*2.3. Test Parameters*

All recorded test parameters are listed in Table 2. The table includes tag names, parameter names, parameter units and specified sensor range. The values for VT.2 and VT.3 are specified values sent to the valves by the controllers, not the actual measured position of the valves.

**Table 2.** Recorded parameters.

As mentioned in Section 2.2, three Coriolis flow meters are used to adjust inlet flow rate and water cut, monitor phase purities, determine the amount of water extracted from the MPPS prototype, and the purity of the extracted water. The WC in the respective flow lines is determined by

$$\text{NCC}\_{i} = \frac{\rho\_{i} - \rho\_{o}}{\rho\_{\text{IV}} - \rho\_{o}} \ . \tag{1}$$

Here, *ρi* is the measured density at DT.1/2/3, while *ρw* and *ρo* are the pre-determined temperature-corrected densities of the water and Exxsol D60, respectively. For pure-phase feed streams, WC1 should be equal to 100% while WC2 should be equal to 0%. From calculated WC and measured flow rates, the actual WC in the multiphase transport line (WC*in*) is calculated as

$$\text{WC}\_{\text{in}} = \frac{\text{WC}\_1 \dot{Q}\_1 + \text{WC}\_2 \dot{Q}\_2}{\dot{Q}\_1 + \dot{Q}\_2},\tag{2}$$

where *Q*˙ 1 and *Q*˙ 2 are the water and oil flow, respectively, before mixing. When running experiments, *Q*˙ 1 and *Q*˙ 2 are adjusted such that the desired total flow and WC*in* are reached.

The amount of water extracted from the MPPS prototype is determined by the extraction rate (ER). The ER is the flow rate through the water extraction line divided by the flow rate in the water feed line:

$$\text{ER} = \frac{\dot{Q}\_3}{\dot{Q}\_1} \,\prime \tag{3}$$

where *Q*˙ 3 is the flow at the water outlet of the MPPS.

As the test loop is a closed system, the water and Exxsol D60 phases will be contaminated over time. Microscopic droplets of water will be dispersed in the oil and vice versa. In order to give a performance measurement that is independent of occurring contamination, the WC ratio is calculated. The WC ratio is equal to the WC at the water extraction line (WC3) divided by the WC at the water feed line (WC1):

$$\text{WC}\_{\text{T}} = \frac{\text{WC}\_{3}}{\text{WC}\_{1}}.\tag{4}$$

A WC*r* equal to 100% means that the extracted water from the MPPS prototype is of equal quality to the water, leaving the baseline separator prior to being mixed with the oil. A WC*r* of 100% is thus the upper limit on the purity that can realistically be achieved by the MPPS prototype.

#### *2.4. System Identification*

A dynamic model of the system is very helpful when designing controllers. A classical way to identify the dynamic relations between a manipulated variable and a control variable is to perform a step response experiment and calculate the transfer function. In this work, the procedure from [16] was followed, and it was assumed that the dynamic model between each input and output could be described by a first-order plus time delay transfer function on the form

$$\frac{y}{u} = G(s) = \frac{k\varepsilon^{-\theta s}}{\tau s + 1},\tag{5}$$

where *y* is the output, *u* is the input, and *s* is the Laplace variable. The transfer function variables describing the dynamic response, *k*, *τ* and *θ* are of special interest. These variables represent the plant gain, the time constant and the time delay, respectively. The plant gain provides the steady-state output of the plant, for a specific input, and is given by

$$k = \frac{\Delta y}{\Delta u} \,. \tag{6}$$

The time constant, *τ*, is the time it takes the output to reach 63% of the steady-state value, and the time delay, *θ*, is the amount of time it takes the input to cause a reaction on the output.

The step response experiments were performed with one valve at a time, with the other valve in a fixed position, and at a fixed inlet flow rate and inlet WC. The valve openings, inlet flow rate and inlet WCs are listed in Table 3.

Some of the measurements contained significant measurement noise, hence the measured values where filtered using a 1st order Butterworth filter before the parameter analysis was performed. The transfer function between each input and output was then calculated and validated by comparing it to the original response. If any deviations were present, the transfer function variables were tuned manually to improve the fit.

**Table 3.** Inlet conditions and changes in valve openings used in step response experiments.

The step response experiments were performed on the two inputs VT.2 and VT.3, and three measurements were chosen as candidate CVs. The pressure PT.1 is a necessary CV for safety reasons. A measurement that gives a direct indication of the separator efficiency is the the water cut ratio WC*r*, and hence this is also a candidate CV. The laboratory is not equipped with a level measurement sensor, instead the pressure drop dPT.2 over the incline is used for this purpose. The level is often used as an CV in previous work, as mentioned in the Introduction, and will also be a candidate CV in this work.

