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 specific orga-nelles and cells, and theranostic (both therapy and diagnostic) properties, as well as their capacity for fine tuning. Polymer nanoparticles are attracting attention due to high efficiency, long-term circulation characteristics, and metabolic discharge mechanisms that are superior to other biomaterials. These beneficial 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 fibrinolysis system [37]. APTT mainly reflects the activity and function of endogenous coagulation factors, PT represents the exogenous coagulation system, TT is the time for conversion of fibrinogen to fibrin, Fbg is the content of fibrinogen, and GMP-140 indicates the activation of platelets. Through the detection of these five indicators, we found that the prepared PLGA nanoparticles did not have a significant 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 significant increase in plaque area and accumulated in the inflammatory sites during 4 weeks and 12 weeks of injection. In addition, PLGA nanoparticles promoted the activation of macrophages, secreting a large number of inflammatory factors, at sites of plaque formation. These inflammatory factors, which include the matrix metalloproteinases, are the major proteins that regulate the activity of inflammatory 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-inflammatory factors TNF-α and IL-6 on the blood vessel walls and no expression of the anti-inflammatory factor IL-10. These observations indicated that in the absence of AS lesions (i.e., under normal physiological conditions) in mice, there was no significant inflammation and inflammatory factors were not activated and released. In the ApoE−/− mice group that could spontaneously form AS plaques, strong positive expression of the pro-inflammatory 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 significantly compared to the control groups. At the same time, IL-10 anti-inflammatory factors also showed strong positive expression. Therefore, during the process of AS plaque formation, PLGA nanoparticles aggravate inflammatory reactions and anti-inflammatory protective effects, causing increased secretion and release of pro-inflammatory factors such as TNF-α and IL-6 and anti-inflammatory factors such as IL-10 to avoid serious tissue damage. These results were consistent with recent studies regarding the relationships between inflammation and anti-inflammatory factors such as TNF-α/IL-10. Internal environmental stability is based on the dynamic balance between inflammatory and anti-inflammatory responses. When the inflammatory response dominates, tissues and cells will be damaged; whereas, a strong anti-inflammatory reaction will inhibit immune function [39]. We initially determined that PLGA nanoparticles enter the body as foreign objects, triggering an inflammatory response in ApoE−/− mice. These nanoparticles were then engulfed by macrophages, migrated to sites of inflammation 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 inflammatory 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 inflammatory 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.