**2. Overview of humoral immunity**

Antibodies are accumulated by the immune system to identify and neutralize foreign objects. They were the first specific product of the adaptive immune response to be identified and are found in the plasma, in the blood, and in extracellular fluids. Immunity mediated by antibodies is known as humoral immunity because of body fluids that were once known as humors [4]. The humoral immune response begins with the recognition of antigens by native B cells. These cells then undergo a process of clonal expansion and differentiation. In this way, the B cell matures into antibody-secreting plasma cells, which secrete antibodies. The activation of B cells and their differentiation into antibody-secreting plasma cells is triggered by antigen and usually requires helper T cells. The term "helper T cell" is often used to mean a cell from the TH2 class of CD4 T cells, but a subset of TH1 cells can also help in B-cell activation [5]. B cells can receive help from helper T cells when antigen bound by surface immunoglobulin is internalized and returned to the cell surface as peptides bound to major histocompatibility complex (MHC) class II molecules. MHC then delivers activating signals to the B cell. Thus, protein antigens binding to B cells both provide a specific signal to the B cell by cross-linking its antigen receptors and allow the B cell to attract antigen-specific T-cell help. These antigens are unable to induce antibody responses in animals or humans who lack T cells, and they are therefore known as thymus-dependent antigens [5]. The first signal required for B-cell activation is delivered through its antigen receptor. For thymus-dependent antigens, the second signal is delivered by a helper T cell that recognizes degraded fragments of the antigen as peptides bound to MHC class II molecules on the B-cell surface; the interaction between CD40 ligand on the T cell and CD40 on the B cell contributes an essential part of this second signal [5]. For thymus-independent antigens, the second signal can be delivered by the antigen itself or by non-thymus-derived accessory cells. The B-cell co-receptor complex of CD19:CD21:CD81 can greatly enhance B-cell responsiveness to antigen. CD21 (=complement receptor 2) is a receptor for the complement fragment C3d. Whether binding of CD21 enhances B-cell responsiveness by increasing B-cell signaling, by inducing co-stimulatory molecules on the B cell, or by increasing the receptor-mediated uptake of antigen is not yet known [5]. Antibodies are the effector products of humoral immunity. Finally, as this response declines, a pool of memory cells remains behind. If the body is reexposed to the antigen, these memory cells will recognize the antigen and respond much more quickly and effectively [6]. There are two purposes of antibodies. The first purpose is to neutralize the target threat, and the second purpose is to recruit other cells or proteins to an antigen so that those cells or proteins can eliminate the antigen [6]. AMR develops when recipient antibody is directed against donor human leukocyte antigens (HLA) on the endothelial layer of the allograft. Antibodies induce fixation and activation of the complement cascade, resulting in tissue injury. Complement and immunoglobulin are deposited within the allograft microvasculature, which results in an inflammatory process that is characterized by endothelial cell activation, upregulation of cytokines, infiltration of macrophages, increased vascular permeability, and microvascular thrombosis. This process ultimately manifests as allograft dysfunction [6].


*Review* **Role of EPA in Inflammation: Mechanisms, Effects, and Clinical Relevance**

**Rosalia Crupi 1 and Salvatore Cuzzocrea 2,\***

**Abstract:** Many chronic inflammatory processes are linked with the continuous release of inflammatory mediators and the activation of harmful signal-transduction pathways that are able to facilitate disease progression. In this context atherosclerosis represents the most common pathological substrate of coronary heart disease, and the characterization of the disease as a chronic low-grade inflammatory condition is now validated. The biomarkers of inflammation associated with clinical cardiovascular risk support the theory that targeted anti-inflammatory treatment appears to be a promising strategy in reducing residual cardiovascular risk. Several literature data highlight cardioprotective effects of the long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA). This PUFA lowers plasma triglyceride levels and has potential beneficial effects on atherosclerotic plaques. Preclinical studies reported that EPA reduces both pro-inflammatory cytokines and chemokines levels. Clinical studies in patients with coronary artery disease that receive pharmacological statin therapy suggest that EPA may decrease plaque vulnerability preventing plaque progression. This review aims to provide an overview of the links between inflammation and cardiovascular risk factors, importantly focusing on the role of diet, in particular examining the proposed role of EPA as well as the success or failure of standard pharmacological therapy for cardiovascular diseases.

**Keywords:** inflammation; eicosapentaenoic acid; atherosclerosis

### **1. Overview of Inflammation**

Inflammation is classified as "normal defense mechanism" able to preserve the host from both infections and insults; the involvement of the inflammatory process helps to restore homeostasis at damaged or infected sites. Cardinal signs of inflammation are redness, swelling, heat, pain, and loss of function, and it sees the presence and interaction of different cell types with recruitment of several chemical mediators. If the inflammatory response is well regulated it resolves quickly and without causing damage to the organism, which overall involve the activation of negative feedback mechanisms such as the secretion of anti-inflammatory mediators, the inhibition of pro-inflammatory signaling cascades, shedding of receptors for inflammatory mediators, and activation of regulatory cells [1]; otherwise damage to organs and tissues can occur. Inflammatory mediators normally play a defensive role in the defense of the host; however, if the production of these mediators is not regulated, tissue damage can be caused, inducing the development of different pathologies. Literature data have reported, in fact, that several chronic inflammatory processes are linked with the continuous release of inflammatory mediators and the activation of harmful signal-transduction pathway that facilitate disease progression. In these cases, there are high concentrations of inflammatory markers and cells both at the primary site of damage and at the systemic level, as occurs in cardiovascular disease or rheumatoid arthritis. Recent studies have shown that the condition of low-grade chronic inflammation is decisive in the obesity state, metabolic syndrome, and cardiovascular diseases [2].

**Citation:** Crupi, R.; Cuzzocrea, S. Role of EPA in Inflammation: Mechanisms, Effects, and Clinical Relevance. *Biomolecules* **2022**, *12*, 242. https://doi.org/10.3390/ biom12020242

Academic Editor: Prakash Kulkarni

Received: 22 December 2021 Accepted: 31 January 2022 Published: 1 February 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

For the aforementioned concepts, inflammation is classified as the lowest common denominator of numerous pathologies (Figure 1).

**Figure 1.** Representative image of inflammatory diseases in which marine n-3 fatty acids might be of benefit.

Classical events that characterized the inflammatory response include the following:

#### *Lipid Mediators*

There are different lipid mediators (e.g., leukotrienes (LTs), prostaglandins (PGs), peptide mediators (e.g., cytokines), amino acid derivatives (e.g., histamine), reactive oxygen species (e.g., superoxide), and enzymes (e.g., matrix proteases) in relationship to the anatomical site involved, cell type involved, nature of the inflammatory stimulus, and the stage during the inflammatory response. Among the mediators involved in the development of inflammatory processes, the first group is represented by toll-like receptors (TLRs), membrane proteins located on macrophages, and dendritic cells. The receptors have the specificity to recognize the molecular patterns that are associated with pathogens (PAMP) and can also identify the endogenous signals that are activated during tissue or cellular damage related to danger (DAMPS). The second group of mediators is represented by the arachidonic acid (AA) mediators. The phosphorylase enzyme is able to act on membrane phospholipids releasing arachidonic acid. The arachidonic acid can metabolize either through the cyclooxygenase pathway or the 5-lipoxygenase pathway. Through the action of cyclooxygenase, the mediators of PGs are formed, which are prostaglandin D2 (PGD2) and thromboxane, which are bronchoconstrictor prostaglandins, and bronchoprotective or inhibitory prostaglandin E2 (PGE2) and prostacyclin. LT is formed from the 5-lipoxygenase pathway [3]. The third group of mediators is represented by mast cells, cells distributed throughout the body.