From the step response experiments, the following transfer functions were identified:

$$\frac{\text{WC}\_{\text{r}}}{\text{VT.2}} = \text{G}\_{1}(s) = \frac{0.2423 \epsilon^{-10s}}{20.9s + 1},\tag{7}$$

$$\frac{\text{PT.1}}{\text{VT.2}} = \text{G2}(s) = \frac{0.0026e^{-10s}}{14.1s + 1},\tag{8}$$

$$\frac{\text{dPT.2}}{\text{VT.2}} = \text{G}\_{\text{3}}(s) = \frac{-0.0739e^{-10s}}{23.1s + 1},\tag{9}$$

$$\frac{\text{WC}\_r}{\text{VT.3}} = \text{G}\_4(s) = \frac{-0.5962\epsilon^{-4s}}{2s+1},\tag{10}$$

$$\frac{\text{PT.1}}{\text{VT.3}} = G\_5(s) = \frac{0.0156 \epsilon^{-4s}}{8.3s + 1},\tag{11}$$

$$\frac{\text{dPT.2}}{\text{VT.3}} = G\_6(s) = \frac{0.2722e^{-4s}}{13.2s+1}.\tag{12}$$

A comparison between the measured response, the filtered response, and the transfer function response is shown in Figure 4. Here, we see that some of the responses could be better described by a second-order transfer function. In particular, the transfer functions between VT.2 and the different outputs (note the second order dynamics in WC*r* in Figure 4a not captured by *G*1(*s*) and the overshoot in dPT.2 in Figure 4e not captured by *G*3(*s*)). However, for the control study in this work, it is assumed that a first order model is sufficient. The fluctuations present are purely caused by measurement and process noise.

#### *2.5. Control Structure Analysis*

The separator is a multiple input multiple output (MIMO) system with two inputs and several possible outputs. It is not straightforward to pair an input with an output, and hence a relative gain array (RGA) analysis ([17], Section 3.4) was performed. The RGA provides a measure of interactions between the inputs and outputs and [17,18] recommends pairing inputs and outputs such that the rearranged system has an RGA matrix close to identity. Furthermore, negative steady-state RGA elements should be avoided. The RGA for a square system on the form

$$y = G(s)u\tag{13}$$

is found by calculating the element-wise matrix product

RGA(*G*) = *G*0*G*−*T* 0 , (14)

where *G*0 = *G*(0) is the steady-state transfer function matrix of *G*(*s*) in Equation (13).

**Figure 4.** Step response comparison between measured signal, filtered signal and transfer functions.

Another parameter to consider when investigating the pairing of CVs and MVs is the Niederlinski index (NI) ([18], Section 2.2.1)

$$\text{NI} = \frac{\det\left[G(0)\right]}{\prod\_{i=1}^{n} g\_{ii}(0)},\tag{15}$$

where *gii* are the diagonal elements of *G*(0). If the open-loop system is stable (which is the case here), one should select pairings corresponding to positive NI values [19]; otherwise, the closed-loop system

will be unstable ([18], Th. 1). The RGA matrices and the NI for the separator is shown in Table 4. From the RGA analysis, it is clear that VT.2 should be paired with WC*r* or dPT.2, and VT.3 with PT.1, as this corresponds to the pairing closest to 1. The NI is positive for both possible pairings, hence no pairing will lead to an unstable system.

**Table 4.** Relative gain array (RGA) and Niederlinski index (NI) for the separator.

#### *2.6. Controller Design*

A multivariable system, such as the one investigated here, could benefit from a multivariable control scheme. However, since this is an initial control study, the developed controllers are decoupled, single-loop controllers.

#### 2.6.1. PI Control

A PI controller has the form

$$
\mu(s) = k\_{\varepsilon} \left( 1 + \frac{1}{\tau\_{I^{\rm S}}} \right) (r - y), \tag{16}
$$

where *kc* is the proportional gain, *τI* is the integral time in seconds, *r* is the reference and *y* is the measured output to be controlled. PI controllers are developed for the separator by applying the SIMC tuning rules [16]. The SIMC tuning rules states that the proportional gain and integral time of the PI controller should be chosen as

$$k\_c = \frac{1}{k} \frac{\tau}{\tau\_c + \theta'} \tag{17}$$

$$
\pi\_l = \min\left(\pi\_\prime \left(\pi\_\varepsilon + \theta\right)\right),
\tag{18}
$$

where *kc* is the proportional gain, *τI* is the integral time, and *τc* is the desired closed-loop time constant, which is the only tuning parameter. For tight and robust control, Ref. [16] recommends choosing *τc* = *θ*. Although the RGA analysis recommended a specific pairing of inputs and outputs, PI controllers are developed and tested for all configurations. The parameters used in the PI controllers are found in Section 3.2.1.

