Dendrimer–tesaglitazar Conjugate Induces A Phenotype Shift Of Microglia And Enhances β-amyloid Phagocytosis† Part 1

Switching microglia from a disease-exacerbating, 'pro-inflammatory' state into a neuroprotective, 'antiinflammatory' phenotype is a promising strategy for addressing multiple neurodegenerative diseases. 

Microglia are a type of cell in the central nervous system that is primarily responsible for maintaining the health and function of neurons. They can secrete a variety of growth factors and neurotrophic factors and can maintain the normal metabolic activity of neurons by clearing waste materials around neurons. Studies in recent years have shown that microglia are closely related to memory. Let's take a look at it together.

First, microglia can stimulate neurons and promote the activation of neurons, thereby enhancing memory. By releasing a series of neurotransmitters, such as glutamate and alanine, microglia can promote signal transmission between neurons and can enhance the glial-neuron signal resonance between presynaptic membranes, further promoting neuronal excitability and memory formation.

Secondly, microglia can also clear waste around neurons, maintain normal metabolic activity of neurons, and reduce neuronal mortality, thereby promoting memory improvement. When too much garbage accumulates around neurons, it will affect the normal metabolic activity of neurons, leading to neuronal death and weakened function, thereby reducing memory performance. Microglia can maintain the normal metabolic environment of neurons and reduce neuronal mortality by engulfing and decomposing surrounding waste, thereby improving memory.

In summary, microglia are closely related to memory. By promoting the excitation of neurons and clearing surrounding waste, microglia can further improve memory and keep our brains healthy and active. We should pay attention to protecting the health of microglia to achieve better memory. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because Cistanche is a traditional Chinese medicinal material with many unique effects, one of which is to improve memory. The efficacy of Cistanche comes from the various active ingredients it contains, including tannic acid, polysaccharides, flavonoid glycosides, etc. These ingredients can promote brain health in a variety of ways.

Click know 10 ways to improve memory

Proinflammatory microglia contribute to disease progression by releasing neurotoxic substances and accelerating pathogenic protein accumulation. PPARα and PPARγ agonists have both been shown to shift microglia from a pro-inflammatory ('M1-like') to an alternatively activated ('M2-like') phenotype. Such strategies have been explored in clinical trials for neurological diseases, such as Alzheimer's and Parkinson's disease, but have likely failed due to their poor blood-brain barrier (BBB) penetration. 

Hydroxyl-terminated polyamidoamine dendrimers (without the attachment of any targeting ligands) have been shown to cross the impaired BBB at the site of neuroinflammation and accumulate in activated microglia. 

Therefore, dendrimer conjugation of a PPARα/γ dual agonist may enable targeted phenotype switching of activated microglia. Here we present the synthesis and characterization of a novel dendrimer-PPARα/γ dual agonist conjugate (D-tesaglitazar). 

In vitro, D-tesaglitazar induces an 'M1 to M2' phenotype shift, decreases secretion of reactive oxygen species, increases expression of genes for phagocytosis and enzymatic degradation of pathogenic proteins (e.g. β-amyloid, α-synuclein), and increases β-amyloid phagocytosis. 

These results support further development of D-tesaglitazar towards translation for multiple neurodegenerative diseases, especially Alzheimer's and Parkinson's Disease.

Introduction

In the United States alone, there are currently over 5.3 million people with Alzheimer's disease (AD) and 1 million people with Parkinson's disease (PD).1 As increased age is the largest risk factor for many neurodegenerative diseases, the prevalence and cost of treatment for these diseases will continue to increase as the population continues to grow older. 

Moreover, the lack of recent success in developing new drugs to treat these diseases has highlighted the need for the development of innovative therapies.2,3 These clinical failures highlight the difficulties in developing a drug, including delivering a high enough concentration of the drug to the brain for efficacy without causing adverse side effects. 

Neurodegenerative diseases such as AD and PD share three major neuropathological components: neuroinflammation, pathogenic protein accumulation, and neuronal death.4–7 In healthy people, the innate immune cell of the brain (the microglia) constantly phagocytose the misfolded proteins (e.g. β-amyloid, α-synuclein) that cause neuronal death as they are produced, which prevents the hallmark aggregates from forming. 