Mast cells are immune cells of the myeloid lineage and are present in connective tissues throughout the body. The activation and degranulation of mast cells significantly modulates many aspects of physiological and pathological conditions in various settings. With respect to normal physiological functions, mast cells are known to regulate vasodilation, vascular homeostasis, innate and adaptive immune responses, angiogenesis, and venom detoxification. On the other hand, mast cells have also been implicated in the

pathophysiology of many diseases, including allergy, asthma, anaphylaxis, gastrointestinal disorders, many types of malignancies, and cardiovascular diseases. The characteristic of these cells is to undergo a degranulation process when activated by tissue damage; proinflammatory molecules are released from the granules such as histamine, tumor necrosis factor (TNF), kinin, and leukotrienes [4].

#### **2. Inflammation in Cardiovascular Diseases: Focus on Atherosclerosis**

All cardiovascular diseases are characterized by the presence of an inflammatory environment often resulting from comorbid pathologies such as diabetes mellitus and arterial hypertension. These are then associated with additional risk factors which are represented by the emotional component and lifestyle. Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of morbidity and mortality globally. In the United States, nearly 808,000 people died from ASCVD in 2014, translating to about 1 in every 3 deaths [5], and the death rate from ASCVD increased by 1% in 2015, the first since 1999 [6]. ASCVD is defined as acute coronary syndrome, stable angina, history of myocardial infarction, coronary or other revascularization, ischemic stroke, transient ischemic attack, or peripheral arterial disease presumably of atherosclerotic origin. Atherosclerosis can also be considered a chronic, immuno-inflammatory, fibroproliferative disease of large and medium-sized arteries fueled by lipids. The study carried out by Anichkov, on rabbits who were fed a diet rich in fatty acids, highlighted the importance and involvement of dyslipidemic phenomena in the development of cardiovascular diseases [7]. Arteriosclerosis is a composite disease in which the inflammatory process is found on the one hand, but on the other, it is also a lipid disorder. Indeed, the Framingham Heart Study in the 1950s demonstrated that hypercholesterolemia accelerates atherosclerosis in humans [3]. In the study of cardiovascular diseases, great importance was given to the thrombotic aspect, which still represents the greatest risk in the management of low-grade systemic inflammation; in fact, there are numerous studies that underline the link between the mediators of inflammation and platelet activation [8].

Formation and rupture of atherosclerotic plaque are closely connected with the development of the inflammatory process. In particular, the lipoprotein stimulates both chemokines and endothelial cells that facilitate the recruitment of lymphocytes and adherent monocytes to the forming lesion site; growth factors and cytokines formed by the inflammatory intima stimulate the differentiation of monocytes into macrophages linked by the upregulation of the TLRs, in particular TLR4 and scavenger receptors; the macrophages activated form foam-like cells or stimulate an inflammatory cascade that lead to atherosclerotic plaque formation [9].

#### **3. Fatty Acids: Composition and Role in Inflammatory Processes**

Fatty acids are long-chain hydrocarbons and important components of lipids in plants, animals, and microorganisms. Among several role of fatty acids, it was demonstrated that they are able to influence inflammatory processes through several mechanisms by acting, for example, via the cell surface and intracellular receptors/sensors that regulate both gene expression patterns and inflammatory cell signaling. Alterations in fatty acid compositions of cell membranes modulates membrane fluidity, lipid raft formation, and cell signaling leading to altered gene expression. A fatty acid consists of a straight chain of an even number of carbon atoms, with hydrogen atoms along the length of the chain and at one end of the chain and a carboxyl group (—COOH) at the other end. They can be separated into four categories: saturated, mono-unsaturated, polyunsaturated, and *trans* fats. In particular, if the carbon-to-carbon bonds are all single, the acid is saturated; if any of the bonds is double or triple, the acid is unsaturated and is more reactive.

A fatty acid containing two or more double bonds is called a polyunsaturated fatty acid (PUFA). Saturated fatty acids do not contain double bonds in the acyl chain. A few fatty acids have branched chains; others contain ring structures (e.g., prostaglandins). Fatty acids are not found in a free state in nature; commonly, they exist in combination with glycerol in the form of triglycerides. Two principal families of PUFAs are known, namely the n-6 (or omega-6) and the n-3 (or omega-3) (Table 1).

**Table 1.** PUFAs n-6 (or omega-3) family.

Linoleic acid (18: 2n-6) and α-linolenic acid (18: 3n-3) belong to these families, which have the particularity of not being able to be synthesized by mammals. Vegetable oils contain high amounts of linoleic acid. Green plants and some vegetable oils, on the other hand, contain α-linolenic acid.

Importantly, although linoleic and α-linolenic acids cannot be synthesized by humans, they can still be metabolized into other fatty acids. In particular, linoleic acid can be converted via γ-linolenic acid (18: 3n-6) and dihomo gamma-linoleic acid (20: 3n-6) into arachidonic acid (20: 4n-6); it is also possible to convert α-linolenic acid in eicosapentaenoic acid (20: 5n-3; EPA). The specific chemical structure of EPA reflects important biological characteristics. EPA can modify the physical properties of cellular membranes by substituting the omega-6 fatty acid arachidonic acid (AA; 20:4, n-6) in membrane phospholipids. Moreover, from the metabolism of EPA, antithrombotic and anti-inflammatory lipid mediators can be generated, which are in stark contrast to the prothrombotic and pro-inflammatory factors that are produced by arachidonic acid (AA) [12,13]. EPA can be further metabolized, giving rise to docosapentaenoic acid (22: 5n-3; DPA) and docosahexaenoic acid (22: 6n-3; DHA).

#### *Fatty Acid Sources*

Sources of fatty acids include fruits, vegetable oils, seeds, nuts, animal fats, and fish oils. Essential fatty acids, such as omega-3 fatty acids, serve important cellular functions. They are a necessary part of the human diet because the body has no biochemical pathway to produce these molecules on its own. Particular linoleic and α-linolenic acids are introduced through diet. Arachidonic acid is found in meats, and intake is estimated to be between 50 and 500 mg/day. Fish, especially fatty fish, are rich in EPA, DPA, and DHA. Membrane phospholipids consist of basic units of polyunsaturated fatty acids. Preclinical studies, conducted on laboratory guinea pigs fed according to standard feeding, showed a high content of arachidonic acid (20: 4n-6) and a low content of eicosapentaenoic acid (20: 5n-3; EPA) and docosahexaenoic acid (22: 6n-3; DHA) in the phospholipid composition of tissue lymphocytes [14], peritoneal macrophages [15,16], alveolar macrophages [17], Kupffer cells, and alveolar neutrophils [18]. If the diet of animals was enriched with fish oil, rich in EPA and DHA, there is an accumulation of these in membrane phospholipids both in lymphocytes [14] and in macrophages [19,20], and in Kupffer cells [17]. The even more interesting data that emerges is represented by the decrease in the content of arachidonic acid. Studies conducted on membrane phospholipids of cells such as neutrophils, lymphocytes, and monocytes of patients who consume typical Western diets contain about 10–20% of fatty acids such as arachidonic acid, with about 0.5–1% EPA and about 24% DHA [21–23]. The fatty acid composition of these cells can vary by increasing the intake of marine n-3 fatty acids [23,24]. This occurs in a dose–response mode [23] and over a period of days to weeks, with a new steady-state composition achieved within approximately 4 weeks. There are several mechanisms through which fatty acids can influence inflammatory processes, in particular the following:

