Forms and Methods for Interferon's Encapsulation

Abstract

Interferons (IFNs) are cytokines involved in the immune response that act on innate and adaptive immunity. These proteins are natural cell-signaling glycoproteins expressed in response to viral infections, tumors, and biological inducers and constitute the first line of defense of vertebrates against infectious agents. They have been marketed for more than 30 years with considerable impact on the global therapeutic protein market thanks to their diversity in terms of biological activities. They have been used as single agents or with combination treatment regimens, demonstrating promising clinical results, resulting in 22 different formulations approved by regulatory agencies. The 163 clinical trials with currently active IFNs reinforce their importance as therapeutics for human health. However, their application has presented difficulties due to the molecules' size, sensitivity to degradation, and rapid elimination from the bloodstream. For some years now, work has been underway to obtain new drug delivery systems to provide adequate therapeutic concentrations for these cytokines, decrease their toxicity and prolong their half-life in the circulation. Although different research groups have presented various formulations that encapsulate IFNs, to date, there is no formulation approved for use in humans. The current review exhibits an updated summary of all encapsulation forms presented in the scientific literature for IFN-α, IFN-ß, and IFN-γ, from the year 1996 to the year 2021, considering parameters such as: encapsulating matrix, route of administration, target, advantages, and disadvantages of each formulation.

Keywords: IFN-α; IFN-β; IFN-γ; PEGylation; antiproliferative; antiviral; drug delivery system; encapsulate IFNs; formulation; immunomodulator; interferons; liposomes; microparticles; nanoparticles; polymeric micelles.

Figure 1

Type I Interferons Induction and Functions. Type I IFNs are first induced intrinsically in infected cells through a process of host cell recognition of segments of DNA or RNA or other viral macromolecules called pathogen-associated molecular patterns (PAMPs). PAMPs recognition as non-self-starts with their binding to specific cellular pathogen recognition receptors (PRRs), such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors. Receptor-ligand binding triggers the type I IFN induction cascade via NFκß, resulting in the activation and translocation to the nucleus of IFN regulatory factors IRF3 and IRF7, which induce the expression of type I IFNs. The expressed cytokine is exported to the extracellular milieu and binds to IFNAR, a heterodimeric receptor consisting of two subunits, IFNAR1 and IFNAR2. The molecule forms a trimeric structure with the receptor, thus activating the proteins Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2). These proteins activate the signal transducers and activators of transcription 1 and 2 (STAT1 and STAT2), which induce the transcription of interferon-stimulated genes (ISGs) by forming a complex with IRF9. These ISGs encode antiviral effectors (PKR, Mx1, OAS, etc.) and activate type I IFN production, thus triggering autocrine and paracrine signaling. Cells from the innate immune system, such as dendritic cells (DCs) and macrophages, produce type I IFN after sensing pathogen components using various PRRs found on the plasma membrane, in endosomes, and throughout the cytosol. Created with BioRender.com.

Figure 2

Roles of Type II Interferons. IFN type II is a pleiotropic cytokine that participates in viral response and regulating innate and adaptive immune responses. NK cells, T cells, B cells, APC release this cytokine to function both as an inducer (pro-inflammatory) and a regulator (anti-inflammatory) of immune responses. Regarding the pro-inflammatory effects, IFN-γ has a strong macrophage-activating activity and induces B cell maturation and IgG2 production. Additionally, this cytokine stimulates antigen presentation via MHC, development of Th1 effector cells, and cell function of T_reg cells. The anti-inflammatory effects of IFN-γ include the inhibition of T cell-dependent osteoclastogenesis and production of IL-17, which leads to decreased levels of neutrophil-specific CXC chemokines and limited mobilization of neutrophils. Furthermore, type II IFN inhibits myelopoiesis of CD11b⁺ leukocytes, T cell proliferation and induces apoptosis by secreting nitric oxide (NO) and indoleamine 2,3-dioxygenase (IDO). All these anti-inflammatory properties contribute to the protective role of IFN-γ against autoimmune diseases. Created with BioRender.com.

