Vitamin D (Vit D) is a steroid hormone that has a well-known physiological role in calcium homeostasis required for the growth and maintenance of healthy bones. Vit D absorbs calcium, magnesium, and phosphate from the gut into the circulatory system. A schematic representation of circulatory Vit D and its biological roles is shown in Figure 1. Most of the bioavailable Vit D is produced in the skin as Vit D3 (Cholecalciferol) from 7-dehydrocholesterol in response to sunlight exposure, and a small amount (~ 10%) is also absorbed from a healthy and balanced diet, both in D3 and D2 (ergocalciferol) forms. Further metabolism of Vit D to its major circulating form, 25-hydroxyvitamin D or 25(OH)D (calcidiol), and biologically active hormonal form, 1,25 dihydroxyvitamin D or 1,25(OH)₂D (calcitriol), take place in the liver and kidney, respectively, but also in other tissues where 1,25(OH)₂D produced serves as paracrine/autocrine function such as the skin, cells of the immune system, parathyroid gland, intestinal epithelium, prostate, breast, brain, colon, and pancreas. Dietary Vit D comes in both D₂ and D₃ forms, but they are metabolized to the same active form of Vit D the body needs, i.e. calcitriol. Vit D receptors are ubiquitously present in the body (1) and thus play a plethora of physiological roles (Figure 1). For instance, Vit D deficiency (VDD), defined as circulatory 25(OH)D levels < 20 ng/ml, is not only linked with rickets or osteomalacia or osteoporosis but also with the risk of cardiovascular diseases, autoimmune diseases (such as type 1 diabetes, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis), impaired mental health conditions (such as depression, anxiety, and Parkinson's disease), tuberculosis, and cancer (breast cancer and colon cancer) (2). Vit D has also shown protective effects against respiratory tract infections (3–5), and there is emerging evidence of a possible association between VDD and progression to severe illness and mortality in patients with COVID-19 (6). In recent years, the role of Vit D in immune and inflammatory responses has been extensively studied. Reported evidence suggests Vit D as an endogenous regulator of both innate and adaptive immunity (3–5, 7–11). Circulatory Vit D levels affect the proliferation and differentiation of immune cells, and its deficiency results in impaired immune response and increases the risk of developing immune-related diseases.

The lack of exposure to sunlight, inadequate nutritional intake, malabsorption syndrome, and increased skin pigmentation have been identified as the main reasons for VDD (12). Vit D deficiency is a common metabolic/endocrine abnormality and a global public health problem. Approximately, over one billion people worldwide are Vit D deficient, and the deficiency is frequently undiagnosed. The prevalence of VDD in Europe, USA, and the Middle East ranges from 20 to 90% (13, 14), with similar trends also reported in Australia, India, Africa, South America, Turkey, and Lebanon (13–16). In South Asia, Pakistan has the highest rate of VDD of 53.5%, mostly in its female population (63%) (17).

Sunlight exposure and dietary intake alone are insufficient to maintain optimal circulatory Vit D levels (30–50 ng/ml) required for physiological functions (13, 18). To achieve and maintain adequate circulatory Vit D levels, over-the-counter Vit D supplements are often consumed, which can be taken daily, weekly, or monthly. Currently, there is no international consensus on the optimal dose of Vit D supplementation. Dosage recommendations vary across countries, ranging from 400 to 2000 IU Vit D per day (10–50 μg). Supplemental Vit D3 form is generally preferred because it appears to be more efficacious in raising circulatory 25(OH)D concentrations than the D2 form. Vit D3 supplement is usually administered orally and produced in various forms, such as oral drops, tablets, soft gel capsules (SGC), and injectable oily solutions. However, despite the widespread consumption of Vit D supplements for treating VDD, the absorption (and hence efficacy) of different Vit D formulations has not been well studied (19). Vit D, being a fat-soluble vitamin, is immiscible in the aqueous gut environment which presents challenges to its absorption. Its absorption in the intestinal mucosa is highly dependent on its food/delivery matrix dissolution in the gastrointestinal (GI) fluid (20–27) (stomach juices, liver bile, liver, and pancreatic secretions) and the integrity of the intestinal wall. The absorption is further limited by multiple factors including the physiochemical state of Vit D3 food/delivery matrix, Vit D interaction with other compounds in the food such as cholesterol, fatty acids, and dietary fibers, and factors associated with humans such as malabsorption syndrome, other chronic diseases, age, medication use, surgery, obesity, and genetic variation (28). To overcome these negative factors and intestinal barriers, the development of more bio-accessible delivery vehicles can enhance absorption and achieve adequately higher Vit D levels in the circulatory system for physiological roles.