A derivative part could have been added to the controllers, but this would have required a measurement of the derivative of the CV. This is not available but could have been calculated numerically. However, as the control objective is to keep the CVs at steady-state, the derivative of the CV would be close to zero when operating at steady-state and the contribution would only be from the measurement noise. Derivative action is uncommon in process control applications where the plants are stable with overdamped responses and first-order dominant dynamics (which is the case here), since the performance improvement is usually too small compared to the added complexity [17,20].

#### 2.6.2. Adaptive Control

As an alternative to conventional PI control, a model reference adaptive controller (MRAC) ([21], Section 6.2.2) was implemented and tested for the two control configurations recommended by the RGA analysis, i.e., VT.3 controls PT.1 and VT.2 controls either WC*r* or dPT.2.

When using MRAC, the controller parameters are automatically updated such that the error between the measured variable and the output of a reference model is reduced. This could be very beneficial if the process parameters change over time or at different operating points, which may lead to poor control when using a controller with fixed gains. The MRAC structure is different from a PI controller structure, i.e., there is no proportional and integral gain in the MRAC, but rather two parameters that try to approximate a value that causes the closed-loop system dynamics to be equal to the reference model dynamics. Hence, using the proportional and integral gain of a PI controller as initial values in an MRAC is not necessarily helpful. A schematic of the MRAC is shown in Figure 5.

**Figure 5.** Model reference adaptive control (MRAC) schematic.

The MRAC has the form

$$u = -k\_a(t)y + l\_a(t)r,\tag{19}$$

where *ka*(*t*) and *la*(*t*) are time-varying gains, updated by the adaptive laws

$$
\dot{k}\_a = \gamma\_k \epsilon y \operatorname{sign}(k),
\tag{20}
$$

$$\dot{l}\_d = -\gamma\_l \varepsilon r \,\text{sign}(k),\tag{21}$$

where *γk*, *γl* are adaptation gains, *y* is the measured value to be controlled, *r* is the reference and sign(*k*) is either 1 or −1. The error signal *e* = *y* − *ym*, where *ym* is the output of the reference model

$$
\dot{\mathbf{x}}\_{\text{m}} = a\_{\text{m}} \mathbf{x}\_{\text{m}} + b\_{\text{m}} r\_{\text{s}} \tag{22}
$$

$$y\_m = x\_{m\prime} \tag{23}$$

where *am* < 0 and *bm* are chosen by the operator and specify the desired closed-loop dynamics of the system.

The only system knowledge required by the MRAC is the sign of *k*. It can be shown ([21], Section 6.2.2) that, if *y* = *x* is the state in a linear system, the controller given by Equations (19)–(23) causes *y* → *ym* asymptotically for *γk*, *γl* > 0. The parameters used in the MRAC are found in Section 3.2.2.

#### **3. Results**

In total, six experiments were carried out in the laboratory: four experiments with PI controllers and two experiments with MRAC. When using PI control, all control pairings were tested, but with MRAC only the pairings recommended by the RGA analysis was carried out.

#### *3.1. LabView Implementation*

The laboratory is controlled through a computer running LabView 2015 (National Instruments, Austin, TX, USA) on Windows 7 (Microsoft, Redmond, WA, USA) with an Intel i7 4770S (Santa Clara, CA, USA), 3.1 GHz processor and 16 GB RAM. The PI controllers could be readily implemented in the LabView 2015 block diagram through existing PI controller blocks.

The MRAC, however, had to be implemented using an add-on for LabView called MathScript Module. This module allows the user to write code, and execute it at each iteration of the LabView program. To calculate the MRAC input in Equation (19), the differential equations in Equations (20)–(22) must be solved. This was done using first-order Euler integration with a step length of 0.0001 s. A dead band was introduced to the adaptation algorithms, i.e., the adaptation was stopped if the error was less than 5% of the setpoint for dPT.2 and PT.1 and less than 0.5% for WC*r*.

