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- Insight into the role of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto poly(ethylenimine): cell viability and gene transfection studiesPublication . Nouri, Alireza; Castro, Rita; Kairys, Visvaldas; Santos, José L.; Rodrigues, João; Li, Yulin; Tomás, HelenaIn the present study, the effect of N,N-dimethylaminoethyl methacrylate (DMAEMA) conjugation onto branched poly(ethylenimine) (PEI) with different grafting degree was examined for gene delivery applications. The DMAEMA-grafted-PEI conjugates were characterized and complexed with plasmid DNA (pDNA) at various concentrations, and the physicochemical properties, cell viability, and in vitro transfection efficiency of the complexes were evaluated in HEK 293T cells. Computational techniques were used to analyze the interaction energies and possible binding modes between DNA and conjugates at different grafting degrees. The cytotoxicity analysis and in vitro transfection efficiency of the conjugate/pDNA complexes exhibited a beneficial effect of DMAEMA conjugation when compared to PEI alone. The computational results revealed that the DNA/vector interaction energy decreases with increasing grafting degree, which can be associated to an enhanced release of the pDNA from the carrier once inside cells. The results indicate the significance of DMAEMA conjugation onto PEI as a promising approach for gene delivery applications.
- Interaction of antimicrobial peptides, BP100 and pepR, with model membrane systems as explored by brownian dynamics simulations on a coarse-grained modelPublication . Alves, Carla S.; Kairys, Visvaldas; Castanho, Miguel A. R. B.; Fernandes, Miguel X.This work focuses on the conformational and dynamic properties of the antimicrobial peptides (AMPs), BP100 and pepR, when confined within model membrane systems. Brownian dynamics (BD) simulations of a coarse-grained model of each respective peptide in an environment reproducing the phospholipid bilayer were carried out. Simple mean-field potentials were used to reproduce three physically different model phosphatidylcholine (PC) membrane systems. Based on the simplicity of the peptide-membrane models used, 1 ls simulations were performed. With the appropriate choice of parameters, the structure and dynamics of each peptide were recovered from each of the simulated BD trajectories. BP100 was observed to adopt a a-helical conformation when confined in each PC membrane. For pepR under the same conditions, the formation of an N-terminal a-helix was detected, whereas the C-terminus appeared to be less ordered. The dynamic properties of each peptide were characterized in terms of local and global motions. BP100 tended to localize with no preferred orientation approximately halfway across each membrane leaflet, whereas pepR localized near the membrane core with no preferred orientation. Overall, the peptide dynamics were found to vary according to the size of the peptide, as well as the width of the membrane environment.
- Polyester Dendrimers Based on Bis-MPA for Doxorubicin DeliveryPublication . Gonçalves, Mara; Kairys, Visvaldas; Rodrigues, João; Tomás, HelenaAlthough doxorubicin (DOX) is one of the most used chemotherapeutic drugs due to its efficacy against a wide group of cancer types, it presents severe side effects. As such, intensive research is being carried out to find new nanoscale systems that can help to overcome this problem. Polyester dendrimers based on the monomer 2,2-bis- (hydroxymethyl)propionic acid (bis-MPA) are very promising systems for biomedical applications due to their biodegradability properties. In this study, bis-MPA-based dendrimers were, for the first time, evaluated as DOX delivery vehicles. Generations 4 and 5 of bis-MPA-based dendrimers with hydroxyl groups at the surface were used (B-G4-OH and B-G5-OH), together with dendrimers partially functionalized with amine groups (B-G4-NH2/OH and B-G5-NH2/OH). Partial functionalization was chosen because the main purpose was to compare the effect of different functional groups on dendrimers’ drug delivery behavior without compromising cell viability, which is often affected by dendrimers’ cationic charge. Results revealed that bis-MPA-based dendrimers were cytocompatible, independently of the chemical groups that were present at their surface. The B-G4-NH2/OH and B-G5-NH2/OH dendrimers were able to retain a higher number of DOX molecules, but the in vitro release of the drug was faster. On the contrary, the hydroxyl-terminated dendrimers exhibited a lower loading capacity but were able to deliver the drug in a more sustained manner. These results were in accordance with the cytotoxicity studies performed in several models of cancer cell lines and human mesenchymal stem cells. Overall, the results confirmed that it is possible to tune the drug delivery properties of bis-MPA-based dendrimers by modifying surface functionalization. Moreover, molecular modeling studies provided insights into the nature of the interactions established between the drug and the bis-MPA based dendrimersDOX molecules attach to their surface rather than being physically encapsulated.
- Enantioseparation and chiral recognition mechanism of new chiral derivatives of xanthones on macrocyclic antibiotic stationary phasesPublication . Fernandes, Carla; Tiritan, Maria Elizabeth; Cass, Quezia; Kairys, Visvaldas; Fernandes, Miguel Xavier; Pinto, MadalenaA chiral HPLC method using four macrocyclic antibiotic chiral stationary phases (CSPs) has been inves tigated for determination of the enantiomeric purity of fourteen new chiral derivatives of xanthones (CDXs). The separations were performed with the CSPs Chirobiotic T, Chirobiotic TAG, Chirobiotic V and Chirobiotic R under multimodal elution conditions (normal-phase, reversed-phase and polar ionic mode). The analyses were performed at room temperature in isocratic mode and UV and CD detection at a wavelength of 254 nm. The best enantioselectivity and resolution were achieved on Chirobiotic R and Chirobiotic T CSPs, under normal elution conditions, with RS ranging from 1.25 to 2.50 and from 0.78 to 2.06, respectively. The optimized chromatographic conditions allowed the determination of the enan tiomeric ratio of eight CDXs, always higher than 99%. In order to better understand the chromatographic behavior at a molecular level, and the structural features associated with the chiral recognition mech anism, computational studies by molecular docking were carried out using VDock. These studies shed light on the mechanisms involved in the enantioseparation for this important class of chiral compounds.