Atomic force microscopy-based exploration of layered double hydroxide nanoparticles adjuvant mechanism

Project Information

AFM-LDH

Grant agreement ID: 101064861

DOI

10.3030/101064861

Project closed

EC signature date 1 August 2022

Start date 1 September 2023

End date 31 January 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 230 774,40

Coordinated by

AARHUS UNIVERSITET
Denmark

Periodic Reporting for period 1 - AFM-LDH (Atomic force microscopy-based exploration of layered double hydroxide nanoparticles adjuvant mechanism)

Reporting period: 2023-09-01 to 2026-01-31

Summary of the context and overall objectives of the project

For more than a century, aluminium-based adjuvants (collectively known as Alum) have been essential components of many licensed vaccines. Their long clinical history and safety profile have made them a cornerstone of modern immunisation programmes. However, traditional Alum adjuvants primarily stimulate antibody-mediated immunity and have limited capacity to induce robust CD8⁺ T-cell responses, which are crucial for protection against intracellular pathogens and for effective cancer immunotherapy. This limitation creates an important gap in current vaccine technology, particularly in a global health landscape increasingly shaped by emerging infectious diseases and the growing need for personalised cancer treatments.

Recent advances in nanotechnology have led to the development of layered double hydroxide (LDH) nano-aluminium adjuvants (NA), engineered from clinically used aluminium hydroxide and magnesium hydroxide materials. These next-generation adjuvants show great promise: they can stimulate both strong antibody responses and potent CD8⁺ T-cell immunity. In addition to their role in vaccine formulations, NA materials can function as immunomodulators capable of reshaping the tumour microenvironment and enhancing CD8⁺ T-cell activity, offering potential therapeutic value in oncology.

Despite these encouraging findings, the fundamental relationship between the chemical composition of NA and their immunological behaviour remains poorly understood. This knowledge gap limits the rational design and optimisation of NA-based vaccines and immunotherapies.

This project aims to address this challenge by applying advanced physicochemical and biological characterisation techniques to systematically investigate how specific chemical features of NA influence their immunomodulatory mechanisms. By elucidating the chemistry-immunology relationship, the project seeks to establish a scientific basis for designing more effective and predictable nano-aluminium adjuvants. The expected outcomes will contribute to the development of next-generation vaccines and immunotherapies with broader efficacy, potentially benefiting public health at large and supporting strategic priorities in infectious disease preparedness and cancer treatment.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

This project investigated how the intrinsic physicochemical properties of nano-aluminium layered double hydroxides (NA) influence their immunomodulatory mechanisms at cellular and tissue levels. Owing to their mild alkalinity, NA particles effectively neutralize acidic microenvironments. At the cellular level, NA rapidly buffers endosomal acidity and releases magnesium ions that increase ionic strength, facilitating endosomal escape and enabling cytosolic delivery of associated antigens. Once in the cytosol, NA particles engage the autophagy pathway and interfere with the acidification of autophagolysosomes, thereby modulating cellular functions. Because the divalent metal cation in NA can be readily substituted with other metals (e.g. zinc), the immunological properties of NA are strongly influenced by their chemical composition. At the tissue level, NA neutralizes extracellular acidity and reshapes the functional state of local immune cells, including CD8⁺ T cells and macrophages.

1. Metal composition of NA regulates dendritic cell antigen presentation.
This project demonstrated that modifying the metal composition of NA markedly alters dendritic cell (DC) activation. By partially or fully substituting magnesium ions with zinc ions to produce zinc-modified NA (ZNA), we observed enhanced DC maturation and activation with increasing zinc content. Mechanistic studies showed that conventional NA primarily inhibits autophagy, whereas the incorporation of zinc ions restores and enhances autophagic flux. This enhancement promotes more efficient antigen cross-presentation, thereby strengthening both humoral responses and CD8⁺ T-cell immunity.

2. Magnesium ions released from NA modulate macrophage inflammatory responses.
We found that magnesium ions liberated from NA exert a notable anti-inflammatory effect on macrophages. In inflamed liver and lung tissues, NA uptake significantly increased intracellular magnesium levels, driving macrophages toward an anti-inflammatory phenotype and reducing local tissue inflammation. This effect is mediated largely through activation of the SIRT1/Nrf2 signalling pathway by released magnesium ions.

3. NA enhances CD8⁺ T-cell function by restoring cellular mechanics and signalling.
A key discovery of this project is that NA directly influences CD8⁺ T-cell activation by modifying cell biomechanical properties. Acidic microenvironments were found to stiffen CD8⁺ T cells and impair their cytotoxic function. NA-mediated neutralization of extracellular acidity effectively restored T-cell softness and improved effector activity. Furthermore, magnesium ions released from NA interacted with the LFA-1 receptor on CD8⁺ T cells, enhancing cytotoxic function via the calcium–NFAT2–IFN signalling pathway. While magnesium alone had a moderate activating effect, its combination with interleukin-12 (IL-12) proved particularly beneficial: magnesium ions mitigated cytokine-induced exhaustion and preserved the functional fitness of CD8⁺ T cells under strong stimulatory conditions.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

This project has advanced the state of the art by establishing, for the first time, a mechanistic framework linking the chemical composition of layered double hydroxide nano-aluminium adjuvants (NA) to their immunomodulatory functions. We discovered that substituting magnesium with zinc ions reprograms dendritic cell biology by restoring and enhancing autophagic flux, which in turn improves antigen cross-presentation and strengthens both humoral and CD8⁺ T-cell immunity. Beyond dendritic cells, we demonstrated that magnesium ions released from NA exert potent anti-inflammatory effects on macrophages in inflamed liver and lung tissues through activation of the SIRT1/Nrf2 pathway. Additionally, we identified a novel mechanobiological mechanism whereby NA neutralises acidic microenvironments to restore CD8⁺ T-cell softness and cytotoxic function, complemented by magnesium-mediated activation of the LFA-1–calcium–NFAT2–IFN signalling axis. Collectively, these findings elevate NA from conventional adjuvants to multifunctional immunomodulators with applications in vaccination, cancer immunotherapy, and inflammatory disease management.

The outcomes of this project have strong potential to influence multiple biomedical fields. Precision engineering of metal-modified NA provides a promising route to next-generation vaccines capable of inducing robust cellular immunity against intracellular pathogens and tumours. The ability of NA to restore CD8⁺ T-cell function under acidic conditions and to limit IL-12–induced T-cell exhaustion highlights its suitability for incorporation into combination immunotherapies. Meanwhile, the anti-inflammatory properties of magnesium-enriched NA formulations expand their potential utility beyond vaccines, offering new opportunities for therapeutic intervention in inflammatory disorders of the liver and lungs. These advances shift the paradigm for aluminium-based adjuvants and open new avenues of translational research.

To ensure the successful uptake and long-term impact of these results, several enabling steps are required. Further preclinical research is needed to examine long-term safety, biodistribution, and dose optimisation for metal-modified NA formulations. Standardised, GMP-compatible production methods and clear material characterisation protocols will be essential for regulatory approval and industrial adoption. Protection of intellectual property relating to NA compositions and their immunological applications will support commercialisation efforts, while partnerships with vaccine manufacturers and biotech companies will accelerate market entry. Engagement with regulatory authorities and international stakeholders will help define safety frameworks and facilitate broader deployment of NA-based technologies in both high-income and resource-limited settings.

Next-generation NA preparation and chemical modification strategies, and their potential immunomodul