(T1030-04-19) Cannabidiol and BDNF-Encapsulated Brain-Targeted Lipid Nanoparticles Reduce Inflammation, Lower Pathological Amyloid and Tau Levels, and Improve Cognition in Transgenic Models of Alzheimer’s disease

Purpose: Neuroinflammation is gaining increasing attention as a key mediator of disease progression in Alzheimer’s disease (AD), in addition to the classical pathological hallmarks, i.e., amyloid plaques and phosphorylated tau¹. In interplay with amyloid plaques and tau, the brain-resident microglia mediate neuroinflammation and aggravate the AD pathology. Targeting neuroinflammation is thus an attractive therapeutic alternative to delay the progression of AD. The Brain-Derived Neurotrophic Factor (BDNF) level involved in synaptic plasticity and neuronal differentiation is depleted in AD, further exacerbating cognitive functions². This research aims to deliver an anti-inflammatory agent in the form of cannabidiol and restore the decreased levels of BDNF through gene delivery for the treatment of AD. Brain-targeted liposomes functionalized with Penetratin and mannose will be used to deliver cannabidiol and pBDNF to the brain, and their efficacy in AD treatment will be assessed in two transgenic mice models of AD, i.e., 5XFAD and PS19.

Methods: Liposomes were prepared by the lipid film hydration method using DOPE: DOPTAP: DSPE PEG2000k: cholesterol at a molar ratio of 45:45:8:2. DSPE PEG Man or DSPE PEG Penetratin were used at a 4% molar ratio each for functionalization. Cannabidiol was loaded at 10% w/w of total lipid weight, and the drug was estimated using HPLC. Further, BDNF was complexed with chitosan (N: P ratio 5) and added to liposomes during hydration. The anti-inflammatory study was performed in an IMG cell line stimulated by 10 ng/ml LPS and 10 µM amyloid beta 42 oligomers for 24 hours. The level of anti-inflammatory markers TNF α and IL-1β was estimated using ELISA, while their relative gene expression was estimated using qPCR. The levels of pathological markers amyloid β 40 and amyloid β 42 were estimated in brain lysates of 6-month-old 5XFAD transgenic mice and phosphorylated tau 181 in 6-month-old PS19 transgenic mice after one month of weekly dosing^3,4. In both transgenic mice models, cognitive function was assessed using a Y maze where mice were allowed to move freely for 5 minutes, and their movement was recorded using a camera for analysis.

Results: Dual functionalized liposomes encapsulating cannabidiol and BDNF reduced the anti-inflammatory cytokine level TNFα (p < 0.05) and IL1β (p < 0.05) 24 hours after the treatment with 10 ng/ml LPS in IMG cell lines compared to its non-modified counterpart. Similarly, the mRNA level of genes TNFα (p < 0.05) and IL1β (p < 0.05) was also reduced significantly. The anti-inflammatory properties similar to LPS were also observed when the IMG cells were challenged by 10 µM amyloid beta 42 oligomers, i.e., reduction of protein and gene levels of TNFα (p < 0.05) and IL1β (p < 0.05) (Figure 1). The levels of Aβ 40 (p < 0.05) and Aβ 42 (p < 0.05) in 6-month-old transgenic 5XFAD mice was reduced significantly after the treatment compared to saline in TBSX and Guanidine Hydrochloride fractions. Similarly, the sarkosyl soluble and sarkosyl insoluble fraction of phosphorylated tau 181 was reduced significantly (p < 0.05) in 6-month-old PS19 mice after the treatment with our formulation compared to saline (Figure 2). Finally, the cognitive function in terms of percentage spontaneous alternation and total arm entries estimated using the Y maze test demonstrated significant improvement (p < 0.05) in our treatment group compared to saline treated group (Figure 3).

Conclusion: Brain-targeted liposomes functionalized with penetratin and mannose demonstrated anti-inflammatory activity in the IMG cell line after being challenged with LPS and amyloid beta 42 oligomers. This formulation reduced the major pathological markers amyloid beta and tau in two different transgenic mouse models, i.e., 5XFAD and PS19, respectively. It improved the cognitive function assessed by the Y maze test. Immunohistochemistry of these pathological markers will be performed further to corroborate our findings. Additionally, neurogenesis markers (BrdU, Ki67) and synaptic markers (Synaptophysin, PSD95) will be studied to assess the neurogenesis effect of our formulation.

References: 1. Mary, Arnaud, Renzo Mancuso, and Michael T. Heneka. "Immune Activation in Alzheimer Disease." Annual Review of Immunology 42 (2024).
2. Peng, Shiyong, Joanne Wuu, Elliott J. Mufson, and Margaret Fahnestock. "Precursor form of brain‐derived neurotrophic factor and mature brain‐derived neurotrophic factor are decreased in the pre‐clinical stages of Alzheimer's disease." Journal of neurochemistry 93, no. 6 (2005): 1412-1421.
3. Youmans, Katherine Lynn, Steffi Leung, Juan Zhang, Erika Maus, Kathleen Baysac, Guojun Bu, Robert Vassar, Chunjiang Yu, and Mary Jo LaDu. "Amyloid-β42 alters apolipoprotein E solubility in brains of mice with five familial AD mutations." Journal of neuroscience methods 196, no. 1 (2011): 51-59.

Acknowledgements: Acknowledgment: This research was supported by the National Institute of Health (NIH) grant R01 AG051574, R01AG083981-01, RF1 AG068034, and 3RF1AG068034-01A1S1.


Figure 1: Anti-inflammatory property of cannabidiol liposomes in Immortalized Microglia (IMG) cell line stimulated by 10 ng/ml LPS and 10 µM amyloid beta 42 oligomers for 24 hours. The treatment consists of free cannabidiol or cannabidiol liposomes equivalent to 10μM of cannabidiol A, E) Protein expression of TNF-α estimated using ELISA kits B, F) Protein expression of IL-1 β estimated using ELISA Kits C, G) Relative gene expression of TNF-α estimated using qPCR. D, H) Relative gene expression of IL-1β estimated using qPCR. The data represents mean ± SD (n=4) (* p<0.05)


Figure 2: Pathological markers amyloid and tau analyzed in brain lysate of 6-month-old 5XFAD and PS19 mice. Amyloid Beta 40 and Amyloid Beta 42 were analyzed in the TBSX fraction (A, C) and the Guanidine Hydrochloride fraction (B, D)using ELISA and normalized by total protein content estimated by the BCA assay. Similarly, the phosphorylated tau 181 content was estimated as sarkosyl soluble (E) and sarkosyl insoluble fraction (F) using ELISA and normalized to total protein content estimated by BCA. The data represents mean ± SD (n=6) (* p<0.05)


Figure 3: Cognitive function tests performed on 5XFAD and PS19 AD mice in the Y maze apparatus. Percentage spontaneous alternation and total arm entries were calculated from the recorded movements of each mouse in the Y maze apparatus for 5 minutes. The data represents mean ± SD (n=6) (* p<0.05)