**4. IOUS of the liver: malignant tumor**

IOUS finds its usefulness in liver surgery for both primary and secondary malignant lesions facilitating the detection, characterization of lesions and guiding the surgical procedure [26, 27]. Most studies have evaluated the role of IOUS for treatment of hepatocarcinomas and hepatic metastases due to colorectal cancer, these pathologies being considered the most common liver malignant lesions. Intraoperative detection and local treatment of these lesions may have a major impact in choosing surgical strategy [28, 29].

#### **4.1. Hepatocarcinoma (HCC)**

from homogeneous to heterogeneous, with their edges becoming irregular. These features make them more difficult to differentiate from malignant tumor formations. When surgery is indicated, IOUS has the role to localize and visualize the relationships of the hemangioma with the intrahepatic structures. The surgeon can trace the hepatic resection line outside the hemangioma, minimizing hemorrhagic risk, and preserving the healthy hepatic parenchyma to its full potential. The CE-IOUS can be useful, capturing the contrast agent by the hemangioma being most of the time characteristic. Differentiation from malignant tumor formation becomes difficult for arterial hem-

For *focal nodular hyperplasia*, the central location of a fibrous scar is characteristic. This tumor appears as a well-defined lesions with variable size, usually unique, of solid consistency and inhomogeneous structure. Rarely, the central scar can be distinguished when using simple ultrasound, without contrast agent. When using CE-IOUS, in the arterial phase, there is a central filling followed by a complete capture in the venous phase. At this stage, the center of the tumor becomes hypoechoic. In the late phase, the tumor remains isogenic together with

*Hepatic adenoma* appears ecographically as a well-defined solid tumor lesion; it may have an inhomogeneous structure in the presence of intratumoral hemorrhage. Doppler ultrasound does not detect a vascular signal. When using CE-IOUS, in the arterial phase, there is a centripetal and inconsistent capture; in the venous phase, a moderate washout may be noted. In

Differentiation between focal nodular hyperplasia and hepatic adenoma is important for establishing the therapeutic indication, surgery being indicated for large adenomas, due to

Because sometimes it is difficult to make a benign-malignant US differentiation, intraoperatory, when the situation imposes, might by necessary to make a bioptic puncture for establishing a correct diagnosis [20]. IOUS has an important guiding role, especially in the case of

*Simple hepatic cysts (biliary cysts)* are benign tumors with no malignant potential, usually asymptomatic, that can be easily diagnosed with ultrasound imaging. They are described by ultrasound as well-defined lesions with very thin walls, no Doppler signal, anechoic, with transonic content due to the liquid composition. Simple hepatic cysts have therapeutic indications only when they become symptomatic, often due to symptoms related to the mass effect

Percutaneous ultrasound guided treatment with cyst evacuation is often possible, but is followed by an increased risk of relapse, with the rebound of collection. In this idea, the laparoscopic surgical resection of the cystic dome is indicated. This technique is easy if the lesions are located superficially, in segments II, III, IVB, V, VI (after Couinaud) [23]. The lesions local-

Depending on the evolutionary stage, *hydatid cysts* may appear as single or multiple lesions, anechoic, with membranes and sediment inside, with thin or calcified walls. They may be multilocular or may contain multiple fluid compartments (daughter vesicles). IOUS helps the surgeon in finding the cysts and in some situations it can detect bile duct communication. These lesions

ized intraparenchymatous can be approached safely only when using IOUS [24].

the hepatic parenchyma, which strengthens the diagnosis of benign lesion [21].

the risk of rupture and hemorrhage as well as due to its malignant potential.

lesions located in the depth of the liver parenchyma, hard to reach when palpating.

the late phase, the appearance is isoechoic or hyperechoic [21].

they have on neighboring structures.

angiomas or for those with arterio-venous shunts [21].

76 Liver Research and Clinical Management

HCC is the most common primary malignancy in the liver, and is frequently associated with cirrhosis [30, 31].

Ecographically, this tumor has the appearance of a solid tumor with irregular contours, heterogeneous, uni-, or multilocular ("encephaloid form"). Typically, it invades the liver vessels, primarily the portal branches, but also the suprahepatic veins. Doppler screening usually highlights a high-speed arterial flow. Vessel distribution is irregular, disordered. CE-US shows hypercaptation in the arterial phase with a specific "washout" of contrast substance in the venous phase. In the late phase, the tumor appears as hypoechoic. This behavior is usually described in tumor nodules larger than 2 cm [21].

In the case of HCC, IOUS is superior in detecting lesions measuring less than 1 cm, preoperative MRI having a lower sensitivity and specificity for these lesions [11, 32]. It has also been shown in several studies that CE-IOUS can modify in 19–29% of the cases the initial treatment plan [33, 34]. CE-IOUS finds its usefulness especially in cirrhotic patients when it comes to differential diagnosis between malignant lesions and regenerative nodules [29, 35]. It has been demonstrated that neoangiogenesis of tumor nodules is a specific criteria for distinguishing hepatocarcinomas from dysplastic or regenerative nodules [35].

CE-IOUS has a sensitivity of 100%, a specificity between 69 and 100% and can modify the surgical strategy in up to 79% of patients [36–38], most frequently by detecting new lesions. The literature emphasizes that the filling pattern of the contrast agent in nodules found by IOUS can guide surgical resection [36]. It has also been shown that the vascular pattern of HCC visualized by using CE-IOUS has been associated with the expression of some genetic profiles, suggesting that CE-IOUS images can be used as an indicator for predicting prognosis of patients [39].

During hepatic resection, which is the standard treatment for HCC, particular attention should be paid in preserving as much hepatic parenchyma as we can, the remaining hepatic volume being an important prognostic factor for the short outcome [37, 39, 40]. Thus, local resection of the tumor formation or its ablation under IOUS guidance may be chosen to minimize the

Several studies in the literature have shown that after the surgical treatment of the primary tumor, the ultrasound of metastasis after colorectal cancer can be correlated with prognosis. Thus, Gruenberger et al. [52] demonstrated that in patients with hyperechoic ultrasound liver metastases, survival is longer than in those with the hypoechoic aspect of the lesions. This suggests that the role of IOUS is more than a diagnostic one and can be useful in establishing prognosis [53].

Intraoperative Ultrasound of the Liver: Actual Status and Indications

http://dx.doi.org/10.5772/intechopen.73856

79

The CE-IOUS applied for colorectal liver metastases has an 96% accuracy, in contrast to 74 and 79%, percentages associated with pre-operative CT and MRI [34, 54]. The fact that undetected preoperative liver metastases represent the main cause of recurrent neoplasia [55] highlights the important role that IOUS has in the management of patients diagnosed with colorectal

Chemotherapy is an important, standardized element in regard with the adjuvant and neoadjuvant therapy in colorectal cancer patients. [57, 58] Regarding hepatic metastases, good results of cytostatic treatment mean either stagnation or regression of these lesions [59, 60]. A particular situation is when liver metastases are no longer visible in CT and/or MRI performed after chemotherapy. Literature indicates that the complete, real response is found in up to 66% of cases [61, 62]. For the rest of the cases (34%), chemotherapy can affect the echogenity of the metastases making them difficult to be identified with preoperative imaging (CT, MRI, even IOUS) [13, 33]. In these situations, CE-IOUS allows the surgeon to check areas where hepatic lesions have been described before chemotherapy [11]. The role of this technique is highlighted in many studies that have shown that only be confirmation given by the CE-IOUS in regard with the lack of lesions can be associated with a complete therapeutic response [59, 62].

Resection or ablation of all lesions is the gold standard in the treatment of colorectal liver metastases [63]. Even in patients with unresectable metastases, local ablation or combination between ablation and surgical resection of the lesions has been shown to be able to locally control the disease [64]. It is obvious that IOUS plays a major role in liver surgery for the

The laparoscopic approach and minimally invasive surgery have more and more indications and thus the role of IOUS in laparoscopic surgery has become increasingly important. Of course, laparoscopic surgery has some disadvantages in assessing the liver because the surgeon loses the advantage of palpating the structures and lesions. IOUS manages to compensate for most of these laparoscopic minuses by providing intraoperative high utility imaging with greater sensitivity in detecting liver lesions than most preoperative imaging techniques [65–69]. Intraoperative laparoscopic ultrasound (LIOUS) has a sensitivity and specificity similar to that in open surgery [69]. Several authors have suggested routine use of LIOUS in laparoscopic colorectal surgery [70] and prior to planned laparotomies for liver resections [71]. In cases where hepatic disease is known, with the help of LIOUS data, around 64% of cases could be exempted from laparotomy [71, 72]. The success of the laparoscopic approach depends primarily on the location of the lesions [73, 74]. Guiding surgical maneuvers by the use of LIOUS is possible especially in superficial tumors on the left lobe or on the anterior segments of the right lobe (hepatic segments II, III,

cancer. This is why routine IOUS is recommended in these patients [56].

detection and localization of metastatic lesions [28].