#### **4. Fatty Acid: Preclinical Studies**

Several animal models were used to study the effects of EPA. As reported in a study conducted on a mouse model of hyperlipidemia, represented by ApoE- and LDL-receptordeficient mice, the administration of EPA reduced the development of atherosclerotic lesions and increased the cell content of omega-3 PUFAs, without changing the total cholesterol or HDL content [25]. The analysis carried out on the atherosclerotic plaques of mice administered with EPA showed a stable morphology associated with a lower deposition of lipids and a reduced accumulation of macrophages accompanied by an increase in smooth muscle cells and collagen content. An anti-inflammatory effect of EPA was also highlighted due to the inhibition of the expression of adhesion molecules and monocyte chemoattractant protein 1 (MCP-1) and inhibition of the production of metalloproteinases by macrophages. The atherosclerotic plaques of mice treated with EPA had a stable morphology, including less lipid deposition, decreased accumulation of macrophages, increased smooth muscle cells, and greater collagen content. In addition, EPA had an anti-inflammatory effect on endothelial cells, inhibiting the expression of adhesion molecules and MCP-1 and by inhibiting production of matrix metalloproteinase by macrophages.

EPA is converted to 18R-hydroxyheicosapentaenoic acid (18R-HEPE) by COX-2 expressing vascular endothelial cells, which is acetylated in the presence of aspirin during inflammation. Endothelial cells expressing COX-2 convert EPA into 18R-HEPE, which is

subsequently released by endothelial cells and converted via neutrophil-derived 5-LOX through a common epoxy intermediate in Resolvin E1 (RvE1) and Resolvin E2 (RvE2).

Data from a study in which they were used have recently been acquired. ApoE 3 Leiden transgenic mice transgenic (known as mice able to develop hyperlipidemia and susceptible to diet-induced atherosclerosis) were used to study RvE1 obtained by the EPA on the development of arteriosclerotic lesion. The animals were given a hypercholesterolemic diet for 9 weeks after which they received the administration of RvE1 (low or high dose) for 16 weeks. A group treated with atorvastatin and one with low-dose RvE1 were also added to the study atorvastatin. Compared to the control group, the group treated with RvE1 (low or high dose) and with atorvastatin reduced the arteriosclerotic lesion from 35% to 27%; the combination of RvE1 and atorvastatin reduced the atherosclerotic lesion area by 51% [26]. Data collected from another study conducted on LDL-receptor-deficient mice showed that EPA significantly reduces the size of atherosclerotic plaques as well as total cholesterol levels [27]. Levels of proinflammatory cytokines and chemokines (TNF alpha; interferon gamma) were lower than in the control group. Another target on which EPA acts is represented by dendritic cells whose phenotype is reorganized after administration of EPA; the number of T cells is reduced in the spleen and lymph nodes [28].

#### **5. Fatty Acids: Clinical Studies**

The importance of the use of omega-3 fatty acids in general, and of EPA in particular, is also linked to the numerous guidelines on their use; for the National Lipid Association (NLA) omega-3 fatty acids represent the first option for the treatment of patients with high triglyceride (TG) levels (≥500 mg/dL) and as an add-on option to statin therapy for those with high TG levels (200–499 mg/dL) [29]. For the Japan Atherosclerosis Society (JAS) the addition of EPA to a statin therapy represents an added value for the treatment of high-risk patients with LDL-C levels ≥140 mg/dL [30]. Numerous clinical studies have reported data on the importance assumed by omega-3 PUFAs on the prevention of ASCVD; in particular, on the international scene, four studies stand out: Gruppo Italiano per Io Studio della Sopravvivenza nell'Infarto Miocardico (GISSI)-Prevention study [3], the Japan Eicosapentaenoic Acid Lipid Intervention (JELIS trial) [31], the GISSI-Heart Failure study [32], and the Reduction of Cardiovascular Events with Icosapent Ethyl-Intervention Trial (REDUCE-IT) [33] (see Table 2). Moreover, other studies were also formulated in which a low dose of omega-3 PUFA was used (in a range of 840 mg/d of EPA + DHA) but which did not produce positive results. In particular, we can count the ORIGIN trial [34], the Risk and Prevention Study [35], the ALPHA OMEGA trial [36], A Study of Cardiovascular Events in Diabetes (ASCEND) trial [37], and the Vitamin D and Omega-3 Trial (VITAL) [38].

**Table 2.** Clinical studies showed data on the relevance of omega-3 PUFAs on the prevention of ASCVD.

**Table 2.** *Cont.*

#### *EPA: Outcomes Studies*

In the Outcomes to Assess Statin Residual Risk Reduction with Epanova in High CV Risk Patients with Hypertriglyceridemia (STRENGTH) study, the effects of a 4 g/day administration of EPA and DHA in a carboxylic acid formulation were analyzed [39]. A meta-analysis conducted on 20 studies that enrolled 68,680 patients showed that the administration of omega-3 PUFAs led to a reduction in the risk of cardiac death (RR 0.91, 95% CI: 0.85–0.98) in secondary prevention [40]. Data obtained from this survey led the American Heart Association (AHA) of 2017 to express a scientific opinion in which it is reported that the low-dose supplementation of omega-3 PUFA was conceivable to prevent the secondary manifestations of coronary diseases in patients with overt coronary heart disease. [41] A recent meta-analysis formulated by expanding the data with those obtained from three recent large-scale RCTs of omega-3 PUFAs (REDUCE-IT, ASCEND, and VITAL) clearly highlighted the beneficial properties of omega-3 PUFAs in limiting the risk of developing cardiovascular events. These reductions appear to be associated with the supplemental omega-3 PUFA dose [42]. In the analysis carried out by the REDUCE-IT study, the effects obtained from the use of the ethyl ester EPA (icosapent ethyl 4 g/day) on the development of high-risk cardiovascular events in patients on drug treatment with statins were examined [33]. The clinical condition of the patients was subsequently assessed with 4.9-year follow-up, which demonstrated a positive effect of using EPA [33]. Data obtained from REDUCE-IT led the National Lipid Association to recommend the administration of icosapent ethyl for patients aged ≥45 years with clinical ASCVD, or aged ≥50 years with diabetes mellitus requiring drugs plus ≥1 factor of additional risk, with fasting triglycerides 135 to 499 mg/dL on high-intensity or maximally tolerated statin therapy to reduce the risk of ASCVD [43]. The FDA has approved the administration of icosapent ethyl to both treat patients with triglyceride levels ≥500 mg/dL, and to reduce the risk of ASCVD in people with diabetes mellitus and two or more additional risk factors for cardiovascular disease in drug therapy with statins [43]. The difference between the positive effect of icosapent ethyl reported in the REDUCE-IT [33] study and the lack of an effect of the mixed carboxylic acids EPA and DHA in the STRENGTH [39] study is still questionable. The studies cited used omega-3 PUFAs at high doses but in different formulations, so comparisons should be made with caution. The studies were designed differently in at least three aspects. First, the omega-3 PUFAs were administered as an ethyl ester formula in the REDUCE-IT study and as non-esterified fatty acids, rapidly ionized transforming into molecules with detergent properties (soaps), in the STRENGTH study. Second, unlike the corn oil used in the STRENGTH study, the placebo mineral oil used in the REDUCE-IT study may have influenced expectations. Third, the DHA component of omega-3 PUFAs may be ineffective or even harmful, although there are currently no studies on the ASCVD outcomes of DHA monotherapy. The significant therapeutic efficacy of EPA in combination with statin on ASCVD was not found in other triglyceride-lowering agents, including fenofibrate and niacin, which failed to reduce cardiovascular events as compared to statin treatment alone [44,45]. With the withdrawal of recommendations by the FDA on the combination of statins with fibrates or niacin in the prevention or treatment of ASCVD, icosapent ethyl

