1. Liposome-based synergy strategies for effective drug delivery across the BBB as a novel treatment for Alzheimer’s disease.

2. Alzheimer's Disease (AD)
Alzheimer's disease (AD) is the most common form of dementia and has become one of the biggest global healthcare challenges.
The pathophysiology of this disease is not fully understood.
Research has shown that progressive production and accumulation of insoluble protein aggregates, such as neurofibrillary tangles of tau and amyloid-β (Aβ) plaques trigger a pathogenic cascade of events that eventually lead to AD.
Common Symptom: Brain atrophy and neurodegeneration.
AD is also characterized by chronic brain inflammation and the action free radicals on brain tissue has been investigated extensively and the problem of oxidative stress is now considered to be one important factor in the progression of AD.

3. Current Treatment & Diagnosis:
A definitive diagnosis of AD can only be made by examining the brain after a person dies. AD diagnosis in the living must be made through differential diagnosis(exclusion) or protein marker tests and some advanced imaging techniques.
The US Food and Drug Administration (FDA) has approved four acetylcholinesterase inhibitors – rivastigmine, galantamine, tacrine and donepezil – for the treatment of AD, which works by increasing the cholinergic action in the brain areas affected by the disease.
It is reported that the administration of these inhibitors is often accompanied with severe side effects.
At present, most of the protective therapeutic strategies against AD are very limited.
An effective strategy would be to develop protective therapeutic drugs which possess both antioxidant and plaque clearing properties.

4. AD & Blood Brain Barrier
Studies show that the BBB plays a critical role in the manifestation and progression of chronic inflammation during earlier stages of AD. During the onset of AD, it is observed that there is a BBB dysfunction manifests 1. Lower Aβ plaque clearance rates 2. Impaired endothelial transport, 3.Changes in pericyte functions, and decreased tight junction (TJ) integrity, 4.Activation of glial cells and neuro-inflammation through the recruitment of leukocytes in the brain. As AD progresses, several components of the neurovascular unit undergo functional /morphological changes and eventually become dysfunctional, this leads to nervous tissue detrition and cognitive deficits.

5. BBB: Challenges
The Blood-Brain-Barrier (BBB) poses a particularly difficult obstacle to drug delivery via traditional means such as IV and oral dosage forms.
BBB averts the penetration of therapeutic agents and diagnostic tools
It is relatively impermeable to various proteins, small peptides, and amino acids
Small lipophilic molecules are able to pass from blood capillaries.

6. Liposomal Drug Delivery System
Liposomal techniques have long been utilized as a potent Drug Delivery System (DDS) to the brain, because the liposomal particles can encapsulate compounds and prevent rapid elimination or degradation.
The liposomal vesicles are non-toxic, non-immunogenic (low allergenicity), non-carcinogenic and biodegradable.
An encapsulated penetration through the BBB increases the effective dose of a compound.

7. The Promise Of Natural Compounds
Many studies have focused on the beneficial use of natural compounds in diets across the world and has shown that consumption of certain compounds increase the Aβ clearance rate and antioxidant activity . These compounds can be considered as potential candidates for neuroprotective agent against AD.
Experimental research has described a few dozen natural compounds as potential candidates for liposomal treatment of AD. However, no synergistic experimentation has been conducted to understand the efficacy of these natural compounds when used together.
It would be extremely beneficial to find viable synergistic combinations of potent neuro-supportive compounds which can ameliorate AD. Some advantages include: Low toxicity rates and low allergenicity.

8. Potential Candidates
Drug Synergism: Two drugs/compounds that produce overtly similar effects will sometimes produce exaggerated or diminished effects when used concurrently.
Potential candidates for the Synergies:
α-Mangostin is a polyphenolic derivative of mangostin that exhibits pharmacological effects, such as anti-inflammation, antioxidant, and antitumor effects. When administered intravenously, α-mangostin liposomes have been shown to protect and improve the neurons against Aβ- oligomer toxicity in rats(Cheng et al.,2016).
Curcumin is a natural compound extract from the plant Curcuma longa. This is a fluorescent molecule with high affinity for the Aβ peptide and able to reduce Aβ aggregation. In prior research, intracranial injection of liposomes encapsulating curcumin efficiently ameliorated Aβ deposits in both human and mice tissues, proving to be an effective formulation for the treatment of AD (Lazar et all.,2013).
Quercetin a flavonoid commonly found in various vegetables and fruits. It was reported to protect against oxidative injury and cytotoxicity (Ishige et al., 2001). In addition, oral administration of quercetin was also able to improve learning and memory ability.
Inductive approach: Prior experimental research has described several potential candidates for liposomal treatment of AD. However, no synergistic experimentation has been conducted to understand the efficacy of these natural compounds when used together.
“ Can a liposomal formulation consisting of a combination of curcumin, α-Mangostin & quercetin can help decrease neuronal oxidative stress and decrease Aβ aggregation and show a synergistic effect.”

