Phagocytosis of Polymeric Nanoparticles Aided Activation of Macrophages to Induce Atherosclerotic Plaques in Apoe-/- Mice

The unique physiochemical properties of nanomaterials have been widely used in drug delivery systems and diagnostic contrast agents. The safety issues of biomaterials with exceptional biocompatibility and hemo-compatibility have also received extensive attention at the nanoscale, especially in cardiovascular disease. Therefore, we conducted a study of the effects of poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) on the development of aortic atherosclerotic plaques in ApoE -/- mice. The particle size of PLGA NPs was 92.69 ± 3.1 nm and the zeta potential were -31.6 ± 2.8 mV, with good blood compatibility. ApoE -/- mice were continuously injected with PLGA NPs intravenously for 4 and 12 weeks. Examination of oil red O stained aortic sinuses conrmed that the accumulation of PLGA NPs promoted the formation of atherosclerotic plaques and increasing the expression of associated inammatory factors, such as TNF-α, IL-6, and IL-10. The combined exposure of ox-LDL and PLGA NPs accelerated the conversion of macrophages to foam cells. Our results highlight the potential risk for PLGA NPs in vivo and further understanding the interaction between PLGA NPs and the atherosclerotic plaques, which we should consider in future nanomaterial design and pay more attention to the process of using nano-medicines on cardiovascular diseases.


