10.57647/jnsc.2026.1603.11

Design of a Calcium-Triggered Nanostructured Delivery System for Controlled In Vitro Release of Trypsin Inhibitor

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  • Yugang Qin1,
  • Wenxuan Zhang1,
  • Bolun Zhang1,
  • Gaoxue Li2,
  • Yongbiao Ma3,
  • Qiang Zhang1,
  • Xiaojun Wei1,
  • Yang Xu*1,
  1. Department of Hepatobiliary Surgery, Aerospace Center Hospital, Beijing, 100049, China
  2. Department of Gastroenterology, Shouguang People’s Hospital, Shouguang, Shandong, 262702, China
  3. Department of Pancreatic Surgery Weifang People’s Hospital, Weifang, Shandong, 261100, China

Received: 11-12-2025

Revised: 07-01-2026

Accepted: 03-02-2026

Published in Issue 30-06-2026

Copyright (c) 2026 Yugang Qin, Wenxuan Zhang, Bolun Zhang, Gaoxue Li, Yongbiao Ma, Qiang Zhang, Xiaojun Wei, Yang Xu (Author)

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This work is licensed under a Creative Commons Attribution 4.0 International License.

How to Cite

Qin, Y., Zhang, W., Zhang, B., Li, G., Ma, Y., Zhang, Q., Wei, X., & Xu, Y. (2026). Design of a Calcium-Triggered Nanostructured Delivery System for Controlled In Vitro Release of Trypsin Inhibitor. Journal of Nanostructure in Chemistry, 16(3 (June 2026). https://doi.org/10.57647/jnsc.2026.1603.11
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Abstract

We report a mesoporous silica nanoparticle platform that exploits physiological divalent ions as a benign trigger to control stage-specific protein release under gastrointestinal-mimicking in vitro conditions, using soybean trypsin inhibitor as a model cargo. Spherical pore-expanded MSNs (~300 nm, pore diameter ~3.8 nm, BET surface area ~800 m2/g) were synthesized, loaded with up to 15.6 wt% inhibitor at 75% encapsulation efficiency, and then capped with an ~8 wt% PVA–borate nanogel gate to yield PB-MSN composites containing 14.5 wt% protein. Structural, textural and dispersion analyses confirmed that the coating partially occluded pore mouths while preserving the mesoporous framework and improving colloidal stability. In a two-stage simulated gastrointestinal protocol, PB-MSNs leaked only 3.8 ± 0.5% cargo after 2 h in pH 1.2 medium, whereas uncoated MSNs released 68 ± 5% under the same conditions. Subsequent exposure to pH 7.4 buffer containing 10 mM CaCl2 triggered a rapid burst, with cumulative release reaching ~40% within 0.5 h, 78 ± 3% at 4 h and 88 ± 4% at 8 h; in Ca2+-free buffer, release remained ≤12.3 ± 1.5% over 8 h, demonstrating a sharp ion-dependent on/off effect. Trypsin activity assays showed that inhibitor liberated under triggering conditions suppressed trypsin activity to a level comparable to free inhibitor, while SGF supernatants exhibited negligible inhibition, indicating substantial retention of inhibitory function in a reductionist activity assay and minimal gastric-phase leakage. The combined data establish PVA–borate-gated MSNs as a modular, ion-responsive platform that decouples protection in acid from fast deployment in near-neutral media. While calcium-responsive carriers and borate-based dynamic networks are well established in other delivery and hydrogel contexts, the present work translates this chemistry into an MSN pore-gating architecture in which Ca²⁺ acts as a competitive borate-binding trigger to dismantle a nanoscale sacrificial gate and thereby generate a sharp ‘off/on’ release response under GI-mimicking conditions.

Keywords

  • Mesoporous silica nanoparticles,
  • PVA–borate gate,
  • Gastrointestinal targeting,
  • Protein nanocarriers,
  • Ion-responsive drug platform
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References

