**2. EV User Behavior Scenario and V2H Charging Strategy**

The availability of EV to be used for V2H services depends on EV user behaviors, which are unpredictable to a large extent. However, some typical user behaviors can be identified on the basis of factors describing the way of EV exploitation, i.e.,:

In this paper, the following scenario of EV exploitation is assumed. The EV is used in urban and non-urban areas for commuting. The vehicle is mainly charged on a private property. The most probable choice is charging in the user's private garage in the evening, less probable is charging in the workplace during the day.

Currently the typical charging solutions may include: AC on-board charger (rectifier), or off-board DC charger, or on-board motor converter (also used for motor propulsion, braking energy regeneration and battery charging). In a further consideration, only the controlled AC charging, with the use of motor inverter, will be dealt with.

The control strategy applied defines charging and control parameters [29] and requires infrastructure. A vehicle used for commuting is parked most of the day and can be connected to the electric installation. Hence, it is justified to extend the charging control strategy in order to use the EV battery as energy storage for the EV user needs.

The control of the charging process can be based on the tariff signal and the power limit value set by the distribution system operator (DSO). This strategy is beneficial for both the EV user and the network operator. The EV user reduces energy costs and the DSO receives network management opportunities (such as network load control).

The utilization of energy storage devices by end-users has been known for a long time and reported in the literature [30–33]. The typical application concerns a peak-shaving. When a distinction is made in tariffs between peak and off-peak hours, it is profitable for an end-users to reduce the peak demand. The EV battery is charged during off-peak hours, when energy is cheaper, and it is discharged during peak load periods. For EV users the reduction of peak power demand means decreasing demand charges. It should be noted that the peak demand occurs usually in the evening time period, so, according to the described scenario, while the EV is parked and its battery is available for ancillary service.

Another useful application of energy storage in LV installations is the supply of loads during dips and short interruptions. Its purpose is lowering the costs of losses incurred by end-users due to such disturbances in the network.

According to authors the infrastructure for the V2H services requires both the EV motor inverter and the building installation equipped with a home energy management system (HEMS) (Figure 1). HEMS is responsible for the calculation of the starting and ending time of charging or discharging processes. It is based on the following parameters:

The vehicle should be connected to the AC power network via dedicated EVSE (Electric Vehicle Suppling Equipment) i.e., socket outlet, connecting cable and wallbox. Communication between EV and EVSE must be bi-directional.

**Figure 1.** Vehicle-to-home (V2H) charging scheme.

### **3. Description of EV Simulation Model**

### *3.1. Asumptions*

During driving, the EV motor inverter provides power to its motor or is used for regenerative braking. While an EV is parked, the motor inverter is connected to 3-phase LV home installation and is used to charge the batteries or for the V2H services such as energy storage operation or uninterruptable power supply (UPS). This functionality requires an appropriate control system and procedures to run depending on the function being performed: drive, energy storage or UPS (when the installation is disconnected from the network). The controller is located on-board the vehicle. Its operation modes and settings should include all limitations regarding all the cooperating devices: battery, vehicle engine, electric loads and the supplying feeder.

During driving the EV driver controls the power fed to the EV motor within the limits of its rated power (using accelerator and brake pedals).

While EV is parked and connected to the home installation, the battery can be charged or discharged according to the user's decision or predefined profile. The amount of power drawn from or returned to the customer installation is limited by EV motor rated power, technical parameters of home installation and should be managed by the HEMS. Additionally, it should be noted that every EV is equipped with the on-board battery management systems (BMS). It is responsible for battery protection, monitoring and controlling its state. The HEMS cannot and should not have priority over BMS so it cannot jeopardize the battery safety. However, HEMS has access to the additional information (the energy tariffs, current energy consumption at home or other EV user preferences). That data can be used to effectively manage services like the peak shaving or planning the charging schedule.

The UPS option requires fully autonomous operation of the controller in order to balance an active and reactive power in the user's home installation (islanded operation). The amount of power drawn from or returned to the customer installation is limited by EV motor rated power and technical parameters of the home installation.

The objective of the presented model is to create a simulation tool to examine and evaluate the EV capability for V2H services. In particular, the model can be used to:


*2.4. Sample Collection*

At the time points indicated in the result section, the mice were anaesthetised by an i.p. injection of ketamine (100 mg/kg b.w.) and xylazine (10 mg/kg b.w.). After the loss of reflexes, blood as well as liver tissue samples were collected. The blood samples were collected from the portal vein, the hepatic vein, and the right heart chamber in EDTA-coated syringes as previously described [15]. The samples were centrifuged at 13,000 rpm for 10 min in order to separate plasma. The collected plasma was stored at −80 ◦C until used for analyses. After blood collection, the remaining blood was removed by perfusion trans-cardially with 40 mL PBS. Subsequently, the whole liver was excised and specimens were collected from defined anatomical positions as follow: (i) a specimen of approximately 1 cm size was taken from the left liver lobe, fixed in 4% paraformaldehyde (PFA) for 2 days and then embedded in paraffin for 2D staining; (ii) a specimen of approximately 0.5 cm size was taken from the left liver lobe and immediately embedded in Tissue-Tek®cryomold in Neg-50 media (ThermoFisher Scientific, Oberhausen, Germany), frozen in 2-methylbutane and stored at −80 ◦C until used for cryosection preparation; (iii) a specimen of approximately 0.5 cm size was taken from the median liver lobe, fixed in 4% PFA for 2 days, incubated in 30% sucrose for 2 days, and then embedded in Tissue-Tek®cryomold in Neg-50 media, frozen in 2-methylbutane and stored at −80 ◦C until used for preparation of liver slices; (iv) a specimen of approximately 20 mg weight was taken from the left liver lobe, snap-frozen in liquid nitrogen and stored at −80 ◦C until used for RNA isolation.


**Jung-Geun Han 1, Jin-Woo Cho 2,\*, Sung-Wook Kim 3, Yun-Suk Park 1 and Jong-Young Lee 1,\***

Received: 24 August 2020; Accepted: 16 September 2020; Published: 21 September 2020

**Abstract:** In this study, to reduce the use of cement and sand, porous feldspar with excellent economic efficiency was used as a substitute in the heat storage concrete layer. When porous feldspar and four other silicate minerals were used as substitute materials for sand in cement mortar, the specimen with the porous feldspar exhibited approximately 16–63% higher compressive strength, thereby exhibiting a higher reactivity with cement compared to the other minerals. To compensate for the reduction in strength owing to the decreased cement content, mechanical and chemical activation methods were employed. When the specific surface area of porous feldspar was increased, the unit weight was reduced by approximately 30% and the compressive strength was increased by up to 90%. In addition, the results of the thermal diffusion test confirmed that thermal diffusion increased owing to a reduction in the unit weight; the heat storage characteristics improved owing to the better porosity of feldspar. When chemical activation was performed after reducing the cement content by 5% and replacing the sand with porous feldspar, the compressive strength was found to be approximately twice that of an ordinary cement mortar. In a large-scale model experiment, the heat storage layer containing the porous feldspar exhibited better heat conduction and heat storage characteristics than the heat storage layer composed of ordinary cement mortar. Additionally, energy savings of 57% were observed.

**Keywords:** porous feldspar; activation; compressive strength; substitute material; energy saving concrete


*2.3. Sintering Process: Thermal Cycles*

The green samples were debinded and pressureless solid state sintered (SSS) in flowing air (LINN Elektronik HT—1800 VAC, LINN HIGH THERM GMBH, Hirschbach, Germany). Samples were dewaxed with a cycle up to 800 ◦C (10 ◦C/h ramp) in flowing air and then pressureless sintered in flowing air in the range of 1450–1570 ◦C for different holding times (2–80 h) depending on the composition. The dewaxing and sintering steps for the 3D-printed green bodies were performed at 1550 ◦C for ATZ for 1 h after a debinding step performed at 800 ◦C.

In addition, the two-step sintering (TSS) process was also tested. In this case, T1 and T2 were in the ranges of 1400–1500 and 1350–1450 ◦C, respectively, with zirconia and ATZ.

#### *2.4. Physical, Microstructural and Mechanical Characterization*

Sintered density was determined by Archimedes' method.

Diffraction patterns were collected by using a Philips X-ray powder diffractometer with Bragg–Brentano geometry and Cu Kα radiation (40 kV and 35 mA) to identify the crystalline phases in the sintered samples and to evaluate the tetragonality of the tetragonal phase.

