**1. Introduction**

In recent years, energy shortages and environmental degradation have attracted increasing attention [1–6]. Increasing car ownership has led to an increase in pollution from automobile exhaust emissions, thereby forcing the acceleration of energy transformation in the automotive industry. As a new type of transportation, electric vehicles (EVs) have great advantages in energy saving, emission reduction and reduction of fossil fuel dependence, which has greatly promoted their development.

The power supply system is the core part of the EV and directly affects the overall performance of the vehicle [7–10] and currently, the power supply problem of electric vehicles is a major obstacle to the development of EVs [11]. To promote and use EVs more widely, electric vehicles need higher specific energy, higher specific power, longer cycle life of energy storage system and higher charging efficiency [12–16].

Conventional EV energy storage systems are battery-based storage devices, which have large deficiencies and limitations [17–20]. First, the power density of the battery is low, thereby it cannot meet the peak power demand of EVs under acceleration or climbing conditions. Although the power demand can be satisfied by increasing the number of battery cells, the EV load will be increased considerably. Second, in battery energy storage systems, frequent current changes generate additional heat, reducing efficiency and battery life.

Therefore, we herein study a hybrid energy storage system (HESS) comprising batteries and supercapacitors (SCs). As a new type of environmentally friendly energy storage device, SC has the advantages of high power density, high efficiency, fast response speed, and long cycle life that can improve energy utilization and dynamic performance to some extent [21–26].

The range of power required for operation under certain operating conditions (such as starting, acceleration, deceleration, stopping, and climbing) is wide. When the SC is directly connected to the battery, stabilizing the output voltage and controlling the energy distribution is difficult. In our research, we adopted the half-bridge of the control topology, wherein a bidirectional DC/DC converter connects the SC to the battery [27–30]. To enhance the discharge persistence of the battery current, the battery current is reduced to a minimum. The entire driving cycle strategy is employed to reduce the power loss in a given load curve. A discrete optimization design is used to improve the control effect of EVs.

This paper analyzes the deficiencies of the topology and control strategies of traditional HESSs, improves the previous topology, and proposes an optimized power allocation strategy based on the current control of EVs, thereby accurately predicting future power requirements. In addition, on the basis of current control, we herein add the SOC control of SCs based on the driving speed of EVs, thereby reducing the overcharge and overdischarge of the battery, improving the climbing performance of the EV, improving the energy utilization rate and reducing the battery aging effect [31–37].

#### **2. Hybrid Energy Storage System**

Energy storage devices can be divided into physical energy storage devices, chemical energy storage devices, and electromagnetic energy storage devices. Table 1 lists the main parameters of the common energy storage devices.

**Table 1.** Main parameters of common energy storage.

Table 1 shows that a single energy storage device cannot unify power density and energy density, and cannot meet the complex and variable power demand of EVs. Combining the advantages of batteries and SCs, it is proposed that batteries and SCs be used as composite energy sources to meet the needs of EV power supply changes. The high energy density of the battery guarantees EV mileage for one charge. SCs can provide instantaneous high current output to meet the peak power (such as for acceleration and climbing) required by EVs, thus ensuring dynamic performance. When the EV decelerates or degrades, the power system is in an energy feedback state; thus, the SC can more efficiently and quickly absorb feedback energy, improve the energy efficiency of the system, and protect the battery from heavy currents. There are four common structures of HESS composed of two energy storage devices and one load device [38–41]:

(1) The SC, battery and load are directly connected in parallel, as shown in Figure 1a. This connection is low cost and fast response, but it also has several limitations: such as the capacity of the energy storage system cannot be fully utilized, and the power allocation is not controlled; the voltage of the SC is not controlled, and it depends on the SOC of the battery, which will affect the optimal utilization of SC.

(2) The SC control structure, as shown in Figure 1b. The SC is connected to the DC/DC bidirectional converter, and the battery is directly connected to the DC bus. This structure can lead to the capacity of the SC being fully utilized, and this is advantageous to the high-power throughput capacity of the SC. However, the charging and discharging and the power of the battery cannot be effectively controlled.

**Figure 1.** (**a**) Direct parallel structure; (**b**) Structure of SC control; (**c**) Structure of battery control; (**d**) Structure of two power supply control.

(3) The battery control structure, as shown in Figure 1c. The power of the battery can be controlled, and the charging and discharging current of the battery can be optimized that can prolong the service life of the battery. The SC can achieve a fast response speed when the peak power output changes, but obtaining a stable voltage is difficult, resulting in poor system stability.

(4) The dual-power control structure, as shown in Figure 1d. The structure can provide flexible voltage control, and can better allocate power to the battery and the SC. The battery can also prolong the service life, and its energy can be fully utilized. Bidirectional DC/DC can be used for an energy management system with a more flexible configuration. However the existence of bidirectional DC/DC will increase the cost and loss of the system, as well as increase the complexity of the system structure and the mass of EVs [42–44].

Currently, SC control structures are widely used in EV composite power supply systems. In this study, a new power allocation strategy is required to accurately control the charging and discharging of the battery and SC. Figure 1d shows a schematic of the structure. In this study, the bidirectional DC/DC converter adopts half-bridge topology structure. The principle of the bidirectional DC/DC converter in HESS of EVs. When the system is discharged, the bidirectional DC/DC controller operates in the Boost mode, and the energy flows from the low-voltage side to the high-voltage side; when the system is in charge, the bidirectional DC/DC controller operates in the Buck mode, and the energy flows from the high-voltage side to the low-voltage side.

A supercapacitor is a type of energy storage device, that stores electric energy converted from various clean energy sources. It is conventionally divided into two types: stacked and wound. Electrode materials are important factors affecting the energy storage characteristics of SCs. Ordered mesoporous carbon materials not only have the characteristics of uniform pore size distribution, large pore volume and high specific surface area, but also have the advantages good chemical inertness, strength, electrical conductivity and thermal stability. In this study, a spiral wound SC with ordered mesoporous carbon electrodes was used. Figure 2 shows the diagram of the SC structure.

**Figure 2.** SC structure diagram.

Figure 3 shows the electrochemical properties of the ordered mesoporous carbon (OMC) electrode. According to the X-ray diffraction (XRD) pattern of the ordered mesoporous carbon, it is known that no impurity peak appears, the full width at half maximum is large, and the degree of crystallization is small, indicating it to be a good electrode material. According to the SEM (scanning electron microscope) image of the ordered mesoporous carbon, the basic unit structure is uniform in size. According to the transmission electron microscope (TEM) image of the ordered mesoporous carbon, the pore structure is arranged in order and the conductivity is strong. The charge and discharge curves of the electrode material at 5 mA show good linearity, the self-discharge current is relatively small, and there is no obvious voltage drop at the initial discharge, indicating that the electrode material has a small internal resistance and ideal capacitance performance; moreover, it is suitable as an energy storage system unit for EVs.

**Figure 3.** Electrochemical properties: (**a**) =XRD spectrum of the synthesized OMC; (**b**) SEM photograph of the OMC; (**c**) and (**d**) =TEM photographs of the OMC.

To detect the characteristics of SCs, constant current charge and discharge experiments were carried out on the SC cells used herein. The charge and discharge voltage range was set to −1.0–0 V, and the charge and discharge currents were 5, 8 and 10 mA. The single charge and discharge curves have good reversibility, and the two sides of the curve are basically symmetrical. The initial voltage has no significant voltage drop and has ideal capacitive characteristics. As shown in Figure 4b, the half-arc of the high-frequency region is small, indicating that the charge transfer resistance at the interface between the electrode and the electrolyte is small. In the intermediate-frequency region, the slope is close to 45◦, which is related to the charge transfer impedance; the straight line in the low-frequency region is similar to the vertical line, demonstrating good capacitance characteristics. As shown in Figure 4c, the curve shows a typical capacitance characteristic. The time constant (the product of capacitance and resistance) determines the steepness of the potential conversion. When the scanning direction changes, the electrode exhibits a fast current response and is rapid in a stable state, indicating that the internal resistance is small, the RC time constant is small, and it is suitable for high current. As shown in Figure 4d, as the number of cycles increases, the attenuation of capacitance weakens. In the initial stage of the cycle, the surface functional groups of the OMC will decompose, thereby consuming part of the capacitance. Second, as the number of cycles increases, the increase in the temperature of the capacitor will decrease the capacitance, causing a partial fragile hole to be broken. Simultaneously, the increase in temperature further exacerbates the decomposition of the surface functional groups. However, the overall fit is good, and it is suitable as an energy storage system unit for EVs.