  1. Dang, Y., Guan, J. Nanoparticle-based drug delivery systems for cancer therapy. Smart Mater. Med. 1, 10–19 (2020).
  2. Gao, Y., Gao, D., Shen, J., Wang, Q. A review of mesoporous silica nanoparticle delivery systems in chemo-based combination cancer therapies. Front. Chem. 8, 598722 (2020).
  3. Lou, J., Best, M. D. Calcium-responsive liposomes: toward ion-mediated targeted drug delivery. Methods Enzymol. 640, 105–129 (2020).
  4. Carneiro, A. A. B., Patekar, S., Goyal, M., De Campos, S. B., Bally, J., Hassanpour, M., Zhang, Z. pH-responsive mesoporous silica nanoparticles from rice husk ash for delivering trypsin inhibitor to control cotton bollworm. Ind. Crops Prod. 228, 120934 (2025).
  5. Nicze, M., Borówka, M., Dec, A., Niemiec, A., Bułdak, Ł., Okopień, B. The current and promising oral delivery methods for protein- and peptide-based drugs. Int. J. Mol. Sci. 25, 815 (2024).
  6. Jain, D., Mahammad, S. S., Singh, P. P., Kodipyaka, R. A. A review on parenteral delivery of peptides and proteins. Drug Dev. Ind. Pharm. 45, 1403–1420 (2019).
  7. Onoue, S., Yamada, K., Sato, H. Advanced oral drug delivery systems: current challenges and emerging technologies. Acta Pharm. Sin. B (2025).
  8. Deraison, C., Vergnolle, N. Proteases in intestinal health and disease. Nat. Rev. Gastroenterol. Hepatol. 22, 1–23 (2025).
  9. Islam, M. R., Ihenacho, K., Park, J. W., Islam, I. S. Plasmid DNA nicking: a novel activity of soybean trypsin inhibitor and bovine aprotinin. Sci. Rep. 9, 11596 (2019).
  10. Kunitz, M. Crystalline soybean trypsin inhibitor. J. Gen. Physiol. 30, 291–310 (1947).
  11. Coria, L. M., Pueblas Castro, C., Bruno, L., Nakaya, H. I., Pasquevich, K. A., Cassataro, J. Discovery of protease inhibitors from bacteria as novel adjuvants for oral vaccine formulations. Front. Immunol. 16, 1679540 (2025).
  12. Sharma, V., Devkota, L., Kishore, N., Dhital, S. Understanding the interplay between dietary fiber, polyphenols, and digestive enzymes. Food Hydrocoll. 166, 111310 (2025).
  13. de Souza Nascimento, A. M., de Oliveira Segundo, V. H., Felipe Camelo Aguiar, A. J., Piuvezam, G., Souza Passos, T., Florentino da Silva Chaves Damasceno, K. S. F. S., de Araújo Morais, A. H. Antibacterial action mechanisms and mode of trypsin inhibitors: a systematic review. J. Enzyme Inhib. Med. Chem. 37, 749–759 (2022).
  14. Bhattacharyya, S., Wang, H., Ducheyne, P. Polymer-coated mesoporous silica nanoparticles for the controlled release of macromolecules. Acta Biomater. 8, 3429–3435 (2012).
  15. Carneiro, A. A. B., Patekar, S., Goyal, M., de Almeida, S., Dayarathne, N. K., De Campos, S. B., Bally, J., Hassanpour, M., Zhang, Z. Lignin-enabled silica hybrid nanoparticles from rice husk for improved biopesticide delivery and cotton bollworm control. Int. J. Biol. Macromol. 309, 142589 (2025).
  16. Pikor, D., Hurła, M., Słowikowski, B., Szymanowicz, O., Poszwa, J., Banaszek, N., Drelichowska, A., Jagodziński, P. P., Kozubski, W., Dorszewska, J. Calcium ions in the physiology and pathology of the central nervous system. Int. J. Mol. Sci. 25, 13133 (2024).
  17. Luan, S., Wang, C. Calcium signaling mechanisms across kingdoms. Annu. Rev. Cell Dev. Biol. 37, 311–340 (2021).
  18. Klipp, A., Greitens, C., Scherer, D., Elsener, A., Leroux, J.-C., Burger, M. Modular calcium-responsive and CD9-targeted phospholipase system enhancing endosomal escape for DNA delivery. Adv. Sci. 12, 2410815 (2025).
  19. Hunt, H., Tilūnaitė, A., Bass, G., Soeller, C., Roderick, H. L., Rajagopal, V., Crampin, E. J. Ca2+ release via IP3 receptors shapes the cardiac Ca2+ transient for hypertrophic signaling. Biophys. J. 119, 1178–1192 (2020).
