Captura magnética e entrega direcionada de híbridos de pre-miRNA-nanotubos de carbono em terapia do cancro do pulmão

Innovative therapeutic strategies are really needed to discover treatments for emergent disorders, or diseases without effective therapies. In this context, miRNAs can become one of the major classes of therapeutic molecules in the future. During the past two decades, numerous miRNAs have been characterized as oncogenic or tumor-suppressive miRNAs in almost all types of cancer and many other human diseases. As a result, many preclinical and clinical trials centered on miRNAs have been carried out (Kim and Croce, 2023). In cancer cases, it was found that the miR-29 family is involved in the regulation of DNA methylation (Kwon et al., 2019), and it was also demonstrated that the expression of miR-29 is downregulated in LC, suggesting that miR-29 levels are inversely correlated to the expression of DNMT3A/-3B in cancer cells (Fabbri et al., 2007). This generates the hypothesis that re-establishing the levels of miR-29 in LC cells can help in the control of cancer growth and facilitate the action of chemical drugs. Despite their potential, miRNA-based therapeutics have not arrived yet, mainly due to limitations on their manufacturing, stabilization, and delivery. Current clinical trials with RNAi-based therapeutic candidates rely on the use of synthetic miRNAs. However, several issues associated with the presence of contaminants restrain the translation of miRNA-based therapeutics into clinical practice. It is evident that establishing a robust miRNA production technology may unlock the possibility of developing effective RNAi-based therapies. This is exactly the challenge expected to be addressed in this project, whose main goal is to establish an integrated, cost-effective platform for pre-miRNA capture, purification, stabilization, and delivery by employing a groundbreaking process where modified CNTs will play a central role. In this context, it is proposed an innovative approach for the in situ recovery of the recombinant pre-miR-29, directly from the fermentation broth, using modified CNTs. To achieve this goal, it will be performed the screening of ONs with complementarity for different regions of the corresponding pre-miRNA, in Task 1. This screening will facilitate selecting the most suitable ligands for the modification of CNTs, to reach an improved selectivity towards the target pre-miRNA, facilitating the posterior purification. Thus, Task 2 aims to synthesize and modify CNTs for further use in pre-miRNA capture. Another feature that will be exploited is the magnetic properties that will be conferred to CNTs, enabling not only a rapid capture of the pre-miRNA-CNTs hybrids from the medium but also the concentration of the sample. The third main goal is related to the establishment of an innovative approach for the direct purification of pre-miR-CNTs hybrids. 3D-printed chromatographic supports will be designed, to explore anion-exchange, hydrophobic, or affinity interactions. Lastly, it is aimed at assessing the bioactivity of pre-miRNA in cancer cell lines. In this step, the integrated process is concluded, and pre-miR-CNT hybrids are directly delivered to cells, exploiting the magnetic properties of the CNTs. This integrated approach will also guarantee RNA stability as the manipulation and exposure to external conditions are much more limited. Overall, understanding the production, recovery, and purification processes could lead to the future implementation of this platform at a higher level.