In this study, we reported the effect of OA-loaded lipid-based nanoparticles on anti-inflammatory potency against neutrophils and the ability to mitigate ARDS in a mouse model. Our ability to control the nanoparticulate size made it possible to elucidate how the specific sizes affected anti-inflammatory efficacy. Our major findings were that the smaller sizes showed greater internalization and cytotoxicity in neutrophils. Nanoparticulate OA inhibited the superoxide and elastase of activated neutrophils with less potency compared to the free control. However, injectable OA-loaded nanoparticles demonstrated greater mitigation on lung injury in vivo than free OA. The larger-sized nanocarriers were beneficial to ameliorate ARDS with greater efficiency. There are limited drugs for treating ARDS-induced inflammation. Current data on the use of steroids in ARDS indicate no definitive evidence of reducing mortality . OA-loaded nanosystems may provide an opportunity to treat ARDS in the future.
Previous study  demonstrated that the particulate diameters ranging between 10 nm and 200 nm are most relevant for physical and biochemical targeting to specific tissues. Our nanoparticles fell within this range. OA was expected to remain in the lipid core of the nanoparticles because of its high lipophilicity capable of mixing with mineral oil, the main ingredient of the lipid matrix. Nevertheless, the emulsifier nature of OA might lead to the possible intercalation of some OA molecules in the emulsifier layer of the particulate surface. The carboxyl moiety of OA is responsible for negative zeta potential since this moiety is deprotonated in the physiological environment . We found that the blank AS without OA displayed a negative surface charge of –35.2 mV, which was less than that of OA-loaded AS (–41.0 mV). This confirmed the coating of some OA on the nanoparticulate shell. Zeta potential serves as a predominant factor to offer electrostatic repulsion among the charged nanoparticles for storage stability . The high zeta potential of our nanosystems signified a great repulsion for increased stability. We developed the nanocarriers with negative surface charges with the aim of injectable anionic nanoparticles being favorable for longer circulation, leading to the possibility of enhanced distribution to the target site . The anionic nanoparticles are also well tolerated in the lung whereas the cationic nanoparticles are detrimental to pulmonary function . Mucin in the mucus layer of the lung is negatively charged. Mucin can capture cationic nanoparticles to retard the penetration into the alveolar space . The negatively charged nanoparticles facilely transport across the mucus barrier into the alveolar space. The various sizes of OA-loaded nanoparticles are expected to penetrate through the mucus layer with a pore size range of 60–300 nm .
The OA nanoparticles were quickly internalized into the neutrophil cytoplasm. The surface functionalization of the nanocarriers is key to their reaction to cellular uptake. OA as the nanoparticulate stabilizer increases the probability of insertion into the cells . OA interaction with membrane phospholipid bilayers can increase and disturb cellular membrane fluidity to increase invagination [1, 22]. This may be one of the reasons for the cytotoxicity induced by OA nanoparticles at a high concentration. Previous study  also suggests the enhancement of the phagocytic capacity of neutrophils by OA. Lipid-based nanocarriers could be recognized as the soft but not rigid nanoparticles. The soft nanoparticles are facilely deformed by the cells for phagocytosis. This internalization process is less energetically favorable . Nanoparticulate size is a physicochemical factor to influence immune cell uptake . It is generally acknowledged that the increased nanoparticulate size correlates with increased uptake by phagocytic cells . This was not the case in the present study. We showed an increased neutrophil ingestion with decreased size of OA-loaded nanoparticles. The size range of our nanocarriers was 105–225 nm. The previous studies that concluded that increased size resulted in increased uptake usually used the particulate size of ≥300 nm as the representative of larger particles, e.g. the microscale versus nanoscale  and 500 nm versus 150 nm . Wang et al.  also demonstrated the inefficient phagocytosis of nanoparticles of <100 nm by phagocytic macrophages. Of course, the different experimental conditions and nanoparticle types might be responsible for the different results. The size range of OA-loaded lipid-based nanoparticles might display a unique property for neutrophil uptake.
Some investigations support the inverse correlation between particle size and neutrophil uptake. Kelley et al.  demonstrated the increased human neutrophil uptake following the size reduction of polystyrene nanoparticles. Gifford et al.  suggested a facile uptake of iron oxide nanoparticles with a mean diameter of 110 nm by leukocytes. The increased total surface area-to-mass ratio of the smaller sizes has promoted the opportunity to interact with cells. The smaller-sized nanoparticles often need less driving energy in the internalization procedure. The role of the surface charge and the molecular environment on neutrophil uptake cannot be ignored. The greater zeta potential causes more hydrophilicity of the nanoparticles, decreasing the cellular uptake through a stealth effect . The negative surface charge and hydrophilicity of OA nanocarriers increased following the size increase, leading to less internalization. Basically, the nanoparticles would fuse with the lysosomes after invagination into the cells. The lysosomes degraded the nanoparticles to release active ingredients for revealing the bioactivity.
