**1. Introduction**

Vitamin D or calciferol is a lipid-soluble vitamin which is a combination of various steroidal derivatives. These derivatives include: cholecalciferol (vitamin D3), which is a derivative of cholesterol, calcidiol (partially active hydroxylated form of cholecalciferol), calcitriol (dihydroxylated active form), ergocalciferol (D2), and its mono and dihydroxylated derivatives [1]. Vitamin D is both endogenous and exogenous. The human skin epidermis, when exposed to ultraviolet-B (UVB) radiation present in sunlight, produces

**Citation:** Ahmed, M.Z.; Gupta, A.; Warsi, M.H.; Ali, A.M.A.; Hasan, N.; Ahmad, F.J.; Zafar, A.; Jain, G.K. Nano Matrix Soft Confectionary for Oral Supplementation of Vitamin D: Stability and Sensory Analysis. *Gels* **2022**, *8*, 250. https://doi.org/ 10.3390/gels8050250

Academic Editors: Kiat Hwa Chan and Yang Liu

Received: 12 March 2022 Accepted: 15 April 2022 Published: 19 April 2022

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

vitamin D3 from 7-dehydrocholesterol endogenously. Exogenous vitamin D is sourced from dietary food, such as dairy, oily fish, liver, egg yolk, etc., and supplements. Since the natural food options which can provide exogenous vitamin D are quite limited, supplementation is increasingly encouraged to counter its deficiency. Vitamin D in general, refers to vitamin D3. Its primary circulating form 25-hydroxyvitamin D is converted to active metabolite 1,25-dihydroxyvitamin D within the human body [2].

Individuals having serum vitamin D levels (i.e., 25-hydroxyvitamin D [25(OH)D]) below 20 ng per milliliter or 50 nmol per liter (deemed appropriate level by the Institute of Medicine in 2011) are considered vitamin D deficient. In 2011, after a comprehensive analysis of the literature, the Institute of Medicine (IOM) reached the conclusion that a 25(OH)D concentration of 20 ng/mL or above in blood was adequate for optimal bone health [3–5]. Vitamin D deficiency is associated with a number of clinical manifestations in people of different age groups. It is predominantly associated with bone health, and vitamin D deficiency is well understood to contribute to musculoskeletal disorders. Without vitamin D, just 10–15% of dietary calcium and about 60% of phosphorus are absorbed. [2]. Vitamin D deficiency results in musculoskeletal fatigue, and low vitamin D levels have been observed in conjunction with rheumatoid arthritis, multiple sclerosis, and arthritis, closely associated with many cardiovascular risk factors, e.g., myocardial heart infarction, congestive heart failure [6], diabetic cardiovascular disease, and peripheral arterial disease. A number of studies have suggested the role of vitamin D deficiency in increased cerebrovascular accident (CVA) risk and it has been linked to an elevated risk of depression and schizophrenia [7–9]. Vitamin D modulates the role of B and T-lymphocytes and regulates cell differentiation and proliferation [10,11]. Multiple studies have acknowledged that women represent a high risk population for vitamin D deficiency, with pregnant women being at higher risks, leading to multiple pregnancy complications, e.g., gestational diabetes and preeclampsia [12]. Vitamin D deficiency is prevalent across the world, with an estimated 1 billion people suffering from the condition [13]. According to several reports, 40–100% of elderly people in the U.S. and European countries are vitamin D deficient. Furthermore, various studies have shown low vitamin D status regardless of age, gender, and geography [2,13–15]. According to a meta-analysis report from 2015, vitamin D deficiency was 35% more frequent among obese people irrespective of age or demography [16]. Vitamin D deficiency has been associated with populations having higher levels of melanin in their skin and the populations that use extensive skin care, notably in Middle East nations [13,17].

The Institute of Medicine (IOM) has published guidelines for vitamin D supplementation, suggesting 600 IU/day vitamin D for most of the population aging between one and 70 years. The Recommended Dietary Allowance (RDA) of vitamin D for individuals above 70 years of age is 800 IU per day and for infants up to one year of age is 400 IU a day [18]. For the treatment and prevention of vitamin D deficiency, the Endocrine Society in the United States recommended more than 30 ng/mL of serum 25(OH)D concentrations to be achieved, more preferably in the range of 40–60 ng/mL [4]. To reach a serum 25(OH)D level of 30 ng/mL, all deficient adults should be provided with either 50,000 IU weekly of vitamin D3 or 6000 IU daily for 8 weeks. A daily maintenance dose of 1000 IU in patients up to 18 years and 1500–2000 IU daily in patients aging 18–50 years is recommended by The Endocrine Society [4,19].

Limitations with supplemental solid oral delivery: Oral delivery is perceived as most reliable route for the administration of active pharmaceutical ingredients (APIs). More than 60% of all medicines comprise of solid oral dosage forms due to ease of manufacture, availability of large number of excipients, cheaper development process, more accurate dosing compared to oral liquids, and ease of handling and dispensing [20]. Since it is non-invasive, patients are more comfortable compared to other routes. Major disadvantages with oral formulations include low gastric and intestinal solubility, which reduces the bioavailability of large number of pharmaceuticals and rapid stomach breakdown, possibly followed by degradation of the API by gastrointestinal (GI) tract enzymes [21]. Another critical concern is patient compliance among children and the elderly majorly due to dysphagia. A questionnaire research revealed that 26% of 6158 patients reported difficulties swallowing

tablets [22]. Another study found that 37.4% of all participants (*n* = 1051) reported having trouble swallowing tablets and capsules [17]. When it comes to supplementation (either prophylactic or treatment), where a medication or dose is not usually regarded equally relevant or critical as other urgent needs or treatment medications, the graph of compliance further falls. Adults with dysphagia or those reluctant to take medications usually ignore the supplementary pills. Parents usually miss the supplements when given as solid orals due to reluctance of children to take the pills.

