Treating AKI is the ultimate challenge. According to the Kidney Disease: Improving Global Outcomes (KDIGO) Clinical Practice Guideline for Acute Kidney Injury [63], there is no strong recommendation for its administration; thus, there is a demand for the development of new AKI therapeutic agents. As regeneration of kidneys can occur, there are several challenges involved. For example, stem cell therapy, including mesenchymal stem cells, kidney stem cells, and induced pluripotent stem (iPS)-cell-derived nephron progenitor cells, has been explored over several decades [14,64,65]. There are reports on direct replacement of transplanted stem cells [15,66], but currently, it appears likely that the dominant effect of transplanted stem cells is derived from the secreted factors from stem cells. For example, MSCs secrete a variety of factors, including soluble factors, cytokines, chemokines, and growth factors [14]. In addition, MSCs secrete extracellular vesicles containing soluble proteins, mRNAs, and miRNAs [13]. These factors transfer to the recipient cells, which mediate renoprotection (anti-apoptosis, anti-nectrosis, anti-inflammation, anti-oxidative stress, and anti-fibrosis) and regeneration (cell proliferation, cell migration, tubular de-differentiation, and angiogenesis) [67]. It is likely that extracellular vesicles are the predominant paracrine effects in AKI [67]. Which factors provide the dominant therapeutic effects? It was reported that EVs derived from MSCs with knockdown of Drosha, essential for miRNA production, failed to ameliorate I/R-induced AKI, while MSC-derived EVs without knockdown ameliorated AKI [68]. These data suggest that miRNAs in EVs might be the most important factors for renal protection and regeneration in AKI. Renoprotective miRNAs from MSCs were previously reported [67]. In the report, miR-21 and miR-30 mediated anti-apoptosis, miR-210 mediated angionegesis, miR-145 mediated autophagy, miR-15 and miR-16 ameliorated kidney fibrosis, and miR-15, miR-16, miR-21, and let-7 ameliorated inflammation through the regulation of macrophage. In addition to MSCs, secreted factors from other stem cells, such as kidney stem cells and iPS-derived nephron progenitor cells, have also been reported to be renoprotective [14,64,65]. These trophic effects might be at least partly delivered via miRNAs. miRNAs from other cells, such as circulating inflammatory cells and tubular cells, might be involved in kidney damage and/or regeneration during AKI. Researchers still need to explore miRNA dynamics. Nevertheless, miRNAs might be novel therapeutic targets for AKI.

In addition, a hypoxia-inducible factor (HIF)-prolyl hydroxylase (HIF-PHD) inhibitor has been developed as a therapy for renal anemia [69]. The pharmacological activation of HIF regulates a variety of genes, including Epo, leading to hematopoiesis. Other than hematopoiesis, the HIF-PHD inhibitor has also been shown to ameliorate AKI in rodent experimental models [70], including I/R-induced and drug-induced models with cisplatin, gentamicin, and lipopolysaccharide (LPS). These protective mechanisms include anti-apoptosis via miR-21, anti-inflammation by macrophage reduction, reduced VCAM1, up-regulation of angiogenesis via VEGF up-regulation, and anti-oxidative stress via up-regulation of Heme Oxygenase 1 (HO-1) [70]. As shown with increases in miR-21, these effects may be mediated at least partly via regulation of miRNAs. Indeed, it is reported that kidney ischemia activates HIF-1α, which in turn up-regulates miR-21, leading to anti-apoptosis through the suppression of pro-apoptotic factor programmed cell death protein 4 (Pdcd4) and phosphatase and tensin homolog deleted from chromosome 10 (PTEN) [71]. HIF1 also increases miR-668 expression, which targets mitochondrial fission process protein 1 (MTP18), leading to the protection of kidney tubular cells via mitochondrial dynamics under ischemic AKI in humans and mice [72]. Activation of HIF also increases miR-489, leading to anti-apoptosis in kidney tubular cells during ischemic AKI though targeting repair sensor poly(ADP-ribose) polymerase 1 (PARP1) [73]. Taken together, these data suggest that the regulation of miRNA might be a novel and specific therapy against AKI.