Introduction
Nanoparticles are ultra ne particles with at least one dimension < 100 nm in size. Nanoparticles possess physical properties, such as macroscopic quantum tunneling, nano size and surface effects, which make them desirable for applications in medicine, materials science and biology [1,2] . Nanoparticles may accumulate within the human body through inhalation, ingestion, skin absorption, and injection [3,4] . The biological safety of nanomaterials has received widespread attention due to their special properties including small size and high speci c surface area [5] . An accumulation of nanoparticles in the lungs will result in passage through the alveolar epithelial cells or lymphatic system into the circulation to be redistributed throughout the body. Therefore, nanoparticles may have a signi cant impact on the cardiovascular system [6,7,8] . Studies have shown that atmospheric particulate matter (PM), composed mainly of nanoparticles, increases cardiovascular disease morbidity and mortality. The cardiovascular system is now recognized as one of the important targets of nano-toxicity [9,10] .
Nanoparticles have more serious biological toxicity and more complex toxicological mechanisms than common chemicals. Studies have shown that nanoparticles can damage vascular endothelial cells and trigger an in ammatory reaction, which in turn may cause platelet aggregation and thrombosis [11,12,13] . Therefore, nanoparticles may be an important risk factor for cardiovascular diseases such as atherosclerosis (AS) [14,15,16] . Medical research has shown that an in ammatory response is an important pathological mechanism for the development of AS, which can cause endothelial cell dysfunction. When nanoparticles adhere to the cell membrane of endothelial cells, they induce the expression and release of in ammatory factors (such as IL-6, IL-8, and TNF-α) [17,18] . Nanoparticles also may promote adhesion of monocytes to endothelial cells, further differentiation into macrophages, and penetration of the blood vessel walls, leading to AS [19] . Accumulating lipids in unstable plaques further exacerbate the in ammatory response, thereby promoting the development of AS [20] . Nanoparticles induce in ammatory reactions, impair lysosomal function, promote abnormal hydrolysis of triglycerides, and lead to an increased lipid load in macrophages, which in turn induces foam cell formation. In the in ammatory state, vascular smooth muscle cells, dendritic cells, and mast cells also may produce foam cells. Nanoparticles activate neutrophil elastase, which degrades elastin and various collagens, damaging vascular endothelial cells (VECs) and basement membranes [21] . Nanoparticles interacts with the complement system, coagulation functions and brinolysis, which aggravates the formation and instability of arterial plaque [22] .
Pober and Cotran examined the relationship between AS and hemodynamics and proposed the shear stress theory to describe the onset of AS [23] . At present, it is well known that atherosclerotic lesions are mainly concentrated in sites with obvious changes in blood ow. In the early stage of plaque development, endothelial cells on the arterial wall attract monocytes, which transform into macrophages and then absorb large amounts of oxidized low-density lipoprotein (ox-LDL) to transform into foam cells.
Therefore, atherosclerotic lesions are complex environments containing lipids, cholesterol crystals, in ammatory cells and secreted cytokines. However, when nanoparticles enter the body, they tend to accumulate in areas of infarction. Studies have found that nanoparticles with longer blood circulation times are more likely to cross the endothelial barrier and accumulate in the infarcted area due to the destruction of the endothelial barrier caused by ischemic injury. This mechanism is similar to enhanced permeability and retention [24,25,26] .
In 2007, Dawson and Linse jointly proposed the concept of a protein corona, which led researchers to study the fate of nanoparticles in vivo [27] . Nanoparticles will rapidly adsorb proteins forming what is known as the protein "corona" after enters the circulation system. The structure and composition of the protein corona depends on the synthetic identity of the nanomaterial, this would in uence the biological identity of nanoparticles [28] . The physiological functions of various proteins that comprise the protein corona generally involve lipid transport, coagulation, complement activation, pathogen recognition, or ion transport [29] . Understanding nanoparticle-protein interactions is a crucial issue in the development of targeted nanomaterial delivery in vivo [30] . The physiological environment to which nanoparticles are rst exposed after intravenous administration is the blood stream, and the cell-free portion of the blood (plasma) contains more than 1000 proteins. These proteins potentially interact with nanoparticles to exert different physiological functions, such as recognition by macrophages, causing in ammatory reactions, thrombosis and allergic reactions [31] .
The safety issues of biomaterials with exceptional biocompatibility and hemo-compatibility have also received extensive attention at the nanoscale. Numerous nanomaterials have been widely used as drug delivery systems and diagnostic contrast agents in treating cardiovascular diseases. This article focuses on polymer nanoparticles and explores their bioactivity impact on the development of AS and physiochemical mechanisms. Therefore, we conducted a study of the effects of poly (lactic-co-glycolic acid) (PLGA) nanoparticles, which are widely used in a variety of Food and Drug Administration (FDA) approved therapeutic devices, on the development of aortic atherosclerotic plaques in ApoE −/− mice.  [32] . Brie y, PLGA (100 mg) was dissolved into 10 mL dimethyl sulfoxide (DMSO). The mixture (2 mL) was precipitated by adding dropwise into 6 mL deionized water with gentle stirring, and further dialyzed using dialysis bag (molecular weight cutoff, MWCO: 3500 Da) against water to remove the free DMSO. The volume was adjusted to 10 mL to obtain PLGA nanoparticles solution (2 mg/mL), collected and preserved at 4 °C. The blood was collected with heparin from the eyeball and stored at 4 °C. The collected blood of the mice was statically placed at 4 ℃ and after 6 hours, centrifuged at 3000 rmp for 15 min to obtain serum. To obtain PLGA + protein corona (PC), 1 mL of solution of PLGA (2 mg/mL) was incubated with 5 µg of serum at 37 °C for 30 min.

Characterization of PLGA Nanoparticles and PLGA + PC
The aqueous phase diameter, size and zeta potential of PLGA nanoparticles and PLGA + PC were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS unit (Nano ZS 90, Malvern, UK) with He-Ne laser (λ = 633 nm) at a scattering angle of 90° at 25 °C. A drop of PLGA nanoparticles or PLGA + PC solution at a concentration of 100 µg/mL was dropped onto a copper mesh (200 mesh), and air-dried naturally. Then stained by 2% phosphotungstic acid for 3 min, air-drying. Subsequently, the morphology of PLGA nanoparticles and PLGA + PC were visually observed using a transmission electron microscope (TEM, Zeiss Germany, Optima 75 KV).

Determination of serum protein adsorbed by PLGA nanoparticles
Determination of serum protein adsorbed by PLGA nanoparticles was carried out according to the standards BCA protein assay kit. PLGA nanoparticles were incubated with 2 mL mouse serum for 30 min, the mixture was centrifuged at 3000 rpm for 20 min, and then the supernatant was collected to determine protein content by the BCA kit. Meantime, untreated serum was the control group.