  20. Liu, C., Tang, X., Huang, G. Biodegradable Ca2+ doped mesoporous silica nanoparticles promote chemotherapy synergism with calcicoptosis and activate anti-tumor immunity. Inorganics 12, 152 (2024).
  21. Choi, E., Lim, D.-K., Kim, S. Calcium-doped mesoporous silica nanoparticles as a lysosomolytic nanocarrier for amine-free loading and cytosolic delivery of siRNA. J. Ind. Eng. Chem. 81, 71–80 (2020).
  22. Dan, N., Samanta, K., Almoazen, H. An update on pharmaceutical strategies for oral delivery of therapeutic peptides and proteins in adults and pediatrics. Children 7, 307 (2020).
  23. Xu, B., Li, S., Shi, R., Liu, H. Multifunctional mesoporous silica nanoparticles for biomedical applications. Sig. Transduct. Target. Ther. 8, 435 (2023).
  24. Zhu, H., Zeng, J., Jiang, S., Huang, S., Xie, Z., Huang, Y., Lin, S., Wang, B. Poly(vinyl alcohol)/borax/modified silver nanowire composite hydrogel with self-healing, high conductivity, and electromagnetic interference shielding properties. J. Alloys Compd. 1011, 178437 (2025).
  25. Wu, H., Su, Q., Zhou, R., Yan, J., Feng, Y., Guan, C., Su, W., Chen, D., Tang, L., Huang, X., et al. Synergistic borate crosslinking and chain entanglement for mechanically robust and water-rich hydrogels. J. Colloid Interface Sci. 702, 138972 (2026).
  26. Taniguchi, T., Urayama, K. Linear dynamic viscoelasticity of dual cross-link poly(vinyl alcohol) hydrogel with determined borate ion concentration. Gels 7, 71 (2021).
  27. Lou, J., Carr, A. J., Watson, A. J., Mattern-Schain, S. I., Best, M. D. Calcium-responsive liposomes via a synthetic lipid switch. Chem. Eur. J. 24, 3599–3607 (2018).
  28. Rogers, H. R., van den Berg, C. M. G. Determination of borate ion-pair stability constants by potentiometry and non-approximative linearization of titration data. Talanta 35, 271–275 (1988).
  29. Hu, C., Lu, W., Mata, A., Nishinari, K., Fang, Y. Ions-induced gelation of alginate: mechanisms and applications. Int. J. Biol. Macromol. 177, 578–588 (2021).
  30. Mao, J., Liu, B., Xiao, Y., Yang, X., Lin, C., Zhang, Y., Wang, Q., Zhang, Q. Study of crosslinker size on the rheological properties of borate crosslinked guar gum. Int. J. Biol. Macromol. 231, 123284 (2023).
  31. Chen, J., Xu, Z., Zhang, M., Ju, Z., Niu, Z., Ma, Y., Xu, Z., Zhang, T., Shi, F. Mesoporous silica nanoparticles: a synthesis guide and research progress in the biomedical field. Mater. Today Chem. 42, 102426 (2024).
  32. Carvalho, G. C., Marena, G. D., Karnopp, J. C. F., Jorge, J., Sábio, R. M., Martines, M. A. U., Bauab, T. M., Chorilli, M. Cetyltrimethylammonium bromide in the synthesis of mesoporous silica nanoparticles: general aspects and in vitro toxicity. Adv. Colloid Interface Sci. 307, 102746 (2022).
  33. Martinez-Ortigosa, J., Millán, R., Simancas, J., Hernández-Rodríguez, M., Vidal-Moya, J. A., Jordá, J. L., Martineau-Corcos, C., Sarou-Kanian, V., Boronat, M., Blasco, T., et al. Crystalline phase transition in as-synthesized pure silica zeolite RTH containing tetra-alkyl phosphonium as organic structure directing agent. J. Mater. Chem. A 12, 876–891 (2024).
  34. Li, X., Yin, H., Zhang, J., Liu, J., Chen, G. Effect of organic template removal approaches on physiochemical characterization of Ni/Al-SBA-15 and eugenol hydrodeoxygenation. J. Solid State Chem. 282, 121063 (2020).
  35. Kong, X., Li, Y., Liu, X. Purification of soybean Kunitz trypsin inhibitor and the mechanism of its passivation by lysine and disulfide bond modifications. Food Biosci. 55, 103042 (2023).
