Dendrimer–tesaglitazar Conjugate Induces A Phenotype Shift Of Microglia And Enhances β-amyloid Phagocytosis† Part 2

In vitro biological assays

Cell culture. BV2 murine microglial cell line was obtained from the Children's Hospital of Michigan Cell Culture Facility. 

In recent years, scientists have found that cell culture is closely related to human memory. Studies have shown that through the study of cell culture, the molecular mechanisms in the memory and learning process can be discovered, thereby improving human cognitive ability and memory.

Cell culture refers to the process of placing biological cells in a culture medium containing essential nutrients to grow and reproduce under in vitro conditions. Cells are the basic units of life, and cell culture provides scientists with a platform to study cell behavior and life activities. Through the study of cells, scientists have discovered a lot of information about neuron growth and synaptic connections. These findings can not only help humans better understand how the brain works but also help develop new treatments and drugs.

Research in recent years has shown that cell culture is also closely related to human memory. Scientists have found that the formation and maintenance of human memory requires a lot of molecular regulation and signal delivery. These signal delivery and regulation mechanisms have many similarities with the mechanisms in cell culture. By studying the signal delivery and regulation mechanisms in cell culture, scientists can better understand human memory and learning mechanisms.

In addition, some studies have also shown that cell culture can enhance human memory and learning ability in certain ways. For example, using a method called "electrical stimulation", the activity of cells and the connection between neurons can be stimulated, thereby improving human cognitive ability and learning effects. Although this method is still in the laboratory research stage, it is expected to become a new cognitive training method in the future.

In summary, there is a close connection between cell culture and human memory. Through the study of cells, we can better understand the human cognitive mechanism and memory mechanism and also help to develop new treatment methods and cognitive training methods. Let us look forward to the contribution of cell culture technology to mankind in the future! It can be seen that we need to improve memory, and Cistanche can significantly improve memory because Cistanche has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.

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BV2 cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) media with 10% heat-inactivated fetal bovine serum (HI FBS) and 1% penicillin/streptomycin (P/S) at 37 °C and 5% CO2. 

Once cells reached confluency in the culturing flask, the cells were passaged into a new flask utilizing 0.05% trypsin–ethylenediamine tetra-acetic acid (EDTA). For the experiments, BV2 cells were seeded in DMEM with 5% HI FBS and 1% P/S. 24–48 hours later, the cells were stimulated with 100 ng ml−1 (300 EU ml−1 ) of LPS for 3 hours to allow the cells to enter a pro-inflammatory (M1) phenotype to simulate the neuroinflammatory environment present in many neurological diseases. 

The cells were then co-treated with LPS and either free Tesa or D-Tesa at varying concentrations for 48 h and then the supernatant and cells were collected for processing. Cells never treated with LPS (No LPS) and cells treated with LPS-only at all times (LPS-only) served as the control groups. Free Tesa and D-Tesa stock solutions were sterilized utilizing poly(ether sulfones) 0.2 µm filters. 

D-Tesa was soluble in the cell media. Tesa was solubilized by utilizing dimethyl sulfoxide (DMSO) at less than 0.1% (v/v) final concentration. Cytotoxicity, nitric oxide assay, and TNF-α ELISA. For the cytotoxicity assay, cells were treated as described above in a 96-well plate, and then cytotoxicity of free Tesa and D-Tesa was assessed by MTT assay following the manufacturer's instructions. 

For the nitric oxide assay, cells were treated as described above in 12-well plates, and then supernatants from treated cells were collected and immediately nitric oxide levels were quantified by following the manufacturer's protocol for the Griess Reagent. 

For the TNF-α ELISA, cells were treated as described above in 12-well plates, and then supernatants from treated cells were collected and stored at −80 °C until ready for further processing. 

Then samples were thawed on ice and the TNF-α ELISA was run by following the manufacturer's protocol. For these studies, both D-Tesa and free Tesa were sonicated and vortexed until they were both completely soluble. To solubilize free Tesa, it was first solubilized in DMSO before being diluted, where the final concentration of DMSO was under 0.1% (v/v) for all free Tesa samples. 