However, in people who eventually develop neurodegenerative diseases, the microglia no longer remove these proteins effectively and shift into a disease-exacerbating, proinflammatory phenotype (typically designated as M1). 

While the predominantly pro-inflammatory/anti-inflammatory (M1/ M2) classification of microglial activation is an oversimplification of the spectrum of macrophage polarization, it is still used as a broad nomenclature to describe the dominant phenotype of microglia in neuroinflammation and response to therapy. 

M1-like microglia release reactive oxygen species and other inflammatory mediators that both induce neuronal death and exacerbate disease pathology by increasing the production of pathogenic proteins (e.g. β-amyloid, α-synuclein). 

Moreover, the major genetic risk factors for developing AD (TREM2 and APOE) are expressed at high levels in microglia, and the TREM2/APOE pathway has been shown to cause a microglial phenotype shift in AD, amyotrophic lateral sclerosis (ALS), and multiple sclerosis animal models.8 

These findings further demonstrate the role of microglia in the pathology of multiple human neurodegenerative diseases. An approach to manipulate the phenotype of microglia would enable researchers to understand their role in neurodegenerative diseases, in addition to potentially being an effective therapeutic. 

Switching microglia from an M1 to an anti-inflammatory and neuroprotective (M2) phenotype has been proposed as a therapeutic strategy to treat multiple neurodegenerative diseases.5,6 Two currently FDA-approved PPARγ agonists, type II diabetes drugs rosiglitazone and pioglitazone, have been shown to induce an M1 to M2 phenotype shift in macrophages and microglia in vitro and in vivo. 

9,10 PPARγ agonists have been shown to reduce the LPS-induced secretion of reactive oxygen species by reducing the activity of NF-κB by inducing NF-κB degradation and export from the nucleus, as well as ligand-dependent transrepression.11 

Moreover, epidemiological studies have shown that diabetes patients who take rosiglitazone or pioglitazone are at a reduced risk for developing AD and PD.12 Subsequently, rosiglitazone and pioglitazone were investigated through phase III clinical trials for AD, but failed.13 

One likely explanation for the failure of the aforementioned clinical trials is the poor transport of these drugs across the blood-brain barrier (BBB), thus limiting the number of drugs that reached the brains of patients enrolled in these clinical trials.14 

Indeed, it is estimated that the BBB prevents about 98% of all small molecule drugs from reaching the brain, and only a fraction of the drug that enters the brain reaches the microglia.15 

In addition, PPARα is another nuclear receptor in the PPAR family.16 It exhibits a role in lipid homeostasis and regulating inflammation, and PPARα agonists have also been shown to exhibit anti-inflammatory effects in microglia.

Clinical studies using neuroimaging, post-mortem tissue analysis, and CSF biomarkers have provided evidence that the BBB is impaired in AD and PD, as well as other neurodegenerative diseases.

17 Generation-4 hydroxyl-terminated polyamidoamine (G4-PAMAM-OH) dendrimers have been shown to intrinsically bypass the impaired BBB and accumulate in activated microglia without the need for targeting ligands, after systemic administration in multiple different neuroinflammation disease models, including in rodents, rabbits, dogs, and nonhuman primates.18–28 Significantly, G4-PAMAM-OH can be administered systemically and cross the BBB in disease models with mild BBB disruption such as Rett Syndrome.29 

Additionally, the extent of uptake of G4-PAMAM-OH into the brain is directly proportional to disease severity in a rabbit model of cerebral palsy.30 Hydroxyl-terminated dendrimers have the advantage of being delivered noninvasively compared to the highly invasive, local delivery through the skull required in previous studies with other nanoparticles such as poly-εcaprolactone and PEG, negatively charged PAMAM dendrimers, quantum dots, and nanoformulations composed of polyethyleneimine (PEI) and dextran sulfate.31–35 

In addition, these hydroxyl PAMAM dendrimers are ideally positioned for translation due to their scalability and well-tolerated in vivo safety profile.36–38 Due to positive pre-clinical efficacy data, a (G4- PAMAM-OH)-N-acetyl-cysteine conjugate is currently being evaluated in early clinical trials for childhood cerebral adrenoleukodystrophy (NCT03500627) and severe coronavirus disease 2019 (COVID-19) associated inflammation (NCT04458298). 