  36. Wang, A., Ma, Y., Zhao, D. Pore engineering of porous materials: effects and applications. ACS Nano 18, 22829–22854 (2024).
  37. Kong, J., Park, S. S., Ha, C.-S. pH-sensitive polyacrylic acid-gated mesoporous silica nanocarrier incorporated with calcium ions for controlled drug release. Materials 15, 5926 (2022).
  38. Wang, Y., Zhao, K., Xie, L., Li, K., Zhang, W., Xi, Z., Wang, X., Xia, M., Xu, L. Construction of calcium carbonate-liposome dual-film coated mesoporous silica as a delayed drug release system for antitumor therapy. Colloids Surf. B Biointerfaces 212, 112357 (2022).
  39. da Silva, J. A. L. Borate esters of polyols: occurrence, applications and implications. Inorg. Chim. Acta 520, 120307 (2021).
  40. Ireland, P., Fordtran, J. S. Effect of dietary calcium and age on jejunal calcium absorption in humans studied by intestinal perfusion. J. Clin. Invest. 52, 2672–2681 (1973).
  41. MacConaill, M. Calcium precipitation from mammalian physiological salines (Ringer solutions) and the preparation of high [Ca] media. J. Pharmacol. Methods 14, 147–155 (1985).
  42. Wang, J., Zhao, M., Liu, T., Feng, F., Zhou, A. Guidelines for the digestive enzymes inhibition assay. eFood 3, e31 (2022).
  43. Polyakov, Y. S., Zydney, A. L. Ultrafiltration membrane performance: effects of pore blockage/constriction. J. Membr. Sci. 434, 106–120 (2013).
  44. Galata, D. L., Péterfi, O., Ficzere, M., Szabó-Szőcs, B., Szabó, E., Nagy, Z. K. The current state-of-the art in pharmaceutical continuous film coating: a review. Int. J. Pharm. 669, 125052 (2025).
  45. Palomeque Chávez, J. C., Mohammed, A. A., Aghajanpour, S., Li, S., Enzo, S., Kelly, N. L., Bradley, D. G., Kerherve, G., Porter, A. E., Hanna, J. V., et al. One-pot sol–gel synthesis of Sr/Ca-doped silica nanoparticles for osteogenic therapy in osteoporosis. Front. Nanotechnol. 7, 1634210 (2025).
  46. Jurašin, D. D., Ćurlin, M., Capjak, I., Crnković, T., Lovrić, M., Babič, M., Horák, D., Vrček, I. V., Gajović, S. Surface coating affects behavior of metallic nanoparticles in a biological environment. Beilstein J. Nanotechnol. 7, 246–262 (2016).
  47. Trayford, C., van Rijt, S. In situ modified mesoporous silica nanoparticles: synthesis, properties and theranostic applications. Biomater. Sci. 12, 5450–5467 (2024).
  48. Xie, X., Yang, D. Self-healing thermally conductive polymer composites based on polyvinyl alcohol with dynamic borate ester and hydrogen bonds. Compos. Part A Appl. Sci. Manuf. 181, 108131 (2024).
  49. Fitzsimons, B., Parry, T. Coating failures and defects. In Protective Organic Coatings; Tator, K. B., Ed.; ASM International, 502–512 (2015).
  50. Krebs, J., Agellon, L. B., Michalak, M. Ca2+ homeostasis and endoplasmic reticulum (ER) stress: an integrated view of calcium signaling. Biochem. Biophys. Res. Commun. 460, 114–121 (2015).
  51. Ye, J., Liu, L., Lan, W., Xiong, J. Targeted release of soybean peptide from CMC/PVA hydrogels in simulated intestinal fluid and their pharmacokinetics. Carbohydr. Polym. 310, 120713 (2023).
  52. Chang, S.-H. A dishing model for STI CMP process. Microelectron. Eng. 82, 136–142 (2005).
  53. Cui, M., Zhang, M., Liu, K. Colon-targeted drug delivery of polysaccharide-based nanocarriers for synergistic treatment of inflammatory bowel disease: a review. Carbohydr. Polym. 272, 118530 (2021).
  54. Khalbas, A. H., Albayati, T. M., Ali, N. S., Salih, I. K. Drug loading methods and kinetic release models using of mesoporous silica nanoparticles as a drug delivery system: a review. S. Afr. J. Chem. Eng. 50, 261–280 (2024).
