Development of Highly Stable Vitamin C Gummies Using Innovative In Situ Soft Sphere Integrated (ISSI) Liposomal Technology: Characterization and In Vitro Release Studies

Abstract

Vitamin-enriched gummies are gaining popularity due to their convenience and consumer appeal; however, incorporating water-soluble and unstable vitamins such as vitamin C remains challenging because of its poor stability and rapid degradation during processing. This study aimed to develop a heat-stable liposomal vitamin C gummy (LVC gummy) using In Situ Soft Sphere Integrated (ISSI) technology with a xyloglucan/trehalose/citric acid cross-linked polymeric matrix. The ISSI system enhanced the thermal, structural, and chemical stabilities of vitamin C during gummy preparation and storage. Characterization by transmission and scanning electron microscopy, FTIR, particle size, and zeta potential analysis confirmed a uniform vesicle formation and stable polymeric coating. The LVC gummies showed desirable physicochemical properties, including optimal dispersion, swelling behavior, and low water activity. Accelerated stability studies demonstrated improved vitamin C retention, while in vitro release testing indicated minimal release under gastric conditions and sustained release in the intestinal phase compared to conventional gummies. Overall, this approach offers a robust and consumer-acceptable delivery system for vitamin C, providing a practical solution for enhancing vitamin stability in functional foods and nutraceuticals.

1. Introduction

Vitamin C (l-ascorbic acid) is an essential water-soluble micronutrient required for numerous physiological functions, including immune defense, collagen biosynthesis, neurotransmitter formation, and maintenance of redox balance. It serves as a potent antioxidant protecting biomolecules from oxidative damage and contributing to enzymatic regulation and iron homeostasis. Despite its well-established health benefits, the oral bioavailability of vitamin C is limited by its poor stability and rapid degradation under environmental factors such as heat, oxygen, moisture, and light. − The recommended daily intake of vitamin C for a healthy adult is approximately 500 mg per day. Fruits and vegetables are the primary dietary sources of vitamin C, and consuming five servings of mixed fruits and vegetables can provide more than 200 mg of vitamin C daily. However, the actual intake and bioavailability of vitamin C from natural sources may vary widely due to food processing, storage losses, and individual dietary habits. In addition, vitamin C is highly sensitive to heat, oxygen, and light, leading to significant degradation during cooking and prolonged storage. Therefore, when dietary intake is inadequate or when physiological demands increase due to stress, illness, or oxidative burden, vitamin C supplementation offers a reliable strategy to maintain optimal plasma levels and ensure consistent antioxidant protection. Conventional vitamin C supplements, including tablets and powders, are prone to oxidative degradation during processing and storage, resulting in reduced potency over time. Furthermore, the intestinal absorption of vitamin C occurs through a sodium-dependent vitamin C transporter (SVCT1), which exhibits saturation kinetics, limiting bioavailability at higher oral doses. − Consequently, only a fraction of orally ingested vitamin C reaches the systemic circulation in its active form.

To overcome these challenges, employing advanced delivery systems, such as liposomes, can significantly enhance vitamin C stability and absorption. Liposomes, composed of phospholipid bilayers encapsulating aqueous cores, protect encapsulated vitamin C from environmental oxidation and enzymatic degradation while promoting sustained release and improved gastrointestinal uptake. − Such systems, therefore, offer a promising strategy for achieving higher plasma retention and improved therapeutic efficacy compared with conventional supplementation. Liposomes offer minimal immunogenicity, low toxicity, and good biocompatibility owing to their structural similarity to biological membranes, which allows for safe encapsulation and delivery of bioactive compounds. − They are very valuable in the domains of agriculture, cosmetics, nutraceuticals, medicines, and functional foods. − In recent years, liposomes have been widely utilized in nutritional and functional food research as delivery systems for diverse bioactives such as essential oils, fatty acids, minerals, proteins, peptides, vitamins, and polyphenols. − Their proven ability to encapsulate and protect sensitive nutrients supports the development of liposomal vitamin C systems aimed at enhancing the stability and bioavailability in nutraceutical applications. The rapid removal of liposomes from blood circulation and reticuloendothelial system macrophages is a significant drawback of employing liposomes as medication carriers. Furthermore, issues including phospholipid oxidation, hydrolysis, aggregation, and degradation are brought on by the physical and chemical instability of liposomes. − In addition, many liposomal products are in liquid or suspension form since phospholipids are stabilized in a water-in-oil emulsion that functions as a kind of carrier for encapsulation. Due to the spontaneous and nonuniform assembly of phospholipid bilayers in aqueous environments, conventional liposomes often exhibit heterogeneity in vesicle size and lamellarity. Such structural irregularities can lead to inconsistent encapsulation efficiency, variable drug release profiles, and reduced formulation stability, thereby limiting reproducibility and controlled delivery performance. To address these problems, the liposomal product in powder form that is obtained after drying might be an effective remedy. Encapsulation of vitamin C by using liposomes through spray drying method could be suitable techniques to preserve all the benefits of vitamin C. Nevertheless, the evaporation of water during the drying activity may lead to destruction of the core structure of liposomes. In this investigation, we have designed and developed a powdered form of liposomal formulation by in situ soft sphere integrated (ISSI) technology under high pressure homogenization and nanomilling processes, and then using a spray drying technique to remove the water.

The ISSI technology represents an integrated self-assembly approach capable of encapsulating a broad range of molecules, such as hydrophilic, lipophilic, and amphiphilic, within a stabilized hybrid vesicular system. It functions through in situ coassembly of phospholipid bilayers, self-emulsifying gum arabic nanospheres, and a cross-linked xyloglucan/trehalose/citric acid polymeric matrix. During the process, phospholipids form nanoscale vesicles that are interpenetrated by gum arabic nanospheres, which act as natural emulsifiers and interfacial stabilizers by forming hydrogen bonds and electrostatic interactions with phospholipid head groups. Concurrently, a polymeric matrix develops at the outer interface, creating a soft and flexible coating that enhances the mechanical and oxidative stability of the vesicles. This multilevel assembly provides strong protection for encapsulated actives against environmental stress and supports sustained release behavior. In this study, the ISSI process was employed to encapsulate vitamin C within a hybrid liposomal–polymeric system, achieving enhanced stability and controlled release in a nutraceutical gummy formulation.

Liposomal vitamin C gummies (LVC gummies) offer a convenient and appealing delivery format for both children and adults. However, developing such systems under elevated temperatures presents challenges, as heat-induced phase transitions and shear forces can destabilize the liposomal bilayer. , Hence, components that enhance membrane protection during gummy processing are essential. Biopolymer coatings cross-linked with phospholipids have been shown to strengthen liposomal membranes, reduce oxygen exposure and leakage, and maintain structural integrity under thermal stress. Such polymer-coated liposomes also exhibit a prolonged circulation time and controlled release behavior due to thicker membranes and modified surface charge, supporting their potential for stable gummy formulations. Biopolymers such as xyloglucan and trehalose can form thermally and mechanically stable cross-linked composites using citric acid as a cross-linking agent, providing structural protection to liposomal formulations under high heat and shear during gummy preparation. Xyloglucan, a hemicellulose found in plant cell walls, comprises xylose, glucose, and galactose residues in a (1 → 4)-β-d-glucan backbone. Its branched, water-soluble structure makes it an excellent matrix or stabilizer for drug delivery and functional food systems. − Trehalose (α, α-d-trehalose), a nonreducing disaccharide widely present in nature, is known for protecting proteins and membranes from denaturation under stress and is extensively used as a stabilizer in pharmaceutical and nutraceutical formulations. , More acid- and heat-stable than maltose or cellobiose, its symmetric molecular structure enables the formation of biocompatible, homogeneous polymeric networks, supporting its suitability for biobased polymer synthesis.