remains a viable non-LDL target therapy for patients with increased ASCVD risk and hyper-triglyceridemia. Pemafibrate to Reduce Cardiovascular Outcomes by Reducing Triglycerides in Patients with Diabetes (PROMINENT; NCT03071692), an ongoing trial of pemafibrate (a selective PPAR *α* modulator that significantly lowers triglyceride) in patients with type 2 diabetes mellitus, mild-to-moderate hypertriglyceridemia, and low HDL-cholesterol might further shed light on the mechanism of triglyceride-lowering agents on ASCVD [46].

#### **6. Role of EPA on Athero-Inflammatory-Thrombotic Processes**

The scientific literature demonstrates how EPA plays a beneficial function in the regulation of endothelial tone. The endothelial cells, in fact, release nitric oxide (NO), which has the ability to modulate the vasomotor tone in response to acetylcholine and other vasoactive agonists [47]. Under physiological conditions, there is, in the body, an endotheliumdependent vasodilation due to the release of NO. If one has endothelial dysfunction, then NO release is reduced or absent. In this case, we are witnessing the appearance of toxic effects due to reactive oxygen costs, including peroxynitrite (ONOO\_1) [47,48]. EPA is able to significantly reduce the formation of reactive oxygen species, as well as the expression of adhesion molecules, the release of pro-inflammatory cytokines, and the apoptotic cascade, as demonstrated by the Din in vitro studies conducted on HUVEC cells [49]. EPA was also found to be able to reduce or inhibit lipid peroxidation in membrane vesicles with even high cholesterol levels. The highlighted effect is also easy due to the presence of statins [44]. It is known that glucose contributes to the development of lipid peroxidation, the direct consequence of which is the appearance of high cholesterol levels. In this process, EPA also plays a key role, since it inhibits the formation of lipids [50]. The antioxidant effect of EPA seems to be due to its ability to intercalate in the lipid bilayer of the membrane while preserving its structural component (Figure 2) [50].

**Figure 2.** Possible effect of EPA on the endothelial membrane: inhibition of the propagation of free radicals. According to this model, it is hypothesized that EPA is able to intercalate between membrane phospholipids, in the central region, inhibiting the propagation of free radicals and thus preserving a more homogeneous distribution of cholesterol.

#### *EPA and Atherosclerotic Plaque*

Recent studies show how EPA is able to reduce the neo vasculogenic process in human endothelial cells by acting on the modulation of the expression of the c-lit protein on the PI3-K/Akt/eNOS pathway, thus preventing ischemic injury [51]. Two studies in particular demonstrate these EPA actions; in the ANCHOR study, the administration of EPA at a dose

of 4 g/day for 12 weeks reduced the oxidation of LDL by 13.3% compared to the placebo group in patients treated with statins and high triglycerides (from 200 to <500 mg/dL). In the MARINE study, there was a reduction of 6.6% (*P* = 0.055) in patients with higher levels of triglycerides (500 to <2000 mg/dL) [52]. In patients with type 2 diabetes mellitus and on drug therapy with statins, the addition of EPA at a dose of 1.8 g/day for 6 months) improved the endothelial function, as well as the conditions of CHD patients [53]. Similarly, in hyperlipidemic patients, the administration of EPA at a dose of 1.8 g/day for 3 months restored endotheliumdependent vasodilation at a superimposable level compared to that found in the group of normolipidemic patients [54]. Co-administration of EPA at a dose of 1.8 g/day for 48 weeks) with statins compared to statin monotherapy significantly inhibited the progression of arterial stiffness as observed in the analysis of the b-index stiffness parameter of the carotid artery in CHD patients (*p* = 0.02) [55]. EPA at a dose of 4 g/day for 12 weeks was shown to be effective in significantly reducing RLP-C by 25.8% in statin-treated patients who exhibited high levels of triglycerides (*p* = 0.0001) and 29.8%, respectively, in patients with even higher triglyceride levels (*p* = 0.0041) than in the placebo group [56]. Analyzing the ANCHOR study even more selectively, it was found that EPA significantly reduces RLP-C by 25.0% (*p* <0.0001) and VLDL triglycerides by 28.9% (*p* < 0.01) compared to the placebo group in the subgroup of patients with type 2 diabetes mellitus and on drug treatment with statins and elevated triglycerides [57]. In another study, in patients with type 2 diabetes mellitus and metabolic syndrome, EPA administered at a dose of 1.8 g/day for 3 months significantly reduced the volume of dense LDL particles (*p* < 0.01) and RLP triglycerides (*p* < 0.05) compared to baseline [58]. Numerous studies have shown that EPA is able to significantly reduce the volume of lipid plaque. By adding EPA at a dose of 1.8 g/day to background drug therapy with high-intensity statins, it significantly reduces the volume of lipid plaque and that of fibrotic plaque after 6 months (both *p* < 0.05) as measured by ultrasound intravascular [59]. Similarly, EPA at a dose of 1.8 g/day in co-administration with pitavastatin significantly reduced coronary plaque volume after 8 months of treatment compared to pitavastatin alone (−24% vs. −2%, *p* < 0.01) in patients with impaired glucose tolerance and angina pectoris [60]. In patients treated with EPA for 1 year, the tomography reported an important reduction in volume of soft plaque, contrary to what was seen in patients undergoing pharmacological treatment with ezetimibe [61]. The aforementioned studies have shown that the EPA, in general, exerts protective effects on the development of arteriosclerosis processes; in particular, it is clear in its involvement in the protection of endothelial function, in the reduction of oxidative stress levels, and in the maintenance of low inflammatory levels, which see the release of cytokines, platelet aggregation, and thrombus formation. Furthermore, EPA is also able to act on the reduction of atherogenic dyslipidemia levels with numerous benefits given by the ability to intercalate in the lipid bilayer of the plasma membrane. Of considerable pharmacological interest is the data that demonstrate how the beneficial effects of EPA are still maintained if administered together with statins. A clinical trial, randomized and controlled, called REDUCE-IT (NCT01492361), is now available, developed to evaluate the administration of EPA in a highly purified formulation [62]. The drug currently under study is called icosapent ethyl and represents a high purity formulation containing the ethyl ester of EPA. The recommended therapeutic dose in the trial is 4 g/day to be taken as two 1 g capsules twice daily with food. Therapeutic results were also achieved in the MARINE and ANCHOR studies. In particular, in the MARINE study, conducted in patients with elevated triglyceride levels, treatment with icosapent ethyl at a dose of 4 g/day for 12 weeks significantly reduced their levels by 33.1% (*p* < 0.0001), VLDL triglycerides by 25.8% (*p* = 0.0023), VLDL cholesterol (VLDL-C) by 28.6% (*p* = 0.0002), non-high density lipoprotein cholesterol (not HDL-C) by 17.7% (*p* < 0.0001), and Apo B by 8.5% (*p* = 0.0019) compared to the placebo values [63]. In the ANCHOR study conducted in patients on drug treatment with statins and elevated triglycerides, treatment with icosapent ethyl at a dose of 4 g/day for 12 weeks significantly reduced triglyceride levels by 21.5% (*p* < 0.0001), LDL-C by 6.2% (*p* = 0.0067), VLDL triglycerides by 26.5% (*p* < 0.0001), C-VLDL by 24.4% (*p* < 0.0001), C-non-HDL by 13.6% (*p* < 0.0001), and Apo B by 9.3% (*p* < 0.0001) compared to placebo [63]. On the basis of the data obtained from the analysis of the different clinical

