Dendrimer–tesaglitazar Conjugate Induces A Phenotype Shift Of Microglia And Enhances β-amyloid Phagocytosis† Part 3
Jul 15, 2024
D-Tesa increased the expression of enzymes responsible for the removal of pathogenic proteins
Insulin degrading enzyme (Ide) and matrix metalloprotease 9 (MMP9) are enzymes secreted by microglia that degrade extracellular β-amyloid and α-synuclein.71,72 D-Tesa treatment significantly increased expression of Ide 3.1-fold (p < 0.001) with a trend towards increasing MMP9 expression (1.8-fold increase, p = 0.057) compared to LPS-only treated controls (Fig. 6A and B).
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Free Tesa significantly increased the expression of Ide 2-fold (p = 0.011), and non-significantly increased MMP9 2-fold (p = 0.35) (Fig. 6A and B). M2 microglia in neurodegenerative diseases can remove β-amyloid, α-synuclein, and other pathogenic proteins via enzymatic degradation or phagocytosis, and D-Tesa upregulates proteins involved in these processes (i.e. Ide and MMP9).71–73
Ide expression is downregulated in AD and PD pathology and is known to be upregulated by PPARγ agonists, which is consistent with our results.
71,72 Functionally, only a 2-fold increase in Ide levels has been shown to decrease β-amyloid accumulation and neuronal death in vivo. 74,75 Therefore, the 3-fold increase in Ide observed by D-Tesa, and 2-fold increase of Ide by free Tesa could be therapeutically efficacious in vivo.
D-Tesa increases phagocytosis of β-amyloid
CD36 is a microglial scavenger receptor that facilitates phagocytosis and degradation of β-amyloid.73 Its downregulation in AD results in decreased removal of β-amyloid, but it is upregulated by PPARγ activation.
Both D-Tesa and free Tesa significantly increased CD36 expression levels when compared to LPS-only exposed cells (p < 0.0001 vs. p < 0.005 for D-Tesa and free Tesa, respectively), but D-Tesa was much more effective than free Tesa (6-fold increase vs. 2.8-fold increase for D-Tesa and free Tesa, respectively, p < 0.0005) (Fig. 6C).
Our results are consistent with previous studies with pioglitazone (another PPARγ agonist) that showed that increased microglial phagocytosis of β-amyloid occurs through a PPARγ and CD36-dependent mechanism.73
To investigate if the upregulation of CD36 from D-Tesa treatment correlated to the increased phagocytic ability of these cells, we performed a functional phagocytosis assay of β-amyloid. 73
Briefly, after treating the cells as we did in the previous in vitro assays, we applied fluorescently labeled β-amyloid1–42 to the cells for two hours, washed the cells, and then performed flow-cytometry to investigate the extent of cellular uptake of β-amyloid. D-Tesa increased both the percentage of cells that phagocytosed β-amyloid and the average amount of β-amyloid internalized per cell (Fig. 7A and B).
In contrast, free Tesa yielded no improvements in phagocytosis of β-amyloid. The superior effects of D-Tesa compared to Tesa are likely attributable to improved cellular internalization enabled by dendrimer conjugation.
This is consistent with previous work that demonstrated over 95% of BV2 cells treated with fluorescently labeled G4-PAMAM-OH had internalized the dendrimer within thirty minutes and continued to internalize the dendrimer for at least 24 hours.76
Likewise, conjugation of the small molecule minocycline to fluorescently-labeled G4- PAMAM-OH demonstrated that 99% of BV2 cells had internalized the fluorescently-labeled dendrimer–drug conjugate within 3 hours.26 They also showed that the conjugate reduced nitric oxide levels superior to the free drug after treating BV2 cells with LPS, consistent with our results.