Hemolysis rate of PLGA nanoparticles
The collected fresh vein blood from healthy rabbits were mixed with sodium citrate in a 9:1 ratio to prevent coagulation. Four microliters anticoagulation was added with 5 mL 0.9% sodium chloride (NaCl) injection to dilute. The rst group as a negative control contained 5% glucose, second group as a positive control contained only deionized water and the last group as experimental group consisting of three subgroups, 2 mg/ml PLGA nanoparticles, 1 mg/mL PLGA nanoparticles and isotonic solution that contained a mixture of PLGA nanoparticles and 5% glucose.
Then, each group above solution (200 µL) was incubated with 0.9% NaCl (2.8 mL) at 37 °C for 30 min in a water bath. The mixture was added 60 µL of diluted anticoagulant solution, after second incubation at 37 °C for 60 min in a water bath and centrifugation 3000 rpm for 10 min, the supernatant was collected and measured absorbance (OD) at 545 nm spectrophotometer.
To quantify percent hemolysis, the hemoglobin concentration measured was divided by the hemoglobin concentration of the diluted blood solution as described by the following equation: Hemolysis rate exceeding 5% is considered hemolysis.

Effects of PLGA nanoparticles on APTT, PT, TT and Fbg
The blood was collected from healthy rabbits, mixed with sodium citrate in a 9:1 ratio to prevent coagulation. Brie y method, the mixture was centrifuged at 3000 rpm for 10 minutes, then the top supernatant was collected as platelet-poor-plasma (PPP). The solution (10 µL) was incubated with PPP (300 µL) at 37 °C for 30 min. Finally, the incubated mixture was conducted evaluation of effects of nanoparticles on plasma coagulation include activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and brinogen (Fbg) levels using a fully automated coagulation apparatus.
2.2.6. Activation of platelet α-granule membrane protein (GMP-140) by PLGA nanoparticles Rabbit venous blood was anticoagulated with sodium citrate in a ratio of 9:1, centrifuged at 1000 rpm for 10 min, and the supernatant was collected to obtain as platelet-rich plasma (PRP). Then, the PLGA nanoparticles solution (10 µL) was incubated with PRP (300 µL) at 37 °C for 30 min, the incubated mixture was tested with ELISA kit.

Animal experiment
Army Medical University Animal Experiment Ethics Committee and Authority approved all animal procedures for Animal Protection. ApoE −/− and C57 mice were used in this study in accordance with the guidelines of the Chinese Animal Care and Use Committee standards.
The experimental animals were fed with an adaptive feeding week. As shown in Table 1, twenty C57 mice were randomized into two groups, and forty ApoE −/− mice were randomized into four groups (10 mice per group). Then, the mice were subjected to the different treatments for 12 weeks. PLGA nanoparticles was injected at a dose of 10 mg/kg and the frequency of injection was once every two days. The control group was injected with an equal volume of 5% glucose isotonic solution. During the experiment, all the experimental animals were fed with high-fat diet, freely drinking water.
After treatment for 12 weeks the serum, from the mice were harvested. Total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) were detected using an automated biochemical analyzer.