Viscosity measurements were performed using a Malvern Kinexus Pro+ rheometer (Kinexus pro+, Malvern Instruments, Ltd., Worcestershire, UK) at 25 ◦C with cone-plate geometry (4◦, 40 mm) in shear rate control from 0.1 to 300 s−1.

The microstructural analysis of both the surface and cross sections of sintered bodies was performed with a scanning electron microscope (SEM, LEO 438 VP).

The flexural strength was determined at room temperature with four-point bending tests (five tests for each composition). Samples, in the form of 2 × 2.5 × 25 mm bars, were prepared and tested in accordance with the standard ENV843-1:2004 (cross head speed of 0.5 mm/min and support span of 20 mm). Hardness (*Hv*) was determined by means of Vickers indentation with a load of 9.8 N, while fracture toughness (*KIC*) was determined by means of Vickers indentation with a load ranging from 9.8 to 98 N. To calculate fracture toughness, the formula proposed by Niihara [45] for Palmqvist cracks was used (Equation (1)):

$$K\_{IC} = \frac{0.035 \left(H\_{\overline{v}} a^{0.5}\right) \left(3 \frac{F}{H\_v}\right)^{0.4} \left(\frac{l}{d}\right)^{-0.5}}{3} \text{ for } 0.25 < \frac{l}{a} < 2.5 \tag{1}$$

where *a* is the indent half-diagonal, *E* is the Young's modulus, and *l* is the Palmqvist crack length.

#### **3. Results and Discussion**

#### *3.1. Stabilization of Zirconia: Variables That Influence Transformability*

3.1.1. Type of Stabilizer

Different zirconia-based materials were produced and characterized to study the effect of the type of the stabilizer on the t–m phase transformation. The dopants could be classified according to their oxidation state (Cu2+, Y+3, Ce4+, and Ta5+). More precisely, they are stabilizers of the tetragonal phase (Y2O3 and CeO2) and toughening oxides (Ta2O5 and CuO) [46].

Cations' valence and size affect the stabilization mechanism of the tetragonal phase [47–50], even if the correlation is not univocal, as suggested by Yoshimura et al. [51].

The phase composition and crystallographic parameters were evaluated for each mixture (lattice constants c and a and their c/a ratio, namely "tetragonality") of doped tetragonal zirconia. The values of fracture toughness and hardness were also determined (Table 2).

**Table 2.** Properties of zirconia-based materials doped with different stabilizers and toughening oxides. DR is the relative density, c/a is the tetragonality, KIC the fracture toughness, and HV the microhardness.

Yttrium oxide (Y2O3) is the most common stabilizer of the tetragonal phase, and Y-TZP is widely used due to its strong mechanical properties [26,52]. The addition of different amounts of yttria influenced the c/a ratio, which indicated the transformability of the available tetragonal phase. In comparing the values in Table 2 for the mixtures of Zr2Y and Zr3Y, it is clear that a higher yttria content (3 vs. 2 mol%) led to a greater stabilization of the tetragonal phase, which corresponded to a decrease in the c/a ratio (1.0159 vs. 1.0166) and a lower toughness (4.0 vs. 9.4 MPa m1/2).

Cerium oxide (CeO2) is another well-known stabilizer of zirconia, and ceria-doped zirconia exhibits very high values of fracture toughness [53]. The ZrCe sample in our study showed toughness value four times higher than that of Zr3Y (18.4 vs. 4.0 MPa m1/2, respectively) as indicated in Table 2. CeO2 is a stabilizer as Y2O3, but its c/a ratio is higher; this means that the tetragonal phase is less stabilized, so its transformation is easier, thus leading to an increase in fracture toughness. On the other hand, as described in the literature [54], CeO2 does not allow one to obtain high values of mechanical resistance due to its limited capability to contain grain growth during sintering. Indeed, ZrCe grains are wider (ca. 2.0 μm) than Y-TZP ones (ca. 0.5–0.8 μm) [27]. As the oxidation state is the same of Zr4+, Ce4+ does not generate oxygen vacancies inside the ZrO2 cell, so, in a humid environment, the t–m spontaneous transformation is not promoted and CeO2-stabilized zirconia shows significantly high resistance to LTD [17,54,55].

Tantalum oxide (Ta2O5) is known in the literature for its toughening effect when added to 3Y-TZP [56,57]. In our study, the addition of Ta2O5 led to a higher value of fracture toughness than 3Y-TZP (9.4 vs. 4.0 MPa m1/2, respectively), as shown in Table 2. The addition of Ta2O5 to 3Y-TZP increased the c/a ratio (1.0173 vs. 1.0159, respectively) such that the chemical driving force for the t–m transformation was enhanced, and this led to a higher value of fracture toughness. On the other hand, the stabilizing effect of Y2O3 was contrasted by the addition of Ta2O5, which is a toughening oxide that increases the t–m martensitic transformation temperature [50], resulting in a toughening effect.

Copper oxide (CuO) was also tested as toughening agent for Y-TZP. The results reported in Table 2 show that the addition of CuO only led to a slight increase in the fracture toughness of the 3Y-TZP (4.6 vs. 4.0 MPa m1/2, respectively). This result is in contrast with the results reported by Ramesh et al. [58], where a different Y-TZP powder was used.

After comparing the fracture toughness values (Table 2) as function of the tetragonality, a linear correlation was obtained, as shown in Figure 1. If the c/a ratio of the tetragonal phase was near 1 (i.e., the c/a value of the cubic phase), the tetragonal phase was more stable and hence the t–m transformation became more difficult and the fracture toughness decreased. On the contrary, if the c/a ratio of the tetragonal phase increased up to 1.022 (which is the b/a value of the monoclinic phase), the t–m transformation was favored and the fracture toughness increased.

**Figure 1.** Fracture toughness vs. tetragonality of the different stabilizers in zirconia-based materials.

#### 3.1.2. Stabilizer Content

The effect of different contents of stabilizer (Y2O3) was studied in ZTA composites with a 60/40 alumina/zirconia weight ratio. The results reported in Table 3 show that the fracture toughness reached a maximum value of 6.2 MPam1/2 with the lowest amount of stabilizer (60402Y). The same results were previously observed in ZTA composites with a 50/50 alumina/zirconia weight ratio, as reported in Table 3 [59].

**Table 3.** Properties of ZTA materials doped with different amounts of stabilizer. DR is the relative density, c/a is the tetragonality, KIC the fracture toughness, HV the microhardness, and MOR is the four-point flexural strength.

1 Reprinted with permission from ref. [59]. Copyright © 2021 Elsevier Ltd.

This behavior can be explained by the analysis of the variation of the tetragonal phase amount and the tetragonality with stabilizer content. In fact, 60403Y had a lower amount of tetragonal phase (less than 80%) and a lower tetragonality than those of 60402Y. This means that a lower quantity of tetragonal phase was available to the toughening t–m transformation in the 60403Y composite. Furthermore, in the same sample, the lower tetragonality enhanced the stability of the tetragonal phase, which caused a decrease in the fracture toughness. These observations are also in line with the study of Yoshimura et al. [60], which reported the dependence of the c/a ratio on stabilizer content.

#### 3.1.3. Critical Grain Size

3Y-TZP was sintered in six different conditions in order to highlight the effect of the grain size variation on tetragonality and, consequently, fracture toughness. The experimental results are reported in the Table 4.

After increasing the sintering time to 60 h at 1550 ◦C, the fracture toughness and grain size increased up to maximum values of 7.7 MPa m1/2 and 1.19 μm, respectively (Figure 2). Furthermore, a strong dependence between the fracture toughness and tetragonality was observed at the microstructural level. Indeed, with the increase in sintering time, tetragonality increased, i.e., the tetragonal cell instability grew. This instability, caused by the distortion of the cell, promoted the t–m transformation and a consequent increase in fracture toughness.

**Table 4.** Properties of zirconia-based (3Y-TZP) samples sintered with different thermal cycles. DR is the relative density, Dm the average grain size, c/a is the tetragonality, KIC the fracture toughness, and HV the microhardness.

**Figure 2.** Fracture toughness vs. average grain size of the different sintered zirconia-based materials.

The sample sintered at 1550 ◦C for 80 h was characterized by the lowest sintered density due to the formation of the monoclinic phase, and it showed many cracks. In fact, XRD analysis confirmed that all the samples were mainly constituted by the tetragonal phase with traces of the cubic phase, while the sample sintered at 80 h showed an increase in monoclinic phase content.