**5. The role of laparoscopic approach**

**Figure 3.** Anatomical resection: ischemic delimitation of sixth and seventh liver segments (intraoperative aspect, from the personal archive of the authors).

volume of resected liver parenchyma, respecting the oncological resection margin. Also, in order to minimize the risk of postoperative complications (hemorrhage, necrosis of the liver parenchyma) and remote relapse (by satellite micrometastases, specific for HCC), the use of IOUS is vital in guiding anatomical resections. These involve the ultrasound identification of vascular pedicles corresponding to the affected hepatic segments and through various associated maneuvers (digital compression, injection of contrast agents) an exact delimitation of the targeted resection area can be obtained (**Figure 3**). More details will be given in the following rows, in the sub-section dedicated to the role of IOUS in guiding hepatic resections.

#### **4.2. Hepatic metastases**

Despite significant advances in preoperative staging diagnostic procedures (conventional CE-US, multi-sliced CT, CE-MRI, and PET-CT), studies have shown that 10–30% of the patients with colo-rectal cancer remain with undiagnosed hepatic metastases during primary tumor surgery [41–46].

In this respect, IOUS and CE-IOUS have a special role in completing the diagnosis, in addition to the liver's palpation technique. IOUS is considered the "gold standard" in open surgery for colorectal cancer since 1980, being able to detect liver metastases that cannot be palpated intraoperatively and that have not been visualized with preoperative imaging techniques [8, 47–50].

Liver metastases have a non-characteristic echographic appearance, being circumscribed lesions with imprecise or halo delineation, with a homogeneous or heterogeneous pattern. They may be solitary (usually liver metastases from colonic neoplasms) or multiple. Their echogenicity is variable. When they are large, they can compress the bile ducts (which may appear to be dilated) and the liver vessels. As for their vascularization, they may be hypovascular (in gastric, colon, pancreatic, or ovarian cancers) with hypoechoic pattern in arterial phase and similar in the venous and late phases or hypervascular (neuroendocrine tumors, malignant melanomas, sarcomas, renal tumors, breast, or thyroids), with a hyperechoic appearance during the arterial phase, with wash out during the venous phase and hypoechoic pattern at about 30 s after the injection of the contrast substance [51].

Several studies in the literature have shown that after the surgical treatment of the primary tumor, the ultrasound of metastasis after colorectal cancer can be correlated with prognosis. Thus, Gruenberger et al. [52] demonstrated that in patients with hyperechoic ultrasound liver metastases, survival is longer than in those with the hypoechoic aspect of the lesions. This suggests that the role of IOUS is more than a diagnostic one and can be useful in establishing prognosis [53].

The CE-IOUS applied for colorectal liver metastases has an 96% accuracy, in contrast to 74 and 79%, percentages associated with pre-operative CT and MRI [34, 54]. The fact that undetected preoperative liver metastases represent the main cause of recurrent neoplasia [55] highlights the important role that IOUS has in the management of patients diagnosed with colorectal cancer. This is why routine IOUS is recommended in these patients [56].

Chemotherapy is an important, standardized element in regard with the adjuvant and neoadjuvant therapy in colorectal cancer patients. [57, 58] Regarding hepatic metastases, good results of cytostatic treatment mean either stagnation or regression of these lesions [59, 60]. A particular situation is when liver metastases are no longer visible in CT and/or MRI performed after chemotherapy. Literature indicates that the complete, real response is found in up to 66% of cases [61, 62]. For the rest of the cases (34%), chemotherapy can affect the echogenity of the metastases making them difficult to be identified with preoperative imaging (CT, MRI, even IOUS) [13, 33]. In these situations, CE-IOUS allows the surgeon to check areas where hepatic lesions have been described before chemotherapy [11]. The role of this technique is highlighted in many studies that have shown that only be confirmation given by the CE-IOUS in regard with the lack of lesions can be associated with a complete therapeutic response [59, 62].

Resection or ablation of all lesions is the gold standard in the treatment of colorectal liver metastases [63]. Even in patients with unresectable metastases, local ablation or combination between ablation and surgical resection of the lesions has been shown to be able to locally control the disease [64]. It is obvious that IOUS plays a major role in liver surgery for the detection and localization of metastatic lesions [28].


**2. Literature Review**

The author of this paper studied the literature on the causes of financial distress in households in European, North and South American, and East Asian countries. The literature review showed that the causes of personal bankruptcy do not depend on the region where consumers are located. In Figure 1, a classification of causes that considers the influence of macro-economic, micro-economic, and social factors on personal bankruptcy risk is presented. The proposed classification considers the entire spectrum of factors leading to an increased risk of consumer bankruptcy as discussed in detail in literature review below.

**Figure 1.** The classification of personal bankruptcy causes. Source: based on the author's own studies.

Generally, the causes of bankruptcy can be divided into two groups. The first consists of exogenous causes. These are the factors shaping the economic conditions affecting a country's households. Households do not have any influence over them and must adapt to the rules they are given for functioning within their country's economy. There is no doubt that the macro-economic situation directly affects the financial standing of consumers, as it influences the availability and cost of credit and the prospective income levels and wealth of households. The second group of causes of bankruptcies includes endogenous causes that can be divided into micro-economic and social factors. Both types of endogenous factors are shaped by the decisions of households, which is why it is important to identify them, as they can then be managed by consumers, leading to a reduced risk of insolvency. It is also important to note that both groups of risk factors overlap, as shown in Figure 1. The social factors are also overlapped by micro- and macro-economic factors, because in the majority of cases these factors are strongly influenced by the environment. The level of education of the consumer is correlated to financial awareness. The health condition of consumer can be shaped by such factors as age, gender, and to some extent even income

level. The level of debts can be affected by religious beliefs as proved in the literature review below.

The results of many studies (e.g., [14–18]) have confirmed the existence of a strong relationship between households' level of financial vulnerability and the unemployment rate, as increasing unemployment results in borrowers being unable to meet their repayment obligations. Shocks to the GDP are the second factor among the macro-economic variables affecting households' insolvency risk. They disrupt the sources of household income, which decreases households' ability to repay their debts. This condition gradually increases the probability of experiencing financial distress, causing foreclosures on the collateral used for securing bank credit, such as homes or cars [19–22]. The third macro-economic factor is the interest rate. When the lending rate increases, the cost of credit card payments, purchase loans, and personal loans also increases, becoming a significant financial burden for borrowers [21,23,24]. The inflation rate directly affects the interest rate, but by influencing the financial situation of enterprises, it can also affect the unemployment rate, as has been discussed [25,26]. The last, most common macro-economic variable affecting the global risk of personal bankruptcy is the exchange rate. The exchange rate clearly has a direct impact on the economic situation of enterprises (both exporters and importers), but it should also be noted that households are also under its influence, either directly (in case of, for example, incurring debts in foreign currencies) or indirectly (through increases in the cost of living in the case of countries dependent on imported goods, e.g., petrol or gas).

Social scientists have long been interested in the determinants of individuals' financial well-being [27]. Most studies have highlighted the following six factors, which are believed to have the biggest influence on an individual's risk of personal bankruptcy [28–33]: level of education, marital status, gender, income level, length of employment, and degree of indebtedness. Caputo [34] studied the influence of marital status and gender on households' risk of financial distress in the USA. He found that formerly married persons (i.e., those who had separated or divorced) were 3.6 times as likely to declare bankruptcy as married persons and that single persons were 4.4 times less likely to declare bankruptcy than married persons. In his study, it was also proven that women were more likely than men to declare personal bankruptcy between 1986 and 2004 in the USA. Traditionally, a person's level of education, income level, and degree of indebtedness have been studied in the literature, with there being clear evidence to support the influence of these factors on an individual's probability of declaring insolvency [35].

Financial vulnerability may also be driven by factors other than macro- and microeconomic ones, including materialism and lifestyle behaviors that may be induced by irresponsibility or short-sightedness on the part of consumers. These may, in turn, dramatically increase the risk of consumers declaring personal bankruptcy. Household attitudes and behavioral characteristics are known to be related to debt-related decisions (e.g., the likelihood of carrying debt and the amount of debt) [36]. For example, spending behaviors, compulsive shopping, saving for a goal, expectations about future income and the economy (e.g., the interest and inflation rates), and credit-related attitudes are commonly reported to be associated with debt-related decisions [36–38]. Among the social factors that may cause personal bankruptcy are health issues, as consumers are sometimes unable to cover huge medical expenses, forcing them to mortgage their homes [11]. Several studies have also been focused on specifically analyzing the relationship between religion and risk-taking attitudes. One of the latest studies conducted in Germany showed that religiously affiliated individuals were, in general, more risk-averse than non-religious people. Furthermore, it was proven that Muslims in Germany exhibited fewer risk-taking behaviors in general than Catholics and Protestants [39].

Nowadays, households encounter an environment characterized by a high level of financial complexity, which requires not only financial knowledge but also appropriate consumption decisions depending on both the macro-economic situation and the consumer's predisposition to make decisions that do not increase their risk of insolvency. Figure 2 presents the common profiles of consumers' financial risk behaviors. This classification

considers a broad spectrum of factors, from level of education, income, and marital status to impatience, snobbery, and financial literacy. Based on consumers' demographic resources (low vs. high) and consumption behaviors, such as self-control and financial awareness (low vs. high), four quadrants to classify the risk-taking behaviors of households were identified.

