Designing advanced functional materials capable of passing through complex biological environments requires a deep understanding of their dynamic structural behavior in situ. We investigate pH-responsive core-shell cubosomes for oral drug delivery applications. These nanoparticles comprise a lipid-based core of cubic Im3m liquid crystalline structure and are coated with a chitosan-N-arginine/alginate polyelectrolyte shell (PS). The cubosomes encapsulate varying concentrations (0-30% w/w) of Aloe vera-derived acemannan, an immunomodulatory macromolecular drug. Utilizing synchrotron small-angle X-ray scattering and cryogenic transmission electron microscopy, we performed an advanced spatiotemporal analysis, which focused on their nanoscale structural evolution under simulated gastric (pH 2.5) and intestinal (pH 7.4) conditions. The interactions with key individual gastrointestinal components, including mucins, pepsin, bile salts, and pancreatin, were systematically examined. Our results demonstrate that acemannan incorporation and environmental pH significantly modulate cubosome structure and heterogeneity (phase coexistence) during disassembly. The pH-responsive polyelectrolyte shell imparts notable structural stability against pepsin and mucins at pH 2.5, ensuring functional gastric protection. However, under intestinal conditions (pH 7.4), bile salt-mediated solubilization caused complete disassembly. Pancreatic lipase-induced digestion triggered a remarkable time-dependent phase transition from a cubic (Im3m) to an inverted hexagonal (HII) topology in PS-cubosomes containing 30% acemannan. A simulated duodenum mixture induced lamellar phases at pH 2.5 for acemannan-loaded systems but led to complete disassembly at pH 7.4, primarily driven by bile salts. Deconvoluting these structural responses over time provides crucial insights into their mechanistic nature. It clarifies pH-dependent stability and component-specific disassembly pathways. The achieved understanding is crucial for designing advanced stimuli-responsive lipid/biopolymer nanomaterials that facilitate efficient oral delivery.

Department of Structural Dynamics Extreme Light Infrastructure ERIC Dolni Brezany 18221 Czech Republic

ESRF The European Synchrotron 71 Avenue des Martyrs Grenoble 38000 France

Institut Galien Paris Saclay CNRS Université Paris Saclay Orsay 91400 France

Institute of Bioscience Sao Paulo State University Botucatu 18618 689 Brazil

Laboratory of NanoBioMaterials LNBM Department of Biophysics Paulista Medical School Federal University of Sao Paulo Sao Paulo 04023 062 Brazil

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ACS Appl Mater Interfaces. 2026 Jan 27. doi: 10.1021/acsami.6c01264. PubMed

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Blanco E., Shen H., Ferrari M.. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 2015;33:941–951. doi: 10.1038/nbt.3330. PubMed DOI PMC

He V., Seibt S., Cadarso V. J., Neild A., Boyd B. J.. Compartmentalised enzyme-induced phase transformations in self-assembling lipid systems. J. Colloid Interface Sci. 2024;672:256–265. doi: 10.1016/j.jcis.2024.05.087. PubMed DOI

Zhao D., Yang N., Xu L., Du J., Yang Y., Wang D.. Hollow structures as drug carriers: Recognition, response, and release. Nano Res. 2022;15:739–757. doi: 10.1007/s12274-021-3595-5. PubMed DOI PMC

Fong W.-K., Hanley T., Boyd B. J.. Stimuli responsive liquid crystals provide “on-demand” drug delivery in vitro and in vivo. J. Controlled Release. 2009;135:218–226. doi: 10.1016/j.jconrel.2009.01.009. PubMed DOI

Moitzi C., Guillot S., Fritz G., Salentinig S., Glatter O.. Phase Reorganization in Self-Assembled Systems Through Interparticle Material Transfer. Adv. Mater. 2007;19:1352–1358. doi: 10.1002/adma.200601679. DOI

Fong W.-K., Sánchez-Ferrer A., Ortelli F. G., Sun W., Boyd B. J., Mezzenga R.. Dynamic formation of nanostructured particles from vesicles via invertase hydrolysis for on-demand delivery. RSC Adv. 2017;7:4368–4377. doi: 10.1039/C6RA26688F. DOI

Yu H., Dyett B., Kirby N., Cai X., Mohamad M. E., Bozinovski S., Drummond C. J., Zhai J.. pH-Dependent Lyotropic Liquid Crystalline Mesophase and Ionization Behavior of Phytantriol-Based Ionizable Lipid Nanoparticles. Small. 2024;20:e2309200. doi: 10.1002/smll.202309200. PubMed DOI