#### *3.2. Controller Tuning*

#### 3.2.1. PI Controllers

A transfer function model is only an approximation of the real system dynamics. It was assumed that the transfer functions were of first order. The controller parameters may require re-tuning if the transfer function models differ significantly from the real dynamics (they may change with operating point and inlet conditions) or if nonlinearities in the valve openings are not considered. Furthermore, there are interactions between the control loops; hence, a multivariable controller would probably be a better choice. This, however, has not been studied in this work.

It was found during initial testing that the choice of *τc* = *θ* was too aggressive for VT.2 and, hence, *τc* = 30 s was chosen for this valve. For VT.3, *τc* = *θ* could only be used when controlling WC*r*. Otherwise, *τc* = 10 s was used. The controller parameters for the different PI controllers are listed in Table 5.

**Table 5.** PI controller parameters.

#### 3.2.2. Model Reference Adaptive Controller

It was found during testing in the laboratory that the initial values *ka*(0) and *la*(0) as well as the adaptation gains *γk*, *γl* had to be chosen with care. This is due to the fact that time delays and measurement noise was ignored when the controller was derived. The adaptive controller parameters used are listed in Table 6.

**Table 6.** Model reference adaptive controller (MRAC) parameter values.

#### *3.3. Test Sequence and Control Objectives*

All experiments presented here were performed on the same day. Prior to each experiment, the lab was operated at nominal inlet conditions for 5 min, i.e., a flow rate of 350 L/min and 60% WC. The valves were manually set to VT.2 = 30% closed and VT.3 = 70% closed. This led to an initial pressure PT.1 ∼ 0.1 barg, an initial WC*r* ∼ 99% and an initial dPT.2 ∼ 1.9 mbar when the controllers were activated. The static pressure is initialized at 0.1 barg in order to see how the controllers, and especially the adaptive controllers, perform during an initial transient. A setpoint of 0.4 barg for PT.1 was necessary as the valve controlling the pressure could then operate in the middle of its range, and not saturate, when the inlet flow rate was high.

The inlet conditions were varied in order to emulate situations that may occur in a subsea oil/water separator. The inlet variables available for manipulation are the total liquid flow and the inlet WC. Table 7 shows the different inlet conditions and the scenarios they emulate.

The main control objectives in all experiments are to maintain the desired pressure PT.1 and to keep WC*r* as high as possible. The latter is important in order to ensure that the water quality is high enough for the downstream water cleaning equipment. A setpoint of 99% is chosen for the WC*r*. Looking only at WC*r* may, however, be misleading, since it says nothing about how much water is extracted. For this, the ER is used. It is important to maintain a high ER while maintaining a high WC*r*. If the ER is very low, almost no liquid is leaving the separator through the water outlet. In this case, the WC*r* may be high, but a lot of water is leaving through the oil outlet.

In Experiments 1 and 2, the WC*r* is controlled directly using either VT.2 or VT.3. In Experiments 3, 4 and 6, the dP between the bottom of the outlet incline and the top of the outlet incline is controlled. This serves as a proxy level measurement of the water level in the incline. When controlling the dP, and the level, by proxy, a buffer of water is built up in the incline, making the WC*r* more robust to inlet variations. It was found through image analysis that a dP of 2 mbar gave stable oil and water layers (see the Appendix A) and the buffer volume was assumed sufficient. Hence, this setpoint is used in the controllers. Figure 6 shows a sketch of this.

**Figure 6.** When controlling WC*r* directly, no buffer volume of water is present in the inclined section. Hence, oil breakthrough into the water outlet is more frequent when comparing to the dP/level control.

**Table 7.** Inlet conditions used in all experiments.


*Edited by Jessica A. Lovich-Sapola,*

*Kelly Lebak, Michael D. Bassett*

*and Kasia P. Rubin*

Tracheal intubation is a common medical procedure. It is relatively simple to accomplish, although there are cases in which tube placement is difficult or even impossible. This book provides a comprehensive overview of endotracheal tube placement with chapters on advanced techniques, ultrasound use for airway management, airway management techniques for obstetric patients, and much more.