**Figure 2.** The common profiles of consumers' financial risk behaviors. Source: based on [17].

The first quadrant (Figure 2) represents the consumers burdened with high-risk behaviors. Such households are characterized, on the one hand, by low levels of financial awareness and self-control, and, on the other hand, by the availability of few demographic resources (e.g., poor education, low income, undesirable jobs, etc.). Thus, quadrant I represents the worst situation for consumers. The opposite of quadrant I is quadrant IV, which represents the group of households that is the least financially vulnerable. The consumers in this group are well-educated, high-income persons with good financial knowledge and reasonable, controlled attitudes toward consumerism.

Although the consumers who fall into quadrants II and III are not as risk-prone as those in quadrant I, these households are also of key interest for risk analysts because of the disconnect between their levels of financial literacy and demographic resources, which leads to moderately risky behaviors. For example, the consumers who fall into quadrant III lack financial awareness, which can present an increased risk of experiencing financial hardships regardless of their demographic status (e.g., level of education or income) because they have a low degree of self-control in the context of consumerism. In turn, the households classified as belonging to quadrant II may have an abundance of demographic resources, but they may still be subject to the risk of insolvency when they lack financial awareness of their uncontrolled consumption.


**3. Results**

#### *3.1. AQ Produced from Di*ff*erent Chickpea Cultivars*

AQ prepared from di fferent chickpea cultivars showed significantly di fferent yields and moisture contents (Figure 1). Liquid AQ yields ranged from 70.90 g/100 g seed to 107.44 g/100 g seed, with the highest yield produced by CDC Luna and the lowest by CDC Leader. AQ moisture content ranged from 92.4% to 94.2%, with the highest moisture content in AQ produced by CDC Luna and the lowest by CDC Leader. Commercially, high yield AQ with low moisture content (high dry matter content) would be of greater economic value.

Colour and turbidity of AQ varied with chickpea cultivar (Figure 2). In the current study, CDC Leader, CDC Orion, CDC Luna and Amit are Kabuli class chickpea cultivars which normally have a white to cream–yellow colour seed, while CDC Consul is a Desi class chickpea with brown to fawn colours. AQ produced from CDC Leader and CDC Orion had similar colour and high turbidity. The AQ samples were pale yellow and cloudy liquids. Whereas, AQ produced from Amit was bright yellow and cloudy. Interestingly, AQ produced by CDC Luna had the lowest turbidity, and was nearly translucent and bright yellow. Also remarkable, AQ produced by CDC Consul was a dark brown colour, and had high turbidity. This dark brown colour might arise from tannins in the CDC Consul seed coat that may have migrated to the water during cooking [23]. AQ colour is also influenced by other water-soluble molecules in chickpea seed such as pigments, vitamins and other plant secondary metabolites. Chickpea contains pigments mainly falling into the carotenoids class (β-carotene, cryptoxanthin, lutein and zeaxanthin) as well as small amounts of chlorophyll [24]. Moreover, there are also water-soluble

vitamins in chickpea, such as thiamin, riboflavin, niacin, vitamin B6, folate and ascorbic acid [25]. Additionally, some flavonoid compounds, including anthocyanin, flavonols, isoflavones, flavonol glucosides, phlobaphenes, proanthocyanidin, leucoanthocyanidin and proanthocyanidin in the seed coat might contribute to AQ colour [26].

**Figure 1.** Fresh Aquafaba (AQ) yield (g/100 g seed) and moisture content (%) prepared from different chickpea cultivars from Crop Development Centre (CDC) in Saskatoon, SK, Canada. Means within the same property without a common letter (a–e) are significantly different according to Tukey's test.

**Figure 2.** (**A**) AQ and seed of different chickpea cultivars prepared in jars. From left to right: CDC Consul, CDC Luna, CDC Orion, CDC Leader, and Amit; (**B**) AQ separated from chickpea seed. From left to right: CDC Consul, CDC Luna, CDC Leader, CDC Orion, and Amit.

In general, hemicellulose [27] and cellulose [28] are probably disrupted by cooking for 30 min at 115–118 ◦C and autogenic pressure (70–80 kPa), leading to partial destruction of the chickpea cell wall and breaking bonds between lignin and hemicelluloses [29]. Therefore, AQ turbidity and colour is a result of the disruption of chickpea seed microstructure during cooking, leaching of organic compounds, pigments, proteins, sugars, starch and vitamins into the cooking water [30].

#### *3.2. AQ Emulsification Properties*

#### 3.2.1. AQ Emulsion Capacity

Today, healthier and nutritious foods are demanded by health-conscious consumers. Food oil-in-water emulsions, such as mayonnaise and salad dressing, are often avoided due to their high fat and cholesterol content. Plant-based protein fractions, including soybean and wheat proteins, are ingredients that may be used in replacing egg as emulsifiers in mayonnaise emulsion systems [31,32]. Nikzade et al. (2012) developed a combination of soy milk, gums and other stabilizers to replace egg in low cholesterol-low fat mayonnaise formulas [18]. The application and development of different ingredients in reduced fat/cholesterol salad dressing and mayonnaise have been summarized by Ma and Boye (2013) [33].

*EAI* values of AQ prepared from each of five chickpea cultivars were measured (Figure 3). *EAI* ranged from 1.10 ± 0.04 to 1.30 ± 0.05 m2/g with the highest *EAI* observed for AQ prepared from CDC Leader (1.30 ± 0.05 m2/g), while the lowest *EAI* occurred with CDC Orion (1.10 ± 0.04 m2/g). The *EAI*s of CDC Consul (1.21 ± 0.02 m2/g), CDC Luna (1.17 ± 0.07 m2/g), and Amit (1.20 ± 0.05 m2/g) were not statistically di fferent from each other.

**Figure 3.** Emulsion capacity and stability of AQ prepared from di fferent chickpea cultivars. Means within the same property without a common letter (a–c) are significantly di fferent according to Tukey's test.

#### 3.2.2. AQ Emulsion Stability

Emulsion stability of AQ from cooked whole seed of five chickpea cultivars was also investigated in this study (Figure 3). AQ emulsion stability ranged from 71.9 ± 0.8% to 77.1 ± 0.5%, with the highest emulsion stability also observed from AQ prepared by CDC Leader and Amit, while the lowest emulsion stability for AQ prepared by CDC Luna. The emulsion stability of AQ from CDC Orion (75.6%) and CDC Consul (74.7%) were not statistically di fferent.

As a novel water-soluble emulsifier, AQ can stabilize oil-in-water emulsions to prepare egg-free vegan food oil emulsions. Additionally, our results showed that AQ prepared from Kabuli type (CDC Leader) chickpea exerted the best emulsion capacity and stability compared to the other chickpea cultivars studied in this research. This indicates the potential for selecting CDC Leader to produce an AQ emulsifier. This di fference in emulsification properties between di fferent chickpea cultivars is probably related to the di fference in the physical properties and chemical compositions of chickpea seed which a ffect the mass transfer to the cooking water during cooking.

To better understand the emulsion properties di fferences of AQ prepared from di fferent chickpea cultivars, we studied the physiochemical properties of chickpea seed, and we tested for correlations between these properties and AQ emulsion properties.

#### *3.3. Chickpea Physical Properties*

Seed coat cracking after soaking and during cooking results from splitting of the outer cell wall layers. During chickpea cooking, this seed coat works as a membrane that controls mass transfer, which would a ffect the composition, and therefore, the functional properties of the resulting cooking water (AQ) [8]. Seed coat physical characteristics depend on the genotype and environmental conditions (temperature, soil and moisture) at the time of seed maturity or during storage. Physical characteristics of seed from di fferent chickpea cultivars are shown in Table 1. Significant di fferences were observed in *HSW*, *ED*, *SCI* and *WSA* among chickpea cultivars. *HSW* and *ED* ranged from 22.43 ± 0.08 g to 42.9 ± 0.3 g and 6.89 ± 0.5 mm to 8.49 ± 0.2 mm, respectively. CDC Leader exhibited heavier (42.90 ± 0.3 g/100 seed) and larger (8.49 ± 0.2 mm) seed compared with the other cultivars.

CDC Orion was not significantly (*p* > 0.05) di fferent from CDC Luna, except for *WSA* (10 ± 1 mg/cm2). CDC Consul exhibited the greatest *SCI* (11.2 ± 2%) and *WSA* (15 ± 1 mg/cm2). Amit showed the highest *SSA* (0.668 ± 0.09 mm2/mg). These observed values are comparable to results reported previously [19], where three Sicilian chickpea cultivars (Calia, Etna and Principe) were evaluated for their *HSW*, *ED*, *SCI*, *SSA* and *WSA*. In their study, the chickpea *HSW* value ranged from 31.3 g/100 seed to 48.8 g/100 seed. Three Sicilian chickpea cultivars had similar *ED* and *SCI* with an average of 7.8 mm and 5.78%, respectively. The *SSA* value di ffered among chickpea cultivars and ranged from 0.45 mm2/mg to 0.6 mm2/mg. The *WSA* value showed a wide range from 8.3 to 11.9 mg/cm2. These di fferences in the physical characteristics between di fferent chickpea cultivars will be reflected in the seed coat behavior during soaking and cooking and might explain the variation in AQ properties. CDC Leader exhibited a good seed weight (42.90 ± 0.3 g) with the lowest *SCI* (3.89 ± 0.3%) which might explain why AQ prepared from this variety has the highest dry material (7.6%) and emulsion capacity and stability. *SCI* reflects fiber content, seed coat thickness and compactness, which is correlated with the di ffusion resistance and leaching of soluble solids during soaking and cooking. The di fferences in the cookability of di fferent chickpea genotypes have been reported previously which they attributed to the di fference in the seed characteristics [8,19].