9. Cell-Based Therapy : 3-D Cell culture model
In 2014, Choi et all developed a three-dimensional human neural cell culture model of Alzheimer’s.
They modified Human neural stem cells in a 3-D gel culture system. The cells were modified to produce high levels of 2 proteins—beta-amyloid precursor protein to mimic Alzheimer’s disease.
This cell-culture system can be utilized to screen and can be used to study the viability and metabolism of the various therapeutic compounds.

10. Challenges with Natural Compounds
Problem: Even though the three candidates have potent neuroprotective benefits, their poor absorption and difficulty to pass Blood-Brain-Barrier (BBB) appear to be the big burden for the therapeutic action.
Solution: Encapsulating the natural compounds via liposomal vesicles will allow them to pass the BBB.
In order to develop a synergy models, we will need to create various combinations of natural substances and create a Matrix model to eliminate outliers and additive effects.

11. Matrix Model Synergy layout 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A B D E F
Controls
Curcumin (C)
α-Mangostin
Quecerticn(Q)
Combination (2 X 2)
Combination (3 X 3)
Negative Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 A C CA
AC1 Q
AQ1 M B CQ AQ
QC1
CM+CQ
CM+ CQ CC AA QQ D E F G
N N
T N EF
N T
T T
S T FE
T S
S S
N N
T N EF
T T
S T SS

12. Matrix Model: Continued
Not all combinations will show synergy
Cellular metabolism is indication of overall cell health and also cell viability(survival rate)

13. Expected Results Future Research:
A synergistic action exists in the liposomal combination of curcumin, α-Mangostin & quercetin.
Can synergistic actions help decrease neuronal oxidative stress and decrease Aβ aggregation.
Potential liposomal candidates which can be tested in-vitro.
Future Research:
Intranasal delivery: It had been demonstrated that nasal route, a noninvasive delivery system, could produce higher bioavailability than oral route due to the decrease hepatic metabolism, shorter distance to brain target and easy penetration through the brain (Wang et al., 2006; Illum, 2004)
A liposomal formulation consisting of a synergistic combinations of natural compounds with the highest in-vivo efficacy to be tested in-vitro via Intranasal delivery in rats.
Potential development of a intranasal nutraceutical

14. References:
Banks, W From blood–brain barrier to blood–brain interface: new opportunities for CNS drug delivery.Nature Reviews Drug Discovery 15, 275– 292 (2016) doi: /nrd
Vieira DB, Gamarra LF. Getting into the brain: liposome-based strategies for effective drug delivery across the blood–brain barrier. International Journal of Nanomedicine. 2016;11: doi: /IJN.S
Hersh DS, Wadajkar AS, Roberts NB, et al. Evolving Drug Delivery Strategies to Overcome the Blood Brain Barrier. Current Medicinal Chemistry. 2016;22(9): doi: /
Lai F, Fadda AM, Sinico C. Liposomes for brain delivery. Expert Opin Drug Deliv. 2013;10:1003–1022.
Chen ZL, Huang M, Wang XR, et al. Transferrin-modified liposome promotes α-mangostin to penetrate the blood-brain barrier. Nanomedicine ;12:421–430.
Phachonpai W, Wattanathorn J, Muchimapura S, Preechagoon D. Neuroprotective effect of quercetin encapsulated liposomes: a novel therapeutic strategy against Alzheimer’s disease. Am J Appl Sci. 2010;7:480–485.
Lazar AN, Mourtas S, Youssef I, et al. Curcumin-conjugated nanoliposomes with high affinity for Aβ deposits: possible applications to Alzheimer disease. Nanomedicine. 2013;9:712–721
Sercombe L, Veerati T, Moheimani F, Wu SY, Sood AK, Hua S. Advances and Challenges of Liposome Assisted Drug Delivery. Frontiers in Pharmacology. 2015;6:286. doi: /fphar