Pimenta B. V., Madrid R. R. M., Mathews P. D., Riske K. A., Loh W., Angelov B., Angelova A., Mertins O.. Interaction of polyelectrolyte-shell cubosomes with serum albumin for triggering drug release in gastrointestinal cancer. J. Mater. Chem. B. 2023;11:2490–2503. doi: 10.1039/D2TB02670H. PubMed DOI

Barriga H. M. G., Holme M. N., Stevens M. M.. Cubosomes: The Next Generation of Smart Lipid Nanoparticles? Angew. Chem., Int. Ed. 2019;58:2958–2978. doi: 10.1002/anie.201804067. PubMed DOI PMC

Sivadasan D., Sultan M. H., Alqahtani S. S., Javed S.. Cubosomes in Drug Delivery-A Comprehensive Review on Its Structural Components, Preparation Techniques and Therapeutic Applications. Biomedicines. 2023;11:1114. doi: 10.3390/biomedicines11041114. PubMed DOI PMC

Mathews P. D., Mertins O., Angelov B., Angelova A.. Cubosomal lipid nanoassemblies with pH-sensitive shells created by biopolymer complexes: A synchrotron SAXS study. J. Colloid Interface Sci. 2022;607:440–450. doi: 10.1016/j.jcis.2021.08.187. PubMed DOI

Madrid R. R. M., Mathews P. D., Pramanik S., Mangiarotti A., Fernandes R., Itri R., Dimova R., Mertins O.. Hybrid crystalline bioparticles with nanochannels encapsulating acemannan from Aloe vera: Structure and interaction with lipid membranes. J. Colloid Interface Sci. 2024;673:373–385. doi: 10.1016/j.jcis.2024.06.073. PubMed DOI

Madrid R. R. M., Mathews P. D., Pimenta B. V., Mertins O.. Biopolymer–Lipid Hybrid Cubosome for Delivery of Acemannan. Mater. Proc. 2023;14:56. doi: 10.3390/IOCN2023-14486. DOI

Mertins O., Mathews P. D., Angelova A.. Advances in the Design of pH-Sensitive Cubosome Liquid Crystalline Nanocarriers for Drug Delivery Applications. Nanomater. 2020;10:963. doi: 10.3390/nano10050963. PubMed DOI PMC

Akanchise T., Angelov B., Deng Y., Fujino T., Bizien T., Angelova A.. Nanostructuring and Antioxidant Activity of Nanotherapeutics Designed by Self-Assembly of Natural Lipids and Phytochemicals. ACS Biomater. Sci. Eng. 2025;11:3488–3502. doi: 10.1021/acsbiomaterials.5c00006. PubMed DOI

Rakotoarisoa M., Angelov B., Espinoza S., Khakurel K., Bizien T., Drechsler M., Angelova A.. Composition-Switchable Liquid Crystalline Nanostructures as Green Formulations of Curcumin and Fish Oil. ACS Sustain. Chem. Eng. 2021;9:14821–14835. doi: 10.1021/acssuschemeng.1c04706. DOI

Angelova A., Angelov B., Garamus V. M., Drechsler M.. A vesicle-to-sponge transition via the proliferation of membrane-linking pores in ω-3 polyunsaturated fatty acid-containing lipid assemblies. J. Mol. Liq. 2019;279:518–523. doi: 10.1016/j.molliq.2019.01.124. DOI

Li Y., Angelova A., Liu J., Garamus V. M., Li N., Drechsler M., Gong Y., Zou A.. In situ phase transition of microemulsions for parenteral injection yielding lyotropic liquid crystalline carriers of the antitumor drug bufalin. Colloids Surf. B Biointerfaces. 2019;173:217–225. doi: 10.1016/j.colsurfb.2018.09.023. PubMed DOI

Sanchez-Ballester N. M., Sciortino F., Mir S. H., Rydzek G.. Weak Polyelectrolytes as Nanoarchitectonic Design Tools for Functional Materials: A Review of Recent Achievements. Molecules. 2022;27:3263. doi: 10.3390/molecules27103263. PubMed DOI PMC

