Nanobiotechnology and Nanomaterials for Gene Expression and Bacterial Growth Control

Résumé

In this thesis, we have initiated four research projects. Three of them related to the development and evaluation of nanomaterials as antimicrobials. The fourth one is devoted to the assessment of gold nanoparticles modified with oligonucleotides as regulators of p21 expression. The first project has been carried out at the Universidad Autónoma de Madrid and deals with the preparation of silver nanoparticles (AgNPs) using broths from bacteria and their evaluation as antimicrobials. Particularly, four psychrophilic bacteria isolated from Antarctica have been evaluated. Different conditions were assessed, including the addition of NaCl and incubation temperatures. The AgNPs obtained were characterized and their antimicrobial properties evaluated against Gram-negative and Gram-positive bacteria. We also have evaluated the antimicrobial properties of silver nanoclusters stabilized by DNA (DNA-AgNCs), which are novel fluorescent materials. After testing 9 oligonucleotides with different sequence and length we found that the antibacterial activity depends on the sequence of the oligonucleotides employed. The sequences tested yielded fluorescent AgNCs, which can be grouped in blue, yellow and red emitters. Interestingly, blue emitters yielded poor antibacterial activity whereas yellow and red emitters afforded an activity similar to AgNO3. Finally, we prepared a trimeric structure containing the sequence that afforded the best antimicrobial activity, which inhibited the growth of Gram positive and negative bacteria in the sub-micromolar range. In the third project, we demonstrated that oligonucleotides modified with a fluorescent dye (HEX) can be used in photodynamic therapy to kill bacteria. Interestingly, the sequence of the oligonucleotide employed modulates the antimicrobial activity of the system. The last project has been done in collaboration with the Centro Nacional de Biotecnología (CNB) and the preliminary results of the regulation of p21 with modified gold nanoparticles are presented. P21 is involved in cell senescence and replication, and its regulation can be done using different oligonucleotides, such as antisense and siRNAs. However, the translocation into the cells of nucleic acids is not easy and transfection systems are required. In this case, oligonucleotides were conjugated to gold nanoparticles (AuNPs) and used in T-cells. The results suggest that the approach employed is valid although further optimization is required.