Despite the widespread availability of fortified foods and supplements, vitamin deficiencies remain prevalent even in developed nations and are considered an epidemic among children in developing countries. − Vitamin-enriched gummies have gained popularity due to their ease of consumption, appealing appearance, and palatable flavors, particularly for children and the elderly. The U.S. gummy market alone is projected to reach 5.8 billion USD by 2029, reflecting strong consumer acceptance. These gummies typically employ polysaccharide-based gelling agents such as pectin, starch, xanthan, agar, and carrageenan, with pectin being the most common due to its gelling, stabilizing, and fat/sugar replacement properties in low-calorie foods, candies, and jellies. Accordingly, the aim of this study was to develop LVC gummies as a functional food model and to evaluate their physicochemical, structural, and sensorial characteristics. A powdered form of liposomal vitamin C was prepared using ISSI technology involving high-pressure homogenization and nanomilling, followed by spray drying and surface coating with a xyloglucan/trehalose/citric acid cross-linked polymeric matrix to obtain a highly stable formulation with enhanced protection and controlled-release potential. Furthermore, structural characterization and simulated gastrointestinal digestion studies were performed to assess the suitability of the developed LVC gummies for functional food and nutraceutical applications.

2. Materials and Methods

2.1. Materials

l-(+)-Ascorbic acid (≥99%, CAS No. 50-81-7), phosphatidylcholine (CAS No. 8002-43-5), gum arabic (CAS No. 9000-01-5), trehalose (CAS No. 99-20-7), citric acid (CAS No. 77-92-9), d-sorbitol (CAS No. 50-70-4), sucrose (CAS No. 57-50-1), glycerol (CAS No. 56-81-5), and pectin (CAS No. 9000-69-5) were of analytical or HPLC grade and were purchased from Merck India. Corn syrup was purchased from Jiaxing Renze Food Ingredients, China. Xyloglucan (CAS No. 37294-28-3) was purchased from Wuhan HengHeDa Pharm Co., Limited, China. All organic solvents were HPLC grade and were procured from Merck India. The entire study was conducted using Millipore–Milli-Q distilled water.

2.2. Preparation of Liposomal Vitamin C by ISSI Technology

In this design, we have developed a powder form of stabilized liposomal vitamin C using high-pressure homogenization, afterward eliminating the water via a spray drying method. The formulation was developed with a combination of vitamin C, gum arabic and phospholipids using homogenization process as follows: initially, 76.8 g of gum arabic was dissolved in 500 mL of water and subjected to a nanomilling process to deliberately generate, self-emulsifying gum arabic nanospheres, , which act as natural interfacial stabilizers for phospholipid dispersion (Figure ). After that, 24.8 g of phospholipids dissolved in 300 mL of water at 80 °C underwent homogenization process for 10 min followed by mixing with 100 g of vitamin C and further homogenized for 10 min at 40 °C. This mixture was added to the self-emulsified nanosphere solution and underwent a high-pressure homogenization process at 4000 rpm for three cycles, and the process was repeated three times and further underwent nanomilling process for 15 min to obtain vitamin C-encapsulated soft spheres integrated into the liposomal formulation by ISSI technology. During homogenization, the self-emulsifying gum arabic nanospheres integrate within the aqueous phase of the developing liposomal vesicles, acting as interfacial stabilizers, while vitamin C is simultaneously encapsulated within the aqueous core of the liposomes. The gum arabic nanospheres at the interface form hydrogen bonding and electrostatic interactions with the phospholipid head groups, thereby improving bilayer stability and preventing vesicle aggregation. This coassembly process enables uniform encapsulation and contributes to the enhanced structural and oxidative stability of the ISSI liposomal system. The developed formulation was dried out by a spray drying method with 180 and 80 °C as inlet and exhaust temperatures, respectively, with drying airflow of 10 kg/cm³ to obtain dry liposomal vitamin C.

1.

Preparation of a xyloglucan/trehalose/citric acid cross-linking biopolymeric matrix-coated liposomal vitamin C gummies (LVC gummies) via nanomilling process using high pressure homogenization by in situ soft spheres integrated (ISSI) technology.

2.3. Preparation of Thermally Stabled Xyloglucan/Trehalose/Citric Acid Cross-Linking Biopolymeric Matrix Coated Liposomal Vitamin C

The xyloglucan solution was prepared by dissolving 10 g of xyloglucan with Milli-Q Millipore water with mechanical stirring. Further, the trehalose was prepared by dissolving 10 g of trehalose at 50 °C in Milli-Q Millipore water. Xyloglucan and trehalose were mixed while continuously stirring for 30 min at 50 °C utilizing a mechanical stirrer. Citric acid (0.5 g/L) was added to the prepared polymeric solution, which was homogenized at 8000 rpm for 10 min. Thereafter, the cross-linking was achieved by heating the polymeric solution at 170 °C for 15 min. Cross-linking was achieved by heating the viscous polymeric mixture in a temperature-controlled oil bath at 170 °C for 15 min using a temperature-controlled oil bath system, allowing esterification reactions between citric acid carboxyl groups and polysaccharide hydroxyl groups of both xyloglucan and trehalose with release of water molecules. The high viscosity of the xyloglucan/trehalose/citric acid cross-linking biopolymeric matrix at this stage minimizes water evaporation, maintaining a uniform reaction medium. The resultant viscous gel was subsequently homogenized at 8000 rpm for 10 min to ensure complete cross-linking and uniform matrix formation. Then, the prepared dry liposomal vitamin C formulation was added into the xyloglucan/trehalose/citric acid matrix solution while being stirred for 15 min. The combined solutions were then homogenized under high pressure in a PRIMIX Homomixer Mark II 2.5 homogenizer operating in the pressure range of 30–300 bar with 8000 rpm, and the procedure was carried out three times to obtain xyloglucan/trehalose/citric acid cross-linking biopolymeric matrix-coated liposomal vitamin C.

2.4. Preparation of Vitamin C Gummies

Vitamin C gummies were prepared by using the facilities of Solistaa Pharmaceuticals Private Limited, Puducherry, India. A schematic representation of the LVC preparation process is provided in Figure . Initially, sorbitol (300 g), sugar (130 g), corn syrup (100 g), and water were combined to produce sugar syrup. This mixture was then heated to 120 °C until a refractometer showed 88–90 °Brix. Melted pectin was prepared by initially granulated pectin (20 g) being properly hydrated using cold water for 30 min and melted at 60 °C in a water bath for 60 min. The melted pectin was mixed with sugar syrup at 100 °C and stirred for 5 min to obtain a completely mixed sugar-pectin mixture. Finally, xyloglucan/trehalose/citric acid cross-linking biopolymeric matrix-coated liposomal vitamin C or vitamin C was incorporated into the sugar-pectin mixture and swirled for 15 min to evenly distribute the vitamin C or liposomal vitamin C formulations. Then, the mixtures were altered to attain pH 3.0 utilizing citric acid, and the mixtures were placed into the silicon molds at 60 °C, which were preserved in a dark room for 24 h at room temperature. The gummies were prepared with conventional vitamin C (CVC gummies) and xyloglucan/trehalose/citric acid cross-linking biopolymeric matrix-coated LVC gummies with the same quantity of vitamin C (mg). The preparation method of the vitamin C gummies is under intellectual property protection and has been filed as a patent application with the Indian Patent Office under application number 202441059155.