[Ca2+]i is a second messenger contributing to neutrophil activation. It is a potential target for anti-inflammation. Our results showed that AS but not AM and AL significantly impeded fMLP-induced Ca2+ influx amplification. This could be due to neutrophils’ facile uptake of AS. All nanoformulations hastened the speed of [Ca2+]i decline. The ability of OA to inhibit Ca2+ influx has been proved in neutrophils and T lymphocytes [2, 7, 32]. The augmentation of [Ca2+]i results in neutrophil activation, producing oxidative stress and degranulation. Excess superoxide production by activated neutrophils contributes to lung tissue damage in ARDS via the release of cytokines and chemokines . Elastase also holds an apparent capacity for the pathogenesis of ARDS . Increased superoxide anion production and elastase release by activated neutrophils were abrogated in neutrophils internalizing OA nanoparticles. It should be noted that the inhibitory effect of the lipid-based nanocarriers at a high OA dose (10 μg/ml) was partly mediated via the cytotoxic result. No significant difference in this inhibition was detected for the nanosystems of different sizes. This suggests that although different particulate sizes showed different levels of neutrophil uptake, this effect did not influence the following alleviation of oxidative stress and degranulation. However, the cellular uptake level did affect the cytotoxicity and peak [Ca2+]i.
NETosis is a process of neutrophil degranulation in response to microbial invasion to release elastase, MPO, histones, and extracellular DNA fibers . The creation of NETs is associated with ARDS severity . Although OA nanoparticles could inhibit the release of elastase, NETs were augmented by this treatment. Some nanoparticles act directly on the neutrophil membrane to activate the generation of NETs. These include lipid-based, silver, polystyrene, and graphene oxide nanoparticles [36–39]. A similar effect might be found in our case. As already mentioned, OA shows controversial data involving inflammation through different mechanisms. Further investigation is necessary to elucidate the detailed mechanisms related to nanoparticle-induced NETosis. It is generally recognized that NETosis is a unique type of neutrophil death via the NADPH-oxidase (Nox)-dependent pathway . Previous evidence  expressed that OA only induces Nox-dependent NETosis in human neutrophils. This explains the enhanced NET formation and cell death by the nanosystems with an OA dose at 10 μg/ml.
We aimed to deliver OA-loaded nanocarriers to the lung to treat ARDS. A rationale for nanoformulation design to achieve this intention was the incorporation of SPC and Poloxamer 188 as the emulsifiers. The activated neutrophils migrate to the alveolar space in ARDS development , promoting a large number of proinflammatory mediators. The nanoparticles should penetrate across the alveolar epithelium to reach the neutrophils. The epithelium is covered with the lining layer containing pulmonary surfactants. The main components of pulmonary surfactants are phospholipids, neutral lipids, and proteins. These surfactants can be used as efficient carriers associated with nanoparticles for delivery into the airspace [42, 43]. Our previous study  showed that the nanovesicles with abundant SPC were beneficial for interacting with pulmonary surfactants for lung targeting. The nanoparticles with a negative surface charge generally have a higher biodistribution than those with a positive charge . The bioimaging assay displayed a broad organ distribution by intravenous OA nanocarriers. A differential response in biodistribution was detected with the different nanocarriers, with larger nanoparticles representing a more-extensive biodistribution in the peripheral organs. This can account for the longer half-life of the smaller nanoparticles in circulation after intravenous injection [26, 45]. Because of the soft and deformable characters of lipid-based nanoparticles, the larger nanoparticles such as AL facilely transported across the biological barriers such as the capillary wall.