Since D-Tesa was soluble in cell culture media, DMSO was not added to those samples. For all in vitro studies, an equivalent amount of free or conjugated drug was applied to cells in both the free Tesa and D-Tesa groups. 

Quantitative real-time PCR (qRT-PCR). Cells were treated as described above in 12-well plates, and after collecting the supernatant, cells were collected in Invitrogen™ TRIzol™ Reagent (from Fisher Scientific) and RNA was extracted by following the manufacturer's protocol. The concentration and the purity of the resulting RNA were analyzed utilizing Nanodrop. 

Equivalent amounts of RNA from each sample were converted to cDNA by following the manufacturer's protocol for the High Capacity cDNA Reverse Transcription Kit (from Applied Biosystems by Thermo Fisher Scientific). 

The resulting cDNA was utilized for qRT-PCR analysis utilizing the FAST-SYBR green reagent and by following the manufacturer's protocol. Ct values were calculated by the machine and data was analyzed utilizing the 2−ΔΔCt method. 

For each different marker, the ΔΔCt value was calculated by subtracting the ΔCt for the No LPS group from the ΔCt for each sample. The ΔCt value was the Ct value for the gene of interest minus the Ct for GAPDH for each given sample. 

Once the 2−ΔΔCt was calculated for all groups, they were then all normalized to the No LPS group, thereby giving the No LPS group a relative expression level of 1.0 for all markers. The forward and backward primer sequences used for qRT-PCR are shown in the below table. All sequences are written from 5′→3′.

Additionally, commercially available primers were purchased from BIORAD for iNOS/Nos2 (qmmuCID0023087), TLR4 (qmmuCID0023548), CD206/Mrc1 (qmmuCID0012670), TGF-β1 (qmmuCID0017320), IL-10 (qmmuCID0015452), SOCS1 (qmmuced0024846), CD36 (qmmucid0014852), Ide (qmmuced0049796), MMP9 (qmmucid0021296), CCL1 (qmmuced0038249), TLR8 (qmmuced0039837), CD86 (qmmucid0006086), STAT6 (qmmucid0006404), and PPARγ (qmmucid0018821). Phagocytosis assay. 

The impact of free Tesa and D-Tesa on phagocytosis of β-amyloid was determined using a previously reported method.47 Briefly, HiLyte™ Flour 488-labeled β-amyloid1–42 (from Anaspec Inc.) was dissolved in DMSO (final DMSO concentration under 0.5% (v/v)) and diluted to 5 µg ml−1 in PBS. 

After treating the cells with Tesa and D-Tesa with the treatment scheme outlined above, the β-amyloid solution was applied to the cells for 2 hours. Then, the cells were washed thrice with Hank's Balanced Salt Solution with divalent cations (calcium and magnesium), removed from the wells using trypsin, and resuspended in FACS buffer (from Invitrogen). 

Samples were stored on ice, and immediately were run on a Sony Cell Sorter SH800 flow cytometry machine, and data was analyzed with the associated software. 

The gate was set using cells not treated with fluorescent β-amyloid, and the results shown are the percent of cells from each group that exhibited fluorescence above the background fluorescence. 

The mean fluorescent intensity (MFA) of HiLyte™ 488-labeled β-amyloid was also reported for each group. This assay was performed in duplicate. Statistics. All data shown is the result of three separate experiments, each performed in triplicate unless otherwise noted. GraphPad Prism 5 and Microsoft Excel were used to perform statistics. 

Two-tailed, paired Student's t-tests, with Bonferroni Correction, were performed. Grubbs' test was used to determine outliers. GraphPad Prism 5 for Windows was used to plot data (San Diego, CA). The data shown is the average + SEM.

Results and discussion

Synthesis and characterization of D-Tesa

To allow for targeted, intracellular delivery to activated microglial cells in the brain, Tesa was covalently conjugated on the surface of G4-PAMAM-OH with cleavable ester bonds between the drug and the dendrimer-linker (Fig. 1). 

