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 - 3 h). Caco-2 monolayers presented 3-fold higher intracellular/basolateral vitamin B12 with gelatinised than with non-gelatinised vitamin B12, without TEER disruption. Conclusion: Gelatin encapsulation confers superior gastric stability and epithelial uptake, supporting progression to pharmacokinetic studies for improved therapeutic vitamin B12 delivery in absorption-compromised populations.
References
[1]
Badar, A. (2022) Neuropsychiatric Disorders Associated with Vitamin B12 Deficiency: An Autobiographical Case Report. Cureus, 14, e21476. https://doi.org/10.7759/cureus.21476
[2]
Halczuk, K., Ka？mierczak-Barańska, J., Karwowski, B.T., Karmańska, A. and Cie？lak, M. (2023) Vitamin B12—Multifaceted in Vivo Functions and in Vitro Applications. Nutrients, 15, Article 2734. https://doi.org/10.3390/nu15122734
[3]
Umekar, M., Premchandani, T., Tatode, A., Qutub, M., Raut, N., Taksande, J., et al. (2025) Vitamin B12 Deficiency and Cognitive Impairment: A Comprehensive Review of Neurological Impact. Brain Disorders, 18, Article ID: 100220. https://doi.org/10.1016/j.dscb.2025.100220
[4]
Al Amin, A.S.M. and Gupta, V. (2025) Vitamin B12 (Cobalamin). StatPearls. http://www.ncbi.nlm.nih.gov/books/NBK559132/
[5]
Obeid, R., Fedosov, S.N. and Nexo, E. (2015) Cobalamin Coenzyme Forms Are Not Likely to Be Superior to Cyano-and Hydroxyl-Cobalamin in Prevention or Treatment of Cobalamin Deficiency. Molecular Nutrition & Food Research, 59, 1364-1372. https://doi.org/10.1002/mnfr.201500019
[6]
Temova Raku？a, ？., Ro？kar, R., Hickey, N. and Geremia, S. (2023) Vitamin B12 in Foods, Food Supplements, and Medicines—A Review of Its Role and Properties with a Focus on Its Stability. Molecules, 28, Article 240. https://doi.org/10.3390/molecules28010240
[7]
Behringer, C.R., Kulkarni, A. and Weinstein, A. (2025) Vitamin B12: A Comprehensive Review of Natural vs Synthetic Forms of Consumption and Supplementation. Cureus, 17, e96258. https://doi.org/10.7759/cureus.96258
[8]
Aroda, V.R., Edelstein, S.L., Goldberg, R.B., Knowler, W.C., Marcovina, S.M., Orchard, T.J., et al. (2016) Long-Term Metformin Use and Vitamin B12 Deficiency in the Diabetes Prevention Program Outcomes Study. The Journal of Clinical Endocrinology & Metabolism, 101, 1754-1761. https://doi.org/10.1210/jc.2015-3754
[9]
Fituri, S., Akbar, Z. and Ganji, V. (2023) Impact of Metformin Treatment on Cobalamin Status in Persons with Type 2 Diabetes. Nutrition Reviews, 82, 553-560. https://doi.org/10.1093/nutrit/nuad045
[10]
Marchi, G., Busti, F., Lira Zidanes, A., Vianello, A. and Girelli, D. (2020) Cobalamin Deficiency in the Elderly. Mediterranean Journal of Hematology and Infectious Diseases, 12, e2020043. https://doi.org/10.4084/mjhid.2020.043
[11]
Wong, C. (2015) Vitamin B12 Deficiency in the Elderly: Is It Worth Screening? Hong Kong Medical Journal, 21, 155-164. https://doi.org/10.12809/hkmj144383
[12]
Niklewicz, A., Hannibal, L., Warren, M. and Ahmadi, K.R. (2024) A Systematic Review and Meta-Analysis of Functional Vitamin B12 Status among Adult Vegans. Nutrition Bulletin, 49, 463-479. https://doi.org/10.1111/nbu.12712
[13]
Fragasso, A. (2013) Vitamin B12 Deficiency in Alcoholics. In: Alcohol, Nutrition, and Health Consequences, Humana Press, 131-134. https://doi.org/10.1007/978-1-62703-047-2_10
[14]