According to these data, the critical grain size can be estimated to be equal or greater than 1.19 μm for this 3Y-TZP material. This value is in agreement with the critical grain observed by Lange [32].

#### *3.2. Parameters That Influence Mechanical Properties*

The relationships between the microstructure and mechanical properties of Y-TZP ceramics have been extensively studied over the past four decades, and different effects have been identified.

It was demonstrated that the toughening effect, related to the t–m transformation mechanism in Y-TZP ceramics, is promoted by larger grain sizes [12,32–36]. On the other hand, some mechanical properties, including flexural strength, are known to be enhanced by fine microstructures [36,61,62].

Indeed, as grain size coarsens, the critical defect enlarges, thus leading to a strength decrease [63]. According to the Griffith (Equation (2)), strength (*σR*), fracture toughness (*KIC*), and failure origin size (*c*) are strictly connected and their control is necessary to obtain reliable structural ceramic materials.

$$
\sigma\_R \sim \frac{K\_{IC}}{\sqrt{\pi c}} \tag{2}
$$

Unfortunately, the best conditions (composition, grain size, transformability, etc.) to reach ceramic strength in zirconia-based materials are not the same for maximizing

fracture toughness, so the reliability of these ceramics comes from the compromise of these two properties [64].

Hardness is also influenced by microstructure [65]. Generally, hardness is strictly related to density, but no univocal correlation between hardness and grain size has been proven. The hardness values of 3Y-TZP do not show the influence of the grain dimension in the submicrometric range [66].

A typical method to obtain ceramics with fine microstructures and improved mechanical properties (flexural strength) is based on the application of innovative sintering processes that limit grain growth. Among the best known sintering methods to refine ceramic microstructures, the spark plasma sintering (SPS) [67,68] and microwave sintering (MWS) [69] methods are the most efficient.

A simple and cost-effective method for industrial applications to obtain near full dense ceramics with controlled grain growth is TSS (two-step sintering) [70], in which the sample is first heated to a higher temperature to achieve an intermediate density and then cooled down and held at a lower temperature until it is fully dense. This sintering method has been successfully applied for ZTA composites [71,72].

The effect of TSS on the 3Y-TZP and ZTA samples (Table 5) was studied and compared to that of classic SSS. In the case of 3Y-TZP, TSS showed an advantageous effect on grain size (almost halved), as shown in Figure 3a,b. However, TSS seemed to have no effect on the fracture toughness. This was probably due to two opposite and concomitant effects of TSS that compensate for each other. The grain size refinement contrasted with the toughening effect achieved when the grain size approached the critical value. On the other hand, the tetragonal phase obtained with the TSS was more transformable, as evidenced by the slight increase in the tetragonality. It is probable that the longer holding time at the higher temperature promoted the migration of the stabilizer (Y3+) [52]; hence, the yttria concentration within the tetragonal phase decreased and enhanced transformability.

**Table 5.** Properties of 3Y-TZP and ZTA materials sintered with the single step (SSS) or two-step cycles (TSS). DR is the relative density, KIC the fracture toughness, HV the microhardness, MOR is the four-point flexural strength, Dm is the average grain size (A refers to alumina and Z to zirconia grains), and c/a is the tetragonality.

Again, the flexural strength values were very similar despite the halved grain size. It is probable that the grain refinement obtained with TSS did not contribute to a decrease in critical defect size. In fact, as observed by Xiong et al. [73], the TSS method could yield the formation of thermodynamically stable large pores, thus showing its limit in eliminating last residual porosity (1–2%). The effects of grain size refinement and critical defect dimension compensate for each other, thus leaving the strength value unaltered (as also described by Trunec [62]).

In the case of the ZTA composites, the TSS method effectively limited grain growth (Figure 3c,d). Comparing two samples with the same stabilizer content, the grain size refinement resulted in a lower toughness, probably due to the average grain dimension being too far from the critical grain size. The strength values of the 60403Y samples were found to be similar, likely because the increase in the critical defect size was not sufficiently compensated for by the refinement of the microstructure, as suggested by Trunec [62]. For the 60402Y samples, dynamic pore coalescence occurred in the second step of TSS, which did not aid the elimination of residual porosity and had detrimental effects on bending strength [73].

**Figure 3.** SEM micrographs of zirconia samples 3Y-TZP SSS (**a**) and 3Y-TZP TSS (**b**), as well as of ZTA samples 60403Y SSS (**c**) and 60403Y TSS (**d**).

Finally, the bending strength was also influenced by the powder preparation technique. Using the freeze-drying technique to dry the slurry, the production of a homogeneous granulate without aggregates was achieved (Figure 4). This granulation process strongly influences the quality of a green and sintered body [74]; in our study, higher values of bending strength were obtained (872 ± 47 MPa for 60402Y TSS-FD; see Table 5).

**Figure 4.** SEM micrograph of 60402Y powder prepared with freeze-dry granulation.

### *3.3. New Manufacturing Techniques: 3D Printing*

The production of ceramic components via the DLP technique is strictly connected to the availability of a suitable ceramic slurry. Nowadays, the most important producers of vat polymerization printers commercialize feedstocks for their 3D printer models with limited possibility to access to other resins available on the market. Another problem for the AM of ceramics with the DLP technique is the low disposability of printable slurries filled with desired ceramic powders.

For the preparation of new resin–ceramic powder mixtures, one of the main problems related to the addition of a high content of ceramic powder to the photopolymeric resin is the increase in the viscosity of the mixture. This drawback was solved here by wisely selecting monomers with different functionalities and molecular weights. The shear thickening behavior that is commonly observed in high solid loaded suspensions was reduced by the use of an appropriate surfactant and a zirconia powder with a lower surface area (7 ± 2 m2/g). In this way, high content ceramic photocurable resins (see Materials and Methods section) with low viscosity, suitable for the DLP printing process, were prepared.

The shear viscosity for two ATZ resins is reported in Figure 5.

**Figure 5.** Photocurable slurry viscosity.

Prototypal dental endosseous implants were obtained via the DLP technique with the developed ZTA resin (Figure 6), which was sintered up to 1550 ◦C for 1 h and reached a final density of 96.8%.

**Figure 6.** Endosseous dental implant in ATZ printed with the DLP technique.

More complex shapes, as the lattice structure shown in Figure 7, were successfully printed with a final relative density of 98%. Layer-by-layer deposition is highlighted in Figure 8. SEM observations revealed a regular lattice structure profile, where the overlapping layers and their homogeneity in thickness were clearly visible. The slicing value was set to 50 μm and fell to 35 μm after sintering shrinkage. Nevertheless, the layer adhesion could be further enhanced to completely avoid the delamination defects partially present in these items.

**Figure 7.** Lattice structure in ATZ printed by the DLP technique.

**Figure 8.** Micrograph of the lattice structure profile.

These preliminary outcomes highlighted the possibility to develop resins with the required ceramic material and the feasibility to print ceramic materials with low cost and widely available DLP printers.


*2.3. Digestibility Trial*

At 10 weeks of age, sixteen male rabbits were randomly taken to determine the nutrient digestion coefficients of the experimental diets. The rabbits were allocated to four different treatments (four rabbits in each group). The rabbits within each treatment were housed individually in metabolic cages that enabled the separation of urine and feces. The preliminary period was 2 days to adapt rabbits to the new cages and then followed by 5 days as a collection period for feces and urine. During the collection period, the total excreted feces and urine of each rabbit are collected daily in buckets before offering a morning meal and weighing them.

Representative samples (10%) of the total quantity of feces from each rabbit were oven-dried daily at 70 ◦C for 48 h to determine the total dry matter (DM) of the feces and to calculate the quantity of feces on a DM basis. At the end of the collection period, the faecal samples from each rabbit were mixed thoroughly, and representative samples (10%) of the mixtures were ground through a 1-mm screen on a Wiley mill grinder and then stored frozen at −20 ◦C prior to the chemical analysis.