In the first step, Tesa (1) was reacted with tetramethylene glycol azide (TEG-azide) (2) to produce Tesa-TEG-azide (3), which has an ester linkage between the drug and linker (Fig. 1A). 

The HPLC of compound (3) showed a retention time of 13.4 minutes with purity greater than 99% (Fig. S1B†). The mass spectrum of (3) showed a peak at 610.13 [M + 1]+ corresponding to the Tesa-TEG-azide molecular weight further confirming the formation of the product (Fig. S2†). Separately, G4-PAMAM-OH dendrimer (4) was reacted with 5-hexynoic acid using an esterification reaction to produce D-YNE (5) (Fig. 1B). 

Lastly, Tesa-TEG-azide (3) and D-YNE (5) were reacted by the highly efficient copper-catalyzed azide-alkyne cycloaddition (CuAAC) click reaction to produce D-Tesa (6) (Fig. 1B).48 The 1 H NMR confirmed the successful synthesis of all intermediates and the final product; D-Tesa (6) contained the characteristic peaks of dendrimer and druglinker protons demonstrating the successful synthesis of D-Tesa (Fig. 2A). 

NMR of the final D-Tesa conjugate revealed that an average of ten molecules of Tesa were attached to each dendrimer. HPLC confirmed successful covalent conjugation, as D-Tesa exhibited a shift from both the D-YNE and TesaTEG-azide (Fig. 2B and S1†). 

Tesa has poor aqueous solubility, estimated to be 0.0035 mg mL−1. 49 The conjugation of Tesa on hydroxyl dendrimer improved its water solubility several orders of magnitude from this estimate. D-Tesa was solubility in water at 22 mg mL−1, and since Tesa comprises ∼19% of the mass of D-Tesa, 4.2 mg ml−1 equivalent of Tesa was solubilized (Fig. 3A). 

The increased solubility afforded by the dendrimer increases the ease of formulation and removes the need for potentially toxic excipients.50 These benefits are all in addition to the dendrimer's superior ability to deliver free drugs across the BBB to microglia in vivo, and potentially reduce the dose needed to achieve therapeutic effect.18–28 Lastly, D-Tesa had an average size of 7.75 ± 0.29 nm (Fig. S3†), and a zeta-potential of 2.86 ± 0.38 mV (N = 5 and N = 3 measurement, respectively).

Drug release

We designed D-Tesa to release Tesa intracellularly within the low pH and high esterase concentration environment in endosomes/lysosomes of activated microglia. We have previously reported that the PAMAM dendrimers mainly enter the cells via fluid-phase endocytosis, and the vesicles that contain the conjugates transform into lysosomes.51 

We incubated D-Tesa at 37 °C in sodium citrate buffer (pH 5.5) in the presence of esterases to mimic the conditions of lysosomes, as performed previously.52–54 We also investigated release under conditions that mimic plasma conditions (phosphate-buffered saline [PBS] buffer, pH 7.4). 

Under plasma conditions, only about 1.5% of Tesa is released after 48 hours, and less than 20% of Tesa is released by day 25 suggesting the plasma stability of the conjugate (Fig. 3B). 

Under lysosomal conditions, about 60% of Tesa is released from D-Tesa within the first 48 hours, and within 19 days, nearly 100% of Tesa is released. These results demonstrate that D-Tesa provides sustained, triggered release of the free drug for the first two weeks incubated under physiologically relevant, lysosomal conditions. 

Additionally, the minimal release of Tesa in the simulated plasma conditions ( pH 7.4 group) over the first 48 hours is significant, since this is the typical G4-PAMAM-OH circulation time before kidney clearance, so minimal Tesa will likely be released from the conjugates before they reach the microglia.24

D-Tesa decreased expression of M1 markers and increased M2 markers

To evaluate the ability of D-Tesa to induce an M1 to M2 phenotype shift, we evaluated D-Tesa's ability to alter the expression of M1 and M2 markers in vitro using a murine microglial cell line (BV2). 

BV2 cells were created by immortalizing murine microglial cells and were shown to be a suitable alternative to primary microglia cells to study the microglial inflammatory response.55,56 In our experiments, to mimic the neuroinflammatory environment present in multiple neurological diseases, we pretreated the microglia with 100 ng ml−1 (300 endotoxin units (EU) per ml) LPS for 3 hours. 