The cells treated with D-Tesa phagocytosed 1.9-fold more β-amyloid than the LPS-treated control cells (p < 0.001), which is comparable to the 2.5-fold increase of β-amyloid phagocytosis by rat primary microglial cells treated with pioglitazone reported by Yamanaka et al.73 The slightly higher extent of β-amyloid phagocytosis by the previous study could be either because they did not co-treat their cells with LPS as we did, or because primary microglia express PPARγ at a higher level than BV2 cells used in this study.77
As such, a dose of D-Tesa lower than that estimated from the in vitro experiments in our study can likely be efficacious in vivo studies and humans, since BV2 cells are known to express PPARγ at lower levels than primary microglia.77
Microglia have been implicated in many neurodegenerative diseases, and the BBB has prevented many drugs that can modify the phenotype of microglia from a pro-inflammatory, neurotoxic M1 phenotype to an anti-inflammatory, neuroprotective M2 phenotype from reaching therapeutic levels in the brain.5,6,14,15
For example, two PPARγ agonists, pioglitazone and rosiglitazone, were each investigated through phase III clinical trials for Alzheimer's disease due to their ability to alter the phenotype of microglia, but failed likely due to poor transport across the BBB.13,14 Thus, there is clinical interest in altering the phenotype of microglia in neurodegenerative diseases.
Such an approach requires delivery of the drug to microglia at sufficient levels to drive a response. Towards this end, G4-OH-PAMAM dendrimers have been shown to deliver drugs to microglia in many animal models after systemic injection, and as a result, are currently being evaluated in clinical trials for the treatment of ccTLD (NCT03500627) and severe COVID-19 associated inflammation (NCT04458298).18–28
To combine the beneficial effects of altering microglial phenotype with the ability to deliver drugs to microglia, we conjugated tesaglitazar (a PPARα/γ dual agonist) to a G4-OH-PAMAM dendrimer (Fig. 1–3). We demonstrated that D-Tesa is capable of altering the phenotype of M1 microglia towards an M2 phenotype (Fig. 4 and 5), resulting in a decrease in the secretion of harmful reactive oxygen species.
Furthermore, we demonstrated that the microglia treated with D-Tesa increase their expression of enzymes that degrade pathological proteins such as α-synuclein and β-amyloid, as well as upregulating the phagocytosis of β-amyloid in a functional assay (Fig. 6 and 7).
Although we do not present data demonstrating the ability of D-Tesa to bypass the BBB and accumulate in microglia, we have previously shown that G4-OH-PAMAM drug conjugates with similar drug loading, size, and zeta potential are capable of passing through the impaired BBB and accumulating in microglia after intravenous administration.26,36,52
These results support the further development of D-Tesa for the treatment of multiple neurological diseases. While we focus on Alzheimer's and Parkinson's diseases in this paper, D-Tesa has the potential for clinical translation in multiple neurological disorders. Due to the similar role of microglia in the pathology of multiple neurodegenerative diseases, the PPARγ agonist pioglitazone was also investigated or is currently being investigated in phase II clinical trials for Parkinson's disease,78 ALS,79 adrenomyeloneuropathy (NCT03864523), multiple sclerosis (NCT03109288), and hematoma resolution in intracerebral hemorrhage (NCT00827892).
If these clinical trials also fail due to poor delivery of pioglitazone across the BBB, D-Tesa could overcome this delivery obstacle and treat patients with these diseases.
Other groups have utilized nanoparticles to improve the delivery of PPAR agonists to macrophages, but they have not attempted to deliver these agonists to microglia. Osinski et al. demonstrated that Tesa-loaded liposomes were mostly taken up by macrophages in visceral white fat in a male leptin-deficient obesity model.80
They additionally demonstrated that Tesa-loaded liposomes did not alter the expression of the M1 marker Mcp-1, but did increase the expression of the M2 marker Arg1, while treatment with free Tesa decreased the total number of M1 macrophages and expression of Mcp-1, and did not increase expression of Arg1.
Nakashiro et al. used poly(lactic-co-glycolic-acid) (PLGA) nanoparticles to deliver pioglitazone (a PPARγ agonist) to macrophages in the context of atherosclerosis.81
In vivo, they demonstrated that PLGA-pioglitazone reduced the levels of immune cells in the blood. In primary bone marrow-derived macrophages treated with LPS and interferon-γ, they found that PLGA-pioglitazone increased IL-4 and IL-10 (M2 markers) and did not decrease IL-6 or TNFα levels.
