**1. Introduction**

Diabetes is a chronic health condition that affects the metabolism of glucose in the body due to decreased insulin secretion from the pancreatic islets. Diabetes is further classified into three main types: (i) Type 1, (ii) Type 2, and (iii) gestational diabetes. Out of these, Type 1 diabetes (T1D) is caused by an autoimmune disorder that inhibits the production of insulin in affected individuals [1,2]. Chronic autoimmune diseases are the consequence of the mistaken recognition of self-proteins (antigens) as foreign by the immune system, which leads to an immune response and subsequent destruction of the targeted tissues [3]. T1D is characterized by selective loss of insulin-producing beta cells of islets of Langerhans in the pancreas. Such a loss disrupts the glycemic homeostasis of the body. The loss of beta cell mass occurs when autoreactive T cells migrate to the Langerhans islets and cause local inflammation [4,5]. This autoimmune-mediated beta cell destruction can be asymptomatic for years prior to the manifestation of clinical symptoms. Currently, there is no clinical "cure" of T1D. The condition is managed using insulin and its variates which attempt to maintain the blood glucose levels within the healthy range. The condition furthers needs to be supplemented with proper diet, exercise, and weight management [6,7]. A big limitation of the conventionally prescribed insulin replacement therapy is its life-long dependence on

**Citation:** Nigam, S.; Bishop, J.O.; Hayat, H.; Quadri, T.; Hayat, H.; Wang, P. Nanotechnology in Immunotherapy for Type 1 Diabetes: Promising Innovations and Future Advances. *Pharmaceutics* **2022**, *14*, 644. https://doi.org/10.3390/ pharmaceutics14030644

Academic Editors: João Paulo Figueiró Longo and Lúis Alexandre Muehlmann

Received: 30 December 2021 Accepted: 8 March 2022 Published: 15 March 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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such treatment [8–10]. Factors including lack of specificity, altered effects, and diminished potency can also hinder the patient's recovery.

In recent years, various studies have been undertaken to limit or reverse this autoimmunemediated beta cell damage and a variety of strategies have been developed to enhance beta cell survival and/or islet regeneration [11–13]. Immunotherapy deals with the development of preclinical therapeutic strategies for disease treatment targeted towards the body's immune system. The immune system comprises a complex collaboration of cells working together to counteract the pathogens which invade the body. This intricacy makes the immunotherapeutic approach a particularly challenging task, as it involves various measures to prevent, diagnose, and treat a variety of disorders [14,15]. These approaches focus on the combination of immunity analysis and clinical chemistry to develop alternative therapeutic methodologies. It has been shown that autoantigen in combination with an appropriate immunomodulator holds potential in tackling a variety of autoimmune diseases [16]. These two compounds, when delivered simultaneously, affect the action of immune cells by modulating their autoreactivity. This immune modulation can play a key role in delaying and reducing the onset of T1D and is being explored using functional nanomaterials [17]. However, the ideal therapeutic approach for T1D should be long-lasting, render elevated antigen-specific tolerance, and integrate personalized or broad antigen specificity.

Towards this end, various nanoparticulate systems have been developed and have been transformative in the field of nanomedicine. Nanotechnology provides us with this tailorable ability to work at the atomic and molecular levels and opportunities to understand and create nanoparticulate platforms with new intrinsic properties attributed to their nanoscale size (<100 nm). For use in biomedical applications, the nanomaterials must exhibit unique properties such as aqueous stability, biocompatibility, and interactive functional groups. A variety of "smart" functional nanomaterials have made significant advances in the areas of drug delivery and drug discovery, biosensing, cell labeling and transplantation, gene therapy, immune therapy, and diagnoses and imaging [18–23]. The most attractive trait of these nanosystems is their large surface area/volume ratio, which allows their surface to carry and deliver multiple molecules to the same site of interest. Recent years have seen extensive evaluation and applications of nanosystems such as polymeric, lipid-based, liposomes, dendrimers, and inorganic nanoparticles (Metal oxides, gold, rare earth metals, graphene etc.). The range of nanoparticles used in recent preclinical studies is enormously extensive.

Various materials and compositions are being explored to both target and modulate the specific immune cell populations for the treatment of T1D. However, an absolute "cure" remains a challenge [24]. Insulin, its variates, and other therapeutic molecules which can modulate hyperglycemia have already been extensively delivered using nanoparticles to remediate the condition [25–29]. However, for the use in immunotherapy of T1D, these functional nanomaterials are required to conjugate and deliver immunomodulators to desired localized sites while maintaining its activity. Combing through a wide variety of targets, immunomodulatory agents accumulate locally and initiate specific immune responses. Development of tolerogenic vaccines for T1D and beta cell replacement therapies have shown promise by using nanoparticulate platforms [30–32]. By delivering tolerogenic agents and autoantigens, these nanomaterials can also be used to restore and enhance immune tolerance against T1D [33].

