Chitosan-Coated Niosomal Nanocarriers for the Co-Delivery of Glibenclamide and Curcumin in Diabetes Mellitus

TL;DR

This study develops a niosomal system to co-deliver glibenclamide and curcumin, improving their effectiveness for treating type 2 diabetes.

Contribution

A novel chitosan-coated niosomal nanocarrier is introduced for co-delivery of glibenclamide and curcumin.

Findings

The niosomal system achieved high encapsulation efficiency for both drugs.

The formulation provided gastric protection and sustained intestinal drug release.

Chitosan coating improved stability and reduced drug leakage.

Abstract

Glibenclamide (Gli), widely used in the management of type 2 diabetes mellitus (T2DM), shows low oral bioavailability, while curcumin (Cur) is limited by poor aqueous solubility and instability. This study reports the development of a niosomal co-delivery system combining hypoglycemic and antioxidant agents to improve formulation performance for T2DM. Gli and Cur were co-encapsulated into niosomal vesicles (NIOs) using the thin-film hydration method, followed by surface coating with chitosan (CS). The formulations were characterized by dynamic light scattering, scanning transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy, complemented by in vitro release studies under simulated gastrointestinal conditions. The prepared NIOs exhibited particle sizes between 413.5 and 576.9 nm, with encapsulation efficiency strongly dependent on formulation composition. The optimized system showed high encapsulation efficiency for both Gli (98.95 ± 0.87%) and Cur (91.09 ± 2.00%). In vitro release studies demonstrated enhanced release compared with the physical mixture, providing gastric protection and sustained intestinal delivery. Release kinetics indicated controlled drug release governed by diffusion- and erosion-based mechanisms. Both uncoated and CS-coated NIOs displayed good physical and osmotic stability, with CS coating further reducing drug leakage. These results highlight the potential of niosomal systems for efficient Gli and Cur administration in T2DM.

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1. Introduction

Diabetes mellitus (DM) is a chronic, multifactorial metabolic disorder characterized by disturbances in carbohydrate, lipid and protein metabolism, affecting multiple organ systems. The World Health Organization (WHO) recognizes diabetes as a major global health challenge due to its high morbidity and mortality rates. The global prevalence of diabetes has increased dramatically, from approximately 200 million in 1990 to 828 million in 2022, and projections indicate that it may exceed 640 million by 2040 [1].

Type 1 diabetes mellitus (T1DM), accounting for 5–10% of all cases, is an autoimmune disorder characterized by the selective destruction of pancreatic β-cells and absolute insulin deficiency. In contrast, type 2 diabetes mellitus (T2DM), which represents 90–95% of all diabetes cases, is primarily associated with insulin resistance in peripheral tissues combined with a progressive decline in β-cell function. The pathogenesis of T2DM involves a complex interplay between genetic predisposition, environmental factors, and metabolic dysfunction, ultimately leading to impaired glucose homeostasis and chronic hyperglycemia [2,3].

Chronic hyperglycemia represents the central pathological feature of DM and plays a pivotal role in disease progression. It arises from reduced insulin sensitivity, impaired insulin signaling, increased hepatic gluconeogenesis, and decreased peripheral glucose uptake. Sustained hyperglycemia promotes excessive production of free fatty acids (FFAs) and induces oxidative stress (OS) through mitochondrial dysfunction and incomplete fatty acid oxidation. The resulting overproduction of reactive oxygen species (ROS) triggers DNA damage and activates pro-inflammatory signaling pathways, leading to the release of cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). These processes further exacerbate insulin resistance, impair antioxidant defense systems, and accelerate pancreatic β-cell dysfunction, establishing a self-perpetuating cycle that drives DM progression [4,5,6,7].

The key pathophysiological mechanisms underlying DM, particularly OS, chronic inflammation, insulin resistance, and β-cell dysfunction, are schematically summarized in Figure 1.

The multifactorial nature of DM and its numerous associated complications require the development of targeted therapeutic strategies that extend beyond conventional pharmacological interventions. Such approaches should address not only achieving optimal glycemic control but also managing OS, chronic inflammation, and pancreatic β-cell dysfunction [8]. The integration of antioxidant supplementation with advanced nanotechnology-based drug delivery systems has the potential to enhance therapeutic outcomes. By enabling site-specific delivery and sustained release of therapeutic agents, these systems may enhance treatment efficacy and contribute to more personalized and sustained long-term metabolic control [9].

In this context, nanotechnology provides innovative platforms for enhancing the bioavailability of antidiabetic drugs and optimizing glycemic control through the development of versatile nanocarriers capable of targeted delivery and controlled drug release [10]. A particularly promising strategy in advanced drug delivery is the design of nanoformulations capable of co-encapsulating two or more drugs, which is essential for the management of complex multifactorial diseases. Unlike the administration of separately encapsulated drugs or their simple co-administration, co-encapsulation enables the simultaneous targeting of the same cells or tissue compartment, resulting in synchronized pharmacodynamic effects and reduced variability in therapeutic response associated with differences in absorption and distribution of individual drugs [11]. The co-delivery of complementary therapeutic agents can enhance overall treatment efficacy through additive or synergistic interactions. Moreover, the presence of multiple drugs within the same nanocarrier can modulate drug–membrane interactions, affecting carrier organization, interfacial properties, and hydration behavior in different biological environments, thereby contributing to a coordinated and controlled release profile [12]. In addition, co-encapsulated nanocarriers may exhibit improved cellular uptake due to enhanced interactions with biological membranes, facilitating simultaneous intracellular drug release and optimized intracellular drug concentrations [13,14]. Collectively, these features position co-encapsulated nanocarriers as a promising strategy for personalized and precision therapy, particularly in multifactorial diseases such as DM.

Several types of nanocarriers have been extensively investigated for their potential, including: solid lipid nanoparticles (SLNs) [15], lipid–drug conjugates (LDCs) [16,17], liposomes [18,19], niosomes (NIOs) [20,21,22], polymeric nanoparticles (PNPs) [23,24], dendrimer-derived nanostructures [25], silver-based nanoparticles (AgNPs) [26], and also gold-based nanoparticles (AuNPs) [27]. NIOs exhibit remarkable formulation versatility, extending beyond conventional delivery systems to advanced platforms such as niosome-loaded hydrogels. These systems have been successfully developed for topical application to diabetic wounds, promoting wound healing while also enabling systemic antidiabetic effects [28,29].

Niosomes (NIOs) exhibit remarkable formulation versatility, extending beyond conventional drug delivery systems to advanced platforms such as niosome-loaded hydrogels. These systems have been successfully developed for topical application in diabetic wounds, promoting local wound healing while also enabling systemic antidiabetic effects [28,29]

NIOs, also known as non-ionic surfactant vesicles, are promising vesicular drug delivery systems (VDDSs) formed through the self-assembly of non-ionic surfactants (NISs) in aqueous media. These systems are composed of amphiphilic surfactants, which contain both a hydrophobic tail and a hydrophilic head, resulting in a lamellar bilayered structure capable of encapsulating both lipophilic and hydrophilic compounds [30]. Hydrophilic molecules are typically located in the internal aqueous core or associated with the bilayer surfaces, while lipophilic compounds are incorporated into the hydrophobic regions of the bilayer. Cholesterol (Chol) is frequently included in NIOs to enhance membrane stability by modulating bilayer rigidity and reducing permeability through interactions with NISs [31,32]. NIOs function as nano-sized carriers for a wide range of therapeutic agents, enhancing bioavailability and prolonging therapeutic efficacy [33,34]. Furthermore, their surfaces can be functionalized through polymer grafting or ligand attachment to optimize pharmacokinetic and pharmacodynamic behavior. These modifications can lead to improved surface charge, enhanced colloidal stability, prolonged systemic circulation, and targeted drug delivery, ultimately increasing therapeutic efficiency while minimizing systemic side effects [35].