**Figure 4.** Electrical performance test: **(a**) Charge–discharge curves; (**b**) Impedance characteristic curves; (**c**) CV curves; (**d**) Cycle performance curves.

Figure 5 shows the state of health (SOH) estimation during the supercapacitor cycle. When the number of cycles was less than 6000, the SOH of the supercapacitor was maintained above 80%, and the cycle performance was relatively good.

**Figure 5.** SC cycle performance.

Currently, SC control structures are widely used in EV composite power systems. Based on the dual-power control structure, a new power allocation strategy is proposed to accurately control the charging and discharging of the battery and the SC. The DC converter used herein a composite converter with an isolated soft-switching symmetrical half-bridge bidirectional converter as a protection structure. The principle of bidirectional DC/DC converter in HESS of Ev is as follows: When the system is discharged, the bidirectional DC/DC controller works in the boost mode, and energy flows from the low-voltage side to the high-voltage side. When the system is charged, the bidirectional DC/DC controller operates in the buck mode, and energy flows from the high-voltage end to the low-voltage end. Figure 6 shows a device used in EV testing.

**Figure 6.** Experimental test device: (**a**) Mixed energy storage unit; (**b**) Vehicle test device.

## **3. Typical Control Policy**

The basic structure of the control strategy in the EV HESSs is shown in Figure 7. The DC/DC converter uses a composite converter with an isolated soft-switching symmetrical half-bridge bidirectional converter as a protection structure when extreme currents occur, as opposed to the non-isolated DC/DC converter used previously. In the event of a sudden increase in current, switching to an isolated converter is possible to avoid damage to the energy storage system due to direct electrical connections. Under normal operating conditions, the DC/DC converter operates in the boost mode during discharge and operates in the buck mode during energy feedback.

**Figure 7.** Conventional control structure.

The power distribution method is as follows. When the EV is driven at a constant speed, the battery unit is separately powered in the HESS. At this time, the battery cells were discharged at a constant current. When the EV accelerates or climbs a hill, the remaining peak power is provided by the SC to meet the power demand. When the EV is decelerated, the feedback power generated by the brake is obtained by the battery, reducing energy waste. When the bus current changes abruptly, the battery unit maintains a constant current through the power distribution module, and the remaining peak current is absorbed and released by the SC.

The driving cycle test was performed under the working condition of 1015 as shown in Figure 8. The simulation results of power distribution are shown in Figure 9. It can be concluded that the operating current of the SC is relatively large under this control mode, and *isc* is 2–3 times that of the *ibat*. The SC acts as a "peak load transfer" to help extend battery life. This control method can

effectively play the role of SC's "peak clipping and valley filling", improve the energy utilization rate of the HESS, and limit the peak current of the battery unit to a safe range.

**Figure 9.** Charge and discharge current distribution of supercapacitors and batteries under the traditional strategy.

As shown in Figure 10, the SOC of the SC demonstrated a large decline at the end of the EV driving cycle and at the lowest point, it dropped to 0.67. This causes the terminal voltage of the SOC of the SC to drop, thereby reducing the transmission efficiency of the DC/DC circuit.

**Figure 10.** SOC curve of supercapacitors in the typical strategy.

This phenomenon occurs owing to some frequent peak acceleration and deceleration during driving, which result in a large discharge current, but the feedback current is small. During the braking process, most feedback current is absorbed by the battery unit, and the SC cannot recover to a higher SOC value in a short time. In response to this drawback, the feedback object is replaced, and the control SC preferentially absorbs the feedback energy. However, this method allows the SC to charge in a short time. If the remaining large current is still absorbed by the battery, it will reduce the battery life and reduce the energy efficiency.

When an EV operates under extreme acceleration or emergency braking, the energy storage system needs to provide a large peak power or a large peak current. If it is through a non-isolated bidirectional DC/DC converter, direct electrical connections can cause significant damage to the energy storage system. When the peak current exceeds a certain threshold, the current in the DC converter passes through the isolated soft-switching symmetric half-bridge bidirectional converter that protects the safe operation of the energy storage system to some extent. Compared with a full-bridge bidirectional converter, the symmetrical half-bridge bidirectional converter is only half of the latter; therefore, the loss is small and the efficiency is high. The protection circuit topology is shown in Figure 11.

**Figure 11.** Isolated soft-switching symmetric half-bridge bidirectional converter.


Author details

detrimental to aero fins. Line-of-sight angular velocities of the ISMCG are less than those of the APNG with the actual target's acceleration. From Figures 2(a), 3(a), 4(a), and 5(a), in case 1 and case 2, ISMCG's guidance commands are smoother than others, which are appropriate for continuous aero surfaces to track. From Figures 2(b), 3(b), 4(b), and 5(b), because the ISMCG uses an adaptive estimation to identify atq's upper boundary, line-of-sight angular velocities of the ISMCG are not as moderate as those of the SMCG; however, from Table 1, line-of-sight angular velocities in the endgame of the ISMCG are less than those of the SMCG and the OSMG in case 1 and case 2. From Figures 6 and 7, the guidance commands and line-of-sight angular velocities of OSMG, SMCG, and ISMCG have little differences and are superior to

From Figure 8, it illustrates the δatq identified by the ISMCG in three cases under two conditions. Compared with ∣atq∣, in the initial 2 s, δatq is larger since q\_ and bk are larger, and then, the tracking error decreases since the ISMCG restrains the line-of-sight angular velocity. Because δatq is not the estimation of atq, tracking phases are considered and tracking errors are not concerned. Tracking phases reflect that estimations lag behind the actual target maneuver; thereby, it decides whether the compensation bk is timely and can influence the guidance precision. With tracking phases under consideration, δatq mostly tracks ∣atq∣ with a tiny time delay. In fact, for the step maneuver target in case 3, from Figure 8(e, f), δatq tracks ∣atq∣ well. As

In this chapter, robust guidance schemes are presented, which require states such as initial relative speed, relative distance, and error boundaries of them besides line-of-sight angular velocity. Proposed schemes' performances are validated by simulating under uncertainties for different target's maneuver modes.  By comparisons with the APNG and OSMG, the ISMCG is superior, and the OSMG and SMCG perform similarly, whereas the APNG's miss distances are greater. Moreover, guidance commands of the APNG and ISMCG are smoother than those of the OSMG and SMCG for complicated maneuver modes of the target. In conclusion, ISMCG's advantage is that the guidance scheme is not required to obtain the target acceleration under

The future work concentrates on adapting the interceptor's maximum maneuverability to be a constraint condition in the proposed guidance scheme. Anti-saturation design is studied to

This chapter is supported by National Key R&D Program of China under Grant Nos. 2017YFD0701000 and 2016YFD0200700, National Natural Science Foundation of China under

shown in Table 1, small tracking phases obtain small miss distances.

uncertain conditions for different target maneuvers.

address the control-saturation problem in Eq. (4) [18–20].

those of the APNG in case 3.

38 Adaptive Robust Control Systems

5. Conclusions

Acknowledgements

Jian Chen1 , Yongjun Zheng1 , Yuan Ren2 \*, Yuan Tian3 , Chen Bai4 , Zhang Ren5 , Guangqi Wang1 , Nannan Du1 and Yu Tan1


**2. Risk management methodology**

Risk management methodology was first described in detail by Wideman in [1]. The methodology was then improved by the PMI [2], adding details based on users' experience.

Project Risk management involves the following steps:

4. A Risk Response plan includes answers to the threats that are identified in the risk assessment phase. There are a number of ways to address these threats.