Some of the most important evidence regarding miRNAs in AKI was reported in 2010, where knockouts of tubular miRNAs were analyzed in the rodent model [74]. The loxp-cre system was used to produce mice lacking Dicer, a key enzyme for miRNA production, predominantly in kidney proximal tubular cells by crossing with phosphoenolpyruvate carboxykinase (PEPCK)-cre mice. The mice showed global down-regulation of miRNAs in the kidney cortex and had normal kidney function and histology under normal conditions. Importantly, the mice were resistant to renal ischemia-reperfusion (I/R) injury, demonstrating the involvement of miRNAs under AKI. Since then, there has been accumulating evidence supporting miRNAs as potential therapeutic targets in AKI. Several miRNAs have been reported to have protective and/or pathogenic roles in AKI, regulating tubular apoptosis, tubulointerstitial fibrosis, and inflammation in a variety of etiologies of AKI, including ischemia, drug, and sepsis. Some miRNAs have common gene targets. For example, miR-30 and miR-26a target Snai1, regulating epithelial–mesenchymal transition (EMT), while miR-21, miR-17, miR-188, and miR-378 target PTEN, which is implicated in cell apoptosis, proliferation, inflammation, and fibrosis [27]. The potential therapeutic targets of miRNAs for AKI are summarized in Table 2.

There are several protective mechanisms of miRNAs under AKI, including regulation of cell apoptosis, inflammation, autophagy, and energy metabolism. For example, miR-30, miR-181, miR-22, and miR-590 are reported to mediate anti-apoptosis and anti-inflammation effects in I/R-AKI rodent models. It was reported that MSC-derived extracellular vesicles ameliorated rat I/R-induced AKI by inhibiting cell apoptosis through miR-30b, miR-30c, and miR-30d, which targeted dynamin-related protein 1 (DRP1), thereby inhibiting mitochondrial fission [75]. In addition, injection of miR-30c-5p agomir, a chemically modified double-stranded small RNA that mimics the miR-30c-5p, ameliorated rat I/R-induced AKI through transformation of M1 to M2 macrophages, mediating an anti-inflammatory effect via changes in inflammatory cytokines [76]. miR-181d-5p overexpression in the mouse model of I/R-induced AKI ameliorated kidney injury by reducing inflammatory mediators and apoptosis through targeting Krueppel-like factor 6 (KLF6) [77]. A renoprotective effect of miR-181 was also shown in a cisplatin-induced mouse AKI model by targeting PTEN [78], as well as in a LPS-induced AKI model through an anti-apoptosis effect by targeting GJB2 [79]. In addition, miR-181a-5p is reported to target NIMA-related kinase 7 (NEK7), thereby inhibiting pyroptosis in sepsis-induced AKI mice [80]. It was reported that in a sepsis-induced AKI mouse model, lncRNA TCONS_00016233, targeting miR-22-3p, was up-regulated in plasma. In the report, TCONS_00016233 overexpression worsened sepsis-induced mouse AKI by down-regulating miR-22-3p, thus increasing the miR-22-3p target, apoptosis-inducing factor mitochondrion-associated 1 (AIFM1), leading to apoptosis [81]. These data suggest an anti-apoptosis effect of miR-22. In addition, miR-22 attenuated sepsis-induced rat AKI, targeting High Mobility Group Box 1 (HMGB1) and inhibiting the HMGB1/TLR4/NF-kB pathway [82]. Adenovirus expressing miR-590-3p via a tail-vein injection in LPS-induced septic AKI mice ameliorated cell apoptosis and inflammation by targeting tumor necrosis factor receptor-associated factor 6 (TRAF6) [83]. miR-590-3p was also shown to possibly increase autophagy and protect kidney injury by targeting TRAF6, which was evaluated in an in vitro I/R model using renal tubular epithelial cell line (HK-2 cells) [84].

There are several reports indicating an anti-apoptosis effect from miRNAs. miR-124 was shown to ameliorate I/R-induced mouse AKI, where miR-124 mimics reduced endoplasmic reticulum stress (ERS)-mediated apoptosis [85]. In addition to the anti-apoptosis mechanism, miR-124 also inhibited necroptosis by targeting PARP1 in an I/R-induced mouse AKI model [86]. miR-489 also targeted PARP1, mediating an anti-apoptosis effect, and miR-489 mimics protected against I/R mouse AKI [73]. miR-17-5p mimics suppressed death receptor 6 (DR6), mediating anti-apoptosis in an I/R-induced mouse AKI model [87]. Likewise, miR-424 mimics inhibited its target gene DR6, mediating anti-apoptosis in an I/R-induced mouse AKI model [88]. A miR-5100 mimic injection into I/R-mice ameliorated kidney injury by inhibiting several apoptotic pathways [45]. miR-486-5p was shown to target PTEN, thereby mediating an anti-apoptosis effect in a mouse I/R-AKI model [89]. A miR-191-5p mimic injection could inhibit cell apoptosis by targeting Oxidative stress responsive 1 (OXSR1) [90]. miR-290-5p activated by propofol ameliorated a sepsis-induced mouse AKI model by targeting C-C motif chemokine ligand 2 (CCL2), thereby mediating an anti-apoptosis effect [91].