Limitations with vitamin D stability and bioavailability: Cholecalciferol is highly susceptible to environmental degradation (sunlight, heat, and oxygen), and readily undergoes isomerization or oxidation when exposed to open air in its raw form, resulting in a loss of its functioning and physiological implications. Several studies report that it is poorly water-soluble and solubilizing vitamin D in some carrier oils protects it from the effects of heat or oxidation [23–25]. Vitamin D has very limited bioavailability, which limits its effectiveness when given as an oral supplement, and higher doses are required to maintain effective levels. It has been observed that only around half (~50%) of the Vitamin D3 consumed orally is absorbed [26].

Lipid-based formulations have gained commercial recognition by improving the oral bioavailability and solubility of poor water-soluble drugs. They enhance the drug absorption by solubilizing the poorly water soluble or insoluble drugs and promoting the emulsification process in the GIT. The application of nano-emulsions (NEs) in the food sector has attracted growing interest owing to the changes in the physicochemical attributes and biological efficiency associated with particle size modifications. Particle size reduction in emulsion-based formulations can have a variety of effects that may benefit various food and supplemental applications: (i) enhanced stability against aggregation and separation of droplets under gravity; (ii) greater visual clarity; and (iii) improved oral bioavailability [27,28]. In one of the studies conducted by Salvia-Trujillo et al., lipid droplets of a relatively smaller size were digested considerably quicker in simulated GI fluid suggesting mixed micelles that solubilize lipophilic vitamins may develop more quickly in the small intestine [27]. Because of their potential to yield robust and transparent delivery systems with excellent oral bioavailability, NEs are particularly well suited for encapsulating lipophilic nutraceuticals [29]. NEs are dual phased thermodynamically stable systems comprising of at least two immiscible liquids having droplet sizes in the nanometer range usually below 200 nm. They have good kinetic stability upon storage and can be fabricated with minimal surfactant concentrations [30,31]. High-energy emulsification technologies, such as using ultrasonic generators or high-pressure homogenizers, can be used to synthesize NEs. Nano emulsion-based soft chewable matrices that are easy to chew and swallow and have the potential to entrap the medication, giving sustained, controlled release while avoiding quick disintegration in the stomach, have shown promise as delivery systems.

Vitamins in chewable gummy form have become increasingly common today among both the young and adults. Gummy supplements are reported to accommodate the 'pill fatigue' of consumers and to mask the 'vitamin taste' that is often very poor or has an odious smell [32]. Nowadays, children with complete dentition readily embrace gelled gummies and candies because of their good taste and chewability, since they are frequently flavored with various fruity and other lucrative flavors and extracts and are sweet in nature. Presently, two out of the top 10 vitamin D supplements are gummies. This is due to the widespread acceptability of gummy soft chew supplements among people of all age groups. It has also been evident in many studies that children's nutritional status has been improved with chewable vitamins [32,33]. In the light of above considerations, a nano-emulsionbased soft confectionary system was developed, having a soft chewable matrix, which was followed by an evaluation of its texture profile, dissolution, and consumer compliance.

### **2. Materials**

Vitamin D (97–99% pure) was procured from HiMedia Laboratories Pvt. Ltd. Mumbai, India. Cold pressed pure corn oil was purchased from Deve herbes, Delhi, India. Gelatin

160 g bloom strength, Poloxamer 188, lecithin, and sucralose were purchased from Sigma-Aldrich, Inc., St. Louis, MO, USA. Transcutol and Poloxamer 407 were purchased from Gattefosse, Saint-Priest, France. Tween 20, Tween 80, Tween 60, and Span 20 were bought from Loba Chemie Pvt. Ltd., Mumbai, India. HPLC grade methanol and water were bought from Merck, Mumbai, India. Food Safety and Standards Authority of India (FSSAI) grade sorbitol/xylitol, sucralose, citric acid, and flavors were purchased from local vendors.

### **3. Experimental Design**

### *3.1. Preparation and Optimization of Vitamin D Nanoemulsion*

The oil phase of nanoemulsion was selected based on the reported solubility of vitamin D in various oils [34–39]. Surfactant and co-surfactant were selected based on their ability to spontaneously emulsify corn oil [40]. Briefly, 100 mg of corn oil was dissolved in 3 mL of methylene chloride and then surfactant/co-surfactant (10 mL) was added and stirred at 200 rpm (at 40 ◦C) using magnetic stirrer to remove methylene chloride. Thereafter, 1 mL of sample was withdrawn and in it 10 mL of distilled water was added gradually, and percentage transmittance was determined at 339 nm wavelength using a UV spectrophotometer [41]. All the measurements were performed in triplicate.

Nanoemulsion optimization was performed by aqueous titration method followed by pseudo-ternary phase diagram as described previously [42,43]. Corn oil containing a weighed quantity of vitamin D was admixed with the combination of surfactant and co-surfactant (Smix) in different ratios (1:1, 2:1, 1:2, 1:3 and 3:1). The mixtures were then titrated with water, vortexed, and analyzed visually for their appearance, clarity, or turbidity (Table 1) and plotted as ternary phase diagram. Clear dispersions (nanoemulsions) with a minimum concentration of Smix were selected for further evaluation [44].

**Table 1.** (**A**). Grading system of oil and Smix based on appearance and emulsification time. (**B**). Visual screening data of nano emulsions using different ratio of Smix and oil.