Data are expressed as mean ± SD (*n* = 3). Value within rows followed by the same letter (e.g., a, b, c, d) indicates no significant difference (*p* > 0.05) between varieties by Tukey's test. CDC: Crop Development Centre (Saskatoon, SK, Canada).

## *3.4. Hydration Kinetics*

Water absorption capacity during soaking is generally related to the physical properties of the seed. Hydration of the testa and swelling of the cotyledons soften the cell walls and change tissue permeability, reduce the cooking time, and affect mass transfer to the cooking water. The relationship between soaking time and cumulative values of water uptake (Figure 4) was described by a nonlinear iterative regression method with an exponential relationship (Equation (8)). The applied model fitted the experimental data with an R2 that was greater than 0.94 for all cultivars. Therefore, a single curve for water uptake was used in further analysis with all data combined. Soaking processes achieve rapid water uptake (*Hrate* = 0.38 g H2O g/min), and after soaking for 6 h, water absorbed reached 90% of seed dry weight. Subsequently, the water absorption rate declined until the hydrated seed weight was 2.06-fold greater than before hydration, where total hydration reached saturation at 1.06 g H2O g/dw (*Hmax*). Similar trends for chickpea seed hydration were described by several authors [19,34,35]. However, water content of chickpea seed exceeded 90% of total water imbibition after4h[19].

**Figure 4.** Chickpea seed water absorption kinetics. A common curve fitted all data (different chickpea cultivars and hydration solutions).

Statistical analysis revealed no significant differences among *Hmax*, except Amit seed which absorbed more water than the other chickpea cultivars (1.20 g H2O g/dw) in all soaking solutions (Tables 1 and 2). The modeled hydration rate (*Hrate*) of these five chickpea cultivars were mostly similar and ranged from 0.328 to 0.417 g H2O g/dw. Interestingly, the *Hmax* and *Hrate* of chickpea seed soaking in NaCl solution was slightly lower compared with that obtained in deionized water. However, seed of CDC Leader, exhibited similar *Hrate* in both deionized water and NaCl solution. Conversely, soaking seed in NaHCO3 solution increased *Hmax* for CDC Luna and *Hrate* for CDC Consul. These results are partially in agreement with previous reports that the presence of salt in soaking solution results in slowed seed hydration [36], but contrast with the findings of Avola and Patanè (2010) and Clemente et al. (1998), where no effect was observed on the hydration of chickpea seed soaking in salt solutions [19,34].

There were two possible explanations for increased *Hmax* results in NaHCO3 solution: (1) the osmotic pressure gradient across membranes of cotyledon cells was decreased [37], (2) there was an interaction of carbonate ions with biopolymers in cotyledon cells which might produce molecular unfolding with a possible exposure of new sites for water binding [38].

**Table 2.** Kinetic constants of the nonlinear regression analysis for chickpea seed hydration.

*Hmax*, Max hydration capacity; *Hrate*, initial hydration rate.

#### *3.5. Chickpea Chemical Properties*

The main chemical constituents (moisture, carbohydrate, protein, fat, ash and fibre content) of different cultivars of chickpea are summarized in Table 1. The moisture content of raw chickpea seed showed significant difference among chickpea cultivars (5.29 ± 0.01% to 10.7 ± 0.1%), with the highest moisture content for CDC Consul and the lowest for Amit. Carbohydrate was the main component in all of the samples, while protein was the second most abundant component. CDC Luna had the highest carbohydrate content (67.4 ± 0.7 g 100 g/dw), followed by Amit (66.8 ± 1 g 100 g/dw) and CDC Leader (65.4 ± 2 g 100 g/dw). It is important to note that CDC Orion had the lowest carbohydrate content, which might be correlated with the lowest emulsion properties observed for AQ prepared from this cultivar.

Protein content ranged from 18.3 ± 0.3 (CDC Luna) to 23.6 ± 0.08 g 100 g/dw (CDC Orion).  Chickpea ash content did not differ with genotype. The mean value of ash content was 3.0 g 100 g/dw. Crude fibre content ranged from 4.32 ± 1 g 100 g/dw to 8.59 ± 0.6 g 100 g/dw. These observations are in agreement with previous studies by Xu et al. (2014), Özer et al. (2010), and de Almeida Costa et al. (2006) for chemical composition of raw chickpea seed from different chickpea cultivars [39–41]. In addition, Khattak et al. (2006) analyzed protein and ash content of seven Kabuli chickpea cultivars, which ranged from 18.08 to 19.22% and 2.45% to 2.94% [42], and thus, was similar to the values in this study.


2.1.2. Turbine Operation

The path of the fluid is inverted with respect to direct operation: the entry coincides with the final diffuser of the machine and is identified by Section 4, while the discharge is identified by Section 1. The speeds are then calculated starting from the inlet section of the machine.


**2. Principles of tracer technology**

Exogenous and endogenous labelling techniques have been used to study the in vivo metabolism of an endogenous molecule, the tracee (see review [4]). In the exogenous method, the same molecule, in form of a usually radioactively labelled tracer, is introduced into the bloodstream [9]. In lipoprotein studies, this methodology first requires purification of the target molecule or particle and ex-vivo radiolabelling followed by reinfusion into the circulation. The physological integrity of the target molecule might, however, suffer from such procedure. Furthermore, in case of multiprotein complexes (which most lipoproteins are), the kinetics of individual protein components cannot be investigated by this approach. As an example, the investigation of in vivo kinetics of both protein components of Lp(a), as described in this article, to study its assembly mechanism would not be possible with the exogenous labelling approach.

In contrast, in endogenous labelling, a labelled precursor of the molecule of interest, in case of proteins usually a labelled amino acid, is used to label the target molecule by infusion into the circulation of a suitable proband. Ideally, the tracer can easily be detected and quantified, has the same kinetic behaviour as the tracee, and does not perturb the system. Usually, kinetic studies are performed in steady state, where the rates of input and output for a given unlabelled tracee substance are equal and time invariant. Thus, the information provided by the tracer reflects the behaviour of the tracee [10, 11]. At various times, the target protein or particle has to be purified from the blood of human probands and the amount of tracer is quantified to provide a kinetic curve. A mathematical model is then constructed to extract all the information contained in the kinetic curve. By fitting a model to the data, it is possible to calculate the parameters of the model that characterize the flux of molecules between kinetically homogeneous pools. For example, it is thus possible to investigate the whole pathway including production, conversion or catabolism of lipoprotein particles, information that cannot be obtained by static measurements alone.

The term stable isotope refers to a non-radioactive isotope of a given atom that is less abundant in a molecule within a biological system than the lightest naturally occurring isotope. The most common stable isotope used as metabolic tracer for apolipoprotein kinetic studies is [2H3]-leucine. Stable isotope tracers are much safer than radioactive tracers for both the study subject and the investigator. Furthermore, the duration of stable isotope experiments is normally less than 24 hours which is much shorter compared to radiotracer techniques which may need up to 14 days of examination [9].

#### **2.1. Tracer administration**

22 Lipoproteins – Role in Health and Diseases

laboratory animals such as mice and rats which – unless genetically modified or induced by special diet - do not develop atherosclerosis (see review [6]). The same argument is valid for investigations using cellular model systems. Since the liver is the central organ responsible for lipoprotein metabolism and primary human hepatocytes are of only limited use in research, most cellular studies in lipoprotein metabolism have been conducted in human hepatoma cells lines. These lines express, secrete and assemble a lipoprotein pattern which

For all these reasons, the in vivo investigation of metabolic pathways in human subjects is the ultimate approach to elucidate physiological or pathological functions of metabolites in the human body. Historically, such human kinetic studies were performed using radioactive tracers; this methodology is, however, nowadays of only restricted use. Therefore, stableisotope tracer kinetic studies in human subjects with clear advantages regarding safety and technical issues have replaced the radiotracer methods to become an important research tool for achieving a quantitative understanding of the dynamics of metabolic processes in vivo. The aim of this review is to shortly describe the methodology and illustrate how the approach has expanded our understanding of physiological mechanisms as well as the pathogenesis of disorders of human lipoprotein metabolism. We will then specifically address the assembly mechanism of the atherogenic Lp(a) complex and focus on the kinetics of apoB-containing lipoproteins in patients with chronic kidney disease. This patient group is well-known for its high risk for atherosclerotic complications and a 10- to 20-fold

Exogenous and endogenous labelling techniques have been used to study the in vivo metabolism of an endogenous molecule, the tracee (see review [4]). In the exogenous method, the same molecule, in form of a usually radioactively labelled tracer, is introduced into the bloodstream [9]. In lipoprotein studies, this methodology first requires purification of the target molecule or particle and ex-vivo radiolabelling followed by reinfusion into the circulation. The physological integrity of the target molecule might, however, suffer from such procedure. Furthermore, in case of multiprotein complexes (which most lipoproteins are), the kinetics of individual protein components cannot be investigated by this approach. As an example, the investigation of in vivo kinetics of both protein components of Lp(a), as described in this article, to study its assembly mechanism would not be possible with the

In contrast, in endogenous labelling, a labelled precursor of the molecule of interest, in case of proteins usually a labelled amino acid, is used to label the target molecule by infusion into the circulation of a suitable proband. Ideally, the tracer can easily be detected and quantified, has the same kinetic behaviour as the tracee, and does not perturb the system. Usually, kinetic studies are performed in steady state, where the rates of input and output for a given unlabelled tracee substance are equal and time invariant. Thus, the information provided by the tracer reflects the behaviour of the tracee [10, 11]. At various times, the target protein or particle has to be purified from the blood of human probands and the

is substantially different from the respective human counterpart [7].

increased cardiovascular mortality compared to the general population [8].

