*1.5. Extracellular Vesicles and miRNA-Based Therapy*

Extracellular vesicles (EVs) are membrane-bound small vesicular bodies released by the cells and are utilized in cell-to-cell communication/signaling. These vesicles are relatively small in size and fall under the nanoscale category. However, they preserve the ability to transport molecular cargo. EVs are categorized into three sub-classes, namely, microvesicles, exosomes, and apoptotic bodies. Distinctions are based on their size, type of originating cells, and formation mechanism. They can be released in response to a variety of external stimuli. Examples of this include changes in cell microenvironments (pH, temperature, irradiation), cellular stress, and chemically-induced activation.

EVs may also serve as a communication bridge between immune cells and beta cells. Pancreatic islets have also been shown to secrete EVs that behave in an autocrine manner to regulate beta cell proliferation and death. These islet mesenchymal stem cell-derived exosomes containing miRNAs, can activate the T cell response and stimulate the release of interferon gamma (IFN-γ) to induce autoimmune responses in T1D [130]. Recently, nucleic acids containing exosomes—especially miRNAs—have been shown to regulate communication networks between organs in pathological processes relating to diabetes. One such example includes influencing metabolic signals and insulin signals in target tissues, affecting cell viability, and modulating inflammatory pancreatic cells [131]. This also opens the possibility for exosomes to be developed and utilized as a tool to improve the islet transplant by modulating the immune response or as a biomarker of recurrent autoimmunity for islet transplant diagnosis.

A class of short noncoding RNAs of 19–22 nucleotides, known as microRNAs (miR-NAs), act as negative regulators of gene expression by partially pairing to the 30 or 50 of the untranslated regions of their target messenger RNAs (mRNAs) [132]. This new and fast rising technology using miRNAs has appealed to many researchers as a potential, minimally invasive biomarker for T1D due to three main reasons: miRNAs are exceptionally stable in cell-free body fluids such as serum, they have high resistance to RNAse digestion, and miRNA molecules have an ability to remain intact in extreme conditions (such as being in extended storage and going through repeated freeze–thaw cycles). Additionally, there is a strong possibility that miRNAs are involved in gene regulation of T1D development [133,134].

According to Scherm et al., miRNA expression differs in peripheral mononuclear cells (PMNC) and specific immune cell subsets, such as regulatory T cells, in T1D patients when compared to healthy individuals [135]. This uncharacteristic expression in miRNA leads to disrupted T cell differentiation and loss of function, subsequently resulting in immune activation and the onset of islet autoimmunity and initiation of T1D [136]. In order to provide an elaborate catalog of coding and noncoding miRNAs in human isletderived exosomes, Krishnan et al. profiled such RNAs in human islet-derived exosomes and identified the RNAs which were aberrantly expressed under cytokine stress [137]. Wang et al. attempted a theranostic approach to deliver miRNA-targeting oligonucleotides conjugated iron oxide nanoparticles in order to modify their expression in pancreatic islets of NOD mice [124]. MiR-216a was identified as a pivotal point in regulating the beta cell proliferation and altering its expression levels significantly affected the progression of T1D (Figure 2). Similarly, modulating the levels of the miR-29 family (miR-29a, miR-29b, and miR-29c) via iron oxide nanoparticles serves to regulate the glucose homeostasis and overcome the hypoglycemic shock induced by diabetes [138]. The levels of miRNA-181a impaired immune tolerance and affect the function of Treg cells. Attempts have been made to successfully block miRNA181a, increasing the Treg induction and reducing the islet autoimmunity in mice [139]. These findings suggest that the identification and subsequent block of trigger markers might allow for the reversal of islet autoimmunity. MiRNAs pertaining to autoantibodies, such as insulin autoantibodies (IAA), islet cell cytoplasmic antibodies (ICA), insulinoma-associated 2, or protein tyrosine phosphatase antibodies (IA-2), zinc transporter8 (ZnT8), and glutamic acid decarboxylase (GAD65), trigger pancreatic T cells to initiate insulitis. when compared to healthy individuals [135]. This uncharacteristic expression in miRNA leads to disrupted T cell differentiation and loss of function, subsequently resulting in immune activation and the onset of islet autoimmunity and initiation of T1D [136]. In order to provide an elaborate catalog of coding and noncoding miRNAs in human isletderived exosomes, Krishnan et al. profiled such RNAs in human islet-derived exosomes and identified the RNAs which were aberrantly expressed under cytokine stress [137]. Wang et al. attempted a theranostic approach to deliver miRNA-targeting oligonucleotides conjugated iron oxide nanoparticles in order to modify their expression in pancreatic islets of NOD mice [124]. MiR-216a was identified as a pivotal point in regulating the beta cell proliferation and altering its expression levels significantly affected the progression of T1D (Figure 2). Similarly, modulating the levels of the miR-29 family (miR-29a, miR-29b, and miR-29c) via iron oxide nanoparticles serves to regulate the glucose homeostasis and overcome the hypoglycemic shock induced by diabetes [138]. The levels of miRNA-181a impaired immune tolerance and affect the function of Treg cells. Attempts have been made to successfully block miRNA181a, increasing the Treg induction and reducing the islet autoimmunity in mice [139]. These findings suggest that the identification and subsequent block of trigger markers might allow for the reversal of islet autoimmunity. MiR-NAs pertaining to autoantibodies, such as insulin autoantibodies (IAA), islet cell cytoplasmic antibodies (ICA), insulinoma-associated 2, or protein tyrosine phosphatase antibodies (IA-2), zinc transporter8 (ZnT8), and glutamic acid decarboxylase (GAD65), trigger pancreatic T cells to initiate insulitis.