Allen, L.H., Miller, J.W., de Groot, L., Rosenberg, I.H., Smith, A.D., Refsum, H., et al. (2018) Biomarkers of Nutrition for Development (BOND): Vitamin B-12 Review. The Journal of Nutrition, 148, 1995S-2027S. https://doi.org/10.1093/jn/nxy201
[15]
Brito, A., Habeych, E., Silva-Zolezzi, I., Galaffu, N. and Allen, L.H. (2018) Methods to Assess Vitamin B12 Bioavailability and Technologies to Enhance Its Absorption. Nutrition Reviews, 76, 778-792. https://doi.org/10.1093/nutrit/nuy026
[16]
Ahmad, I. (2012) Effect of Riboflavin on the Photolysis of Cyanocobolamin in Aqueous Solution. The Open Analytical Chemistry Journal, 6, 22-27. https://doi.org/10.2174/1874065001206010022
[17]
Chalella Mazzocato, M., Thomazini, M. and Favaro-Trindade, C.S. (2019) Improving Stability of Vitamin B12 (Cyanocobalamin) Using Microencapsulation by Spray Chilling Technique. Food Research International, 126, Article ID: 108663. https://doi.org/10.1016/j.foodres.2019.108663
[18]
Ahmad, I., Qadeer, K., Zahid, S., Sheraz, M.A., Ismail, T., Hussain, W., et al. (2014) Effect of Ascorbic Acid on the Degradation of Cyanocobalamin and Hydroxocobalamin in Aqueous Solution: A Kinetic Study. AAPS PharmSciTech, 15, 1324-1333. https://doi.org/10.1208/s12249-014-0160-5
[19]
Krawczyk-Coda, M., Zgo？a-Grze？kowiak, A. and Stanisz, E. (2025) Quality of Vitamin B12 Supplements regarding Vitamin Assay and Content of Heavy Metals. Molecules, 30, Article 3808. https://doi.org/10.3390/molecules30183808
[20]
Abdullah, M.S.P., et al. (2018) Recent Advances in the Use of Animal-Sourced Gelatine as Natural Polymers for Food, Cosmetics and Pharmaceutical Applications. Sains Malaysiana, 47, 323-336.
[21]
Echave, M.C., Hernáez-Moya, R., Iturriaga, L., Pedraz, J.L., Lakshminarayanan, R., Dolatshahi-Pirouz, A., et al. (2019) Recent Advances in Gelatin-Based Therapeutics. Expert Opinion on Biological Therapy, 19, 773-779. https://doi.org/10.1080/14712598.2019.1610383
[22]
Pateiro, M., Gómez, B., Munekata, P.E.S., Barba, F.J., Putnik, P., Kova？evi？, D.B., et al. (2021) Nanoencapsulation of Promising Bioactive Compounds to Improve Their Absorption, Stability, Functionality and the Appearance of the Final Food Products. Molecules, 26, Article 1547. https://doi.org/10.3390/molecules26061547
[23]
Goudie, K.J., McCreath, S.J., Parkinson, J.A., Davidson, C.M. and Liggat, J.J. (2023) Investigation of the Influence of pH on the Properties and Morphology of Gelatin Hydrogels. Journal of Polymer Science, 61, 2316-2332. https://doi.org/10.1002/pol.20230141
[24]
Lin, J., Pan, D., Sun, Y., Ou, C., Wang, Y. and Cao, J. (2019) The Modification of Gelatin Films: Based on Various Cross-Linking Mechanism of Glutaraldehyde at Acidic and Alkaline Conditions. Food Science & Nutrition, 7, 4140-4146. https://doi.org/10.1002/fsn3.1282
[25]
Milano, F., Masi, A., Madaghiele, M., Sannino, A., Salvatore, L. and Gallo, N. (2023) Current Trends in Gelatin-Based Drug Delivery Systems. Pharmaceutics, 15, Article 1499. https://doi.org/10.3390/pharmaceutics15051499
[26]
Naharros-Molinero, A., Caballo-González, M.á., de la Mata, F.J. and García-Gallego, S. (2024) Shell Formulation in Soft Gelatin Capsules: Design and Characterization. Advanced Healthcare Materials, 13, Article ID: 2302250. https://doi.org/10.1002/adhm.202302250
[27]