Chitosan (CS), a natural and biocompatible polymer obtained by the deacetylation of chitin, is widely used to improve the properties of NIOs due to its strong mucoadhesive capacity and its ability to impart a positive surface charge [36]. Its mucoadhesive properties arise from ionic interactions between the protonated amino groups and the negatively charged functional groups on the epithelial cell surface, leading to prolonged residence time and controlled drug release, enhanced intestinal permeability, and ultimately improved drug absorption in the gastrointestinal tract (GIT) [37,38]. Moreover, CS has been shown to enhance the aqueous solubility of NPs, thereby reinforcing its role as an effective absorption enhancer. However, its applicability is limited by its pH-dependent solubility: CS is soluble and biologically active only under acidic conditions, where protonation of amino groups confers a positive charge, whereas in neutral or alkaline environments, deprotonation results in polymer precipitation and reduced solubility [35].

Glibenclamide (Gli), a second-generation sulfonylurea, has been widely used in the management of T2DM. It induces hypoglycemia through two main mechanisms: (i) stimulation of insulin secretion from pancreatic β-cells via inhibition of ATP-sensitive potassium channels, leading to membrane depolarization, calcium influx, and subsequent insulin release, and (ii) enhancement of peripheral insulin sensitivity by increasing insulin receptor binding on target cells, thereby improving cellular responsiveness to insulin [39,40]. Belonging to the Biopharmaceutics Classification System (BCS) Class II [41], Gli exhibits poor aqueous solubility and high membrane permeability, resulting in low and variable oral bioavailability [42,43]. Its solubility and ionization are pH-dependent, owing to its weakly acidic nature (pKa~5.3–5.5), which confines its absorption primarily to the proximal GIT [44,45]. Consequently, drug absorption declines significantly in the distal GIT, further reducing systemic availability. Despite these pharmacokinetic limitations, Gli remains a potent hypoglycemic agent that is effective at low doses. However, its narrow therapeutic index increases the risk of dose-dependent, severe, and prolonged hypoglycemia, particularly if dosing is not properly individualized [46]. These challenges underscore the need for pH-independent or targeted drug delivery systems to enhance solubility, improve bioavailability, and ensure safer and more consistent glycemic control.

Curcumin (Cur) is a natural polyphenolic compound derived from the rhizomes of Curcuma longa L. and other Curcuma spp. It is the major constituent of the curcuminoid family, which also includes demethoxycurcumin and bisdemethoxycurcumin, two structural analogues that differ from Cur in the number and position of methoxy groups on the aromatic rings (Figure 2) [47]. Collectively, these compounds are known as curcuminoids, and their structural diversity contributes to distinct yet complementary biological effects. Extensive in vitro and in vivo studies have demonstrated a wide range of pharmacological activities of Cur, including antioxidant, anti-inflammatory, antimicrobial, cardioprotective, nephroprotective, antineoplastic, hepatoprotective, immunomodulatory, hypoglycemic, and antirheumatic properties [48].

In the context of T2DM, Cur has demonstrated beneficial effects in multiple experimental models by modulating pathological processes through various mechanisms and molecular targets. One of its primary actions involves regulating lipid metabolism, achieved by downregulating transcription factors involved in hepatic lipogenesis, such as sterol regulatory element-binding protein 1c (SREBP1c) and carbohydrate response element-binding protein (ChREBP), which are known to promote hepatic cholesterol and triglyceride synthesis [49]. Cur also improves insulin sensitivity by enhancing insulin signaling pathways and reducing systemic insulin resistance, effects that are closely linked to the normalization of blood glucose levels and correction of lipid imbalances characteristic of diabetic dyslipidemia [50]. Moreover, its potent antioxidant and anti-inflammatory properties, mediated through the restoration of enzymatic antioxidant defenses and inhibition of pro-inflammatory cytokines such as IL-6, TNF-α, and monocyte chemoattractant protein-1 (MCP-1), contribute significantly to the reduction of OS and chronic inflammation associated with T2DM progression [51,52].

The clinical application of Cur is significantly limited by its poor oral bioavailability, primarily due to its low water solubility, poor gastrointestinal absorption, and rapid systemic elimination through metabolic reduction and conjugation. To address these challenges, recent studies have focused on the development of advanced drug delivery systems capable of enhancing Cur’s pharmacokinetic profile. These include micelles, NPs, liposomes, NIOs and phospholipid complexes such as phytosomes, all of which have shown potential in improving the oral bioavailability and systemic retention of curcuminoids [52].

The aim of this study was to develop and optimize a niosomal co-delivery system for simultaneous encapsulation of Gli and Cur, targeting improved formulation performance for antidiabetic therapy. Co-loaded niosomal systems were prepared using the thin-film hydration method (TFH) and optimized in terms of encapsulation efficiency (EE%). The optimized formulation was further modified by CS coating to enhance resistance to osmotic stress and improve storage stability. The uncoated and CS-coated NIOs were subjected to comprehensive physicochemical characterization using dynamic light scattering (DLS), scanning transmission electron microscopy (STEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC), together with evaluation of osmotic stress resistance, storage stability, and in vitro drug release under simulated gastrointestinal conditions.

2. Materials and Methods

2.1. Materials

Sorbitan monostearate (Span 60), Tween 60, cholesterol (Chol) from sheep wool (≥92.5%), dihexadecyl phosphate (DCP), glibenclamide (Gli) (≥99%), curcumin (Cur) (≥94% curcuminoid content; ≥80% Cur), chitosan (CS) (75–85% deacetylated, low molecular weight), sodium taurocholate, L-α-lecithin (≥97% choline basis) and pepsin (from porcine gastric mucosa, ≥400 units/mg protein) were purchased from Sigma Aldrich (St. Louis, MO, USA). Sephadex G-25 M column (Cytiva, Global Life Science Solutions Operations, Buckinghamshire, UK) and Spectra/Por 4 dialysis membrane with MWCO of 12–14 kD (St. Louis, MO, USA) were also used. All solvents, chloroform (CHCl_3_, 99.8%), methanol (MeOH, 99.9%), ethanol (EtOH, ≥99.9%), acetonitrile (MeCN, ≥99.5%), and acetic acid (98–100%) were from HPLC quality.

2.2. Preparation of Co-Drug-Loaded NIOs (APIs@NIOs)

The thin-film hydration method [53,54,55] was used for the preparation of NIOs loaded with Gli and Cur (Gli-Cur@NIOs). The preparation is schematically illustrated in Figure 3.

Briefly, the weighed amounts of NISs (Span 60 and Tween 60), Chol, and DCP, as specified in Table 1, were dissolved in a CHCl_3_:MeOH mixture (3:1, v/v) in a round-bottom flask. The pre-solubilized Active Pharmaceutical Ingredients (APIs) were incorporated into this lipid phase (Gli in MeOH/MeCN, 2:1, v/v; Cur in MeOH/EtOH, 1:1, v/v). The resulting organic solution was subjected to solvent evaporation under reduced pressure at room temperature using a Rotavapor^®^ R-114 (Büchi-Italia S.r.l., Assago, MI, Italy), forming a uniformly distributed thin lipid film on the inner wall of the flask. To remove residual solvents, the film was kept under vacuum overnight. The dry lipid film was hydrated with preheated deionized water (60 °C) to yield a dispersion of multilamellar vesicles (MLVs). The size reduction and enhancement of vesicle uniformity were accomplished through sonication of the MLV suspension, resulting in the formation of small unilamellar vesicles (SUVs). The resulting niosomal dispersion was purified by gel filtration chromatography using a Sephadex G-25 M column (5 × 1.45 cm) to remove unentrapped drugs. The Gli-Cur@NIOs were stored at 4 °C in tightly sealed containers until further analysis. Blank niosomes (NIOs), prepared under identical conditions without APIs, were used as a control in subsequent experiments.