  55. Shalon, D., Culver, R. N., Grembi, J. A., Folz, J., Treit, P. V., Shi, H., Rosenberger, F. A., Dethlefsen, L., Meng, X., Yaffe, E., et al. Profiling the human intestinal environment under physiological conditions. Nature 617, 581–591 (2023).
  56. Fatima, R., Katiyar, P., Kushwaha, K. Recent advances in mesoporous silica nanoparticle: synthesis, drug loading, release mechanisms, and diverse applications. Front. Nanotechnol. 7, 1564188 (2025).
  57. Mace, O. J., Morgan, E. L., Affleck, J. A., Lister, N., Kellett, G. L. Calcium absorption by Cav1.3 induces terminal web myosin II phosphorylation and apical GLUT2 insertion in rat intestine. J. Physiol. 580, 605–616 (2007).
  58. Niewiadomski, K., Szopa, D., Pstrowska, K., Wróbel, P., Witek-Krowiak, A. Comparative analysis of crosslinking methods and their impact on the physicochemical properties of SA/PVA hydrogels. Materials 17, 1816 (2024).
  59. Al-Emam, E., Soenen, H., Caen, J., Janssens, K. Characterization of polyvinyl alcohol-borax/agarose (PVA-B/AG) double network hydrogel utilized for the cleaning of works of art. Herit. Sci. 8, 106 (2020).
  60. Li, J., Liu, Y., Chen, Q. Conformation of dilute poly(vinyl alcohol)-borax complex by asymmetric flow field-flow fractionation. J. Chromatogr. A 1624, 461260 (2020).
  61. Khalil, A. K. A., Teow, Y. H., Takriff, M. S., Ahmad, A. L., Atieh, M. A. Recent developments in stimuli-responsive polymer for emerging applications: a review. Results Eng. 25, 103900 (2025).
  62. Guo, P., Liang, J., Li, Y., Lu, X., Fu, H., Jing, H., Guan, S., Han, D., Niu, L. High-strength and pH-responsive self-healing polyvinyl alcohol/poly 6-acrylamidohexanoic acid hydrogel based on dual physically cross-linked network. Colloids Surf. A Physicochem. Eng. Asp. 571, 64–71 (2019).
  63. Fu, S., Rempson, C. M., Puche, V., Zhao, B., Zhang, F. Construction of disulfide containing redox-responsive polymeric nanomedicine. Methods 199, 67–79 (2022).
  64. Nakagawa, T., Wang, X., Miguez-Cabello, F. J., Bowie, D. The open gate of the AMPA receptor forms a Ca2+ binding site critical in regulating ion transport. Nat. Struct. Mol. Biol. 31, 688–700 (2024).
  65. Nair, A., Chandrashekhar H., R., Day, C. M., Garg, S., Nayak, Y., Shenoy, P. A., Nayak, U. Y. Polymeric functionalization of mesoporous silica nanoparticles: biomedical insights. Int. J. Pharm. 660, 124314 (2024).
  66. Lérida-Viso, A., Estepa-Fernández, A., García-Fernández, A., Martí-Centelles, V., Martínez-Máñez, R. Biosafety of mesoporous silica nanoparticles: towards clinical translation. Adv. Drug Deliv. Rev. 201, 115049 (2023).
  67. Adepu, S., Ramakrishna, S. Controlled drug delivery systems: current status and future directions. Molecules 26, 5905 (2021).
  68. Deluque, A. L., Dimke, H., Alexander, R. T. Biology of calcium homeostasis regulation in intestine and kidney. Nephrol. Dial. Transplant. 40, 435–445 (2025).
  69. Cui, Y., Dong, H., Cai, X., Wang, D., Li, Y. Mesoporous silica nanoparticles capped with disulfide-linked PEG gatekeepers for glutathione-mediated controlled release. ACS Appl. Mater. Interfaces 4, 3177–3183 (2012).
  70. Mas, N., Agostini, A., Mondragón, L., Bernardos, A., Sancenón, F., Marcos, M. D., Martínez-Máñez, R., Costero, A. M., Gil, S., Merino-Sanjuán, M., et al. Enzyme-responsive silica mesoporous supports capped with azopyridinium salts for controlled delivery applications. Chem. Eur. J. 19, 1346–1356 (2013).
  71. Haq, I. U., Liu, H., Ghafar, M. A., Zafar, S., Subhan, M., Abbasi, A., Hyder, M., Basit, A., Rebouh, N. Y., Hou, Y. Mesoporous silica nanoparticles impair physiology and reproductive fitness of Tuta absoluta through plant-mediated oxidative stress and enzymatic disruption. Insects 16, 877 (2025).