(L-474) The endocytosis of the macrophage foam cell to nanoparticles based on THP-1 model

Introduction: Oxidized low-density lipoprotein (oxLDL) is a modified form of low density lipoprotein (LDL). As a type of cholesterol carrying lipoprotein, it can be taken up by macrophages and results in the formation of foam cells that are seen in Type 2 diabetes. Thus, the formation of cells contributes to hyperglycemia and insulin resistance, both symptoms of diabetes [1]. Because of this, one of the possible therapies to treat
diabetes could be related to the reduction of foam cells. Metformin which is a type of biguanides drug. It can reduce foam cell production; however, metformin has poor solubility. Nanoparticle encapsulation can improve metformin uptake into macrophages and reduce the pro-inflammatory activation of foam cell formation. Polylactic-co-glycolic acid (PLGA) is being widely used due to its low toxicity and biocompatible and has been used in diabetic applications previously. Another possible nanoparticle is polystyrene which also has good versatility and stability. In this abstract, both nanoparticles are considered to test the ability of reduceing macrophage foam cells. We developed a THP-1 based model for studying the endocytosis of the macrophage foam cell to nanoparticles.

Material & Method:  Poly lactic-co-glycolic acid and metformin were purchased from Sigma-Aldrich. (St. Louis, MO, USA). Metformin-loaded PLGA particles are formed at a size around 100 nm by using microfluidics system from Sigma-Aldrich. (St. Louis, MO, USA). Cy5.5 amine is used to make fluorescent conjugated PLGA. NHS/EDC is treated with PLGA terminate with carboxylate group to conjugate with Cy5.5 amine. The experiment of treating PLGA particles to cells is not able to be done at this time point. Thus, polystyrene-fluorescent with a size of 100nm is used instead of PLGA particles. The goal of using polystyrene is to have a comparison about the uptake of nanoparticles between wild type and oxLDL-treated cells. A group of wild type cells were used to be the control blank cell. Human leukemia monocytic cells (THP-1) were trained with oxLDL (Figure 1). THP-1 is firstly incubated in RPMI with 10% FBS in 6-well-culture plate overnight. The monocytes are then incubated with oxLDL (1-10 μg) for 24 hours. PMA (10ng/ml) is used for the differentiation process after the incubation with oxLDL. After 7 days of incubation in the RPMI medium, LPS (10 ng/ml) is added. After 24 hours of incubation, cells and cell culture supernatant were collected to perform nanoparticles endocytosis experiment and detection of pro-inflammatory cytokines respectively. The formation of the foam cells is detected by Oil Red O. Flow cytometry is performed to observe the uptake of polystyrene and PLGA particles. Production of IL-8, TNF-α, IL-1β, and MCP-1 are measured by ELISA from supernatants.

Results & Discussions: Metformin-loaded PLGA was tested by DLS after the dialysis process. The average diameter is 104 nm and from the size distributaion graph, it can infer that the polydispersity index (PDI) has a low value which is 0.159 (Figure 2). The size of the PLGA nanoparticles is in the expected range. Brightfield images of THP-1 monocytes during the cell culture process reveal obvious morphological differences after adding PMA on Day 3. On Day 4, THP-1 monocytes transformed from a round or monocyte-like shape to a spread-out and flattened associated with macrophages (Figure 3.A). Furthermore, cell adhesion was also observed. However, the effect of adding LPS did not show any observable morphological changes on Day 12 (Figure 3.B). The oxLDL trained and LPS restimulated macrophages were treated with fluorescently labeled polystyrene nanoparticles (Figure 4). The percentage positive data is determined. The histograms calculated the percentage of cells that have a higher fluorescence than the control group removing auto-fluoresence from cells and determining cells which took up nanoparticles. With a higher number, the cells can take up more polystyrene particles. From figure 4.B, it can infer that the cells treated with 0 ug oxLDL would have the highest number. However, from the other three groups, they give a result that treated with more oxLDL will obtain a higher positive percentage. It can infer that oxLDL-treated could be a factor that affects the uptake of nanoparticles in cells. However, this data is only representative of one sample for each condition. There are no repetitions for experimental groups. Thus, no final conclusions can be determined from this data. Oil Red O staining was applied to OxLDL treated THP-1. Based on the fluorescence microscope, all the samples shows out highlight red particles which should represent foam cell. However, the result was not correlated with hypothesis that with more OxLDL more foam cell should be appeared. Based on the result, it shows out THP-1 macrophage with 0ug OxLDLD also has lipid.

Conclusions: From the images of cell culture, it is evident that THP-1 monocytes change into macrophages. However, Oil Red O staining process shows out that four group of THP-1 with different amount OxLDL all have lipid formed. There is no significant difference between four groups of cell. The cell culture process needs to be modified in the future experiment or different types of cell lines would be considered to be used. The flow cytometry result is not significant enough to have a certain conclusion on the effect of polymers to macrophage. Future work for it is to have more replication to determine significance. The next step for the research would be applying the metformin-loaded PLGA particles to macrophages and having ELISA test to the cells.