Nutritive values in terms of total digestible nutrients (TDN) and digestible crude protein (DCP) were calculated according to the classic formula [31] as follows:

$$\text{TDN\%} = \text{DCP\%} + \text{DCF\%} + \text{DNNFE\%} + \text{(DEE\%} \times \text{2.25)} \tag{1}$$

where DCP is the digestible crude protein, DCF is the digestible crude fiber, DNFE is the digestible nitrogen-free extract, and DEE is the digestible ether extract.

$$\text{NR: Multiitive value} = \text{(TDN (\%)/DCP (\%))} - 1 \tag{2}$$

Digestible energy (DE) was calculated using the equation according to [32], as follows:

$$\text{DE (kcal/kg diet)} = 44.3 \times \text{TDN}\% \tag{3}$$

#### *2.4. Lipid Content and Fatty Acid Profile of Rabbit Meat*

The lipid content and fatty acid profile of rabbit meat were determined in the musculus semitendinosus of three slaughtered rabbits per group. Total lipids were extracted with chloroform:methanol (2:1 *v*/*v*) from 0.8 g of meat, according to the procedure of Folch et al. [33].

Lipid extraction from the meat samples was performed according to the procedure of Pearson [34]. About 10 g of the sample was weighed in a 250 mL centrifuge bottle. The total volume was completed to 16 mL with distilled water, then 40 mL of methanol and 20 mL of chloroform were added and macerated for 2 min. After that, 20 mL of chloroform was added and macerated for 30 s, then 20 mL of water was added and macerated again for 30 s. The mixture was centrifuged for 10 min at 2000–2500 rpm. The bottom layer of chloroform was removed and filtered through a coarse filter paper into a dry-weight flask or beaker. Then, the chloroform was evaporated to dryness.

Preparation of fatty acid methyl esters from the total lipids of the sample was performed according to the procedure of Radwan [35]. A sample of total lipids (50 mg) was transferred into a Screw-Cap flask, then 2 mL of benzene and 5 mL of methanolic sulphuric acid (1 mL of conc sulphuric acid and 100 mL of methanol) were added. The vial was covered under a stream of nitrogen gas, then placed in a water bath at 90 ◦C for 90 min. The flask was cooled, then 10 mL of distilled water was added and the methyl esters in each flask were extracted with 5 mL of petroleum ether three times. The petroleum ether extracts were combined and concentrated to their minimum volume using a stream of nitrogen. The analysis of fatty acids was carried out by gas-liquid chromatography (HP, Hewlett Packard 6890 GC model) equipped with a flame ionization detector (FID). Separation was achieved in a column HP-INWAX (cross linked polyethylene glycol, 60 m, 0.25 mm ID, 0.25 μm film thickness) under the following conditions: Detector temperature, 250 ◦C; injector temperature, 220 ◦C; injection volume, 3 μL; split ratio, 50:1; carrier gas, nitrogen; gas flow, 1.5 mL/min. Before running the samples, a standard mixture of methyl esters was analyzed under identical conditions. The retention times of the unknown sample of methyl esters were compared with the standard. The proportions of methyl esters were calculated by the triangulation method.

#### *2.5. Serum Biochemical Parameters*

Before slaughter, 4 mL of blood sample was taken with a sterile syringe from the ear vein of five growing rabbits from each group. The blood sample was placed into a sterile vacutainer tube without an anticoagulant for the serum biochemical analysis.

The serum total protein, lipid profile, and urea were estimated colorimetrically using commercial kits produced by Bio Diagnostic Co., Giza, Egypt. The serum total protein and albumin were determined according to Doumas et al. [36]. The serum globulin concentration was calculated by the difference between the total protein and albumin [37].

Total lipids were estimated by the reaction with sulphuric and phosphoric acids and vanillin to form a pink chromophore [38]. Triglycerides were measured colorimetrically using the quadruple enzymatic reaction [39]. Cholesterol was determined after enzymatic hydrolysis and oxidation as described by Allain et al. [40]. High-density lipoproteincholesterol (HDL-c) was determined according to the methods of Grove [41]. Low-density lipoprotein-cholesterol (LDL-c) was determined using the following calculation according to Warnick et al. [42] using the following equation:

$$\text{LDL-c} = \text{cholesky-t} \text{-} (\text{FDL-c} + \text{vLDL-c}).\tag{4}$$

The very low-density lipoprotein-cholesterol (vLDL-c) was calculated by dividing the value of TG by a factor of 5 according to the method of Warnick, Benderson, and Albers [42]. Serum urea was assayed according to Chaney and Marbach [43].

The triiodothyronine (T3) and thyroxine (T4) hormones were determined in the serum by a direct radioimmunoassay technique. Kits from the Diagnostic Products Corporation (Los Angles, CA, USA) with ready, antibody-coated tubes were used based on the manufacturer's instructions, according to Kubasik et al. [44].


**Abstract**

The autonomous navigation technology uses the multiple sensors to percept and estimate the spatial locations of the aerospace prober or the Mars rover and to guide their motions in the orbit or the Mars surface. In this chapter, the autonomous navigation methods for the Mars exploration are reviewed. First, the current development status of the autonomous navigation technology is summarized. The popular autonomous navigation methods, such as the inertial navigation, the celestial navigation, the visual navigation, and the integrated navigation, are introduced. Second, the application of the autonomous navigation technology for the Mars exploration is presented. The corresponding issues in the Entry Descent and Landing (EDL) phase and the Mars surface roving phase are mainly discussed. Third, some challenges and development trends of the autonomous navigation technology are also addressed.

**Keywords:** autonomous navigation, deep space exploration, inertial navigation, celestial navigation, visual navigation, integrated navigation

#### **1. Introduction**

The Mars is an attractive planet in the space, which looks red, faraway, and mysterious. It is believed that the Mars is the second planet, which is suitable for people to live [1]. After many years of probing, it has been found that the Mars has plenty of minerals, its average surface temperature is about −60°C, it has thin atmosphere, and it even has water [2]. The scientific fiction is if we deploy some solar reflectors in Mars' orbit and build many greenhouse gas factories in its surface, the temperature there will increase gradually, and then the thick atmosphere [3] will be created, the ice in its polar and underground will melt, and finally, the primitive life may appear. Nowadays, to land that red planet and build new base, there is the competitive focus of aerospace technology for many countries. However, it is known that the distance between the Earth and the Mars is between 5.5 × 107 and 4 × 108 km, and it will cost us almost 7 months to reach that place. To reach that red planet in the vast and boundless universe safely, all the solutions point to the navigation technology [4].

In general, the flight mission of Mars exploration [5] can be divided into four phases. The first one is the launch phase. The rocket will send the Mars prober into the space and escape from the gravitational constraint of the Earth. The second one is the cruise flight phase. The prober will fly to the Mars by several times of orbit transfer [6]. The third one is the Entry, Descent, and Landing (EDL) phase [7]. This is the most dangerous flight stage in this mission because of the influences of the thin Mars atmosphere, the Mars gravitation, and its complex terrain. The last one is the roving phase. After a safe landing, the rover will walk on the surface of

**12**

*Mars Exploration - A Step Forward*

[1] https://mars.nasa.gov/#mars\_

[3] https://www.hq.nasa.gov/office/pao/

[4] http://www.jpl.nasa.gov/history/60s/

[5] https://edition.cnn.com/2013/10/21/ world/mars-exploration-fast-facts/

[6] http://nssdc.gsfc.nasa.gov/image/

[7] http://nssdc.gsfc.nasa.gov/image/ spacecraft/mars3\_lander\_vsm.jpg

[9] http://photojournal.jpl.nasa.gov/

[10] http://photojournal.jpl.nasa.gov/

[11] http://photojournal.jpl.nasa.gov/

[12] https://www.nasa.gov/specials/

[13] https://www.esa.int/Science\_ Exploration/Human\_and\_ Robotic\_Exploration/Mars500/ Mars500\_study\_overview

[14] https://earthobservatory. nasa.gov/images/92630/

living-the-mars-life-on-mauna-loa

[8] https://nssdc.gsfc.nasa.gov/

exploration\_program/3

**References**

[2] https://mars.nasa.gov/ all-about-mars/facts/

History/SP-4212/p164.html

Mariner4\_1965.htm

spacecraft/mars3\_iki.jpg

planetary/viking.html

index.html

index.html

artemis/

catalog/PIA04413

catalog/PIA19920

Mars and look around here and there. Clearly, different navigation technologies should be employed in different flight phases. The satellite navigation [8], the inertial navigation [9], the celestial navigation [10], the visual navigation [11], and the integrated navigation [12] are the most popular navigation techniques in the aerospace engineering research field. **Figure 1** presents the sketch map and an example of Mars exploration. In **Figure 1**, (a) is the sketch map of Mars exploration flight mission, and (b) gives out a kind of landing method in the EDL phase.