Selecting a Response Plan Under Budget Constraints 57

al. in [4] describe the use of a methodology for the risk ranking of projects by some subjective judgment; this method has been implemented in construction projects and multiproject environments. Engert from MITRE [3] wrote a user's manual for an Excel application for risk management. The application includes a ranking method of risks based on Borda's method. The Borda method is more quantitative than the subjective judgment method, but still includes some fuzzy ranking when it combines the rank of risk probability with the rank of impact. Ochsner [5] emphasizes the limited attention to risk-based priorities and the growing consensus among industries that risk considerations need to be better integrated into decisions. He agrees that although money is not always the best way to measure risks, no better alternative has thus far been suggested. His ranking method is based on discussions with consultants and experts, assigning scores from 1 to 10 for each category. Li et al. [6] present a ranking method for multiple hazard risks; the method is based on screening all the risks with experts and weighting the risks according to frequency, severity,

In [7] the author presents the difficulties involved in ranking risks. He utilizes the following framework: Risk = Threat×Vulnerability×Consequence, which is usually used in military operations research. The Threat*×*Vulnerability framework actually reflects the probability to damage a target, when the consequence is the damage impact. Our study is important in that for some qualitative measures, it presents counter examples that highlight the limitation of this measurement type. Klein [8] developed a conceptual model for analyzing alternative risk mitigation responses, while accounting for the possibility of trade-off risk among the three main success criteria: cost, duration and scope (or quality). He showed that, given the numerical estimates of risks probabilities and impacts, of all the relevant responses, mathematical techniques - such as dynamic programming or integer programming - could be applied to find the best combination of responses that minimizes project uncertainty. This

Ben-David et al. [9] analyzes a problem that is similar to the current one, but takes a different approach. Assuming that several risk mitigation responses can be implemented with different costs and different expected results, a selection of the best combination of responses is needed. All of the responses are broken down to their work elements, so that each risk can belong to several of them. The Total Risk Cost (TRC) is minimized by two heuristic algorithms; the greedy and the naïve, after which a comparison is presented. The current manuscript does not take into account the budget limitation, and assumes that as long as risk

There are many studies that use subjective judgment to rank risks in different areas, industries, projects and programs. However, none of these ranking methods take into account *the response capability to risks*. There might be a huge difference between two risks that have the same probability to occur and the actual impact*,* when one of the risks occurs. However, for the first risk there is a mitigation plan that reduces its effect substantially and costs \$1,000, while for the second risk, any type of mitigation plan costs more than \$100,000. The study of Gonen et al. [10] proposes an additional criterion for the assessment of risks – that of *controllability*. The introduction of this criterion adds a third dimension to the risk

can be mitigated and it is worthwhile from the budget point of view – it will be done.

availability of warning, awareness, etc.

approach analyzes trade-off among success criteria.

Out of the five responses, only three (avoidance, transfer and mitigation) involve a real investment and require budget allocation.

5. A Control Plan is a series of course adjustments within the project's main objectives. These adjustments include scheduling and tracing the advance of risk situations. The control plan defines indicators that provide warnings regarding the realization of specific risks.

Continuously assessing program risks is the implementation of the control plan by checking any changes in the assessment of risks, and conducting a continuous search for warning signs that indicate any realization of known risks.

This part of the project plan includes the updating of the risk management plan.

The current study concentrates on allocating a budget to the response plan in an optimal manner.

This paper includes a literature review, problem definition, algorithms' definition, an example that is solved by all algorithms, and a comparison among the algorithms by simulation results. The research is quantitative and presents simulation results. Since the difference among the algorithms for different budgets are so big, statistical analysis is unnecessary.

The simulation and algorithms were verified by solving known problems and their solutions.

### **3. Literature review**

Project risk management literature commonly describes the need to rank and prioritize project risks in order to focus the risk management effort on the higher risks. Baccarinia et al. in [4] describe the use of a methodology for the risk ranking of projects by some subjective judgment; this method has been implemented in construction projects and multiproject environments. Engert from MITRE [3] wrote a user's manual for an Excel application for risk management. The application includes a ranking method of risks based on Borda's method. The Borda method is more quantitative than the subjective judgment method, but still includes some fuzzy ranking when it combines the rank of risk probability with the rank of impact. Ochsner [5] emphasizes the limited attention to risk-based priorities and the growing consensus among industries that risk considerations need to be better integrated into decisions. He agrees that although money is not always the best way to measure risks, no better alternative has thus far been suggested. His ranking method is based on discussions with consultants and experts, assigning scores from 1 to 10 for each category. Li et al. [6] present a ranking method for multiple hazard risks; the method is based on screening all the risks with experts and weighting the risks according to frequency, severity, availability of warning, awareness, etc.

56 Risk Management – Current Issues and Challenges

agreements.

manner.

unnecessary.

**3. Literature review**

solutions.

preventing the risk.

4. A Risk Response plan includes answers to the threats that are identified in the risk

b. Transfer – transfer responsibility for the particular risk to a third party, either by utilizing insurance or, in the international arena, by forming treaties and international

c. Acceptance - a rational decision to accept a known risk without taking any action to prevent its outcome or deal with its consequences. The risk is usually dealt with when it is recognized as a risk. An acceptance of risk is recommended in situations where the consequences of the risk are less costly or less traumatic than the effort required

d. Mitigation - refers to action taken to reduce either the probability of occurrence of an unfavorable event or the impact of this event. Mitigation is usually executed in the form

e. Contingency Planning – refers to specific actions to be taken when a potential risk event occurs. In general, contingency plans should be developed in advance in preparation

Out of the five responses, only three (avoidance, transfer and mitigation) involve a real

5. A Control Plan is a series of course adjustments within the project's main objectives. These adjustments include scheduling and tracing the advance of risk situations. The control plan defines indicators that provide warnings regarding the realization of specific risks.

Continuously assessing program risks is the implementation of the control plan by checking any changes in the assessment of risks, and conducting a continuous search for warning

The current study concentrates on allocating a budget to the response plan in an optimal

This paper includes a literature review, problem definition, algorithms' definition, an example that is solved by all algorithms, and a comparison among the algorithms by simulation results. The research is quantitative and presents simulation results. Since the difference among the algorithms for different budgets are so big, statistical analysis is

The simulation and algorithms were verified by solving known problems and their

Project risk management literature commonly describes the need to rank and prioritize project risks in order to focus the risk management effort on the higher risks. Baccarinia et

This part of the project plan includes the updating of the risk management plan.

assessment phase. There are a number of ways to address these threats.

a. Avoidance - generate a course of action that eliminates the risk.

of a plan designed to handle high-threat possible events.

for the moment when risk events are realized.

investment and require budget allocation.

signs that indicate any realization of known risks.

In [7] the author presents the difficulties involved in ranking risks. He utilizes the following framework: Risk = Threat×Vulnerability×Consequence, which is usually used in military operations research. The Threat*×*Vulnerability framework actually reflects the probability to damage a target, when the consequence is the damage impact. Our study is important in that for some qualitative measures, it presents counter examples that highlight the limitation of this measurement type. Klein [8] developed a conceptual model for analyzing alternative risk mitigation responses, while accounting for the possibility of trade-off risk among the three main success criteria: cost, duration and scope (or quality). He showed that, given the numerical estimates of risks probabilities and impacts, of all the relevant responses, mathematical techniques - such as dynamic programming or integer programming - could be applied to find the best combination of responses that minimizes project uncertainty. This approach analyzes trade-off among success criteria.

Ben-David et al. [9] analyzes a problem that is similar to the current one, but takes a different approach. Assuming that several risk mitigation responses can be implemented with different costs and different expected results, a selection of the best combination of responses is needed. All of the responses are broken down to their work elements, so that each risk can belong to several of them. The Total Risk Cost (TRC) is minimized by two heuristic algorithms; the greedy and the naïve, after which a comparison is presented. The current manuscript does not take into account the budget limitation, and assumes that as long as risk can be mitigated and it is worthwhile from the budget point of view – it will be done.