2.5. Typical Commercial-Scale Preparation of LVC Gummies

A commercial-scale process was established to validate the scalability of the optimized LVC gummy formulation, as described in Section . The same composition, ingredient quantities, and formulation ratios were used for the 150 L pilot-scale batch to ensure direct comparability with the R&D trials. Initially, the required quantities of sorbitol, corn syrup, sugar, and purified water were loaded into a 150 L steam-jacketed mixing vessel and heated to approximately 120 °C with continuous agitation for 20–30 min until the mixture reached 85–88 °Brix, forming the sugar syrup. In parallel, the weighed quantity of pectin was dispersed in purified water and heated to 60 °C under stirring for 20–30 min to obtain a fully hydrated pectin base. The pectin base was then transferred into the sugar syrup and blended for 10 min, maintaining a temperature range of 60–80 °C. The predispersed biopolymeric matrix-coated liposomal vitamin C formulation was introduced into the combined sugar–pectin mixture and homogenized for 10 min at 60–80 °C to ensure uniform distribution. A 50% w/w citric acid solution was subsequently added to adjust the pH (3.0–4.0) and °Brix (65–68), followed by continued mixing for 10–15 min. Color and flavoring agents were added and blended for 5 min to achieve complete uniformity. The final slurry was transferred to a deposition tank and deposited into prelubricated silicone molds using a gummy depositor to obtain units of approximately 3.5 g each. The molds were passed through a cooling chamber (5–10 °C) to facilitate demolding, and the ejected gummies were dried in a hot-air chamber at 40–50 °C until the residual moisture content fell below 15%. The dried gummies were inspected, packed in suitable containers, and subjected to quality control and stability studies.

2.6. Physical Parameters

2.6.1. Organoleptic Observations

Organoleptic analysis was performed on the prepared LVC and CVC gummies to determine color, taste, shape, texture, and clarity. To determine the texture of the gummies, lightly rub the surface and rub them between two fingers.

2.6.2. Weight Variation Test

The content homogeneity of each gummy was ascertained by measuring the weight variation of the gummies. Initially, the average weight was determined by weighing a minimum of 20 individual gummies. If there is a weight variation of no more than 7.5% from the average, then the gummy is said to have met the predetermined standard. If even one gummy deviated from this range, the test proceeded to the second phase, which involved a minimum of 20 prepared gummies. In this step, a gummy is deemed to have met the requirements if its weight does not differ by more than 10% from the average weight.

2.6.3. Gummy Dimension Test

The size of the gummies was assessed to ascertain size uniformity and the required dimensions of the main packaging to shield the gummies from the circumstances. For this justification, a vernier caliper was used to measure the height and diameter of ten gummy samples. If the dimensions of the gummies have a standard variation of less than 5%, it satisfies the criteria.

2.6.4. Swelling Ratio Test

Each prepared gummy weighing 1.0 g was immersed in deionized water for 8 h. After removing the excess surface water, the swollen gummies were weighed, and the swelling ratio was calculated using eq .

$$\mathrm{Swelling}\mathrm{ratio}(\%)=\frac{w_2-w_1}{w_1}\times 100$$ 1

where w ₁ and w ₂ are the initial and swollen weights of the gummies (g), respectively.

2.6.5. Dispersion Time Test

A flask of purified water held at 37 °C was used to conduct the dispersion test. Each prepared gummy was kept in the flask and continuously swirled with a magnetic stirrer. It was observed how long it took the particle to disseminate entirely. Standard gummy dispersion time is 10–30 min.

2.6.6. Water Activity and pH Analysis

The water activity of the gummies was determined using USP42-NF 37, 2019, with the appropriate adjustments. After the gummy was cut into circular discs that were 2 mm thick, the pieces were put in disposable sample containers. A benchtop water activity meter was used for the measurement at 25 °C. The pH of the gummies was determined using appropriate modifications in accordance with USP42-NF37, 2019. A 1.5 g gummy slice and an equivalent volume of water were placed in a centrifuge tube and centrifuged for 15 min. The sample was diluted to 15 mL and melted for one h at 60 °C in a water bath (Rotax, India) until it was totally dissolved. After the melted gummy was cooled down to 27 ± 2 °C, a pH meter was used to determine the pH of the gummies (Electronics India model 111, India).

2.6.7. Color Characterization

The color of the gummy was evaluated using a spectrophotometer (Sensegood instrument) standardized versus a standard white tile set to a D65 illuminant/2° observer angle. In a transparent circular container set against a white backdrop, gummy slices with a thickness of 2 mm were arranged. The gummy surface was used to quantify the reflectance.

$$C^{*}={(a^{*2}+b^{*2})}^{1/2}$$ 2

$$h={\tan }^{-1}(b^{*}/a^{*})$$ 3

$$\mathrm{\Delta }{E}^{*}={(\mathrm{\Delta }{L}^{*2}+\mathrm{\Delta }{A}^{*2}+\mathrm{\Delta }{b}^{*2})}^{1/2}$$ 4

The result was stated as CIE values of lightness (L ^∗), redness (+a∗) or greenness (−-a∗), and yellowness (+b∗) or blueness (−b∗). The chroma (C∗), hue angle (h), and total color difference ΔE were calculated using eqs –, respectively, where ΔL*, Δa*, and Δb* are the luminosity, redness, and yellowness intensity differences from the initial samples. All measurements were performed in triplicates.

2.7. Analysis and Characterization

2.7.1. Analysis of Ascorbic Acid (Vitamin C)

Ascorbic acid content in the prepared LVC gummy was performed according to a method reported by Yan et al. with suitable amendments. Briefly, vitamin C was measured by ultra performance liquid chromatography (UPLC) (Agilent 1260 Infinity II, Germany) using a ZORBAX Eclipse Plus C18 column, 5 μm, with a volume of 4.6 × 250 mm. The mobile phase was acetonitrile: 0.05 M ammonium dihydrogen phosphate buffer adjusted to pH 3 using orthophosphoric acid (90:10). The flow was isocratic at a rate of 1 mL/min at 32 °C. The eluate was detected using an Agilent VWD detector set at 245 nm. All the samples were analyzed in triplicate.

2.7.2. Fourier Transform Infrared Spectroscopy (FTIR-ATR)

The FTIR-ATR spectra, obtained with 32 scans per sample, were recorded by JASCO ATR-FT/IR-4700 for phospholipids, GA, vitamin C, liposomal vitamin C, and LVC gummies in the 400 to 4000 cm^–1 range.

2.7.3. Particle Size Distribution and Zeta Potential Studies

The dynamic light scattering (DLS) technique was used to ascertain the mean particle sizes and zeta potentials of the liposomal vitamin C and LVC gummies, utilizing DLS-nanoZS, Zetasizer Nanoseries, and Malvern Instruments. The dispersions of liposomal vitamin C and LVC gummies were measured at 25 °C after being appropriately diluted with water. The Stokes–Einstein equation was utilized to ascertain the particle sizes. With the help of Nano DTS (version 6.34), the zeta potentials were calculated. Each measurement was carried out in a minimum of three sets of 10 runs.

2.7.4. Transmission Electron Microscopy (TEM)

A TEM instrument (JEM-2100, JEOL, USA) was utilized to evaluate the morphology of liposomal vitamin C and LVC gummies. The samples were distributed by placing them in a sonicator for 10 min. A few drops of liposomal vitamin C or LVC gummies were distributed on glow-discharged thin carbon-coated TEM microgrids and allowed to dry out at room temperature.

2.7.5. Scanning Electron Microscopy (SEM)

The surface morphology of the liposomal vitamin C powder and LVC gummies was examined by using a scanning electron microscope (Vega3 Tescan, Germany). Samples were air-dried at ambient temperature (25 ± 2 °C) to remove surface moisture and gently fractured to expose internal structures. The fragments were mounted on aluminum stubs using double-sided carbon adhesive tape and sputter-coated with a thin layer of gold to ensure surface conductivity. The coated samples were scanned at an accelerating voltage of 30 kV under high vacuum.