ISBN 978-1-80356-860-7

Advances in Tracheal Intubation

Published in London, UK © 2023 IntechOpen © chaikom / iStock


**Chapter 8 125** Conversion of Cellulose into Value-Added Products *by Lethiwe D. Mthembu, Rishi Gupta and Nirmala Deenadayalu*

Preface

Recent developments connected to increased environmental awareness, recognition of global climate problems, and the advent of bioeconomies and biorefineries have brought cellulosic products back into the public consciousness as valuable biomaterials and chemical feedstocks. In this context, the pulp and paper industries are

The development of biorefinery methods has highlighted the characteristics of celluloses such as biomineralization, recycling, conversion, and permanence. Today, the cellulose discipline is one of the most rapidly advancing fields in the natural and physical sciences. Cellulose plays and will continue to play a dominant role in the universal appearance of bioeconomies, leading the way from fossil-based chemical industries to true biorefineries. Today's uses for cellulose, tissues, fibers, and cellulose derivatives, including paper products, are only the beginning.

This book begins by addressing the conversion of biomass waste products into valuable products like cellulose hydrogel films, which are important in cell regeneration. The other chapters explain the application of cellulose derivatives in mineral processing, cellulose derivatives (CDs) that have been recognized as anionic water-soluble, non-toxic, biocompatible, and biodegradable polysaccharides. Also one chapter deals with de-inking of MOW type paper using enzymes and. One chapter covers bacterial cellulose, an alternative ecological materials have been used as viscosity regulators, thickening agents, sizing agents, coating agents,

This book conveys the fascination of contemporary cellulose science, including its

**Arpit Sand**

Faridabad, India

**Sangita Banga**

Faridabad, India

Department of Chemistry, Manav Rachna University,

Manav Rachna University,

emulsion stabilizers, and electrode binders in various industries.

versatility, applicability, challenges, and bright future.

increasingly regarded as businesses engaged in high-tech innovation.


**1. Introduction**

76 New Approaches to the Study of Marine Mammals

Research 2008;650 96–103.

[46] Platt KL, Aderhold S, Kulpe K, Fickler M. Unexpected DNA damage caused by polycyclic aromatic hydrocarbons under standard laboratory conditions. Mutation

[47] Gravato C, Teles M, Oliveira M, Santos MA. Oxidative stress, liver biotransformation and genotoxic effects induced by copper in *Anguilla anguilla* L. – the influence of pre-

exposure to b-naphthoflavone. Chemosphere 2006;65 1821–1830.

Bioaccumulation of persistent organic pollutants (POPs) represents a risk to the marine environment and wildlife, including marine mammals and birds [1-4]. Biomagnification is a special case of bioaccumulation and is defined as the process by which concentrations of contaminants or chemical substances (i.e. thermodynamic activities of chemical substances often measured by the lipid normalized concentration) in consumer and higher trophic level organisms exceed those concentrations in the diet or organism's prey [5-7]. This process can occur at each step in a food chain, potentially producing very high and toxic concentrations in upper-trophic-level species [7].

Bioaccumulation and biomagnification are important considerations in the categorization and risk assessment of chemical compounds under the treaty of the Stockholm Convention for POPs and regulatory and management efforts in several nations such as the Canadian Environmental Protection Act Canada (CEPA [8]), the Toxic Substances Control Act (TSCA [9]) in the United States and the Registration, Evaluation, Authorisation and Restriction of Chemicals program (REACH) in the European countries [10]. Due to the long-range atmospheric transport and global fractioning of POPs northward from low or mid latitudes [11, 12], the Arctic and northern hemisphere have remained as active regions of research to study biomagnification of POPs in trophic chains and food webs [2, 13-15]. However, very little is known about the bioaccumulative behaviour and fate of these substances in tropical zones of the planet.

© 2012 Alava and Gobas, 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.

There are several measures that have been used to express the degree of biomagnification. The simplest measure is the Biomagnification Factor (BMF), which is described as the ratio of the chemical concentrations in the organism (*C*B) and the diet of the organism (*C*D), i.e., BMF = *C*B/*C*D, where the chemical are usually expressed in units of mass of chemical per kg of the organism (in wet weight or in a lipid basis) and mass chemical per kg of food (in wet weight or in a lipid basis) [6]. Biomagnification of organic contaminants and foraging preferences in aquatic and marine food webs can also be investigated using stable nitrogen isotope as biomarkers of trophic level [15-20]. Stable isotope analysis (SIA) has emerged as a tool in foraging ecology/habitat use, physiology and ecotoxicology, and is applied widely to study marine mammal ecology [21]. Stable nitrogen isotope analysis is a known well established technique for assessing predator–prey interactions and organism trophic levels (TL) in food webs [22-25]. Specifically,*δ*15*N*, the concentration ratio of 15*N*/14*N*, expressed relative to a standard (i.e., atmospheric *N*2), has been shown to increase with increasing trophic level due to the preferential excretion of the lighter nitrogen isotope [26]. Likewise, carbon isotope signatures (*δ*13C) provide information on habitat use and general sources of diet of organisms, i.e., marine/freshwater, coastal/oceanic, pelagic/benthic [27].