Analysis of Atherosclerotic Plaques
ORO staining of the cross-sections of the aortic roots was performed as previously described [33] . After treatment for 12 weeks the aortas, from the heart to the iliac bifurcation, from the mice were harvested.
Aortas were xed by perfusion with paraformaldehyde (4% in PBS). After removing the periadventitial tissue, aortas were dissected longitudinally, and then stained with oil red O (ORO) to quantify the plaque area. The extent of atherosclerotic plaque at the aortic root was also determined by ORO staining.
2.2.9. Histology and Immunohistochemistry Staining of the Aortic Root Histology and Immuno uorescence staining of the cross-sections of the aortic roots was performed as previously described [33] . The aortic roots were xed with paraformaldehyde (4% in PBS) for 1 h, and then prepared to para n sections. After dewaxing, Masson's trichrome and Hematoxylin and eosin (H&E) staining were used to observe the collagen, lipid core and some plaque ruptures. For immunohistochemistry analysis, the activity of the endogenous peroxidase was inhibited by immersion into 3% hydrogen peroxide and 100% methanol for 20 min. Then, the sections were blocked with 5% bovine serum albumin in PBS for 60 min. Antibodies to TNF-α, IL-6, IL-10. Sections of the main organs including heart, liver, spleen, lung, and kidney were also analyzed by H&E staining.
2.2.10. PLGA nanoparticles co-localization with the in ammatory plaque site DiI@PLGA nanoparticles solution (2 mg/mL) was prepared by the similar preparation method of PLGA nanoparticles described in previous part, mixing DiI solution (1 mM, 15 µL) and PLGA (15 mg) dissolved in 1 mL DMSO. Then the DMSO is removed.The volume was adjusted to 7.5 ml to obtain 2 mg/ml DiI@PLGA nanoparticles solution. C57 mice were control group, and ApoE −/− mice were experimental group (3 mice per group). The experimental animals were fed with HFD for 12 weeks, then DiI@PLGA (200 µL) was injected through the tail vein. After 24 h, mice were euthanized, perfused with PBS containing 4% paraformaldehyde and heparin sodium, and the heart was isolated. Immuno uorescence staining of the cross-sections of the aortic roots was performed as previously described [33] . The frozen sections of carotid roots were incubated with 5% serum. Then, the sections were incubated with anti-CD68 and CD11b antibody overnight at 4 °C, followed by Donkey anti-rabbit IgG H&L for 2 h at room temperature. Samples were stained with DAPI to show the cell nucleus. The sections were observed by the confocal laser scanning microscopy (SP8, Leica, Germany). and 48 h), 20 µL of MTS assay solution was added to each well, and incubation was continued for 1 h. The absorbance (OD) of each well was measured at a wavelength of 490 nm using a microplate reader, and repeated six times at each time point. Cell viability was obtained by the following equation: Cell viability = (ODT -ODB) / (ODC -ODB) × 100% (2) ODT indicates the absorbance of the experimental group; ODC indicates the absorbance of the control group; and ODB indicates the blank absorbance.

Characterization and blood compatibility of PLGA nanoparticles
The DLS results showed that the diameters of PLGA and PLGA + PC in the water phase were 92.7 ± 3.1 nm and 123.8 ± 5.3 nm, respectively. The Zeta potentials were − 31.6 ± 2.8 mV and − 12.0 ± 3.5 mV, respectively ( Fig. 1C and D). PLGA + PC particles in an aqueous solution were larger than PLGA nanoparticles alone and were less stable. Under dry conditions, TEM (Fig. 1Aand B) showed that PLGA and PLGA + PC were spherical particles consistent in size with the DLS measurements. Serum protein concentrations, measured by a bicinchoninic acid (BCA) protein assay, decreased after incubation and con rmed the presence of a protein corona on the PLGA nanoparticles due to the formation of PLGA + PC (Fig. 1E).
As shown in Fig. 1F, the hemolysis rates of 1 mg/ml PLGA nanoparticles, 2 mg/ml PLGA nanoparticles, and PLGA + Glu were 2.96 ± 0.10%, 3.24 ± 0.14%, and 2.95 ± 0.29%, respectively. Hemolysis rates in three experimental groups were less than 5%, according to the national standard for the hemolysis rate of medical biological materials. There were no signi cant differences in GMP-140, APTT, PT, TT and Fbg values between the negative control group and the experimental groups, indicating that PLGA nanoparticles had no signi cant effect on coagulation and did not induce platelet activation ( Fig. 1G and H). Atherosclerotic plaque formation was detected in the aortic sinus, with a large number of red fat granules accumulating in the ApoE −/− mice groups compared to the C57 control groups ( Fig. 2A). The areas of plaque and lipid deposition were severe in each PLGA nanoparticle injection group, 23.24 ± 0.8% vs. 16.99 ± 1.8%, 22.03 ± 1.4% vs. 16.95 ± 1.1% (Fig. 2D). These results suggest that the combination of PLGA nanoparticles and HFD promote the formation of atherosclerotic plaques in ApoE −/− mice.
Next, we examined the composition of atherosclerotic plaques in aortic root sections by immunohistochemistry staining. Hematoxylin and eosin (H&E) staining of aortic sinuses in ApoE −/− mice revealed extensive plaque formation and severe stenosis in the lumen (Fig. 2B). Thickening of the intima and irregular bulging in the lumen, an increase in cell hyperplasia and atherosclerotic plaque formation below the intima were observed. The structure of the arterial wall was disordered, the media was contracted and atrophied, and ruptured plaques with lipid cores were apparent. The collagen arrangement in the aortic sinuses of each group of ApoE −/− mice was disordered, and collagen bers in the long-term injection of PLGA nanoparticles group were the most scattered. In addition, blue staining was observed at the edges of the plaques (Fig. 2C).
Hyperlipidemia is known to play an important role in the process of plaque formation. Serum TC, TG, LDL-C and HDL-C were signi cantly higher in the PLGA nanoparticle groups compared with the HFD ApoE −/− mice group (Fig. 2E). Serum TC in the ApoE −/− mice group was approximately 7-fold higher than in the C57 wild type group; TG was nearly 50-fold higher, and LDL-C and HDL-C were 4-fold higher. However, with the exception of HDL-C, there were no signi cant differences in other lipid pro les between the HFD and PLGA nanoparticle groups. These observations indicate that PLGA nanoparticles do not cause signi cant changes in blood serum lipids during the formation of AS in ApoE −/− mice.