In this regard, this manuscript summarizes our knowledge of diabetic etiology, clinical scenarios, and the application of nanoparticle-based delivery approaches in targeting the immune system towards the treatment of T1D. Figure 1 illustrates various immunotherapeutic strategies currently under exploration to combat T1D discussed in the current review.

**Figure 1.** Schematic depiction of immunotherapies for T1D described below. T Cell Therapy: T cells modulated by nanoparticles conjugated to siRNA/miRNA leads to immunosuppression. B Cell Therapy: Modulation of B cells via monoclonal antibodies and/or nanoparticles conjugated to siRNA/miRNA leads to B cell apoptosis and depletion. Immune Checkpoint therapy: Nanoparticles conjugated to siRNA/miRNA induces PD-L1/CTLA4 expression resulting in abrogation of proximal T cells. Stem Cell Therapy: Differentiated stem cells transform into insulin producing beta cells. **Figure 1.** Schematic depiction of immunotherapies for T1D described below. T Cell Therapy: T cells modulated by nanoparticles conjugated to siRNA/miRNA leads to immunosuppression. B Cell Therapy: Modulation of B cells via monoclonal antibodies and/or nanoparticles conjugated to siRNA/miRNA leads to B cell apoptosis and depletion. Immune Checkpoint therapy: Nanoparticles conjugated to siRNA/miRNA induces PD-L1/CTLA4 expression resulting in abrogation of proximal T cells. Stem Cell Therapy: Differentiated stem cells transform into insulin producing beta cells.

#### *1.1. Understanding T1D: Etiology and Current Clinical Scenario 1.1. Understanding T1D: Etiology and Current Clinical Scenario*

T1D is a chronic autoimmune disorder characterized by the loss of insulin-producing beta cells within pancreatic islets. The immune-mediated destruction of beta cells results in insufficient levels of insulin production, creating a dependence on exogenous sources of insulin in affected individuals [2,34]. Both genetic and environmental factors contribute to the development of T1D. Typically, genetic predispositions are the result of polymorphic alleles coding for human leukocyte antigen (HLA), insulin gene promoter, and cytotoxic T lymphocyte antigen-4. In-fact these genetic disturbances account for 55–65% of T1D cases [35–37]. In the vast majority of T1D cases, pathogenesis is identified by the presence of several pancreatic autoantibodies. These autoantibodies include antibodies to insulin (IAA), islet cell cytoplasmic antibodies (ICA), insulinoma-associated 2 or protein tyrosine phosphatase antibodies (IA-2), zinc transporter8 (ZnT8), and glutamic acid decarboxylase (GAD65). The number of autoantibodies present in circulation and their abundance contribute directly to an individual's likelihood for developing T1D [38]. Autoantibodies provoke CD4+ and CD8+ T cells to migrate into pancreatic islets, triggering insulitis. Once these lymphocytes infiltrate the intra-islet space, they proliferate and attack endogenous beta cells, leading to the cessation of insulin production within pancreatic islets and subsequent loss of glycemic homeostatic mechanisms [39–41]. Insufficient insulin production prevents the inhibition of lipolysis leading to uncontrolled fat metabolism and the accumulation of ketone bodies in the blood. The buildup of ketone bodies such as aceto-T1D is a chronic autoimmune disorder characterized by the loss of insulin-producing beta cells within pancreatic islets. The immune-mediated destruction of beta cells results in insufficient levels of insulin production, creating a dependence on exogenous sources of insulin in affected individuals [2,34]. Both genetic and environmental factors contribute to the development of T1D. Typically, genetic predispositions are the result of polymorphic alleles coding for human leukocyte antigen (HLA), insulin gene promoter, and cytotoxic T lymphocyte antigen-4. In-fact these genetic disturbances account for 55–65% of T1D cases [35–37]. In the vast majority of T1D cases, pathogenesis is identified by the presence of several pancreatic autoantibodies. These autoantibodies include antibodies to insulin (IAA), islet cell cytoplasmic antibodies (ICA), insulinoma-associated 2 or protein tyrosine phosphatase antibodies (IA-2), zinc transporter8 (ZnT8), and glutamic acid decarboxylase (GAD65). The number of autoantibodies present in circulation and their abundance contribute directly to an individual's likelihood for developing T1D [38]. Autoantibodies provoke CD4<sup>+</sup> and CD8<sup>+</sup> T cells to migrate into pancreatic islets, triggering insulitis. Once these lymphocytes infiltrate the intra-islet space, they proliferate and attack endogenous beta cells, leading to the cessation of insulin production within pancreatic islets and subsequent loss of glycemic homeostatic mechanisms [39–41]. Insufficient insulin production prevents the inhibition of lipolysis leading to uncontrolled fat metabolism and the accumulation of ketone bodies in the blood. The buildup of ketone bodies such as acetoacetate and β-hydroxybutyrate

acetate and β-hydroxybutyrate causes ketoacidosis. In the absence of compensatory

causes ketoacidosis. In the absence of compensatory mechanisms, ketoacidosis results in loss of consciousness, cerebral edema, mental confusion, coma, and even death [42].

