Optimizing vitamin B12 delivery in solid pharmaceutical formats: The Role of Gelatin in Stability and Formulation Performance

Optimizing vitamin B12 delivery in solid pharmaceutical formats: The Role of Gelatin in Stability and Formulation Performance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimizing vitamin B12 delivery in solid pharmaceutical formats: The Role of Gelatin in Stability and Formulation Performance Garima Chauhan, Tarun Goel, Pankaj Attarde, Heather Rodebush, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8670841/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Cyanocobalamin delivery in solid oral formulations is limited by gastric acid instability and the dependence on impaired haptocorrin/ intrinsic factor/ pathways in groups at high-risk of vitamin B12 deficiency such as metformin-treated diabetic patients (14–41% vitamin B12 deficiency) and older adults (10–40%). Gelatin encapsulation may enhance vitamin B12 delivery at therapeutic doses via acid protection and mucoadhesion. Methods In vitro studies compared 1% w/w gelatinised cyanocobalamin with non-gelatinised vitamin B12 using simulated gastric fluid (SGF, pH 1.2; HPLC stability 0–3 h) and differentiated Caco-2 monolayers (24 h apical uptake ± SGF/SIF preexposure; LC17 MS/MS; TEER). Results Gelatinised vitamin B12 retained 97.1% to 98.3% whereas non-gelatinised vitamin B12 retained 90.0% to 93.7% of the total vitamin B12 content in SGF (3-fold less degradation, 1-3h). Caco-2 monolayers presented 3˜-fold higher intracellular/basolateral vitamin B12 with gelatinised than with non-gelatinised vitamin B12, without TEER disruption. Conclusions Gelatin encapsulation confers superior gastric stability and epithelial uptake, supporting progression to pharmacokinetic studies for improved therapeutic vitamin B12 delivery in absorption-compromised populations. vitamin B12 gelatin encapsulation gastric stability Caco-2 passive absorption Figures Figure 1 Figure 2 1 Introduction Vitamin B12 (cobalamin) is an essential water-soluble vitamin required for red blood cell maturation, DNA synthesis, methylation pathways, and normal neurological function ( 1 – 3 ). A deficiency disrupts erythropoiesis, impairs myelin integrity, and can manifest as megaloblastic anaemia, peripheral neuropathy, cognitive decline, or neuropsychiatric symptoms ( 1 , 4 , 5 ). Cobalamin exists in several chemical forms: cyanocobalamin and hydroxocobalamin are synthetic or modified forms widely used pharmaceutically, whereas methylcobalamin and adenosylcobalamin are the metabolically active cofactors in human tissue ( 2 , 6 , 7 ). Among these different chemical forms, cyanocobalamin remains the most commonly used form in oral supplements, medicinal products and fortified foods because of its cost-effectiveness and greater chemical stability, with comparable absorption profiles when given at supplementation and therapeutic doses ( 6 , 8 – 10 ). Clinically significant vitamin B12 deficiency is common in multiple risk groups: type 2 diabetes patients especially those treated with metformin (14–41%), pernicious anaemia and post-gastrectomy patients (approximately 100%), and older adults (10–15% in community-dwelling; up to 40% in institutionalised), vegetarians or vegan, chronic alcoholism and others ( 11 – 16 ). If left untreated, vitamin B12 deficiency can lead to serious haematological and neurological complications ( 3 , 5 ). Standard management of vitamin B12 deficiency and peripheral neuropathy includes high-dose oral cyanocobalamin or intramuscular injections, in combination with other neurotropic B vitamins (B1, B6). Under normal physiological intake, vitamin B12 is absorbed through a combination of high-affinity, receptor-mediated routes with a low-efficiency passive diffusion at low microgram doses; at larger pharmacologic doses used in many oral neurotropic B-complex products, overall uptake is predominantly by passive diffusion, with approximately 1–2% of the ingested dose crossing the intestinal epithelium into the circulation ( 3 , 6 ). However, conventional cyanocobalamin is chemically labile under strongly acidic conditions, such as those found in the gastric environment, and has a markedly shorter half-life at gastric pH than under near-neutral conditions ( 17 – 19 ). In addition to gastric acidity, cobalamin is sensitive to light and humidity in solid pharmaceutical forms, and the relatively low amount used in solid pharmaceutical formats (microgram range per unit) creates additional challenges for ensuring content uniformity and maintaining potency throughout manufacturing and storage ( 20 , 21 ). Gelatin is a collagen-derived, biodegradable excipient that is generally recognised as safe (GRAS) status and has a long history of use in pharmaceutical dosage forms, including capsules and matrix systems to improve ingredient stability and help with dosing uniformity, especially at small amounts ( 22 – 24 ). Recent work has highlighted the ability of gelatin to form dense, pH-responsive matrices, interact with mucus, and act as a platform for controlled release and targeted delivery in oral preparations ( 25 – 27 ). In commercial pharmaceutical products, gelatin-based capsules are used to maintain content uniformity and product stability ( 28 ). Although gelatin is widely used in pharmaceutical formulations, the protective effects of gelatin encapsulation specifically on oral cyanocobalamin stability and bioavailability remain poorly characterised ( 27 , 29 , 30 ). Recent research has highlighted the potential of gelatin in vitamin B12 delivery systems, particularly through nanofiber formulations and sustained-release mechanisms ( 29 ) and related lipid-based carriers have shown enhanced passive absorption of vitamin B12 via P-glycoprotein inhibition ( 31 ). However, the dual protective effects of gelatin—acid stabilization combined with improved cellular uptake or absorption—have not been comprehensively characterised in complementary in vitro models. Gelatinised vitamin B12 technology is designed to address several of these constraints simultaneously: by embedding cyanocobalamin (a common pharmaceutical form of vitamin B12) within a gelatin matrix, it can enhance stability against gastric acid and maintain higher free vitamin B12 concentrations in proximity to the intestinal epithelium, thereby maximising the contribution of passive diffusion at pharmacologic oral doses. This study therefore aims to characterise the role of gelatin in the protection of vitamin B12 during gastric transit using in vitro models by evaluating the impact of gelatin encapsulation on acid stability and epithelial uptake. 2 Materials and Methods 2.1 Materials Cyanocobalamin active pharmaceutical ingredient (API, Hebei Huarong Pharmaceutical Company Ltd, China) was used as the reference vitamin B12 form. Gelatin (Rama Industries Ltd, bovine source) suitable for pharmaceutical use was employed to manufacture gelatinised vitamin B12 granules containing approximately 1% w/w vitamin B12 in a 99% gelatin matrix, which is consistent with established commercial formulations. Simulated gastric fluid (SGF; pH 1.2) was prepared according to the USP (US Pharmacopeial), and other reagents were of analytical grade. 2.2 Preparation of gelatinised Vitamin B12 Gelatinised vitamin B12 can be manufactured via various methods such as spray drying or fluid-bed granulation. The gelatinised vitamin B12 granules (1%, Supreem Pharmaceuticals, India) used in this study were produced via fluid-bed granulation, in which small gelatin cores were sprayed with a solution of cyanocobalamin and gelatin under heated air (45°C), followed by drying and application of a final gelatin coating (Fig. 1). This process yielded granules with consistent particle sizes (80–100% passes through mesh 60#) and uniform vitamin B12 contents (1% loading, hereinafter referred to as 1% gelatinised vitamin B12) embedded within a continuous gelatin matrix and is analogous to the process used in marketed Neurobion® formulations containing 100mg of B1, 100-200mg of B6, 200mcg − 5000mcg of B12. For in vitro experiments, 1% gelatinised vitamin B12 granules were dispersed and diluted to the target vitamin B 12 concentration in the relevant test media, and non-gelatinised vitamin B12 solutions were prepared at matched cyanocobalamin concentrations Figure 1: Manufacturing process flow chart for vitamin B12 embedding in gelatin. 2.3 Simulated Gastric Fluid (SGF) Stability Studies The concentration of vitamin B12 (both non-gelatinised and 1% gelatinised) was adjusted based on the gastric volume, and label claim of vitamin B12 in the formulation (200 mcg). Stock solutions of 1% gelatinised vitamin B12 and non-gelatinised vitamin B12 were prepared in SGF pH 1.2 media to achieve concentration of 0.4 ppm (equivalent to 200 mcg dose, n = 3), similar to the concentration from Neurobion® and other preparations on the market containing 200 mcg vitamin B12. The vitamin B12 content was quantified via reversed-phase high-performance liquid chromatography (HPLC) with 0.1% trifluoracetic acid (TFA) solution and acetonitrile as the mobile phase with a gradient pump mode via a phenyl column (150 mm × 4.6 mm) and UV detection at 361 nm. The amount of vitamin B12 was measured at various time points (initial, 1 hour, 2 hour and 3 hour) in the simulated setup until gastric emptying for both 1% gelatinised vitamin B12 and non-gelatinised vitamin B12. 2.4 Caco-2 cell culture and uptake assays Human Caco-2 cells (American Tissue Culture Collection, Manassas VA USA, ATCC cat# HTB-37) were cultured on permeable inserts (growth area 3.14 cm 2 , pore size 0.4 µm) at a density of approximately 1 × 10 5 cells per insert and maintained in standard growth medium (EMEM ATCC cat#30-2003 supplemented with 10% FBS- ATCC cat#30-2020). The medium was changed 24 h after seeding and every other day thereafter until the monolayers reached differentiation and confluence (18 to 21 days), as evidenced by stable transepithelial electrical resistance (TEER) readings of approximately 750. Before the experiments, the baseline TEER was recorded to confirm barrier integrity. Gelatinised and non-gelatinised vitamin B12 formulations were prepared in cell culture medium with or without prior exposure to simulated gastric and intestinal fluids to mimic gastrointestinal transit ( 27 ). The formulations were applied to the apical (mucosal) side (n = 4 for each treatment group), with fresh medium in the basolateral compartment, and the cells were incubated for 24 h at 37℃ ( 32 ). After incubation, the cells were washed to remove extracellular vitamin B 12 and lysed, and the basolateral vitamin B12 content was quantified via LC to MS/MS ( 27 ). TEER was remeasured to assess whether barrier integrity was maintained during exposure ( 32 ). SGF (VWR cat# RC7108-16) was prepared according to USP guidelines, with 3.2 g of pepsin (VWR cat#M142-250G)/Liter added to make complete SGF. For simulated intestinal fluid (VWR cat# RC7109-16), SIF, 10 g/Liter of pancreatin (VWR cat # 75875-592) was used. Prior to the application of SGF/IF-treated vitamin B12 solutions to Caco-2 cells, 30 µL of Protease Inhibitor Cocktail (ThermoFisher cat# 78429) was added to mitigate the deleterious impact of pepsin/pancreatin on Caco-2 cells. 2.5 Data Handling and Analysis For each experiment, values were generated from replicate samples or inserts per condition ( 18 , 27 , 32 ). The results are reported descriptively as approximate fold differences between gelatinised and non-gelatinised vitamin B12 based on mean values, reflecting the mechanistic focus and in vitro nature of the work ( 27 ). Where relevant, literature was used to contextualise the magnitude of observed effects relative to physiological constraints and clinical dosing paradigms ( 6 , 33 ). 