Balan？, B., Salevi？-Jeli？, A., ？or？evi？, V., Bugarski, B., Nedovi？, V., Petrovi？, P., et al. (2024) The Application of Protein Concentrate Obtained from Green Leaf Biomass in Structuring Nanofibers for Delivery of Vitamin B12. Foods, 13, Article 1576. https://doi.org/10.3390/foods13101576
[28]
Farzanfar, S., kouzekonan, G.S., Mirjani, R. and Shekarchi, B. (2020) Vitamin B12-Loaded Polycaprolacton/Gelatin Nanofibrous Scaffold as Potential Wound Care Material. Biomedical Engineering Letters, 10, 547-554. https://doi.org/10.1007/s13534-020-00165-6
[29]
Jia, C., Wang, S., Yuan, Y., Wu, Y., Cai, Y., Liu, J., et al. (2023) The Passive Diffusion Improvement of Vitamin B12 Intestinal Absorption by Gelucire That Fit for Commercialized Production. Saudi Pharmaceutical Journal, 31, 962-971. https://doi.org/10.1016/j.jsps.2023.04.024
[30]
Pires, C.L., Pra？a, C., Martins, P.A.T., Batista de Carvalho, A.L.M., Ferreira, L., Marques, M.P.M., et al. (2021) Re-use of Caco-2 Monolayers in Permeability Assays—Validation regarding Cell Monolayer Integrity. Pharmaceutics, 13, Article 1563. https://doi.org/10.3390/pharmaceutics13101563
[31]
Mucha, P., Kus, F., Cysewski, D., Smolenski, R.T. and Tomczyk, M. (2024) Vitamin B12 Metabolism: A Network of Multi-Protein Mediated Processes. International Journal of Molecular Sciences, 25, Article 8021. https://doi.org/10.3390/ijms25158021
[32]
Maciejewski, B., Str？m, A., Larsson, A. and Sznitowska, M. (2018) Soft Gelatin Films Modified with Cellulose Acetate Phthalate Pseudolatex Dispersion—Structure and Permeability. Polymers, 10, Article 981. https://doi.org/10.3390/polym10090981
[33]
Cao, H., Wang, J., Hao, Z. and Zhao, D. (2024) Gelatin-Based Biomaterials and Gelatin as an Additive for Chronic Wound Repair. Frontiers in Pharmacology, 15, Article 1398939. https://doi.org/10.3389/fphar.2024.1398939
[34]
Jang, Y., Jang, J., Kim, B., Song, Y. and Lee, D.Y. (2024) Effect of Gelatin Content on Degradation Behavior of PLLA/Gelatin Hybrid Membranes. Tissue Engineering and Regenerative Medicine, 21, 557-569. https://doi.org/10.1007/s13770-024-00626-4
[35]
Dressman, J.B. and Fleisher, D. (1986) Mixing-Tank Model for Predicting Dissolution Rate Control of Oral Absorption. Journal of Pharmaceutical Sciences, 75, 109-116. https://doi.org/10.1002/jps.2600750202
[36]
Varum, F.J.O., Merchant, H.A. and Basit, A.W. (2010) Oral Modified-Release Formulations in Motion: The Relationship between Gastrointestinal Transit and Drug Absorption. International Journal of Pharmaceutics, 395, 26-36. https://doi.org/10.1016/j.ijpharm.2010.04.046
[37]
Davis, S.S., Hardy, J.G. and Fara, J.W. (1986) Transit of Pharmaceutical Dosage Forms through the Small Intestine. Gut, 27, 886-892. https://doi.org/10.1136/gut.27.8.886
[38]
Yu, L.X., Amidon, G.L., Polli, J.E., Zhao, H., Mehta, M.U., Conner, D.P., et al. (2002) Biopharmaceutics Classification System: The Scientific Basis for Biowaiver Extensions. Pharmaceutical Research, 19, 921-925. https://doi.org/10.1023/a:1016473601633
[39]
da Silva, L. and McCray, S. (2009) Vitamin B12: No One Should Be Without It. Nutrition Issues in Gastroenterology, No. 70, 34-46. https://med.virginia.edu/ginutrition/wp-content/uploads/sites/199/2014/06/PG_Jan09_daSilvaArticle.pdf
[40]
Chu, J.N. and Traverso, G. (2022) Foundations of Gastrointestinal-Based Drug Delivery and Future Developments. Nature Reviews Gastroenterology & Hepatology, 19, 219-238. https://doi.org/10.1038/s41575-021-00539-w