trials, a new pharmacoeconomic model was prepared that highlighted how the combination of EPA and statin for the secondary prevention of cardiovascular diseases is now associated with important economic savings and utility therapeutics when compared with single statin treatment [64].

#### **7. Conclusions**

Numerous studies have now shown that the inflammatory process is the basis of various diseases; in this context, it is now recognized that the role of fatty acids is to control inflammation by changing their composition in cell membranes, for example making it more fluid or altering its gene expression. The key cells of the inflammatory process are rich in arachidonic acid n-6 fatty acid, but their content can vary through oral administration of EPA and DHA. The increase in the membrane content of EPA and DHA causes a change in the production pattern of eicosanoids and probably also of resolvins. Given the involvement that n-3 marine PUFAs have in modulating inflammatory responses, it is understood how these can be decisive in inflammatory process and resolution. The clinical data obtained from the anti-inflammatory evaluations obtained thanks to the role of EPA have raised the awareness that an increase in the diet could bring a clinical benefit. There have been numerous studies that have provided encouraging data in patients with rheumatoid arthritis [65], though fewer in patients suffering from inflammatory bowel diseases [2]. With regard to these diseases, the data between adults and children are conflicting; in the latter, there are greater therapeutic successes [66]. A very interesting study was conducted in pregnant women where the administration of EPA determined beneficial effects on both the maternal and fetal immune systems [67,68], so as to reduce the risk of onset of allergic diseases during early childhood [69]. These first evidences opened the field to study the effects of EPA on the immune system [70]. In the plethora of inflammatory diseases, an exception is represented by cardiovascular disease, of which, to date, studies on EPA have been most concentrated [71,72], whose effects are particularly evident in the stabilization of atherosclerotic plaques [73]. A univocal and clear dose of EPA to be used has not yet been identified, although it is clear that the therapeutic effects are strictly dose-dependent [23]. Studies conducted on volunteers have highlighted that an intake of >2 g EPA + DHA/day is required to modulate inflammatory processes. Studies in patients with rheumatoid arthritis used 1.5 to 7 g EPA + DHA/day (average about 3.5 g/day) and were long-lasting (3 to 12 months), with effects which appear after several months [74]. One study compared two different doses [75], showing that the greatest benefits were obtained with the higher dose. Reading data clearly demonstrate that fatty acids modulate inflammatory phenomena through various motive mechanisms related to changes in the fatty acid composition of cell membranes. Inflammatory cells are rich in n-6 arachidonic acid, but the content of arachidonic acid and n-3 fatty acids EPA and DHA may vary after oral administration of EPA and DHA. The EPA in itself can be a source of eicosanoids with characteristics different from those deriving from arachidonic acid. In fact, both EPA and DHA are sources of recently discovered resolvins with anti-inflammatory properties. The altered fatty acid composition of inflammatory cells also alters the synthesis of peptide mediators of inflammation such as adhesion molecules or cytokines.

Summarizing, data related to clinical investigation reported the following:

**Author Contributions:** Conceptualization, R.C. and S.C.; investigation (literature review), R.C.; writing—original draft preparation, R.C.; writing—review and editing, S.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

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**6.  They are usually very painful because of their somatic innervations, the pain resulting from spasm of the anal sphincter in response to stretching and tearing during passage of stool. A well developed anal fissure rests directly over the internal sphincter and the circular fibers of this sphincter are visible on the floor of

ANAL FISSURE - ANATOMY

Anal fissures presents mostly in children aged 6-24 months. The overall incidence in children is not well described. Anal fissures are located in the posterior midline in 90% of the cases, although 10-20% in women and 1-10% in men are located in the anterior midline (Notaras, 1988). The posterior commissure of the anoderm is less well perfused than other anodermal regions (Schouten et al., 1994). Pressure over the branches of the inferior rectal artery (increased tone at the internal sphincter and high canal pressures) causes relative ischemia (Klosterhalfen et al., 1989). First described as a disease entity in 1934, the cause of

**3. Definition**

**4. Anatomy**

the fissure on naked eye inspection.

Fig. 1. Anal fissure anatomy.

**5. Incidence and etiology**

The pathophysiology is fairly complex and multifactorial, with anodermal ischemia, infection, chronic constipation, hypertonicity of the smooth muscle of the internal anal sphincter (IAS) and elevated maximal anal resting pressure (MARP) being involved (Gillet & Padias, 2006; Schouten et al., 1996). The exact mechanism surrounding the pathophysiology of anal fissures has not been clearly established, but current theories involve the tonicity of the anal sphincter and anal blood flow. A relative lack of nitrate oxide synthase, as found in other spasmodic states of the gastrointestinal tract, has been suggested as a possible mechanism for IAS hypertonia (Lund, 2006). As fissures are most commonly seen in the posterior midline, inadequate blood flow to this region has been hypothesized to play a role in the development of fissures. End arterioles from the inferior rectal artery pierce both sphincters to reach the submucosa of the anal canal and travel cephalad in this plane. Klosterhalfen et al. (1989) suggested that hypertonic sphincter decreases blood flow in these terminal vessels as they pass through the IAS fibers.


**2. The double face of NOx in the eukaryotic world**

To better understand why NOx impact on so many organs, it is essential to focus on the units forming each part of our body: the eukaryotic cells themselves.


3.2.2. Payne Effect

Payne [108] reported that the effects of changes due to deformation of the microstructure of the material were due to breakage and reform of weak physical bonds connecting neighboring filler clusters. This analysis has been applied for the elastomeric composites where the filler particles are connected to the elastomer in the microstructural viewpoint. As the strain amplitude increases, the distance between the CI particles increases and the bond between the elastomer and the particle breaks. For this reason, G' decreases as the structure breaks. In other words, if the bond strength of particles and matrix is strong, the change of G' will be small due to the change of strain amplitude. If the bond strength of particles and elastomer is weak, the change of G' will be large due to strain amplitude. In other words, the Payne effect represents the bond strength between the elastomeric medium and the filler particles. Many researchers have evaluated the properties of MREs through the Payne effect of anisotropic and isotropic MREs [109–112]. The Payne effect is measured by the change of G' according to the change of strain amplitude under cyclic loading conditions and calculated using Equation (1),

$$\text{Payne Effect} = \frac{\text{G}\_0'-\text{G}\_\text{\textphi}'}{\text{G}\_0'} \times 100\tag{1}$$

where *G*0 is the G' at an initial strain and *G*∞ is the G' at the infinite strain. Figure 4 is a graph showing the G' of both pure CI based MRE and PGMA coated CI-based MRE as a function of strain [60]. The Payne effect of the CI-based MRE was calculated to be 84%, while that of PGMA coated CI-based MRE was 78%. MRE with PGMA coated CI had a lower Payne effect. This confirms that CI coated with PGMA has a stronger bond strength with the rubber matrix than the CI-based MRE.

