Protective Effect of Doxycycline-Loaded Calcium Phosphate Nanoparticles on LPS-Induced Neuroinflammation in Mice

In the field of neuroscience research, neuroinflammation is an important pathological feature of many neurological diseases. Neuroinflammation is a complex response of the central nervous system (CNS) to various damaging triggers (such as ischemia, infection, trauma, immune response, or exposure to toxic proteins). When this inflammatory response cannot be effectively terminated, it leads to persistent inflammation, causing severe neurological damage and related diseases such as depression, Alzheimer’s disease, Parkinson’s disease, and dementia. In this context, the authors conducted a study to evaluate the effects of doxycycline (DX) and its loaded calcium phosphate nanoparticles (dx@cap) and pectin-coated doxycycline calcium phosphate nanoparticles (pec/dx@cap) on lipopolysaccharide (LPS)-induced neuroinflammation in mice, and explored the role of adenosine monophosphate-activated protein kinase (AMPK) in this process.

Research Background and Objectives

The main purpose of the current study is to evaluate the potential therapeutic effect of pectin-coated, doxycycline-loaded calcium phosphate nanoparticles (pec/dx@cap) on LPS-induced neuroinflammation in mice, and to confirm the roles of IL-6, SOD, TLR-4, AMPK, and NRF2 in this process. LPS, a component of Gram-negative bacterial cell walls, is closely associated with various neurodegenerative diseases. Peripheral injection of LPS can induce elevated cytokines within the CNS, thus simulating a model of neuroinflammation, providing an experimental basis for studying new therapies.

Author and Institution Information

This study was jointly completed by six authors: Sahar A. Harby, Suzan Awad Abdelghany Morsy, Mona Hassan Fathelbab, Norhan S. El-Sayed, Salma E. El-Habashy, and Rania G. Aly, who are from the Departments of Clinical Pharmacology, Biochemistry, Physiology, Pharmaceutics, and Pathology at the Faculty of Medicine, Alexandria University, Egypt. This research paper was published in the Journal of Neuroimmune Pharmacology, Volume 19, Issue 2, 2024, and was accepted on December 6, 2023.

Research Methods

1. Preparation of Calcium Phosphate Nanoparticles (CAP)

Doxycycline calcium phosphate nanoparticles (dx@cap) were prepared using the wet chemical precipitation method. First, calcium chloride (CaCl2) and sodium dihydrogen phosphate (Na2HPO4) were mixed in deionized water, adjusting the pH to around 11. The Na2HPO4 solution was added dropwise to the CaCl2 solution and stirred at 25°C. The formed CAP precipitate was separated by centrifugation and washed with ethanol.

2. Preparation of Doxycycline-Loaded Calcium Phosphate Nanoparticles (dx@cap)

The CAP precipitate was dispersed in a doxycycline ethanol solution, mixed at a weight ratio of 2:1, and stirred for 24 hours, followed by centrifugation to obtain dx@cap particles.

3. Preparation of Pectin-Coated Doxycycline-Loaded Calcium Phosphate Nanoparticles (pec/dx@cap)

The separated dx@cap particles were dispersed in deionized water and then used to titrate pectin aqueous solution, further stirred for 30 minutes to obtain pec/dx@cap particles.

Research Results

1. Characterization of Nanoparticles

The nanoparticles were characterized by Zeta potential measurement, Transmission Electron Microscopy (TEM), and Fourier Transform Infrared Spectroscopy (FTIR). The results showed that CAP particles had a negative surface charge, dx@cap particles had a positive surface charge, indicating successful loading of doxycycline. After further coating with pectin, the particle surface charge turned negative again.

2. Behavioral Assessment

Cognitive function was assessed through T-maze spontaneous alternation test and Novel Object Recognition (NOR) test. LPS-treated mice showed memory impairment, while doxycycline and its two nanoparticle formulations significantly improved the cognitive function of mice, with pec/dx@cap showing the best effect, approaching that of the normal control group.

3. Histological and Biochemical Analysis

Histological examination showed inflammatory infiltration, cellular degeneration, and edema in the brain tissue of LPS-induced mice. Doxycycline and its nanoparticle treatment groups all reduced these pathological changes, especially the pec/dx@cap group, which showed near-normal tissue structure.

Biochemical analysis results indicated that after LPS treatment, the antioxidant enzyme activity in mouse brain tissue decreased, with SOD and AMPK significantly reduced, while TLR-4 and IL-6 significantly increased. Doxycycline and its two nanoparticle formulations all increased SOD and AMPK levels and decreased TLR-4 and IL-6 levels, with pec/dx@cap showing the best effect.

Conclusions and Significance

This study demonstrates that pectin-coated, doxycycline-loaded calcium phosphate nanoparticles (pec/dx@cap) can significantly reverse LPS-induced neuroinflammation and improve cognitive function by enhancing AMPK and NRF2 expression and inhibiting TLR-4. This research not only showcases the potential of nano-drug delivery systems in the treatment of neurodegenerative diseases but also provides important references for future drug development and design.