Methods for Evaluating the Stimuli-Responsive Delivery of Nucleic Acid and Gene Medicines

Abstract

In this review, we have summarized evaluation methods for the analysis of external stimuli-mediated nucleic acid and gene delivery. Prior to reviewing these evaluation methods, we describe various delivery processes of nucleic acid and gene medicines (small interfering RNA (siRNA), micro RNA, mRNA, plasmid DNA, etc.), which include interaction with blood components, bio-distribution, disposition in the target tissue, cell entry, intracellular trafficking, nuclear localization, and dissociation from the carriers. Next, we discuss the advantages of external stimuli-mediated nucleic acid and gene delivery. External stimuli enable us to effectively deliver nucleic acids and genes to targeted regions. Evaluation methods are required to elucidate the behaviors of nucleic acid and gene medicines in the body. Quantitative analyses of the bio-distribution and in situ disposition in perfused organs, as well as visualization of bio-distribution, transgene expression in the body, and intracellular trafficking of nucleic acid and gene medicines, are all useful in evaluating not only the efficacy and safety of delivery, but also serve as guidelines for the further development of nucleic acid and gene medicines by elucidating delivery problems. Progress in evaluation methods, including tissue optical clearing and super resolution microscopy, will help to better elucidate the in vivo fate of nucleic acid and gene medicines.

Establishment of a cell line derived from the gills of Gymnocypris przewalskii, an endemic Schizothoracine fish from Qinghai Lake of Tibet Plateau

“…Transfection is a key technology for studying gene expression, protein transport and localization. Effective transfection methods are the basis of many studies (Faneca et al ., 2010; Fumoto & Nishida, 2017). In this study, the transfection efficiency was not significantly improved by adjusting the proportion of plasmid and liposome, transfection time or composition of transfection reagent (data not shown).…”

Monitoring Translation Activity of mRNA-Loaded Nanoparticles in Mice

“…38−40 Furthermore, positron-emission tomography of radiolabeled mRNAs and carriers or the detection of specific mRNA-encoded reporter proteins have been used. 38 However, the informative value of bioluminescence imaging and positron-emission tomography is limited, because both methods will not generate high-resolution images or detect single cells. Only when combined with more sophisticated methods such as photoacoustic tomography serial block-face imaging 41,42 or optical-sectioning microscopy, 43 high-resolution 3D images can be produced.…”

“…The gold standard for visualizing mRNA delivery in mice has been bioluminescence imaging of luciferase activity or the labeling of mRNA and/or carrier molecules with fluorescent dyes. − Furthermore, positron-emission tomography of radiolabeled mRNAs and carriers or the detection of specific mRNA-encoded reporter proteins have been used . However, the informative value of bioluminescence imaging and positron-emission tomography is limited, because both methods will not generate high-resolution images or detect single cells.…”

Understanding In Vivo Fate of Nucleic Acid and Gene Medicines for the Rational Design of Drugs

“…In the development of non-viral vectors, it is necessary to evaluate not only the efficiency of gene transfer, but also the formulation characteristics, internal and cellular behavior, and safety [ 309 ]. We summarized methods for evaluation of nucleic acid and gene medicines in Table 1 .…”