3 Results 3.1 Gelatin Encapsulation Enhances Vitamin B12 Stability in Simulated Gastric Fluid The stability results of the sample solutions, with a concentration of 0.4 ppm (equivalent to a 200 mcg dose) of non-gelatinised vitamin B12 and 1% gelatinised vitamin B12 prepared in Simulated Gastric Fluid (SGF) pH 1.2 media were analysed by HPLC at different time intervals: initial, 1 hour, 2 hour and 3 hour. As shown in Table 1 , for 1% gelatinised vitamin B12, 1.7%, 2.3% and 2.9% losses were observed at 1 hour, 2 hour and 3 hour respectively, whereas for non-gelatinised vitamin B12, 6.3%, 8.6% and 10.0% loss were observed from the initial assay at 1 hour, 2 hour and 3 hour respectively. The test data demonstrate that gelatin encapsulation (1% vitamin B12 and 99% gelatin matrix) protects vitamin B12 from degradation under acidic stomach conditions, with a mean result of 3.6 times less loss of vitamin B12 due to gelatin encapsulation. Table 1 Gelatinised vitamin B12 loss vs non-gelatinised vitamin B12 loss at different time points Duration in SGF 1% Gelatinised vitamin B12 loss % Solution Concentration (200mcg), n = 3 (A) Non-Gelatinised vitamin B12 loss % Solution Concentration (200mcg), n = 3 (B) Total loss ratio [B/A] 1 hr. Mean: 1.7% loss SD: 2.2 Mean: 6.3% loss SD: 4.4 3.7 2 hr. Mean: 2.3% loss SD: 2.9 Mean: 8.6% loss SD: 5.9 3.7 3 hr. Mean: 2.9% loss SD: 3.2 Mean: 10.0% loss SD: 6.7 3.4 Mean: 3.6 SD: 0.2 The percentage loss (%) of vitamin B12 at 1 hour, 2 hour and 3 hour was compared against the initial assay results for both gelatinised and non-gelatinised vitamin B12. The HPLC method used for assessing vitamin B12 stability in simulated gastric fluid is operating at a low sample concentration of 0.4 ppm, which can lead to variability in the results, hence the probability of higher standard deviations cannot be eliminated. 3.2 Gelatinised Vitamin B12 Increases Cellular Passage in Caco-2 Monolayers Without Compromising Barrier Integrity Figure 2 shows that 1.2% of a dose of non-gelatinised vitamin B12 is absorbed which is in line with clinical observations showing that 1–2% of a dose of vitamin B12 is absorbed via the passive absorption pathway. When the same amount of vitamin B12 embedded in gelatin (1% vitamin B12 and 99% gelatin matrix) was added to the cells, a 3-fold increase in absorption was noted (3.6%). This result was achieved both when gelatinised vitamin B12 was added directly to cells or when it was pre-incubated with simulated gastric and simulated intestinal fluids for 1 hour each (to approximate the conditions of the stomach and first parts of the small intestine). Figure 2: Intracellular vitamin B12 levels in Caco-2 monolayers after 24 h of incubation with gelatinised vitamin B12 vs pure vitamin B12, with and without simulated gastric fluid (GF) and intestinal fluid (IF) 4 Discussion Exposure to simulated gastric fluid (SGF; pH 1.2, 37℃) for 3 hour led to substantial degradation of non-gelatinised vitamin B12, with only approximately 90.0% to 93.7% of the initial vitamin B12 content remaining at matched starting concentrations. In contrast, gelatinised vitamin B12 granules (1% vitamin B12 in a 99% gelatin matrix) retained approximately 97.1% to 98.3% of their initial vitamin B12 content under identical conditions, corresponding to an approximate threefold reduction in vitamin B12 loss compared with non-gelatinised vitamin B12 (cyanocobalamin). This observation aligns with the hypothesised dual protection mechanism, which comprises first a dense, minimally swollen gelatin matrix that physically limits hydrogen ion penetration into the core for approximately 60 min at pH 1.2 ( 34 ) and, second, proton-accepting amino acid residues (approximately 13% of the polypeptide chain of gelatin are positively charged) within the gelatin that buffer the local microenvironment around the encapsulated vitamin B12 ( 27 , 35 ). Gelatin-based matrices have been reported to provide sustained protection over several hours; with crosslinked gelatin hydrogels remaining mechanically stable for 4 to 6 hr under physiological conditions, which is consistent with the gastric residence time of 1 to 3 h ( 35 , 36 ). Scintigraphic and transit studies indicate that, depending on the prandial state and formulation, the upper gastrointestinal residence time (stomach plus small intestine) for solid and drinkable dosage forms typically lies within a 1–3 hr window in adults, extending toward 3–4 hr in the fed state ( 37 – 40 ). The observed threefold reduction in cyanocobalamin degradation within 1–3 hours in SGF is regarded as clinically significant, as this period corresponds to the typical duration during which most of an orally administered dose is subjected to gastric conditions prior to reaching the small intestine, where predominantly passive absorption occurs at high oral doses ( 41 , 42 ). As the product is taken with or shortly before a meal, the gastric pH may be transiently elevated, but tablet disintegration and early dissolution still occur during this 1–3 hr residence window, thus reducing acid-mediated cyanocobalamin degradation over this period is expected to translate into a higher intact dose reaching the small intestine for absorption ( 37 , 43 ). The observed threefold reduction in degradation is in line with literature showing greater cyanocobalamin stability at moderately acidic pH and with reports of pH-responsive behaviour in gelatin hydrogels ( 17 – 19 , 25 ). Taken together, these data indicate that improving cyanocobalamin stability over the physiologically relevant 1–3 hr gastric residence period may increase the amount of intact vitamin 12 reaching the small intestine, where absorption at pharmacologic doses is driven predominantly by passive diffusion. The Caco-2 experiments evaluate the next step of the physiological journey of vitamin B12 toward absorption by demonstrating approximately threefold greater transcellular vitamin B12 uptake from gelatinised formulations compared with non-gelatinised vitamin B12 formulations, without evidence of reduced transepithelial electrical resistance (TEER) or barrier breakdown ( 27 , 32 ). This experimental model approximates the gastrointestinal tract through three key features: the epithelial monolayer reflects the absorptive surface; pretreatment with simulated gastric and intestinal fluids mimics physiological pH conditions and the enzymatic milieu; and the measurement of TEER confirms preservation of tight junction integrity throughout the incubation period. The observed threefold increase in transcellular vitamin B12 uptake with gelatin encapsulation may be attributed to three complementary mechanisms: first, prolonged contact of the gelatinised formulation with the apical epithelial surface due to mucoadhesive interactions ( 44 ). Second, sustained local vitamin B12 availability from the protective gelatin matrix, maintains a favourable concentration gradient for passive diffusion ( 27 ). Third, the chemical integrity of vitamin B12 is protected during transit through the acidic gastric phase, preventing premature degradation ( 18 ). This pattern suggests that gelatin promotes epithelial transport under preserved tight junction function, likely via prolonged apical surface contact, maintenance of local vitamin B12 gradients, and protection of vitamin B12 integrity during simulated gastric transit ( 27 , 44 ). Gelatin peptides contain collagen-derived motifs (Pro-Gly sequences and RGD-like epitopes, which are arginine-glycine-aspartic acid sequences) that may interact with epithelial integrins and subtly modulate claudin-2 tight junction proteins, potentially enhancing paracellular transport; however, this mechanism remains speculative and requires targeted experimental validation ( 45 – 47 ). This work provides convergent in-vitro evidence that encapsulating cyanocobalamin (a common pharmaceutical form of vitamin B12) in a gelatin matrix confers meaningful advantages for gastric stability, improved availability for gastric absorption and enhanced epithelial uptake, mechanisms that are directly relevant to oral vitamin B12 replacement at therapeutic doses ( 18 , 27 , 32 ). Clinical relevance The clinical relevance of these findings lies in the different absorption pathways of vitamin B12. In the stomach, vitamin B12 binds to a glycoprotein called haptocorrin (also referred to as R-factor or R-protein), forming the haptocorrin-vitamin B12 complex which protects vitamin B12 from acid degradation. Once the haptocorrin-vitamin B12 complex reaches the duodenum, pH changes favour the degradation of haptocorrin and cleavage of the haptocorrin-vitamin B12 complex, releasing the free form of vitamin B12. In the active absorption pathway, the free vitamin B12 can then bind to intrinsic factor (IF), a glycoprotein secreted by gastric parietal cells to form an IF-vitamin B12 complex and that travels to the ileum for receptor-mediated absorption into the blood ( 48 , 49 ). While the active mechanism saturates at low physiological levels and therefore contributes only a small fraction of the total absorbed vitamin B12, passive diffusion plays an important role in uptake at pharmacological doses. At pharmacological oral doses of vitamin B12, such as those used in neurotropic B-complex preparations (200–5000 mcg), total absorption is achieved primarily through passive diffusion across the intestinal epithelium. In this dose range, the amount of vitamin B12 exceeds the haptocorrin binding capacity, leaving significant unbound vitamin B12 vulnerable to acid degradation ( 42 ). Clinical effectiveness therefore depends on the amount of intact vitamin that reaches the ileum ( 3 ). By stabilizing cyanocobalamin throughout production and storage, ensuring consistent microgram-level dosing of vitamin B12, protecting it from stomach acid during digestion, and maintaining higher concentrations near the absorption sites, gelatinised vitamin B12 formulations are specifically designed to support its absorption ( 27 ). Formulations that reliably deliver vitamin B12 and support intestinal absorption are important to restoring deficient vitamin B12 levels and contribute to improving deficiency-related neuropathic symptoms. These benefits are particularly pertinent for common patient groups in whom therapeutic doses of vitamin B12 are frequently indicated. These include patients with a clinical or subclinical deficiency in vitamin B12 and patients suffering from peripheral neuropathy. Common target groups requiring a therapeutic dose of vitamin B12 are patients with type 2 diabetes treated with metformin, who have a higher prevalence of vitamin B12 deficiency and peripheral neuropathy; older adults, in whom multiple age-related factors reduce vitamin B12 status; individuals following vegetarian or vegan diets, who have low dietary intake of cobalamin; people with chronic alcoholism, where malnutrition and malabsorption coexist; and patients receiving chronic haemodialysis, who often exhibit low or borderline serum vitamin B12 levels and others ( 11 – 16 , 50 ). In these populations, high-dose oral vitamin B12 is routinely used to correct deficiency and support nerve function, and a gelatinised formulation that stabilises vitamin B12 in the stomach and enhances passive uptake in the small intestine can provide a practical advantage over non-gelatinised preparations ( 51 , 52 ). Metformin-treated type 2 diabetic patients : Approximately 14 to 41% of patients on long-term metformin therapy develop biochemical vitamin B12 deficiency, with the risk increasing with increasing metformin dose and cumulative exposure ( 11 , 12 , 53 – 55 ). The underlying mechanism involves calcium antagonism: the positive charge of metformin competes with vitamin B12 for the calcium binding sites required for intrinsic factor-B12-Ca2+-cubilin complex formation in the terminal ileum ( 54 ). Consequent impairment of active absorption forces greater reliance on passive diffusion through the gastrointestinal tract. Additionally, metformin use decreased plasma levels of total cobalamin ( 56 , 57 ), total haptocorrin and haptocorrin-bound cobalamin in diabetic patients taking metformin, revealing the effect of metformin on haptocorrin glycoprotein leading to vitamin B12 degradation in the stomach. Gelatinised vitamin B12 addresses these absorption barriers through mechanisms independent of haptocorrin status, including acid protection of unbound vitamin B12, mucoadhesion-mediated epithelial contact, and sustained release that maintains concentration gradients for passive absorption ( 27 ). The passive pathway becomes functionally relevant when gelatin-encapsulated cyanocobalamin is provided at pharmacologic concentrations ( 6 ). This mechanism is particularly valuable in populations where active absorption is compromised, such as metformin-treated diabetic patients (calcium antagonism), pernicious anaemia and post-gastrectomy patients (impaired haptocorrin and absent intrinsic factor), and older adults (age-related receptor density reduction) ( 14 , 54 ). Older adults with age-related absorption decline Approximately 10 to 15% of community-dwelling individuals and up to 40% of institutionalised older persons exhibit vitamin B12 deficiency ( 13 , 14 ). While the haptocorrin level might be lower in healthy elderly people, it is generally not considered the primary cause of vitamin B12 deficiency. Age-related absorption impairment is multifactorial, with increased gastric hypochlorhydria impairing food bound vitamin B12 release, reduced ileal cubilin receptor density, and altered intestinal microbiota composition ( 13 , 14 , 33 ). Through the three complementary mechanisms identified in the Caco-2 model (prolonged apical contact via mucoadhesion, sustained local vitamin B12 availability, and acid protection), gelatin-mediated enhancements in passive uptake, mucoadhesion, and acid protection collectively address these multifactorial absorption deficits ( 6 , 27 ). In these high-risk groups, gelatinised vitamin B12 may enable lower effective supplemental doses while maintaining therapeutic vitamin B12 status, with implications for improved adherence, reduced pill burden, improved treatment compliance, and enhanced clinical outcomes ( 27 , 58 ). 