There are many studies that use subjective judgment to rank risks in different areas, industries, projects and programs. However, none of these ranking methods take into account *the response capability to risks*. There might be a huge difference between two risks that have the same probability to occur and the actual impact*,* when one of the risks occurs. However, for the first risk there is a mitigation plan that reduces its effect substantially and costs \$1,000, while for the second risk, any type of mitigation plan costs more than \$100,000. The study of Gonen et al. [10] proposes an additional criterion for the assessment of risks – that of *controllability*. The introduction of this criterion adds a third dimension to the risk

evaluation process, in addition to its probability and impact. The controllability of a given risk reflects the ability to control it, mitigate it, or even prevent it. Assessing controllability may reduce the efforts and spending of managerial time and expenses on non-controllable risks and, in the end, direct the attention of management solely to controllable risks.

Selecting a Response Plan Under Budget Constraints 59

Let B=50. In this case, we can either choose the second or the third row. If we choose the second row, we reduce the expected damage of Risk 1 to 100 and stay with Risk 2 at an expected damage of 210. All together, the expected damage of both risks is 310. The same is true if we choose to handle Risk 2 and reduce the expected damage to 110. Since the expected damage of Risk 1 is 200, the total is 310. Let B=80. In this case, we can choose the first row or the previous option of B=50. Choosing Row 1 derives the total expected damage to 90+210=300 (the 210 is from R2). If we choose the second row (mitigating R1), our total expected damage will be 310, and the third row (mitigating R2) will be the same - 310. However, in both mitigation plans we only invest 50, while in the transfer policy we invest a minimum of 80. People who are risk-averse will prefer this option, while others who are attracted to risk might prefer the second or third row. If B=100, then an additional option is open which allows us to choose Rows 2 and 3 and reduce the expected damage to 210. If B=130 and up, we can choose Rows 1 and 3 and reduce the

If we try to minimize the expected damage when B=80, then transferring R1 would be optimal, although usually risk management methods will rank R2 higher and recommend

In order to define the optimal response problem, we will use the following terminology and

There are *n* risks R1,…,R*n*. For each risk R*i*, the probability of its occurrence is Pi and the damage when it occurs is Di. Therefore, for each risk R*i*, the expected damage is *Q*i *= P*i*·D*i*.*

For each risk Ri, there are *k* responses (some can be empty; others can be transfer or mitigation) out of which we can choose, at most, **one**. This can be done by combining

The response *j* to risk Ri costs Cij; after its implementation, the probability of its occurrence is Pij and the corresponding damage is Dij. The expected damage after its implementation is

Qij= Pij·Dij.

(Only if the investment + the expected damage after the implementation are lower than the original expected damage). A response plan that is not worthwhile will not be included in

ܳ െ ൫ܥ ܳ൯. Let us now define the decision variables *X*ij as 1, if response j is selected for risk Ri, and 0,

the list of possible responses. Actually, the savings in selecting response *j* to risk R*i* is:

ܳ ൫ܥ ܳ൯݂ݎ݅ ൌ ͳǡ ǥ ǡ ݊ (1)

mitigation plans together. Index *j will be used for a response plan*.

A response plan is defined as "worthwhile" only if

expected damage to 200.

Index *i will be used for risks.*

treating it first.

symbolization:

otherwise.

Controllability adds a new criterion that takes into account the response capability, but still does not propose a method to quantitatively rank the risks. In the current paper, we overcome the problem of ranking risks by utilizing a method of selecting the optimal mitigation plan for a given budget, and therefore, the risks to be mitigated or transferred.

Kutsch et al. [11] have investigated the type of risks that can be deliberately ignored. In the current study, we deal with risks that are not supposed to be ignored.


**2. Extracellular matrices contribute to the viscoelastic properties of connective tissues including the dermis**

The viscoelastic properties of human tissues are principally governed by the nature of the extracellular matrix (ECM). The ECM comprises secreted proteins that are deposited into the extracellular space. Although cell-ECM interaction and growth factor-ECM interactions have recently been investigated, the ECM represents the fundamental architecture of tissue. Some ECM proteins supply the connective tissues with hydration and resiliency through their characteristic molecular properties and assembly. ECM proteins such as collagen, proteoglycans, and glycoproteins are classified by their biochemical properties rather than their physical properties. Numerous ECM proteins are currently known and have been well characterized biologically. However, the contribution of each ECM protein to the viscoelastic properties of tissues has not been fully investigated due to the lack of a proper experimental system. Therefore, the contribution of each ECM protein to viscoelasticity in human tissue should be determined based on biological, physical, and clinical studies.

© 2012 Nemoto et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**Preparation and Characterization of Self-Assembled Poly(**l**-Lactide) on the Surface of** β**-Tricalcium Diphosphate(V) for Bone Tissue Theranostics**

### **Jan A. Zienkiewicz 1, Adam Strzep 1, Dawid Jedrzkiewicz 2, Nicole Nowak 1, Justyna Rewak-Soroczynska 1, Adam Watras 1, Jolanta Ejfler 2 and Rafal J. Wiglusz 1,3,\***

Received: 23 January 2020; Accepted: 11 February 2020; Published: 15 February 2020

**Abstract:** This work was aimed to obtain and characterize the well-defined biocomposites based on β-tricalcium diphosphate(V) (β-TCP) co-doped with Ce3+ and Pr3+ ions modified by poly(l-lactide) (PLLA) with precise tailored chain length and different phosphate to polymer ratio. The composites as well as β-tricalcium diphosphate(V) were spectroscopically characterized using emission spectroscopy and luminescence kinetics. Morphological and structural properties were studied using X-Ray Diffraction (XRD) and Scanning Electron Microscopy (SEM). The self-assembled poly(l-lactide) in a shape of rose flower has been successfully polymerized on the surface of the β-tricalcium diphosphate(V) nanocrystals. The studied materials were evaluated in vitro including cytotoxicity (MTT assay) and hemolysis tests. The obtained results suggested that the studied materials may find potential application in tissue engineering.

**Keywords:** β-tricalcium diphosphate(V); Ce3+ and Pr3+ ions co-doping; poly(l-lactide); theranostics

#### **1. Introduction**

Theranostics is a field of modern nanomedicine that is based on targeted therapy and medical diagnostic tests. This approach is involved with a dynamic development of chemistry, biology, biotechnology, physics and last, but not least—nanotechnology [1].

In the case of biomaterial, some requirements should be met to be treated as a theranostics agent, but one is obligatorily related to similarity to the original tissue [2]. Moreover, the bone minerals are mostly formed by calcium hydroxyapatites (herein CaHAp) [3]. There are a lot of materials related to the apatite family that could be used in the theranostics applications. Among them is β-tricalcium diphosphate (herein β-TCP) that could be a promising agent. This material crystallizes in rhombohedral structure in *R3C* space group [4]. Furthermore, the CaHAp and β-TCP are biocompatible, osteoconductive, and bioactive biomaterials. Comparing the Ca:P ratios in both materials, hydroxyapatite is more similar to mineralized bone, however resorption time of the β-TCP-based materials is much shorter due to its much greater solubility in water in 37 ◦C [5].

The simplest way to use this material as a bio-imaging agent is a structural modification in order to observe light emission in the range of one of biological optical windows. Our research has showed that β-TCP doped with Pr3+ ions exhibits an intense emission band near 650 nm. It has been shown that luminous efficiency of Pr3+ ions could be further enhanced by Ce3+ co-doping. The literature reports that significant increase of Pr3+ emission via energy transfer from excited Ce3+ions is observed i.e., in YAG (Y3Al5O12, Yttrium Aluminum Garnet) crystals [6]. Efficient luminescent properties are needed to precisely monitor temporal stability and spatial migration of introduced material in the bone tissue.

To insert inorganic material into the bone tissue, a carrier agent is needed. In human bone tissue, calcium hydroxyapatite crystals are distributed in the collagen fibrils forming a complex structure. Another way to obtain biomaterial similar to bone tissue is to use bioresorbable polymer. One of the most used ones is polylactide (PLA).