Studies investigating the molecular mechanisms behind T1D progression have determined that an individual's predisposition towards developing this autoimmune disorder is controlled by complex interactions between a multitude of genetic loci and environmental factors. As previously mentioned, polymorphic alleles at the HLA locus account for nearly half the familial clustering of T1D. HLA molecules are divided into class I (A, B, and C), and class II (DR, DQ, and DP). While HLA class I molecules are expressed on the surface of nearly all cells, HLA class II molecules are only expressed on the surface of activated T cells, B cells, and antigen-presenting cells [36,43]. Abnormalities affecting the genes which code for class II molecules contribute significantly towards T1D susceptibility, specifically those mapped to HLA-DQ/DR. Haplotypes corresponding to DR3-DQ2 and DR4-DQ8 have been observed in approximately 90% of patients diagnosed with T1D [44,45]. One study revealed that expression of human DR3 and/or DQ8 in non-diabetes-prone mice generated a loss of immune tolerance to the beta cell autoantigen glutamic acid decarboxylase, resulting in spontaneous insulitis [46]. Prior to insulitis, antigen-presenting cells (APCs) carrying beta cell peptides migrate towards lymph nodes located near the pancreas. There they present autoantigens to autoreactive CD4<sup>+</sup> T lymphocytes. This interaction triggers a signaling cascade leading to the activation of autoreactive CD8<sup>+</sup> T cells, which in turn infiltrate pancreatic islets. Once inside, CD8<sup>+</sup> T cells begin lysing beta cells, thereby eliminating the islets' ability to produce insulin [1,47,48]. Despite this, residual levels of beta cell mass have been detected long after diagnosis, however, the cause of this remains unknown [49].

Prevalence of T1D is dependent on several environmental factors including geographical location [50]. When examining the various components necessary for T1D development, the conjunction between epigenetic forces and genetic predisposition must be properly assessed. Epidemiological studies designed to assess regional T1D pervasiveness have observed elevated prevalence of the autoimmune disease in northern populations. Countries including the U.S., Canada, Finland, and Sweden suffer the highest rates of T1D; this phenomenon is believed to be the result of vitamin D deficiency [51–53]. Vitamin D serves an important role in the regulation of immune activity. Metabolites of vitamin D, such as 1,25-dihydroxyvitamin D3 suppress T cell proliferation and alter cytokine expression resulting in a more calculated immune response. Furthermore, vitamin D is known to heighten tolerance towards self-antigens and the development of autoantibodies [54,55]. Such factors should be taken into consideration when developing therapeutics or treatments for T1D. T1D continues to affect a growing population of affected individuals around the world and as a result, more therapies are needed to accommodate this growing complication.

The current clinical scenario for treating T1D is centered around intensive diet treatments and exogenous insulin administration. Clinicians currently utilize a conjunction of insulin pumps and continuous glucose monitors (CGM) to enhance the precision of glycemic control. Despite this, hypoglycemia remains one of the most consequential acute complications associated with insulin replacement therapy. Severe hypoglycemia, often occurring during nighttime hours, can result in seizures, coma, and death. In fact, nearly 6% of T1D related deaths are the result of nocturnal hypoglycemia [56]. Innovations in CGMs and insulin pumps have made significant progress in reducing nocturnal hypoglycemia. The implementation of threshold systems designed to suspend insulin delivery for up to 2 hours until CGM glucose is at a low threshold have displayed promising results in reducing nocturnal hypoglycemia [57]. Although significant progress has been made in this regard, insulin replacement therapy is only so effective at preventing prolonged periods of hyperglycemia and continues to suffer from the risks associated with hypoglycemia. Another roadblock to insulin replacement therapy stems from the complicated processes by which insulin is manufactured and distributed. More recently, insulin analogues such as Lispro, Aspart, and Delgludec insulins have been developed. Such analogues bear higher efficacy, exhibiting less latency and providing a longer duration of action [58,59]. However, in the U.S., the price of insulin and insulin analogues has increased exponentially over the past several decades. Today, monthly out-of-pocket costs for T1D patients can range between \$75–2000 depending on insurance coverage and insulin requirements, making proper treatment inaccessible for some individuals [60]. With the life-long insulin replacement therapy required to treat T1D patients, alternative forms of treatment must be developed to address the growing cohort.

T1D is more uncommon than Type 2 diabetes, resulting in a limited understanding of the immunology of T1D when compared to its counterpart. A variety of cell types are involved in the onset of T1D and involve an intricate cascade of interactions between immune cells and the islet beta cells. Immune cells responsible for this autoimmunity participate in both innate and adaptive immune responses. The initial trigger responsible for directing the autoimmune attack is still unclear, however, most researchers attribute it to an individual's genetic predisposition. Understanding the role genetically directed antigens play is necessary for identifying checkpoints which can be targeted to generate a cure or reversal of T1D phenotype. In T1D, APCs and varying lymphocytes interact to generate immune responses when presenting pathogens [61,62]. These lymphocytes include the more commonly explored T cells, as well as the more unexplored class of B cells.