Rydzek G., Pakdel A., Witecka A., Awang Shri D. N., Gaudière F., Nicolosi V., Mokarian-Tabari P., Schaaf P., Boulmedais F., Ariga K.. pH-Responsive Saloplastics Based on Weak Polyelectrolytes: From Molecular Processes to Material Scale Properties. Macromolecules. 2018;51:4424–4434. doi: 10.1021/acs.macromol.8b00609. DOI

Ramirez C. A. B., Mathews P. D., Madrid R. R. M., Garcia I. T. S., Rigoni V. L. S., Mertins O.. Antibacterial polypeptide-bioparticle for oral administration: Powder formulation, palatability and in vivo toxicity approach. Biomaterials Advances. 2023;153:213525. doi: 10.1016/j.bioadv.2023.213525. PubMed DOI

Veloso S. R. S., Vázquez-González M., Ribeiro M. O., Hilliou L., Amorim C. O., Amaral V. S., Correa-Duarte M. A., Castanheira E. M. S.. Chitosan/Alginate Polyelectrolyte Magnetic Gel Nanoarchitectonics with Tunable Mechanical Properties for Magnetic Hyperthermia and Sustained Release of 5-Fluorouracil. ACS Appl. Mater. Interfaces. 2025;17:61731–61741. doi: 10.1021/acsami.5c15863. PubMed DOI PMC

Lai W.-F., Shum H. C.. Hypromellose-Graft-Chitosan and Its Polyelectrolyte Complex as Novel Systems for Sustained Drug Delivery. ACS Appl. Mater. Interfaces. 2015;7:10501–10510. doi: 10.1021/acsami.5b01984. PubMed DOI

Macierzanka A., Torcello-Gómez A., Jungnickel C., Maldonado-Valderrama J.. Bile salts in digestion and transport of lipids. Adv. Colloid Interface Sci. 2019;274:102045. doi: 10.1016/j.cis.2019.102045. PubMed DOI

Salvati Manni L., Duss M., Assenza S., Boyd B. J., Landau E. M., Fong W.-K.. Enzymatic hydrolysis of monoacylglycerols and their cyclopropanated derivatives: Molecular structure and nanostructure determine the rate of digestion. J. Colloid Interface Sci. 2021;588:767–775. doi: 10.1016/j.jcis.2020.11.110. PubMed DOI

Phan S., Salentinig S., Hawley A., Boyd B. J.. Immobilised Lipase for In Vitro Lipolysis Experiments. J. Pharm. Sci. 2015;104:1311–1318. doi: 10.1002/jps.24327. PubMed DOI

Hong L., Sesen M., Hawley A., Neild A., Spicer P. T., Boyd B. J.. Comparison of bulk and microfluidic methods to monitor the phase behaviour of nanoparticles during digestion of lipid-based drug formulations using in situ X-ray scattering. Soft Matter. 2019;15:9565–9578. doi: 10.1039/C9SM01440C. PubMed DOI

Brennan J. L., Kanaras A. G., Nativo P., Tshikhudo T. R., Rees C., Fernandez L. C., Dirvianskyte N., Razumas V., Skjo̷t M., Svendsen A., Jo̷rgensen C. I., Schweins R., Zackrisson M., Nylander T., Brust M., Barauskas J.. Enzymatic Activity of Lipase–Nanoparticle Conjugates and the Digestion of Lipid Liquid Crystalline Assemblies. Langmuir. 2010;26:13590–13599. doi: 10.1021/la1018604. PubMed DOI

Borné J., Nylander T., Khan A.. Effect of Lipase on Monoolein-Based Cubic Phase Dispersion (Cubosomes) and Vesicles. J. Phys. Chem. B. 2002;106:10492–10500. doi: 10.1021/jp021023y. DOI

Boyd B. J., Clulow A. J.. The influence of lipid digestion on the fate of orally administered drug delivery vehicles. Biochem. Soc. Trans. 2021;49:1749–1761. doi: 10.1042/BST20210168. PubMed DOI PMC

Lee J. K., Lee M. K., Yun Y. P., Kim Y., Kim J. S., Kim Y. S., Kim K., Han S. S., Lee C. K.. Acemannan purified from Aloe vera induces phenotypic and functional maturation of immature dendritic cells. Int. Immunopharmacol. 2001;1:1275–1284. doi: 10.1016/S1567-5769(01)00052-2. PubMed DOI