Formulation optimization was achieved through the systematic modulation of key formulation variables, including the total lipid content, the molar ratios of Span 60:Tween 60:Chol:DCP, the lipid-to-drug ratio, and the Gli:Cur molar ratio (Table 1). Based on EE% evaluation, the optimal niosomal formulation was selected and subsequently subjected to CS surface coating to enhance structural stability and overall formulation performance.

2.3. Formulation Optimization of APIs@NIOs Based on Encapsulation Efficiency (EE%)

2.3.1. HPLC Method

EE% was determined using a HPLC method on a Shimadzu Nexera LC-40-XR system (Shimadzu, Kyoto, Japan) equipped with a serial dual-plunger pump, an autosampler (SIL 40 XR), an SPD-40V series UV-Vis, and an RF-20Axs fluorescence detector. Chromatographic separation of the APIs was performed in a C18 column (2.1 × 100 mm, Waters CORTECS 2.7 μm) using two mobile phases: A (water/formic acid—99.9/0.1, v/v) and B (MeCN). Before use, the solvents were filtered through a 0.22 μm filter and degassed by ultrasonication. The injection sample was 10 μL, the run time was 16 min in isocratic mode (0.5 mL/min), and the optimal mobile phase ratio was A (50%) to B (50%). The column temperature was kept at 30 °C during the chromatographic operation with UV-Vis detection for Gli (λ = 230 nm) and fluorescence detection for Cur (λex = 420 nm and λem = 550 nm).

The qualitative and quantitative analysis of the APIs (Gli and Cur) was carried out based on the retention times and peak areas, respectively. The LabSolutionDB software 6.106SP1 was used for the peak integration and all samples were analyzed in triplicate.

2.3.2. EE% of APIs into Niosomal Matrix

As described in the preparation section, the niosomal dispersion was purified by gel filtration chromatography using a Sephadex G-25 M column (5 × 1.45 cm) to remove unentrapped drugs. Then, 1 mL of purified APIs@NIOs was completely lysed with 1 mL MeOH:MeCN mixture (1:1, v/v), followed by sonication for 10 min in an ultrasonic bath (Elmasonic P, Singen (Hohentwiel), Germany) to ensure complete vesicle disruption. The resulting mixture was centrifuged at 15,000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm membrane filter prior to HPLC analysis. The API content (Gli and Cur) was quantified using an HPLC method [56] and the quantified drug amounts were subsequently used for the calculation of EE%, according to the following equation [57]:

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2.4. Preparation of Chitosan-Coated Drug-Loaded NIOs (APIs@NIOs-CS)

The CS solution was prepared at a concentration of 1% (w/v) by dissolving CS in a 1% (v/v) acetic acid aqueous solution, followed by filtration through a 0.45 μm Millipore membrane. The filtered CS solution was then added dropwise to the niosomal dispersion at a rate of 0.2 mL/min, maintaining a 1:1 (v/v) ratio between the two phases, under magnetic stirring (MS-300HS, Misung Scientific Co., Ltd., Republic of Korea). The mass ratio of CS to total lipids was maintained at 1:1 (w/w). The resulting mixture was stirred at room temperature for 2 h at 100 rpm and subsequently left overnight to ensure complete interaction between CS and the vesicle surface [58].

2.5. Characterization of APIs@NIOs(CS)

2.5.1. Physical Parameters

The particle size (PS), polydispersity index (PDI) and zeta potential (ZP) of the developed niosomal formulations (NIOs, Gli-Cur@NIOs and Gli-Cur@NIOs-CS) were determined by the dynamic light scattering (DLS) technique, using a Malvern Zeta Sizer (Malvern Instruments Ltd., UK). Samples were diluted appropriately with deionized water prior to measurement and analyzed at 25 °C. Results are expressed as the mean ± standard deviation (SD) of at least three measurements (n = 3).

2.5.2. Morphological Characterization

The morphological characteristics of the developed NIOs were evaluated using scanning transmission electron microscopy (STEM), enabling high-resolution visualization of vesicle architecture. Samples were deposited onto carbon-coated copper grids and allowed to dry under ambient conditions. The acquired micrographs were analyzed using ImageJ 1.54g software to determine vesicle size distribution, and the mean vesicle area ± standard deviation (SD) was calculated for each type of NIOs.

2.5.3. Fourier Transform Infrared Spectroscopy (FT-IR)

The presence of APIs within the niosomal scaffold was confirmed using Fourier-transform infrared (FTIR) spectroscopy. Spectra were recorded using an ABB-MB3000 FT-IR spectrophotometer (MIRacleTM Single Bounce ATR accessory with ZnSe crystal, ABB, Zurich, Switzerland), over the spectral range of 400–4000 cm^−1^, at a resolution of 4 cm^−1^. Each spectrum represents the average of 32 scans to improve the signal-to-noise ratio.

2.5.4. Differential Scanning Calorimetry (DSC)

DSC measurements were performed using a Maia F3 200 DSC device (Netzsch–Gerätebau GmbH, Selb, Germany). Approximately 5 mg of each sample was placed in aluminum crucibles with pierced and sealed lids and heated at a rate of 10 °C min^−1^ and under a nitrogen flow of 50 mL min^−1^. The device was calibrated with indium in accordance with standard procedures.

2.5.5. X-Ray Diffraction (XRD)

The crystalline properties of the developed niosomal formulations (NIOs, Gli-Cur@NIOs and Gli-Cur@NIOs-CS) as well as those of the raw materials (Gli, Cur, CS) were investigated using XRD. Measurements were performed using a RigakuMiniflex 600 diffractometer (Rigaku, Tokyo, Japan), with CuKα radiation (λ = 1.5418 Å), over an angular range of 2–50° (2θ), a scanning step of 0.02° and a scanning rate of 5°/min.

2.5.6. Osmotic Stress Resistance

The resistance of uncoated (Gli-Cur@NIOs) and CS-coated (Gli-Cur@NIOs-CS) niosomal formulations to osmotic stress was evaluated by exposing the vesicles to hypotonic (0.6% NaCl) and hypertonic (1.2% NaCl) medium for 10 min at room temperature under gentle stirring. Changes in PS and PDI were assessed by DLS measurements as described in Section 2.5.1. Samples maintained under isotonic conditions (0.9% NaCl) were used as a control in order to compare the structural stability of the niosomal formulations [59]. The results are expressed as the mean ± standard deviation (SD) of at least three measurements (n = 3).

2.5.7. Stability to Storage

The long-term storage stability of both niosomal formulations (Gli-Cur@NIOs and Gli-Cur@NIOs-CS), in terms of EE%, was evaluated over a 70-day storage period at two different conditions: refrigeration (4 ± 1 °C) and room temperature (25 ± 1 °C), in the absence of light. EE% was determined at predefined time points (0, 7, 14, 21, 28, 42, 56 and 70 days) using the HPLC method described in Section 2.3.1.

