**4.3 miRNA delivery systems**

An important aspect that is worth considering in miRNA cancer therapy aims to use miRNA delivery systems. One of these delivery systems including microvesicles and exosomes aim to block miRNA-entrapped exosomes released by tumors. It is already demonstrated that miRNA-entrapped exosomes secreted by tumor cells can regulate gene expression in the receiving cells by binding to their target mRNAs [37]. The use of some agents that block specific miRNAs (such as LNA anti-miR-21 and LNA anti-miR-29a) in tumor cells could lead to the reduction in miRNAentrapped exosomes, released by cancer cells [38]. However, an ideal delivery system meets the following criteria: protects the miRNAs from early degradation in the bloodstream, efficient distribution to the target cells, facilitates cellular uptake, does not induce an immune response, and made of biocompatible and biodegradable materials [39].

The most commonly used vectors for miRNA delivery include viral and nonviral vectors. Previous data demonstrated that viral vectors mainly caused an immune response; therefore, the focus of the actual studies is on developing efficient nonviral vectors. Nonviral vectors are classified into three main groups, including polymeric vectors (polyethyleneimines, atelocollagen, polylactic-coglycolic acid, polyamidoamine dendrimers), lipid-based carriers (positively, negatively or neutral charged), and inorganic materials (gold, diamond, silica, and ferric oxide) [40].

The delivery system based on viral vectors transfers the pri-miRNA or mature miRNAs, usually a TS-miR, into a plasmid, which contains a viral promoter, an antibiotic resistance gene, and a restriction enzyme gene, to the tumor cells. After nuclear integration of the miRNA and further transcription, the mature miRNA represses the translation and/or induces the degradation of the target mRNA [41].

## *4.3.1 Discussion*

One of the most studied classes of polymeric vectors was represented by polyethylenimines (PEIs) but was removed from clinical studies due to their high toxicity, given by an excessive positive charge, low biological degradation, and inactivation in serum caused by a nonspecific protein. Ibrahim et al. [42] have demonstrated that by using low molecular weight, PEIs as system delivery for miR-145 and miR-33a would

decrease the toxicity and increase the antitumor effect, in a model of colon carcinoma. Recent studies [43, 44] have proved that codelivery of miR-200c with chitosan, a cationic polymer with a high specificity for nucleic acid binding, decreased the angiogenesis, invasion, EMT, and metastasis and increased the apoptosis, highlighting the role of miRNA concentration in treatment effectiveness. Hao et al. [45] used miRNA (MiR-15a, miR16-1)/ATE-APT complex formed by atelocollagen (ATE), a type I collagen positively charged polymer, in combination with a RNA aptamer (APT) used as a ligand to target PCa cells that express prostate-specific membrane antigen (PSMA). Their study concluded that miRNA/ATE-APT complex was more efficient than an ATE-miRNA complex and that by using a PSMA-targeted system, the chances for selective killing of prostate cancer cells significantly would increase.

Moreover, it is worth into consideration the administration methods used for synthetic miRNAs (miRNA mimics) delivery into cells. Previous studies of Trang et al. have shown that both intratumoral and intravenous administration of let-7a mimics lead to the diminishing of non-small-cell lung cancer (NSCLC) tumor size in mouse models [46, 47].

As a future improvement in miRNA delivery systems, it is recommended to be synthetized proteins or peptides in order to be used as vector polymeric due to their low cytotoxicity and immunogenicity. Finding a suitable delivery system for a specific miR according to tumor cell type and the development of systems to target specific cancer membrane antigens still represent major challenges.