Figure 3

Type III Interferons Production and Activity. Type III IFNs act on epithelial cells and tissue-resident neutrophils, dendritic cells, macrophages, B cells, and plasmacytoid dendritic cells. Like the type I IFN pathway, IFN-λ induction starts with PRR (TLR and RLR) recognition of the respective PAMPs. Receptor-ligand binding triggers the IFN-λ induction cascade via NFκß, resulting in the activation and translocation to the nucleus of IFN regulatory factors IRF3 and IRF7, which induce the expression of type III IFN. After its release to the extracellular milieu, IFN-λ binds to its heterodimeric receptor (IFNLR), which consists of two subunits: α-subunit (IL28RA) and β-subunit (IL10RB). IFN-λ-IFNLR trimeric complex formation leads to the activation of JAK1 and TYK2, followed by the phosphorylation of STAT1 and STAT-2. Afterward, STAT1 and STAT-2 translocate to the nucleus and induce the expression of hundreds of ISGs with antiviral activity. Type I and III IFNs both show a complex mechanism of feedback loops, leading to autocrine and paracrine signaling. Even though epithelial cells are the primary source of type III IFN, macrophages, monocytes, and dendritic cells can also secrete them. Created with BioRender.com.

Figure 4

Encapsulation methods and IFN-delivery system. Summary of the different transport systems for type I and II IFNs, including PEGylation, liposomes, micellar systems, self-assembled nanostructures, microparticles, and nanoparticles. Created with BioRender.com.

Figure 5

Activation pathways are regulated by type I interferons (IFNs) in the antitumor response. The antiproliferative activity of type I IFNs has anti-angiogenic effects on tumor vascularization, increased cytotoxicity, and survival of NK cells. These cytokines induce the generation and survival of cytotoxic T lymphocytes, memory CD8 T, and maturation of dendritic cells. Type I IFNs influence the maturation, homeostasis, and activation of NK cells, eliminating tumor cells through other immune cells or cells of the tumor microenvironment. Dendritic cells play an essential role in recognizing and presenting the various antigens that trigger the activation cascade. Another indirect effect of type I IFNs on NK cells in a tumor environment is the modulation of surface molecules on CD8 + cytotoxic T lymphocytes (CTL) [NCR1 ligands; classical and non-classical major histocompatibility complex class I (MHC I)] with evasion of CTLs from NK cell-mediated killing. Created with BioRender.com.

Figure 6

Role of IFN-γ in the antiproliferative response. Interferon-γ interacts with various cells in a tumor microenvironment to initiate the production of the cytokine itself. Some of these cells are T lymphocytes, macrophages, and dendritic cells. Macrophages: the protein stimulates the polarization of macrophages towards a proinflammatory phenotype by increasing the secretion of chemokines. Dendritic cells: increases the maturation of these cells, positive regulation of MHC I and II with increased IRF1 expression, and decreased IFN-γ-dependent dendritic cell survival. T cells: stimulates their differentiation with Th1 polarization. IFN-γ causes positive feedback, increasing their production in Th1 cells and inhibiting differentiation towards Th2 and Th17. Maturation of virgin T cells to effector CD8 + T cells requires IFN-γ. IFN-γ is the primary cytotoxic molecule secreted by these cells. IFN-γ inhibits immunosuppressive regulatory T cells. Created with BioRender.com.

Figure 7

Biological activity of IFNβ in autoimmune diseases. Its action directly increases the expression and concentration of anti-inflammatory agents and down-regulates the expression of proinflammatory cytokines. The same pathway that enables interferon beta’s biological effects mediates its mechanism of action in MS. This cytokine binds to its specific receptors IFNR I and IFNRII on the surface of the main cells of the immune system (DC, TH1 TH2, and B cells). Ligand-receptor binding triggers a cascade of events within these cells that results in positive feedback from the molecule, increasing IFNβ levels and producing the expression of multiple ISGs such as MHC Class I, Mx protein, 2′/5′-oligoadenylate synthetase (OAS), β2-microglobulin and neopterin. These products have been found in serum and cellular fractions of blood from patients treated with interferon-beta. Created with BioRender.com.