4.1 Limitations This work has recognised limitations typical of mechanistic in vitro studies. Simulated gastric fluid assays use simplified media lacking dynamic peristalsis, enzymatic processes and variable pH microenvironments characteristic of living gastric tissue ( 32 ). Caco-2 monolayers, although widely predictive of intestinal permeability in vivo, lack full mucus layers, peristaltic stimulation, and complete proteolytic milieu of the living intestinal tract; consequently, the observed threefold increase in cellular uptake should be interpreted as a qualitative indicator of potential bioavailability enhancement rather than a direct in vivo prediction ( 32 ). Despite these constraints, the data provide internally consistent mechanistic support that warrants progression to in vivo studies ( 27 ). Another limitation of the HPLC method used for assessing vitamin B12 stability in simulated gastric fluid is that operating at a low sample concentration of 0.4 ppm can lead to variability in results. With a small sample size (n = 3), the standard deviation of the sample result tends to be wider with more variability and can lead to less precise outputs in terms of mean vitamin B12 loss. 5 Conclusions This study evaluated the impact of gelatin encapsulation on cyanocobalamin stability and cellular update via two complementary in vitro systems: simulated gastric fluid stability assays, and Caco-2 intestinal epithelial monolayers. Compared with non-gelatinised vitamin B12, gelatinised vitamin B12 resulted in approximately threefold less vitamin B12 loss via acid-mediated degradation, and approximately threefold greater intracellular uptake through Caco-2 cells without compromising barrier integrity. These converging findings support gelatin as a multifunctional excipient that simultaneously protects vitamin B12 from gastric acid, sustains availability via controlled release and mucoadhesion, and enhances passive epithelial transport-all mechanisms particularly relevant to populations dependent on passive absorption pathways, including metformin treated diabetic patients, older adults and peripheral neuropathy patients. The in vitro evidence suggests that progression to human pharmacokinetic studies in healthy volunteers and clinical efficacy trials in target populations are needed to establish the magnitude of benefit and to evaluate the role of gelatinised vitamin B12 in improving formula performance in vivo. Abbreviations API Active pharmaceutical ingredient B1 Thiamine (Vitamin B1) B6 Pyridoxine (Vitamin B6) B12 Cobalamin (Vitamin B12) ATCC American Type Culture Collection Caco-2 Human colorectal adenocarcinoma cells DNA Deoxyribonucleic acid EMEM Eagle’s Minimum Essential Medium FBS Fetal bovine serum GRAS Generally recognized as safe HC-Cbl Haptocorrin-bound cobalamin HPLC High-performance liquid chromatography IF Intrinsic factor LC-MS/MS Liquid chromatography-tandem mass spectrometry pH Potential of hydrogen ppm Parts per million SGF Simulated gastric fluid SIF Simulated intestinal fluid TEER Transepithelial electrical resistance TFA Trifluoroacetic acid USP United States Pharmacopeia UV Ultraviolet w/w Weight per weight Declarations Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable Competing Interests GC, TG, PA, HR, JZW, TL, LL and YL are employed by and own stocks in Procter and Gamble. JTS is a former employee of Procter and Gamble and own stocks in Procter and Gamble. Funding: This study was funded by Procter and Gamble Health. Author Contribution Study conceptualization: GC, YL, JTS. Design of study: GC, TG, PA, JZW, TL, LL, JTS. Acquisition and analysis: HR, JZW. Interpretation of data: GC, YL, TG, PA, HR, JZW, TL, LL, JTS. Drafted work: GC, YL, TG, PA, JZW, TL, LL. Revision and review: GC, YL, TG, PA, TL, LL, JTS. Acknowledgement The authors would like to thank Jia Hao Wong, Bhuvaneswari S and Adrien Gras from Ipsos Healthcare Singapore for supporting the medical writing. Data Availability All data supporting the findings of this study are available within the manuscript. References Badar A. Neuropsychiatric Disorders Associated With Vitamin B12 Deficiency: An Autobiographical Case Report. Cureus 14(1):e21476. Halczuk K, Kaźmierczak-Barańska J, Karwowski BT, Karmańska A, Cieślak M. Vitamin B12—Multifaceted In Vivo Functions and In Vitro Applications. Nutrients. 2023 June;13(12):2734. National Institues of Health. Vitamin B12 [Internet]. [cited 2026 Jan 20]. Available from: https://ods.od.nih.gov/factsheets/VitaminB12-HealthProfessional/ Umekar M, Premchandani T, Tatode A, Qutub M, Raut N, Taksande J, et al. Vitamin B12 deficiency and cognitive impairment: A comprehensive review of neurological impact. Brain Disorders. 2025 June;18:100220. Singh N. Vitamin B12-Associated Neurological Diseases: Background, Pathophysiology, Epidemiology. 2025 Aug 26 [cited 2026 Jan 20]; Available from: https://emedicine.medscape.com/article/1152670-overview?form=fpf Al Amin ASM, Gupta V. Vitamin B12 (Cobalamin). In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 [cited 2026 Jan 20]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK559132/ Seeking Health. Seeking Health. 700 [cited 2026 Jan 20]. The Ultimate Guide to Vitamin B12: Forms, Benefits, Supplements, & Mor. Available from: https://www.seekinghealth.com/blogs/education/the-ultimate-guide-to-vitamin-b12-forms-benefits-supplements Octagon Chem. Hydroxocobalamin Vs Cyanocobalamin: Your Comprehensive Guide [Internet]. 2025 [cited 2026 Jan 20]. Available from: https://octagonchem.com/blog/hydroxocobalamin-vs-cyanocobalamin/ DrugBank. DrugBank. [cited 2026 Jan 20]. Cyanocobalamin. Available from: https://go.drugbank.com/drugs/DB00115 Ajmera R, Healthline. 2020 [cited 2026 Jan 20]. Methylcobalamin vs. Cyanocobalamin: What’s the Difference? Available from: https://www.healthline.com/nutrition/methylcobalamin-vs-cyanocobalamin Aroda VR, Edelstein SL, Goldberg RB, Knowler WC, Marcovina SM, Orchard TJ, et al. Long-term Metformin Use and Vitamin B12 Deficiency in the Diabetes Prevention Program Outcomes Study. J Clin Endocrinol Metab. 2016;101(4):1754–61. Fituri S, Akbar Z, Ganji V. Impact of metformin treatment on cobalamin status in persons with type 2 diabetes. Nutr Rev. 2023;82(4):553–60. Marchi G, Busti F, Zidanes AL, Vianello A, Girelli D. Cobalamin Deficiency in the Elderly. Mediterr J Hematol Infect Dis. 2020 July 1;12(1):e2020043. Wong C. Vitamin B12 deficiency in the elderly: is it worth screening? Hong Kong Med J [Internet]. 2015 Mar 10 [cited 2026 Jan 20]; Available from: http://www.hkmj.org/earlyrelease/hkmj144383.htm Niklewicz A, Hannibal L, Warren M, Ahmadi KR. A systematic review and meta-analysis of functional vitamin B12 status among adult vegans. Nutr Bull. 2024;49(4):463–79. Fragasso A. Vitamin B12 Deficiency in Alcoholics. In: Watson RR, Preedy VR, Zibadi S, editors. Alcohol, Nutrition, and Health Consequences [Internet]. Totowa, NJ: Humana Press; 2013 [cited 2026 Jan 22]. pp. 131–4. Available from: https://doi.org/10.1007/978-1-62703-047-2_10 Ahmad I, Hafeez A, Akhter N, Vaid F, Qadeer K. Effect of Riboflavin on the Photolysis of Cyanocobolamin in Aqueous Solution. TOACJ. 2012;6(1):22–7. Mazzocato M, Thomazini M, Favaro-Trindade CS. Improving stability of vitamin B12 (Cyanocobalamin) using microencapsulation by spray chilling technique. Food Res Int. 2019;126:108663. Ahmad I, Qadeer K, Zahid S, Sheraz MA, Ismail T, Hussain W et al. Effect of Ascorbic Acid on the Degradation of Cyanocobalamin and Hydroxocobalamin in Aqueous Solution: A Kinetic Study. AAPS PharmSciTech. 2014 June 12;15(5):1324–33. Ankar A, Kumar A. Vitamin B12 Deficiency. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 [cited 2026 Jan 21]. Available from: http://www.ncbi.nlm.nih.gov/books/NBK441923/ Temova Rakuša Ž, Roškar R, Hickey N, Geremia S. Vitamin B12 in Foods, Food Supplements, and Medicines—A Review of Its Role and Properties with a Focus on Its Stability. Molecules. 2022;28(1):240. Abdullah MSP, Noordin MI, Ismail SIM, Mustapha NM, Jasamai M, Danik MF, et al. Recent Advances in the Use of Animal-Sourced Gelatine as Natural Polymers for Food, Cosmetics and Pharmaceutical Applications. Sains Malaysiana. 2018;47(2):323–36. Echave MC, Hernáez-Moya R, Iturriaga L, Pedraz JL, Lakshminarayanan R, Dolatshahi-Pirouz A, et al. Recent advances in gelatin-based therapeutics. Expert Opin Biol Ther. 2019;19(8):773–9. Pateiro M, Gómez B, Munekata PES, Barba FJ, Putnik P, Kovačević DB, et al. Nanoencapsulation of Promising Bioactive Compounds to Improve Their Absorption, Stability, Functionality and the Appearance of the Final Food Products. Molecules. 2021;26(6):1547. Goudie KJ, McCreath SJ, Parkinson JA, Davidson CM, Liggat JJ. Investigation of the influence of pH on the properties and morphology of gelatin hydrogels. J Polym Sci. 2023;61(19):2316–32. Lin J, Pan D, Sun Y, Ou C, Wang Y, Cao J. The modification of gelatin films: Based on various cross-linking mechanism of glutaraldehyde at acidic and alkaline conditions. Food Sci Nutr. 2019;7(12):4140–6. Milano F, Masi A, Madaghiele M, Sannino A, Salvatore L, Gallo N. Current Trends in Gelatin-Based Drug Delivery Systems. Pharmaceutics. 2023;15(5):1499. Naharros-Molinero A, Caballo‐González MÁ, de la Mata FJ, García‐Gallego S. Shell Formulation in Soft Gelatin Capsules: Design and Characterization. Adv Healthc Mater. 2024;13(1):2302250. Balanč B, Salević-Jelić A, Đorđević V, Bugarski B, Nedović V, Petrović P et al. The Application of Protein Concentrate Obtained from Green Leaf Biomass in Structuring Nanofibers for Delivery of Vitamin B12. Foods [Internet]. 2024 May 18 [cited 2026 Jan 20];13(10). Available from: https://www.mdpi.com/2304-8158/13/10/1576 Farzanfar S, kouzekonan GS, Mirjani R, Shekarchi B. Vitamin B12-loaded polycaprolacton/gelatin nanofibrous scaffold as potential wound care material. Biomed Eng Lett 2020 Sept 15;10(4):547–54. Jia CQ, Wang SY, Yuan Y, Wu YQ, Cai Y, Liu JW, et al. The passive diffusion improvement of Vitamin B12 intestinal absorption by Gelucire that fit for commercialized production. Saudi Pharm J. 2023 June;31(6):962–71. Pires CL, Praça C, Martins PAT, Batista de Carvalho ALM, Ferreira L, Marques MPM et al. Re-Use of Caco-2 Monolayers in Permeability Assays—Validation Regarding Cell Monolayer Integrity. Pharmaceutics. 