The present study aimed to assess the GI absorption (hence efficacy) of a high-dose (200,000 IU) supplementation of an innovative oral phospholipids-sucrester matrix delivery vehicle-based, also known as sucrosomial^® (29) orodispersible Vit D3 formulation vs. a reference marketed chewable tablet and soft gel capsule (SGC) Vit D3 formulation in raising circulatory 25(OH)D levels, as well as safety, and tolerability in Vit D deficient but otherwise healthy adults. It is anticipated that the unique structural, physiochemical, and pharmacokinetic characteristics of the sucrosomial^® Vit D3 preparation could offer several advantages over the conventional oral Vit D3 formulations such as stability in the GI tract, efficient intestinal absorption, increased Vit D3 bioavailability, and improved patient compliance, particularly in pediatric and geriatric patients, and those with malabsorption syndrome.

This exploratory prospective clinical trial for the first time assessed the efficacy of an innovative sucrosomial^® orodispersible Vit D3 formulation vs. a reference chewable tablet (study 1) and SGC (study 2) Vit D3 preparation in Vit D-deficient healthy adults and revealed that the sucrosomial^® Vit D3 is more efficacious in raising the circulating 25(OH)D levels owing to its efficient absorption. Moreover, the results of study 1 were validated by the results in study 2 which was conducted in a different province (Khyber Pakhtunkhwa) of Pakistan and involved participants of different ethnic background (Pashtuns) as compared to study 1 which was conducted in Balochistan province of Pakistan and involved participants of Baloch ethnicity, demonstrating the generalization of the results of efficient absorption/efficacy of sucrosomial^® Vit D3 supplementation. Vit D3 in the sucrosomial^® formulation is protected by a phospholipids, made mainly from sunflower lecithin plus a sucrester (sucrose ester) matrix (Figure 5). Additional stability and coating are achieved thanks to the presence of other ingredients as starch and tricalcium phosphate and forming a “sucrosome” delivery vehicle. The gastro-resistant properties of sucrosome (34) are believed to protect Vit D3 from degradation through its journey in the GI tract, decrease its interactions with food in the stomach, and promote transport and absorption across the intestinal epithelium to the circulatory system. The intact sucrosome allows the Vit D3 to reach the intestinal mucosa where it is absorbed as a vesicle-like structure through para-cellular and trans-cellular (M-cells) routes (29, 34–36). The unique structural, physicochemical, and pharmacokinetic properties make sucrosomial^® technology of significant interest in drug delivery applications. Reported evidence suggests that sucrester enhances the accumulation of drugs in CACO-2 cells (37) and acts as an enhances of intestinal permeability, as shown in animal model studies (38). In previous studies, with iron as an active agent, sucrosomial^® biodelivery vehicle has been shown to achieve high iron bioavailability and low gastrointestinal toxicity including in patients with chronic kidney disease, cancer, and bariatric surgery (29, 34, 36). In a recent study by Bertani et al. (39), an oral sucrosomial^® iron formulation was shown as effective as intravenous iron in treating anemia in patients with ulcerative colitis.