Then, we co-treated the cells with LPS and either free Tesa or D-Tesa for 48 hours, and then collected the samples. We treated the cells at concentrations of 1.5, 15, and 150 µM free Tesa or D-Tesa, on an equivalent drug basis. 

The MTT assay demonstrated that free Tesa or D-Tesa at these concentrations did not cause cytotoxicity (Fig. S4†). An initial dose-finding study was done with Tesa and D-Tesa to determine the most effective dose of Tesa and D-Tesa. 

Treatment with 1.5 and 15 µM free Tesa and D-Tesa did not alter the secretion of nitric oxide or TNF-α, as determined by the Griess Reagent and a TNF-α ELISA, respectively (Fig. S5†). Based on these results, we did not analyze these lower concentrations in our qRT-PCR assays.

In many neurodegenerative diseases, one major neurotoxic function of proinflammatory, M1 microglia is their secretion of reactive oxygen species (e.g. nitric oxide) that directly kill neurons.57,58 Subsequently, it has been postulated that one potential therapeutic strategy would be to decrease the secretion of these molecules. 

We evaluated D-Tesa's ability to achieve this effect. After LPS stimulation, D-Tesa decreased secreted nitric oxide and inducible nitric oxide synthase (iNOS) mRNA levels 3.7-fold (p < 0.0001) and 2-fold (p = 0.011), respectively, compared to cells only treated with LPS (LPS-only) (Fig. 4A and B). 

On the other hand, free Tesa decreased the secreted nitric oxide more moderately (1.4-fold, p = 0.004) and did not lead to a significant decrease in the iNOS mRNA expression (Fig. 4A and B). 

D-Tesa was much more effective than free Tesa in suppressing nitric oxide secretion. Next, depending on if microglia are in an M1 or M2 phenotype, microglia upregulate either iNOS or Arginase 1 (Arg1) to metabolize L-arginine to produce nitric oxide for their M1 pathogen-killing response, or ornithine and urea for the M2 wound healing response, respectively.59 

Consistent with its downregulation of iNOS, D-Tesa increased Arg1 mRNA levels 2-fold (p = 0.011) compared to the LPS-only control, while free Tesa did not increase expression significantly (p = 0.40) (Fig. 4C). 

D-Tesa, and to a lesser extent free Tesa, switched microglia from releasing cytotoxic nitric oxide to metabolizing L-arginine for wound healing, which has implications in reducing and potentially reversing, the neurotoxicity caused by microglia in neurodegeneration.5–7,10 

The downregulation of iNOS and secreted nitric oxide levels with D-Tesa treatment is by previous work that demonstrated PPARγ's natural ligand (15-deoxy-Δ12,14-prostaglandin J2) decreased iNOS and nitric oxide expression and secretion in LPS treated primary microglia.60 

IL-10, IL-4, and TGF-β1 are all cytokines that are secreted by alternatively activated M2 microglia that can induce a neuroprotective, anti-inflammatory environment in the brain.58,61,62 Towards this end, D-Tesa increased the expression of IL-10 5.5-fold (p = 0.011) and IL-4 8.2-fold (p = 0.013) compared to the LPS-only control (Fig. 4D and E). 

Free Tesa did not significantly increase either IL-10 or IL-4 levels, although the averages were 1.7-fold (p = 0.066) and 2.4-fold (p = 0.11) higher, respectively, for Tesa-treated microglia compared to LPS-only-controls (Fig. 4D and E). Additionally, D-Tesa increased the expression of TGF-β1 2.3-fold (p = 0.015) and free Tesa did not significantly change expression (Fig. 4F). 

