Metformin turned into a "God Assist" in the treatment of Alzheimer's disease!

How many people, like the singularity cake, usually have a super low laugh, and they are always there hahaha! It is said that people who love to laugh are not bad luck, but a study once suggested that a low laugh point may be a precursor to dementia
.

For a time, laughter and laughter all turned into forced smiles.
.
.
After reading carefully, Singularity Cake was relieved.
It turns out that "low laughing point" does not mean that you are prone to dementia.
Dementia shows a kind of "morbid laughter".
', rather than the humorous laughter that we have on a daily basis
.

So, everyone still has to laugh and keep humorous! However, it is also true that the number of people diagnosed with dementia in recent years has been huge
.

Alzheimer's disease (AD), commonly known as senile dementia, is one of them.
AD ​​patients show memory loss, confusion, cognitive impairment and changes in personality and behavior, which greatly affect the daily life of themselves and their families.
Life also poses a serious challenge to the development of global health
.

Therefore, dementia must be known and must be prevented
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Recently, researcher Yang Lichao from Xiamen University led a team to design and develop an on-demand dual-drug (donepezil + metformin) supramolecular nano-delivery system for synergistic treatment of AD
.

They found that this structurally stable inclusion complex not only selectively penetrates the blood-brain barrier (BBB), prolonging the accumulation time of drugs in the brain, but also increases the ability of microglia to react to amyloid beta (Aβ).
Clearance efficiency, showing significant anti-dementia effect [1]
.

AD is a neurodegenerative brain disease.
Although its pathogenesis has not been fully elucidated, scientists have found that the brains of AD patients usually show the characteristics of intracellular neurofibrillary tangles and extracellular Aβ deposition[2], The imbalance between the production and clearance of Aβ is one of the factors driving AD [3]
.

Therefore, promoting Aβ clearance is a promising research direction in the field of AD treatment
.

Microglia are equivalent to macrophages in the brain, and they also perform the duty of "cleaning up"
.

Using small-molecule drugs to promote the clearance of Aβ by microglia is one of the main options for improving AD, but the application of small-molecule drugs faces many challenges, such as non-specific distribution, poor targeting, rapid clearance, and severe toxicity [4] ]
.

In contrast, nanomedicines with suitable particle size (< 120 nm) can effectively deliver small-molecule drugs to the brain through the BBB, improving the therapeutic efficiency
.

In addition, the development of an intelligent and responsive dual-drug nanosystem not only solves the problems of traditional nanodrugs such as high dose requirements, large toxic and side effects, and easy drug resistance, but also the advantage of on-demand release at the lesion site effectively avoids the drug being released in the blood circulation process.
of leaks [5-7]
.

Donepezil (Don) is an acetylcholinesterase (AchE) inhibitor approved by the US Food and Drug Administration (FDA), which is usually used to improve the symptoms of AD patients [8]
.

However, due to the strong hydrophobicity of Don, the Don-PC inclusion complexes self-assembled through the supramolecular recognition between Don and phospholipids (PC) are prone to serious drug leakage and physiological instability
.

Metformin (Met) was originally the first-line clinical drug for the treatment of type 2 diabetes.
With the deepening of research, scientists found that metformin can significantly improve AD-related neuropathological changes, such as abnormal Aβ deposition, Tau phosphorylation, and memory deficits [9].
, the guanidine group of Met can also significantly enhance the structural stability of nanomedicines
.

Based on this, the research team of Yang Lichao used the interaction between host and guest molecules to prepare an Aβ-responsive dual-drug inclusion complex (MPD), which not only has superior biological stability and targeted release, but also It has synergistic anti-dementia effect by increasing the clearance rate of Aβ and inhibiting the activity of AchE
.

Schematic diagram of the preparation of Aβ-responsive inclusion complexes and their synergistic anti-dementia treatment effect After many attempts, the researchers successfully prepared MPD inclusion bodies by thin film dispersion method, and used molecular docking (AutoDock Vina program), 1H-NMR ( 1H-NMR) and Fourier transform infrared spectroscopy (FT-IR) were used to explore the formation mechanism of MPD inclusion bodies
.

The results show that the guanidine group of Met can interact with the phosphate group of PC through hydrogen bonds and salt bridges, while the aromatic group of Don can interact with the long-chain fatty acid of PC through π-π stacking to form MPD inclusion complexes structure
.

In addition, the researchers also revealed the distribution state of Met and Don by X-ray diffraction (XRD) and transmission electron microscopy (TEM)
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They found that Met and Don are uniformly distributed in the PC matrix in crystalline form, with the polar head of PC facing inward towards Met, the inward nonpolar tail of PC towards Don, and the outward nonpolar tail in nonpolar solvents.
The tails are oriented towards the organic phase to create spherical nanostructures
.

XRD and TME characterization of MPD inclusion bodies In order to further optimize the formulation process to obtain ideal MPD, Yang Lichao's team characterized the morphology, hydrodynamic diameter, dispersion, zeta potential and drug encapsulation efficiency of three different ratios of MPD inclusion bodies
.

After comparative screening, they finally determined the weight ratio of Met : Don : PC to be 1 : 1 : 4.
At this time, the particle size of MPD was about 110 nm, and the zeta potential was about -21.
3 mV, which was conducive to penetrating the BBB and had good colloidal stability.
and monodispersity
.

The drug loading rates of Met and Don were 13.
7% and 12.
1%, and the encapsulation efficiencies were 82.
3% and 72.
5%, respectively
.

Afterwards, the researchers verified the performance of MPD inclusion body disassembly and Aβ-responsive on-demand release
.