Many research works have been done in the aerospace navigation research field. In [13], the authors proposed a new method, which could use the impulses in the solar light and the time difference of arrival method to improve the navigation accuracy. In [14], an inertial measurement unit and the orbits/aerostats beacons-based integrated navigation scheme were developed. The range and the Doppler information were integrated in the extended Kalman filter. In [15], a navigation method, which could use the inter-satellite link and the starlight angle, was simulated. The prober could determine its position by communicating with other space crafts and observing the stars. In [16], a norm-constrained unscented Kalman filter-based navigation scheme

#### **Figure 1.**

*The sketch map and an example of Mars exploration: (a) is the sketch map of the flight mission; (b) is a landing example in the EDL phase.*

**15**

**Figure 2.**

*The popular autonomous navigation methods.*

*Autonomous Navigation for Mars Exploration DOI: http://dx.doi.org/10.5772/intechopen.92093*

navigation methods.

**2.1 The inertial navigation**

was proposed. This method could be used for the spacecraft attitude quaternion estimation during the Mars powered descent. After a comparable study of the popular navigation methods, it can be found that the navigation technique has the development trend of high accuracy, multi-function, mini-size, low power consumption, and long service life. In addition, the Artificial Intelligence (AI) technique [17] also

Different from the traditional navigation techniques, the autonomous navigation [18] can realize the perception, the computation, and the decision making of the flight trajectory all by the aerospace prober itself. No exterior information is needed. Sometimes the autonomous navigation system can also emit some state information to the ground for the safety monitoring purpose. Many methods can be used to assist the autonomous navigation flight. The popular methods include the inertial navigation [19], the celestial navigation [20], the visual navigation, and the integrated navigation. **Figure 2** shows the sketch map of the popular autonomous

The inertial navigation can measure the acceleration of prober and use the integral computations to estimate its transient flight speed and spatial position. During that process, no energy will be radiated from the inertial navigation device, and no exterior auxiliary information is needed. The inertial navigation adopts the devices of the accelerometer [21] and the gyroscope [22] to realize the state estimation. The accelerometer [23] is a device, which can measure the acceleration of the carrier. In general, it can be classified into the linear accelerometer and the angular accelerometer. In the practical application, the linear accelerometer is commonly used because of its high precision and excellent stability. Many accelerometers have been designed, such as the piezoelectric accelerometer, the piezoresistive accelerometer, or the capacitive accelerometer. **Table 1** shows a kind of classification of the linear accelerometer. The gyroscope is a device, which can measure the rotation rate. **Table 2** illustrates some gyroscope products. They are the mechanical rotor gyroscope, the electrostatic suspended gyroscope, the vibratory gyroscope [24], the

The inertial navigation is the most mature and popular method in the navigation

research field. Comparing with other navigation techniques, the inertial navigation at least has three advantages. First, it is independent to the environment.

has the potential application value in the navigation research field.

**2. The key autonomous navigation techniques**

laser gyroscope, and the fiber optic gyroscope [25].

*Autonomous Navigation for Mars Exploration DOI: http://dx.doi.org/10.5772/intechopen.92093*

*Mars Exploration - A Step Forward*

Mars and look around here and there. Clearly, different navigation technologies should be employed in different flight phases. The satellite navigation [8], the inertial navigation [9], the celestial navigation [10], the visual navigation [11], and the integrated navigation [12] are the most popular navigation techniques in the aerospace engineering research field. **Figure 1** presents the sketch map and an example of Mars exploration. In **Figure 1**, (a) is the sketch map of Mars exploration

flight mission, and (b) gives out a kind of landing method in the EDL phase.

*The sketch map and an example of Mars exploration: (a) is the sketch map of the flight mission; (b) is a*

Many research works have been done in the aerospace navigation research field. In [13], the authors proposed a new method, which could use the impulses in the solar light and the time difference of arrival method to improve the navigation accuracy. In [14], an inertial measurement unit and the orbits/aerostats beacons-based integrated navigation scheme were developed. The range and the Doppler information were integrated in the extended Kalman filter. In [15], a navigation method, which could use the inter-satellite link and the starlight angle, was simulated. The prober could determine its position by communicating with other space crafts and observing the stars. In [16], a norm-constrained unscented Kalman filter-based navigation scheme

**14**

**Figure 1.**

*landing example in the EDL phase.*

was proposed. This method could be used for the spacecraft attitude quaternion estimation during the Mars powered descent. After a comparable study of the popular navigation methods, it can be found that the navigation technique has the development trend of high accuracy, multi-function, mini-size, low power consumption, and long service life. In addition, the Artificial Intelligence (AI) technique [17] also has the potential application value in the navigation research field.


**1. Introduction**

With the tightening of resource and environmental constraints, problems such as disordered spatial distribution of rural land, inefficient use of resources, and deterioration of the ecological environment have become increasingly prominent [1–3]. The traditional land consolidation model that takes a single element as the consolidation object has been unable to cope with the continuous comprehensive problems in the process of rural development [4,5]. Under the background of the rural revitalization strategy, land consolidation has expanded from the single agricultural land consolidation to the comprehensive consolidation of the whole elements of "mountains, rivers, forests, fields, lakes, grasses and sand" [6–10]. The overall promotion of comprehensive land consolidation will help gradually narrow the gap between urban and rural development, stimulate the internal driving force of rural development, coordinate the harmonious development of man and nature, and ultimately achieve comprehensive rural revitalization [11–14].

As an important part of land consolidation project management, the decision-making is the basis of scientifically selecting land consolidation projects, ensuring the quality of project, and making the project advance in an orderly manner [15,16]. In recent years,

**Citation:** Xia, W.; Yang, G.

Decision-Making Evaluation of the Pilot Project of Comprehensive Land Consolidation from the Perspective of Farmers and Social Investors: A Case Study of the Project Applied in Xianning City, Hubei Province, in 2020. *Land* **2022**, *11*, 1534. https:// doi.org/10.3390/land11091534

Academic Editors: Yongsheng Wang, Qi Wen, Dazhuan Ge and Bangbang Zhang

Received: 26 August 2022 Accepted: 8 September 2022 Published: 10 September 2022

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

**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/).

China's annual investment in land consolidation has reached hundreds of billions of Yuan [17]. However, due to the characteristics of large capital demand and long return time for comprehensive land consolidation, the supply of consolidation funds is still difficult to meet its demand. Western countries also face the limitation of funds [18], so they will strictly allocate the limited funds to the most suitable areas in the land consolidation project initiation stage to ensure effective resource management and successful financial support [2,19]. In order to solve the problem of the shortage of funds for comprehensive land consolidation, the Ministry of Natural Resources strongly advocates and encourages social capital to participate in comprehensive land consolidation and ecological restoration, and local governments also actively explore ways to attract social capital to comprehensive land consolidation. In the case of insufficient government financial funds, how to leverage or attract social capital to participate and ensure the high-quality implementation of comprehensive land consolidation projects has become an important issue to be solved in the decision-making of comprehensive land consolidation projects.

To attract social capital to participate in comprehensive land consolidation projects, the most important thing is to understand the interests of social investors and set up projects with high investment willingness of social investors so as to attract investment from social investors. At present, the academic research on the decision-making of land consolidation project mainly starts from the perspective of land [9,19,20], and there is relatively little literature on the decision-making of land consolidation projects from the perspective of the microsubject of the social capital. With the gradual development of land consolidation work, some scholars have found that mandatory land consolidation has adverse effects on farmers [21], and land consolidation should fully respect the dominant position of farmers [19,22–24]. In some places, in the process of social investors' participation in comprehensive land consolidation, the phenomenon of damage to the rights of farmers also appeared. Therefore, the decision-making of a comprehensive land consolidation project should not only consider the interests of the investor, namely the social capital, but also the rights of local farmers. Farmers are the ultimate beneficiaries of comprehensive land consolidation [25], and social investors are an important force to promote comprehensive land consolidation projects. It has important theoretical and practical significance to construct a comprehensive land consolidation project decision-making evaluation index system from the perspective of farmers and social investors.