**2. Principles of tracer technology**

exogenous labelling approach.

A tracer can be administered intravenously as either a single bolus injection, a primed constant infusion (i.e., a constant infusion given immediately after a priming bolus), or as a combination of both. The tracer bolus administration offers superior dynamics compared with the primed constant infusion, because the enrichment curves (the tracer tracee ratios) after a bolus injection correspond to the impulse response of the system. It is therefore suitable to study components of lipoprotein metabolism with a slow rate of turnover. Another advantage of bolus administration is that it facilitates the determination of newly synthesized particles, as the intracellular precursor enrichment is greater at the start of the study. This argument therefore counts particularly when investigating kinetics of particle assembly, as described in 3.1.1. Practically, the bolus infusion is also most convenient for both subjects and investigators.

#### **2.2. Multicompartment models for data analysis**

Multicompartment modelling is a superior method to dissect the complexities of lipoprotein metabolism, and has been widely applied to systems in which material is transferred over time between compartments connected in a specific structure to permit the movement of material amongst the compartments [12].

Each compartment is assumed to be a homogenous entity within which the entities being modelled are equivalent. For instance, the compartments may represent different types of lipoprotein particles that are kinetically homogeneous and distinct from other material in the system. Very often, the data can be described by more than one model. To ensure that the best model is selected, it is necessary to carefully examine the fitting of the kinetic curve, to determine the precision of the parameter estimates, and to perform statistical tests to compare results obtained with different models. However, the complexity of a multicompartment model is usually a compromise for what is practically possible. A very simple model may not adequately describe the kinetic heterogeneity present within the system. A model that is too complex, on the other hand, will not be supported by

experimental data and, hence, will have little predictive value. Furthermore, even if the development of models is based on experimental data, several assumptions are required in order to derive the model that is to be used. Thus, mathematical models do not determine the kinetics of lipids directly; rather, they derive an indirect approximation.

The software SAAM (Epsilon Group, Charlottesville, VA, USA) has become the first choice for modelling lipoprotein kinetic studies. The SAAM II program was recently developed by SAAM Inst., Inc., Seattle, WA, USA, and is frequently used to analyse lipoprotein tracer data using compartmental models [13, 14]. The primary kinetic parameter resulting after modelling with SAAM II is the fractional synthesis rate (FSR) which, under steady state conditions, is identical to the fractional catabolic rate (FCR) and has the dimension of pools/day. The reciprocal value of FSR/FCR is called retention time (RT, given in days) and indicates the residence time of the investigated tracee (the target apolipoprotein in our cases) in the circulation. The product of FSR multiplied by the concentration of tracee is called production rate (PR) and is usually expressed as mg/kg body weight/day.


*Article* **MPC Polymer Promotes Recovery from Dry Eye via Stabilization of the Ocular Surface**

**Noriaki Nagai 1,\*, Shunsuke Sakurai 2 , Ryotaro Seiriki 1 , Misa Minami 1 , Mizuki Yamaguchi 1 , Saori Deguchi 1 and Eiji Harata 2**

**Abstract:** The polymer that includes 2-methacryloyloxy ethyl phosphorylcholine (MPC) is wellknown as an effectively hydrating multifunction agent. In this study, we prepared an MPC polymer (MPCP) using radical polymerization with co-monomers—MPC/Stearyl Methacrylate/*N*,*N*dimethylacrylamide—and evaluated the MPCP's usefulness for dry eye treatment using a rabbit model treated with *N*-acetylcysteine. The MPCP particle size was 50–250 nm, and the form was similar to that of micelles. The MPCP viscosity (approximately 0.95 mPa·s) was 1.17-fold that of purified water, and a decrease in the transepithelial electrical resistance value (corneal damage) was not observed in the immortalized human corneal epithelial cell line HCE-T cell (HCE-T cell layer). The MPCP enhanced the water maintenance on the cornea, and the instillation of MPCP increased the lacrimal fluid volume and prolonged the tear film breakup time without an increase in total mucin contents in the lacrimal fluid of the normal rabbits. The therapeutic potential of the MPCP for dry eye was evaluated using an *N*-acetylcysteine-treated rabbit model, and, in our investigation, we found that MPCP enhanced the volume of lacrimal fluid and promoted an improvement in the tear film breakup levels. These findings regarding the creation and characteristics of a novel MPCP will provide relevant information for designing further studies to develop a treatment for dry eyes.

**Keywords:** MPC polymer; dry eye; ocular surface; lacrimal fluid; mucin

#### **1. Introduction**

Dry eye disease is a complex and multifactorial ocular disease, and tear film instability, visual disturbance, and discomfort are commonly observed, with potential damage to the ocular surface [1]. It was reported that dry eye disease has multiple causes, such as air pollution, medication, androgen deficiency, contact lens usage, and excessive computer use [2]. Inflammation and enhanced osmolarity on the ocular surface are also important factors in the onset of dry eye disease [3]. It has also been reported that oxidant stress and aging are other factors that contribute to dry eye disease [4]. The prevalence of dry eye disease is estimated at approximately 5–50% of the global adult population [5], and it is expected that the economic burden from the disease will increase as the population ages [6].

Current treatments for dry eye disease aim to prevent the objective signs and clinical symptoms of the disease and recover the quality of life of the patients. The main approved treatments are as follows: Mucosta® (rebamipide ophthalmic suspension 2%, Otsuka Pharmaceutical, Tokyo, Japan) and Diquas® (diquafosol ophthalmic solution 3%, Santen Pharmaceutical, Osaka, Japan) in Asia; Xiidra® (lifitegrast ophthalmic solution 5.0%, Shire, Lexington, KY, USA) and Restasis® (Cyclosporine ophthalmic emulsion 0.05%, Allergan,

**Citation:** Nagai, N.; Sakurai, S.; Seiriki, R.; Minami, M.; Yamaguchi, M.; Deguchi, S.; Harata, E. MPC Polymer Promotes Recovery from Dry Eye via Stabilization of the Ocular Surface. *Pharmaceutics* **2021**, *13*, 168. https://doi.org/10.3390/ pharmaceutics13020168

Academic Editor: Yolanda Diebold Received: 13 January 2021 Accepted: 25 January 2021 Published: 27 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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/).

Irvine, CA, USA) in North America; and Ikervis® (CsA cationic emulsion, Santen Pharmaceutical, Osaka, Japan) in Europe. In addition, CequaTM (ananomicellar formulation of CsA 0.09%, Sun Pharmaceuticals, Mumbai, India) has been used in the US since 2018. CequaTM enhances lacrimal fluid production in patients with dry eye disease. Overall, these medicines are used to stabilize the tear film and/or prevent the inflammation of the ocular surface [7].

The polymer including 2-methacryloyloxy ethyl phosphorylcholine (MPC) is wellknown for being not only an antiadhesive and antithrombogenetic agent, but also a significant hydrating multifunction agent [8–10]. These characteristics are due to the polymer's phosphorylcholine group and the water structure surrounding the polar group. To date, soft contact lenses containing this MPC have been approved by the Food and Drug Administration (FDA) and Pharmaceuticals and Medical Devices Agency (PMDA). Hall et al. have reported that the dehydration of this soft contact lens is significantly less than that of other conventional lenses [11]. Eye drops, soft contact lens care products, oral care products, and cosmetics containing the MPC-BMA polymer have also been approved by the PMDA. Ayaki et al. reported that, after treatment with eye drops containing MPC polymer (MPC-BMA), cell viability rates were maintained at over 80%. Moreover, the conformation of proteins did not change, even when they were adsorbed on the surface or came into contact with the surface, and were similar to those of clinically approved artificial tear products [12]. In this way, the polymer including MPC, which is significantly hydrating and safe, could be used to improve the ocular surface and treat dry eye disease with the potential to be added to dry-eye drugs.

In this study, we prepared a novel MPC polymer using radical polymerization with co-monomers, MPC/stearyl methacrylate/*N*,*N*-dimethylacrylamide (MPCP), and evaluated the polymer's usefulness for dry eye treatment using a rabbit model treated with *N*-acetylcysteine.