Thus, D-Tesa induces the secretion of anti-inflammatory cytokines after LPS treatment of microglia, which can mitigate the neurotoxic, pro-inflammatory environment present in neurodegenerative diseases.5–7,10

D-Tesa-induced expression of M2-subtype markers

CD206, Ccl1, and TLR8 are specific markers for the M2a, M2b, and M2c microglia subtypes, respectively.58 D-Tesa upregulated CD206 expression 4.5-fold (p < 0.001), Ccl1 3.5-fold (p < 0.005), and TLR8 5-fold (p < 0.01), while free Tesa did not significantly increase expression of any of these three markers (Fig. 5A–C). 

These data show that D-Tesa-treated microglia demonstrate significant upregulation of markers of all three M2 subtypes, which agrees with the understanding that microglia phenotype is plastic and not binary.63–65 

The M1/M2 nomenclature oversimplifies the complexity that microglia can exist in a spectrum of activation states; along this continuum, three distinct M2 phenotypes (M2a, M2b, and M2c) have been characterized, and D-Tesa induces expression of markers consistent with each of these states.58,61,63,66,67 M2a microglia are involved in increased phagocytosis of pathogenic proteins by upregulating scavenger receptors, tissue repair, and anti-inflammatory actions. 

M2b microglia are like M1 microglia in that they express IL-1β, TNF-α, and IL-6; however, M2b are distinct from M1 microglia in that they express IL-10 at high levels and downregulate iNOS expression. M2b macrophages and microglia stimulate Th2 T-cells, which is indicative of one of the roles of M2b in inducing an anti-inflammatory response. 

M2c microglia are involved in wound healing, tissue remodeling, iron sequestration, and STAT3 activation that reduces proinflammatory signaling.58,61,63,66 The PPARγ activity of Tesa stimulates a feedforward loop that results in increased expression of PPARγ and its upstream signaling protein (STAT6), which are both M2a markers.58 D-Tesa increased the expression of PPARγ 2.3-fold (p = 0.0036) and STAT6 3.4-fold (p < 0.001), while free Tesa increased STAT6 1.8-fold (p = 0.011) without a significant increase in PPARγ expression (1.58-fold increase, p = 0.17) (Fig. 5D and E). 

The increased expression of STAT6 and PPARγ results in antiinflammatory gene production and inhibition of pro-inflammatory signals by inhibiting NF-κB activity. Since M2a microglia can be induced by treating microglia with IL-4, which signals through STAT6 and PPARγ, the upregulation of these two downstream signaling proteins can result in feedforward M2a polarization.68 

In our assays, LPS activation increased IL-6, TNF-α, and IL-1β levels, and treatment with D-Tesa further upregulated the expression of these cytokines (Fig. S6A–D†), which is likely due to D-Tesa inducing the M2b phenotype (Fig. 5B). 

Free Tesa increased the expression of IL-6 and did not significantly change TNF-α and IL-1β expression (Fig. S6A–D†). While these cytokines are secreted by M1 microglia, M2b microglia also express these cytokines.58,61 CD86 is a marker of both M1 and M2b microglia, and is increased with D-Tesa treatment (Fig. S6E†). 

Additionally, the suppressor of cytokine signaling (SOCS) is a family of intracellular proteins that regulate the phenotypic polarization of microglia by suppressing multiple signaling pathways activated by cytokines.69 SOCS1 is one member of this family, and it has been shown to inhibit LPS/ TLR4 mediated NF-κB and JAK2 signaling, thereby reducing the amount of TNF-α, IL-1β, and IL-6 released by M1 microglia through the LPS/TLR4 pathway. 

D-Tesa upregulates expression of SOCS1 compared to LPS-only control (Fig. S6F†), suggesting that the increase in IL-6, TNF-α and IL-1β exhibited by D-Tesa could be due to induction of the M2b phenotype and not via enhancement of the LPS/TLR4, M1-inducing phenotype. 

Moreover, LPS signals through TLR4 and to prevent excessive cellular activation, microglia possess a negative feedback mechanism whereby TLR4 activation leads to decreased expression of TLR4.70 

Treatment with free Tesa and D-Tesa increased TLR4 levels 3.6-fold (p < 0.001) and 3.7-fold (p < 0.001), respectively (Fig. 5F), suggesting that Tesa and D-Tesa altered the typical LPS/TRL4 negative feedback signaling pathway.

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