Assessing Biomagnification and Trophic Transport of Persistent Organic Pollutants in the Food Chain of the Galapagos Sea Lion (*Zalophus wollebaeki*): Conservation and Management Implications 79

local sentinels of food web contamination [33-35]. Concentrations of polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT) were recently detected in this species, underlying the health risk due to the toxicity and bioaccumulation potential of these contaminants in the Galapagos food web [34, 35]. Thus, equivalent to the role of killer whales as global sentinels of pollution in the Northeastern Pacific [1], the Galapagos sea lion can be used as an eco-marker of environmental pollution and a key indicator of not only the

With the aim to contribute to the understanding of the behaviour and fate of POPs in marine food webs of tropical regions, this chapter provides an advanced primer on biomagnification assessment of POPs in the Galapagos Islands based on the existing literature on baseline levels of DDT detected in Galapagos sea lions [35] and recent unpublished data on organochlorine pesticides (i.e. mirex, dieldrin, chlordanes, β-HCH) and PCBs in Galapagos sea lions and fish preys. To accomplish this work, we made use of concentration data measured in Galapagos sea lions and their fish prey and determination of predator-prey biomagnification factors to assess biomagnification in this tropical system. Insights on the impact of biomagnification and conservation and management implications

In a recent study [35], blubber biopsy and hair samples of 20 Galapagos sea lion pups of 2– 12 months of age were obtained from four rookeries in the Galapagos Archipelago (3°N−4°S, 87°−94°W) between 24-29March 2008. Briefly, pups were sampled at Isabela (Loberia Chica, *n* = 5), Floreana, (Loberia, *n* =6) and Santa Cristobal (Puerto Baquerizo, *n* = 4; Isla Lobos, *n* = 5) islands. Pups were captured with hoop nets and manually restrained. Age was estimated by visual observation of both the size and weight of the animal. In all circumstances, capture stress and holding time were minimized (< 10-15 min). Hair samples were obtained using a sterile scissor to trim or a scalpel to shave the region to be used prior to the biopsy collection and deposited into labelled zipper bags. Biopsies (100 mg; 6mm−Miltex biopsy punch) were collected from an area 10-20 cm lateral to the spinal column and anterior to the pelvis. The biopsy site was pre-cleaned with alcohol and betadine. Biopsies were wrapped in hexanerinsed aluminum foil and placed in a cooler with wet ice and transferred into cryovals placed in a cryoship (-20°C) during the field sampling, and, afterwards stored at -80C in the

Pups were chosen because (a) the animals are readily accessible and relatively easy to capture in most of the rookeries of the Galapagos Islands year round; (b) the animals are of similar age (3-10 months), minimizing the influence of life history parameters on contaminant concentrations; (c) because they are nursed by adult reproductive females they have a high trophic position because they are feeding on mother's milk, ingesting energy and pollutants and analogous to a predator–prey relationship [35]. The rationale of the

coastal marine health, but the public health in the region.

at the ecosystem level in the Galapagos are discussed.

**2.1. Tissue collection from Galapagos sea lion pups**

**2. Materials and methods**

laboratory until chemical analysis.

Studies of the biomagnification and food web transport of POPs in tropical systems such as remote islands around the equatorial Pacific Ocean are lacking. Due to the remoteness and isolation of the Galapagos Islands relative to other better studied geographical areas, the Galapagos Island food web offers a unique opportunity to undertake research related to the transport, bioaccumulative nature and biomagnification of globally distributed contaminants in tropical environments at the ecosystem level. The low population levels and generally good environmental control and management practices on the islands ensures that local pollutant sources are in most cases insignificant compared to global sources. These conditions provide a unique mesocosm to study the behaviour of global pollutants in marine mammalian food-chains.