PLGA nanoparticle co-localization within the in ammatory plaque site
Atherosclerosis is characterized by plaque formation and chronic in ammation of the arterial wall. We detected co-localization of PLGA nanoparticles at sites of in ammation by immuno uorescence staining. DiI@PLGA nanoparticles were detected in arterial plaque 24 h after injection into the blood stream. CD68 is a marker for a wide range of macrophages that can effectively label monocytes and macrophages (Fig. 3A). CD11b can label neutrophils, monocytes, and macrophages, and function in adhesion and signal transduction during the in ammatory response (Fig. 3B). The results showed that the PLGA nanoparticles co-localized in the plaque sites with CD68 and CD11b positive cells, indicating that the PLGA nanoparticles had a close relationship with AS in ammation, especially the macrophages. We speculate that the nanoparticles co-localized in the plaque sites because they were detected as foreign objects that stimulated macrophages, causing an in ammatory reaction, and increasing phagocytosis inside the plaque.

PLGA nanoparticles cause in ammatory factor release
After con rming that PLGA nanoparticles co-localized with in ammatory cells in atherosclerotic plaques, we investigated the effects of PLGA nanoparticle injection on the expression of the in ammatory factors TNF-α, IL-6 and IL-10 ( Fig. 4A). Positive expression of TNF-α and IL-6 was indicated by brown particles located mainly in the cytoplasm of endothelial and smooth muscle cells. Expression was pronounced in the plaques of the 12-week PLGA nanoparticle injection groups as indicated by dark brown staining. The expression of IL-10 was strongly positive at plaque sites in the ApoE −/− mice groups compared with that in the C57 mice groups, but no signi cant differences were found between each PLGA nanoparticle injection group and their respective controls (Fig. 4B). These observations indicate that under the in uence of a HFD, continuous long-term injection of PLGA nanoparticles can promote an in ammatory response in the plaques of ApoE −/− mice, and PLGA nanoparticles coupled with a HFD had a long-term synergistic effect on the production of AS lesions.

PLGA nanoparticles were phagocytized by macrophages and decreased cell viability
Macrophages are key contributors to the atherosclerotic process due to their in ammatory and phagocytosis inducing properties. The dynamic phagocytosis of nanoparticles by macrophages was investigated using DiI loaded nanoparticles and observed under confocal laser scanning microscopy (CLSM) and ow cytometry. Raw 264.7 cells began to phagocytize PLGA nanoparticles and PLGA + PC from 0.5 h and increased over time (Fig. 5A, B and C). The red uorescence in the PLGA + PC group was more evident in the nucleus compared to the PLGA nanoparticle group. Presumably, the protein corona on the PLGA nanoparticle surface was more likely to help the nanoparticles enter the nucleus (Fig. 5A). The amount of phagocytosis after treatment with nanoparticles for 0.5 h by DiI@PLGA and DiI@PLGA + PC was 35.8% and 2.57%, respectively; after 2 h, phagocytosis increased to 88.2% and 7.72%, respectively; then increased to 92.0% and 22.9% after 4 h, respectively ( Fig. 5B and C).
To con rm whether the accumulated PLGA nanoparticles would be phagocytized by macrophages and in uence their function, we conducted cell viability assays of Raw 264.7 macrophages co-cultured with different PLGA nanoparticle concentrations. The activity of Raw 264.7 cells decreased but remained greater than 60% using different concentrations of PLGA nanoparticles and PLGA + PC treated cells ( Fig. 5A and B). When the concentration was less than 100 µg/ml, we observed no signi cant effects on the viability of Raw 264.7 cells. However, when the concentration was greater than 200 µg/ml, the viability of Raw 264. Raw 264.7 cells at the same concentration ( Fig. 6A and B). Nanoparticles also increased the Raw 264.7 CE/TC values (%) at higher concentrations but showed no signi cant differences at concentrations less than 100 µg/mL (Fig. 6C). These ndings con rmed that PLGA nanoparticles and PLGA + PC will accelerate the transformation of Raw 264.7 macrophages into foam cells, and that PLGA + PC has a stronger effect than PLGA nanoparticles.