In addition to facilitating efficient intestinal absorption, another possible benefit of sucrosomial^® orodispersible Vit D3 could be its absorption in the oral cavity. Our study showed that the sucrosomial^® Vit D3 when taken inside the mouth, dissolves within moments when combined with saliva, and the fine micro-sized droplets containing Vit D3 are then possibly (and efficiently) absorbed through the buccal mucosa, palatal membranes, and sublingually into numerous capillaries and veins, close to the tissue surface. The oral cavity has a rich blood supply and is relatively permeable, facilitating an efficient route for Vit D absorption into the circulatory system. In the oral cavity route, Vit D3 bypasses the GI absorption pathway and is protected from degradation due to the pH and digestive enzymes of the GI tract (40), thus contributing to increased circulatory levels. It is speculated that the sucrosomial^® Vit D3 oral cavity absorption properties may well prove superior for those with GI malabsorption syndromes such as individuals with cystic fibrosis (41), Crohn's disease (42), intestinal reresection (43, 44), ulcerative colitis, and liver disease (45, 46), who fail to achieve adequate circulatory 25(OH)D levels, despite routine supplementation and for individuals with difficulty swallowing such as the elderly, young children, and babies.

The absorption of Vit D3 preparations in the oral cavity has been investigated in several studies. In study by Khazi et al. involving cystic fibrosis patients, a greater bioavailability of powder Vit D preparation was observed compared with an oil-soluble vehicle (22). In study by Satia et al., a buccal spray Vit D3 preparation led to significantly higher circulatory 25(OH)D concentration as compared with SGC Vit D3 preparation, in both healthy subjects and patients with malabsorption syndrome (26). In study by Cupone et al., an orodisintegrable film Vit D3 preparation showed disintegration of the vehicle in less than 1 min and the Vit D3 release was ≥75% after 15 min (20). In a similar study by Radicioni et al., a higher Vit D3 bioavailability was achieved with an orodispersible Vit D3 preparation compared with an oral solution preparation in healthy subjects (25). In general, among most of the previously reported studies of Vit D3 oral cavity absorption, the sucrosomial^® Vit D3 preparation appears to be the most effective for achieving increased bioavailability of Vit D, owing to its unique structural and physiochemical characteristics. However, further studies are required to establish the absorption of sucrosomial^® Vit D3 in the oral cavity.

This study also demonstrated excellent safety and tolerability profile of the sucrosomial^® Vit D3 supplement in high dosage, and no cases of hypercalcemia occurred. There were no negative effects on the liver function enzymes and kidney function (creatinine) as well as hematology in any of the study participants throughout the study 6-weeks period. The adequately higher circulatory Vit D levels achieved with high-dose sucrosomial^® Vit D3 supplementation in this study, alongside excellent safety and tolerability, could provide a rationale for clinical trials to assess whether such Vit D levels are associated with immunomodulatory pharmacological effects in autoimmune diseases and infection risk (47–49).

Our study is not free from limitations. The relatively small sample size, open-label nature, and absence of diseased participants such as frail elderly, those with chronic conditions, e.g. chronic liver or kidney diseases, and malabsorption syndrome are some of the drawbacks of this study. Nevertheless, those involved in the randomization, enrolment of participants, drawing the participants' blood samples, clinical diagnostic laboratories evaluating the serum 25(OH)D levels, and investigators assessing the outcomes were blinded to the intervention. Moreover, the results of study 1 were validated by results of the study 2 conducted in a different geographic location and involving participants of different ethnic background, demonstrating the wider applicability of the results.

According to the results of our study, the sucrosomial^® orodispersible Vit D3 preparation dissolves and absorbs efficiently in the GI system, leading to adequately higher and sustained circulatory Vit D levels as compared to a chewable tablet and soft gel capsule forms of Vit D3 preparations in Vit D-deficient healthy adults, and thus could contribute effectively to body protection against diseases associated with VDD.

Data supporting the findings of this study are included within the article and/or Supplementary material, further inquiries can be directed to the corresponding author.

The study was reviewed and approved by the BMCH Ethical Board Committee (Ref. no. BMCH/EBC/5500) and LRH Institutional Review Board (Ref. no. 696/LRH/MTI). The patients/participants provided their written informed consent to participate in this study.

AK, AB, AAB, and SAb: conceptualization, methodology, resources, supervision, and project administration. AB, SAb, AAB, FA, MB, SM, and HZ: investigation and data curation. MF and GC: statistical analysis. AK: writing—original draft preparation, conception and coordination of the study, full access to all data in the study, and responsibility for the integrity of the data and the accuracy of the data analysis. ID, SAs, EB, GT, AG, ST, and SK: writing—reviewing and editing. All authors have read and approved the final version of the manuscript for submission.