**Figure 4.** The storage moduli CI/PGMA and pure CI based magnetorheological (MR) elastomers [60].


**Abstract**

Maternal Diabetes is one of the most common metabolic disorders resulting an increased risk of abnormalities in the developing fetus and offspring. It is estimated that the prevalence of diabetes during pregnancy among women in developing countries is approximately 4.5 percent and this range varies between 1 to 14 percent in different societies. According to earlier studies, diabetes during pregnancy is associated with an increased risk of maternal and child mortality and morbidity as well as major congenital anomalies including central nervous system (CNS) in their offspring. Multiple lines of evidence have suggested that infants of diabetic women are at risk of having neurodevelopmental sequelae. Previous studies reveal that the offspring of diabetic mothers exhibit disturbances in behavioral and intellectual functioning. In the examination of cognitive functioning, a poorer performance was observed in the children born to diabetic mothers when compared with the children of non-diabetic mothers. Therefore, it is important to study the possible effects of maternal diabetes on the hippocampus of these infants.

**Keywords:** Maternal diabetes, Central nervous system, Hippocampus, Hyperglycemia


**4. Conclusions**

An alginate lyase-producing bacterium was isolated and identified as *Serratia marcescens* NJ-07. The alginate lyase AlgNJ-07 was purified by anion-exchange chromatography. It had a molecular weight of approximately 25 kDa and exhibited the maximal activity of 2742.52 U/mg under 40 ◦C at pH 9.0. Additionally, AlgNJ-07 could retain more than 95% of its activity at pH range of 8.0–10.0, which indicates it possesses excellent pH-stability. It showed high activity and affinity toward polyM block and no activity on polyG block, suggesting it is a strict polyM-specific alginate lyase. TLC and ESI-MS analysis indicated that it can hydrolyze substrates in a unique endolytic manner and produce oligosaccharides with Dp of 2–5 and a large fraction of monosaccharides. Therefore, it may be a potent tool to produce alginate oligosaccharides with lower Dps.

**Acknowledgments:** The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (No. 31601410).

**Author Contributions:** B.Z. and F.H. conceived and designed the experiments; B.Z. and F.H. performed the experiments; Y.S., H.Y. and Z.Y. analyzed the data; B.Z. wrote the paper. All authors reviewed the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.


**2.1 Main features of NSL**

To obtain desired nanostructures by NSL, one monolayer of self-assembled nanospheres is always obtained first and served as a mask for the subsequent fabrication of nanostructures. The material of the nanosphere can be nanoscale polystyrene (PS), SiO2, or polydimethylsiloxane (PDMS) (Choi et al, 2009), and etc. The shape of the resulting pattern is most often spherical. Using PDMS, the shape and the feature size of the pattern can be modulated by changing the stretching axis and ratio of the PDMS replica. The nonspherical shaped patterns, such as rectangular or elongated hexagonal shaped patterns, can then be obtained (Choi et al, 2009). An additional noteworthy feature of the PDMS is that different pattern can be produced from a single PDMS replica mold (Choi et al, 2009). In addition, binary nanospheres composed of two different-size colloidal particles can be self-assembled both in hexagonal lattices via a two-step process (Kim et al., 2009), forming binary colloid crystals (BCCs). Such BCCs may have potential applications in the fabrication of photonic crystal structures, theoretical models of phase transition, and templates of inverse structure (Kim et al., 2009). The arrangement of selfassembled nanospheres can be close-packed or non-close-packed (Vogel et al., 2011). In general, the self-assembled nanospheres are arranged in a hexagonal lattice. Using more sophisticated processes, squarely ordered array of nanospheres can also be realized, which is speculated to be metastable structures between more stable hexagonal structures (Sun et al., 2009).

In the simplest NSL, a monolayer of close-packed nanospheres in a hexagonal lattice is first obtained on the substrate, which can be served as a mask for the subsequent deposition of desired materials. For generally vertical deposition, the three-fold interstices allow deposited material to reach the substrate, giving rise to an array of triangularly shaped nanoparticles with P6mm symmetry (Haynes & Van Duyne, 2001). The perpendicular bisector of the triangular nanoparticles, *a*, and the interparticle spacing, *dip*, are proportional to the nanosphere diameter, *D*, which can be simply calculated by,

Enormous efforts have been devoted to investigate alternative nanolithography approaches. One of promising methods is nanosphere lithography (NSL) (Fuhrmann et al., 2005; Haynes & Van Duyne, 2001; Hulteen & Van Duyne, 1995; Kosiorek et al., 2004; Sinitskii et al., 2007), which is a highly accessible, low cost, parallel fabrication process capable of producing nanostructured surfaces over large areas and with high resolution. In NSL, self-assembled nanospheres can be served directly as ordered nanostructures (Park et al., 1998) or a mask for the subsequent fabrication of nanostructures, which can be realized by deposition of desired materials, or by etching on desired substrates. A rich varieties of ordered nanostructures have been achieved by NSL, such as triangular structures (Winzer et al., 1996), metallic rings (Boneberg et al., 1997), nanopillars (Weeks et al., 2004), and multilayer with modified topography (Albrecht et al., 2005), nanodots (Chen et al., 2009; Weekes et al., 2007), 3D nanostructure (Zhang et al., 2007), discs (Hanarp et al., 2003) and nanoscale crescents (Gwinner et al., 2009; Retsch et al., 2009; Vogel et al, 2011). The shape, the size and the arrangement of ordered nanostructures can be readily controlled in combination of NSL and the subsequent deposition of desired materials (Haynes & Van Duyne, 2001; Zhang et

To obtain desired nanostructures by NSL, one monolayer of self-assembled nanospheres is always obtained first and served as a mask for the subsequent fabrication of nanostructures. The material of the nanosphere can be nanoscale polystyrene (PS), SiO2, or polydimethylsiloxane (PDMS) (Choi et al, 2009), and etc. The shape of the resulting pattern is most often spherical. Using PDMS, the shape and the feature size of the pattern can be modulated by changing the stretching axis and ratio of the PDMS replica. The nonspherical shaped patterns, such as rectangular or elongated hexagonal shaped patterns, can then be obtained (Choi et al, 2009). An additional noteworthy feature of the PDMS is that different pattern can be produced from a single PDMS replica mold (Choi et al, 2009). In addition, binary nanospheres composed of two different-size colloidal particles can be self-assembled both in hexagonal lattices via a two-step process (Kim et al., 2009), forming binary colloid crystals (BCCs). Such BCCs may have potential applications in the fabrication of photonic crystal structures, theoretical models of phase transition, and templates of inverse structure (Kim et al., 2009). The arrangement of selfassembled nanospheres can be close-packed or non-close-packed (Vogel et al., 2011). In general, the self-assembled nanospheres are arranged in a hexagonal lattice. Using more sophisticated processes, squarely ordered array of nanospheres can also be realized, which is speculated to be metastable structures between more stable hexagonal structures

In the simplest NSL, a monolayer of close-packed nanospheres in a hexagonal lattice is first obtained on the substrate, which can be served as a mask for the subsequent deposition of desired materials. For generally vertical deposition, the three-fold interstices allow deposited material to reach the substrate, giving rise to an array of triangularly shaped nanoparticles with P6mm symmetry (Haynes & Van Duyne, 2001). The perpendicular bisector of the triangular nanoparticles, *a*, and the interparticle spacing, *dip*, are proportional to the nanosphere diameter, *D*, which can be simply

**2. Nanosphere lithography (NSL)**

al., 2007; Vogel et al, 2011).

