(T1530-07-37) Attenuating Neuroinflammation and Promoting Neurogenesis through Co-delivery of Curcumin and Brain Derived Neurotrophic Factor (BDNF) for the Management of Alzheimer’s disease

Purpose: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by loss of memory, cognition and functional abilities accounting for 60 – 80 % of the dementia cases worldwide. Amyloid hypothesis has been at the forefront of research to develop therapies directed towards AD. It proposes the extracellular amyloid plaques as the central cause of pathology and subsequent events like neurofibrillary tangle formation, inflammation, neuronal death and synaptic dysfunction as the outcome of plaque formation. Inflammation is one such strategy gaining increasing attention. The post mortem brain samples of AD patients have revealed sustained inflammatory response with activated microglia and elevated inflammatory cytokines. Additionally, observational studies have reported relatively lower risk of AD in subjects consuming NSAIDS for long term indicating anti-inflammatory agents to play a role in preventing the aggravation of AD. Targeting neuroinflammation, thus, could be a promising approach to mitigate the severities of AD. We plan to use curcumin, a natural polyphenolic compound, widely known for its anti-inflammatory properties to tackle the elevated neuroinflammation. Neurotrophin, Brain Derived Neurotrophic Factor (BDNF), promotes neuronal survival, differentiation and plasticity in brain and is reported to have reduced in AD patients. Supplementing BDNF in AD patients could ameliorate their pathology and enhance their cognitive ability. Therefore, we hypothesize co-delivery of curcumin and BDNF gene to brain reduces neuroinflammation, promotes neurogenesis, survival and maintenance of neuron for management of AD. We propose the use of liposomes modified for brain specific delivery using Transferrin and Penetratin for delivery of curcumin and BDNF.

Methods: Liposomes were prepared using lipids DOPE: DOPTAP: DSPE PEG_2000k: Cholesterol at molar ratio of 45:45:8:2 through lipid film hydration method. Ligand modified lipid conjugate DSPE PEG Tf or DSPE PEG Penetratin were used at 4% molar ratio each to prepare functionalized liposomes. Curcumin was loaded 30% w/w of total lipid quantity in each formulation and drug content of the purified formulation was calculated using HPLC. BDNF was complexed with chitosan at N:P ratio of 5 and encapsulated in liposomes during hydration. The liposomes were characterized for its size, PDI and zeta potential using zetasizer. SEM was used for morphological evaluation of the liposomes formed. Release studies were performed using 1.5 ml of curcumin formulation in 12-14 kD dialysis membrane with 2% Tween-80 in phosphate buffer of pH 6.4 as release medium. Amount of gene encapsulated by liposomes were calculated with Hoechst dye fluorescence method. DNAse protection assay was performed in each formulation to test the ability of formulation to protect from nuclease activity. Finally, transfection was performed in IMG cell line using GFP as reporter plasmid to observe gene expression of formulation. LPS solution at 10 ng/ml were used to stimulate IMG cell line for production of inflammatory cytokines. TNF alpha level were measured six hours after the treatment from cell culture supernatants using ELISA kit.

Results: The lipid film hydration method formed curcumin liposomes of size 231.9 ± 12.69 nm with PDI of 0.278 ± 0.053 having zeta potential of 5.92 ± 0.83 mV. Encapsulation efficiency of Curcumin was found to be 86.06 ± 5.77 %. SEM revealed the spherical morphology of the liposomes (Figure 1A). The formulation released 79.34 ± 2.07 % of the encapsulated curcumin over seven days (Figure 1B). Plain liposomes as well as functionalized liposomes were able to encapsulate more than 75% of the plasmid DNA and was robust enough to prevent the encapsulated DNA from the nuclease action as seen from the DNAse protection assay (Figure 2B). Transfection of IMG cell with reporter plasmid GFP revealed expression of green fluorescent protein 48 hours after the transfection (Figure 2C). Dual modified liposomes significantly reduced the expression of TNF alpha in cell culture supernatant (p value 0.0131) of microglial cell line after six hours of treatment as shown in Figure 3.

Conclusion: Brain specific liposomes were successfully formulated encapsulating curcumin and pBDNF. The encapsulated curcumin was released over one week allowing us to leverage less frequent dosing. Additionally, our formulation successfully encapsulated the plasmid DNA, protected the gene and expressed the reporter protein in IMG cell line. Finally, it also reduced the pro-inflammatory cytokine TNF alpha level in LPS activated microglial cell line to demonstrate its anti-inflammatory potential. Further, studies will be performed to test its neurogenesis potential and cognitive function improvement in relevant AD mouse models.

Acknowledgements: This research was supported by the National Institute of Health (NIH) grant RF1 AG068034.


Figure 1: A) Curcumin liposomes observed under Scanning Electron Microscope (SEM) B) Cumulative percentage release of Curcumin liposomes observed for a period of seven days. The data represents mean mean ± SD (n = 4)


Figure 2: A) Percentage DNA encapsulation determined by Hoechst dye fluorescence method of functionalized liposomes B) Gel electrophoresis diagram showing DNASe protection assay of different liposome formulation C) GFP expression observed in IMG cell line under fluorescence microscope 48 hours after transfection. The data represents mean mean ± SD (n = 4)


Figure 3: TNF alpha expression on cell culture supernatant of IMG cell line after six hours of treatment with 10 μM equivalent of curcumin formulation . The data represents mean ± SD (n = 4).