They used PBS without or containing Aβ (30 U/mL) to simulate the physiological environment of normal human and AD patients, respectively.
The results showed that after incubation with PBS containing Aβ, the morphology of MPD was no longer uniform and regular, and the particle size also showed signs of increasing.
, indicating that MPD can partially disassemble and aggregate in response to Aβ
.

TME images and hydrodynamic diameters of MPD inclusion bodies after incubation with Aβ-containing PBS.
In addition, the release profile of the PBS group showed that within 72 h, less than 32% and 37% of Met and Don were released from MPD, respectively.
Indirectly indicates that MPD has excellent physiological stability
.

In contrast, the cumulative release of Met and Don in MPD was greater than 83% and 82%, respectively, after 72 hours of incubation in Aβ-containing PBS, showing the characteristics of responsive release
.

The above findings confirm that the advantage of MPD-responsive on-demand release is expected to achieve simultaneous release of Met and Don in Aβ-rich brain regions, improve synergistic therapeutic efficiency, and reduce adverse effects on normal cells and tissues
.

Next, the researchers verified the targeting ability of MPD and its ability to penetrate the blood-brain barrier
.

The design method for in vivo verification of the penetration of the blood-brain barrier is shown in the figure below.
The structure of the monolayer of bEnd.
3 cells on the top and N2a and BV2 cells on the bottom is used to simulate the structure of the blood-brain barrier, and the MPD is labeled with a fluorescent group to obtain fluorescent properties.
endosomal complex (MNPD) for subsequent detection
.

The results of the in vitro blood-brain barrier penetration model showed that, compared with the control group (Control) and the naked drug group (MN), MNPD exhibited strong fluorescent signals in both N2a and BV2 cells after 12 hours, indicating that MPD can effectively penetrate the blood-brain barrier.
It is taken up by neuronal cells and microglia across the monolayer bEnd.
3 cell barrier
.

In vivo imaging results showed that the fluorescence signal in the MNPD group was stronger than that in the MN group in the brains of BALB/c nude mice at the same time point, and reached a maximum 24 hours after injection, indicating that MPD can effectively penetrate the BBB and accumulate in the brain.
brain
.

Interestingly, compared with the MN group, the retention time of MNPD in the brain was significantly prolonged, which was consistent with the results of pharmacokinetic studies in mice.
The blood clearance curve showed that the blood circulation half-life of MPD was significantly prolonged, relatively The long circulation time provides a realistic basis for the penetration and accumulation of drugs in the brain
.

Encouraged by the results of in vitro studies on the plasma concentration-time curves of Met and MPD, researcher Yang Lichao's team continued to conduct in-depth research and systematically explored the effect of MPD's synergistic anti-dementia effect in vivo
.

First, by analyzing AD-related genes, they found that MPD identified a total of 838 AD-related genes, significantly more than the 44 genes identified when Don treatment was given alone, indicating that Met can enhance the efficacy of Don in AD treatment
.

In addition, analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that AD, cholinergic synapse and neurotrophin signaling pathways, and glutathione metabolism were significantly enriched in the dataset; analysis by Gene Ontology (GO) revealed that , Aging, neuronal death, phagocytosis, learning or memory, neuroinflammation, and oxidoreductase activity were significantly enriched in AD pathways
.

The above gene network analysis indicated that MPD may achieve synergistic anti-dementia effect by regulating learning and memory-related pathways
.

MPD may play an anti-dementia effect by regulating learning and memory-related pathways.
Next, researcher Yang Lichao and others injected Aβ25-35 into the bilateral hippocampus of mice to establish a mouse AD model.
Mice treated with different treatments were required to receive Morris water for 22 days.
Maze (MWM) training
.

Observing the behavior of the mice in each group, they found that the swimming traces of the mice in the AD group were evenly distributed around the four quadrants, while the traces of the mice in the MPD group were mainly distributed in the target area.
The number of platform crossings, time spent in the target quadrant, and distance in the target quadrant all increased
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The above results demonstrate that MPD can improve cognitive deficits induced by Aβ and exert a synergistic anti-dementia effect
.

In addition, they confirmed by Congo red staining and confocal microscopy (CLSM) observations that MPD can significantly reduce the deposition of Aβ in the brain, while also improving the efficiency of microglia clearance of Aβ through the lysosomal pathway
.

The results of Congo red staining in the brain tissue of mice in each group showed that the deposition of Aβ in the MPD group was significantly reduced.
Studies have shown that the increase of BACE-1 and PS-1 can increase the expression level of Aβ in the brain, while ADAM-10 can effectively prevent the production of Aβ [10]
.

Therefore, researcher Yang Lichao and others used Western blotting to detect the expression of the above proteins to characterize the Aβ levels in the brains of mice in each group
.

They found that the expressions of BACE-1 and PS-1 in the hippocampus of MPD mice were significantly reduced, while ADAM-10 did not change much, suggesting that MPD may eliminate excessive Aβ by regulating the expression of Aβ-related secretases BACE1 and PS1
.

Finally, the researchers analyzed the morphology of neuronal cells and the expression of neuron-specific protein NeuN in each group of mice, confirming that MPD has a good neuroprotective effect and can effectively improve the cognitive function of AD mice
.

Overall, this dual-drug supramolecular nanomedicine developed by Yang Lichao's team provides a set of research ideas for "decompression and synergy" for AD treatment
.

Due to its structural advantages, MPD can effectively penetrate the BBB and accumulate in the brain, and release Met and Don synchronously on demand under the stimulation of Aβ, which can not only reduce the pressure of AD treatment by reducing the level of Aβ in the brain, but also increase the acetylcholine in the brain.
Active, dedicated to improving cognitive function
.

The two-pronged approach greatly enhanced the synergistic effect of Met and Don against dementia
.

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