We studied a dendrimer–drug conjugate of tesaglitazar (Tesa) attached to generation-4 hydroxyl-terminated PAMAM dendrimer. Tesaglitazar is a potent PPARα/γ dual agonist that combines the beneficial effects of PPARα and PPARγ agonists. It contains a carboxylic acid functional group for covalent conjugation to the dendrimer and for subsequent release. 

Additional guitars have been developed and tested clinically, but Tesa was chosen due to its larger PPARγ to PPARα activity ratio, relatively simple chemical structure, and safety profile.39–43 

Tesa has previously reached phase III clinical trials for type 2 diabetes in the United States of America but failed due to dose-dependent toxicity, which may be prevented by decreasing the necessary dose of administration by the controlled dendrimer delivery.39,40,44 Since Tesa is a PPARα/γ dual agonist, its targeted delivery to activated microglia at the site of neuroinflammation could be highly beneficial. 

Herein, we demonstrate the synthesis and characterization of a dendrimer–tesaglitazar conjugate (D-Tesa) and demonstrate this compound's ability to induce a 'M1 to M2' phenotype shift in microglia and enhance phagocytosis of fluorescently labeled β-amyloid.

Materials and methods

Materials

1-[3-(Dimethylamino)propyl]-3-ethyl carbodiimide methiodide (EDC), 4(dimethylamino)pyridine (DMAP), CuSO4·5H2O, sodium ascorbate, hexynoic acid and bovine serum albumin (BSA) were purchased from Sigma Aldrich US and used as received (St Louis, MO). 

Tesaglitazar was obtained from AstaTech Inc. (Bristol, PA). Ethylenediamine-core PAMAM dendrimer (generation 4 with 64 hydroxyl end-groups) was received from Dendritech Inc. (Midland, MI) as a solution in methanol. 

The dendrimer was stored in methanol at 4 °C and methanol was evaporated before use. A dialysis membrane with a molecular weight cut-off (MWCO) of 1 kDa was purchased from Spectrum Laboratories Inc. (New Brunswick, NJ). 

All other solvents were used as received in their anhydrous forms. All reactions, except the copper(I), catalyzed alkyne–azide cycloaddition (CuAAC) click reactions, were conducted under anhydrous conditions in an organic medium with oven-dried glassware under an inert nitrogen atmosphere. 

For cell culture: Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P/S), 0.05% trypsin-EDTA, and MTT reagent were obtained from Invitrogen (Carlsbad, CA, USA). 

Griess reagent was obtained from Promega (Madison, WI) and TNF-α ELISA was obtained from R&D Systems (Minneapolis, MN). Analytical grade methanol was purchased from Sigma-Aldrich. Trypan blue was obtained from Corning (Manassas, VA, USA).

Synthesis procedures for D-Tesa conjugates
Tetraethylene glycol mono azide (2) was synthesized using a previously published protocol.45

Synthesis and purification of Tesa-TEG-azide (3).
Tesa (950 mg, 2.32 mmol) was dissolved in 10 ml dimethylformamide (DMF). To this stirred solution, tetramethylene glycol mono-azide (2, 662.1 mg, 3.02 mmol) in DMF (1 ml) was added dropwise. DMAP (255.4 mg, 2.09 mmol) and EDC (577.5 mg, 3.02 mmol) were then added to the reaction mixture, and the reaction was stirred under nitrogen purge at room temperature for 24 hours. 

The reaction was monitored using thin-layer chromatography and high-performance liquid chromatography (HPLC). The reaction mixture was diluted with 100 ml dichloromethane (DCM) and the crude reaction mixture was shifted into a separatory funnel, and the organic layer was subsequently washed thrice with saturated sodium bicarbonate solution, followed by saturated ammonium chloride solution and finally with brine. 