Based on the perspective of farmers and social investors, this paper constructs a comprehensive land consolidation project decision-making evaluation index system and evaluation method. We performed an empirical analysis by using the survey data of seven pilot projects of comprehensive land consolidation in Xianning, Hubei Province, in 2020 and the entropy weight TOPSIS method. It provides the theoretical basis and case support for standardizing the decision-making of the pilot project of comprehensive land consolidation and promoting the pilot work of comprehensive land consolidation.

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

#### *2.1. Study Area and Data Sources*

In April 2020, the Office of the Leading Group for Comprehensive Land Consolidation of Hubei Province issued the "Notice on Application for Comprehensive Land Consolidation Projects", requiring the province to carry out the application of comprehensive land consolidation projects. Xianning City, located in the Wuhan urban circle, organized the following 7 projects to apply for the 2020 Hubei Province Comprehensive Land Consolidation Pilot Project: Zhaoliqiao Town Project in Chibi City (Project A), Henggouqiao Town Project in Xian'an District (Project B), Xiangyanghu Town Project in Xian'an District (Project C), Dupu Town Project in Jiayu County (Project D), Daping Township Project in Tongcheng County (Project E), Tiancheng Town Project in Chongyang County (Project F), Honggang Town Project in Tongshan County (Project G) (see Appendix A: Figure A1 and Table A1). This paper takes 7 applied projects as examples to conduct empirical research (Figure 1).

**Figure 1.** Survey area.

In order to obtain empirical data, the research group of more than 10 people conducted a questionnaire survey on farmers and social investors (new business entities) in the above-mentioned 7 project areas in January 2022. After removing invalid questionnaires, a total of 306 valid farmers questionnaires were obtained, including 47 items A, 35 items B, 45 items C, 47 items D, 43 items E, 41 items F, and 48 items G; and 20 valid social investor questionnaires, including 3 items A, 2 items B, 2 items C, 4 items D, 3 items E, 3 items F, and 3 items G (The details see Appendix A: Tables A2 and A3).


**HOLOGRAPHY – BASIC PRINCIPLES AND CONTEMPORARY APPLICATIONS**

Edited by **Emilia Mihaylova**

#### **Holography - Basic Principles and Contemporary Applications**

http://dx.doi.org/10.5772/46111 Edited by Emilia Mihaylova

#### **Contributors**

Anette Gjörloff Wingren, Olga Andreeva, Yuri Leonidovich Korzinin, Boris Glebovich Manuhin, Ngoc Diep Lai, Vladimir A. Postnikov, Aleksander Kraiskii, Valeri Sergienko, Francisco Palacios, Ligia Ferreira, Oneida Font, Guillermo Palacios, Jorge Ricardo, Miriela Escobedo, Isis Vasconcelos, Mikiya Muramatsu, Diogo Soga, José Luis Valin Rivera, Aline Ambrogi, Flavia Oliveira, Young-Ho Seo, Dong-Wook Kim, Hyun-Jun Choi, Gabriel Martinez-Niconoff, Elena Stoykova, Hoonjong Kang, Jiyung Park, Sung Hee Hong, Youngmin Kim, David Brotherton-Ratcliffe, Eduardo Acedo Barbosa, Danilo Silva, Merilyn Ferreira, Dagmar Senderakova, Luciano Lamberti, Cesar A. Sciammarella, Federico Sciammarella, Izabela Naydenova, Emilia Mihaylova

#### **© The Editor(s) and the Author(s) 2013**

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#### **Notice**

Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book.

First published in Croatia, 2013 by INTECH d.o.o. eBook (PDF) Published by IN TECH d.o.o. Place and year of publication of eBook (PDF): Rijeka, 2019. IntechOpen is the global imprint of IN TECH d.o.o. Printed in Croatia

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**5. Characterization of liposomes**

After preparation and before application, liposomes have to be characterized in order to ensure their in vitro and in vivo performance. Liposomal properties that are commonly discussed include lamellarity (the number of bilayers present in liposomes), diameter and size distribu‐ tion, lipid composition and concentration determination, the encapsulant concentration and its encapsulation efficiency.

For the characterization of chemical properties, phospholipids can be quantitatively in terms of concentration either by Bartlett Assay or Stewart Assay. The phospholipid hydrolysis might be followed by HPLC where the column outflow can be monitored continuously by UV absorbance to obtain a quantitative record of the eluted components. Moreover, the phospho‐ lipid oxidation can also be followed by a number of techniques i.e., UV absorbance method, TBA method (2-thiobarbutiric acid) (for endoperoxides), iodometric method (for hydroper‐ oxides) and GLC (gas-liquid chromatography) method [93].

of preparation methods is done through gas chromatography (GC) [100-101] This is a very rapid and reliable method and most analytical and organic laboratories are equipped with a

**Figure 2.** Cryo-TEM pictures of Size Exclusion Chromatography fractions eluted at 90 minutes and prepared from egg-

Adopted from Holzer, M., Barnert, S., Momm, J., Schubert, R., 2009. Preparative size exclusion chromatography com‐ bined with detergent removal as a versatile tool to prepare unilamellar and spherical liposomes of highly uniform size

Liposomes as Potential Drug Carrier Systems for Drug Delivery

http://dx.doi.org/10.5772/58459

15

An important feature of liposomes is the existence of a temperature dependant, reversible phase transition, where the hydrocarbon chains of the phospholipid structures undergo a transformation from an ordered gel state to a more disordered fluid, liquid crystalline, state. This transition temperature is important in optimizing the storage conditions (i.e Temperature) to minimize fusion and drug leakage. These changes have been monitored by freeze fracture electron microscopy and much more easily by differential scanning calorimetry (DSC)

Entrapped volume is a crucial parameter that governs the morphology of liposomes. This internal volume is defined as the aqueous entrapped volume per unit quantity of lipids. The most promising way to determine the internal volume is to measure the quantity of water by replacing external medium (water) with a spectrophotometrically inert fluid (i.e. deuterium

It is essential to measure the quantity of the encapsulated material inside liposomal structures before studying the behavior of this encapsulated material physically and biologically since the effects observed experimentally will be dose related. After the removal of the nonencapsulated material by the separation techniques the quantity of material remained can be assumed as 100% encapsulated. Minicolumn centrifugation and protamine aggregation

Methods for determining the amount of material encapsulated within the liposomes typically rely on the destruction of the lipid bilayer and subsequent quantification of the released material [105]. In these measurements, the signal due to intact liposomes is typically monitored

methods are the general separation procedures that are commonly used [93].

oxide) and then measuring water signal by NMR [93].

distribution. J. Chromatogr. A 1216, 5838–5848.

GC.

[102-104, 93].

phosphatidylcholine.

The most direct method for determination of liposome size is the electron microscopy due to the possibility of viewing the liposomes individually and obtaining the exact information about the liposome population over the whole range of sizes [94]. As liposomes do not naturally create a contrast to be visible by electron microscopy, either cryo-TEM (Figure 2) should be used or staining of the liposome sample is required. Either way, it is a very timeconsuming method and it requires equipments that may not always be immediately accessible. The other method for the determination of liposome size, dynamic light scattering [95, 96], is very simple and rapid to perform but it measures an average size of liposome bulk. More recently, atomic force microscopy is also used to determine the morphology, size and stability of liposomal structures. All these size determination methods are very expensive. If only an approximate size range is required, gel exclusion chromatography might be suitable.

Electrostatic stabilization of liposomes may be a desirable feature to prevent fusion. The surface charge on the liposomes is measured by zeta-potential measurements [98]. These measure‐ ments are useful in determination of the in vivo behavior of liposomes. Often zeta potential values <-25 mV or >+25 mV are considered stable [99]. However, as mentioned earlier, charged liposomes have the disadvantage of being unstable in biological conditions.