Chelu M., Musuc A. M., Popa M., Calderon Moreno J.. Aloe vera-Based Hydrogels for Wound Healing: Properties and Therapeutic Effects. Gels. 2023;9:539. doi: 10.3390/gels9070539. PubMed DOI PMC

Gao Y., Kuok K. I., Jin Y., Wang R.. Biomedical applications of Aloe vera . Crit. Rev. Food Sci. Nutr. 2019;59:S244–S256. doi: 10.1080/10408398.2018.1496320. PubMed DOI

Angelov B., Angelova A., Filippov S. K., Drechsler M., Štěpánek P., Lesieur S.. Multicompartment Lipid Cubic Nanoparticles with High Protein Upload: Millisecond Dynamics of Formation. ACS Nano. 2014;8:5216–5226. doi: 10.1021/nn5012946. PubMed DOI

Mathews P. D., Patta A. C. M. F., Madrid R. R. M., Ramirez C. A. B., Pimenta B. V., Mertins O.. Efficient Treatment of Fish Intestinal Parasites Applying a Membrane-Penetrating Oral Drug Delivery Nanoparticle. ACS Biomater. Sci. Eng. 2023;9:2911–2923. doi: 10.1021/acsbiomaterials.1c00890. PubMed DOI

Madrid R. R. M., Mathews P. D., Patta A. C. M. F., Gonzales-Flores A. P., Ramirez C. A. B., Rigoni V. L. S., Tavares-Dias M., Mertins O.. Safety of oral administration of high doses of ivermectin by means of biocompatible polyelectrolytes formulation. Heliyon. 2021;7:e05820. doi: 10.1016/j.heliyon.2020.e05820. PubMed DOI PMC

Chokboribal J., Tachaboonyakiat W., Sangvanich P., Ruangpornvisuti V., Jettanacheawchankit S., Thunyakitpisal P.. Deacetylation affects the physical properties and bioactivity of acemannan, an extracted polysaccharide from Aloe vera . Carbohydr. Polym. 2015;133:556–566. doi: 10.1016/j.carbpol.2015.07.039. PubMed DOI

Simões J., Nunes F. M., Domingues P., Coimbra M. A., Domingues M. R.. Mass spectrometry characterization of an Aloe vera mannan presenting immunostimulatory activity. Carbohydr. Polym. 2012;90:229–236. doi: 10.1016/j.carbpol.2012.05.029. PubMed DOI

Yılmaz H., Lee S., Chronakis I. S.. Interactions of β-Lactoglobulin with Bovine Submaxillary Mucin vs. Porcine Gastric Mucin: The Role of Hydrophobic and Hydrophilic Residues as Studied by Fluorescence Spectroscopy. Molecules. 2021;26:6799. doi: 10.3390/molecules26226799. PubMed DOI PMC

Stie M. B., Cunha C., Huang Z., Kirkensgaard J. J. K., Tuelung P. S., Wan F., Nielsen H. M., Foderà V., Ro̷nholt S.. A head-to-head comparison of polymer interaction with mucin from porcine stomach and bovine submaxillary glands. Sci. Rep. 2024;14:21350. doi: 10.1038/s41598-024-72233-1. PubMed DOI PMC

Johansson M. E. V., Hansson G. C.. Immunological aspects of intestinal mucus and mucins. Nat. Rev. Immunol. 2016;16:639–649. doi: 10.1038/nri.2016.88. PubMed DOI PMC

Ambort D., Johansson M. E. V., Gustafsson J. K., Nilsson H. E., Ermund A., Johansson B. R., Koeck P. J. B., Hebert H., Hansson G. C.. Calcium and pH-dependent packing Enzymatic Activity of Lipase and release of the gel-forming MUC2 mucin. Proc. Natl. Acad. Sci. U.S.A. 2012;109:5645–5650. doi: 10.1073/pnas.1120269109. PubMed DOI PMC

Johansson M. E. V., Sjövall H., Hansson G. C.. The gastrointestinal mucus system in health and disease. Nat. Rev. Gastroenterol. Hepatol. 2013;10:352–361. doi: 10.1038/nrgastro.2013.35. PubMed DOI PMC

Ghosh S., Dolai S., Patra T., Dey J.. Solution Behavior and Interaction of Pepsin with Carnitine Based Cationic Surfactant: Fluorescence, Circular Dichroism, and Calorimetric Studies. J. Phys. Chem. B. 2015;119:12632–12643. doi: 10.1021/acs.jpcb.5b07072. PubMed DOI