2.8. Encapsulation Efficiency and Loading Capacity

The encapsulation efficiency (EE) and loading capacity (LC) of vitamin C in the LVC gummies were determined by using a differential quantification method. A known amount (100 mg) of the sample was dispersed in 100 mL of Milli-Q water, followed by vortexing and sonication to ensure complete vesicle disruption. The total vitamin C content was then determined using the UPLC method described in Section . To quantify the free (nonencapsulated) vitamin C, an equivalent sample dispersion was centrifuged at 10,000 rpm for 10 min, and the supernatant was filtered through a 0.2 μm membrane to remove intact vesicles and gummy matrix debris. The vitamin C content in this filtrate corresponded to that of the free fraction. The encapsulated amount was calculated by subtracting the free fraction from the total vitamin C content. ,, EE and LC were calculated according to eqs and :

$$\mathrm{EE}(\%)=\left(\frac{\mathrm{Total}\mathrm{vitamin}\mathrm{C}\mathrm{content}-\mathrm{Free}\mathrm{vitamin}\mathrm{C}\mathrm{content}}{\mathrm{Total}\mathrm{vitamin}\mathrm{C}\mathrm{content}}\right)\times 100$$ 5

$$\mathrm{LC}(\%)=\left(\frac{\mathrm{Total}\mathrm{vitamin}\mathrm{C}\mathrm{content}}{\mathrm{Weight}\mathrm{of}\mathrm{the}\mathrm{sample}}\right)\times 100$$ 6

2.9. Determination of Storage Stability

The stability study was conducted in accordance with the International Council for Harmonization (ICH) Q1A­(R2) guidelines, providing a standardized framework for evaluating both long-term and accelerated stability of nutraceutical formulations under controlled temperature and humidity conditions. The LVC gummies were stored at three temperature settings, such as 4, 25, and 45 °C, for a period of 180 days to assess their stability. Samples were withdrawn at predetermined intervals, and the vitamin C content was quantified before and after storage. Each measurement was performed in triplicate. The stability of vitamin C was calculated using eq :

$$\mathrm{Stability}\mathrm{of}\mathrm{vitamin}\mathrm{C}(\%)=\frac{\mathrm{V}{\mathrm{C}}_t}{\mathrm{V}{\mathrm{C}}_0}\times 100$$ 7

where VC₀ is the initial concentration of vitamin C and VC_t represents the concentrations of vitamin C at different sampling points in time.

2.10. In Vitro Release Study

The in vitro release of vitamin C from CVC gummies and LVC gummies was performed with appropriate modifications according to USP 42-NF 37 (2019). The digestion media compositions and pH conditions were prepared following the standardized static INFOGEST 2.0 protocol, with suitable modifications to accommodate the semisolid gummy matrix and maintain physiological relevance. Simulated gastric fluid (SGF) consisted of 100 mL of Milli-Q water, 0.7 mL of 37% w/w HCl, and 0.2 g of NaCl. Using either NaOH (2 M) or HCl (2 M), the final pH was lowered to 1.2. Simulated intestinal fluid (SIF) was composed of 7.7 mL of 0.2 M NaOH, 0.68 g of monobasic potassium phosphate dissolved in 25 mL of water, and water added to reach a final volume of 100 mL. Using 1 M NaOH, the final pH was increased to 6.8. One gummy, weighing roughly 2.8 g, was submerged in 900 mL of SGF or SIF in a beaker with a filtration bag inside. For 12 h, the solution was magnetically agitated while being maintained at 37 °C. The samples (3 mL) were taken at predetermined intervals of time, namely, 0, 0.5, 1, 1.5, 2, 4, 6, 8, 10, and 12 h. To maintain the total capacity of the flask, an equal volume of fresh medium was introduced. As mentioned in Section , UPLC was used to estimate the total amount of vitamin C. The release rate was calculated by eq .

$$\mathrm{Release}\mathrm{rate}(\%)=\frac{\mathrm{V}{\mathrm{C}}_t}{\mathrm{V}{\mathrm{C}}_0}\times 100$$ 8

where VC₀ is the initial concentration of vitamin C and VC _t represents the concentrations of vitamin C at different sampling points in time.

3. Results and Discussion

3.1. Mechanistic Overview of ISSI Technology

The present study demonstrates the successful development of LVC gummies using ISSI technology, an advanced nanoliposomal encapsulation approach that integrates in situ phospholipid self-assembly with interfacial and polymeric stabilization. During the ISSI process, gum arabic undergoes nanomilling to form self-emulsifying nanospheres, which act as natural emulsifiers and interfacial stabilizers by interacting with phospholipid head groups through hydrogen bonding and electrostatic attraction. Such behavior aligns with previous findings demonstrating that the amphiphilic glycoprotein–polysaccharide architecture of gum arabic enables the formation of nanoscale emulsifying domains capable of stabilizing lipid interfaces. , Simultaneously, a cross-linked xyloglucan/trehalose/citric acid polymeric matrix develops around the vesicles, forming a flexible coating that reinforces membrane integrity and minimizes oxidative or pH-induced degradation. This multilayered soft-sphere configuration, comprising a liposomal core, gum arabic nanosphere interface, and polymeric outer shell, provides enhanced physicochemical stability and controlled-release behavior, offering a robust platform for the delivery of sensitive bioactives in functional food and nutraceutical systems. The following sections detail the physicochemical, morphological, and release characteristics that confirm the successful formation and performance of the ISSI-based LVC gummies.

Importantly, ISSI technology differs fundamentally from classical liposomal encapsulation followed by spray drying. In ISSI, the self-emulsified gum arabic nanospheres, phospholipid vesicles, and the xyloglucan/trehalose/citric acid polymeric network coassemble in situ, forming a unified three-layered hybrid vesicle prior to dehydration. This integrated assembly minimizes bilayer collapse during drying and imparts thermal and oxidative stability not achievable with conventional liposome preparation methods. To the best of our knowledge, no prior studies have reported this tricomponent coassembly strategy for stabilizing vitamin C or other labile actives within heat-processed gummy delivery systems.

3.2. Physicochemical Properties

The prepared CVC and LVC gummies were analyzed by various physicochemical parameters, and the results are listed in Table . Organoleptically, both CVC and LVC gummies exhibited a uniform drop shape, transparent orange color, orange aroma, and sweet taste, which are desirable attributes for consumer acceptance. This uniform appearance demonstrates a favorable influence on the perception and marketability of the product. The texture of both formulations was elastic, chewy, and nonsticky, exhibiting suitable gummy strength, which refers to the optimal balance between mechanical firmness and chewability required for nutraceutical gummies. In general, gummy strength values in the range of 5–8 N cm^–2, as determined in literature by compression or puncture testing, are considered ideal for maintaining structural integrity during handling while ensuring comfortable mastication. ,, In this study, the observed textural characteristicselasticity, cohesiveness, and nonstickinesscorrespond to this desirable range, indicating that the polymeric matrix provided adequate gel strength and viscoelastic stability. The formulation composition, particularly the cross-linked pectin–xyloglucan–trehalose–citric acid network, is known to yield gummies with mechanical resilience and controlled hydration behavior consistent with such strength parameters.

1. Physicochemical Characteristics of CVC and LVC Gummies.

Parameters	LVC gummies	CVC gummies
Organoleptic
Color	Orange	Orange
Shape	Drop	Drop
Taste	Sweet and sour	Sweet and Sour
Flavor	Orange	Orange
Texture	Elastic and nonsticky	Elastic and nonsticky
Average weight (g)	2.77 ± 0.14	2.76 ± 0.23
Dimension
Diameter (cm)	1.86 ± 0.03	1.85 ± 0.04
Height (cm)	2.12 ± 0.06	2.11 ± 0.07
Swelling ratio (%)	1.95 ± 0.05	1.63 ± 0.06
Dispersion time (min)	11.45 ± 0.05	10.62 ± 0.07
Water activity	0.65 ± 0.04	0.54 ± 0.05
pH	3.13 ± 0.06	2.13 ± 0.04