The organic layer was then dried with anhydrous sodium sulfate. The solvent in the organic layer was then removed using a rotary evaporator, and the solution was redissolved in 3 ml DCM and absorbed onto silica gel to be purified with a CombiFlash® chromatography system using a gradient method with ethyl acetate/hexane as the solvents to produce 3 as clear-yellow oil. 

The desired, pure product is eluted at approximately 30–40% ethyl acetate. (Yield: 70%.) 1 H NMR (500 MHz, CDCl3) δ 7.27 (d, J = 8.6 Hz, 2H), 7.15 (d, J = 1.9 Hz, 2H), 7.08 (d, J = 8.6 Hz, 2H), 6.73 (d, J = 8.6 Hz, 2H), 4.25–4.14 (m, 2H), 4.07 (t, J = 6.8 Hz, 2H), 3.94 (dd, J = 7.8, 5.2 Hz, 1H), 3.67–3.47 (m, 14H), 3.30 (dd, J = 8.7, 3.8 Hz, 2H), 3.06 (s, 3H), 3.02 (t, J = 6.7 Hz, 2H), 2.91–2.84 (m, 2H), 1.08 (t, J = 7.0 Hz, 3H). ESI-MS: theoretical C28H39N3O10S: 609.24, obtained (M + 1): 610.13.

Synthesis and purification of D-YNE (5).

(480 mg, 4.22 mmol) was added to a stirred solution of G4- PAMAM-OH (2.5 g, 0.176 mmol) in 20 ml anhydrous DMF. To this mixture, DMAP (430 mg, 3.52 mmol) and EDC (1 g, 5.28 mmol) were added. 

The reaction mixture was stirred under nitrogen purge for 24 hours at room temperature. On completion of the reaction, the reaction mixture was transferred to a 1000 MWCO dialysis tube DMF dialysis was performed for 24 hours, and the DMF was changed about every six hours. 

Then dialysis with deionized (DI) water was performed for 24 hours, with the water being changed about every six hours. Lastly, the resulting dialysis tube contents were lyophilized for 48 hours, yielding a white, fluffy powder. (Yield: 61%.) 1 H NMR (500 MHz, DMSO) δ 8.10–7.67 (m, dendrimer internal amide H), 4.72 (s, dendrimer surface OH), 4.01 (t, ester –CH2), 3.32 (m, dendrimer –CH2), 3.06 (m, dendrimer and linker –CH2), 2.85–2.58 (m, dendrimer –CH2), 2.56–1.89 (m, dendrimer and linker –CH2), 1.78–1.61 (m, linker –CH2). Synthesis and purification of D-Tesa (6). 

Tesa-TEG-azide (3, 177.8 mg, 0.303 mmol) was added to a stirred mixture of D-YNE (5, 350 mg, 0.023 mmol) in 5 ml of a 1: 1 mixture of tetrahydrofuran (THF) and water with 0.5 ml of DMF in a microwave reactor safe 20 ml vial. For the CuAAC click reaction, copper sulfate pentahydrate (11.6 mg, 0.0467 mmol) and (+)-sodium-Lascorbate (9.3 mg, 0.0467 mmol) were added to the reaction mixture. 

The vial was sealed, and the reaction vessel was then placed in a Biotage® Initiator microwave reactor and reacted under 20 W microwave radiation with stirring for 8 hours at 50 °C. The reaction mixture was transferred to a 1000 MWCO dialysis tube, and DMF dialysis was performed for 24 hours, with DMF being replaced by fresh solvent approximately every 4 hours. 

Then, the contents of the dialysis tube were transferred to a falcon tube, and an equivalent amount of DI water was added. Additionally, 200 µl of ethylenediaminetetraacetic acid disodium salt solution was added to the contents of the falcon tube. 

This mixture was then placed into a new 1000 MWCO dialysis tube, and dialysis was performed for 12 hours in 1000 ml DI water with EDTA solution added followed by the water dialysis for 12 hours. 