Residual solvent is very unacceptable for drug delivery applications, therefore residual solvent should be kept at a minimum in the formulations. Quantification of residual solvents as a result

Filtration sterilization is relatively time-consuming and not efficient for the removal of viruses [91]. Studies have shown that polycarbonate membranes are less effective than hydrophobic Fluoropore membrane and cellulose acetate/surfactant-free membrane filtration units [91]. Although the limitations of filtration provoked researches on other sterilization methods, all resulted in the formation of degradation products via the previously mentioned degradation pathways. Filtration and the other methods are summarized according to their applicability

After preparation and before application, liposomes have to be characterized in order to ensure their in vitro and in vivo performance. Liposomal properties that are commonly discussed include lamellarity (the number of bilayers present in liposomes), diameter and size distribu‐ tion, lipid composition and concentration determination, the encapsulant concentration and

For the characterization of chemical properties, phospholipids can be quantitatively in terms of concentration either by Bartlett Assay or Stewart Assay. The phospholipid hydrolysis might be followed by HPLC where the column outflow can be monitored continuously by UV absorbance to obtain a quantitative record of the eluted components. Moreover, the phospho‐ lipid oxidation can also be followed by a number of techniques i.e., UV absorbance method, TBA method (2-thiobarbutiric acid) (for endoperoxides), iodometric method (for hydroper‐

The most direct method for determination of liposome size is the electron microscopy due to the possibility of viewing the liposomes individually and obtaining the exact information about the liposome population over the whole range of sizes [94]. As liposomes do not naturally create a contrast to be visible by electron microscopy, either cryo-TEM (Figure 2) should be used or staining of the liposome sample is required. Either way, it is a very timeconsuming method and it requires equipments that may not always be immediately accessible. The other method for the determination of liposome size, dynamic light scattering [95, 96], is very simple and rapid to perform but it measures an average size of liposome bulk. More recently, atomic force microscopy is also used to determine the morphology, size and stability of liposomal structures. All these size determination methods are very expensive. If only an

approximate size range is required, gel exclusion chromatography might be suitable.

liposomes have the disadvantage of being unstable in biological conditions.

Electrostatic stabilization of liposomes may be a desirable feature to prevent fusion. The surface charge on the liposomes is measured by zeta-potential measurements [98]. These measure‐ ments are useful in determination of the in vivo behavior of liposomes. Often zeta potential values <-25 mV or >+25 mV are considered stable [99]. However, as mentioned earlier, charged

Residual solvent is very unacceptable for drug delivery applications, therefore residual solvent should be kept at a minimum in the formulations. Quantification of residual solvents as a result

on liposomal preparations in Table 1 [92], given above.

oxides) and GLC (gas-liquid chromatography) method [93].

**5. Characterization of liposomes**

14 Application of Nanotechnology in Drug Delivery

its encapsulation efficiency.

Adopted from Holzer, M., Barnert, S., Momm, J., Schubert, R., 2009. Preparative size exclusion chromatography com‐ bined with detergent removal as a versatile tool to prepare unilamellar and spherical liposomes of highly uniform size distribution. J. Chromatogr. A 1216, 5838–5848.

**Figure 2.** Cryo-TEM pictures of Size Exclusion Chromatography fractions eluted at 90 minutes and prepared from eggphosphatidylcholine.

of preparation methods is done through gas chromatography (GC) [100-101] This is a very rapid and reliable method and most analytical and organic laboratories are equipped with a GC.

An important feature of liposomes is the existence of a temperature dependant, reversible phase transition, where the hydrocarbon chains of the phospholipid structures undergo a transformation from an ordered gel state to a more disordered fluid, liquid crystalline, state. This transition temperature is important in optimizing the storage conditions (i.e Temperature) to minimize fusion and drug leakage. These changes have been monitored by freeze fracture electron microscopy and much more easily by differential scanning calorimetry (DSC) [102-104, 93].

Entrapped volume is a crucial parameter that governs the morphology of liposomes. This internal volume is defined as the aqueous entrapped volume per unit quantity of lipids. The most promising way to determine the internal volume is to measure the quantity of water by replacing external medium (water) with a spectrophotometrically inert fluid (i.e. deuterium oxide) and then measuring water signal by NMR [93].

It is essential to measure the quantity of the encapsulated material inside liposomal structures before studying the behavior of this encapsulated material physically and biologically since the effects observed experimentally will be dose related. After the removal of the nonencapsulated material by the separation techniques the quantity of material remained can be assumed as 100% encapsulated. Minicolumn centrifugation and protamine aggregation methods are the general separation procedures that are commonly used [93].

Methods for determining the amount of material encapsulated within the liposomes typically rely on the destruction of the lipid bilayer and subsequent quantification of the released material [105]. In these measurements, the signal due to intact liposomes is typically monitored prior to bilayer disruption. The techniques used for this quantification depend on the nature of the encapsulant and include spectrophotometry [106, 107], fluorescence spectroscopy [108], enzyme-based methods [109] and electrochemical techniques. If a separation technique such as HPLC of field-flow fractionation (FFF) is applied, the percent encapsulation can be ex‐ pressed as the ratio of the unencapsulated peak area to that of a reference standard of the same initial concentration [110, 111]. This method can be applied if the liposomes do not undergo any purification following preparation. Either technique serves to separate liposome encap‐ sulated materials from those that remain in the extravesicular solution and hence can also be used to monitor the storage stability in terms of leakage or the effect of various disruptive conditions o the retention of encapsulants. Some authors have combined the size distribution and encapsulation efficiency determination in one assay by using FFF-MALS (multi angled light scattering) coupled to a concentration detector suitable for the encapsulant [112].

carrier, as well as their biological interactions between liposomes and cells. Based on these liposome properties, several modes of drug delivery can be listed: the major ones are enhanced drug solubilization (e.g. amphotericin B, minoxidil), protection of sensitive drug molecules (e.g. cytosine arabinose, DNA, RNA, antisense olgionucleotides, ribozymes), enhanced intracellular uptake (all agents, including antineoplastic agents, antibiotics and antivirals) and

Liposomes as Potential Drug Carrier Systems for Drug Delivery

http://dx.doi.org/10.5772/58459

17

Although lipid based formulations have advantages as drug carriers, drug-delivery systems based on unmodified liposomes are limited by their short blood circulation time, instability in vivo and lack of target selectivity [119, 120]. To increase accumulation of liposomal formula‐ tions in the desired cells and tissues, the use of targeted liposomes including surface-attached ligands such as; antibodies, folates, peptides and transferrin that are capable of recognizing and binding to the desired cells. Despite of some improvements in targeting efficiency by these immunoliposomes, the majority of these modified liposomes were still eliminated rapidly by the reticulo endothelial system, primarily in the liver [120]. Better target accumulations are

Schematic drawing of cytosolic delivery and organelle-specific targeting of drug loaded nanoparticles (i.e. most frequently liposomes) via receptor-mediated endocytosis is shown in

**Figure 3.** Schematic drawing of the cytosolic delivery and organelle-specific targeting of drug loaded nanoparticles via receptor-mediated endocytosis. After receptor mediated cell association with nanoparticles, the nanoparticles are engulfed in a vesicle known as an early endosome. Nanoparticles formulated with an endosome disrupting property disrupt the endosomes followed by cytoplasmic delivery. On the other hand, if nanoparticles are captured in early en‐ dosomes, theymaymake theirway to lysosomes as late endosomes where their degradation takes place. Only fraction of non-degraded drug released in the cytoplasm interacts with cellular organelles in a random fashion. However, cyto‐ solic delivery of a fraction of organelle-targeted nanoparticles via endosomal escape or from lysosomes travel to the

targeting organelles to deliver their therapeutic cargo [121].

altered pharmacokinetics and biodistribution of the encapsulated drug.

expected if liposomes can be made to remain in the circulation long enough.

Figure 3.

Since techniques used to separate free materials from liposome-encapsulated contents can potentially cause leakage of contents and, in some cases, ambiguity in the extent of separation, research using methods that do not rely on separation are of interest. Reported methods have included 1H NMR where free markers exhibited pH sensitive resonance shifts in the external medium versus encapsulated markers [113]; diffusion ordered 2D NMR which relied on differences in diffusion coefficients of entrapped and free marker molecules [114]; fluorescence methods where the signal from unencapsulated fluorophores was quenched by substances present in the external solution [115]; electron pin resonance (ESR) methods which rely on the signal broadening of unencapsulated markers by the addition of a membrane-impermeable agent [116, 117].

The drug release from liposomes can be followed by the usage of a well calibrated in vitro diffusion cell in order to predict pharmacokinetics and bioavailability of drug before expensive and time-consuming in vivo studies. For the determination of pharmacokinetic performance of liposomal formulations, dilution-induced drug release in buffer and plasma was employed and for the determination of drug bioavailability, another procedure is followed which involves the liposome degradation in the presence of mouse-liver lysosome lysate [93].