In addition to the anti-apoptosis effect, an anti-inflammation effect by miRNAs has also been explored. miR-204/miR-211 mimics, targeting H6 Family Homeobox 1 (Hmx1), reduced kidney injury via immune suppression in candidemia-induced AKI mice [92]. A miR-195-5p mimic injection ameliorated rat I/R-AKI by targeting vascular endothelial growth factor A (VEGFA) via anti-inflammatory and anti-oxidative stress [93]. miR-140-5p up-regulation by apigenin ameliorated I/R-induced AKI mice through targeting Chemokine (C-X-C Motif) Ligand 12 (CXCL2), thereby reducing inflammation [94]. In addition, miR-140-5p was shown to activate nuclear factor erythroid 2-related factor (Nrf2) pathway, thereby mediating anti-oxidative stress in cisplatin-induced AKI mice [95]. miR-27a targeting Toll-like receptor 4 (TLR4) inhibited inflammation in I/R-induced AKI [96]. In addition, overexpression of LINC00520, targeting miR-27b-3p, activated Oncostatin M Receptor (OSMR), leading to the PI3K/AKT pathway to aggravate kidney injury in I/R-induced AKI. In contrast, up-regulation of miR-27b-3p could accelerate recovery from AKI [97]. miR-146a-5p derived from human urine-derived stem cells protected against rat I/R-induced AKI by targeting interleukin 1 receptor associated kinase 1 (IRAK1), thereby inhibiting NF-κB signaling and infiltration of inflammatory cells [98]. Mice lacking miR-146 showed more extensive tubular injury, inflammatory infiltrates, and fibrosis than wild-type mice after I/R-induced AKI [99]. Furthermore, the renoprotective effect of miRNAs involves vascular, mitochondrial, and podocyte protection. miR-210 was shown to activate VEGF signaling to regulate angiogenesis in I/R-induced AKI mice [100]. Overexpression of miR-126 in the hematopoietic compartment promoted vascular integrity and supported the recovery of kidney injury after I/R-induced AKI mice [101]. miR-668 was shown to target mitochondrial protein 18 kDa (MTP18) to preserve mitochondrial dynamics and tubular cell survival in I/R-AKI mice [72]. A miR-187 agomir injection in I/R protected against I/R-induced AKI and mediated podocyte protection, evaluated by nephrin expression by targeting acetylcholinesterase (AChE) [102].

There are several reports indicating MSC-related miRNAs as providing renoprotection via anti-apoptosis. MSC-derived exosome containing miR-199a-3p targeted semaphorin3A (SEMA3A), leading to the AKT and Extracellular signal-regulated kinase (ERK) pathway activation and ameliorated apoptosis [103]. Bone-marrow-derived MSCs ameliorated I/R-induced mouse AKI via miR-223, targeting NLR family pyrin domain containing 3 (NLRP3), thereby inhibiting apoptosis [104]. Bone-marrow-derived MSCs ameliorated cisplatin-induced AKI by inhibiting fibrosis through the regulation of miR-146a-5p and its target transcription Factor Dp-2 (Tfdp2) [105].

In addition to these renoprotective miRNAs, there are several pathogenic miRNAs under AKI, aggravating kidney injury by causing apoptosis, inflammation, mitochondrial damage, and fibrosis. It is reported that the administration of an antisense oligonucleotide inhibiting miR-182-5p ameliorated an I/R-induced AKI rat model [106]. In another report, an miR-182 inhibitor ameliorated I/R-induced rat AKI and apoptosis by regulating the transcription factor 7-like-2 (TCF7L2)/ Wnt/β-catenin pathway [107]. miR-182 was also shown to target Forkhead box O3 (FoxO3), leading to cell apoptosis in an I/R-induced AKI rat model [108]. Similarly, there are several miRNAs reported to promote cell apoptosis under AKI, including miR-122, miR-301, miR-375, miR-188, miR-687, miR-24, and miR-218. miR-122 was suggested to target FoxO3, thereby causing cell apoptosis in cisplatin-induced AKI mice [109]. miR-301a-5p inhibition ameliorated vancomycin-induced AKI by reducing apoptosis [110]. miR-375 was reported to be induced in a cisplatin-induced mouse AKI model to repress hepatocyte nuclear factor 1 homeobox B (HNF-1β), leading to the promotion of tubular cell apoptosis [111]. miR-188 was shown to aggravate contrast-induced AKI by targeting Serine and Arginine Rich Splicing Factor 7 (SRSF7), leading to cell apoptosis [112]. miR-687 was shown to be interfered with by lncRNA TCONS_00016406, leading to an anti-apoptosis effect [113]. miR-24 silencing protected against I/R-induced mouse AKI by inhibiting cell apoptosis by targeting H2A histone family, member X, (H2A.X), and HO-1 [114]. It was also suggested that honokiol inhibited miR-218-5p, leading to an increase in its target HO-1, thereby mediating an anti-apoptosis effect in sepsis-induced mouse AKI [115].