#### **2. Materials and Methods**

#### *2.1. Animals*

Male adult rabbits (weight 2.58 ± 0.75 kg) were obtained from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan), and the protocol was approved by the Kindai University (KAPS-31-002, 1 April 2019). The experiments using the rabbits were performed according to the Association for Research in Vision and Ophthalmology (ARVO) and Kindai University guidelines. Thirty microliters of 0.1% MPCP were instilled in single applications at 14:00 h. For repeat applications, 0.1% MPCP (30 µL) was instilled once a day (14:00 h) for 5 days, and the measurement of the levels of lacrimal fluid, mucin, tear film breakup time (TBUT), ocular surface, and tear film breakup began at 16:00 h (single application) and 18:00 h (repetitive application). The dry eye model using rabbits was conducted via the instillation (30 µL) of 10% *N*-acetylcysteine six times per day (at 9:00, 11:00, 13:00, 15:00, 17:00, and 19:00 h). This protocol was performed following our previous report [13].

#### *2.2. Chemicals*

Dulbecco's modified Eagle's medium/Ham's F12, penicillin, streptomycin, and fetal bovine serum were provided from GIBCO (Tokyo, Japan). Cell Count Reagent SF was purchased from Nacalai Tesque, Inc. (Kyoto, Japan), and the Tear mucin assay ELISA kit was obtained from Cosmo Bio Co., Ltd. (Tokyo, Japan). Transwell-ClearTM (polyester filters, surface area 1.0 cm2 and 0.4 µm pore size) and rat tail collagen type 1 were purchased from Costar (Cambridge, MA, USA) and Sigma (Tokyo, Japan), respectively. All other chemicals used were of the highest purity commercially available.

#### *2.3. Preparation of MPCP*

MPCP was obtained via radical polymerization with co-monomers, MPC/stearyl methacrylate/*N*,*N*-dimethylacrylamide, with a composition ratio of 50:5:45, and purified using the dialysis method. After polymerization, we measured the residual monomers and calculated that the conversion rate of this polymer in each monomer was >99%. We thus confirmed that the target polymer was obtained. In this study, we used MPCP diluted to a 1% aqueous solution with purified water. To confirm the MPCP concentration of the aqueous solution, we studied the residue upon drying. The structural formula of the MPCP is illustrated in Figure 1.

**Figure 1.** The structural formula for MPCP in this study. The MPCP based on MPC/stearyl methacrylate/*N*,*N*dimethylacrylamide was prepared via radical polymerization. MPC—2-methacryloyloxy ethyl phosphorylcholine; MPCP— MPC polymer.

#### *2.4. Measurement of Characteristics in MPCP*

A NANOSIGHT LM10 (Quantum Design Japan, Tokyo, Japan) was used to measure the size and number of MPCP nanoparticles—the measurement was performed for 60 s at 405 nm. Atomic force microscopy (AFM) images were obtained via an A SPM-9700 (Shimadzu Corp., Kyoto, Japan), and AFM images were created by combining the phase and height images. The viscosity of MPCP was measured with an SV-1A at 10–40 ◦C (A&D Company, Limited, Tokyo, Japan) [14]. To confirm the wettability, defined as the tendency of one fluid to spread on or adhere to a solid surface, we performed contact angle measurements in 0.1% MPCP and 0.1% MPC-BMA aqueous solutions. In total, 1 µL of each of the 0.1% MPCP and 0.1% MPC-BMA aqueous solutions (or purified water as a control) was dropped onto a slide glass, and the contact angle was measured using a contact angle meter (DropMaster500, Kyowa Interface Science Co., Ltd., Saitama, Japan).

#### *2.5. Cell Culture and Treatment*

The immortalized human corneal epithelial cell line (HCE-T cell) used in this study were developed by Araki-Sasaki et al. [15]. The HCE-T cells were cultured in Dulbecco's modified Eagle's medium/Ham's F12 with heat-inactivated fetal bovine serum (5%), penicillin (1000 IU/mL), and streptomycin (0.1 mg/mL).

#### *2.6. Measurement of Cell Adhesion*

The MPCP treatment was carried out by seeding HCE-T cells (1 × 104 cells) in a culture medium containing 0.1% MPCP and incubating the medium for 12 h. Then, a Cell Count Reagent SF was added, and the absorbance (Abs) at 450 nm was measured according to the manufacturer's protocol. The Cell Count Reagent SF is based on the conversion of the reagent to formazan salts according to mitochondrial activity and, as a metabolic assay, is related to the cell number and its metabolic state and efficiency. Therefore, the cell number was also measured via counting under a microscope (Olympus Corporation, Tokyo, Japan), and the cell adhesion was evaluated using a combination of data on the cell

number and Cell Count Reagent SF. The cell adhesion (%) was represented as the Abs ratio of the MPCP treatment and non-treatment [16].

#### *2.7. Measurement of Cell Proliferation*

The cell cultures were treated with MPCP 1 d after seeding (1 × 104 cells) by changing to a culture medium with 0.1% MPCP and incubating the medium for 24 h. Then, the Cell Count Reagent SF was added, and the Abs at 450 nm was measured according to the manufacturer's protocol. Cell proliferation (%) was recorded as the Abs ratio of the MPCP treatment and non-treatment [16]. The cell proliferation was also evaluated by a combination of the data on the cell number and Cell Count Reagent SF in this study.

#### *2.8. Preparation of HCE-T Cell Layer Model*

The cell layer models (multilayer) consisting of one cell (only HCE-T cells) were cultured by following our previous reports [17,18]. The HCE-T cells were seeded onto Transwell-ClearTM (90,000 cells/cm2 ) coated with rat tail collagen type 1 (71.5 µg/cm2 ) and grown for seven days until the cells reached confluency. The HCE-T cells were then exposed to an air–liquid interface for two weeks with the culture medium containing the vehicle or 0.1% MPCP replaced every other day. In this process, 50 µL of the medium solution with or without MPCP was dropped onto the donor side twice a day (9:00 and 19:00 h) so that the donor would not dry completely. In this study, chopstick electrodes connected to an epithelial Volt–Ohm meter Millicell-ERS (Millipore Co., Bedford, MA, USA) were used to measure the transepithelial electrical resistance (TER) and followed the differentiation stages during cultivation.

#### *2.9. Cell Toxicity of MPCP*

Differentiated cells with TER values greater than 350 Ω·cm2 were used for the cell toxicity analysis. The HCE-T cell layer model was treated with the vehicle and 0.1% MPCP, and the changes in TER values were measured for 60 min [17].

#### *2.10. Measurement of Water Retention in the Cornea*

The rabbits were euthanized by injecting a lethal dose of pentobarbital, and the corneas were carefully separated from other ocular tissues. The individual corneas were treated with the vehicle, 0.1% MPC-BMA, and 0.1% MPCP for 1 min. After that, the cornea was placed on a plastic cell, and the changes in the weight and number of MPCP particles were measured at 22 ◦C. In this study, the changes in weight were expressed as water retention in the cornea. Moreover, the samples (vehicle, MPC-BMA, and MPCP) on the cornea were collected by pipette, and the number of MPCP particles in the samples was measured with NANOSIGHT LM10, as described above.

#### *2.11. Monitoring the Ocular Surface of Rabbits Instilled with MPCP*

The ocular surface was monitored by using a dry eye monitor DR-1 (KOWA Co., LTD., Aichi, Japan), and the TBUT and changes in the ocular surface were monitored following our previous study [13]. Each rabbit treated with a fluorescein strip was allowed to blink several times to distribute the fluorescein. The time from the opening of the eyes to the appearance of the first dry spot in the central cornea was analyzed. The changes in tear film after blinking were monitored by the dry eye monitor DR-1. The TBUT and the tear film breakup level changes in the ocular surface were evaluated 2 h (16:00 h) and 4 h (18:00 h) after the application of the MPCP, respectively. The tear film breakup levels (area) were measured 2 s after the last blink using the Image J software (ver. 1.51, NIH, USA), and the measurement was performed three times; the mean was used as the value.

#### *2.12. Lacrimal Fluid and Mucin Levels in Rabbits Instilled with MPCP*

The volume of lacrimal fluid in rabbits instilled with MPCP was measured using Schirmer tear test strips (AYUMI Pharmaceutical Corporation, Tokyo, Japan), and the

mucin was measured using a Tear mucin assay ELISA kit according to the manufacturer's instructions. Briefly, lacrimal fluid was collected with Schirmer tear test strips, and the Schirmer tear test strips were added into elution buffer of a tear mucin assay ELISA kit to extract the mucin. After that, the extracted mucin was measured by the Tear mucin assay ELISA kit, and a fluorescence microplate reader (Absorption/Emission = 336/383 nm) [13]. The mucin levels in the total lacrimal fluid volume of the eye are expressed as total mucin content (µg). The mucin concentration in lacrimal fluid (mg/mL) is estimated from the mucin level/lacrimal fluid volume.

#### *2.13. Statistical Analysis*

Differences between the mean values were analyzed with an ANOVA, followed by Student's *t*-test, and Dunnett's multiple comparisons test was used for the statistical analysis, where *p* < 0.05 was considered significant. The data are expressed as the mean ± standard error (S.E.).