Discussion
Due to their unique physico-chemical characteristics, advantages of NPs in this context include their ability to easily penetrate across cell barriers, preferential accumulation in speci c orga-nelles and cells, and theranostic (both therapy and diagnostic) properties, as well as their capacity for ne tuning. Polymer nanoparticles are attracting attention due to high e ciency, long-term circulation characteristics, and metabolic discharge mechanisms that are superior to other biomaterials. These bene cial properties have resulted in the widespread use of polymer nanoparticles as drug delivery systems and diagnostic contrast agents for medical applications. Despite their good biocompatibility, there are also disadvantages of polymer biomaterials in nano scale, especially under pathological conditions and the interactions of NPs with living cells are complex and still far from fully understood [35] .This article focuses on polymer nanoparticles and explores their impact on the development of cardiovascular diseases such as AS and possible mechanisms of function.
Current research has shown that nanoparticles < 100 nm in size are easily absorbed by tissues [36] . PLGA nanoparticles prepared by dialysis were characterized by DLS and TEM and found to possess the expected size (nanoscale) and useful characteristics such as good dispersion, uniform size and spherical shape. In addition to the standard physical criteria, medical biomaterials must also exhibit a high degree of compatibility with the circulatory system. Therefore, we evaluated blood compatibility of PLGA nanoparticles from three aspects, hemolysis rate and coagulation function and platelet activation. The hemolysis rate of PLGA nanoparticles was < 5%, in accordance with international standards. The physiological anticoagulant function is mainly achieved through the joint action of the coagulation system, platelets and the brinolysis system [37] . APTT mainly re ects the activity and function of endogenous coagulation factors, PT represents the exogenous coagulation system, TT is the time for conversion of brinogen to brin, Fbg is the content of brinogen, and GMP-140 indicates the activation of platelets. Through the detection of these ve indicators, we found that the prepared PLGA nanoparticles did not have a signi cant impact on coagulation and had excellent blood compatibility.
In 2017, Miller et al. studied the effects of gold nanoparticles on cardiovascular disease, and discovered that red and purple particles accumulated in foam cells at sites of atherosclerotic plaque in ApoE −/− mice treated with gold nanoparticles [38] . Furthermore, gold nanoparticles could be detected in surgical specimens of carotid artery disease from patients at risk of stroke. Based on previous research, in this paper we investigated the effects of PLGA nanoparticles on atherosclerosis by administrating PLGA nanoparticles to ApoE −/− mice by intravenous injection. The ApoE −/− mice were fed a HFD and injected with PLGA nanoparticles for 12 weeks to investigate the effects of nanoparticles on the development of atherosclerosis. In a second experimental group, ApoE −/− mice were fed a HFD for 8 weeks to form atherosclerotic plaques, then PLGA nanoparticles were injected simultaneously for 4 weeks. Wild type C57 mice were used as a healthy vascular control group. We observed that PLGA nanoparticles caused a signi cant increase in plaque area and accumulated in the in ammatory sites during 4 weeks and 12 weeks of injection. In addition, PLGA nanoparticles promoted the activation of macrophages, secreting a large number of in ammatory factors, at sites of plaque formation. These in ammatory factors, which include the matrix metalloproteinases, are the major proteins that regulate the activity of in ammatory cells. Matrix metalloproteinases degrade the extracellular matrix and reduce the collagen and elastin content in the plaque lipid core. In the presence of atherosclerotic lesions, PLGA nanoparticles promote these pathological phenomena.