**2.1 Main features of NSL**

(Sun et al., 2009).

calculated by,

$$a = \frac{3}{2}(\sqrt{3} - 1 - \frac{1}{\sqrt{3}})D, \quad d\_{\circ p} = \frac{1}{\sqrt{3}}D^2$$

If double layer of nanospheres are employed as the mask, both *a* and *dip* will be changed (Haynes & Van Duyne, 2001). In addition, circular shaped interstice particles are frequently obtained in the case of small nanospheres mainly because some materials are not perpendicularly deposited in the interstices, and the general hot materials can diffuse in some region. More interestingly, for angle resolved deposition, some particularly-shaped nanostructures can be realized, such as nanochain structures (Haynes & Van Duyne, 2001) and nanocrescents (Vogel et al., 2011). If the monolayer of nanospheres is non-close-packed, more complex nanostructures can be obtained by changing the incidence angle of the material vapor beam and the azimuth angle of the vapor beam with respect to the normal direction of the nanospheres mask (Zhang et al., 2007 ).

The critical step of NSL is to form monolayer of ordered nanospheres on desired substrates. Several methods have been developed to form regularly arranged nanospheres on substrates, including transferal coating (Weekes et al., 2007), vertically dipping coating (Choi et al., 2009), spin coating (Hulteen & Van Duyne, 1995), drop coating (Hulteen et al., 1999), and thermoelectrically cooled angle coating (Micheletto et al., 1995). All of these formation methods are based on the ability of the nanospheres to freely diffuse to seek their lowest energy configuration. The diffusion processes and the interaction among nanospheres can be influenced by chemically modifying the nanosphere surface with a negatively charged functional group such as carboxylate or sulfate. Such a modification of the surface features of nanospheres can be easily realized for polystyrene (PS) spheres (Weekes et al., 2007). The self-assembled monolayer of nanosphere masks always include a variety of defects that arise as a result of nanosphere polydispersity, site randomness, point defects (vacancies), line defects (slip dislocations), and polycrystalline domains (Haynes & Van Duyne, 2001). These defects are always remained in the finally obtained nanostructures, which will degrade the properties of the ordered nanostructures. Therefore, it is important to try to get rid of those defects in the monolayer of self-assembled nanospheres.

#### **2.2 NSL based on transferal coating**

It was found that the transferal coating is much easier in operation to obtain ordered nanospheres in large areas than the other methods. The domain size of ordered PS spheres can be up to 1 cm2 (Weekes et al., 2007). The key step of the transferal coating is to selfassemble highly ordered monolayer of PS spheres at the interface between water and air (or oil). In general, the suspensions (1-10 wt%) of PS spheres in de-ionized (DI) water are diluted in a 1:1 ratio in some spreading agent, such as ethanol or methanol. Drops of the diluted suspension of PS sphere are then introduced into water surface via a titled glass from a pipet. On contact with the water, the PS spheres immediately form a momolayer and start to assemble. The inherent mechanism for the ordering of PS spheres at a liquid interface has been studied by several groups (Aubry & Singh, 2008; Boneva et al., 2009; Larsen & Grier, 1997; Nikolaides et al., 2002; Pieranski, 1980; Trau et al., 1996; Yeh et al., 1997). A reasonable model has been provided to account for the ordering of PS spheres at the interface (Nikolaides et al., 2002). It was suggested that the ordering arrangement of PS spheres resulted from the balance between an electrostatic repulsion and an additional capillary attraction among PS spheres. The former is originated from the negative charges on PS spheres (Weekes et al, 2007). The latter is due to the deformation of liquid meniscus

A Feasible Routine for Large-Scale Nanopatterning via Nanosphere Lithography 537

the interaction among PS spheres. It has been found that the ordering of self-assembled PS spheres at interface can be considerably improved by adding suitable acid (H2SO4) in water (Sirotkin et al., 2010), as shown in Fig. 2. Given that the charges on PS spheres are related to the diameter of PS spheres,the suitable amount of acid or other electrolyte is dependent on the size of PS spheres。In addition, the temperature of the water to some degree also affect the self-assembly of the PS spheres at the interface. It was found that ordering of PS was improved on the water of ~ 4 oC. Such an improvement may be related to the increase of water surface tension and the suppression of the Brownian motion of the PS spheres and

Fig. 2. Self-assembled monolayer of PS spheres (5 x 5 m2) at the interface of air and the solution of de-ionized water and H2SO4 with PH value of, (a) ~7, (b) ~ 5.3. The diameter of PS sphere is 240 nm. The H2SO4 can provide some additional ions around PS spheres, which can effectively change the interaction among PS spheres. Under certain PH value of the

The self-assembled monolayer of PS spheres can be finally transferred to varieties of smooth substrates underneath the water by draining off the water. This process can be affected by some charges on the substrates. In addition, some cracks may appear once the monolayer was disturbed by movements of the water during draining. The PS spheres nearby the cracks slightly displaced from the ideal sites of a hexagonal lattice. In this case, the longrange ordering of the subsequent structure is degraded. The monolayer of ordered PS spheres on substrates can then serve as a mask for the subsequent fabrication of ordered

Periodic pit-pattern can be obtained in combination of NSL and selective chemical etching (Chen et al., 2009). The processes mainly involves three steps: (i) self-assembling monolayer of PS spheres on hydrogenated Si surface; (ii) forming a novel net-like Au-Oxide mask via Au catalyzed oxidation; (iii) resulting in periodic pits by selective chemical etching of Si in

solution, considerable improvement of ordering of PS spheres can be made.

nanostructures by deposition of varieties of materials or by etching.

**2.3 Periodic pit-pattern obtained by NSL and chemical etching**

dust clusters in the water.

KOH solution.

by electrostatic stresses at interface. Both of these two forces were associated with an electric dipolar field, which resulted from an asymmetric charge distribution on particles at the interface due to mismatch in dielectric constant of adjacent fluids. Such a creation of the attractive capillary force is crucial because spheres with diameters of less than 5 m generally do not have sufficient weight to deform the liquid meniscus by means of gravity (Kralchevsky & Nagayama, 2000).

Considering the importance of the electrical field in self-assembling PS spheres at interface, it is natural to find ways to change the electric field to control the arrangement of PS spheres. One way is to apply an external electric field during the self-assembly of PS spheres at the interface (Aubry & Singh, 2008; Boneva et al., 2009; Nikolaides et al., 2002; Trau et al., 1996). This external electrical field will change the charge distribution on the surface of PS Spheres, leading to the change of the electric dipole field around PS spheres. It may also exert an additional electric force on the negatively charged PS spheres, which will affect the deformation of the surface. In a word, the external electric field will affect both the attraction force and the repulsion force mentioned above. It has been found that the inter-particle distance can be remarkably changed by the electric field (Nikolaides et al., 2002). As a result, the ordering of self-assembled PS spheres can be improved considerably, as shown in Fig. 1. In addition, the effect of an external electric field on the arrangement of PS spheres is more pronounced for smaller spheres.