Several miRNAs, namely, miR-494, miR106, miR-155, and miR-152, were shown to promote inflammation as well as apoptosis. It is reported that miR-494-3p down-regulation by lncRNA TUG1 reduced I/R-induced mouse AKI and cell apoptosis by regulating E-cadherin [116]. In addition, overexpression of miR-494 was shown to reduce activating transcription factor 3 (ATF3), leading to an increase in inflammatory mediators, such as IL-6 and monocyte chemotactic protein-1, exacerbating inflammation in I/R-induced mouse AKI [43]. An injection of miR-494 antagomir, a chemically modified miR-494 antagonist, ameliorated LPS-induced mouse AKI via anti-apoptosis and anti-inflammation mechanisms by regulating the NF-κB signaling pathway [117]. It was reported that serum miR-106a was increased in sepsis-induced AKI mice, and miR-106a was suggested to target thrombospondin 2 (THBS2), leading to inflammation and apoptosis [118]. miR-155 inhibitor ameliorated LPS-induced mouse AKI through the reduction in inflammatory cells by regulating the target Suppressor Of Cytokine Signaling 1 (SOCS1) and Signal Transducer And Activator Of Transcription (STAT)1 mRNAs [119]. It was also reported that macrophage-derived exosomal miR-155 promoted tubular injury by targeting SOCS1 [120]. In addition, miR-155^−/− mice were made resistant to cisplatin-induced AKI by reducing tubular cell apoptosis [120]. miR-155 was shown to be up-regulated in rat I/R-induced AKI, and suggested to promote kidney injury and apoptosis by targeting Transcription factor 4 (TCF4)/Wnt/β-catenin signaling pathway [121]. miR-152-3p was suggested to promote cell apoptosis by silencing Sirtuin 7 (SIRT7) in I/R-induced rat AKI [122]. miR-152-3p was shown to promote sepsis-induced rat AKI by targeting ERBB receptor feedback inhibitor 1 (ERRFI1), leading to an increase in STAT3 expression, resulting in promoting cell apoptosis and inflammation [123]. An miR-709 antagomir injection attenuated cisplatin-induced mouse AKI via a reduction in mitochondrial dysfunction by regulating miR-709 target gene mitochondrial transcriptional factor A (TFAM) [124].

miR-21 is one of the most analyzed miRNAs, described as having double-edged-sword effects in kidneys [67] and both protective and pathogenic effects in kidney diseases. As a protective effect, miR-21 ameliorates I/R-induced AKI by inhibiting tubular cell apoptosis in I/R- and LPS-induced AKI mice [71,125], targeting PTEN/Akt/mammalian target of rapamycin (mTOR) signaling and Cyclin-dependent kinase 6 (CDK6). miR-21 is also shown to inhibit maturation of dendritic cells through the PDCD4/ NFκ-B pathway [71] and CCR7 [126], thereby mediating anti-inflammatory effects in I/R-induced AKI mice. In addition, miR-21 is also shown to target mitogen-activated protein kinase kinase 3 (MKK3), inhibiting the downstream factors IL-6 and TNF-α levels, mediating anti-inflammatory effects [127]. On the other hand, as a pathogenic effect, miR-21 inhibits autophagy in I/R-AKI rats by targeting Rab11a [128]. In addition, long-term elevation of miR-21 might promote kidney fibrosis, including via PPARα [129]. Furthermore, miR-21-3p was shown to regulate energy metabolism via AKT/Cyclin-dependent kinase 2 (CDK2)-FOXO1 in a sepsis-induced rat AKI model, while it was unclear whether the regulation was protective or harmful for a long-term prognosis [130]. Taken together, these data suggest that miR-21 may target several signaling pathways, involving cell apoptosis, inflammation, and autophagy, as well as energy metabolism under AKI.

