Studies have revealed that metformin’s antidiabetic role, by suppressing hepatic glucose production (45). Metformin is widely prescribed for type 2 diabetes as it effectively controls blood sugar levels and provides cardioprotective protection (45). Moreover studies suggest that metformin can help manage metabolism and potentially treat conditions like RA by regulating metabolism (46). Enhancing the effects of metformin using NPs for drug delivery shows promise by optimizing controlled release improving drug targeting and reducing effects (47).
These carriers enhance drug penetration into tissues, specificity and drug availability making them ideal for targeted delivery (48). Nanocarriers also allow drug release, boosting treatment effectiveness (49). iosomes, a type of ionic surfactant vesicles, have garnered attention in the pharmaceutical industry due to their ability to encapsulate both hydrophilic and hydrophobic agents (50). These synthetic vesicles, formed by self-assembly of nonionic surfactants, cholesterol, and some other lipids, offer benefits like breakdown in the body and compatibility with living tissue (51). Researchers have utilized these nanoparticles in various applications, including transdermal drug delivery (52), gene delivery (53), and targeted drug delivery to immune-privileged tissues (54). Scientists have investigated methods to enhance nano carriers, for drug delivery purposes. For example, the development of folic acid-modified nano-drug carriers has shown promise in the targeted delivery of specific drugs (55). Various characteristics affects the properties of the NPs. The size of nanoparticles plays a crucial role in their uptake and retention by cells and tissues (56). For intravenously administered nanoparticles, diameter is a key factor affecting pharmacokinetics and bio-distribution pores in blood vessels (57). The size of nanoparticles influences their ability to overcome transport barriers in biological tissues, affecting their tissue penetration efficacy (58). Additionally, nanoparticle size impacts their biological activity, with factors like concentration and size playing primary roles (59). The DLS analysis showed that the blank niosome NPs had an average diameter of 151 ± 6.2 nm (Table 3). However, encapsulation of metformin inside these NPs causes an increase in their average diameter to 168 ± 10.2 nm. The highest diameter among those fabricated belongs to Hyalo-Nio-met NPs (Fig. 2) with 179 ± 8.5 nm, due to coating with hyaluronic acid and encapsulation of metformin inside these NPs.
The zeta potential of nanoparticles is a critical parameter for characterizing their surface charge properties. It is determined by the presence of charged ions at the NPs surface and in the surrounding solution (60). Zeta potential plays a key role in expressing the stability of nanoparticles in suspension by influencing the electrostatic repulsion between particles, thus preventing aggregation (61). Zeta potential values between − 30 mV and + 30 mV are generally considered ideal for achieving better physical stability of nanoparticles (62). Table 3 lists the obtained zeta potential values for blank niosomes, Nio-met, and Hyalo-Nio-met nanoparticles.
PDI is recognized as another key factor in assessing the stability and functionality of NPs for drug delivery applications (63). PDI significantly influences various aspects of nanoparticles, including their stability, drug release kinetics, cellular uptake, and biodistribution (64). Maintaining a low PDI (< 0.4) is crucial to achieve a narrow size distribution, which is essential for effective tissue accumulation and renal clearance (65). The reproducibility and quality of nanoparticles are greatly affected by PDI, as it indicates the width of the particle size distribution and the uniformity of the nanoparticles (66). Additionally, measuring PDI is vital for evaluating the colloidal properties of nanoparticles, especially in understanding their ability to penetrate biological barriers (67). Particularly in pharmaceutical applications, the size uniformity of nanoparticles is crucial to ensure consistent performance and efficacy (68). According to the DLS results listed in Table 3, the blank niosome, Nio-met, and Hyalo-Nio-met NPs are all within the acceptable range in terms of size, zeta potential, and PDI values.
Table 3
The evaluated size, zeta potential, and PDI values of blank niosome, Nio-met, and Hyalo-Nio-met NPs using DLS.
Groups | Size (nm) | Polydispersity Index | Zeta potential (mV) |
Blank niosome | 151 ± 6.2 | 0.426 | −13.47 ± 3.8 |
Nio-met | 168 ± 10.2 | 0.453 | −15.21 ± 2.9 |
Hyalo-Nio-met | 179 ± 8.5 | 0.663 | −9.76 ± 3.4 |
The interaction of nanoparticles with biological systems is controlled by various factors, including size, shape, and surface properties (69). The shape of nanoparticles has been shown to have significant effects on their interactions with biological materials such as cells and tissues (70). The morphology of NPs can impact their biological activities, such as cell membrane penetration, wetting, and interactions with proteins (71, 72). Furthermore, the morphology of biologically synthesized nanoparticles can influence their toxicity, mechanism of action, and applications as antibacterial and antifungal agents (73, 74). Figure 3 demonstrates the SEM images of fabricated blank niosome, Nio-met, and Hyalo-Nio-met NPs. All these nanoparticles show a spherical morphology, with the only difference between them being their size. The AFM images of nanoparticles are shown in Fig. 3, and these images are consistent with previous DLS results.