Fig. 1. Self-assembled monolayer of PS spheres (5 x 5 m2) with an external electric field of, (a) 0 v/m, (b) 5 x 104 v/m. The diameter of PS sphere is 200 nm. The electric field is upward and perpendicular to the interface of water and air. The external electric field can efficiently reduce defects in the regular arrangement of PS spheres, remarkably improving the ordering of the self-assembled monolayer of PS spheres.

Another way to modify the balance between the attraction force and the repulsion force among PS spheres is to change the surface chemistry of PS spheres or the electrostatic environment of the water-air interface (Sirotkin et al., 2010). By adding some electrolyte, e.g. acid (H2SO4) and NaCl, into the water, effective surface charge density of PS spheres and /or effect of electric screen of PS spheres can be changed, which give rise to the change of

by electrostatic stresses at interface. Both of these two forces were associated with an electric dipolar field, which resulted from an asymmetric charge distribution on particles at the interface due to mismatch in dielectric constant of adjacent fluids. Such a creation of the attractive capillary force is crucial because spheres with diameters of less than 5 m generally do not have sufficient weight to deform the liquid meniscus by means of gravity

Considering the importance of the electrical field in self-assembling PS spheres at interface, it is natural to find ways to change the electric field to control the arrangement of PS spheres. One way is to apply an external electric field during the self-assembly of PS spheres at the interface (Aubry & Singh, 2008; Boneva et al., 2009; Nikolaides et al., 2002; Trau et al., 1996). This external electrical field will change the charge distribution on the surface of PS Spheres, leading to the change of the electric dipole field around PS spheres. It may also exert an additional electric force on the negatively charged PS spheres, which will affect the deformation of the surface. In a word, the external electric field will affect both the attraction force and the repulsion force mentioned above. It has been found that the inter-particle distance can be remarkably changed by the electric field (Nikolaides et al., 2002). As a result, the ordering of self-assembled PS spheres can be improved considerably, as shown in Fig. 1. In addition, the effect of an external electric field on the arrangement of PS spheres is more

Fig. 1. Self-assembled monolayer of PS spheres (5 x 5 m2) with an external electric field of, (a) 0 v/m, (b) 5 x 104 v/m. The diameter of PS sphere is 200 nm. The electric field is upward and perpendicular to the interface of water and air. The external electric field can efficiently

Another way to modify the balance between the attraction force and the repulsion force among PS spheres is to change the surface chemistry of PS spheres or the electrostatic environment of the water-air interface (Sirotkin et al., 2010). By adding some electrolyte, e.g. acid (H2SO4) and NaCl, into the water, effective surface charge density of PS spheres and /or effect of electric screen of PS spheres can be changed, which give rise to the change of

reduce defects in the regular arrangement of PS spheres, remarkably improving the

ordering of the self-assembled monolayer of PS spheres.

(Kralchevsky & Nagayama, 2000).

pronounced for smaller spheres.

the interaction among PS spheres. It has been found that the ordering of self-assembled PS spheres at interface can be considerably improved by adding suitable acid (H2SO4) in water (Sirotkin et al., 2010), as shown in Fig. 2. Given that the charges on PS spheres are related to the diameter of PS spheres,the suitable amount of acid or other electrolyte is dependent on the size of PS spheres。In addition, the temperature of the water to some degree also affect the self-assembly of the PS spheres at the interface. It was found that ordering of PS was improved on the water of ~ 4 oC. Such an improvement may be related to the increase of water surface tension and the suppression of the Brownian motion of the PS spheres and dust clusters in the water.

Fig. 2. Self-assembled monolayer of PS spheres (5 x 5 m2) at the interface of air and the solution of de-ionized water and H2SO4 with PH value of, (a) ~7, (b) ~ 5.3. The diameter of PS sphere is 240 nm. The H2SO4 can provide some additional ions around PS spheres, which can effectively change the interaction among PS spheres. Under certain PH value of the solution, considerable improvement of ordering of PS spheres can be made.

The self-assembled monolayer of PS spheres can be finally transferred to varieties of smooth substrates underneath the water by draining off the water. This process can be affected by some charges on the substrates. In addition, some cracks may appear once the monolayer was disturbed by movements of the water during draining. The PS spheres nearby the cracks slightly displaced from the ideal sites of a hexagonal lattice. In this case, the longrange ordering of the subsequent structure is degraded. The monolayer of ordered PS spheres on substrates can then serve as a mask for the subsequent fabrication of ordered nanostructures by deposition of varieties of materials or by etching.


**4. Acknowledgment**

Special thanks should be given to **Rector of National Aviation University (Kyiv, Ukraine) – Mykola Kulyk.** We would not have finished this chapter without his support.

## **5. References**

Other secure quantum technologies in practice have not been extended beyond laboratory experiments yet. But there are many theoretical cryptographic schemes that provide high information security level up to the information-theoretic security. QSDC protocols remove the secret key distribution problem because they do not use encryption. One of these is the ping-pong protocol and its improved versions. These protocols can provide high information security level of confidential data transmission using the existing level of technology with security amplification methods. Another category of QSDC is protocols with transfer qubits by blocks that have unconditional security, but these need a large quantum memory which is out of the capabilities of modern technologies today. It must be noticed that QSDC protocols are not suitable for the transfer of a high-speed flow of confidential data because there is low data transfer rate in the quantum channel. But when a high information security level is more important than transfer rate, QSDC protocols should

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**10**

*México*

**Web-Based Laboratory**

**Using Multitier Architecture**

Actuality, Internet provides a convenient way to develop a new communication technology for several applications, for example remote laboratories. The remote access to complex and expensive laboratories offers a cost-effective and flexible means for distance learning, research and remote experimentation. In the literature, some works propose platforms based on the Internet in order to access experimental laboratories; nevertheless it is necessary that the platform provides a good architecture, clear methodology of operation, and it must facilitate the integration between hardware (HW) and software (SW) elements. In this work, we present a platform based on "multitier programming architecture" which allows the easy integration of HW and SW elements and offers several schemes of tele-

The remote access to complex and expensive laboratory equipment represents an appealing issue and great interest for research, learning education and industrial applications. The range potentially involved is very large, including among others, applications in all fields of

It is well known that several experimental platforms are distributed in different laboratories in the world, and all of them are on-line accessible through the Internet. Since those laboratories require specific resources to enable a remote access, several solutions for harmonizing the necessary software and hardware have been proposed and described. Furthermore, due to their versatility, these platforms provide user services which allow the transmission of information in a simply way, besides being available to many people,

The potentiality of remote laboratories (Gomez & Garcia, 2007) and the use of the Internet, as a channel of communication to reach the students at their homes, were soon recognized (Basigalup et al., 2006; Davoli et al., 2006; Callangan et al., 2005; Imbre & Spong, 2006;

Several works based on remote experimentation, which are used as excellent alternatives to

access remote equipment, have been published (Costas et al., 2008).

presence: teleoperation, telecontrol and teleprogramming.

engineering (Restivo et al., 2009; Wu et al., 2008).

having many multimedia resources.

Rapuano & Soino, 2005).

**1. Introduction**

C. Guerra Torres and J. de León Morales *Facultad de Ingenieria Mecánica y Eléctrica Universidad Autónoma de Nuevo León*