2.6. In Vitro API Release Profiles and Kinetic Analysis

The release profiles of Gli and Cur from uncoated (Gli-Cur@NIOs) and CS-coated (Gli-Cur@NIOs-CS) niosomal formulations were evaluated independently in simulated gastric fluid (SGF, pH 1.6) and simulated intestinal fluid (SIF, pH 6.5) using fresh NIO samples. This experimental design was intentionally selected to assess drug release behavior under distinct gastrointestinal-like conditions, reflecting the intrinsic release properties of the formulations and the environment-specific responsiveness of the niosomal matrix (Table 2) [60,61,62]. Both media were supplemented with 0.1% (w/v) Tween 80 to maintain sink conditions [63].

Freshly prepared samples were introduced into dialysis bags (Spectra/Por 4 membrane, molecular weight cut-off 12–14 kDa) and immersed in the simulated media using a USP Type II (paddle) Dissolution Apparatus (Distek 2500, Scientific, North Brunswick, NJ, USA) operated at 100 rpm and maintained at 37 ± 0.5 °C [64]. At predefined time points (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 7.5, 11, and 22 h), aliquots of 1 mL were collected and replaced with an equivalent volume of fresh medium pre-equilibrated at 37 °C to keep a constant release volume. The released APIs (Gli and Cur) were quantified using the HPLC method described in Section 2.3.1. All experiments were performed in triplicate, and results are expressed as mean ± standard deviation (SD).

To investigate the release pattern and underlying mechanism of API release, the experimental data were fitted to several kinetic mathematical models [65,66,67], as presented in Table 3. The model that exhibited the highest coefficient of determination (R^2^) was considered the most appropriate for describing the drug release mechanism from the niosomal formulations.

2.7. Statistical Analysis

Results are expressed as mean ± standard deviation (SD). Statistical analyses were performed using Student’s t-test or analysis of variance (ANOVA), as appropriate. Differences were considered statistically significant at p < 0.05.

3. Results and Discussion

3.1. Preparation and Optimization of Co-Drug-Loaded NIOs (APIs@NIOs)

The optimization of co-drug-loaded niosomes (APIs@NIOs) was assessed using EE% as a key response parameter. The effects of total lipid concentration (Figure 4a), the molar ratio of the niosomal membrane components, including the NISs-to-Chol and Span 60-to-Tween 60 ratios (Figure 4b), as well as the lipid-to-drug (L/D) molar ratios and the Gli:Cur molar proportions (Figure 4c) on EE% were investigated to identify the optimal formulation for further surface modification.

3.1.1. Effect of Total Lipid Content

The lipid concentration in niosomal formulations was varied in the range of 25–125 mM. Two-way ANOVA revealed a significant interaction between the NIO formulation and type of APIs for EE% (F(4,20) = 4.32, p = 0.011), indicating that the effects of formulation on EE% depend on the APIs encapsulated. For Cur, the highest EE% was obtained for Gli-Cur@NIOs(2) (93.36 ± 1.78%) and Gli-Cur@NIOs(4) (93.18 ± 1.02%), both being significantly higher than those observed for Gli-Cur@NIOs(1) and Gli-Cur@NIO(3) (p < 0.001). These results indicate that formulations (2) and (4) are the most efficient for Cur encapsulation. In the case of Gli, the maximum EE% was achieved with Gli-Cur@NIOs(2) (83.95 ± 2.01%), which was significantly higher than that of Gli-Cur@NIOs(3) (p < 0.003). Gli-Cur@NIOs(4) also exhibited relatively high EE% (82.40 ± 2.41%), although it was slightly lower than Gli-Cur@NIOs(2). Based on EE% evaluation, Gli-Cur@NIOs(2) emerged as the optimal formulation, providing the highest EE% for both Cur and Gli.

3.1.2. Effect of Niosomal Membrane Components

Two-way ANOVA revealed a statistically significant interaction between the niosomal composition (Span 60:Tween 60:Chol:DCP) and the EE% of the APIs (F(2,12) = 9.041, p = 0.004) (Figure 4b), indicating that the effect of membrane composition on EE% depends on the APIs encapsulated. The most important differences were observed for formulations Gli-Cur@NIOs(7) and Gli-Cur@NIOs(10), in which Gli exhibited markedly higher EE% than Cur. When the effect of formulation was evaluated within each API, it was observed that for Gli, Gli-Cur@NIOs(11) showed the highest EE% (98.74 ± 0.85%), followed by Gli-Cur@NIOs(10) (97.66 ± 1.66%), whereas Gli-Cur@NIOs(9) exhibited significantly lower EE% (89.83 ± 0.64%, p = 0.001). In contrast, for Cur, Gli-Cur@NIOs(6) achieved the highest EE% (92.62 ± 1.69%), significantly exceeding the other formulations (p < 0.001). Notably, Gli-Cur@NIOs(10) demonstrated a clear selectivity for Gli encapsulation (97.66 ± 1.66%), while Gli-Cur@NIOs(9) showed a more balanced but overall lower encapsulation performance.

Considering EE% values for both APIs, Gli-Cur@NIOs(11) emerged as the most balanced and efficient formulation overall, providing consistently high encapsulation efficiency for both Gli and Cur. The reduction in EE% observed at higher Chol levels (Gli-Cur@NIOs(6), Gli-Cur@NIOs(7), and Gli-Cur@NIOs(8)) can be explained by the hydrophobic nature of both Chol and the encapsulated APIs, which compete for space within the bilayer. As Chol content increases, it progressively occupies the hydrophobic regions of the membrane, thereby limiting the available sites for API entrapment [68].

3.1.3. Effect of Lipid-to-Drug and Gli:Cur Molar Ratios

In the subsequent formulation series (Figure 4c), two-way ANOVA demonstrated formulation-dependent differences in EE% as well as significant differences between APIs within the same formulation.

When formulation-dependent effects were evaluated within each API, for Gli, formulations Gli-Cur@NIOs(18), Gli-Cur@NIOs(20), and Gli-Cur@NIOs(21) exhibited significantly higher EE% than formulations Gli-Cur@NIOs(23), Gli-Cur@NIOs(24) and Gli-Cur@NIOs(25) (p < 0.05), while, for Cur, the highest EE% values were observed for formulations Gli-Cur@NIOs(12), Gli-Cur@NIOs(13), and Gli-Cur@NIOs(15) (p < 0.05).

In addition, significant differences between APIs were observed within the same formulation, with Gli generally exhibiting higher EE% than Cur (p < 0.001). Among these, Gli-Cur@NIOs(21), Gli-Cur@NIOs(20), and Gli-Cur@NIOs(18) displayed the highest EE% values for Gli (99.26 ± 0.18%, 98.95 ± 0.87% and 97.85 ± 0.80%, respectively).

When both APIs were considered simultaneously, Gli-Cur@NIO(20) provided the most balanced EE% values for Gli and Cur (98.95 ± 0.87% and 91.09 ± 2.00%, respectively), which justified its selection for subsequent chitosan surface coating. Both the uncoated and CS-coated niosomal formulations (Gli-Cur@NIOs and Gli-Cur@NIOs-CS) were then further evaluated through physicochemical characterization and in vitro release studies.

The optimization of the Gli:Cur molar ratio was primarily guided by formulation-related criteria, as quantified by the main evaluation parameter, namely, EE%). However, the selection of drug ratios was also informed by pharmacological considerations. Specifically, preclinical studies indicate that Gli is typically administered at relatively low doses (approximately 2.5–5 mg/kg), whereas Cur generally requires substantially higher doses (typically 10–80 mg/kg) to achieve biological effects, a difference attributable to its broader therapeutic window and limited oral bioavailability [42,50]. The optimized formulation ratio established in this study represents a rational starting point that balances formulation constraints with pharmacological relevance, ensuring efficient co-encapsulation while providing a robust basis for subsequent in vitro and in vivo investigations aimed at evaluating potential synergistic therapeutic interactions.