Both the CVC and LVC gummies are prepared in the same weight which are shown as 2.76 ± 0.23 and 2.77 ± 0.14 g, respectively, which exhibited that not at all distinct gummy surpassed the weightiness in the pharmacopoeia necessity, and there is no difference in the diameter and height in both the gummies (Table ). The fractional weight expansion of the gummy structure because of water absorption is known as the swelling ratio. The intention of the swelling ratio test was to assess how effectively the gummies could hold onto water molecules inside of them. The greater the swelling ratio, the more capable the gummies are of trapping molecules of water. Pectin was used in the production of both gummies, which stabilized or created new hydrogen bonds with the water molecules. However, the LVC gummies have a higher swelling ratio (1.95 ± 0.5%) compared to CVC gummies (1.63 ± 0.06%) (Table ) because of which has higher number of hydrophilic groups, such as −OH, −COOH in the pectin and xyloglucan/trehalose/citric acid cross-linking biopolymeric matrix, that enables a hydrogen bond with water molecules, leading to the observation of a greater swelling ratio. Moreover, the xyloglucan/trehalose/citric acid cross-linking biopolymeric network also elevates through the absorption of water, which evident by the higher swelling ratio for LVC gummies rather than the CVC gummies. The swelling ratio is a key determinant of the matrix hydration, mechanical strength, and diffusion behavior of the gummies. A moderate swelling capacity facilitates gradual water penetration, allowing sustained diffusion of vitamin C from the hydrated matrix, whereas excessive swelling could cause rapid disintegration and burst release. Therefore, the slightly higher swelling ratio observed in LVC gummies indicates an optimal balance between structural integrity and controlled vitamin C release performance. ,

To ensure that the prepared gummies dissolved when they came into contact with saliva, a dispersion time test was used to measure how quickly the gummies dissolved in aqueous media. Faster absorption. beginning at the point of contact with aqueous solutions. and faster release of the active components from a dosage form are both indicated by faster dispersion times. Kadhim and Ali explained that pharmaceutically customary gummies must fragment within 15 min. Accordingly, the water dispersion times of the developed CVC gummies and LVC gummies showed as 10.62 ± 0.17 and 11.45 ± 0.19 min, respectively.

Gumming agent and its concentration are the main factors to influence the water dispersion time. LVC gummies showed a higher dispersion time (11.45 ± 0.09) because of increasing and strengthening the cross-links between the pectin matrix and xyloglucan/trehalose/citric acid cross-linking biopolymeric matrix due to the hydrogen bonding would create a more robust gummy matrix. The stronger structure of LVC gummies due to the well-encapsulation of vitamin C by ISSI technology and well-protected by the xyloglucan/trehalose/citric acid cross-linking biopolymeric matrix, the longer it takes for the gummies to dissolve, further supporting the previously described suitable gummy strength. Meanwhile, CVC gummies registered a lower dispersion time (10.62 ± 0.17), which are readily absorbed and retained water and rapidly dispersed and released the vitamin C.

The water activity of a gummy is a ratio of its own vapor pressure in perfect equilibrium with the ambient air medium to the vapor pressure of distilled water under ideal conditions. LVC gummies registered slightly higher water activity (0.65 ± 0.04) due to the hydrogen bonding between pectin and the xyloglucan/trehalose/citric acid cross-linking biopolymeric matrix, whereas CVC gummies registered lower water activity (0.54 ± 0.05). Water activity is directly related to the availability of free moisture, which governs the microbial stability and oxidative degradation during storage. Maintaining water activity values below 0.6 ensures a reduced level of microbial growth and enhanced retention of vitamin C potency. The slightly higher water activity of LVC gummies, while still within the safe range, reflects the presence of hydrophilic functional groups within the cross-linked polymeric network, which contributes to improved moisture retention and textural stability without compromising product shelf life. − Furthermore, CVC gummies showed lower pH (2.92 ± 0.04) due to rapid release of vitamin C, whereas LVC gummies showed slightly higher pH (3.13 ± 0.06) due to the slow release of vitamin C because of well encapsulation by the liposomal formulation and further coated by xyloglucan/trehalose/citric acid cross-linking biopolymeric matrix. The commercial-scale batches also exhibited physicochemical and vitamin C retention profiles comparable to those of the laboratory-scale formulation, confirming that the ISSI liposomal–polymeric system retains its integrity and stability under industrial processing conditions.

Conventional gummy formulations still prevalent in the market are typically produced by directly incorporating vitamins into a gelatin- or pectin-based matrix, a process that often involves elevated heat and exposure to oxygen, thereby compromising the potency of sensitive compounds such as vitamin C. ,, In contrast, the ISSI-based system developed here employs a hybrid liposomal–polymeric encapsulation strategy, wherein vitamin C is entrapped within phospholipid vesicles, further stabilized by gum arabic nanospheres and a cross-linked xyloglucan/trehalose/citric acid coating. This multilayered architecture offers enhanced protection during processing and storage, resulting in improved retention and controlled release properties, thereby distinguishing the present LVC gummies from conventional formulations in functional food applications.

Although the individual components of this coating matrix have well-established applications in food and biomaterial systems, the tricomponent combination of xyloglucan, trehalose, and citric acid offers synergistic functionalities ideally suited for gummy formulations. Citric acid acts as a safe, biocompatible cross-linker that esterifies polysaccharide hydroxyl groups, thereby enhancing mechanical integrity and moisture resistance. Xyloglucan contributes film-forming and gel-forming capabilities that strengthen matrix cohesion and barrier properties, , while trehalose functions as a glass-forming stabilizer that protects sensitive actives and lipid membranes during drying and storage. These complementary roles are supported by earlier studies on citric acid–cross-linked polysaccharides and the stabilizing effects of trehalose in solid matrices. Notably, although similar cross-linking chemistries have been explored in edible films and hydrogel systems, no previous studies have reported the use of a xyloglucan/trehalose/citric acid cross-linked network specifically in gummy (chewable) delivery formats. Therefore, this work represents, to the best of our knowledge, the first application of this polymeric combination as a protective coating for LVC gummies.

3.3. Color Change

The color changes in both the gummies stored at 30 ± 2 °C with normal relative humidity (RH) for 12 weeks are given in Table . The C* values for the CVC gummies significantly increased after 12 weeks, which indicates that the CVC gummies turned over to the darker in color because of degradation of vitamin C owing to the oxidative and nonoxidative degradation. Whereas the LVC gummies showed slight increases in the C* value after 12 weeks, indicating that the vitamin C is well preserved inside the liposomal vesicles, which retained the gummy color and texture. Moreover, CVC gummies registered a higher ΔE* value representing the higher amount of color changes, whereas LVC gummies showed a lower ΔE* value indicating the enhanced protection of vitamin C by the liposomal form of vitamin C inside the gummies.

The characteristic color changes of the gummies were assessed with different temperature and RH conditions, such as 40 ± 2 °C with RH 30%, 40 ± 2 °C with RH 75%, and 60 ± 2 °C with RH 75% for 2 weeks, and the findings are given in Table . The CVC gummies showed significant changes in the C*, h, and ΔE* values under the studied conditions, indicating the degradation of vitamin C and gummies structure. , However, the LVC gummies showed there is no significant changes in the a*, b*, C*, h, and ΔE* values in assessed conditions indicating that there is no degradation in vitamin C and gummy structure in the LVC gummies due to the well-encapsulation of vitamin C in the liposomal formulation by ISSI technology and further protected by xyloglucan/trehalose/citric acid cross-linked polymeric matrix, which might protect the vitamin C against different humid and hot conditions.