FTIR is an effective analytical method used for detecting functional chemical groups and characterizing covalent bonding information. The characteristic peaks in the Span 60 spectrum appeared at 3389, 2916, and 1736 cm− 1, corresponding to OH stretching, carbonyl dimer, and C = O stretching, respectively (Fig. 4). For cholesterol, a notable peak at 3435 cm− 1 indicated OH stretching. In the blank niosomes' spectrum, the OH stretching peak from Span 60 was seen at 3388 cm− 1, while the carbonyl dimer shifted to 2918 cm− 1 and the C = O stretching peak shifted to 1737 cm− 1. These shifts in the carbonyl groups' peaks suggest hydrogen bonding between Span and cholesterol, indicative of niosome formation. In the spectrum of drug-loaded niosomes, significant peaks were observed at 3370, 2920, and 1738 cm− 1, likely representing OH stretching, carbonyl dimer, and C = O stretching. These shifts are similar to those seen in blank niosomes and point to interactions that facilitate niosomal formation (75). The FTIR spectrum of cholesterol exhibited a broad band at 3432 cm− 1, indicating OH stretching vibration. Symmetric and asymmetric stretching vibrations in CH2 groups of alkyl chains were observed at 2989 cm− 1 and 2882 cm− 1, respectively. A strong band at 1716 cm− 1 was attributed to the double bond in the second ring of the cholesterol structure (76).
Drug release patterns play a key role in the effectiveness of drug therapy. V Different controlled release systems have been created to improve the therapeutic performance of drugs (77). Factors, like particle size, surface properties and the porous structure of nanoparticles all affect how drugs are released from nanoparticles (78–80). It's highly desirable for drugs to be released steadily from nanoparticles for medical purposes (81). Niosomes, which are composed of biodegradable and non-immunogenic components, can carry both amphiphilic and lipophilic drugs, making them appealing for drug delivery (82, 83). Niosomes are praised for their ability to offer an controlled release of drugs because of their characteristics (31). Figure 5 shows the 120 hours' release pattern of metformin from the Nio-met, and Hyalo-Nio-met NPs at 37°C and pH 7.4. Both Nio-met, and Hyalo-Nio-met NPs shows biphasic release pattern. The maximum release rates reached 42% and 47% within the first 12 hours of the experiment, followed by a subsequent decrease. This initial high release is attributed to the drug being weakly bound to the surface of the niosomal nanoparticles rather than being encapsulated inside them.
MTT is a widely used method for assessing cytotoxicity, viability, and proliferation studies in cell biology (84). This is based on the ability of mitochondrial enzymes in viable cells to reduce the MTT yellow tetrazolium salt to purple formazan crystals (85). The effect of metformin, Nio-met, and Hyalo-Nio-met NPs on PBMCs are illustrated in Fig. 6. The Hyalo-Nio-met composed of Span 60, cholesterol, metformin, and hyaluronic acid. Hyaluronic acid and cholesterol are both natural components find in human body and studies confirmed their safety to normal cells (86). As illustrated in the Fig. 6, metformin, Nio-met, and Hyalo-Nio-met NPs exhibit a negligible and insignificant proliferation effect on PBMCs at concentrations of 5 mM. As previously described, niosomal NPs can enhance treatment effectiveness by increasing the solubility and bioavailability of drugs. Additionally, the decoration of hyaluronic acid on their surface facilitates the localization of drug into the PBMCs, thereby enhancing the likelihood of cellular uptake and therapeutic impact. The highly significant result (p < 0.1 *) is observed in the Hyalo-Nio-met treated group at a 15 mM concentration. Based on these findings, 15 mM of metformin, Nio-met NPs, and Hyalo-Nio-met NPs have been selected for use in the subsequent experiments of this study.
Reactive Oxygen Species (ROS) are highly reactive molecules that can be generated in cells through both enzymatic and non-enzymatic mechanisms (87). These molecules have the ability to interact with active substances, organic compounds, and environmental pollutants, outside the cell (88). Elevated levels of ROS can cause stress, a factor associated with aging and the onset of human illnesses (89). Oxidative stress happens when there is an imbalance, between ROS and antioxidants, leading to tissue damage and the chronicity of diseases (90). Furthermore, ROS has been linked to the progression and severity of RA, affecting joint tissue injury (91). Figure 7 illustrates the ROS level of untreated and treated PBMCs. The treatment of PBMCs with metformin in free form could successfully reduce the ROS level in these cells. The Nio-met treated group showed increased reduction compared to the free form of metformin and eventually, the highest reduction belonged to Hyalo-Nio-met treated group. These results can be interpreted by the role of niosomes in increasing drug solubility and bioavailability, as well as the role of hyaluronic acid in placing these nanoparticles in the vicinity of cells.