*5.4. Toxicological Studies of Reproductive, Hypothalamic, and Pituitary Gland Tissues* 5.4.1. Tissue Samples

Five prepubertal gilts from every group were euthanized on analytical date 1 (D1 exposure day 7), date 2 (D2—exposure day 21), and date 3 (D3—exposure day 42). Initially, general sedation was performed by intravenous administration of pentobarbital sodium (Fatro, Ozzano Emilia BO, Italy) and bleeding. Immediately after cardiac arrest, tissue samples (approximately 1 × 1.5 cm) were collected from the following segments: entire left ovary; left uterine horn (from the ovarian and uterine sections); middle part of the cervix; the entire hypothalamus; and the pituitary gland. The samples were rinsed with phosphate

buffer and prepared for analyses. The collected samples were stored at a temperature of −20 ◦C.

#### 5.4.2. Extraction Procedure

The presence of ZEN, α-ZEL, and β-ZEL in tissue samples was determined in accordance with the with the study protocol described previously [54].

#### 5.4.3. Chromatographic Quantification of Zearalenone and Its Metabolites

Zearalenone and its metabolites were quantified at the Institute of Dairy Industry Innovation in Mr ˛agowo, Poland. The biological activity of ZEN, α-ZEL, and β-ZEL in the tissues was determined in accordance with the study protocol described previously [54].

Mycotoxin concentrations were determined with an external standard and were expressed in ng/mL. Matrix-matched calibration standards were applied in the quantification process to eliminate matrix effects that can reduce sensitivity. Calibration standards were dissolved in matrix samples based on the procedure that was used to prepare the remaining samples. The material for the calibration standards was free of undesirable substances. The limits of detection (LOD) for ZEN, α-ZEL, and β-ZEL were determined as the concentration at which the signal-to-noise proportion decreased to 3. The concentrations of ZEN, alfa-ZEL and beta-ZEL were determined in each group and on 3 analytical dates (see Table 1).

5.4.4. Mass Spectrometry Conditions

The electrospray ionization (ESI) mass spectrometer was operated in the negative ion operation. MS/MS parameters were optimized for every compound. Linearity was tested with a calibration curve including 6 levels. The optimal analytical conditions for the tested mycotoxins are presented in Table 5.

**Table 5.** Optimized conditions for the tested mycotoxins [15].

#### 5.4.5. CF

Carry-over toxicity takes place when an organism is able to survive under exposure to low doses of mycotoxins. Mycotoxins can exert a negative effect on tissues or organ function [56] and modify their activity [10,37]. The carry-over was determined in the examined tissues when the daily dose of zearalenone (5, 10, or 15 µg ZEN/kg BW) administered to each animal was equivalent to 560-32251.5 µg zearalenone/kg of the complete diet, depending on daily feed consumption. Mycotoxin contents in tissues were expressed in terms of the dry matter content of the samples.

The CF was calculated as follows:

CF = toxin concentration in tissue [ng/g]/toxin concentration in diet [ng/g]

#### 5.4.6. Statistical Analysis

Data were processed statistically at the Department of Discrete Mathematics and Theoretical Computer Science, of the Faculty of Mathematics and Computer Science of the University of Warmia and Mazury in Olsztyn, Poland, as described previously [37].

#### *5.5. Toxicological Studies of Blood*

#### Blood samples collection

Blood was sampled in vivo from 5 gilts from every group on each analytical date. The first analytical date was exposure day 7 (D1); the second analytical date was exposure

day 14 (D2); the third analytical date was exposure day 21 (D3); the fourth analytical date was exposure day 28 (D4); the fifth analytical date was exposure day 35 (D5), and the sixth analytical date was exposure day 42 (D6). Blood samples of 20 mL each were collected from all gilts (blood was sampled within 20 seconds after immobilization [57]) directly before slaughter by jugular venipuncture with a syringe containing 0.5 mL of heparin solution. The collected blood was centrifuged at 3000 rpm for 20 minutes at 4 ◦C. The obtained plasma samples were stored at −18 ◦C until estradiol (E2) and progesterone (P4) concentration analyses were performed.


**5. Conclusions**

The results of our long-term research carried out in the mountainous areas of southern Poland revealed that yield of SCMs and leaf area index in pure and mixed sowing in the organic crop rotation was lower than in the integrated one, by 18% and 16%, respectively. At the same time, the average yield of spring cereal mixtures with barley was higher than that of pure cereal sowing. Under the conditions studied, the highest yield was obtained for a mixture of oats and spring barley, which could be partially explained by the higher leaf area index value for this mixture (LAI = 2.19). The yields of the other mixtures (oats with spring triticale and spring triticale with spring barley) were higher than the yield of the one of the components of the mixture in pure sowing. Moreover, the average sum of LERs for mixtures in the organic system was 1.11 and was significantly higher than for mixtures in the integrated system. In mixtures, barley displayed the highest competitiveness ratio. The analysis of the land equivalent ratio (LER) also showed that, under the examined conditions, spring cereal mixtures are a more effective form of cultivation than pure sowings. Each time, LER values for the mixtures exceeded 1. At the same time, despite lower yields of spring cereals in the organic crop rotation, the average value of standard gross margin without subsidies in the organic crop rotation was almost twice as high as in the integrated one. Among pure and mixed sowings, the highest value of standard gross margin without subsidies was found for spring barley cultivation. Summing up, ecological cultivation of spring cereal mixtures, having many pro-environmental values and showing a standard gross margin, should be recommended especially in the mountainous areas of southern Poland.

**Author Contributions:** Conceptualization, K.K.; methodology, K.K.; validation, A.L.; formal analysis, K.K. and A.S.; investigation, K.K., M.C., and P.G.; resources, A.L.; data curation, K.K., P.G., and A.K.; writing—original draft preparation, A.S., J.P., K.P., and B.J.; writing—review and editing, T.D., A.K., and D.G.-C.; visualization, K.K., A.S., and T.D.; supervision, K.K.; project administration, K.K. and A.L.; funding acquisition, K.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Science and Higher Education of Republic of Poland, grant number DS 3124.

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


Preface

A biofilm is a microbial form of life formed by the organic exopolysaccharide structures produced by microorganisms, irreversibly attached to a living or inanimate interface. Today, biofilms are widespread in nature and produced by many bacteria.

Biofilms protect bacteria from changes in environmental conditions and the harmful effects of ultraviolet light, making it easier for the bacteria to store food and remove waste materials. Due to biofilms, it is difficult for microorganisms to undergo phagocytosis by the immune system cells. When favorable conditions occur, biofilms can be created by pathogenic microorganisms. Bacterial attachment levels, surface properties, temperature, type and number of bacteria, pH of the environment, cell-wall structure of the bacteria, mobility, amount and content of the nutrients in the environment, and ion concentration all affect biofilm formation. Microorganisms create biofilms for reasons such as protection from the harmful effects of the environment as well as obtainment of nutrients and new genetic features. Biofilm formation is not a random event, and many microorganisms signal each other using small diffuse molecules to coordinate activities.

The intercellular communication required for biofilm formation is provided by a system called "quorum sensing." This system provides many advantages to bacteria, but it also controls the formation of biofilms through signals transmitted from cell to cell. While the quorum sensing system detects the population density around the bacteria, the bacteria provide gene control. In addition to developing coordination between food sources and bacteria, quorum sensing also increases communication with the bacteria during infection to develop resistance to the immune response in the host.

Studies have shown that bacteria living in biofilms are more resistant to antibacterial agents, iodine, the iodine polyvinylpyrrolidone complex, chlorine, monochloramine, pyrogens and biocides, and heat.

Biofilms are known to cause economic losses due to their negative effects in many areas, especially in health and food. New methods incorporating enzymes, detergents, ultrasound, and electricity are being used to control and prevent bacteria via mechanical cleaning, use of biocides, preventing biofilm development, preventing microbial adhesions, and supporting biomass extraction. However, the effectiveness of each methods varies according to the applied surface, the type of bacteria that forms the biofilm, and the application methods.

Biofilms can cause great industrial losses and threaten public health. Therefore, more research is needed to understand biofilms and develop efficient ways of preventing their formation.

> **Sadık Dincer** Cukurova University, Adana, Turkey

**Melis Sümengen Özdenefe** Near East University,

Nicosia, Turkish Republic of Northern Cyprus

**Afet Arkut**

**1**

Section 1

Biofilm in Wastewater

Treatment

Cyprus International University, Nicosia, Turkish Republic of Northern Cyprus Section 1