2021 Sept 26;13(10):1563. Mucha P, Kus F, Cysewski D, Smolenski RT, Tomczyk M. Vitamin B12 Metabolism: A Network of Multi-Protein Mediated Processes. Int J Mol Sci. 2024 July 23;25(15):8021. Maciejewski B, Ström A, Larsson A, Sznitowska M. Soft Gelatin Films Modified with Cellulose Acetate Phthalate Pseudolatex Dispersion—Structure and Permeability. Polymers. 2018 Sept 3;10(9):981. Cao H, Wang J, Hao Z, Zhao D. Gelatin-based biomaterials and gelatin as an additive for chronic wound repair. Front Pharmacol. 2024;15:1398939. Jang Y, Jang J, Kim BY, Song YS, Lee DY. Effect of Gelatin Content on Degradation Behavior of PLLA/Gelatin Hybrid Membranes. Tissue Eng Regen Med. 2024;21(4):557–69. Dressman JB, Fleisher D. Mixing-tank model for predicting dissolution rate control of oral absorption. J Pharm Sci. 1986;75(2):109–16. Varum FJO, Merchant HA, Basit AW. Oral modified-release formulations in motion: The relationship between gastrointestinal transit and drug absorption. Int J Pharm. 2010;395(1):26–36. Davis SS, Hardy JG, Fara JW. Transit of pharmaceutical dosage forms through the small intestine. Gut. 1986;27(8):886–92. Yu LX, Amidon GL, Polli JE, Zhao H, Mehta MU, Conner DP, et al. Biopharmaceutics Classification System: The Scientific Basis for Biowaiver Extensions. Pharm Res. 2002 July;19(7):921–5. Allen LH, Miller JW, de Groot L, Rosenberg IH, Smith AD, Refsum H, et al. Biomarkers of Nutrition for Development (BOND): Vitamin B-12 Review. J Nutr. 2018;148(Suppl 4):S1995–2027. da Silva L, McCray S. Vitamin B12: No One Should Be Without It. Nutrition Issues in Gastroenterology [Internet]. 2009;(70). Available from: https://med.virginia.edu/ginutrition/wp-content/uploads/sites/199/2014/06/PG_Jan09_daSilvaArticle.pdf Chu JN, Traverso G. Foundations of gastrointestinal-based drug delivery and future developments. Nat Rev Gastroenterol Hepatol. 2022;19(4):219–38. Shaikh R, Raj Singh TR, Garland MJ, Woolfson AD, Donnelly RF. Mucoadhesive drug delivery systems. J Pharm Bioallied Sci. 2011;3(1):89–100. Günzel D, Yu ASL. Claudins and the Modulation of Tight Junction Permeability. Physiol Rev. 2013;93(2):525–69. Jiang X, Du Z, Zhang X, Zaman F, Song Z, Guan Y et al. Gelatin-based anticancer drug delivery nanosystems: A mini review. Front Bioeng Biotechnol [Internet]. 2023 Mar 21 [cited 2026 Jan 20];11. Available from: https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/ 10.3389/fbioe.2023.1158749/full Peterson RJ, Reed RC, Zamecnik CR, Sallam MA, Finbloom JA, Martinez FJ, et al. Apical integrins as a switchable target to regulate the epithelial barrier. J Cell Sci. 2024;137(24):jcs263580. Brito A, Habeych E, Silva-Zolezzi I, Galaffu N, Allen LH. Methods to assess vitamin B12 bioavailability and technologies to enhance its absorption. Nutr Rev. 2018;76(10):778–92. Infante M, Leoni M, Caprio M, Fabbri A. Long-term metformin therapy and vitamin B12 deficiency: an association to bear in mind. WJD 2021 July 15;12(7):916–31. Mushtaq M, Usmani MR, Hameed N, Anwar A, Hashmi AA. Serum Vitamin B12 Deficiency in Chronic Hemodialysis Patients. Cureus 16(4):e58751. Baltrusch S. The Role of Neurotropic B Vitamins in Nerve Regeneration. Biomed Res Int 2021 July 13;2021:9968228. Pinzon R, Schellack N, Matawaran BJ, Tsang MW, Deerochanawong C, Hiew FL, et al. Clinical Recommendations for the use of Neurotropic B vitamins (B1, B6, and B12) for the Management of Peripheral Neuropathy: Consensus from a Multidisciplinary Expert Panel. J Assoc Phys India. 2023;71(7):11–2. Huynh DT, Nguyen NT, Do MD. Vitamin B12 deficiency in diabetic patients treated with metformin: A cross-sectional study. PLoS ONE. 2024;19(4):e0302500. Miyan Z, Waris N. Association of vitamin B12 deficiency in people with type 2 diabetes on metformin and without metformin: a multicenter study, Karachi, Pakistan. BMJ Open Diabetes Res Care. 2020;8(1):e001151. Sayedali E, Yalin AE, Yalin S. Association between metformin and vitamin B12 deficiency in patients with type 2 diabetes. World J Diabetes. 2023;14(5):585–93. Leung S, Mattman A, Snyder F, Kassam R, Meneilly G, Nexo E. Metformin induces reductions in plasma cobalamin and haptocorrin bound cobalamin levels in elderly diabetic patients. Clin Biochem. 2010 June;43(9):759–60. Greibe E, Miller JW, Foutouhi SH, Green R, Nexo E. Metformin increases liver accumulation of vitamin B12 – An experimental study in rats. Biochimie. 2013;95(5):1062–5. Lacombe V, Vinatier E, Roquin G, Copin MC, Delattre E, Hammi S, et al. Oral vitamin B12 supplementation in pernicious anemia: a prospective cohort study. Am J Clin Nutr. 2024 July;120(1):217–24. Additional Declarations Competing interest reported. GC, TG, PA, HR, JZW, TL, LL and YL are employed by and own stocks in Procter and Gamble. JTS is a former employee of Procter and Gamble and own stocks in Procter and Gamble. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8670841","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":599891878,"identity":"63c890a0-2231-425f-a815-256a70ac6c0b","order_by":0,"name":"Garima Chauhan","email":"","orcid":"","institution":"Grocter \u0026 Gamble","correspondingAuthor":false,"prefix":"","firstName":"Garima","middleName":"","lastName":"Chauhan","suffix":""},{"id":599891879,"identity":"ab84d87e-a1d8-4103-b112-bdc9faf93dad","order_by":1,"name":"Tarun Goel","email":"","orcid":"","institution":"Procter \u0026 Gamble (India)","correspondingAuthor":false,"prefix":"","firstName":"Tarun","middleName":"","lastName":"Goel","suffix":""},{"id":599891880,"identity":"26bf4154-e0d1-4c74-b5b1-cea0309b956b","order_by":2,"name":"Pankaj Attarde","email":"","orcid":"","institution":"Procter \u0026 Gamble (India)","correspondingAuthor":false,"prefix":"","firstName":"Pankaj","middleName":"","lastName":"Attarde","suffix":""},{"id":599891881,"identity":"4fb105d5-f758-4457-bf20-e2d8a5f0cfd7","order_by":3,"name":"Heather Rodebush","email":"","orcid":"","institution":"Procter \u0026 Gamble (United States)","correspondingAuthor":false,"prefix":"","firstName":"Heather","middleName":"","lastName":"Rodebush","suffix":""},{"id":599891882,"identity":"95420216-a3e5-4aa3-9fed-0b0efe0cf748","order_by":4,"name":"Jiazhen Wang","email":"","orcid":"","institution":"Procter \u0026 Gamble (United States)","correspondingAuthor":false,"prefix":"","firstName":"Jiazhen","middleName":"","lastName":"Wang","suffix":""},{"id":599891883,"identity":"7353aede-57dd-4bce-b6b5-bf5b9b6a95e9","order_by":5,"name":"Timothy Laughlin","email":"","orcid":"","institution":"Procter \u0026 Gamble (United States)","correspondingAuthor":false,"prefix":"","firstName":"Timothy","middleName":"","lastName":"Laughlin","suffix":""},{"id":599891884,"identity":"2dd84ea1-a26f-4222-ab2e-dac1f4e85f65","order_by":6,"name":"Lijuan Li","email":"","orcid":"","institution":"Procter \u0026 Gamble (United States)","correspondingAuthor":false,"prefix":"","firstName":"Lijuan","middleName":"","lastName":"Li","suffix":""},{"id":599891885,"identity":"6ec000d6-c25a-433e-810d-8047e2be6ed8","order_by":7,"name":"John T. Stickney","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"T.","lastName":"Stickney","suffix":""},{"id":599891886,"identity":"f20860c2-1a0a-4334-a04a-f5fa26f57ac3","order_by":8,"name":"Yan Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqUlEQVRIiWNgGAWjYBACPmYQaSBhx0+0FjawlgKbZMkGorWAyQ9pjBsOEK2FnYFNgsHgMLPx7fYLDD8qthHlMLAWPrM7ZwoYe87cJl4Ls9mNnARmxjYStDBunkGiFqD3JdIPEKuFsdkiwcAmWeLOGYaDRPmFn//wwRsf/gCjcnb7wwc/KojQwsDA2CKRAKIleAwOEKMeBJg/gCkJ9gfE6hgFo2AUjIIRBgCQwDIk7jNGjQAAAABJRU5ErkJggg==","orcid":"","institution":"Grocter \u0026 Gamble","correspondingAuthor":true,"prefix":"","firstName":"Yan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-01-22 14:23:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8670841/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8670841/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104181929,"identity":"373f570a-c5a3-4e63-85a1-48fd9738ed2d","added_by":"auto","created_at":"2026-03-08 17:32:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23082,"visible":true,"origin":"","legend":"\u003cp\u003eManufacturing process flow chart for vitamin B12 embedding in gelatin.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8670841/v1/eeb81362cfe896cd75bf44f0.png"},{"id":104181930,"identity":"36d0f8ed-fce2-4888-a04c-032ba54784d4","added_by":"auto","created_at":"2026-03-08 17:32:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110310,"visible":true,"origin":"","legend":"\u003cp\u003eIntracellular vitamin B12\u003csub\u003e \u003c/sub\u003elevels in Caco-2 monolayers after 24 h of incubation with gelatinised vitamin B12\u003csub\u003e \u003c/sub\u003evs pure vitamin B12\u003csub\u003e,\u003c/sub\u003e with and without simulated gastric fluid (GF) and intestinal fluid (IF)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8670841/v1/6765bfe6ffadaa7842c518ca.png"},{"id":107499668,"identity":"2001b8af-afc6-4ba8-8edc-24eae2edea77","added_by":"auto","created_at":"2026-04-22 05:41:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":560469,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8670841/v1/682a964d-df15-49b3-a51d-d2b571f711c9.pdf"}],"financialInterests":"Competing interest reported. GC, TG, PA, HR, JZW, TL, LL and YL are employed by and own stocks in Procter and Gamble. JTS is a former employee of Procter and Gamble and own stocks in Procter and Gamble.","formattedTitle":"Optimizing vitamin B12 delivery in solid pharmaceutical formats: The Role of Gelatin in Stability and Formulation Performance","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eVitamin B12 (cobalamin) is an essential water-soluble vitamin required for red blood cell maturation, DNA synthesis, methylation pathways, and normal neurological function (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). A deficiency disrupts erythropoiesis, impairs myelin integrity, and can manifest as megaloblastic anaemia, peripheral neuropathy, cognitive decline, or neuropsychiatric symptoms (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Cobalamin exists in several chemical forms: cyanocobalamin and hydroxocobalamin are synthetic or modified forms widely used pharmaceutically, whereas methylcobalamin and adenosylcobalamin are the metabolically active cofactors in human tissue (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Among these different chemical forms, cyanocobalamin remains the most commonly used form in oral supplements, medicinal products and fortified foods because of its cost-effectiveness and greater chemical stability, with comparable absorption profiles when given at supplementation and therapeutic doses (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eClinically significant vitamin B12 deficiency is common in multiple risk groups: type 2 diabetes patients especially those treated with metformin (14\u0026ndash;41%), pernicious anaemia and post-gastrectomy patients (approximately 100%), and older adults (10\u0026ndash;15% in community-dwelling; up to 40% in institutionalised), vegetarians or vegan, chronic alcoholism and others (\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). If left untreated, vitamin B12 deficiency can lead to serious haematological and neurological complications (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Standard management of vitamin B12 deficiency and peripheral neuropathy includes high-dose oral cyanocobalamin or intramuscular injections, in combination with other neurotropic B vitamins (B1, B6). Under normal physiological intake, vitamin B12 is absorbed through a combination of high-affinity, receptor-mediated routes with a low-efficiency passive diffusion at low microgram doses; at larger pharmacologic doses used in many oral neurotropic B-complex products, overall uptake is predominantly by passive diffusion, with approximately 1\u0026ndash;2% of the ingested dose crossing the intestinal epithelium into the circulation (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, conventional cyanocobalamin is chemically labile under strongly acidic conditions, such as those found in the gastric environment, and has a markedly shorter half-life at gastric pH than under near-neutral conditions (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). In addition to gastric acidity, cobalamin is sensitive to light and