3.2. Characterization of APIs@NIOs(CS)

3.2.1. Physical Parameters

The PS, PDI, and ZP values for the developed niosomal formulations are summarized in Table 4. All niosomal systems exhibited PS values ranging from 413.5 to 576.9 nm, with increased PS observed for CS-coated formulations. This increase is expected and can be attributed to the presence of the additional polymeric layer on the vesicle surface, in agreement with previously reported findings [69]. The relatively larger PS observed for blank NIOs can be attributed to the use of Span 60 and Tween 60, both characterized by long hydrophobic alkyl chains (C18), which promote the formation of rigid and stable bilayer membranes. The synergistic effect of these long-chain surfactants facilitates the self-assembly of vesicular structures with increased dimensions, consistent with data reported in the literature [70]. Overall, these results support the established relationship between surfactant alkyl chain length and the PS.

The PS of the Gli-Cur@NIOs was 479.2 ± 6.26 nm, representing an increase of approximately 66 nm (~16%) compared with blank NIOs (413.5 ± 22.80 nm), which was statistically significant (p < 0.05) and can be considered expected and within an acceptable range. This increase is primarily attributed to the simultaneous incorporation of two hydrophobic APIs (Gli and Cur) into the niosomal bilayer. These compounds preferentially partition into the hydrophobic domain of the membrane, leading to bilayer expansion. In addition, interactions between the encapsulated APIs and Chol may contribute to increased membrane rigidity and thickness, thereby promoting vesicle enlargement. For CS-coated APIs-NIOs (Gli-Cur@NIOs-CS), a more pronounced increase in PS was observed, of approximately 163 nm (~39%) relative to blank NIOs and of 97.7 nm (~20%) compared with uncoated Gli-Cur@NIOs, with highly significant differences (p < 0.001). This behavior is consistent with the formation of CS coating and the associated hydration layer on the vesicle surface. Importantly, all formulations exhibited PS values below 600 nm. Together with the observed PDI values, ranging from 0.177 ± 0.026 to 0.626 ± 0.046 mV, these findings indicate acceptable size homogeneity and the absence of significant vesicle aggregation [55].

The relatively higher PDI value observed for Gli-Cur@NIOs-CS indicates a relatively heterogeneous particle size distribution, likely reflecting the coexistence of multiple size populations. This observation, together with the increased PS, may be a consequence of the combined effects of dual loading with hydrophobic APIs and the presence of the CS coating. The simultaneous incorporation of Gli and Cur into the niosomal bilayer may lead to a heterogeneous drug distribution and local variations in membrane packing, resulting in a broader PS distribution. Furthermore, hydration and partial swelling of the CS coating can additionally contribute to the increased PDI of the vesicle system.

Analysis of ZP values indicated that the uncoated niosomal formulations exhibited negative surface charges, ranging from −36.42 ± 2.80 mV for blank NIOs to −39.67 ± 1.50 mV for Gli-Cur@NIOs, with no statistically significant difference, supporting good colloidal stability due to electrostatic repulsion between vesicles. In contrast, the CS-coated NIOs showed a positive ZP value of +50.60 ± 1.80 mV, which was significantly different from the uncoated system (p < 0.001). This shift can be explained by electrostatic interactions between the positively charged amino groups of CS and the negatively charged components present at the vesicle bilayer interface. The presence of the CS coating therefore confers electrostatic stabilization, contributing to enhance overall stability of the niosomal vesicles [38,57].

3.2.2. Morphological Characterization

STEM images of blank NIOs and APIs-NIOs(CS) confirmed the predominantly spherical and homogeneous morphology of the vesicles, characterized by smooth surfaces and well-defined boundaries (Figure 5).

In the case of APIs-NIOs, the co-encapsulation of Gli and Cur did not induce significant morphological alterations, as the overall vesicular architecture remained intact. In addition, localized regions of increased contrast were observed, which may be attributed to the locally higher content of Span 60 within the bilayer. This observation is consistent with previous reports associating similar contrast features with the dense molecular organization of Span 60 in niosomal membranes [71,72].

In contrast, Gli-Cur@NIOs-CS presented fewer regions of increased contrast than the uncoated formulations, most likely due to the presence of the polymeric layer covering the vesicle surface. Anyway, the CS coating induced a tendency of the vesicles to form compact aggregates, which can be attributed to inter-vesicular hydrogen bonding and electrostatic interactions. CS is well recognized for enhancing the physical stability of nanocarriers by increasing membrane rigidity and reducing bilayer fluidity, thereby contributing to improved vesicle robustness and structural integrity under both physiological and stress conditions [73].

Furthermore, PS distribution analysis performed using ImageJ (Figure 5d–f) supported the microscopic observations, revealing relatively narrow size distributions and mean PS in good agreement with the hydrodynamic diameters determined by DLS (Figure 5g). A slight increase in the mean PS was observed after Gli-Cur loading, with a further enlargement after CS coating, which can be attributed to the formation of a CS layer surrounding the vesicles.

3.2.3. Fourier Transform Infrared Spectroscopy (FT-IR)

The FT-IR spectra of blank NIOs, Gli-Cur@NIOs and Gli-Cur@NIOs-CS in comparison with a physical mixture of APIs (Gli-Cur) and pure CS are shown in Figure 6.

In the FT-IR spectrum of blank NIOs, characteristic absorption bands corresponding to the functional groups of the surfactants (Span 60, Tween 60) and Chol were identified as follows: 2920 and 2853 cm^−1^ (asymmetric and symmetric C–H stretching vibrations of -CH_2_-), 1734 cm^−1^ (C=O stretching vibration of ester group) and 1097 cm^−1^ (C–O–C stretching vibration of ether bonds from the polyoxyethylene chains of Tween 60 as well as C–O stretching vibration of Span 60). These assignments are in agreement with reported data in the literature [55,74].

For the Gli-Cur physical mixture, the characteristic absorption bands of both APIs were identified in accordance with the literature [43,75]. Gli exhibited intense bands at 3312 cm^−1^ (N–H stretching of the sulfonylurea/amide group), 3119 cm^−1^ (aromatic C–H stretching), 2932 cm^−1^ (aliphatic C–H stretching), 1715 cm^−1^ (C=O stretching of the sulfonylurea carbonyl), 1616 cm^−1^ (aromatic C=C stretching), and 1340 cm^−1^ and 1155 cm^−1^ (asymmetric and symmetric stretching vibrations of SO_2_). Cur displayed characteristic absorption bands at 3510 cm^−1^ (O–H stretching of phenolic hydroxyl groups), 1626 cm^−1^ (stretching vibration of the C=C and C=O of the inter-ring chain), 1506 cm^−1^ (aromatic ring C=C stretching), 1429 cm^−1^ (aromatic C–H bending), 1275 cm^−1^ (aromatic C–O stretching), 1028 cm^−1^ and 1155 cm^−1^ (symmetric and asymmetric C–O–C stretching vibrations of methoxy groups), and 964 cm^−1^ (=C–H out-of-plane bending vibrations).