[41]
Donnelly, R., Shaikh, R., Raj Singh, T., Garland, M. and Woolfson, A. (2011) Mucoadhesive Drug Delivery Systems. Journal of Pharmacy and Bioallied Sciences, 3, 89-100. https://doi.org/10.4103/0975-7406.76478
[42]
Günzel, D. and Yu, A.S.L. (2013) Claudins and the Modulation of Tight Junction Permeability. Physiological Reviews, 93, 525-569. https://doi.org/10.1152/physrev.00019.2012
[43]
Peterson, R.J., Reed, R.C., Zamecnik, C.R., Sallam, M.A., Finbloom, J.A., Martinez, F.J., et al. (2024) Apical Integrins as a Switchable Target to Regulate the Epithelial Barrier. Journal of Cell Science, 137, jcs26358. https://doi.org/10.1242/jcs.263580
[44]
Foox, M. and Zilberman, M. (2015) Drug Delivery from Gelatin-Based Systems. Expert Opinion on Drug Delivery, 12, 1547-1563. https://doi.org/10.1517/17425247.2015.1037272
[45]
Benoit, Y.D., Groulx, J., Gagné, D. and Beaulieu, J. (2012) RGD-Dependent Epithelial Cell-Matrix Interactions in the Human Intestinal Crypt. Journal of Signal Transduction, 2012, 1-10. https://doi.org/10.1155/2012/248759
[46]
Infante, M., Leoni, M., Caprio, M. and Fabbri, A. (2021) Long-Term Metformin Therapy and Vitamin B12 Deficiency: An Association to Bear in Mind. World Journal of Diabetes, 12, 916-931. https://doi.org/10.4239/wjd.v12.i7.916
[47]
Mushtaq, M., Usmani, M.R., Hameed, N., Anwar, A. and Hashmi, A.A. (2024) Serum Vitamin B12 Deficiency in Chronic Hemodialysis Patients. Cureus, 16, e58751.
[48]
Baltrusch, S. (2021) The Role of Neurotropic B Vitamins in Nerve Regeneration. BioMed Research International, 2021, Article ID: 9968228. https://doi.org/10.1155/2021/9968228
[49]
Pinzon, R., et al. (2023) 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. The Journal of the Association of Physicians of India, 71, 11-12.
[50]
Huynh, D.T., Nguyen, N.T. and Do, M.D. (2024) Vitamin B12 Deficiency in Diabetic Patients Treated with Metformin: A Cross-Sectional Study. PLOS ONE, 19, e0302500. https://doi.org/10.1371/journal.pone.0302500
[51]
Miyan, Z. and Waris, N. (2020) Association of Vitamin B12 Deficiency in People with Type 2 Diabetes on Metformin and without Metformin: A Multicenter Study, Karachi, Pakistan. BMJ Open Diabetes Research & Care, 8, e001151. https://doi.org/10.1136/bmjdrc-2019-001151
[52]
Sayedali, E., Yalin, A.E. and Yalin, S. (2023) Association between Metformin and Vitamin B12 Deficiency in Patients with Type 2 Diabetes. World Journal of Diabetes, 14, 585-593. https://doi.org/10.4239/wjd.v14.i5.585
[53]
Leung, S., Mattman, A., Snyder, F., Kassam, R., Meneilly, G. and Nexo, E. (2010) Metformin Induces Reductions in Plasma Cobalamin and Haptocorrin Bound Cobalamin Levels in Elderly Diabetic Patients. Clinical Biochemistry, 43, 759-760. https://doi.org/10.1016/j.clinbiochem.2010.02.011
[54]
Greibe, E., Miller, J.W., Foutouhi, S.H., Green, R. and Nexo, E. (2013) Metformin Increases Liver Accumulation of Vitamin B12—An Experimental Study in Rats. Biochimie, 95, 1062-1065. https://doi.org/10.1016/j.biochi.2013.02.002
[55]
Lacombe, V., Vinatier, E., Roquin, G., Copin, M., Delattre, E., Hammi, S., et al. (2024) Oral Vitamin B12 Supplementation in Pernicious Anemia: A Prospective Cohort Study. The American Journal of Clinical Nutrition, 120, 217-224. https://doi.org/10.1016/j.ajcnut.2024.05.019