#### **3. Results**

#### *3.1. Design of the MPCP*

Figure 1 shows the structural formula of the MPCP. We designed the MPCP structure based on three points: (1) the MPC, indicating the zwitterionic group, shows the hydrophilic part and gives hydrophilicity to MPCP; (2) stearyl methacrylate including the long-chain alkyl group shows its hydrophobic part and forms a hydrous polymer nano-sphere in an aqueous solution; (3) coupled with the acryl group as a highly reactive functional group, *N*,*N*-dimethylacrylamide accelerates the polymerization between MPC and stearyl methacrylate and strengthens the structure of polymer nano-sphere in an aqueous solution. Figure 2A,B shows the particle distribution and an AFM image of the MPCP. This polymer includes monomer forms similar to micelles (polymer nanosphere), with a hydrophilic outer-most layer and a hydrophobic inner-most layer in an aqueous solution, featuring a particle size of from 50 to 250 nm. In addition, the particle number was 349 ± 0.336 (×108 particles/mL). Figure 2C,D shows the viscosity of MPCP at 10–40 ◦C. The viscosity of MPCP was 1.17- and 0.92-fold that of the purified water and MPC-BMA reported previously (preexisting MPC polymer) [12], respectively, and the viscosity levels of MPCP were similar under 10–40 ◦C conditions. We also measured the hydrophilicity of MPCP and MPC-BMA. The contact angle using MPCP was 25.5 ± 1.3◦ (*n* = 3), the contact angle using MPC-BMA was 26.2 ± 2.0◦ (*n* = 3), and the control was 29.2 ± 0.4◦ (*n* = 3). These results indicate that the MPCP was slightly hydrophilic and had a higher wettability than the MPC-BMA and the control.

#### *3.2. Changes in Cell Conditions in the Immortalized Human Corneal Epithelial Cell Line (HCE-T Cell) Treated with MPCP*

Figure 3A,B shows the effects of MPCP treatment on cell adhesion and growth in the HCE-T cells. No significant differences were observed between the cell adhesion and growth of HCE-T cells treated with and without MPCP. Figure 3C shows the effect of MPCP treatment on the TER during the HCE-T cell layer model preparation. The TER values of HCE-T cells increased to over 380 Ω·cm2 when we exposed them to an air–liquid interface for two weeks. The TER in the MPCP-treated HCE-T cells was similar to non-treated values before exposure to the air–liquid interface; however, during exposure to the air–liquid interface, the TER values in the MPCP-treated HCE-T cells were significantly higher than those found in the HCE-T cells without MPCP treatment. Figure 3D shows the cell toxicity caused by MPCP treatment. The cell toxicity caused by the MPCP was mild, since the decrease in TER in the MPCP-treated HCE-T cells was similar to that in the vehicle-treated HCE-T cells.

**Figure 2.** Particle size and viscosity of the MPCP. (**A**,**B**) Particle distribution (**A**) and atomic force microscopy (AFM) images (**B**) of MPCP. (**C**) Image of MPCP. (**D**) Changes in the viscosity of the MPCP at 10–40 ◦C. *n* = 8–10. The particle size of MPCP was 50–250 nm, and the viscosity of MPCP was 0.95 mPa·s at 20 ◦C. The temperature (10–40 ◦C) did not affect the viscosity.

**Figure 3.** Corneal epithelial cell adhesion, growth, and stimulation of MPCP in the immortalized human corneal epithelial cell line (HCE-T cells). (**A**) Effect of MPCP on the adhesion of HCE-T cells. (**B**) Effect of MPCP on the growth of HCE-T cells. (**C**) Changes in transepithelial electrical resistance (TER) values during the formation of the HCE-T cell layer model treated with MPCP. The arrow indicates the beginning of the air–liquid interface. (**D**) Effect of MPCP on the TER values in the HCE-T cell layer model. *n* = 10–12. \* *p* < 0.05 vs. the vehicle for each group. The treatment with MPCP did not affect the cell adhesion and growth of the HCE-T cells, and no stimulation was observed; however, the TER values in the HCE-T cell layer model were enhanced by the MPCP treatment.

#### *3.3. Effect of MPCP on the Ocular Surface Stability in the Normal Model*

As shown in Figure 3, the treatment with MPCP enhanced cell stability in the HCE-T cell layer model. We investigated whether the ocular surface stability in a normal rabbit was increased via the instillation of MPCP. The lacrimal fluid volume and TBUT were significantly increased via treatment with MPCP (Figure 4A,D); however, the mucin concentration in the lacrimal fluid decreased in the MPCP-instilled rabbits; for the total mucin levels in the lacrimal fluid, no difference between the vehicle- and MPCP-treated groups was found (Figure 4B,C). On the other hand, the lacrimal fluid volume (18.4 ± 1.9 µL, *n* = 5) and TBUT (24.1 ± 2.0 s, *n* = 5) in the rabbits instilled with MPC-BMA tended to increase, although both the lacrimal fluid volume and TBUT were significantly lower than those in the MPCP. Figure 5 shows the effects of MPCP on moisture retention in the excised rabbit cornea. The water content in the cornea instilled with the vehicle decreased with time and completely evaporated after 90 min. The MPCP instillation prolonged the time needed for water evaporation from the cornea and the number of MPCP nanoparticles to decrease on the cornea. Preexisting MPC-BMA prolonged the time needed for water maintenance in comparison with the vehicle, but this ability was significantly lower than that in MPCP.

**Figure 4.** Effect of a single application of MPCP on lacrimal fluid volume, mucin levels, and tear film breakup time (TBUT) in normal rabbits. (**A**,**B**) Changes in lacrimal fluid volume (**A**) and mucin concentration in lacrimal fluid (**B**) after the application of MPCP. (**C**) Changes in total mucin levels in the lacrimal fluid after the application of MPCP. (**D**) Changes in the TBUT in rabbits instilled with the vehicle and MPCP. These measurements were performed 2 h after the instillation of MPCP (16:00). *n* = 6–8. \* *p* < 0.05 vs. the vehicle for each group. Although the application of MPCP induced an increase in lacrimal fluid volume and TBUT in the rabbit eye, the total mucin contents in the rabbits instilled with MPCP were similar to those in the vehicle.

#### *3.4. Therapeutic Potential of the MPCP for Dry Eye Disease*

Next, we investigated the usefulness of MPCP as a therapy for dry eye using the *N*-acetylcysteine-treated rabbit model (Figure 6). The levels of lacrimal fluid and mucin decreased under treatment with *N*-acetylcysteine, with levels 0.8- and 0.67-fold greater than those of normal rabbits, respectively (Figure 6A,B). Both the lacrimal fluid volume and mucin levels were approximately 2.5-fold greater in comparison with the control group (non-instillation group) (Figure 6A,B). Figure 6C,D show the changes in the levels of tear film breakup in the *N*-acetylcysteine-treated dry eye model rabbits instilled with (or without) MPCP. Strong tear film breakup levels were observed and still persisted five days later. Repeat treatment with MPCP attenuated the tear film breakup levels five days

post-*N*-acetylcysteine treatment, and the therapeutic effect of MPCP was significantly higher than that in MPC-BMA (tear film breakup levels at 5 days, 4.26 ± 1.09 mm2 , *n* = 5).

**Figure 5.** Changes in moisture retention in the cornea instilled with the vehicle, MPC-BMA, and MPCP. (**A**) Image of the cornea at 0 and 60 min after the instillation of MPCP. (**B**) Moisture retention curve in the extracted cornea instilled with the vehicle, MPC-BMA, and MPCP for 150 min. (**C**) the number of nanoparticles on the cornea at 10 min after the instillation of MPCP. Vehicle, vehicle-treated cornea. MPC-BMA, MPC-BMA-treated cornea. MPCP, MPCP-treated cornea. *n* = 5–7. \* *p* < 0.05 vs. the vehicle for each group. # *p* < 0.05 vs. MPCP at 0 min. The water content in the cornea instilled with MPCP was higher than that found in the cornea instilled with the vehicle, and the time needed for water maintenance was prolonged in comparison with the vehicle and MPC-BMA. The number of MPCP particles on the cornea decreased after treatment.

**Figure 6.** The therapeutic effect of the repetitive application of MPCP on dry eye in the *N*acetylcysteine-treated rabbit model (dry eye model). (**A**,**B**) The effect of MPCP on the lacrimal fluid volume (**A**) and mucin levels (**B**) in the dry eye model. The mucin levels in the lacrimal fluid are expressed as the ratios of the mucin contents at the start of the experiment. (**C**) Images of the ocular surface in the dry eye model after repetitive applications of MPCP. The bar indicates 1 mm. The dark spots reflect the tear film breakup. (**D**) Effect of MPCP on tear film breakup levels in the dry eye model. Rabbits were instilled with MPCP at 14:00 h once a day for five days, and the experiments were performed at 18:00 h. This protocol was performed by following our previous reports [13]. *n* = 9–12. N.D., not detectable. \* *p* < 0.05 vs. none for each group. The application of MPCP increased the volume of lacrimal fluid and normalized the decreased mucin levels in the dry eye model. The levels of tear film breakup were attenuated by the application of MPCP.

#### **4. Discussion**

In this study, we designed a novel MPCP that includes the zwitterionic group stearyl methacrylat, and the acryl group, and found that the MPCP has a high affinity for water and moisturizes the ocular surface in comparison with the MPC-BMA reported previously (preexisting MPC polymer) [12]. We also showed that the instillation of MPCP appears to provide a useful therapy for dry eye (Figure 7).