In the Gli-Cur@NIOs spectrum, the characteristic absorption bands of both APIs remained detectable; however, they appeared broadened and slightly shifted, compared to those of pure APIs, suggesting the presence of hydrogen bonding and hydrophobic interactions with the niosomal bilayer. For Gli, the main bands were observed at ~3310 cm^−1^ (N–H stretching), ~1715 cm^−1^ (C=O stretching), ~1616 cm^−1^ (aromatic C=C), and 1340/1155 cm^−1^ (SO_2_ stretching), although with reduced intensity and peak broadening. In the case of Cur, the principal absorption bands were shifted to ~1516 cm^−1^ (aromatic C=C with C–O stretching), ~1429 cm^−1^ (olefinic C–H bending), and ~1277 cm^−1^ (phenolic C–O stretching), also exhibiting noticeable broadening. In addition, bands corresponding to the bilayer components were clearly observed at ~2920/2853 cm^−1^ (aliphatic C–H stretching of Span 60, Tween 60 and Chol), ~1734 cm^−1^ (ester C=O stretching of Span 60 and Tween 60), and ~1095 cm^−1^ (C–O stretching vibrations). The preservation of these signals, together with peak broadening and slight shifts relative to the spectra of pure APIs, supports the occurrence of noncovalent interactions between the drugs and the NISs–Chol bilayer, thereby confirming their successful entrapment in the niosomal matrix.

According to the literature [76], the FT-IR spectrum of CS is characterized by a broad absorption band at 3372 cm^−1^ attributed to overlapping O–H and N–H stretching vibrations, indicative of extensive intra- and intermolecular hydrogen bonding. Bands at 2922 and 2876 cm^−1^ correspond to aliphatic C–H stretching vibrations of –CH_2_– groups. The amide I band was identified at 1647 cm^−1^ (C=O stretching), while the amide II band appeared at 1591 cm^−1^ (N–H bending), reflecting the presence of residual N-acetylated groups. The absorption band at 1151 cm^−1^ is assigned to the asymmetric stretching of the C–O–C bridge, where bands at 1065 and 1025 cm^−1^ correspond to C–O stretching vibrations of the polysaccharide backbone. The presence of CS-specific absorption bands in the spectra of CS-coated Gli-Cur@NIOs, with slight shifts and overlaps, further indicates interactions between the polymeric coating, the niosomal membrane and the encapsulated drugs.

3.2.4. Differential Scanning Calorimetry (DSC)

The DSC was employed to evaluate the phase behavior, bilayer fluidity, and drug–membrane interactions of the niosomal systems. The DSC thermograms of the APIs (Gli, Cur) and APIs-loaded NIOs, including CS-coated formulation (APIs-NIOs-CS), are presented in Figure 7.

The APIs exhibited sharp endothermic events during the first heating runs, corresponding to their melting transition (Tm), with peak temperatures at 178.8 °C for Cur (Figure 7a) and 175.5 °C for Gli (Figure 7b). These values are in good agreement with the literature data, which report melting temperatures in the range of 175–200 °C for Cur and approximately 175 °C for Gli [77,78]. In addition, the melting endotherms were associated with high melting enthalpy (ΔHm) values of 102.1 J/g for Cur and 84.36 J/g for Gli, indicating a high degree of crystallinity. Furthermore, both APIs exhibited a glass transition temperature (Tg) during the second heating runs, observed at approximately 68 °C for Cur and 57 °C for Gli, the recorded values being in good agreement with the reported data [79,80].

The thermogram of the blank NIOs, obtained during the first heating run, exhibited two sharp endothermic transitions at 31.9 °C and 50.6 °C, attributed to the melting of the surfactants (Span 60 and Tween 60) within the niosomal bilayer (Figure 7c) [65]. These transitions were followed by broader endothermic events at 80.5 °C and 122.6 °C, with a total enthalpy change of 128.2 J/g, which are associated with Chol intercalation within bilayers and a consequent reduction in membrane fluidity, in agreement with literature data [81].

During the second heating run, the endothermic transitions shifted to lower temperatures (27 °C and 44 °C) and exhibited a markedly reduced enthalpy (51.97 J/g), indicating irreversible bilayer reorganization with disruption of ordered lamellar domains, accompanied by increased membrane flexibility. This behavior is consistent with the role of Chol as a bilayer modulator, disrupting tight hydrocarbon chain packing, reducing transition cooperativity, and promoting conformational disorder within the surfactant alkyl chain [82].

Incorporation of APIs into the niosomal matrix was associated with marked alterations in the thermal behavior of the NIOs (Figure 7d–f). During the first heating cycle, the Gli-Cur@NIOs exhibited a main endothermic transition with an adjacent shoulder peak, observed at 35.1 °C and 48.8 °C, respectively (Figure 7d). In addition, two more pronounced endothermic transitions appeared at 83 °C and 116 °C, leading to an increase in the total enthalpy change to 141.8 J/g. This thermal behavior suggests that API incorporation alters surfactant–Chol interactions and promotes the formation of new microdomains within the bilayer, in agreement with other findings reported [83].

Upon the second heating cycle, multiple endothermic transitions re-emerged, corresponding to the previously identified events, but shifted to lower temperatures (26.3 °C, 34.8 °C, 41.9 °C and 70.4 °C), accompanied by a pronounced reduction in enthalpy (21.55 J/g). These changes indicate disruption of lamellar order and reduced transition cooperativity, consistent with strong drug–bilayer interactions.

The CS-coated formulation (Gli-Cur@NIOs-CS) exhibited a complex thermal behavior, characterized by two major endothermic transitions at −0.8 °C and 110.1 °C (Figure 7e), accompanied by the highest enthalpy change (1780 J/g), indicating the presence of additional interactions introduced by the polymer layer. According to the reported data [84,85], the endothermic peak observed at 110.1 °C is attributed to the thermal response of CS, primarily associated with the release of bound water from its hydrophilic domains. The increased enthalpy suggests that CS contributes to the stabilization of the amorphous state of the encapsulated drugs by hindering recrystallization and reinforcing bilayer integrity through hydrogen bonding and electrostatic interactions [86]. Following the second heating cycle, these thermal events collapsed into two low-intensity peaks at 26.7 °C and 36.4 °C, accompanied by a pronounced reduction in enthalpy (9.97 J/g). This behavior indicates irreversible bilayer reorganization induced by thermal stress, resulting in a more disordered and flexible system compared to Gli-Cur@NIOs.

The cooling cycles of all developed niosomal formulations exhibited exothermic transitions of much lower intensity compared to the endothermic events observed, indicating incomplete structural recovery upon cooling. For blank NIOs, exothermic peaks were observed at 18.8 °C and 33.7 °C, with an associated enthalpy change of ΔH = −41.52 J/g. In contrast, the Gli-Cur@NIOs and Gli-Cur@NIOs-CS displayed highly diffuse exothermic peaks with lower enthalpy values (ΔH = −21.66 J/g for Gli-Cur-NIOs and ΔH = −7.87 J/g for Gli-Cur-NIOs-CS), reflecting a reduced tendency to recrystallize and confirming irreversible thermal alterations associated with the progressive disruption of ordered domains. Moreover, the absence of sharp melting endotherms corresponding to crystalline Gli (Tm = 175 °C) and Cur (Tm = 179 °C) in the loaded formulations further supports the amorphous dispersion of APIs within the niosomal matrix, indicative of successful drug entrapment [87]. Significant differences were also observed in the Tg of the systems, which occurred at significantly lower and negative temperatures (Figure 7d–f), confirming an increased amorphous fraction in the drug-loaded formulations.

3.2.5. X-Ray Diffraction

The XRD spectra of APIs-NIOs(CS) are presented alongside those of the physical mixtures of APIs and native CS in Figure 8.

The blank NIOs displayed a broad amorphous diffraction pattern, with a weak reflection at approximately 2.00° 2θ, attributed to residual lamellar ordering characteristic of the niosomal bilayer structure [88]. Additional very low-intensity reflections at 5.20° and 14.14° 2θ were observed and associated with the partial ordering of Chol within the bilayer structure [89]. Overall, the X-ray diffraction pattern of the blank NIOs indicates a predominantly amorphous system with limited short-range order arising from the disordered arrangement of NISs (Span 60, Tween 60) and Chol.