2. Color Characteristics of CVC and LVC Gummies at Different Storage Conditions.

Conditions	Samples	L*	a*	b*	C*	h	ΔE*
Initial	CVC gummies	48.19 ± 0.04	–0.89 ± 0.03	1.86 ± 0.05	2.06 ± 0.07	–1.12 ± 0.04	-
	LVC gummies	43.51 ± 0.03	–0.56 ± 0.02	1.96 ± 0.07	2.04 ± 0.05	–1.29 ± 0.03	-
12 weeks storage at 30 ± 2 °C	CVC gummies	42.64 ± 0.04	–0.96 ± 0.04	3.86 ± 0.06	3.98 ± 0.08	–1.33 ± 0.03	2.13 ± 0.06
	LVC gummies	44.38 ± 0.05	–0.91 ± 0.04	2.24 ± 0.04	2.42 ± 0.05	–1.18 ± 0.02	0.98 ± 0.04
2 weeks at 40 ± 2 °C with RH 30%	CVC gummies	50.62 ± 0.07	–1.68 ± 0.05	3.36 ± 0.08	3.76 ± 0.07	–1.11 ± 0.04	7.33 ± 0.12
	LVC gummies	44.04 ± 0.04	–1.39 ± 0.04	1.50 ± 0.04	2.05 ± 0.04	–0.82 ± 0.03	1.09 ± 0.05
2 weeks at 40 ± 2 °C with RH 75%	CVC gummies	52.87 ± 0.08	–2.74 ± 0.06	6.34 ± 0.07	6.91 ± 0.09	–1.16 ± 0.05	10.56 ± 0.23
	LVC gummies	44.01 ± 0.05	–1.50 ± 0.04	2.36 ± 0.05	2.80 ± 0.05	–1.00 ± 0.04	1.14 ± 0.06
2 weeks at 60 ± 2 °C with RH 75%	CVC gummies	55.39 ± 0.07	–3.96 ± 0.05	4.89 ± 0.06	6.29 ± 0.06	–0.89 ± 0.03	12.70 ± 0.31
	LVC gummies	43.75 ± 0.06	–1.76 ± 0.07	2.82 ± 0.03	3.32 ± 0.04	–1.01 ± 0.05	1.50 ± 0.04
4 weeks at Sun light	CVC gummies	48.37 ± 0.05	–0.84 ± 0.05	2.94 ± 0.05	3.06 ± 0.05	–1.29 ± 0.04	4.97 ± 0.11
	LVC gummies	43.06 ± 0.06	–0.49 ± 0.04	2.31 ± 0.04	2.36 ± 0.03	–1.36 ± 0.05	0.57 ± 0.03

The gummies further stored under sun light at 24 ± 3 °C for 4 weeks in November 2023 are given in Table . The orange color of CVC gummies was turned into slight brown color, whereas there is no visible alteration in color was detected in the LVC gummies, which indicating that the exposure of the LVC gummies under sun light over the study period did not affect the visible color change if the gummies are stored in a normal storage condition that helps retain its moisture and structure. Furthermore, the values of a*, b*, C*, h, and ΔE* also supported the stability of the LVC gummies.

3.4. IR Studies

To investigate the interactions of gummies with vitamin C, FT-IR analysis was performed with phospholipids, gum arabic, vitamin C, liposomal vitamin C, and prepared LVC gummy, and the results are exhibited in Figure . In the spectrum of phospholipids (Figure a), the peaks at 2924 and 2854 cm^–1 accredited to the C–H stretching vibration, and the peaks at 1735 and 1664 cm^–1 associated with CO stretching vibration of ester in the polar head groups of phospholipids, and the hydrocarbon chains observed at 1467 and 1378 cm^–1. The asymmetric PO₂ stretching peak appeared at 1229 cm^–1 and a symmetric PO₂ stretching peak was observed at 1060 cm^–1 and a characteristic PO₂ stretching frequency was observed at 970 cm^–1. The asymmetric O–P–O stretching peak appeared at 821 cm^–1. , In the spectrum of gum arabic (GA) (Figure b), a broad band between 3650 and 3000 cm^–1, centered at 3368 cm^–1 corresponds to O–H stretching, and a peak at 2918 cm^–1 is associated with C–H vibrational stretching mode of out-of-phase stretching of R–CH₂–R. , The peaks at 1596 and 1439 cm^–1 demonstrate the descriptive O–H in-plane bending band of carboxylic groups of residues or uronic acid of gum polysaccharides. The peaks observed between 1200 and 800 cm^–1 associated with C–O–C, C–C, and C–O stretching frequency and C–H and C–O–H bending modes of the backbone of GA. , A band at 1031 cm^–1 indicates the −C–O–C– stretching vibration of various sugar moieties present in the gum arabic. A peak identified at 780 cm^–1 ascribed to 1–4 linkage of galactose and 1–6 linkage of mannose, and the bands among 700–550 cm^–1 were linked to the skeletal mode vibrations of the pyranose rings.

2.

FTIR spectra of (a) phospholipids, (b) GA, (c) vitamin C, (d) liposomal vitamin C, and (e) LVC gummy.

In the spectrum of vitamin C (Figure c), the peaks at 3525, 3409, 3311, and 3213 cm^–1 are corresponded to the stretching frequency of O–H group and the peaks at 3021 and 2915 cm^–1 attributed to the stretching frequency of C–H in vitamin C. The CO and C–C­(O)–O stretching modes appeared in 1754 and 1274 cm^–1 and the peak at 1666 cm^–1 belongs to CC bonds of vitamin C. The peaks at 1199 and 1112 cm^–1 were attributed to the stretching vibration of C–O–C. The C–OH bending vibration of vitamin C appeared at 1451, 1318, and 1026 cm^–1, whereas the C–H bending detected in sequences of wavenumbers such as 819, 755, 683, and 624 cm^–1. A peak at 991 cm^–1 related to the O–H and C–H bending vibration, and the C–C vibration peak found at 871 cm^–1. ,

In the liposomal vitamin C spectrum (Figure d), a without shift of the CO band from at 1754 cm^–1, however, attenuation of its intensity was observed, indicating hydrogen-bond formation between the carbonyl groups of vitamin C and the polar phosphate headgroups of the phospholipids. Additionally, a slight shift in the PO stretching vibration from 1222 to 1224 cm^–1 confirmed electrostatic interactions and encapsulation of vitamin C within the lipid bilayer. , These spectral changes corroborate the formation of stable liposomal assemblies via the ISSI technology.

In the LVC gummy spectrum (Figure e), the carbonyl band remained at 1753 cm^–1, while the O–H stretching region (3700–3000 cm^–1) became broader and more intense, reflecting enhanced hydrogen bonding among vitamin C, phospholipids, and the polymeric coating components (xyloglucan, trehalose, citric acid). A new peak at 1074 cm^–1 appeared, corresponding to C–O–C and C–O stretching associated with polysaccharide cross-linkages. The broad band near 1753 cm^–1 also represents ester carbonyl groups formed via citric acid cross-linking with hydroxyl groups of xyloglucan and trehalose. These characteristic bands collectively indicate the development of a cohesive liposomal-polymeric hybrid network that effectively encapsulates and stabilizes vitamin C within the LVC gummies. Overall, the observed peak shifts and the emergence of new functional group signals confirm the molecular integration between the liposomal core and the biopolymeric coating, demonstrating the success of the ISSI encapsulation mechanism in producing structurally stable, chemically protected vitamin C gummies.

3.5. Particle Size Distribution and Zeta Potential Measurements

Particle size is one of the most important characteristics of liposomal and gummy formulations. The particle size of the prepared liposomal vitamin C and LVC gummies was analyzed by the DLS method. The mean particle size of the liposomal vitamin C was found to be in the range of 130 to 260 nm with an average particle size of 206.8 ± 17.4 nm (Figure a), whereas LVC gummies registered slightly higher in particles as found in the range of 170 to 320 nm with an average particle size as 247.5 ± 22.4 nm (Figure b) due to the coating of xyloglucan/trehalose/citric acid cross-linked polymeric coating on the liposomal vitamin C, furthermore the results clearly revealed that the sizes of the particles exist in a narrow range and are uniform.

3.

Particle size distribution of (a) liposomal vitamin C and (b) LVC gummies; zeta potential measurements of (c) liposomal vitamin C and (d) LVC gummies.