**Figure 7.** Schematic illustration for the amelioration of dry eye via the instillation of the MPCP. DMA—*N*,*N*dimethylacrylamide; SMA—stearyl methacrylate.

First, we designed MPCP and evaluated its characteristics. MPC/stearyl methacrylate/*N*,*N*-dimethylacrylamide was used to prepare the MPCP in this study, and MPCP was purified using the dialysis method. The over-30-nm nanoparticles reported previously were not detected in the MPC-BMA [12]. In contrast with the results for MPC-BMA, this polymer (MPCP) included monomer forms similar to micelles (polymer nanosphere), with particle sizes of 50–250 nm (Figure 2A,B). The viscosity of MPCP was 0.92-fold that of MPC-BMA and was not different under 10–40 ◦C conditions (Figure 2D). On the other hand, the contact angle value of MPCP tended to decrease compared to that of the MPC-BMA. These results suggest that the MPCP has different physicochemical properties from preexisting MPC-BMP due to the formation of nanoparticles.

Next, we investigated the effects of MPCP on corneal epithelial cells using HCE-T cells. We previously reported that MPC-BMA does not affect the cell adhesion and growth of HCE-T cells, with little toxicity in HCE-T cells [19]. The MPCP did not affect the cell adhesion and growth of the HCE-T cells (Figure 3A,B), and no cell toxicity was observed (Figure 3D); however, we know that the barrier properties of the HCE-T cell layer model are very similar to those in rabbit corneas, and an increase in the barrier properties of the HCE-T cell layer model is expressed as an enhancement of the TER value [15,18]. To prepare the HCE-T cell layer model, the cells were exposed to air in order to increase the TER value, as this step was important to instill the medium solution to the cells twice a day (9:00 and 19:00 h), ensuring that the cell layer model would not dry completely. As a result, we demonstrated that the stabilization of the cell layer model increased via the instillation of a drop of MPCP to the therapeutic target. The MPCP enhanced the TER values of HCE-T cells during exposure to the air–liquid interface (Figure 3C). Taken together, the MPCP may attenuate the desiccation on the donor side, resulting in the enhancement of the TER value in the HCE-T cell layer model.

Further, we investigated the changes in the ocular surface in normal rabbits instilled with MPCP (Figure 4). The instillation of MPCP enhanced the lacrimal fluid volume and prolonged the TBUT (Figure 4A,D). These results show that the instillation of MPCP stabilized the ocular surface conditions; thus, the mucin level relates to the increase in

lacrimal fluid and TBUT. The mucins composed of numerous sugar chains linked to an apomucin, which is a core protein, were heavy molecular glycoproteins, and 50–80% of their mass was comprised of carbohydrates. It was reported that mucin leads to the formation of a smooth spherical surface, offering good vision, the provision of a barrier for the ocular surface, lubrication of the ocular surface to facilitate smooth blinking, and maintenance of the lacrimal fluid on the ocular surface [20,21]. Moreover, the mucin layer spreads lacrimal fluid over the surface of the eye by decreasing the surface tension of the water content. In addition, the mucin can remove foreign materials and prevent damage and infections in the eye [22]; therefore, an increase in mucin may enhance the lacrimal fluid volume and TBUT. Based on previous studies, we also measured the mucin levels in the lacrimal fluid of normal rabbits instilled with MPCP. Against all expectations, the total mucin content in the lacrimal fluid of the MPCP-instilled rabbits was similar to that of the vehicle-instilled rabbits, and the concentration of lacrimal fluid of the MPCP-instilled rabbits was lower than that of the vehicle-instilled rabbits (Figure 4B,C). These results suggest that MPCP is not affected by mucin production, since the total mucin levels were similar in rabbits instilled with or without MPCP. On the other hand, the mucin levels may be diluted by the enhanced lacrimal fluid volume via MPCP instillation, resulting in an apparent reduction in the mucin concentration in lacrimal fluid. To understand this result, we investigated whether MPCP enhanced the moisture retention in the excised rabbit cornea, and the results showed that the MPCP instillation prolonged the time needed for water evaporation from the cornea (Figure 5A,B). Although the preexisting MPC-BMA [12] also prolonged the time needed for water maintenance in comparison with the vehicle, this ability was significantly lower than that in MPCP. These results suggest that the preexisting MPC-BMA and MPCP exert similar effects. However, the MPCP has a strong ability to retain water on the cornea in comparison with MPC-BMA, and this high water affinity may enhance the lacrimal fluid volume and TBUT in normal rabbits. Moreover, this water affinity on the cornea may be enhanced by the formulation of hydrous polymer nano-spheres and polymerization of the MPC, since the time needed for water maintenance was prolonged in comparison with the MPC-BMA.

We found that the number of MPCP nanoparticles decreased on the cornea after treatment (Figure 5C). The MPCP may bind to the cornea, resulting in a decrease in forms like micelles in the solution. Further studies are needed to demonstrate the binding of MPCP on the cornea.

It is important to elucidate the therapeutic effect of MPCP in a dry eye model. The *N*-acetylcysteine, a reducing agent and mucolytic [23], acts on corneal and conjunctival epithelial cells and decreases mucin production and retention. Further, the instillation of 20% *N*-acetylcysteine caused a decrease in the mucin layer in the conjunctiva and cornea, desquamation of conjunctival and corneal epithelial cells, elimination of microvilli [24], and decreased thickness of the tear fluid layer [25]. Because of these actions, a rabbit model treated with *N*-acetylcysteine is widely applied as a mucin-reduced model and may be useful for evaluating the therapeutic effect of the MPC polymer for dry eye. Therefore, we treated a rabbit model with *N*-acetylcysteine and measured the effect of MPCP on the ocular surface. First, we attempted to measure the tear film break-up time (as in Figure 4D). However, the tear film in the *N*-acetylcysteine-treated rabbit model was broken 1–2 s after the last blink, so that the TBUT could not be detected. Therefore, we measured the break up level to evaluate the therapeutic effect of the repetitive application of MPCP (Figure 6). The instillation of *N*-acetylcysteine decreased the lacrimal fluid volume and tear film breakup level (Figure 6). The instillation of MPCP significantly increased both the lacrimal fluid volume and tear film breakup level in comparison with the vehicle and MPC-BMA (Figure 6A,C,D). In this study, we showed that the MPCP has a strong ability to retain water on the cornea in comparison with MPC-BMA (Figure 5). The in vitro study illustrated in Figure 5 supports the in vivo study shown in Figure 6. In contrast with the results of the normal rabbits (Figure 4), the mucin levels were found to be approximately 2.5-fold greater when compared with the control (non-instillation group) (Figure 6B). Urashima et al. reported that *N*-acetylcysteine reduces the mucin-like substances in the cornea and conjunctiva and that the mucin levels are enhanced by positive feedback during the healing process [26]. Therefore, mucin production in rabbits instilled with MPCP may be overexpressed, since MPCP accelerates the normalization of the ocular surface in the dry eye model by keeping the tear film on the cornea. Taken together, we hypothesized that the MPCP would show a high affinity for lacrimal fluid on the cornea after instillation and prolonged moisture retention in the rabbit cornea, resulting in an acceleration of the improvement in tear film breakup levels. In addition, normalization of the ocular surface conditions may induce high mucin production in dry eye models.

In this study, rabbit body weight did not change with the repetitive application of MPCP over 30 d (once a day), although it remains important to assess the systemic parameters and pharmakokinetics in the rabbit model. In addition, further studies are needed to apply the MPCP as dry eye drops. In future work, we intend to measure the therapeutic effect under a combination of MPCP and commercially available eye drops, such as Mucosta®, Diquas®, Xiidra® Restasis®, and Ikervis®, in a rabbit model treated with *N*-acetylcysteine.

#### **5. Conclusions**

In this study, we prepared a novel MPCP using MPC/stearyl methacrylate/*N*,*N*dimethylacrylamide (50:5:45) and demonstrated that this MPCP provides high water affinity in comparison with the preexisting MPC polymer. The instillation of the MPCP prolonged the retention of lacrimal fluid in the eye and enhanced the TBUT in the rabbits. In addition, the MPCP accelerated the normalizing of the ocular surface in the dry eye model by keeping the tear film on the cornea. Moreover, the therapeutic effect of MPCP was significantly higher than that of the MPC-BMA reported previously [12]. Thus, this MPCP may provide an effective therapeutic treatment for dry eye.

**Author Contributions:** conceptualization, N.N. and S.S.; data curation, R.S., M.M., M.Y., and S.D.; formal analysis, R.S., M.M., M.Y., and S.D.; funding acquisition, N.N.; investigation, S.S., R.S., M.M., M.Y., and E.H.; methodology, N.N., S.S., and E.H.; supervision, N.N. and E.H.; visualization, N.N. and S.S.; writing—original draft, N.N. and S.S.; writing—review and editing, N.N. and S.S. All authors have read and agreed to the published version of the manuscript.

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

**Institutional Review Board Statement:** The protocol was conducted according to the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) and Kindai University, and approved by the Kindai University (KAPS-31-002, 1 April 2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** Shunsuke Sakurai and Eiji Harata work for the NOF Corporation. The authors report no other conflict of interest in this work.

#### **References**