In wild-type C57 mice with no plaque formation, immuno-histochemical staining detected only small amounts of the pro-in ammatory factors TNF-α and IL-6 on the blood vessel walls and no expression of the anti-in ammatory factor IL-10. These observations indicated that in the absence of AS lesions (i.e., under normal physiological conditions) in mice, there was no signi cant in ammation and in ammatory factors were not activated and released. In the ApoE −/− mice group that could spontaneously form AS plaques, strong positive expression of the pro-in ammatory cytokines TNF-α and IL-6 could be clearly observed at plaque sites. After 12 weeks of injection of PLGA nanoparticles, TNF-α and IL-6 levels increased signi cantly compared to the control groups. At the same time, IL-10 anti-in ammatory factors also showed strong positive expression. Therefore, during the process of AS plaque formation, PLGA nanoparticles aggravate in ammatory reactions and anti-in ammatory protective effects, causing increased secretion and release of pro-in ammatory factors such as TNF-α and IL-6 and antiin ammatory factors such as IL-10 to avoid serious tissue damage. These results were consistent with recent studies regarding the relationships between in ammation and anti-in ammatory factors such as TNF-α/IL-10. Internal environmental stability is based on the dynamic balance between in ammatory and anti-in ammatory responses. When the in ammatory response dominates, tissues and cells will be damaged; whereas, a strong anti-in ammatory reaction will inhibit immune function [39] . We initially determined that PLGA nanoparticles enter the body as foreign objects, triggering an in ammatory response in ApoE −/− mice. These nanoparticles were then engulfed by macrophages, migrated to sites of in ammation and eventually aggravated the formation of plaque.
During the injection process, nanoparticles are rapidly coated with macromolecules forming a "protein crown or corona (PC)", which alters the size, aggregation state, surface charge and interfacial properties of the nanomaterials to create a biological identity that is distinct from its original synthetic identity. PLGA nanoparticles adsorbed a certain amount of proteins to form the PLGA + PC, with larger diameter and less stability, after incubation with mouse serum. Nanoparticles with protein coronas show completely different cell recognition or biological effects in vitro compared with in vivo [30] .
Macrophages are the most important in ammatory cells in the process of AS lesion formation, are important components of lipid plaques, and serve as an important source of foam cells [40] . Therefore, we studied the effects of PLGA nanoparticles on macrophages in vitro. According to the MTS assay results, the activity of Raw 264.7 cells decreased with increasing concentrations of PLGA nanoparticles and PLGA + PC. The presence of the protein corona inhibited the phagocytosis of PLGA nanoparticles by Raw 264.7 cells. Studies have shown that the role of the protein corona in biological systems can be divided into "opsonins" and "dysopsonins" [41] . Opsonins promote macrophage phagocytosis, while dysopsonins inhibit phagocytosis. The structure and composition of the corona depend on the synthetic identity of the nanomaterial, which includes the chemistry, topography and curvature of the nanomaterial. Polymer nanoparticles possess various chemical compositions, free residues and morphologies (such as spheres, rods, vesicles, tubules and lamellae), which provide them with more diverse synthetic identities. After PLGA nanoparticles enter the blood stream, the surface-adsorbed dysopsonins may be more abundant and more stable.
The physiological functions of proteins that comprise the protein corona include lipid transport, blood coagulation, complement activation, pathogen recognition and ion transport [42] . In the early stages of AS, ox-LDL acts as an in ammatory medium, promoting foam cell development and cholesterol-rich lipid core formation [43] . Cell ORO staining and CE/TC% suggested that PLGA nanoparticles and PLGA + PC accelerated the conversion of Raw 264.7 cells to foam cells and that PLGA + PC had a stronger effect than PLGA nanoparticles. Therefore, the protein corona absorbed on the surface of PLGA nanoparticles may possess a stronger atherogenic potential. This phenomenon may explain many existing inconsistencies between in vitro toxicity screening and in vivo studies, and necessitate a re-evaluation of the toxicity of polymer nanoparticles, even for polymer materials with good biocompatibility.