The lung accumulation of the OA nanoparticles was size-dependent, with the larger sizes exhibiting greater uptake. The nanoparticles even with a large diameter can enter the lung tissue since the pulmonary epithelium in ARDS reveals an elevated microvascular permeability . Dysfunction of the epithelial barrier facilitates the diffusion of macromolecules and nanoparticles into the alveolar space. It is well known that the lung can act as the first mechanical filter in the circulation. The lung vasculature has an extensive vascular network with 30% of the total endothelial surface. This unique characteristic enables larger nanoparticles to deposit in the lung [45, 47]. Another possibility for greater lung accumulation of larger OA-loaded nanocarriers could be that hydrophilic nanoparticles such as AL tend to rapidly penetrate across pulmonary surfactant film . The plasma proteins easily adsorbed onto the intravenous nanoparticle surface to form a protein corona. This opsonization facilitated the recognition of nanoparticles by a mononuclear phagocyte system and was then engulfed by a reticuloendothelial system such as the liver, spleen, and lung. Previous investigations [47, 49] inferred that the nanoparticles between 100 nm and 200 nm were largely stored in the liver, whereas the nanoparticles of <50 nm were mainly delivered to the spleen. Our data fitted this criterion, demonstrating that the OA nanocarriers showed a notable liver accumulation but a low spleen uptake. The high level of OA nanoparticles in the GI may suggest the large excretion through the biliary system from the liver to the GI tract. It is possible that nanoparticles deliver epithelial cells and hepatocytes into the bile via the bile duct .
MPO and cytokines in ARDS-like pulmonary tissue were analyzed to examine the anti-inflammatory effect of OA nanoparticles. MPO expressed in neutrophil granules is a major mediator of lung injury. Neutrophils also secrete some cytokines such as TNF-α, IL–1β, and IL–6 to deteriorate ARDS . An attenuation of ARDS by nanoparticulate OA was found, based on the reduction of MPO and cytokines. Histological observation also confirmed a suppression of neutrophil infiltration in mice receiving OA nanoparticles. Neutrophils could be the predominant cells affected. The larger nanoparticles demonstrated greater improvement according to the data of MPO, TNF-α, and IL–6. This result correlated well with the biodistribution in the lung. Though the larger nanoparticles exhibited less capability for neutrophil uptake, the superoxide and elastase inhibition was comparable to that of the smaller sizes. The ARDS mitigation was far superior with nanocarriers than with free OA. By using nanoparticles, a robust increase of OA in the pulmonary tissue could be accomplished. Because of the extremely lipophilic feature, OA was tightly loaded in the lipid matrix with minimal leakage over an extended period, increasing the stability to prevent enzymatic attack. OA encapsulated in lipid-based nanoparticles safely delivered into the lung for neutrophil internalization, resulting in anti-inflammatory action. Free OA possibly even damaged the alveolar endothelium to evoke pulmonary capillary permeability, causing lung injury .
ARDS is highly associated with the activation of neutrophils. Our data verified that the inhibition of neutrophil stimulation lessened the signs of pulmonary inflammation. Although this study focused on the role of neutrophils in lung injury, the other cells related to ARDS cannot be overlooked. Lung injury involves a mixture of cells with origins of neutrophils, alveolar macrophages, dendritic cells, and T lymphocytes . Cytokines can be produced by different immune cells. For instance, IL–6 is a key proinflammatory mediator secreted by neutrophils, macrophages, and T cells to induce an inflammatory cascade in ARDS . Alveolar macrophages are important phagocytic cells in the lung for nanoparticle internalization. Though the larger OA nanoparticles showed less uptake by neutrophils, the larger sizes present a great opportunity to be endocytosed by macrophages . Fromen et al.  also indicated that the nanoparticles with a high negative charge are beneficial for internalization by alveolar macrophages. Our results demonstrated the higher negative zeta potential of AL compared to that of AS and AM. NETs appear in the pulmonary microvasculature of ALI patients . However, NETs can be phagocytosed and cleared by macrophages . Although OA nanoparticles elicited the NET creation, this negative effect might be absent in the in vivo ARDS model due to the participation of the macrophages.
Another concern is the CXCL–2 upregulation by OA. This suggests the mechanisms other than cytokine inhibition mediated neutrophil chemoattraction. OA is proved to increase chemokines CXCL–8 and MIP–1α in lung tissue . Oral OA induces CXCL–3 release and neutrophil-endothelium interaction in the air pouch . The increase of CXCL–2 did not affect the overall improvement of the ARDS-like lesion by OA-loaded nanoparticles. There are some limitations in the present study. The mouse neutrophils behave differently from human neutrophils. This can cause difficulty in directly linking or correlating the in vitro results with the in vivo profiles. According to previous and present investigations, OA treatment demonstrated both advantageous and detrimental impacts on pulmonary tissue. The independent studies are hard to compare due to the variation in experimental setups such as nanoparticle types, cell models, animal models, and the administered doses. Although our nanoformulations were validated as being useful for inhibiting pulmonary inflammation, whether this effect is still maintained with different doses and clinical status remains uncertain. Further study is needed to clarify these questions.