According to Scherm et al., miRNA expression differs in peripheral mononuclear cells (PMNC) and specific immune cell subsets, such as regulatory T cells, in T1D patients

*Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 12 of 23

**Figure 2.** Fluorescence microscopy of consecutive frozen pancreatic sections from STZ-induced diabetic mice injected with MN-miRNA, MN-ASO, MN-miRNAscr, and MN-ASOscr. Animals injected with MN-miRNA showed higher insulin expression in pancreatic islets (top: green, insulin; red, Cy5.5; blue, cell nucleus) when compared to the animals injected with MN-ASO or control nanodrugs. These animals also showed downregulated PTEN expression in their islets (middle: green, PTEN; red, Cy5.5; blue, cell nucleus) when compared to the animals injected with MN-ASO or control nanodrugs. Finally, there was a notably higher cell proliferation in the islets of these animals when compared to controls (bottom green, Ki67; red, Cy5.5; blue, cell nucleus); Magnification bar = 40 μm. All experiments were performed in triplicates, reproduced with permission from Springer Nature [124]. **Figure 2.** Fluorescence microscopy of consecutive frozen pancreatic sections from STZ-induced diabetic mice injected with MN-miRNA, MN-ASO, MN-miRNAscr, and MN-ASOscr. Animals injected with MN-miRNA showed higher insulin expression in pancreatic islets (top: green, insulin; red, Cy5.5; blue, cell nucleus) when compared to the animals injected with MN-ASO or control nanodrugs. These animals also showed downregulated PTEN expression in their islets (middle: green, PTEN; red, Cy5.5; blue, cell nucleus) when compared to the animals injected with MN-ASO or control nanodrugs. Finally, there was a notably higher cell proliferation in the islets of these animals when compared to controls (bottom green, Ki67; red, Cy5.5; blue, cell nucleus); Magnification bar = 40 µm. All experiments were performed in triplicates, reproduced with permission from Springer Nature [124].

Levels of miRNA in systemic circulation have been proposed as a new class of biomarkers for diagnosis and prognosis of T1D and this has also presented itself as a new target for modulations and therapeutics [140]. There are alterations in serum levels in newly diagnosed T1D patients, with some specific miRNAs appearing to be related to glycemic controls [141]. This newer class of potential circulating biomarkers for T1D have narrowed down their source and improved our knowledge related to the understanding of the molecular functions of these biomarkers. Akerman et al. studied the possible deviations of miRNA levels in the serum of children. They found the serum to be positive for multiple IAAs, and considered these individuals to be at high risk for T1D development [142]. They found that the serum miRNA profiles and autoantibody-positive individuals with high risk of T1D did not differ with respect to healthy, age-matched controls. Some studies have determined that beta cells initiate T1D progression through the activation of various stress pathways. This accelerates the autoimmune-mediated destruction of beta cells and the subsequent loss of insulin-producing mechanisms [130]. The aforementioned study focuses on the need to identify biomarkers in healthy beta cells, which serve as the guiding markers in identifying and monitoring dysfunctional cells. These approaches can not only help to monitor dysfunctional beta cells, but also improve the diagnostics for early detection of T1D. In this context, Bertoccini et al. focused on levels of circulating miR-375, an alleged biomarker of beta-cell death. They observed that an increase in miR-375 was indicative of later onset of T1D, suggesting residual beta-cell function [143]. MiR-375 was directly correlated to the population of viable beta cells that were under autoimmune attack. These results strongly support the potential of miR-375 as an efficient biomarker for T1D diagnosis and prognosis. Bearing this in mind, Lakhter et al. have analyzed the effects of miR-21-5p upregulation on beta cell survival and functionality [144]. Their study determined that the levels of extravesicular-associated miR-21-5p increase significantly in the T1D developing microenvironment and thus, can serve as an efficient biomarker in early T1D detection. However, they noted that utilizing miR-21-5p as an identifying biomarker has limitations due to the abundance of miR-21-5p in circulation, as well as its presence in multiple tissue types. This limits our capability to extrapolate the exact source/reason of the increased levels. Along similar lines, Santos et al. investigated the roles of circulating miR-101-3p and miR-204-5p with respect to T1D progression [134]. Their work concluded that circulating levels of miR-101-3p are higher in T1D patients and healthy individuals with autoantibodies. Based on this data they inferred that miR-101-3p plays an important role in pathways preceding the onset of T1D and can function as an important marker for diagnosis of T1D.

EVs and miRNAs serve as promising biomarker candidates with potential to assist in early T1D diagnosis and prognosis. In comparison to using naked miRNAs, the methods utilizing the conjugated complexes to nanoparticles or nanoscale vesicles have an advantage in terms of ease of administration and in vivo imaging. Although in its nascent stage, this theranostic approach is gaining increased attention and relevance in present-day studies. Table 4 presents variety of miRNA targeting strategies recently studied for T1D.

It has been reported that miRNA expression differs in peripheral blood mononuclear cells and in specific immune cell subsets, such as regulatory T cells, in T1D patients when compared to healthy individuals [135]. This uncharacteristic expression of miRNA leads to disrupted T cell differentiation and loss of function, which subsequently results in immune activation and the onset of islet autoimmunity and initiation of T1D. Researchers have also found that EVs such as islet mesenchymal stem cell-derived exosomes containing miRNAs can activate the T cell response and stimulate the release of interferon gamma (IFN-γ) to induce autoimmune responses in T1D [131].


**Table 4.** Summary of strategies for targeting microRNAs towards T1D.