The Galapagos sea lion(*Zalophus wollebaeki*) is an endemic marine mammal residing year round in the islands and exhibiting a high degree of dietary plasticity, consuming several groups of fish prey (99% of the diet). The Galapagos sea lion diet includes Cupleidae (thread herrings and sardines), Engraulidae (anchovies), Carangidae (bigeye scads), Serranidae (groupers, whitespotted sand bass or camotillo), Myctophidae (lantern fishes), Mugilidae (mullets) and Chlorophtalmidae fishes, and a low proportion of squid, as reported in the existing literature [28-31]. Although the information about diet and trophic level is limited for sea lions at several rookeries in the Galapagos Islands, it is known that the dietary preferences of Galapagos sea lions are also a function of the local variation in prey availability and regional climate-oceanic variability such as the El Niño events, when sea lions can switch their diet composition to more abundant fish items [30, 32, 33]. The Galapagos sea lion has been recognized as a key species for the functioning and health of the marine ecosystem of the islands under the environmental management action plan of the Galapagos Marine Reserve (GMR) [33]. Because of its high trophic position, relative abundance in the islands and non-migratory behaviour, Galapagos sea lions can serve as local sentinels of food web contamination [33-35]. Concentrations of polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT) were recently detected in this species, underlying the health risk due to the toxicity and bioaccumulation potential of these contaminants in the Galapagos food web [34, 35]. Thus, equivalent to the role of killer whales as global sentinels of pollution in the Northeastern Pacific [1], the Galapagos sea lion can be used as an eco-marker of environmental pollution and a key indicator of not only the coastal marine health, but the public health in the region.

With the aim to contribute to the understanding of the behaviour and fate of POPs in marine food webs of tropical regions, this chapter provides an advanced primer on biomagnification assessment of POPs in the Galapagos Islands based on the existing literature on baseline levels of DDT detected in Galapagos sea lions [35] and recent unpublished data on organochlorine pesticides (i.e. mirex, dieldrin, chlordanes, β-HCH) and PCBs in Galapagos sea lions and fish preys. To accomplish this work, we made use of concentration data measured in Galapagos sea lions and their fish prey and determination of predator-prey biomagnification factors to assess biomagnification in this tropical system. Insights on the impact of biomagnification and conservation and management implications at the ecosystem level in the Galapagos are discussed.


*2.3. Data Processing*

Statistical analysis and organization were applied to the study data using Microsoft Excel 2016, and SPSS 25.0 software was also employed for statistical analysis. Origin 2021 was adopted for drawing. The least significant difference (LSD) method was used for multiple comparisons, and two-way analysis of variance was performed for different concentrations and types of plant growth hormones.


**2. Experimental procedure**

Commercial soda-lime-silicate float glass sheets with nominal thickness of 4 mm were used. Table 1 summarizes the composition of the glass.

**Table 1.** Composition (wt%) of the float glass

The sheets, obtained from one single original plate, were cut into square samples of 50 mm × 50 mm. The edges of the specimens were rectified and polished with SiC abrasive paper. The samples were then rinsed and cleaned gently in water, avoiding any damage on the glass surface.

A semi-automatic chemical strengthening lab scale furnace was used for the ion-exchange treatment. In each run, 20 samples were placed in a stainless steel basket to be treated at 450 °C for 4 h, with 30 min preheating and 30 min post-cooling above the bath. At least 60 samples were treated in each bath with the same thermal and time conditions.

Pure KNO3 and NaNO3 salts from Sigma Aldrich were used. Pure potassium nitrate was systematically poisoned by adding a specific amount of NaNO3 as reported in Table 2.

**Table 2.** Amount of NaNO3 added to pure KNO3 in the considered salt baths

After each treatment, the samples were rinsed with water and carefully dried.

The surface residual stress and the case depth were optically measured by surface stress-meter (FSM-60LE, Luceo Co., Ltd., Japan). Bi-axial flexural test [7] was used to measure the strength. This was carried out with a ring-on-ring configuration with the upper loading ring and the lower support ring having a diameter of 8 mm and 40 mm, respectively. The actuator speed was 1 *mm min* . The strength was determined from the maximum load (F) as:

$$
\sigma\_F = K \frac{F}{h^2} \tag{2}
$$

where *h* is the thickness of sample and

knowledge. It was in the 20th century that scientist started to understand the surface of chemically tempered glasses and figured out an exchange between monovalent cations in glasses with silver and/or potassium cations in the molten salts [2, 3]. These investigations increased the industrial application of the ion-exchange process, especially with the aim of

Glass strength can be largely increased by the ion-exchange process, otherwise called chemical

glass, is responsible for the creation of bi-axial residual compressive stress in the surface layers of the material. Because glass products generally break due to excessive tension applied at a surface flaw, the introduction of surface compression strengthens the glass component.