Similarly to miR-21, several miRNAs were shown to mediate both protective and pathogenic effects under AKI. A miR-34b-3p agomir injection ameliorated sepsis-induced mouse AKI via an anti-inflammatory mechanism by targeting ubiquitin-like protein 4A (UBL4A) [131]. miR-34a induced via p53 was suggested to play a cytoprotective role in cell survival in cisplatin-induced mouse AKI [132]. In contrast to these renoprotective effects of miR-34, miR-34 was also shown to promote kidney injury under AKI. lncRNA HOX transcript antisense RNA (HOTAIR) overexpression ameliorated sepsis-induced rat AKI via an anti-apoptosis mechanism by targeting miR-34a and regulating its target B-cell lymphoma-2 (Bcl-2) [133]. It is also reported that increased miR-34a promoted acetylation of FOXO3 by repressing Sirtuin 1 (SIRT1), leading to p53 activation and cell apoptosis in the cisplatin-induced mouse AKI model [109]. miR-34a was also suggested to suppress autophagy in kidney tubular cells by targeting autophagy-related 4B cysteine peptidase (ATG4B) in I/R-induced mouse AKI [134]. miR-34a mimics prevented Nicotinamide phosphoribosyltransferase (NAMPT) expression, which suggest that they affected oxidized NAD (NAD⁺) metabolism, leading to kidney dysfunction in I/R-induced AKI mice [135]. Human umbilical-cord-derived MSCs containing miR-125b-5p suppressed p53, leading to an anti-apoptosis effect in I/R-induced AKI mice [136]. In contrast, miR-125 was also shown to disrupt mitochondrial dynamics by targeting modulate mitofusin1 (MFN1) in cisplatin-induced AKI mice, where anti-miR-125b treatment reduced cisplatin-induced mitochondrial fragmentation and kidney injury [137]. miR-150-5p agomir was shown to ameliorate sepsis-induced mouse AKI via an anti-apoptosis mechanism by targeting mitogen-activated protein kinase kinase 3 (MEKK3)/JNK pathway [138]. On the other hand, it was also reported that the deletion of miR-150 in mice protected against myocardial-infarction-induced AKI through an anti-apoptosis and anti-fibrosis mechanism by targeting insulin-like growth factor-1 receptor (IGF-1R) [139]. In addition, pretreatment with exosomes enriched in miR-150 worsened kidney fibrosis in I/R-induced AKI mice [140]. Similarly, kidney fibrosis was reduced by miR-150-5p-deficient tubular-cell-derived exosome in I/R-induced AKI mice by regulating the miR-150 target gene, SOCS1 [141]. miR-214 is also shown to mediate both protective and pathogenic effects under AKI. Regarding renoprotective effects, a miR-214 injection in I/R-induced mice ameliorated kidney injury by inhibiting apoptosis through targeting Dickkopf WNT signaling pathway inhibitor 3 (Dkk3) [142]. Adenovirus-mediated miR-214 treatment inhibited excess autophagy through regulation of the PTEN/AKT/mTOR pathway, thereby limiting kidney injury in sepsis-induced AKI mice [143]. On the other hand, regarding pathogenic effects, it is reported that kidney proximal-tubular-cell-specific miR-214 knockout mice showed less kidney damage and less apoptosis after I/R-induced AKI by targeting mitofusin-2 (Mfn2) [144]. miR-214-5p antagomir ameliorated LPS-induced kidney inflammation and oxidative stress, while miR-214-5p agomir aggravated kidney injury, presumably by targeting glucagon-like peptide-1 receptor (GLP-1R) [145]. miR-214-3p antagomir protected against cisplatin-induced AKI in mice by inhibiting tubular cell ferroptosis by targeting Glutathione Peroxidase 4 (GPX4) [146]. Microvesicles from human Wharton’s Jelly MSCs ameliorated I/R-induced rat AKI by suppressing C-X3-C Motif Chemokine 1 (CXCL1), and ameliorated inflammation and fibrosis partly via miR-16, miR-15a, and miR-15b [147]. On the other hand, urine miR-16 transactivated by CCAAT enhancer binding protein beta (C/EBP-β) worsened I/R-induced AKI, causing apoptosis [41]. Bone-marrow-derived MSCs reduced miR-107, increasing the expression of the miR-107 target, Ribosomal Protein S19 (RPS19), and protected from cisplatin-induced apoptosis [148]. In contrast, miR-107 was shown to induce TNF-α by targeting dual specificity phosphatase 7 (DUSP7), causing tubular injury [149]. Collectively, miRNAs have several gene targets, thus regulating a variety of mechanisms that may mediate protective and/or pathogenic effects under AKI, and it may depend on the etiologies of AKI and evaluating methods.

