Knockdown of microRNA-135b in Mammary Carcinoma by Targeted Nanodiamonds: Potentials and Pitfalls of In Vivo Applications

Abstract

Nanodiamonds (ND) serve as RNA carriers with potential for in vivo application. ND coatings and their administration strategy significantly change their fate, toxicity, and effectivity within a multicellular system. Our goal was to develop multiple ND coating for effective RNA delivery in vivo. Our final complex (NDA135b) consisted of ND, polymer, antisense RNA, and transferrin. We aimed (i) to assess if a tumor-specific coating promotes NDA135b tumor accumulation and effective inhibition of oncogenic microRNA-135b and (ii) to outline off-targets and immune cell interactions. First, we tested NDA135b toxicity and effectivity in tumorospheres co-cultured with immune cells ex vivo. We found NDA135b to target tumor cells, but it binds also to granulocytes. Then, we followed with NDA135b intravenous and intratumoral applications in tumor-bearing animals in vivo. Application of NDA135b in vivo led to the effective knockdown of microRNA-135b in tumor tissue regardless administration. Only intravenous application resulted in NDA135b circulation in peripheral blood and urine and the decreased granularity of splenocytes. Our data show that localized intratumoral application of NDA135b represents a suitable and safe approach for in vivo application of nanodiamond-based constructs. Systemic intravenous application led to an interaction of NDA135b with bio-interface, and needs further examination regarding its safety.

Surface Functionalization of the Biological Gold Nanoparticles for Micro-Rna Targeting

“…Unbound antimiR-135b molecules were removed by centrifugation at 9000g for 60 min. The pellet was suspended in 400 µl RNase-free ddH2O and used for in vitro assay [24]. To detect the conjugation of antimiR-135b to AuNPs, different methods were used.…”

“…Excitation/emission parameters: Alexa Fluor 488 473/520 nm; TexasRed 595/615 nm. Results were analyzed using Olympus FluoView 2.0 software [24].…”

Why nanodiamond carriers manage to overcome drug resistance in cancer

“…Additionally, nanocarriers also exhibit decreased off-target toxicity in comparison with free drugs. So far, it seems that interactions of nanoparticles with immune cells (excluding the monocyte/macrophage lineage) are rare [5][6] . Among other nanomaterial that were developed for biomedical use, carbon material (nanotubes, graphene, fullerenes, carbon dots, films), and particularly nanodiamonds caught scientists' attention due to their potential use in drug delivery and bioimaging [5,[7][8][9][10][11][12][13] .…”

“…Thus, the effect of the nanodiamondcomplex on macrophages differs from the in vivo situation and may be a concern due to PEG-mediated hypersensitivity [49] . Křivohlavá et al [5] used an alternative approach; here, the studied complex contained a nanodiamond core, poly (ethylene imine), interfering RNA, and transferrin as a tumor-specific marker. The authors showed effective accumulation of loaded carriers in tumors after systemic administration followed by the specific knockdown of oncogenic microRNA-135b in tumor cells.…”

“…Protein corona forms on the surface of unshielded nanodiamonds after exposure to peripheral blood, or to a lesser extent, after exposure to serum-supplemented culture media [ 23 ] . The protein structure added as part of the nanodiamond coating will also shield nanodiamond complexes from uptake by peripheral macrophages [ 5 ] . Conveniently, if we coat nanodiamonds with a tumor-specific antibody, the nanodiamond will effectively accumulate at the tumor site [ 5 , 24 ] .…”