Their findings are similar to ours; M2 markers were increased by nanoparticle-PPAR agonist treatment, while IL-6 and TNF-α levels did not decrease. Di Mascolo et al. used PLGA-polyvinyl alcohol nanoparticles to deliver rosiglitazone (another PPARγ agonist).82
In vitro they demonstrated their nanoparticle-drug complexes decreased iNOS, TNF-α, and IL-1β expression in bone marrow-derived macrophages. They pretreated their cells with the nanoparticle drug before stimulating with LPS, while we pretreated cells with LPS before treating with D-Tesa, which could be a reason we did not observe a decrease in TNF-α and IL-1β, although we also observed a decrease in iNOS expression.
Conclusion
There is currently no pathology-altering therapy for many neurodegenerative diseases, and as the population continues to age, the prevalence and cost of treating these diseases will continue to rise, highlighting the urgent need for a solution to be developed. Recently, pro-inflammatory M1 microglia are critical in the pathology of multiple neurodegenerative diseases.
Subsequently, being able to deliver a drug across the blood-brain barrier that can induce an 'M1 to M2' phenotype shift in microglia has therapeutic potential for multiple diseases, especially Alzheimer's and Parkinson's disease.
D-Tesa was designed to deliver an 'M1 to M2' inducing drug to microglia after systemic administration to reduce microglial secretion of neurotoxic substances, while also inducing an anti-inflammatory state that increases degradation and phagocytosis of pathogenic proteins in the brain. We successfully synthesized D-Tesa using a highly efficient click chemistry approach.
The drug is attached to the dendrimer via an ester bond that is cleavable intracellularly at lysosomal conditions, with approximately 60% of Tesa being released in the first 48 hours under lysosomal conditions.
D-Tesa was shown to be superior to Tesa in vitro in inducing an M1 to M2a/M2b/M2c phenotype shift, which resulted in reduced nitric oxide secretion, increased expression of α-synuclein and β-amyloid degrading enzymes, and increased phagocytosis of β-amyloid.
Thus, D-Tesa combines the beneficial delivery properties of the dendrimer, with the M1 to M2 switching properties of Tesa. Due to the common role of microglia and the common therapeutic benefit of inducing an M1 to M2 phenotype shift, D-Tesa has the potential to treat many neurological disorders when administered at the right stage of disease progression.
Author contributions
L. D., A. S., K. L., R. S., S. K., and R. M. K. conceptualized the experiments. L. D., A. S., and R. S. performed synthesis and characterization of the dendrimer–drug conjugate. L. D., K. L., and J. J. performed the cell experiments. L. D. and K. L. performed the statistics. L. D. and A. S. wrote the manuscript, and all authors edited the manuscript.
Conflicts of interest
R. M. K. and S. K. are co-inventors of using the hydroxyl-terminated dendrimer for targeted delivery to microglia in neurological diseases, as well as patents related to the dendrimer technology described in this paper.
They are co-founders of Ashvattha Therapeutics, Orpheris Inc., and RiniSight Inc., which are companies leading the clinical development of the platform. S. K. and R. M. K. are Board Members of Ashvattha Therapeutics Inc. R. S. is currently employed by Ashvattha Therapeutics and owns shares in the company; the work R. S. performed for this paper was done before he joined Ashvattha. The conflict of interest is managed by the Johns Hopkins University.
Acknowledgments
We would like to thank Elizabeth Smith Khoury for helpful discussions of the in vitro experiments. This project was funded by the Patz Distinguished Professorship Endowment from Johns Hopkins and NICHD (grant number HD076901) (RMK). We thank the Wilmer Core Grant for Vision Research, Microscope, and Core Module (grant number EY001865) for access to the Sony flow cytometer.
We thank Servier Medical Arts for the use of their collection of images (http://smart.servier.com/) that is licensed under a Creative Common Attribution 3.0 Generic License, which was modified to make the graphical abstract.
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