(dimethylamino)ethyl methacrylate (DMAEMA) as functional monomer and ethylene glycol dimethacrylate (EGDMA) as cross-linker, and ibuprofen as molecular recognition template. The imprinted matrix was able to show a higher affinity towards ibuprofen than its corresponding non-imprinted polymer (NIP) meaning that the molecular imprinting in scCO(2) was efficient even using a low crosslinking degree. MIP showed a significant molecular recognition towards the template, presenting higher drug uptake ability in the supercritical impregnation step, loading 33.1wt% of ibuprofen compared to only 10.2wt% for the NIP polymer. In vitro drug release experiments, simulating an oral administration, showed different release profiles at pH 2.2 and pH 7.4. Zeta potential measurements were performed to both MIP and NIP showing that the imprinting process has a significant influence on the charge of the polymeric particles. Cytotoxicity assays performed with human colorectal carcinoma-derived Caco-2 cells demonstrated that the polymers are biocompatible and could be potentially used in drug delivery applications.

Clean synthesis of molecular recognition polymeric materials with chiral sensing capability using supercritical fluid technology. Application as HPLC stationary phases

Molecularly imprinted polymers (MIPs) of poly(ethylene glycol dimethacrylate) and poly(N-isopropylacrylamide-co-ethylene glycol dimethacrylate) were synthesized for the first time in supercritical carbon dioxide (scCO(2)), using Boc-L-tryptophan as template. Supercritical fluid technology provides a clean and one-step synthetic route for the preparation of affinity polymeric materials with sensing capability for specific molecules. The polymeric materials were tested as stationary HPLC phases for the enantiomeric separation of L- and D-tryptophan. HPLC results prove that the synthesized MIPs are able to recognize the template molecule towards its enantiomer which opens up potential applications in chromatographic chiral separation.

Boron trifluoride catalyzed polymerisation of 2-substituted-2-oxazolines in supercritical carbon dioxide

In the last few years there has been an intensive research on multifunctional polymers for therapeutical applications. Poly(2-substituted-2-oxazolines) are strong candidates for the development of new polymeric therapeutics. In this work, boron trifluoride etherate was used, for the first time, as initiator for the polymerisation of three substituted 2-oxazolines in supercritical carbon dioxide. The effect of temperature, pressure and initial monomer/initiator molar ratio on the yield, average molecular weight and polydispersity of the synthesized polymers was investigated. The products of the reaction were characterized by NMR, FT-IR and MALDI-TOF mass spectrometry. In all reactions, low molecular weight polymers were obtained in high yield with narrow molecular weight distribution. 2-Methyl- and 2-ethyl-2-oxazolines yielded water soluble products with degrees of polymerisation varying from 18 to 27 and 17 to 24, respectively, while poly(2-phenyl-2-oxazoline) was found to be insoluble in water and the degree of polymerisation varying from 10 to 12. In each polymerisation, there was an unexpected CO2 insertion in 10–25% of the overall polymer. A possible explanation for this insertion is given.

Supercritical fluid technology as a new strategy for the development of semi-covalent molecularly imprinted materials

Molecularly imprinted polymeric particles with molecular recognition towards Bisphenol A (BPA) were synthesized for the first time using the semi-covalent imprinting approach in supercritical carbon dioxide (scCO2). The materials affinity to BPA was achieved by co-polymerizing ethylene glycol dimethacrylate (EGDMA) with a template-containing monomer, Bisphenol A dimethacrylate (BPADM) in scCO2. Bisphenol A is then cleaved from the polymeric matrix by hydrolysis with tetrabutylammonium hydroxide (n-Bu4OH) also in a supercritical environment, taking advantage of the high diffusivity of scCO2. The selectivity of the molecular imprinted polymer (MIP) was assessed by evaluating its capability to bind BPA in comparison with progesterone and α-ethinylestradiol. In addition, the cross-linked particles were used to prepare a PMMA-based hybrid imprinted membrane by a scCO2-assisted phase inversion method. Results show that the incorporation of MIP particles was able to confer molecular affinity to BPA to the membrane and that at dynamic conditions of filtration, this imprinted porous structure was able to adsorb a higher amount of BPA than the corresponding non-imprinted hybrid membrane. Our work represents a valuable greener alternative to conventional methods, for the synthesis of affinity materials which are able to maintain molecular recognition properties in water.

Integrated desulfurization of diesel by combination of metal-free oxidation and product removal by molecularly imprinted polymers

The production of ultra-low-sulfur diesel is an important worldwide demand. In this work a novel integrated method for desulfurization of diesel is proposed based on the combination of Bronsted acid catalyzed oxidation and the selective removal of the oxidized products using a molecularly imprinted polymer (MIP) produced in supercritical carbon dioxide (scCO2). The biphasic oxidation reaction of dibenzothiophene sulfone (DBT), as model substrate, and H2O2 as oxidant, was optimized by testing different acid catalysts, and also different phase transfer catalysts (PTC), including two different ionic liquids (ILs) trihexyl(tetradecyl)phosphoniumchloride [P6,6,6,14]Cl and Aliquat®. The products of the efficient oxidation of DBT, dibenzothiophene sulfoxide (DBTSO) and dibenzothiophene sulfone (DBTSO2), were then selectively removed from real diesel using the MIP.

First insights of peptidoglycan amidation in Gram-positive bacteria - the high-resolution crystal structure of Staphylococcus aureus glutamine amidotransferase GatD.

Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.

High Affinity Polymers by Molecular Imprinting for Drug Delivery

Molecular recognition processes found in nature have always inspired scientists to mimic these systems in synthetic materials such as molecular imprinted polymers. Selective receptors within a polymer have a huge range of applications such as in separation processes, analytical chemistry, sensors, catalysis, drug discovery and therapeutics. In this last area in particular, polymers are having a central role in recent developments and materials with molecular recognition ability can respond to new challenges and opportunities.