The zeta potential describes the surface charge of nanoparticles and is a key indicator of colloidal stability, with large absolute values associated with reduced aggregation tendencies. A zeta potential magnitude of at least ±30 mV is generally required for a physically stable nanosuspension stabilized predominantly through electrostatic repulsion. In the present study, liposomal vitamin C exhibited a strongly negative zeta potential of −45.47 mV (Figure c), indicating high electrostatic stability. The LVC gummies showed a slightly less negative zeta potential of −31.98 mV (Figure d). The reduction in zeta potential from −45.47 to −31.98 mV cannot be attributed solely to differences in particle size. Instead, this shift primarily results from surface masking of phospholipid headgroups by the xyloglucan/trehalose/citric acid polymeric shell, partial esterification with citric acid, and the presence of abundant neutral hydroxyl groups contributed by the polysaccharides. These interactions reduce the density of exposed negative charges on the vesicle surface, leading to a moderately lower but still sufficiently high negative zeta potential. Importantly, the zeta potential value of −31.98 mV remains above the commonly accepted stability threshold of −30 mV, indicating that the LVC vesicles retain adequate electrostatic repulsion to remain well dispersed without aggregation. This confirms that the polymer-coated liposomal system maintains colloidal stability while achieving the intended surface modification.

3.6. TEM Studies

The morphological appearances of liposomal vitamin C and LVC gummies were observed by TEM, as shown in Figure . Liposomal vitamin C exists as nanosized particles with a range of 150 to 220 nm with mean size of 173.2 ± 14.6 nm (Figure a), spherical shapes, without any aggregation, demonstrating the stability of the liposomal formulation. These data agree well with the particle size data (Figure a) and the zeta potential measurements (Figure c). The TEM micrograph of liposomal vitamin C (Figure a) revealed spherical vesicles with a darker central region corresponding to the encapsulated hydrophilic vitamin C with gum arabic nanospheres surrounded by a lighter, electron-transparent phospholipid bilayer. Such contrast arises due to the differential electron density between the aqueous core and the lipid phase, a well-recognized feature of negatively stained liposomal systems. ,, The uniform spherical morphology without aggregation further confirms the colloidal stability of the formulation.

4.

Transmission electron micrographs showing the morphology of (a) liposomal vitamin C and (b) LVC gummies prepared by ISSI technology (both at a 50 nm scale). The dark central core corresponds to encapsulated vitamin C and gum arabic nanospheres, surrounded by a lighter phospholipid bilayer. In LVC gummies, an additional darker peripheral layer indicates the presence of the xyloglucan/trehalose/citric acid polymeric coating, confirming successful encapsulation and surface modification. Both images are representative of multiple TEM fields (>100 vesicles examined) and confirm structural integrity of liposomes following gummy incorporation.

The TEM image of LVC gummies (Figure b) showed a distinct dark interface inside, which is attributed to the good encapsulation of vitamin C with self-emulsified nanospheres and which was well surrounded by a transparent phospholipid layer. Furthermore, a dark-colored coating around the surface of the phospholipid layer revealed that the visual evident of the successful coating of cross-linked polymeric matrix in the LVC gummy samples. Moreover, the particle size of the LVC gummies was significantly larger (215.1 ± 9.7 nm) than the liposomal vitamin C as confirmed by ImageJ-based analysis of multiple TEM micrographs, this value aligns with the mean particle size range (150–275 nm) determined by DLS and TEM, confirming that the visualized structure is representative of the LVC vesicles coated with the cross-linked polymeric matrix due to the electrostatic attraction and hydrogen bonding. ,

In both the liposomal vitamin C and LVC gummies, the darker central region represents the encapsulated vitamin C with gum arabic nanospheres surrounded by a relatively electron-transparent phospholipid bilayer. In the LVC gummies, an additional dark outer rim is visible, which is attributed to the higher electron density of the xyloglucan/trehalose/citric acid polymeric coating. This multilayer structure is consistent with previous reports of polysaccharide-coated liposomes, where differences in staining affinity and material density yield distinct contrast boundaries. ,

Although only representative TEM micrographs are shown, more than 100 vesicles were examined across multiple fields of view, all exhibiting a consistent spherical morphology, uniform bilayer structure, and well-defined polymeric coatings. These findings validate that the images presented are representative of the overall sample population and confirm that the liposomal vesicles retained their structural integrity after incorporation into the gummy matrix.

3.7. SEM Studies

SEM analysis was conducted to investigate the morphology of liposomal vitamin C and LVC gummies. The SEM images of the liposomal vitamin C formulation (Figure a) revealed predominantly spherical vesicles with smooth surfaces and no evidence of aggregation. The particle size ranged from 100 to 300 nm, with a mean diameter of 167.7 ± 19.2 nm, consistent with the DLS (Figure a) and TEM (Figure a) results, confirming the efficient encapsulation of vitamin C within the vesicles. SEM imaging of the LVC gummies (Figure b) showed well-dispersed, nearly spherical structures with characteristic fold-like surface features, indicating the presence of the cross-linked xyloglucan/trehalose/citric acid polymeric coating. The coated vesicles exhibited a larger size distribution (150–450 nm; mean 331.8 ± 21.4 nm), in agreement with the increased hydrodynamic diameter observed by DLS (Figure b) and the thicker outer shell visualized in TEM (Figure b).

5.

Scanning electron microscopic photographs of (a) liposomal vitamin C and (b) LVC gummy.

The larger apparent particle sizes observed in SEM relative to those in TEM are expected and arise from fundamental differences in imaging principles and sample preparation. TEM captures internal vesicle morphology under partially hydrated conditions, revealing the true liposomal core diameter, whereas SEM visualizes fully dried, polymer-coated samples. During SEM sample preparation, dehydration, polymeric shell thickness, matrix contraction, and surface roughening contribute to the increased apparent diameters. Such technique-dependent discrepancies are well documented for polymer-coated liposomes and do not indicate inconsistencies in vesicle formation. Moreover, the appearance of discrete nanospherical domains in the SEM micrographs is consistent with partial matrix contraction upon drying, which exposes individual coated vesicles. These observations align with the particle size data from DLS and confirm the structural uniformity and stability of both liposomal vitamin C and LVC gummy formulations.

3.8. Encapsulation Efficiency and Loading Capacity

The encapsulation efficiency (EE) and loading capacity (LC) of vitamin C in the LVC gummies were evaluated to determine the capability of the liposomal–polymeric system to retain and stabilize the bioactive compound. The results showed an EE of 85.72 ± 2.11% and an LC of 70.18 ± 1.36%. These values confirm that a large proportion of vitamin C was successfully entrapped within the liposomal vesicles and retained during the gummy preparation process. The high EE observed can be attributed to the strong affinity of vitamin C for the hydrophilic core of the vesicles, combined with the protective effect of the polymeric cross-linked coating. This liposome–polymer hybrid matrix minimized premature leakage and degradation during formulation, in agreement with previous reports on liposomal encapsulation approaches. Furthermore, the relatively high LC suggests efficient utilization of the gummy matrix for active loading, which is essential for ensuring an adequate dosage in small serving sizes. The differentiation between encapsulated and free vitamin C was achieved using a differential quantification method in which total vitamin C was measured following complete vesicle disruption, and the free fraction was obtained after centrifugation and membrane filtration. This analytical approach ensures that the calculated EE accurately reflects the vitamin C confined within liposomal vesicles rather than the unbound fraction in the gummy matrix. Similar quantification strategies have been validated in prior liposome-based encapsulation studies for hydrophilic bioactives. ,, Overall, these findings highlight that the ISSI-based encapsulation method effectively overcomes one of the primary challenges of vitamin C supplementation, its chemical instability, by maximizing retention and providing a robust system for controlled release and long-term protection.

Furthermore, the degradation of vitamin C during each stage of LVC gummy preparation was quantitatively monitored to assess the stabilizing efficiency of the ISSI liposomal–polymeric system. The results showed that vitamin C degradation was limited to 3% during liposome formation, 4% during polymeric coating, and 3% during gummy processing, resulting in a total loss below 10%. In comparison, CVC gummies exhibited nearly 25% degradation under similar processing conditions. This clearly demonstrates that the ISSI approach, integrating phospholipid vesicles with a xyloglucan/trehalose/citric acid polymeric matrix, effectively shields vitamin C from thermal and oxidative stress throughout the entire preparation process, preserving its structural and functional integrity.