Grahame D. S. A., Dupuis J. H., Bryksa B. C., Tanaka T., Yada R. Y.. Comparative bioinformatic and structural analyses of pepsin and renin. Enzyme Microb. Technol. 2020;141:109632. doi: 10.1016/j.enzmictec.2020.109632. PubMed DOI

Grahame D. A. S., Dupuis J. H., Bryksa B. C., Tanaka T., Yada R. Y.. Improving the alkaline stability of pepsin through rational protein design using renin, an alkaline-stable aspartic protease, as a structural and functional reference. Enzyme Microb. Technol. 2021;150:109871. doi: 10.1016/j.enzmictec.2021.109871. PubMed DOI

Roy A., Kundu S., Dutta R., Sarkar N.. Influence of bile salt on vitamin E derived vesicles involving a surface active ionic liquid and conventional cationic micelle. J. Colloid Interface Sci. 2017;501:202–214. doi: 10.1016/j.jcis.2017.04.051. PubMed DOI

Fatouros D. G., Deen G. R., Arleth L., Bergenstahl B., Nielsen F. S., Pedersen J. S., Mullertz A.. Structural development of self nano emulsifying drug delivery systems (SNEDDS) during in vitro lipid digestion monitored by small-angle X-ray scattering. Pharm. Res. 2007;24:1844–1853. doi: 10.1007/s11095-007-9304-6. PubMed DOI

Vithani K., Hawley A., Jannin V., Pouton C., Boyd B. J.. Solubilisation behaviour of poorly water-soluble drugs during digestion of solid SMEDDS. Eur. J. Pharm. Biopharm. 2018;130:236–246. doi: 10.1016/j.ejpb.2018.07.006. PubMed DOI

Sadeghpour A., Rappolt M., Misra S., Kulkarni C. V.. Bile Salts Caught in the Act: From Emulsification to Nanostructural Reorganization of Lipid Self-Assemblies. Langmuir. 2018;34:13626–13637. doi: 10.1021/acs.langmuir.8b02343. PubMed DOI

Zhang X., Caner S., Kwan E., Li C., Brayer G. D., Withers S. G.. Evaluation of the Significance of Starch Surface Binding Sites on Human Pancreatic α-Amylase. Biochemistry. 2016;55:6000–6009. doi: 10.1021/acs.biochem.6b00992. PubMed DOI

Wang T. Y., Liu M., Portincasa P., Wang D.Q.-H.. New insights into the molecular mechanism of intestinal fatty acid absorption. Eur. J. Clin. Invest. 2013;43:1203–1223. doi: 10.1111/eci.12161. PubMed DOI PMC

Whitcomb D. C., Lowe M. E.. Human pancreatic digestive enzymes. Dig. Dis. Sci. 2007;52:1–17. doi: 10.1007/s10620-006-9589-z. PubMed DOI

Ulleberg E. K., Comi I., Holm H., Herud E. B., Jacobsen M., Vegarud G. E.. Human Gastrointestinal Juices Intended for Use in In Vitro Digestion Models. Food Dig. 2011;2:52–61. doi: 10.1007/s13228-011-0015-4. PubMed DOI PMC

Ways T. M. M., Lau W. M., Khutoryanskiy V. V.. Chitosan and Its Derivatives for Application in Mucoadhesive Drug Delivery Systems. Polymers. 2018;10:267. doi: 10.3390/polym10030267. PubMed DOI PMC

Fernandes Patta A. C. M., Mathews P. D., Madrid R. R. M., Rigoni V. L. S., Silva E. R., Mertins O.. Polyionic complexes of chitosan-N-arginine with alginate as pH responsive and mucoadhesive particles for oral drug delivery applications. Int. J. Biol. Macromol. 2020;148:550–564. doi: 10.1016/j.ijbiomac.2020.01.160. PubMed DOI

Briggs J., Caffrey M.. The temperature-composition phase diagram and mesophase structure characterization of monopentadecenoin in water. Biophys. J. 1994;67:1594–1602. doi: 10.1016/S0006-3495(94)80632-0. PubMed DOI PMC

Mistry S., Fuhrmann P. L., de Vries A., Karshafian R., Rousseau D.. Structure-rheology relationship in monoolein liquid crystals. J. Colloid Interface Sci. 2023;630:878–887. doi: 10.1016/j.jcis.2022.10.115. PubMed DOI