Biodegradable aliphatic polyesters, for example polylactide (PLA), are widely used polymers in a variety of bioapplications such as controlled drug release, gene therapy, regenerative medicine, or implants [7–11]. The most effective and controlled method for PLA synthesis is metal-catalyzed/-initiated ring-opening polymerization (ROP) of lactide [12–14]. Among the wide variety of catalytic systems, the most attractive so far are the single-site initiators based on structural motif L-M-OR, where L is the ancillary ligand, M is the metal center, and OR is the initiating group [15–23]. Alternative, similarly attractive in the controlled synthesis of PLA are binary catalytic systems based on the homoleptic complexes and external alcohol combination [24–26]. The most widely used system includes commercially available bis(2-ethylhexanoate)tin(II) (Sn(Oct)2) catalyst, commonly applied in industry [12]. However, in the context of medical applications, biologically benign complexes (Zn, Mg, Ca) are the most searched due to their innocuous nature, ready availability, and their effectiveness for polymerization, both in terms of activity and stereoselectivity. Recently, polymer/inorganic nanocomposite materials have attracted considerable interest because of their excellent properties through synergism of polymer and inorganic nanoparticle (iNPs) components. The key to obtaining suitable in bioaplications polymer/iNPs composites is achieving the fine dispersion of inorganic nanoparticles in the polymer matrix. In terms of variety of synthetic strategies, some of them are more favorable for the formation of polymer/iNPs composites because of the simplicity in materials processing. Among others, it is worth mentioning about binding of polymer chains to iNPs by coating modification, dispersion of iNPs in polymer matrix, and formation of stabilizing polymer shell. PLA is suitable for surface coating of iNPs because of their biocompatibility and versatility while providing a platform for further biological modifications.

The proposed research has been intended to consider the well-characterized theranostics materials for bone damage tissue. Nanocrystalline β-tricalcium diphosphate (β-TCP) (so-called therapeutic part) has been co-doped with Ce3+ and Pr3+ ions (diagnostic part) and coated with poly(l-lactide) (so called a carrier). Moreover, they have been tested in vitro to examine their cytotoxicity using human chondrocyte cell line and mouse osteoblast cell line (MTT assay) and a standard hemolysis test.

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

#### *2.1. Materials*

All reactions and operations that required an inert atmosphere of N2 (synthesis of zinc complex L2Zn and polymers PLA) were performed using a glovebox (MBraun, Garching, Germany) or standard Schlenk-like apparatus (ILT&SR PAS, Wroclaw, Poland) and vacuum line techniques. The solvents for synthesis were purified by standard methods before use: n-hexane (VWR, Radnor, PA, USA) distilled from Na; MeOH (HPLC, VWR, Radnor, PA, USA) distilled from CaH2; CH2Cl2 (99.8% VWR, Radnor, PA, USA) distilled from P2O5; deuterated solvents (C6D6), distilled from NaH. Unless otherwise stated, all chemicals were purchased from commercial sources and used without further purification:

Calcium carbonate (99.5%, Alfa Aesar, Haverhill, MA, USA), ammonium dihydrogen phosphate (99+%, for analysis, ACROS Organics, Geel, Belgium), cerium(III) nitrate hexahydrate (99.999%, Alfa Aesar, Haverhill, MA, USA), praseodymium(III, IV) oxide (99.999%, Sigma-Aldrich, Saint Louis, MO, USA), nitric acid (65%, Suprapure, Merck, Darmstadt, Germany), citric acid (99%+, Alfa Aesar, Haverhill, MA, USA), ethylene glycol (ultrapure, Avantor Performance Materials Poland S.A., Gliwice, Poland) 2,4-di-*tert*-buthylphenol (99%, Sigma-Aldrich, Saint Louis, MO, USA), formaldehyde (37% solution in H2O, Sigma-Aldrich, Saint Louis, MO, USA). *N*-methylcyclohexylamine (99%, Sigma-Aldrich, Saint Louis, MO, USA), ZnEt2 (1.0 M solution in *n*-heptane, Sigma-Aldrich, Saint Louis, MO, USA). Proligand [*LCy-H*] *N*-[methyl(2-hydroxy-3,5-di-*tert*-butylphenyl)]-*N*-methyl-*N*-cyclohexylamine was synthesized according to a literature procedure [24].

#### *2.2. Synthesis of* β*-tricalcium diphosphates (*β*-TCP) Doped with Pr3*+*and Co-Doped with Ce3*+ *Ions*

The nanocrystalline β-Ca3(PO4)2 calcium phosphate co-doped with Ce3+ and Pr3+ ions were prepared by modified Pechini's method, using 18 mmol of CaCO3, 12 mmol of NH4H2PO4, 0.09 mmol of Ce(NO3)3·4H2O, and 0.015 mmol of Pr6O11. Intentional concentration of the Ce3+ and Pr3+ ions was set to 0.5 mol%, in replacement of overall molar content of Ca2+ ions.

In this method, stoichiometric amounts of CaCO3 and Pr6O11 were weighed and digested in excess of HNO3 (Suprapure, Merck) in order to transform them into nitrates. Subsequently, cerium nitrate was dissolved in deionized water together with calcium and praseodymium (III) nitrates. Afterwards, the excess (12,5-fold relative to the total amount of cations) of citric acid as well as ethylene glycol were added under constant stirring at 60 ◦C, resulting in a viscous mixture. Finally, a suitable amount of ammonium hydrogen phosphate was added. The temperature was raised up to 120 ◦C. The heating was continued until a white voluminous foam was obtained. The mixture was further dried for 3 days at 90 ◦C. Afterwards, the resin thus obtained was calcinated in a temperature of 900 ◦C. As a result, a white powder (β-TCP) was obtained.

#### *2.3. Synthesis of poly(*l*-lactide) (PLLA)*

Synthesis of PLLA was divided into two parts. At first, the initiator was synthetized. Afterwards, ring opening polymerization of L-lactide was carried out. Oligomeric chains based on L-lactide with methyl ending group were prepared in ring opening polymerization (ROP) reaction. Homoleptic zinc aminophenolate complex type ZnL2 (*L* = *N*-methyl-(2-hydroxy-3,5-di-tert-buthylphenyl)]-*N*-cyclohexylamine) as initiator was used. High efficiency of this catalyst was described in the literature [24–26].

#### 2.3.1. Synthesis of Initiator

[(LCy)2Zn] was synthesized following a modified procedure of our previous report [25].

To a stirred solution of 0.66 g (2.00 mmol) of proligand LCy-H in *n*-hexane (20 mL), 1.00 mL (1.00 mmol) of ZnEt2 (1.0 M solution in *n*-heptane) was added dropwise at ambient temperature. Next, the solution was stirred for 12 h until a crude product precipitated. The white powder of [(LCy)2Zn] was collected by filtration, washed with cold n-hexane, and dried in vacuo. Yield: 92% (0.67 g, 0.92 mmol). Anal. Calcd (Found) for C44H72N2O2Zn (726.41): C 72.75 (72.83), H 9.99 (9.86), N 1.96 (1.85)%. 1H NMR for major form (500 MHz, C6D6, 298 K): δ = 7.72 (2H, d, ArH, *J*HH = 2.6 Hz), 7.09 (2H, d, ArH, *J*HH = 2.6 Hz), 4.16 (2H, d, N-CH2-Ar, *J*HH = 12.1 Hz), 3.47 (2H, d, N-CH2-Ar, *J*HH = 12.1 Hz), 3.20 (2H, m, C6H11), 2.48 (6H, s, NCH3), 1.74 (18H, s, C(CH3)3), 1.66 (10H, m, C6H11), 1.59 (18H, s, C(CH3)3), 1.05 (10H, m, C6H11). 13C NMR (500 MHz, C6D6, 298 K) δ = 164.3, 138.4, 135.5, 125.9, 124.4, 120.5 (12C, Ar), 65.3 (N-CH2-Ar), 61.9 (2C, C6H11), 36.8 (C(CH3)3), 35.5 (C(CH3)3), 33.8 (NCH3), 32.3 (C(CH)3)3), 30.2 (C(CH)3)3), 26.9, 26.1, 24.4 (10C, C6H11).

#### 2.3.2. Representative Procedure for Solution Polymerization

ROP polymerization of l-Lactide while using binary catalytic system [(*LCy*)2Zn]/MeOH.

