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Influence of physico-chemical properties of two lipoxin emulsion-loaded hydrogels on pre-polarized macrophages: a comparative analysis

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Abstract

Inflammation, a crucial defense mechanism, must be rigorously regulated to prevent the onset of chronic inflammation and subsequent tissue damage. Specialized pro resolving mediators (SPMs) such as lipoxin A4 (LXA4) have demonstrated their ability to facilitate the resolution of inflammation by orchestrating a transition of M1 pro-inflammatory macrophages towards an anti-inflammatory M2 phenotype. However, the hydrophobic and chemically labile nature of LXA4 necessitates the development of a delivery system capable of preserving its integrity for clinical applications. In this study, two types of emulsion were formulated using different homogenization processes:mechanical overhead stirrer (MEB for blank Emulsion and MELX for LXA4 loaded-Emulsion) or Luer-lock syringes (SEB for blank Emulsion and SELX for LXA4 loaded-Emulsion)). Following characterization, including size and droplet morphology assessment by microscopy, the encapsulation efficiency (EE) was determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To exert control over LXA4 release, these emulsions were embedded within silanized hyaluronic acid hydrogels. A comprehensive evaluation, encompassing gel time, swelling, and degradation profiles under acidic, basic, and neutral conditions, preceded the assessment of LXA4 cumulative release using LC-MS/MS. Physicochemical results indicate that H-MELX (Mechanical overhead stirrer LXA4 Emulsion loaded-Hydrogel) exhibits superior efficiency over H-SELX (Luer-lock syringes LXA4 Emulsion loaded-Hydrogel). While both formulations stimulated pro-inflammatory cytokine secretion and promoted a pro-inflammatory macrophage phenotype, LXA4 emulsion-loaded hydrogels displayed a diminished pro-inflammatory activity compared to blank emulsion-loaded hydrogels. These findings highlight the biological efficacy of LXA4 within both systems, with H-SELX outperforming H-MELX in terms of efficiency. To the best of our knowledge, this is the first successful demonstration of the biological efficacy of LXA4 emulsion-loaded hydrogel systems on macrophage polarization. These versatile H-MELX and H-SELX formulations can be customized to enhance their biological activity making them promising tools to promote the resolution of inflammation in diverse clinical applications.

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Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

DMTMM:

4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride

EE:

Encapsulation Efficiency

FBS:

Fetal Bovine Serum

H-MEB:

Mechanical overhead stirrer Blank Emulsion loaded Hydrogel

H-MELX:

Mechanical overhead stirrer LXA4 Emulsion loaded-Hydrogel

H-SEB:

Luer-lock syringes Blank Emulsion loaded-Hydrogel

H-SELX:

Luer-lock syringes LXA4 Emulsion loaded Hydrogel

HA:

Hyaluronic Acid

HPMC:

Hydroxypropylmethylcellulose

IL-1β:

Interleukin-1β

IL-1RA:

Interleukin-1 Receptor Antagonist

IL-4:

Interleukin-4

IL-6:

Interleukin-6

IL-10:

Interleukin-10

IL-12p40:

Interleukin-12p40

IL-12p70:

Interleukin-12p70

IL-13:

Interleukin-13

IL-23:

Interleukin-23

IP-10:

Interferon-gamma-induced Protein-10

LC-MS/MS:

Liquid chromatography-tandem mass spectrometry

LPS:

Lipopolysaccharide

LXA4:

Lipoxin A4

MEB:

Mechanical overhead stirrer Blank Emulsion

MELX:

Mechanical overhead stirrer LXA4 loaded-Emulsion

MMP:

Matrix MetalloProteinases

MES:

2-(N-morpholino)ethanesulfonic acid

RPMI:

Roswell Park Memorial Institute

ROS:

Reactive Oxygen Species

SEB:

Luer-lock syringes Emulsion Blank

SELX:

Luer-lock syringes LXA4 loaded-Emulsion

SPM:

Specialized Pro resolving Mediators

TARC:

Thymus and Activation Regulated Cytokine

TNF-α:

Tumor Necrosis Factor-α

UPLC/MS:

Ultra-Performance Liquid Chromatography and Tandem Mass Spectrometry

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Acknowledgements

The authors are grateful to the Plateforme Spectrométrie de masse (PFSM) – and the cytometry Facility “Cytocell” facilities for expert technical assistance. The authors gratefully acknowledge Christine Herrenknecht (laboratoire Mer Molécules Santé (MMS)) for her technical help.

Funding

This work was supported by Agence Nationale de la Recherche (ANR) for funding the project Grant numbers JCJC BILIPOX (ANR-19-CE19-0006).

Author information

Authors and Affiliations

  1. Nantes Université, Oniris CHU Nantes, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229, F-44000, Nantes, France

    Léna Guyon, Solène Tessier, Marianne Lafont, Florian Segeron, Pierre Weiss & Alexis Gaudin

  2. Nantes Université, CNRS, INSERM, l′institut du thorax, Nantes, France

    Mikaël Croyal & Mathilde Gourdel

  3. Nantes Université, CHU Nantes, Inserm CNRS, SFR Santé, Inserm UMS 016, Nantes, France

    Mikaël Croyal

  4. CRNH-Ouest Mass Spectrometry Core Facility, Nantes, France

    Mikaël Croyal & Mathilde Gourdel

  5. Nantes Université, Oniris CHU Nantes, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229, F-44000, Nantes, France

    Lionel Chabaud & Hélène Gautier

  6. Nantes Université, UFR Sciences Biologiques et Pharmaceutiques, F-44035, Nantes, France

    Lionel Chabaud & Hélène Gautier

  7. Department of Endodontics, University of Nantes, 1 place Alexis Ricordeau, 44093 Nantes Cedex 01, Nantes, France

    Alexis Gaudin

Authors
  1. Léna Guyon
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  2. Solène Tessier
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  3. Mikaël Croyal
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  4. Mathilde Gourdel
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  5. Marianne Lafont
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  6. Florian Segeron
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  7. Lionel Chabaud
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  8. Hélène Gautier
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  9. Pierre Weiss
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  10. Alexis Gaudin
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Léna Guyon, Solène Tessier, Mikaël Croyal, Mathilde Gourdel, Marianne Lafont, Florian Segeron, Lionel Chabaud, Hélène Gautier, Pierre Weiss, Alexis Gaudin. The first draft of the manuscript was written by Léna Guyon, Solène Tessier and Alexis Gaudin and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Alexis Gaudin.

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Guyon, L., Tessier, S., Croyal, M. et al. Influence of physico-chemical properties of two lipoxin emulsion-loaded hydrogels on pre-polarized macrophages: a comparative analysis. Drug Deliv. and Transl. Res. (2024). https://doi.org/10.1007/s13346-024-01588-9

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