Nucleic acid drugs can treat adults at the level of RNA. They are usually composed of oligonucleic acids linked by ten to several tens of bases, and act directly on the body without gene expression [151,152]. Nucleic acid drugs include antisense, siRNA, miRNA, decoy, aptamer, and CpG oligodeoxynucleotides [153]. Among these, two approaches can be used for targeting miRNAs: antisense for inhibition-specific miRNA and miRNA-mimic injection. Clinical trials targeting miRNAs as nucleic acid medicine are currently still limited. Anti-miR was first conducted with miR-122 with locked nucleic acid in the form of Miravirsen for treating type C hepatitis [154]. Subsequently, antisense miR-155 (MRG-106) for T-cell lymphoma and mycosis fungoides was tested in a clinical trial [155]. A miR-10b antisense was used in preclinical trials with dextran-coated iron oxide nanoparticles for multiple cancers [156,157,158,159]. miRNA mimic treatment was conducted with miR-34 in lipid nanoparticles in the form of MRX34 for targeting multiple cancers, including hepatic cancers [160,161,162]. Subsequently, a clinical trial (Phase 1) with miR-16 mimics (TargomiR) was conducted for mesothelioma and lung cancer using the bacterial minicell EnGeneIC delivery system [163].

Currently, treatment targeting miRNAs in kidney diseases in the clinical stage is limited. A phase 2 placebo-controlled randomized controlled trial for Alport syndrome (NCT02855268) using oligonucleotides of miR-21 is currently ongoing [164], where the efficacy for the progression of kidney dysfunction, as well as the safety, pharmacodynamics, and pharmacokinetics, are being evaluated. A phase 1 clinical trial in polycystic kidney disease (PKD) patients using anti-miR-17 oligonucleotide RGLS4326 is also being conducted (NCT04536688) [165], where the primary objective is to assess the dose–response relationship between RGLS4326 and biomarkers of PKD. Referring to the miRNA treatment for AKI, miR-5100 was recently detected as a potential AKI biomarker as well as a therapy target [45]. miR-5100 was down-regulated in a rodent AKI model, and a miR-5100 mimic ameliorated I/R-induced AKI. In addition, in human serum samples, miR-5100 was down-regulated; thus, it is possibly both a biomarker and a therapy target.

In spite of these potentials of miRNAs as the therapeutic targets under AKI, there are several limitations and problems relating to clinical use. One of the most important challenges for miRNA-targeting therapy is the use of a drug delivery system (DDS) to transfer these anti-miR or miR-mimics more efficiently. Antisense drugs composed of single-stranded oligonucleic acids must be translocated into cells because they function by complementary binding to intracellular RNA. However, the antisense is large in size and has a negative charge due to the phosphodiester bond, making it difficult for it to pass through biological membranes. Therefore, many antisense miRNAs are attached with a phosphorothioate modification (Sylation). In addition, chemical modifications are also introduced into the sugar moiety of nucleic acids, thereby exerting efficacy without using carriers, such as liposomes. In contrast, miRNA mimics are normally composed of a double strand, and are thus larger than antisense miRNAs, leading to further difficulty with cell membrane permeability. Therefore, miRNA mimic therapy in general requires a DDS, such as lipid nanoparticles, polyplexes, and polymeric micelles [166]. Exosome may be the natural DDS to transfer anti-miR or miR-mimics. There are two approaches indicated, post-loading and pre-loading, both of which still have difficulties in the efficient incorporation of target nucleic acids [167]. Receptors such as GalNACs can be used as asialoglycoporotein receptors [168]. In addition, miRNA mimics need to be recognized by RISC, and thus the degree of nucleic acid modification possible is also limited. Treatment with nucleic acid drugs may also cause on-target toxicity (toxicity due to binding to target RNA) and off-target toxicity (toxicity due to binding to non-target RNA) [152]. It is notable that treatment targeting miRNAs might cause unexpected side effects [169], considering that one type of miRNA may regulate more than one hundred genes, including genes of interest.