The solution of zinc complex [(*LCy*)2Zn] in CH2Cl2 (15 mL) was placed in a Schlenk flask, and MeOH and l-lactide in molar ratio [(*LCy*)2Zn]/MeOH/l-Lactide 1/1/15 was added. The obtained solution was stirred for 5 h. At certain time intervals, about 1 mL aliquots were removed, precipitated with hexanes, and dried in vacuo. The obtained precipitates were dissolved in C6D6 and used for the conversion monitoring, which was determined by 1H NMR. After the reaction was completed, an excess of hexanes was added to the reaction mixture. The obtained crude polymer was next filtered off and dried in vacuo. The resulting powder was dissolved in dichloromethane and the PLA was precipitated with excess of cold hexanes. The PLA was collected by filtration, washed with hexanes, and dried in vacuo. The reaction mixture was prepared in glovebox, and the next subsequent operations for the isolation of pure PLLA were performed by using standard Schlenk apparatus and vacuum line techniques.

#### *2.4. Representative Procedure for Preparation of* β*-TCP@PLLA Composites*

TCP@PLA composites were obtained by using precipitation method.

To the sample of polymer, PLLA (50 mg) dissolved in 1 mL of CH2Cl2 nanocrystalline β-TCP (5 mg) was added and the mixture was stirred at room temperature for 0.5 h. The β-TCP@PLLA composites were precipitated with an excess of hexanes (50 mL), which was added dropwise over 10 min. Next the hexane was removed by decantation and composite material was purified by washing in hexane (3 × 50 mL) and isolated by drying the precipitate afterwards under reduced pressure during 72 h in the temperature 60 ◦C.

#### *2.5. Physicochemical Characterization*

1H, 13C NMR spectra were obtained using Bruker Avance 500 MHz spectrometer (Bruker, Billerica, MA, USA). The chemical shifts are given in ppm and referenced to the residual protons in the deuterated solvents. Microanalyses were conducted with an Elementar CHNS Vario EL III analyzer (Elementar, Langenselbold, Germany).

X-Ray Powder Diffraction (XRD) studies were carried out using PANalytical X'Pert Pro diffractometer (Malvern Panalytical Ltd, Malvern, UK) equipped with Ni-filtered Cu Kα radiation (*V* = 40 kV, *I* = 30 mA, λ = 1.5406 Å). The XRD patterns were collected during 3 h in the 2θ range of 10–60◦.

The microstructure investigations and elemental analysis were carried out using a scanning electron microscope FEI Nova NanoSEM 230 (FEI Company, Hillsboro, OR, USA) equipped with EDS spectrometer (EDAX PegasusXM4) (EDAX Inc., Mahwah, NJ, USA). TCP sample was attached to the measuring table using graphite tape and imaged at an accelerated voltage 10 kV. In order to eliminate the effect of collecting charge on the surface of material, PLLA and composite samples were imaged at an accelerated voltage 5 kV and 10 kV.

High-resolution emission spectra and luminescence kinetics curves were recorded using excitation wavelength 445 nm. Opolette Nd:YAG Laser-OPO system (Opotek INC, CA, USA) was used as an excitation source. Emission spectra were recorded with use of DongWooOptron d750 monochromator (DongWoo Optron, Maesan-ri, South Korea). Light was collected by Hamamatsu R928 photomultiplier (Hamamatsu Photonics K.K., Hamamatsu, Japan). Signal from the photomultiplier was analyzed in parallel by SR250 Gated Integrator (Stanford Research System, Sunnyvale, CA, USA) for integration of signal and a Tektronix TDS 3050 digital oscilloscope (luminescence kinetics) (Tektronix Inc., Beaverton, OR, USA).

#### *2.6. Evaluation of Biological Properties*

Potential non cytotoxic properties of obtained materials were evaluated via MTT assay by using human chondrocyte cell line (TC28A2) and mouse osteoblast (7F2) cell lines. Hemolysis assay by using ram blood cells was performed to estimate possible hemolytic activity of our composites.

#### 2.6.1. Human Chondrocytes Cell Line

TC28A2 human chondrocyte cell line was maintained in high glucose Dulbecco's Modyfied Eagle Medium (DMEM) with l-glutamine (Biowest, Nuaillé, France) and supplemented with 10% Fetal Bovine Serum (FBS) South America Heat Inactivated (Biowest, Nuaillé, France), 200 U/mL penicillin, and 200 μg/mL streptomycin. Mouse osteoblast cell line (7F2) was cultured in Minimum Essential Medium Eagle–alpha modification (α-MEM) without nucleosides (Biowest, Nuaillé, France). To obtain a full cultured medium, α-MEM was supplemented with 10% FBS and 2mM stable glutamine (Biowest, Nuaillé, France). TC28A2 and 7F2 cell lines were incubated in standard conditions at 37 ◦C in humidified atmosphere of 5% CO2 and 95% air. Passive cells were used three times in the experiments.

#### 2.6.2. Influence of Obtained Composites on Chondrocytes Proliferation Rate

Proliferation capacity of human chondrocytes and mouse osteoblast were evaluated via performing MTT cytotoxicity assay Tc28A2 and 7F2 cells were seeded at density 10,000 cells per well in 96-well plates and allowed to attached and grow for 24 h. Then, cells were washed with sterile PBS (Biowest, Nuaillé, France), and fresh medium and adequate concentration of tested compounds, (50 μg/mL and 100 μg/mL of β-Ca3(PO4)2, PLLA and composite) was added. MTT (BioReagent, >97.5%, Sigma-Aldrich, Saint Louis, MO, USA) assay was performed 24 h after cells treatment. Treatment medium was removed and sterile PBS containing 0.5 mg/mL MTT (tiazol blue tertazolium) was added, and cells were incubated 3 h at 37 ◦C. After incubation, medium containing MTT was removed without washing, and formed formazan crystals were dissolved in DMSO (99.5%, Sigma-Aldrich, Saint Louis, MO, USA). Absorbance was read with a Varioskan LUX plate reader (ThermoFisher Scientific, Waltham, MA, USA) at 560 nm with background reference at 670 nm. The experiment was performed three times. Percentage of cell viability was calculated using the following formula:

$$\text{Cells viability} = \frac{\text{sample absorbance}}{\text{control absorbance}} \times 100\%. \tag{1}$$

As a reference control (100% of cells viability), samples of non-treated cells were used in both cell lines.

#### 2.6.3. Hemolysis Assay

Hemolysis assay was performed according to the protocol described elsewhere with the slight modification [27]. Ram blood (ProAnimali, Wroclaw, Poland) was centrifuged (3000 RPM, 10 min) in order to obtain erythrocyte fraction, which was washed with PBS (phosphate-buffered saline, pH 7.4) and mixed with fresh PBS (1:1 *v*/*v*). β-Ca3(PO4)2; PLLA and the β-Ca3(PO4)2/PLLA (1:10) composite were mixed with erythrocytes at final concentration of 50 and 100 μg/mL and incubated in 37 ◦C for 2 h. Then, samples were centrifuged in order to obtain supernatant (5000 RPM, 5 min) and the optical density was measured at 540 nm with a Varioskan LUX plate reader (ThermoFisher Scientific, Waltham, MA, USA). As a reference control (100% of hemolysis), the 1% solution of SDS (sodium dodecyl sulfate) was used and as negative control the solution of PBS (phosphate-buffered saline) was applied. Obtained results were compared with the absorbance of SDS sample and shown as a percentage of hemolysis and the hemolysis percentage was calculated as using following formula:

$$\text{Hemolyis} = \frac{\text{sample absorbance} - \text{negative control absorbance}}{\text{positive control absorbance} - \text{negative control absorbance}} \times 100\tag{2}$$

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

#### *3.1. Structural Properties*

#### 3.1.1. β-tricalcium diphosphate (β-TCP)

Structure and phase purity were checked with the powder XRD technique and were compared with the reference standard of the tetragonal β–Ca3(PO4)2 lattice ascribed to the *R-3c* space group. Diffractograms of β-TCP co-doped with Ce3+ and Pr3+ and TCP@PLLA composite as well as the

theoretical XRD pattern of β–Ca3(PO4)2 (ICSD 97500) and the diffractogram of pure PLLA are shown in Figure 1.