humidity in solid pharmaceutical forms, and the relatively low amount used in solid pharmaceutical formats (microgram range per unit) creates additional challenges for ensuring content uniformity and maintaining potency throughout manufacturing and storage (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGelatin is a collagen-derived, biodegradable excipient that is generally recognised as safe (GRAS) status and has a long history of use in pharmaceutical dosage forms, including capsules and matrix systems to improve ingredient stability and help with dosing uniformity, especially at small amounts (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Recent work has highlighted the ability of gelatin to form dense, pH-responsive matrices, interact with mucus, and act as a platform for controlled release and targeted delivery in oral preparations (\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). In commercial pharmaceutical products, gelatin-based capsules are used to maintain content uniformity and product stability (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Although gelatin is widely used in pharmaceutical formulations, the protective effects of gelatin encapsulation specifically on oral cyanocobalamin stability and bioavailability remain poorly characterised (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Recent research has highlighted the potential of gelatin in vitamin B12 delivery systems, particularly through nanofiber formulations and sustained-release mechanisms (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) and related lipid-based carriers have shown enhanced passive absorption of vitamin B12 via P-glycoprotein inhibition (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). However, the dual protective effects of gelatin\u0026mdash;acid stabilization combined with improved cellular uptake or absorption\u0026mdash;have not been comprehensively characterised in complementary in vitro models.\u003c/p\u003e \u003cp\u003eGelatinised vitamin B12 technology is designed to address several of these constraints simultaneously: by embedding cyanocobalamin (a common pharmaceutical form of vitamin B12) within a gelatin matrix, it can enhance stability against gastric acid and maintain higher free vitamin B12 concentrations in proximity to the intestinal epithelium, thereby maximising the contribution of passive diffusion at pharmacologic oral doses. This study therefore aims to characterise the role of gelatin in the protection of vitamin B12 during gastric transit using in vitro models by evaluating the impact of gelatin encapsulation on acid stability and epithelial uptake.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eCyanocobalamin active pharmaceutical ingredient (API, Hebei Huarong Pharmaceutical Company Ltd, China) was used as the reference vitamin B12 form. Gelatin (Rama Industries Ltd, bovine source) suitable for pharmaceutical use was employed to manufacture gelatinised vitamin B12 granules containing approximately 1% w/w vitamin B12 in a 99% gelatin matrix, which is consistent with established commercial formulations. Simulated gastric fluid (SGF; pH 1.2) was prepared according to the USP (US Pharmacopeial), and other reagents were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of gelatinised Vitamin B12\u003c/h2\u003e \u003cp\u003eGelatinised vitamin B12 can be manufactured via various methods such as spray drying or fluid-bed granulation. The gelatinised vitamin B12 granules (1%, Supreem Pharmaceuticals, India) used in this study were produced via fluid-bed granulation, in which small gelatin cores were sprayed with a solution of cyanocobalamin and gelatin under heated air (45\u0026deg;C), followed by drying and application of a final gelatin coating (Fig.\u0026nbsp;1). This process yielded granules with consistent particle sizes (80\u0026ndash;100% passes through mesh 60#) and uniform vitamin B12 contents (1% loading, hereinafter referred to as 1% gelatinised vitamin B12) embedded within a continuous gelatin matrix and is analogous to the process used in marketed Neurobion\u0026reg; formulations containing 100mg of B1, 100-200mg of B6, 200mcg \u0026minus;\u0026thinsp;5000mcg of B12. For in vitro experiments, 1% gelatinised vitamin B12 granules were dispersed and diluted to the target vitamin B\u003csub\u003e12\u003c/sub\u003e concentration in the relevant test media, and non-gelatinised vitamin B12 solutions were prepared at matched cyanocobalamin concentrations\u003c/p\u003e \u003cp\u003eFigure 1: Manufacturing process flow chart for vitamin B12 embedding in gelatin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Simulated Gastric Fluid (SGF) Stability Studies\u003c/h2\u003e \u003cp\u003eThe concentration of vitamin B12 (both non-gelatinised and 1% gelatinised) was adjusted based on the gastric volume, and label claim of vitamin B12 in the formulation (200 mcg). Stock solutions of 1% gelatinised vitamin B12 and non-gelatinised vitamin B12 were prepared in SGF pH 1.2 media to achieve concentration of 0.4 ppm (equivalent to 200 mcg dose, n\u0026thinsp;=\u0026thinsp;3), similar to the concentration from Neurobion\u0026reg; and other preparations on the market containing 200 mcg vitamin B12. The vitamin B12 content was quantified via reversed-phase high-performance liquid chromatography (HPLC) with 0.1% trifluoracetic acid (TFA) solution and acetonitrile as the mobile phase with a gradient pump mode via a phenyl column (150 mm \u0026times; 4.6 mm) and UV detection at 361 nm. The amount of vitamin B12 was measured at various time points (initial, 1 hour, 2 hour and 3 hour) in the simulated setup until gastric emptying for both 1% gelatinised vitamin B12 and non-gelatinised vitamin B12.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Caco-2 cell culture and uptake assays\u003c/h2\u003e \u003cp\u003eHuman Caco-2 cells (American Tissue Culture Collection, Manassas VA USA, ATCC cat# HTB-37) were cultured on permeable inserts (growth area 3.14 cm\u003csup\u003e2\u003c/sup\u003e, pore size 0.4 \u0026micro;m) at a density of approximately 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per insert and maintained in standard growth medium (EMEM ATCC cat#30-2003 supplemented with 10% FBS- ATCC cat#30-2020). The medium was changed 24 h after seeding and every other day thereafter until the monolayers reached differentiation and confluence (18 to 21 days), as evidenced by stable transepithelial electrical resistance (TEER) readings of approximately 750. Before the experiments, the baseline TEER was recorded to confirm barrier integrity.\u003c/p\u003e \u003cp\u003eGelatinised and non-gelatinised vitamin B12 formulations were prepared in cell culture medium with or without prior exposure to simulated gastric and intestinal fluids to mimic gastrointestinal transit (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The formulations were applied to the apical (mucosal) side (n\u0026thinsp;=\u0026thinsp;4 for each treatment group), with fresh medium in the basolateral compartment, and the cells were incubated for 24 h at 37℃ (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). After incubation, the cells were washed to remove extracellular vitamin B\u003csub\u003e12\u003c/sub\u003e and lysed, and the basolateral vitamin B12 content was quantified via LC to MS/MS (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). TEER was remeasured to assess whether barrier integrity was maintained during exposure (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). SGF (VWR cat# RC7108-16) was prepared according to USP guidelines, with 3.2 g of pepsin (VWR cat#M142-250G)/Liter added to make complete SGF. For simulated intestinal fluid (VWR cat# RC7109-16), SIF, 10 g/Liter of pancreatin (VWR cat # 75875-592) was used. Prior to the application of SGF/IF-treated vitamin B12 solutions to Caco-2 cells, 30 \u0026micro;L of Protease Inhibitor Cocktail (ThermoFisher cat# 78429) was added to mitigate the deleterious impact of pepsin/pancreatin on Caco-2 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Data Handling and Analysis\u003c/h2\u003e \u003cp\u003eFor each experiment, values were generated from replicate samples or inserts per condition (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). The results are reported descriptively as approximate fold differences between gelatinised and non-gelatinised vitamin B12 based on mean values, reflecting the mechanistic focus and in vitro nature of the work (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Where relevant, literature was used to contextualise the magnitude of observed effects relative to physiological constraints and clinical dosing paradigms (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Gelatin Encapsulation Enhances Vitamin B12 Stability in Simulated Gastric Fluid\u003c/h2\u003e \u003cp\u003eThe stability results of the sample solutions, with a concentration of 0.4 ppm (equivalent to a 200 mcg dose) of non-gelatinised vitamin B12 and 1% gelatinised vitamin B12 prepared in Simulated Gastric Fluid (SGF) pH 1.2 media were analysed by HPLC at different time intervals: initial, 1 hour, 2 hour and 3 hour. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, for 1% gelatinised vitamin B12, 1.7%, 2.3% and 2.9% losses were observed at 1 hour, 2 hour and 3 hour respectively, whereas for non-gelatinised vitamin B12, 6.3%, 8.6% and 10.0% loss were observed from the initial assay at 1 hour, 2 hour and 3 hour respectively. The test data demonstrate that gelatin encapsulation (1% vitamin B12 and 99% gelatin matrix) protects vitamin B12 from degradation under acidic stomach conditions, with a mean result of 3.6 times less loss of vitamin B12 due to gelatin encapsulation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGelatinised vitamin B12 loss vs non-gelatinised vitamin B12 loss at different time points\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration in SGF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1% Gelatinised vitamin B12 loss %\u003c/p\u003e \u003cp\u003eSolution Concentration (200mcg), n\u0026thinsp;=\u0026thinsp;3 (A)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNon-Gelatinised vitamin B12 loss %\u003c/p\u003e \u003cp\u003eSolution Concentration (200mcg), n\u0026thinsp;=\u0026thinsp;3 (B)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal loss ratio [B/A]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1 hr.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean: 1.7% loss\u003c/p\u003e \u003cp\u003eSD: 2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean: 6.3% loss\u003c/p\u003e \u003cp\u003eSD: 4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2 hr.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean: 2.3% loss\u003c/p\u003e \u003cp\u003eSD: 2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean: 8.6% loss\u003c/p\u003e \u003cp\u003eSD: 5.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3 hr.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean: 2.9% loss\u003c/p\u003e \u003cp\u003eSD: 3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean: 10.0% loss\u003c/p\u003e \u003cp\u003eSD: 6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eMean: 3.6\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eSD: 0.2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe percentage loss (%) of vitamin B12 at 1 hour, 2 hour and 3 hour was compared against the initial assay results for both gelatinised and non-gelatinised vitamin B12. The HPLC method used for assessing vitamin B12 stability in simulated gastric fluid is operating at a low sample concentration of 0.4 ppm, which can lead to variability in the results, hence the probability of higher standard deviations cannot be eliminated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Gelatinised Vitamin B12 Increases Cellular Passage in Caco-2 Monolayers Without Compromising Barrier Integrity\u003c/h2\u003e \u003cp\u003eFigure 2 shows that 1.2% of a dose of non-gelatinised vitamin B12 is absorbed which is in line with clinical observations showing that 1\u0026ndash;2% of a dose of vitamin B12 is absorbed via the passive absorption pathway. When the same amount of vitamin B12 embedded in gelatin (1% vitamin B12 and 99% gelatin matrix) was added to the cells, a 3-fold increase in absorption was noted (3.6%). This result was achieved both when gelatinised vitamin B12 was added directly to cells or when it was pre-incubated with simulated gastric and simulated intestinal fluids for 1 hour each (to approximate the conditions of the stomach and first parts of the small intestine).