In contrast, the XRD of the Gli-Cur physical mixture showed multiple intense and sharp diffractions, confirming the crystalline nature of both APIs. Characteristic peaks of Gli were observed at 11.72°, 19.42°, and 21.30° 2θ, while Cur displayed prominent peaks at 7.72° and 16.22° 2θ along with additional peaks in the 20–30° 2θ range. These diffraction features are in agreement with reported crystalline patterns for Gli [43,90] and Cur [91], respectively. Upon encapsulation within the niosomal matrix (Gli-Cur-NIOs), the characteristic diffraction peaks of both APIs remained detectable but showed a marked reduction in intensity, indicating a partial loss of crystallinity and a transition toward an amorphous or molecularly dispersed state within the niosomal matrix.

The diffraction pattern of native CS exhibited two characteristic peaks at approximately 10.1° and 20° 2θ, reflecting its semi-crystalline structure. The low-angle peak is associated with hydrogen-bonded water molecules within the polymer matrix, while the higher-angle peak corresponds to partial alignment of CS chains within an orthorhombic arrangement, according to the literature data [92,93]. Interestingly, the XRD pattern of CS-coated NIOs (Gli-Cur@NIOs-CS) was predominantly diffuse, with the absence of distinct crystalline peaks, indicating a largely amorphous organization. This enhanced amorphous character can be attributed to the presence of CS coating, which forms a stabilizing surface layer that reduces drug recrystallization and maintains the APIs in the molecularly dispersed state through hydrogen bonding and electrostatic interactions. Consistent with DSC findings, this amorphous organization is associated with improved solubility and potential bioavailability of the encapsulated drugs, in line with findings reported for similar polymer-coated niosomal systems [94].

3.2.6. Osmotic Stress Resistance

Under osmotic stress conditions (hypotonic solution, NaCl 0.6%, and hypertonic solution, NaCl 1.2%), the niosomal nanocarriers exhibited distinct morphological and physicochemical responses, as reflected by changes in PS and PDI, as illustrated in Figure 9.

For the Gli-Cur@NIOs exposed to a hypotonic medium, a pronounced swelling behavior was observed, with PS increasing from 479.4 ± 6.26 nm to 797.7 ± 11.55 nm (p < 0.001). This effect could be attributed to osmotic water influx across the niosomal bilayer, leading to membrane stretching, mechanical weakening, and partial destabilization of the vesicular architecture. In contrast, exposure to hypertonic medium resulted in a marked decrease in PS, from 479.4 ± 6.26 nm to 322.5 ± 5.44 nm (p < 0.001), indicative of vesicle shrinkage and bilayer deformation driven by osmotic water efflux. In both osmotic stress conditions, the PDI increased significantly compared to isotonic medium (0.9% NaCl, 0.177 ± 0.026), reaching 0.383 ± 0.075 in hypotonic medium (0.6% NaCl, p < 0.05) and 0.843 ± 0.029 in hypertonic medium (1.2% NaCl, p < 0.001). These changes reflect increased vesicle heterogeneity and non-uniform structural responses, ultimately compromising colloidal stability.

In contrast, the CS-coated formulation (Gli-Cur@NIOs-CS) displayed a markedly attenuated response to osmotic stress, highlighting the protective effect of the polymeric layer. Under hypotonic conditions, a moderate but significant increase in PS was observed (from 576.9 ± 12.06 nm to 637.4 ± 10.19 nm, p < 0.01), consistent with controlled swelling [95]. Conversely, exposure to a hypertonic medium resulted in only a slight PS reduction to 547.9 ± 6.53 nm (p < 0.05). These responses can be attributed to electrostatic interactions and hydrogen bonding between CS and the bilayer, as well as to the retention of a hydration layer that buffers osmotic stress and limits vesicle aggregation, thereby enhancing colloidal stability [96].

Notably, the PDI values of Gli-Cur@NIOs-CS remained relatively stable under osmotic stress, showing a significant decrease under hypotonic conditions (0.195 ± 0.066, p < 0.001) and no significant change under hypertonic medium (0.577 ± 0.047), compared to the isotonic reference (0.626 ± 0.046). These results indicate preserved vesicle homogeneity and enhanced colloidal stability conferred by the CS coating. Overall, the CS layer provides improved structural integrity under osmotic stress by acting as an electrostatic and steric barrier that limits osmotic perturbations and maintains a stable PS distribution.

3.2.7. Stability to Storage

The stability of uncoated and CS-coated niosomal formulations (Gli-Cur@NIOs and Gli-Cur@NIOs-CS) over a period of 70 days at two storage temperatures: refrigerated (4 ± 1 °C) and room temperature (25 ± 1 °C), expressed in terms of EE% for Gli and Cur, is presented in Figure 10.

The Gli-Cur@NIOs exhibited a progressive decline in EE% for both APIs during storage, with a more pronounced decrease observed at 25 °C compared to 4 °C. After 70 days, EE% decreased by 53.99% and 59.90% for Gli and by 50.15% and 61.00% for Cur at 4 °C and 25 °C, respectively. This behavior indicates gradual drug leakage from the niosomal matrix, which is accelerated at higher temperatures due to increased bilayer fluidity and reduced vesicular integrity [97].

In contrast, the Gli-Cur@NIOs-CS showed a significantly slower reduction in EE% under the same storage conditions, confirming the stabilizing effect of the polymeric coating. Over the 70-day period, EE% for Gli decreased by 40.85% and 50.80%, while EE% for Cur declined by 39.17% and 54.43% at 4 °C and 25 °C, respectively. The improved stability of the coated system can be attributed to the presence of the CS layer, which enhances surface charge density and structural rigidity while reducing bilayer permeability, thereby limiting drug leakage during storage [98].

It has been reported in the literature that elevated storage temperatures decrease niosomal stability by increasing bilayer fluidity and promoting drug leakage, whereas the CS-coated system exhibits improved structural integrity due to the restricted bilayer mobility of the bilayer [97,99]. In addition to drug retention, commonly expressed as EE%, the physical stability of vesicle systems is also reflected by a change in hydrodynamic diameter and PDI. Several studies have reported that CS-coated niosomal formulations remain stable under refrigerated storage (4 °C) for up to 12 weeks, displaying only minor and statistically insignificant variations in PS and PDI, along with minimal drug leakage, as indicated by a slight decrease in EE% [38]. These reported findings are consistent with our results, which demonstrate that refrigerated storage (4 ± 1 °C) is associated with a lower loss of EE% compared to room temperature (25 ± 1 °C), thereby indicating improved vesicle integrity.

3.3. In Vitro API Release Profiles and Kinetic Analysis

The release behavior of APIs may be influenced by several factors, including drug solubility, the pH of the dissolution medium, the vesicle size and the permeability of the niosomal membrane. The release profiles of Gli and Cur from the niosomal matrices (Gli-Cur@NIOs and Gli-Cur@NIOs-CS) under simulated gastrointestinal pH conditions are presented in Figure 11.

Both uncoated (Gli-Cur@NIOs) and CS-coated (Gli-Cur@NIOs-CS) niosomal formulations exhibited pronounced gastric stability during the initial 0–3 h in SGF (Figure 11a,b), with minimal drug release, indicating preservation of vesicular integrity under acidic conditions. At 3 h, Gli release remained low for both systems (1.44 ± 0.90% for Gli-Cur@NIOs-CS and 4.12 ± 2.30% for Gli-Cur@NIOs), while Cur showed similarly limited release (4.57 ± 1.70% and 4.52 ± 1.40%, respectively). The lower release observed for the CS-coated formulation confirms the protective barrier effect of the polymer layer during gastric transit.