**Figure 1.** X-ray diffraction patters of poly(l-lactide) (PLLA), β-Ca3(PO4)2 (β-TCP) co-doped with Ce3+ and Pr3+ ions and PLLA with β-Ca3(PO4)2 composite compared to theoretical pattern of β-Ca3(PO4)2 (ICSD-97500).

β-TCP crystallized in rhombohedral structure, in the space group *R3c*. Due to Shannon, Ca2+, Ce3+, and Pr3+ effective ionic radii were 1.00 Å, 1.01 Å, and 0.99 Å, respectively [28]. Due to similar ionic radiilanthanide, ions substituted Ca2+ sites in the β-TCP matrix lattice.

#### 3.1.2. Poly(l-lactide) (PLLA)

The activity and effectiveness of binary catalytic system [(*LCy*)2Zn]/MeOH to synthesis of PLLA in living ROP of lactides from high and ultra-low molecular weight was recently published. That catalytic system is well suited especially for the synthesis of precisely defined oligolactides in comparison with the commercial Sn(Oct)2, which is not as selective as [(*LCy*)2Zn]/ROH. Additionally, the most popular Sn(Oct)2 used for the synthesis of low molecular PL*L*A produces the oligolactides with the fraction of alkyl-(*S,S*)-O-lactyllactate. Therefore, the precisely defined polymer matrix containing planned 15 lactide units has been obtained by using [(*LCy*)2Zn]/MeOH catalytic system. Under selected molar ratio of ROP components [(*LCy*)2Zn]/MeOH/*L*-LA = 1/1/15, the oligomer 15-PL*L*A-Me were obtained (Figure 2).

The end groups and the number of lactide units were detected by using NMR spectroscopy. The 1H NMR spectra for 15-PLLA-Me oligomer showed expected resonances for both chains end, methyl ester and hydroxyl groups, and oligolactide backbone chain (Figure 3). The most intense signals A3 and B3 corresponded to methine and methyl groups of repetitive central open lactide units. The adequate resonances of open lactide units close to end groups were denoted as A1-2 B1-2 (a couple of signals coming from neighboring mer to hydroxy end) and A4-5 B4-5 (the first open lactide molecule close to ester chain end) (see Figure 2).

**Figure 2.** Synthesis of 15-PL*L*A-Me oligomer.

**Figure 3.** 1H NMR spectrum of 15-PL*L*A-Me oligomer (*C6D6*).

#### *3.2. Morfological Properties*

SEM images of uncoated β-TCP (A) and poly(l-lactide) (B) compared to β-TCP coated with PLLA in two β-TCP:PLLA ratios: 1:10 (C) and 1:20 (D) are presented in Figure 4.

**Figure 4.** SEM images of (**A**) β-TCPco-doped with Ce3+ and Pr3+ ions, (**B**) PLLA, (**C**) PLLA with β-TCP composite with phosphate:polymer 1:10 ratio, and (**D**) PLLA with β-TCP composite with phosphate:polymer 1:20 ratio.

The surface of the sample coated with 1:20 ratio (D) clearly showed new morphology with the shape of rose flower, while surfaces of pure PLLA (B) and the sample coated with 1:10 ratio (C) were much smoother.

#### *3.3. Spectroscopic Properties*

#### 3.3.1. Emission Spectra

Emission spectra were measured in the range of wavelength from 500 nm to 700 nm after excitation with 445 nm. Emission spectra of β-TCP:0.5%Ce; 0.5%Pr sintered at 900 ◦C for 3 h, pure and coated with poly(l-lactide) upon excitation at 445 nm, are presented in Figure 5.

**Figure 5.** Emission spectra of PLLA with β-TCP co-doped with Ce3+ and Pr3+ ions (**a**) and PLLA with β-TCP composite with phosphate:polymer 1:20 ratio (**b**). Spectra were recorded after excitation with 445 nm.

The Ce3+ and Pr3+-co-doped tricalcium β-diphosphate showed prominent 1D2 → 3H4 and 3P0 → 3F2 transitions. The 3P0 → 3F2 transition was less intense than the 1D2 → 3H4, but it was observed near 650 nm—in the range of first optical window for biological tissues. Spectrum of pure β-TCP co-doped with Ce3+ and Pr3+ ions was identical to the spectrum of coated with PLLA. This means that the presence of the coating layer did not change luminescent properties of β-TCP core. Furthermore, resemblance of both spectra suggests that Pr3+ ions remained in phosphate structure and did not diffuse to polymer body.

#### 3.3.2. Luminescence Kinetics

Luminescence kinetics curves were recorded after excitation with 445 nm. Decays from the *1D2* and *3P0* state were observed at subsequently 605 and 650 nm.

Lifetimes of the *1D2* and *3P0* states of Pr3+ ions were not mono-exponential. Average lifetimes of all materials were calculated from the equation (Table 1):

$$
\pi\_{w\circ\!g} = \frac{\int I(t)t dt}{\int I(t)dt} \tag{3}
$$

where: *I*—intensity; *t*—time; τ*avg*—average lifetime.

**Table 1.** Calculated lifetimes of β-TCP co-doped with Ce3+ and Pr3+ ions PLLA with β-Ca3(PO4)2 composite with phosphate:polymer 1:10 ratio and PLLA with β-TCP composite with phosphate:polymer 1:20 ratio. Delays from the state 3P0 were observed at 650 nm and delays from the state 1D2 were observed at 605 nm.

*1D2* lifetimes in pure TCP and 10% composite with PLLA were the same, but in the 5% composite, they were a little bit shorter; however, differences between samples were too scarce to consider them as a significant influence of polymer coating. Decay curves observed at 650 nm and 605 nm are presented in Figure 6.

**Figure 6.** Luminescence kinetics curves of β-TCP co-doped with Ce3+ and Pr3+ ions PLLA with β-TCP composite with phosphate: polymer 1:10 ratio and PLLA with β-TCP composite with phosphate: polymer 1:20 ratio, recorded at 650 nm (*3P0*) and recorded at 605 nm (*1D2*).

Decay time of the luminescence from the state *3P0* observed at 650 nm was about 1 μs in each studied case, which was much longer than the luminescence lifetime of emission exhibited by organic molecules from biological tissue [29,30]. This feature could be used to easily discern whether observed luminescence comes from tissue-related molecules or artificially introduced β-TCP. Time dependency and spectral shape of luminesce from introduced material could be used to precisely monitor temporal stability and spatial migration of β-TCP in the bone tissue. Location and stability monitoring are needed to fulfil diagnostic part of β-TCP/PLLA composite possible theranostics application.

#### *3.4. Biological Features*

#### 3.4.1. Cytotoxicity Assay

Our results clearly showed that some of the tested materials increased cell proliferation rate in both concentrations 50 μg/mL and 100 μg/mL in the TC28A2 cell line, which may indicate enhancement of metabolic activity of treated cells. PLLA seems to be degraded in an extracellular environment to l-lactide and then may be transported via MTCs (monocarboxylate transporters) and used by chondrocytes as an energetic fuel [31]. Additionally, chondrocytes proliferation rate was higher than 50% even in the 100 μg/mL concentration of all tested composites. Obtained results were collected in Figure 7. Intriguingly, in comparison with chondrocytes, viability of 7f2 cells treated with PLLA maintain edat 74% in concentration 50 μg/mL and seemed to gradually decrease in higher concentrations. The lower proliferation rate of 7F2 cells may be explained by increased lactate production in osteoblasts, which was caused by the aerobic glycolysis even in the presence of oxygen. This type of metabolism was similar to the Warburg effect, the major hallmark of cancer. It transpired that specific metabolic needs of osteoblasts demanded this particular type of metabolism. Elevated concentrations of lactic acid may additionally acidify extracellular environment, thus osteoblast cells do not use PLLA as an energy fuel like chondrocytes [32]. When compared to other compounds, the viability of mouse osteoblasts maintains around 75% when treated with β-TCP and the composite at a concentration of 50 μg/mL and around 60% at a concentration of 100 μg/mL. This may indicate that the osteoblast cell line is more sensitive to alterations in extracellular environment. However, what is the most important is that proliferation of TC28A2 and 7F2 cell lines was maintained above 50% in each sample and both concentrations.