3.9. Stability Studies

Accelerated color-stability tests performed under elevated temperature (40–60 °C) and humidity (30–75% RH) conditions revealed that the ISSI-based LVC gummies exhibited superior oxidative resistance and negligible color degradation compared with conventional vitamin C gummies (Table ). To substantiate these observations, long-term stability studies were conducted according to ICH Q1A­(R2) guidelines under controlled storage conditions (4, 25, and 45 °C for 180 days). The LVC gummies retained more than 90% of their initial vitamin C content throughout the 180-day period and maintained excellent physicochemical integrity with no detectable discoloration or structural deterioration (Table ). This performance greatly exceeds that of conventional vitamin C gummies, which typically lose a substantial proportion of vitamin C within 2–4 weeks due to oxidative and hydrolytic degradation. Conventional formulations are produced under high temperatures and lack protective encapsulation, leaving ascorbic acid directly exposed to oxygen and moisture. ,, In contrast, the ISSI technology used here forms a multilayer protective architecture in which phospholipid vesicles encapsulate vitamin C, gum-arabic nanospheres enhance interfacial stability, and a cross-linked xyloglucan/trehalose/citric acid matrix provides a dense outer barrier. This hybrid network restricts oxygen diffusion, modulates water mobility, and minimizes molecular degradation during storage. Similar multilayer nanoliposomal systems have been shown to markedly improve vitamin C stability in dry and semisolid matrices. These results collectively confirm that the ISSI-based liposomal–polymeric platform provides long-term stabilization far superior to conventional gummy technology, supporting the development of next-generation functional foods with extended shelf life and consistent bioactive retention. To further support the stability claims, vitamin C loss was quantified at each major processing stage. Less than 3% degradation occurred during liposome formation, 4% during polymeric coating, and approximately 3% during gummy preparation, resulting in a total loss of <10%. In comparison, conventional vitamin C gummies exhibited ∼25% degradation under comparable processing conditions. These findings reinforce that the ISSI hybrid vesicle–polymer system provides substantial protection not only during storage but also throughout manufacturing.

3. Vitamin C Content in the LVC Gummies under Different Stability Conditions.

	Vitamin C content (%)
Stability condition	Initial	30 days	60 days	90 days	120 days	150 days	180 days
4 ± 2 °C	96.74 ± 3.67	95.28 ± 2.32	94.19 ± 3.21	93.58 ± 2.78	92.18 ± 4.26	91.34 ± 3.48	90.41 ± 3.19
25 ± 2 °C	96.74 ± 3.67	94.48 ± 3.41	93.19 ± 2.94	92.12 ± 3.51	91.46 ± 4.36	90.45 ± 3.72	89.36 ± 3.24
45 ± 2 °C	96.74 ± 3.67	93.72 ± 3.76	92.65 ± 3.56	91.34 ± 4.31	90.74 ± 4.82	89.39 ± 3.35	88.82 ± 4.15

3.10. In Vitro Release Study

The release behavior of vitamin C from CVC gummies and LVC gummies was investigated under simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8) conditions for 12 h (Figure ). Both formulations (2.8 g) were incubated in 900 mL of medium at 37 °C with gentle stirring (120 rpm) using dialysis bags. In SGF (Figure a), CVC gummies exhibited an immediate burst release, with ∼58% of vitamin C released within 30 min, increasing to 67% at 2 h and reaching 72% at 12 h. This rapid release reflects the absence of a protective matrix, allowing free diffusion of vitamin C in an acidic medium. In contrast, LVC gummies released only 22% of vitamin C in the first 2 h and maintained a near-steady profile thereafter, indicating that the liposomal core and polymeric cross-linked coating effectively protected vitamin C from acid-catalyzed degradation. Although a slight reduction in the apparent cumulative release of LVC was observed after 2 h, this represents a pseudodecline resulting from transient vesicle aggregation and limited oxidation of the minor nonencapsulated vitamin C fraction rather than degradation of the encapsulated content. This behavior differs mechanistically from the decline observed for CVC under intestinal conditions, which is caused by actual oxidative degradation of unprotected vitamin C molecules. The lower cumulative release of LVC in SGF compared to CVC is therefore attributable to the dual barrier formed by the phospholipid bilayer and xyloglucan/trehalose/citric acid polymeric shell, which restricts diffusion and prevents premature release in gastric conditions. The slight decline observed in cumulative release after 2 h may arise from transient vesicle aggregation or limited oxidation of nonencapsulated vitamin C, rather than matrix collapse. The encapsulated fraction remains stable within the ISSI structure, minimizing proton- and oxygen-mediated degradation. ,

6.

Vitamin C release from CVC gummies and LVC gummies in (a) simulated gastric fluid (SGF) and (b) simulated intestinal fluid (SIF).

In SIF (Figure b), CVC gummies again showed a rapid initial release (∼61% within 1 h) followed by a decline to ≈43% at 12 h, which is attributed to oxidative degradation of free vitamin C under alkaline and oxygenated conditions. Conversely, LVC gummies exhibited a controlled and sustained release profile, increasing from 64% at 2 h to 78% at 12 h. This behavior results from gradual swelling and erosion of the polymeric coating at near-neutral pH, which facilitates the diffusion of vitamin C from intact liposomal vesicles. The biphasic pattern imited release in SGF and sustained release in SIF demonstrates the efficacy of ISSI-based encapsulation in providing gastric protection and promoting intestinal availability. , Collectively, these findings confirm that the ISSI-based LVC gummies possess enhanced physicochemical robustness and superior vitamin C retention compared to conventional formulations, positioning them as a promising functional nutraceutical for improved intestinal availability.

4. Conclusion

In this study, a novel liposomal vitamin C gummy (LVC gummy) was successfully developed using in situ soft sphere integration (ISSI) technology, followed by surface coating with a xyloglucan/trehalose/citric acid cross-linked polymeric matrix. The LVC system achieved a high encapsulation efficiency and loading capacity, confirming the effective entrapment of vitamin C within liposomal vesicles and its stabilization by the polymeric coating. Structural characterization by TEM and SEM revealed spherical vesicles with uniform polymeric layers, while particle size, zeta potential, and FTIR analyses verified successful encapsulation and matrix integration. The LVC gummies demonstrated desirable physicochemical properties, including optimal swelling ratio, dispersion time, pH, and water activity, confirming their suitability as consumer-friendly nutraceutical formulations. Accelerated stability studies indicated excellent vitamin C retention for up to 180 days under an elevated temperature, humidity, and light exposure. In vitro release studies further showed that LVC gummies minimized release in simulated gastric fluid while promoting a controlled and sustained release in simulated intestinal fluid, thereby enhancing the potential for improved bioavailability. The combined use of gum arabic nanospheres, phospholipid vesicles, and a citric acid cross-linked xyloglucan/trehalose matrix in a single in situ assembly process represents a distinct advancement in nutraceutical formulation technology. To our knowledge, this hybrid encapsulation strategy is the first to be applied specifically to heat-processed gummy delivery systems, providing thermal and oxidative protection not achievable with conventional liposomal methods. Overall, these findings establish ISSI-based hybrid encapsulation as a promising strategy for formulating robust and intestinally available vitamin C gummies. By combining high encapsulation efficiency with long-term stability and targeted release, this approach provides a scalable platform for developing functional gummies enriched with vitamin C and other sensitive bioactives, advancing innovation in functional foods and nutraceuticals. While the present study focused on the formulation, stability, and in vitro characterization of LVC gummies, future work will include comprehensive sensory evaluation to assess consumer acceptability, taste masking efficiency, and overall palatability of the developed formulation.

The authors declare no competing financial interest.