In SIF, marked differences in release behavior were observed between the two systems. Gli release from uncoated NIOs increased substantially, reaching 55.75 ± 2.10% after 22 h, whereas the CS-coated formulation showed a delayed and limited release (4.11 ± 2.30%). This difference can be attributed to increased bilayer permeability and partial destabilization of the uncoated NIOs at alkaline pH, while the CS coating initially acts as a diffusion barrier that progressively relaxes upon hydration and partial swelling, resulting in sustained Gli release over time [95].

Under identical intestinal conditions, Cur exhibited a distinct release profile, with a delayed but pronounced increase in release for both formulations after approximately 6 h. At 22 h, cumulative Cur release reached 30.22 ± 2.10% for Gli-Cur@NIOs and 32.02 ± 2.20% for Gli-Cur@NIOs-CS. This behavior is consistent with Cur’s poor aqueous solubility and high lipophilicity, which initially limit diffusion [100], followed by enhanced release upon membrane hydration and partial vesicle destabilization. In the CS-coated system, swelling and loosening of the polymer layer further facilitate Cur diffusion, resulting in a slightly more prolonged and controlled intestinal release profile [101].

In the case of the physical mixture of APIs, a limited and pH-dependent release of Gli was observed, with lower release in SGF (1.12 ± 2.50%) compared with SIF (13.64 ± 2.10%) at 22 h. This behavior is consistent with the acidic character of Gli (pKa = 5.3), which reduces its solubility under low-pH conditions [102]. In contrast, Cur exhibits only minor differences between the two media, with cumulative release values of 15.00 ± 0.90% in SGF and 19.97 ± 0.90% in SIF at 22 h. This profile reflects Cur’s poor aqueous solubility and high lipophilicity under acidic to neutral conditions, indicating that its release is primarily governed by intrinsic solubility rather than medium pH.

These findings demonstrate that both niosomal formulations (Gli-Cur-NIOs and Gli-Cur-NIOs-CS) significantly improved the release profile compared to the physical mixture and the individually encapsulated drugs, providing enhanced gastric protection and sustained intestinal release for both encapsulated APIs.

The obtained results are consistent with the literature, which indicate that co-encapsulation of drugs within the same niosomal carrier offers multiple advantages, including synchronized release at the same target cells or tissue compartment, with simultaneous gastric protection of both drugs and sustained intestinal release profile, benefits that extend beyond those achieved by the concomitant administration of individually encapsulated drugs or their simple co-administration [11,12].

Although Gli and Cur exhibit distinct release kinetics governed by their individual physicochemical properties, their incorporation within the same vesicular structure ensures functional synchronization during intestinal transit. Such coordinated release behavior is particularly relevant for combined therapies, as it may facilitate complementary pharmacological actions within overlapping therapeutic time windows rather than sequential drug exposure.

To further elucidate the underlying release mechanisms, the experimental data were fitted to four kinetic models (zero-order, first-order, Higuchi and Hixson–Crowell) under both SGF and SIF conditions. For each formulation and medium, the most appropriate model was identified based on the highest coefficient of determination (R^2^), revealing distinct kinetic behaviors among the tested systems (Table 5).

In SGF, Gli release from the CS-coated NIOs (Gli-Cur@NIOs-CS) showed an excellent fit to the first-order model (Figure 12b) (R^2^ = 0.974) and a strong correlation with the Higuchi model (Figure 12e) (R^2^ = 0.969), indicating diffusion-controlled release through the CS barrier. This confirms the role of the polymer coating as an effective diffusional shield under acidic conditions. In contrast, uncoated NIOs (Gli-Cur@NIOs) exhibited a weaker first-order fit (R^2^ = 0.857), consistent with a limited and predominantly concentration-dependent diffusion process [64]. For Cur, release from both niosomal formulations was best described by the zero-order model (R^2^ = 0.994 for Gli-Cur@NIOs-CS and R^2^ = 0.995 for Gli-Cur@NIOs (Figure 12a), indicating a nearly constant release rate. Comparable fits to the Hixson–Crowell model (Figure 12g) (R^2^ = 0.987 and 0.991, respectively) suggest that Cur release is governed by a combination of membrane diffusion and surface structural changes in the vesicles, consistent with a predominantly non-Fickian, erosion-assisted mechanism in acidic medium [64,103]. As expected, the Gli-Cur physical mixture showed poor kinetic correlations in SGF (R^2^ < 0.700 for both APIs), reflecting solubility-limited and poorly controlled release.

In SIF, Gli release from both uncoated and CS-coated NIOs (Gli-Cur@NIOs and Gli-Cur@NIOs-CS) showed an excellent fit to the first-order kinetic model (Figure 12d) (R^2^ = 0.991 and 0.958, respectively), indicating a release rate dependent on the remaining drug fraction. For Gli-Cur@NIOs-CS, a strong correlation with the Higuchi model (Figure 12f) (R^2^ = 0.962) further suggests that diffusion through the swollen CS layer contributes to the overall release mechanism. For Cur, release from both niosomal systems followed the zero-order model (Figure 12b) (R^2^ = 0.994 for Gli-Cur@NIOs and 0.993 for Gli-Cur@NIOs-CS), with comparable fits to the Hixson–Crowell model (Figure 12h) (R^2^ = 0.990 and 0.988, respectively). This behavior indicates a near-constant release rate governed by a combination of membrane diffusion and vesicle surface or structural changes, consistent with a predominantly non-Fickian, erosion-assisted mechanism under intestinal conditions. In contrast, the Gli-Cur physical mixture was model-dependent (the Higuchi model for Gli and the first-order model for Cur), reflecting the solubility-driven dispersion of the unencapsulated drugs in the intestinal medium.

Based on the release kinetics analysis, the first-order and Higuchi models were identified as the most suitable to describe Gli release from niosomal systems, whereas Cur release was best described by the zero-order and Hixson–Crowell models. These findings indicate that the release mechanism is strongly influenced by the physicochemical properties of the encapsulated drug.

4. Conclusions

In this study, niosomal and CS-coated niosomal systems co-loaded with glibenclamide and curcumin (Gli-Cur-NIOs and Gli-Cur-NIOs-CS) were successfully developed using the thin-film hydration method based on the self-assembly of ionic surfactants and cholesterol. The optimized formulation exhibited high EE%, while DLS and STEM confirmed the formation of nanosized, predominantly spherical vesicles.

Spectroscopic and thermal analyses (FT-IR, DSC, and XRD) confirmed the successful co-encapsulation of both APIs within the niosomal bilayer, evidenced by reduced crystallinity and the presence of intermolecular interactions with the surfactant-cholesterol membrane and chitosan layer. Both uncoated and CS-coated NIOs demonstrated good physical and osmotic stability, with the coated system exhibiting superior resistance to osmotic stress and reduced leakage during storage. In vitro release studies under simulated gastrointestinal conditions and kinetic modeling revealed that the developed niosomal systems provide a controlled and sustained drug release, governed by diffusion- and erosion-based mechanisms, strongly influenced by the physicochemical properties of the encapsulated drugs and the structural characteristics of the vesicular carriers.

Overall, the CS-coated niosomal formulation preserved the intrinsic advantages of niosomal delivery while providing enhanced stability and surface functionality, supporting its potential as an advanced oral co-delivery platform for Gli and Cur in T2DM management.