**Figure 7.** 7F2 and TC26A2 cells viability after 24 h of incubation and exposition on β-TCP co-doped with Ce3+ and Pr3+ ions, PLLA, and PLLA with β-TCP composite with phosphate:polymer 1:20 ratio, in concentrations of 50 μg/mL and 100 μg/mL.

#### 3.4.2. Hemolysis

Tested compounds at concentrations of 50 and 100 μg/mL did not cause the hemolysis of ram erythrocytes. Obtained results were compared with the hemolysis caused by 1% of SDS solution (complete damage of the cellular membrane and hemoglobin release; data not shown) and are presented in Figure 8. Statistical analysis was also performed (*p* < 0.05) using one-way ANOVA test.

**Figure 8.** Activity of β-TCP co-doped with Ce3+ and Pr3+ ions, PLLA, and PLLA with β-TCP composite with phosphate:polymer 1:20 ratio, in concentrations 50 μg/mL and 100 μg/mL in reference to 1% of SDS solution (mean ± SD, *n* = 3; all results were statistically significant). The red line indicates the acceptable hemolysis level.

Performing such an experiment is essential for chemical compounds designed to be applied in direct contact with the human body and its fluids. The acceptable range of hemolysis is set below 5% [33], therefore tested substances could be regarded as erythrocyte safe. Similar results were obtained by Zibiao et al. who proved that selected polymers based on PLLA and PEG did not cause an hemolysis higher than 5%, even in higher concentrations [34]. The researchers proved that coating some commercial metal alloys with PEO/PLLA (plasma electrolytic oxidized/poly(l-lactide) composites can help reduce their hemolytic potential [35]. β-TCP was also tested before in order to check its hemolytic potential, but such effect was not observed [36].

#### **4. Conclusions**

In this work, the novel and self-assembled poly(l-lactide) (PLLA)/β-tricalcium diphosphate (β-TCP) composite has been prepared. In the first step, the well-β-TCP co-doped with Ce3+ and Pr3+ ions was obtained. Further, it was prepared precisely tailored to low-molecular mass PLLA on the β-TCP surface using biocatalysts from the group of zinc aminophenolates. The obtained materials may be potentially used to bone grafting and in vivo bioimaging in the near-infrared (NIR) window (also known as "optical window" or "therapeutic window"). PLLA has been used to enhance biocompatibility of the obtained composite for further application. Moreover, the polymer did not affect the luminescent properties of Ce3+ and Pr3+ ions co-doped β-TCP.

Furthermore, the hemolysis higher than 5% was not observed for all studied materials. It has been suggested that the obtained materials could be safely used in vivo because the cell proliferation and mitochondrial activity have not been disrupted. In conclusion, it might not interrupt the OXPHOS (Oxidative phosphorylation) and energy production in chondrocytes.

**Author Contributions:** R.J.W. conceived and designed the experiments as well as analyzed all data; J.A.Z., D.J., and J.E. contributed reagents/materials/analysis tools as well as analyzed data; A.W. and A.S. designed the experiments as well as analyzed luminescence data; N.N. and J.R.-S. contributed reagents/materials/analysis tools and analyzed biological data; all authors contributed to the writing of the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** Financial support of the National Science Centre in course of realization of the Projects "Preparation and characterization of biocomposites based on nanoapatites for theranostic (no. UMO-2015/19/B/ST5/01330) and "Biocompatible polyesters with accurately coded sequences: engineering, functionalization and applications" (no. UMO-2017/25/B/ST5/00597) and are gratefully acknowledge.

**Acknowledgments:** The authors would like to acknowledge Piotr Kuropka (Wrocław University of Environmental and Life Sciences) for the human chondrocyte cell line TC28A2 and Ewa Bukowskafor performing XRD measurements as well as Damian Szyma ´nski for SEM images (Institute of Low Temperature and Structure Research, Polish Academy of Sciences).

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

#### **References**

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

*Article*


**4.1** *Acinetobacter baumannii*

MDR *A. baumannii* is a well-recognized current global health threat that needs urgent effective solutions [1, 2]. Last-resort treatments such as colistin are no longer effective in an increasing number of cases, leading to a mortality rate of around 35–60% in hospitalized patients with ventilator-associated pneumonia [54, 55]. The number of antibiotics approved by the FDA cannot keep up with the pace at which resistance is acquired by *A. baumannii*. The therapeutic potential of different repurposing drugs against *A. baumannii* has been tested in preclinical models, most of them in combination with polymyxins (colistin and polymyxin B) or its prodrug colistimethate sodium (CMS).

#### **Table 2.**

*List of repurposing drugs under clinical trial development against Gram-negative critical-priority pathogens.*

**105**

*Drugs Repurposing for Multi-Drug Resistant Bacterial Infections*

The antibacterial activity of anticancer drugs has been reported *in vitro* and *in vivo* non-vertebrate and vertebrate models by *A. baumannii*. Gallium nitrate has demonstrated an inhibitory effect on bacterial growth in a collection of 58 MDR clinical isolates of *A. baumannii in vitro* [33]. This antibacterial activity is maintained in *G. mellonella* model. The administration of this drug alone and in combination with colistin, at concentrations mimicking the human therapeutic dose of gallium nitrate used for cancer patients (28 μM), significantly increased the survival of larvae after infection by A. baumannii [33]. When a vertebrate model was used such as murine models of acute and chronic lung infections by *A. baumannii*, gallium nitrate has reduced lung injury and bacterial loads in tissues [32]. Moreover, the combination of mitomycin C with tobramycin and ciprofloxacin together has increased *in vitro* the activity of this anticancer drug against MDR clinical isolates of *A. baumannii* [17]. Whereas, mitotane combined with polymyxin B against polymyxin B-resistant *A. baumannii* has presented synergy with polymyxin B, increasing the activity of polymyxin B *in vitro* and in murine model of burn wound infection by reducing the bacterial load in wounds [49]. Another group of anticancer drugs developed to combat breast cancer is the SERMs. Tamoxifen has been reported to exhibit activity in the immunocompetent and neutropenic murine model of peritoneal sepsis by ATCC 17978 strain by decreasing the bacterial loads in spleen, lungs and blood and increasing the mice survival [36]. Tamoxifen metabolites (N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen), produced after tamoxifen metabolizing by cytochrome P450 [56], have presented antibacterial activity in vitro with MIC50 and MIC90 of 8 and 16 mg/L, respectively, against a collection 100 MDR and pan-drug resistant (PDR)

The potential activity of the anthelmintic drug has been also tested *in vitro* and in animal models by *A. baumannii*. Niclosamide, oxyclozanide, rafoxanide and ivermectin have been shown to potentiate the activity of colistin against clinical isolates of Col-R *A. baumannii in vitro* [30, 46–48, 57]. In the murine model of peritoneal sepsis by Col-R *A. baumannii* clinical isolate, rafoxanide plus CMS, a prodrug of colistin, compared with CMS alone increased mice survival to 53.8% and reduced bacterial loads in tissues and blood between 3 and 4 log10cfu/g or mL, respectively [47]. Only, rafoxanide has exhibited antibacterial activity in monotherapy in this

As is the case with anthelmintic drugs, anti-inflammatory drugs have demonstrated antibacterial activity against *A. baumannii* in monotherapy and in combination with polmyxins *in vitro*. Glatiramer acetate has presented activity against reference strains and clinical bacteremic isolates of A. baumannii by disrupting the biofilm formation [42]. In addition, ebselen has presented antibacterial effect against *A. baumannii* by reducing their bacterial growth at MICs of 32 μM due to the inhibition of the siderephore TonB [51]. In combination with polymyxins, auranofin, a drug used for the treatment of rheumatoid arthritis, and celecoxib have exhibited synergy with polymyxin B and colistin against reference strains of

*DOI: http://dx.doi.org/10.5772/intechopen.93635*

clinical isolates of *A. baumannii* [36].

model of infection, but not *in vitro* [47].

*4.1.3 Anti-inflammatory drugs*

*A. baumannii* respectively [19, 58].

*4.1.2 Anthelmintic drugs*

*4.1.1 Anticancer drugs*