The mixture was then lyophilized for 48 hours and resulted in a white, fluffy powder. (Yield: 64%.) 1 H NMR (500 MHz, DMSO) δ 8.2–7.6 (m, dendrimer internal amide H), 7.36 (d, Tesa ArH), 7.21 (d, Tesa ArH), 7.03 (d, Tesa ArH), 6.76 (d, Tesa ArH), 4.37 (s, Tesa H), 4.18–4.04 (m, linker H), 3.96 (dd, Tesa and linker H), 3.70 (m, Tesa and linker H), 3.58–3.14 (m, dendrimer –CH2), 3.14–2.86 (m, dendrimer –CH2), 2.87–2.49 (m, dendrimer and linker –CH2), 2.25 (m, dendrimer –CH2), 1.82–1.67 (m, linker –CH2), 0.97 (t, Tesa –CH3).

Characterization techniques

Nuclear magnetic resonance (NMR). NMR spectra were recorded on a Bruker 500 MHz spectrometer at room temperature. Proton chemical shifts (δ) are reported in ppm. 

1 H NMR was used to determine the number of Tesa molecules attached to each molecule of D-Tesa by proton integration method, by comparing the peaks of internal amide protons of dendrimer at δ 7.6–8.2 ppm with aromatic protons of Tesa at δ 7.36–6.76 ppm and methyl protons of Tesa in the aliphatic region. High-performance liquid chromatography (HPLC). 

HPLC (Waters Corporation, Milford, Massachusetts) equipped with a 1525 binary pump, an In-Line degasser AF, a 717 plus autosampler, a 2998 photodiode array detector, and a 2475 multi λ fluorescence detector interfaced with Waters Empower software was used. 

A Symmetry C18 reverse phase column (Tosoh, Japan) having 5 μm particle size, 25 cm length, and 4.6 mm internal diameter was used. Compounds were monitored at 210 nm and 254 nm using the PDA detectors. 

Solvent A was HPLC-grade water with 0.1% trifluoroacetic acid (TFA), and solvent B was acetonitrile (ACN) with 5% water and 0.1% TFA. The method used started at 100 : 0 (ACN: water), decreased to 10: 90 (water: ACN) in 5 minutes, stayed at that polarity for 15 minutes, and returned to 100 : 0 (ACN: water) in 5 minutes. The flow rate was maintained at 1 ml min−1. Mass spectroscopy. 

ESI-MS was performed on Bruker microTOF-II mass spectrometer using acetonitrile/water (9: 1) as solvent system. The molecular ions as protonated peaks [M + nH]n+ or adducts [M + nX]n+ (X = Na, K, or NH4) were used to confirm the empirical formula. 

Dynamic light scattering and ζ-potential. A Zetasizer Nano ZS (Malvern Instrument Ltd, Worchester, U.K) equipped with a 50 mW He–Ne laser (633 nm) was utilized to determine particle size and ζ-potential distribution. D-Tesa was dissolved in DI water to a concentration of 0.2 mg ml−1 for DLS and in 10 mM sodium chloride to a concentration of 0.1 mg ml−1 for ζ-potential. 

The measurements were made at 25 °C, using a scattering angle of 173° as previously described.27,46 Drug release study. D-Tesa was dissolved at a concentration of 1 mg ml−1 in either phosphate buffer solution (pH 7.4) to mimic plasma conditions or sodium citrate solution (pH 5.5) to mimic lysosomal conditions. 

Esterases from porcine liver (from Sigma Aldrich) were added to the sodium citrate solution at the start of the release study and were replenished approximately every 3 days during the study. 

Each vial contained 15 ml sample and they were continuously shaken at 37 °C for the duration of the experiment. At different time points, duplicate 200 µl samples from each pH were collected and the esterase activity was subsequently quenched by adding 200 µl of methanol. Zero-hour time point samples served as the control. 

The samples were stored at −80 °C to further avoid any hydrolysis. The samples were further analyzed by HPLC and the area under the curve (at 210 nm) for the free drug peak was calculated. The area under the curve was correlated to the amount of drug released by utilizing a calibration curve where known concentrations of free Tesa were run on the HPLC at 210 nm.

For more information:1950477648nn@gmail.com