\u003c/p\u003e \u003cp\u003eFigure 2: Intracellular vitamin B12 levels in Caco-2 monolayers after 24 h of incubation with gelatinised vitamin B12 vs pure vitamin B12, with and without simulated gastric fluid (GF) and intestinal fluid (IF)\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eExposure to simulated gastric fluid (SGF; pH 1.2, 37℃) for 3 hour led to substantial degradation of non-gelatinised vitamin B12, with only approximately 90.0% to 93.7% of the initial vitamin B12 content remaining at matched starting concentrations. In contrast, gelatinised vitamin B12 granules (1% vitamin B12 in a 99% gelatin matrix) retained approximately 97.1% to 98.3% of their initial vitamin B12 content under identical conditions, corresponding to an approximate threefold reduction in vitamin B12 loss compared with non-gelatinised vitamin B12 (cyanocobalamin). This observation aligns with the hypothesised dual protection mechanism, which comprises first a dense, minimally swollen gelatin matrix that physically limits hydrogen ion penetration into the core for approximately 60 min at pH 1.2 (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) and, second, proton-accepting amino acid residues (approximately 13% of the polypeptide chain of gelatin are positively charged) within the gelatin that buffer the local microenvironment around the encapsulated vitamin B12 (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Gelatin-based matrices have been reported to provide sustained protection over several hours; with crosslinked gelatin hydrogels remaining mechanically stable for 4 to 6 hr under physiological conditions, which is consistent with the gastric residence time of 1 to 3 h (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eScintigraphic and transit studies indicate that, depending on the prandial state and formulation, the upper gastrointestinal residence time (stomach plus small intestine) for solid and drinkable dosage forms typically lies within a 1\u0026ndash;3 hr window in adults, extending toward 3\u0026ndash;4 hr in the fed state (\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). The observed threefold reduction in cyanocobalamin degradation within 1\u0026ndash;3 hours in SGF is regarded as clinically significant, as this period corresponds to the typical duration during which most of an orally administered dose is subjected to gastric conditions prior to reaching the small intestine, where predominantly passive absorption occurs at high oral doses (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). As the product is taken with or shortly before a meal, the gastric pH may be transiently elevated, but tablet disintegration and early dissolution still occur during this 1\u0026ndash;3 hr residence window, thus reducing acid-mediated cyanocobalamin degradation over this period is expected to translate into a higher intact dose reaching the small intestine for absorption (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). The observed threefold reduction in degradation is in line with literature showing greater cyanocobalamin stability at moderately acidic pH and with reports of pH-responsive behaviour in gelatin hydrogels (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Taken together, these data indicate that improving cyanocobalamin stability over the physiologically relevant 1\u0026ndash;3 hr gastric residence period may increase the amount of intact vitamin 12 reaching the small intestine, where absorption at pharmacologic doses is driven predominantly by passive diffusion.\u003c/p\u003e \u003cp\u003eThe Caco-2 experiments evaluate the next step of the physiological journey of vitamin B12 toward absorption by demonstrating approximately threefold greater transcellular vitamin B12 uptake from gelatinised formulations compared with non-gelatinised vitamin B12 formulations, without evidence of reduced transepithelial electrical resistance (TEER) or barrier breakdown (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). This experimental model approximates the gastrointestinal tract through three key features: the epithelial monolayer reflects the absorptive surface; pretreatment with simulated gastric and intestinal fluids mimics physiological pH conditions and the enzymatic milieu; and the measurement of TEER confirms preservation of tight junction integrity throughout the incubation period. The observed threefold increase in transcellular vitamin B12 uptake with gelatin encapsulation may be attributed to three complementary mechanisms: first, prolonged contact of the gelatinised formulation with the apical epithelial surface due to mucoadhesive interactions (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Second, sustained local vitamin B12 availability from the protective gelatin matrix, maintains a favourable concentration gradient for passive diffusion (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Third, the chemical integrity of vitamin B12 is protected during transit through the acidic gastric phase, preventing premature degradation (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis pattern suggests that gelatin promotes epithelial transport under preserved tight junction function, likely via prolonged apical surface contact, maintenance of local vitamin B12 gradients, and protection of vitamin B12 integrity during simulated gastric transit (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Gelatin peptides contain collagen-derived motifs (Pro-Gly sequences and RGD-like epitopes, which are arginine-glycine-aspartic acid sequences) that may interact with epithelial integrins and subtly modulate claudin-2 tight junction proteins, potentially enhancing paracellular transport; however, this mechanism remains speculative and requires targeted experimental validation (\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis work provides convergent in-vitro evidence that encapsulating cyanocobalamin (a common pharmaceutical form of vitamin B12) in a gelatin matrix confers meaningful advantages for gastric stability, improved availability for gastric absorption and enhanced epithelial uptake, mechanisms that are directly relevant to oral vitamin B12 replacement at therapeutic doses (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eClinical relevance\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe clinical relevance of these findings lies in the different absorption pathways of vitamin B12. In the stomach, vitamin B12 binds to a glycoprotein called haptocorrin (also referred to as R-factor or R-protein), forming the haptocorrin-vitamin B12 complex which protects vitamin B12 from acid degradation. Once the haptocorrin-vitamin B12 complex reaches the duodenum, pH changes favour the degradation of haptocorrin and cleavage of the haptocorrin-vitamin B12 complex, releasing the free form of vitamin B12. In the active absorption pathway, the free vitamin B12 can then bind to intrinsic factor (IF), a glycoprotein secreted by gastric parietal cells to form an IF-vitamin B12 complex and that travels to the ileum for receptor-mediated absorption into the blood (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile the active mechanism saturates at low physiological levels and therefore contributes only a small fraction of the total absorbed vitamin B12, passive diffusion plays an important role in uptake at pharmacological doses. At pharmacological oral doses of vitamin B12, such as those used in neurotropic B-complex preparations (200\u0026ndash;5000 mcg), total absorption is achieved primarily through passive diffusion across the intestinal epithelium. In this dose range, the amount of vitamin B12 exceeds the haptocorrin binding capacity, leaving significant unbound vitamin B12 vulnerable to acid degradation (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Clinical effectiveness therefore depends on the amount of intact vitamin that reaches the ileum (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBy stabilizing cyanocobalamin throughout production and storage, ensuring consistent microgram-level dosing of vitamin B12, protecting it from stomach acid during digestion, and maintaining higher concentrations near the absorption sites, gelatinised vitamin B12 formulations are specifically designed to support its absorption (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Formulations that reliably deliver vitamin B12 and support intestinal absorption are important to restoring deficient vitamin B12 levels and contribute to improving deficiency-related neuropathic symptoms.\u003c/p\u003e \u003cp\u003eThese benefits are particularly pertinent for common patient groups in whom therapeutic doses of vitamin B12 are frequently indicated. These include patients with a clinical or subclinical deficiency in vitamin B12 and patients suffering from peripheral neuropathy. Common target groups requiring a therapeutic dose of vitamin B12 are patients with type 2 diabetes treated with metformin, who have a higher prevalence of vitamin B12 deficiency and peripheral neuropathy; older adults, in whom multiple age-related factors reduce vitamin B12 status; individuals following vegetarian or vegan diets, who have low dietary intake of cobalamin; people with chronic alcoholism, where malnutrition and malabsorption coexist; and patients receiving chronic haemodialysis, who often exhibit low or borderline serum vitamin B12 levels and others (\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). In these populations, high-dose oral vitamin B12 is routinely used to correct deficiency and support nerve function, and a gelatinised formulation that stabilises vitamin B12 in the stomach and enhances passive uptake in the small intestine can provide a practical advantage over non-gelatinised preparations (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetformin-treated type 2 diabetic patients\u003c/b\u003e: Approximately 14 to 41% of patients on long-term metformin therapy develop biochemical vitamin B12 deficiency, with the risk increasing with increasing metformin dose and cumulative exposure (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). The underlying mechanism involves calcium antagonism: the positive charge of metformin competes with vitamin B12 for the calcium binding sites required for intrinsic factor-B12-Ca2+-cubilin complex formation in the terminal ileum (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Consequent impairment of active absorption forces greater reliance on passive diffusion through the gastrointestinal tract. Additionally, metformin use decreased plasma levels of total cobalamin (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e), total haptocorrin and haptocorrin-bound cobalamin in diabetic patients taking metformin, revealing the effect of metformin on haptocorrin glycoprotein leading to vitamin B12 degradation in the stomach.