IL 23 is a cytokine plays an essential role in different inflammatory and autoimmune conditions. Research indicates that IL 23 can activate shared receptors structurally to induce inflammatory responses (92). It is also recognized as a factor in the development of RA (93). In RA there is an increased presence of M1 macrophages that produce levels of IL 23 highlighting its involvement in inflammation (94). Studies have shown elevated levels of IL 23 in the blood of RA patients closely linked to disease activity (93). TGF beta serves as a regulator in cellular functions like proliferation, differentiation, migration, cell survival, angiogenesis and immunesurveillance (95). The TGF beta signaling pathway plays a key role in cancer development and progression by influencing interactions, within the tumor microenvironment (96). TGF-β contributes to increased production of extracellular matrix components and mesenchymal cell activities post inflammatory responses (97). It regulates fibroblast function, influences inflammatory responses, and modulates tissue repair processes (98, 99). The IL-23 and TGF-β levels in PBMCs are illustrated in Fig. 8. These results show a decrease in IL-23 level and an increase in TGF-β level in treated cells.
Catalase is a tetrameric enzyme that plays a crucial role in protecting aerobic cells from oxidative stress by catalyzing the decomposition of hydrogen peroxide into water and oxygen (100). Various research studies have explored the significance of catalase in RA and its potential as a treatment target. They discovered catalase activity in the blood plasma of RA patients compared to those who're healthy hinting at a possible imbalance in antioxidant defenses (101). likewise there have been observations of reduced catalase activity among individuals with RA indicating a connection, between stress and the development of RA (102). Table 4 listed the catalase level in PBMCs isolated from healthy individuals, RA patients, and RA patients with T2DM. Generally, the treated PBMCs demonstrated higher level of catalase compared to untreated group. The highest increase in catalase level belongs to the Hyalo-Nio-met NPs treated group.
Table 4
Catalase level in PBMCs isolated from healthy individuals, RA patients, and RA patients with T2DM
Catalase level (U/mg protein) | Untreated | Metformin | Nio-met | Hyalo-Nio-met |
Healthy | 75 ± 9.3 | 81 ± 7.3 | 99 ± 11.2 | 87 ± 9.5 |
Rheumatoid Arthritis | 43 ± 8.1 | 64 ± 5.8 | 93 ± 7.4 | 109 ± 6.2 |
Rheumatoid Arthritis + T2DM | 28 ± 4.9 | 62 ± 6.2 | 85 ± 3.9 | 117 ± 10.4 |
The expression of various genes plays a role in RA, so the expression of some of these genes was investigated by real-time PCR. The NFATc1 gene, encoding the nuclear factor of activated T-cells c1, plays a crucial role in various biological processes, particularly in bone homeostasis and cancer. It is essential for osteoclast differentiation (103–105). NFATc1 also interacts with other transcription factors like AP-1 and Mitf to stimulate gene expression in osteoclast precursors (106). Studies have shown that NFATc1 expression is increased in synovial osteoclast precursors of RA patients, indicating a potential link to the enhanced osteoclast differentiation observed in RA (107). RANKL, which stands for Receptor activator of NF-κB ligand, is a crucial molecule involved in various physiological processes. It is known to play a significant role in bone homeostasis and the formation of lymphoid tissues (14). It is encoded by a gene located on chromosome 13q14 and is primarily expressed by osteocytes, activated T-cells, and bone marrow stromal cells (108). This molecule exists in two forms: membrane-bound RANKL (mRANKL) and soluble RANKL (sRANKL) (109). It also plays a crucial role in bone erosion in RA by promoting osteoclast formation, function, and survival (110). Studies have shown that RANKL is highly expressed in synovial fluid B cells of RA patients and is a key cytokine involved in bone destruction (111). Anti-RANKL antibody treatment has been proposed as a strategy to protect against joint destruction in RA (112). COX-2 is an inducible enzyme that is involved in pathophysiological processes such as pain, inflammation, and fever (113). COX-2, expressed in synovial cells of RA patients, plays a crucial role in the inflammatory process within the joints (114). Selective COX-2 inhibitors have emerged as important options in RA treatment due to their efficacy and reduced gastrointestinal toxicity compared to traditional non-selective nonsteroidal anti-inflammatory drugs (115, 116). Changes in the expression of NFATc1, RANKL, and COX-2 genes in untreated and treated PBMCs with metformin, Nio-met NPs, and Hyalo-Nio-met NPs are shown in Fig. 9 for both RA patients and RA patients with T2DM. In both groups there is no statistically significant change in NFATc1 gene expression following treatment with metformin and Nio-met NPs. In RA patients, there is a significant decrease in RANKL gene expression following treatment with Hyalo-Nio-met NPs same results are repeated in RA patients with T2DM. In RA patients, there is a statistically significant decrease in COX-2 gene expression in treatment with metformin and Nio-met NPs. Treatment with Hyalo-Nio-met NPs resulted in a significant decrease in COX-2 gene expression in this group. In RA patients with T2DM, there is a statistically significant decrease in COX-2 gene expression between treatment with Nio-met NPs and Hyalo-Nio-met NPs. The results revealed that when metformin was exposed to PBMCs the NFATc1, RANKL, and COX-2 expressions were reducing.