\u003c/p\u003e \u003cp\u003eGelatinised vitamin B12 addresses these absorption barriers through mechanisms independent of haptocorrin status, including acid protection of unbound vitamin B12, mucoadhesion-mediated epithelial contact, and sustained release that maintains concentration gradients for passive absorption (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The passive pathway becomes functionally relevant when gelatin-encapsulated cyanocobalamin is provided at pharmacologic concentrations (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). This mechanism is particularly valuable in populations where active absorption is compromised, such as metformin-treated diabetic patients (calcium antagonism), pernicious anaemia and post-gastrectomy patients (impaired haptocorrin and absent intrinsic factor), and older adults (age-related receptor density reduction) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eOlder adults with age-related absorption decline\u003c/strong\u003e \u003cp\u003eApproximately 10 to 15% of community-dwelling individuals and up to 40% of institutionalised older persons exhibit vitamin B12 deficiency (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). While the haptocorrin level might be lower in healthy elderly people, it is generally not considered the primary cause of vitamin B12 deficiency. Age-related absorption impairment is multifactorial, with increased gastric hypochlorhydria impairing food bound vitamin B12 release, reduced ileal cubilin receptor density, and altered intestinal microbiota composition (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Through the three complementary mechanisms identified in the Caco-2 model (prolonged apical contact via mucoadhesion, sustained local vitamin B12 availability, and acid protection), gelatin-mediated enhancements in passive uptake, mucoadhesion, and acid protection collectively address these multifactorial absorption deficits (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003eIn these high-risk groups, gelatinised vitamin B12 may enable lower effective supplemental doses while maintaining therapeutic vitamin B12 status, with implications for improved adherence, reduced pill burden, improved treatment compliance, and enhanced clinical outcomes (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Limitations\u003c/h2\u003e \u003cp\u003eThis work has recognised limitations typical of mechanistic in vitro studies. Simulated gastric fluid assays use simplified media lacking dynamic peristalsis, enzymatic processes and variable pH microenvironments characteristic of living gastric tissue (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Caco-2 monolayers, although widely predictive of intestinal permeability in vivo, lack full mucus layers, peristaltic stimulation, and complete proteolytic milieu of the living intestinal tract; consequently, the observed threefold increase in cellular uptake should be interpreted as a qualitative indicator of potential bioavailability enhancement rather than a direct in vivo prediction (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Despite these constraints, the data provide internally consistent mechanistic support that warrants progression to in vivo studies (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Another limitation of the HPLC method used for assessing vitamin B12 stability in simulated gastric fluid is that operating at a low sample concentration of 0.4 ppm can lead to variability in results. With a small sample size (n\u0026thinsp;=\u0026thinsp;3), the standard deviation of the sample result tends to be wider with more variability and can lead to less precise outputs in terms of mean vitamin B12 loss.\u003c/p\u003e \u003c/div\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eThis study evaluated the impact of gelatin encapsulation on cyanocobalamin stability and cellular update via two complementary in vitro systems: simulated gastric fluid stability assays, and Caco-2 intestinal epithelial monolayers. Compared with non-gelatinised vitamin B12, gelatinised vitamin B12 resulted in approximately threefold less vitamin B12 loss via acid-mediated degradation, and approximately threefold greater intracellular uptake through Caco-2 cells without compromising barrier integrity. These converging findings support gelatin as a multifunctional excipient that simultaneously protects vitamin B12 from gastric acid, sustains availability via controlled release and mucoadhesion, and enhances passive epithelial transport-all mechanisms particularly relevant to populations dependent on passive absorption pathways, including metformin treated diabetic patients, older adults and peripheral neuropathy patients. The in vitro evidence suggests that progression to human pharmacokinetic studies in healthy volunteers and clinical efficacy trials in target populations are needed to establish the magnitude of benefit and to evaluate the role of gelatinised vitamin B12 in improving formula performance in vivo.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAPI Active pharmaceutical ingredient\u003c/p\u003e\n\u003cp\u003eB1 Thiamine (Vitamin B1)\u003c/p\u003e\n\u003cp\u003eB6 Pyridoxine (Vitamin B6)\u003c/p\u003e\n\u003cp\u003eB12 Cobalamin (Vitamin B12)\u003c/p\u003e\n\u003cp\u003eATCC American Type Culture Collection\u003c/p\u003e\n\u003cp\u003eCaco-2 Human colorectal adenocarcinoma cells\u003c/p\u003e\n\u003cp\u003eDNA Deoxyribonucleic acid\u003c/p\u003e\n\u003cp\u003eEMEM Eagle\u0026rsquo;s Minimum Essential Medium\u003c/p\u003e\n\u003cp\u003eFBS Fetal bovine serum\u003c/p\u003e\n\u003cp\u003eGRAS Generally recognized as safe\u003c/p\u003e\n\u003cp\u003eHC-Cbl Haptocorrin-bound cobalamin\u003c/p\u003e\n\u003cp\u003eHPLC High-performance liquid chromatography\u003c/p\u003e\n\u003cp\u003eIF Intrinsic factor\u003c/p\u003e\n\u003cp\u003eLC-MS/MS Liquid chromatography-tandem mass spectrometry\u003c/p\u003e\n\u003cp\u003epH Potential of hydrogen\u003c/p\u003e\n\u003cp\u003eppm Parts per million\u003c/p\u003e\n\u003cp\u003eSGF Simulated gastric fluid\u003c/p\u003e\n\u003cp\u003eSIF Simulated intestinal fluid\u003c/p\u003e\n\u003cp\u003eTEER Transepithelial electrical resistance\u003c/p\u003e\n\u003cp\u003eTFA Trifluoroacetic acid\u003c/p\u003e\n\u003cp\u003eUSP United States Pharmacopeia\u003c/p\u003e\n\u003cp\u003eUV Ultraviolet\u003c/p\u003e\n\u003cp\u003ew/w Weight per weight\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eGC, TG, PA, HR, JZW, TL, LL and YL are employed by and own stocks in Procter and Gamble. JTS is a former employee of Procter and Gamble and own stocks in Procter and Gamble.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThis study was funded by Procter and Gamble Health.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eStudy conceptualization: GC, YL, JTS. Design of study: GC, TG, PA, JZW, TL, LL, JTS. Acquisition and analysis: HR, JZW. Interpretation of data: GC, YL, TG, PA, HR, JZW, TL, LL, JTS. Drafted work: GC, YL, TG, PA, JZW, TL, LL. Revision and review: GC, YL, TG, PA, TL, LL, JTS.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors would like to thank Jia Hao Wong, Bhuvaneswari S and Adrien Gras from Ipsos Healthcare Singapore for supporting the medical writing.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBadar A. Neuropsychiatric Disorders Associated With Vitamin B12 Deficiency: An Autobiographical Case Report. Cureus 14(1):e21476.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalczuk K, Kaźmierczak-Barańska J, Karwowski BT, Karmańska A, Cieślak M. Vitamin B12\u0026mdash;Multifaceted In Vivo Functions and In Vitro Applications. Nutrients. 2023 June;13(12):2734.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNational Institues of Health. Vitamin B12 [Internet]. [cited 2026 Jan 20]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ods.od.nih.gov/factsheets/VitaminB12-HealthProfessional/\u003c/span\u003e\u003cspan address=\"https://ods.od.nih.gov/factsheets/VitaminB12-HealthProfessional/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUmekar M, Premchandani T, Tatode A, Qutub M, Raut N, Taksande J, et al. Vitamin B12 deficiency and cognitive impairment: A comprehensive review of neurological impact. Brain Disorders. 2025 June;18:100220.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh N. Vitamin B12-Associated Neurological Diseases: Background, Pathophysiology, Epidemiology. 2025 Aug 26 [cited 2026 Jan 20]; Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://emedicine.medscape.com/article/1152670-overview?form=fpf\u003c/span\u003e\u003cspan address=\"https://emedicine.medscape.com/article/1152670-overview?form=fpf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl Amin ASM, Gupta V. Vitamin B12 (Cobalamin). In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 [cited 2026 Jan 20]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/books/NBK559132/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/books/NBK559132/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeeking Health. Seeking Health. 700 [cited 2026 Jan 20]. The Ultimate Guide to Vitamin B12: Forms, Benefits, Supplements, \u0026amp; Mor. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.seekinghealth.com/blogs/education/the-ultimate-guide-to-vitamin-b12-forms-benefits-supplements\u003c/span\u003e\u003cspan address=\"https://www.seekinghealth.com/blogs/education/the-ultimate-guide-to-vitamin-b12-forms-benefits-supplements\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOctagon Chem. Hydroxocobalamin Vs Cyanocobalamin: Your Comprehensive Guide [Internet]. 2025 [cited 2026 Jan 20]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://octagonchem.com/blog/hydroxocobalamin-vs-cyanocobalamin/\u003c/span\u003e\u003cspan address=\"https://octagonchem.com/blog/hydroxocobalamin-vs-cyanocobalamin/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrugBank. DrugBank. [cited 2026 Jan 20]. Cyanocobalamin. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://go.drugbank.com/drugs/DB00115\u003c/span\u003e\u003cspan address=\"https://go.drugbank.com/drugs/DB00115\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAjmera R, Healthline. 2020 [cited 2026 Jan 20]. Methylcobalamin vs. Cyanocobalamin: What\u0026rsquo;s the Difference? Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.healthline.com/nutrition/methylcobalamin-vs-cyanocobalamin\u003c/span\u003e\u003cspan address=\"https://www.healthline.com/nutrition/methylcobalamin-vs-cyanocobalamin\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAroda VR, Edelstein SL, Goldberg RB, Knowler WC, Marcovina SM, Orchard TJ, et al. Long-term Metformin Use and Vitamin B12 Deficiency in the Diabetes Prevention Program Outcomes Study. J Clin Endocrinol Metab. 2016;101(4):1754\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFituri S, Akbar Z, Ganji V. Impact of metformin treatment on cobalamin status in persons with type 2 diabetes. Nutr Rev. 2023;82(4):553\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarchi G, Busti F, Zidanes AL, Vianello A, Girelli D. Cobalamin Deficiency in the Elderly. Mediterr J Hematol Infect Dis. 2020 July 1;12(1):e2020043.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong C. Vitamin B12 deficiency in the elderly: is it worth screening? Hong Kong Med J [Internet]. 2015 Mar 10 [cited 2026 Jan 20]; Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.hkmj.org/earlyrelease/hkmj144383.htm\u003c/span\u003e\u003cspan address=\"http://www.hkmj.org/earlyrelease/hkmj144383.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiklewicz A, Hannibal L, Warren M, Ahmadi KR. A systematic review and meta-analysis of functional vitamin B12 status among adult vegans. Nutr Bull. 2024;49(4):463\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFragasso A. Vitamin B12 Deficiency in Alcoholics. In: Watson RR, Preedy VR, Zibadi S, editors. Alcohol, Nutrition, and Health Consequences [Internet]. Totowa, NJ: Humana Press; 2013 [cited 2026 Jan 22]. pp. 131\u0026ndash;4. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-62703-047-2_10\u003c/span\u003e\u003cspan address=\"10.1007/978-1-62703-047-2_10\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad I, Hafeez A, Akhter N, Vaid F, Qadeer K. Effect of Riboflavin on the Photolysis of Cyanocobolamin in Aqueous Solution. TOACJ. 2012;6(1):22\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazzocato M, Thomazini M, Favaro-Trindade CS. Improving stability of vitamin B12 (Cyanocobalamin) using microencapsulation by spray chilling technique. Food Res Int. 2019;126:108663.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad I, Qadeer K, Zahid S, Sheraz MA, Ismail T, Hussain W et al. Effect of Ascorbic Acid on the Degradation of Cyanocobalamin and Hydroxocobalamin in Aqueous Solution: A Kinetic Study. AAPS PharmSciTech. 2014 June 12;15(5):1324\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnkar A, Kumar A. Vitamin B12 Deficiency. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 [cited 2026 Jan 21]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/books/NBK441923/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/books/NBK441923/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTemova Rakuša Ž, Roškar R, Hickey N, Geremia S. Vitamin B12 in Foods, Food Supplements, and Medicines\u0026mdash;A Review of Its Role and Properties with a Focus on Its Stability. Molecules. 2022;28(1):240.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdullah MSP, Noordin MI, Ismail SIM, Mustapha NM, Jasamai M, Danik MF, et al. Recent Advances in the Use of Animal-Sourced Gelatine as Natural Polymers for Food, Cosmetics and Pharmaceutical Applications. Sains Malaysiana. 2018;47(2):323\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEchave MC, Hern\u0026aacute;ez-Moya R, Iturriaga L, Pedraz JL, Lakshminarayanan R, Dolatshahi-Pirouz A, et al. Recent advances in gelatin-based therapeutics. Expert Opin Biol Ther. 2019;19(8):773\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePateiro M, G\u0026oacute;mez B, Munekata PES, Barba FJ, Putnik P, Kovačević DB, et al. Nanoencapsulation of Promising Bioactive Compounds to Improve Their Absorption, Stability, Functionality and the Appearance of the Final Food Products. Molecules. 2021;26(6):1547.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoudie KJ, McCreath SJ, Parkinson JA, Davidson CM, Liggat JJ. Investigation of the influence of pH on the properties and morphology of gelatin hydrogels. J Polym Sci. 2023;61(19):2316\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin J, Pan D, Sun Y, Ou C, Wang Y, Cao J. The modification of gelatin films: Based on various cross-linking mechanism of glutaraldehyde at acidic and alkaline conditions. Food Sci Nutr. 2019;7(12):4140\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMilano F, Masi A, Madaghiele M, Sannino A, Salvatore L, Gallo N. Current Trends in Gelatin-Based Drug Delivery Systems. Pharmaceutics. 2023;15(5):1499.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaharros-Molinero A, Caballo‐Gonz\u0026aacute;lez M\u0026Aacute;, de la Mata FJ, Garc\u0026iacute;a‐Gallego S. Shell Formulation in Soft Gelatin Capsules: Design and Characterization. Adv Healthc Mater. 2024;13(1):2302250.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalanč B, Salević-Jelić A, Đorđević V, Bugarski B, Nedović V, Petrović P et al. The Application of Protein Concentrate Obtained from Green Leaf Biomass in Structuring Nanofibers for Delivery of Vitamin B12. Foods [Internet]. 2024 May 18 [cited 2026 Jan 20];13(10). Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.mdpi.com/2304-8158/13/10/1576\u003c/span\u003e\u003cspan address=\"https://www.mdpi.com/2304-8158/13/10/1576\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarzanfar S, kouzekonan GS, Mirjani R, Shekarchi B. Vitamin B12-loaded polycaprolacton/gelatin nanofibrous scaffold as potential wound care material. Biomed Eng Lett 2020 Sept 15;10(4):547\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia CQ, Wang SY, Yuan Y, Wu YQ, Cai Y, Liu JW, et al. The passive diffusion improvement of Vitamin B12 intestinal absorption by Gelucire that fit for commercialized production. Saudi Pharm J. 2023 June;31(6):962\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePires CL, Pra\u0026ccedil;a C, Martins PAT, Batista de Carvalho ALM, Ferreira L, Marques MPM et al. Re-Use of Caco-2 Monolayers in Permeability Assays\u0026mdash;Validation Regarding Cell Monolayer Integrity. Pharmaceutics. 2021 Sept 26;13(10):1563.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMucha P, Kus F, Cysewski D, Smolenski RT, Tomczyk M. Vitamin B12 Metabolism: A Network of Multi-Protein Mediated Processes. Int J Mol Sci. 2024 July 23;25(15):8021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaciejewski B, Str\u0026ouml;m A, Larsson A, Sznitowska M. Soft Gelatin Films Modified with Cellulose Acetate Phthalate Pseudolatex Dispersion\u0026mdash;Structure and Permeability. Polymers. 2018 Sept 3;10(9):981.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao H, Wang J, Hao Z, Zhao D. Gelatin-based biomaterials and gelatin as an additive for chronic wound repair. Front Pharmacol. 2024;15:1398939.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJang Y, Jang J, Kim BY, Song YS, Lee DY. Effect of Gelatin Content on Degradation Behavior of PLLA/Gelatin Hybrid Membranes. Tissue Eng Regen Med. 2024;21(4):557\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDressman JB, Fleisher D. Mixing-tank model for predicting dissolution rate control of oral absorption. J Pharm Sci. 1986;75(2):109\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarum FJO, Merchant HA, Basit AW. Oral modified-release formulations in motion: The relationship between gastrointestinal transit and drug absorption. Int J Pharm. 2010;395(1):26\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavis SS, Hardy JG, Fara JW. Transit of pharmaceutical dosage forms through the small intestine. Gut. 1986;27(8):886\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu LX, Amidon GL, Polli JE, Zhao H, Mehta MU, Conner DP, et al. Biopharmaceutics Classification System: The Scientific Basis for Biowaiver Extensions. Pharm Res. 2002 July;19(7):921\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAllen LH, Miller JW, de Groot L, Rosenberg IH, Smith AD, Refsum H, et al. Biomarkers of Nutrition for Development (BOND): Vitamin B-12 Review. J Nutr. 2018;148(Suppl 4):S1995\u0026ndash;2027.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eda Silva L, McCray S. Vitamin B12: No One Should Be Without It. Nutrition Issues in Gastroenterology [Internet]. 2009;(70). Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://med.virginia.edu/ginutrition/wp-content/uploads/sites/199/2014/06/PG_Jan09_daSilvaArticle.pdf\u003c/span\u003e\u003cspan address=\"https://med.virginia.edu/ginutrition/wp-content/uploads/sites/199/2014/06/PG_Jan09_daSilvaArticle.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChu JN, Traverso G. Foundations of gastrointestinal-based drug delivery and future developments. Nat Rev Gastroenterol Hepatol. 2022;19(4):219\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaikh R, Raj Singh TR, Garland MJ, Woolfson AD, Donnelly RF. Mucoadhesive drug delivery systems. J Pharm Bioallied Sci. 2011;3(1):89\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG\u0026uuml;nzel D, Yu ASL. Claudins and the Modulation of Tight Junction Permeability. Physiol Rev. 2013;93(2):525\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang X, Du Z, Zhang X, Zaman F, Song Z, Guan Y et al. Gelatin-based anticancer drug delivery nanosystems: A mini review. Front Bioeng Biotechnol [Internet]. 2023 Mar 21 [cited 2026 Jan 20];11. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/\u003c/span\u003e\u003cspan address=\"https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fbioe.2023.1158749/full\u003c/span\u003e\u003cspan address=\"10.3389/fbioe.2023.1158749/full\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeterson RJ, Reed RC, Zamecnik CR, Sallam MA, Finbloom JA, Martinez FJ, et al. Apical integrins as a switchable target to regulate the epithelial barrier. J Cell Sci. 2024;137(24):jcs263580.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrito A, Habeych E, Silva-Zolezzi I, Galaffu N, Allen LH. Methods to assess vitamin B12 bioavailability and technologies to enhance its absorption. Nutr Rev. 2018;76(10):778\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInfante M, Leoni M, Caprio M, Fabbri A. Long-term metformin therapy and vitamin B12 deficiency: an association to bear in mind. WJD 2021 July 15;12(7):916\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMushtaq M, Usmani MR, Hameed N, Anwar A, Hashmi AA. Serum Vitamin B12 Deficiency in Chronic Hemodialysis Patients. Cureus 16(4):e58751.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaltrusch S. The Role of Neurotropic B Vitamins in Nerve Regeneration. Biomed Res Int 2021 July 13;2021:9968228.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinzon R, Schellack N, Matawaran BJ, Tsang MW, Deerochanawong C, Hiew FL, et al. Clinical Recommendations for the use of Neurotropic B vitamins (B1, B6, and B12) for the Management of Peripheral Neuropathy: Consensus from a Multidisciplinary Expert Panel. J Assoc Phys India. 2023;71(7):11\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuynh DT, Nguyen NT, Do MD. Vitamin B12 deficiency in diabetic patients treated with metformin: A cross-sectional study. PLoS ONE. 2024;19(4):e0302500.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyan Z, Waris N. Association of vitamin B12 deficiency in people with type 2 diabetes on metformin and without metformin: a multicenter study, Karachi, Pakistan. BMJ Open Diabetes Res Care. 2020;8(1):e001151.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSayedali E, Yalin AE, Yalin S. Association between metformin and vitamin B12 deficiency in patients with type 2 diabetes. World J Diabetes. 2023;14(5):585\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeung S, Mattman A, Snyder F, Kassam R, Meneilly G, Nexo E. Metformin induces reductions in plasma cobalamin and haptocorrin bound cobalamin levels in elderly diabetic patients. Clin Biochem. 2010 June;43(9):759\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreibe E, Miller JW, Foutouhi SH, Green R, Nexo E. Metformin increases liver accumulation of vitamin B12 \u0026ndash; An experimental study in rats. Biochimie. 2013;95(5):1062\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLacombe V, Vinatier E, Roquin G, Copin MC, Delattre E, Hammi S, et al. Oral vitamin B12 supplementation in pernicious anemia: a prospective cohort study. Am J Clin Nutr. 2024 July;120(1):217\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"vitamin B12, gelatin encapsulation, gastric stability, Caco-2, passive absorption","lastPublishedDoi":"10.21203/rs.3.rs-8670841/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8670841/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCyanocobalamin delivery in solid oral formulations is limited by gastric acid instability and the dependence on impaired haptocorrin/ intrinsic factor/ pathways in groups at high-risk of vitamin B12 deficiency such as metformin-treated diabetic patients (14\u0026ndash;41% vitamin B12 deficiency) and older adults (10\u0026ndash;40%). Gelatin encapsulation may enhance vitamin B12 delivery at therapeutic doses via acid protection and mucoadhesion.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn vitro studies compared 1% w/w gelatinised cyanocobalamin with non-gelatinised vitamin B12 using simulated gastric fluid (SGF, pH 1.2; HPLC stability 0\u0026ndash;3 h) and differentiated Caco-2 monolayers (24 h apical uptake\u0026thinsp;\u0026plusmn;\u0026thinsp;SGF/SIF preexposure; LC17 MS/MS; TEER).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eGelatinised vitamin B12 retained 97.1% to 98.3% whereas non-gelatinised vitamin B12 retained 90.0% to 93.7% of the total vitamin B12 content in SGF (3-fold less degradation, 1-3h). Caco-2 monolayers presented 3˜-fold higher intracellular/basolateral vitamin B12 with gelatinised than with non-gelatinised vitamin B12, without TEER disruption.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eGelatin encapsulation confers superior gastric stability and epithelial uptake, supporting progression to pharmacokinetic studies for improved therapeutic vitamin B12 delivery in absorption-compromised populations.\u003c/p\u003e","manuscriptTitle":"Optimizing vitamin B12 delivery in solid pharmaceutical formats: The Role of Gelatin in Stability and Formulation Performance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-08 17:32:41","doi":"10.21203/rs.3.rs-8670841/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f2ece6ad-38ae-4102-9c88-5c9f803b1da4","owner":[],"postedDate":"March 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T05:40:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-08 17:32:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8670841","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8670841","identity":"rs-8670841","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}