During the ion-exchange treatment, the glass matrix is considered as a solid negatively charged

The replacement takes place through an interdiffusion process, according to the Nernst–Planck

*Na K Na Na K K D D*

coefficient. One important parameter in the interdiffusion phenomenon is clearly the concen‐ tration of ions in the molten salt. Some studies have shown that the presence of poisoning elements (already present in the salt or coming out from the glass as in the case of Na) can hinder the diffusion of K+ into the glass and slow down the exchange process, thus reducing the strengthening efficiency. An important issue in industrial practice is also the replacement/ renewal of the molten salt which is time and money consuming [5]. Some researchers [3, 6], on the other hand, believe that the ion-exchange process is not affected in the presence of

In the present work, a systematic analysis of the effect of small amount of sodium as poisoning element in the molten bath on the performances of the strengthened soda-lime-silicate float

Commercial soda-lime-silicate float glass sheets with nominal thickness of 4 mm were used.

**SiO2 Al2O3 Na2O K2O MgO CaO** 71 1 13 1 4 10

equations. The flux of the ion species scales with the interdiffusion coefficient [2]:

is the fractional concentration of alkali ion *i* (Na or K) and D*i*

*D*

or Na+

from a molten KNO3 bath at temperatures below the strain point of the

from molten KNO3) responsible for the generation of a compressive stress.

in soda-lime-silicate glass) can be replaced by larger

*D N DN* = + (1)

in an alkali-containing glass, with

the self-diffusion

enhancing the optical and mechanical properties of glass [4].

tempering. The exchange of small ions such as Li+

structure where some mobile ions (Na+

specific amount of poisoning elements.

**2. Experimental procedure**

**Table 1.** Composition (wt%) of the float glass

Table 1 summarizes the composition of the glass.

glass was carried out.

larger ions such as K+

154 Ion Exchange - Studies and Applications

monovalent ions (K+

where N*i*

$$K = \frac{3\left(1+\nu\right)}{2\pi} \left( \ln \frac{D\_{\text{S}}}{D\_{\text{L}}} + \frac{\left(1-\nu\right)\left(D\_{\text{S}}^2 - D\_{\text{L}}^2\right)}{0.72D^2\left(1+\nu\right)} \right) \tag{3}$$

where *DS* and *DL* are the radius of the upper and lower supporting ring, respectively, *D* the sample size (50 mm), and *ν* the glass Poisson's ratio. A certain number of as-cut samples were also tested in this way for comparison.

Some fragments were collected from the broken samples and used for determining the potassium penetration profile. The fragments were attached on an aluminum disk by conduc‐ tive adhesive tape and then coated by sputtering with Au-Pd alloy. Clean and flat portions of the fracture surface were analyzed in a Scanning Electron Microscope (SEM) (JSM5500, Jeol, Japan) and the potassium Kα signal was recorded on a path of ~30 μm long by using the Energy Dispersion X-ray Spectroscopy (EDXS) (EDS2000, IXRF System, USA) probe. The chemical composition of the external surface of the glasses after the ion-exchange process was analyzed in the same way in a region of about 0.5 mm2 .


**1. Introduction**

The extensive mining throughout the globe leads to generate a huge quantity of sulfides caused by weathering process (O2, moisture, and microbes), which promotes an acidic environment. The static/stream water in contact with mines and mines waste becomes a reaction site where it also acts as a reagent for deferent chemical reactions, i.e., metal/metalloid solubilization. Moreover, in-stream water, it also turns out to be a transport media for reaction products [1]. Such water constituents are called "acid mine drainage" or "acid and metalliferous drainage" (AMD) as shown in **Figure 1**. This water is generally characterized as lower pH value as well as a high heavy metals concentration such as iron, manganese, lead, chromium, mercury, cadmium, and arsenic and sulfate content [2]. AMD has a severe impact on the environment, including neighboring surfaces, groundwater,

and soil properties. Various reports suggest that heavy metals transmit to the human body cause severe illness and death in AMD-contaminated areas. Therefore, to ensure human health safety and to control the environmental risk, AMD must be treated.

Various alkaline materials such as CaO, NaOH, CaCO3 have been appropriately examined in this chapter. The use of CaCO3 is more than other neutralizing material due to its cost-effectiveness. It produces less amount of sludge but the reaction period is comparably high than other chemical reagents. The major drawback of these alkaline reagents are high in cost and effectiveness in long-term periods is low. To avoid these major drawbacks, there is always a need for a better reagent, which is cheaper, eco-friendly, easy to handle, and the effectiveness in mitigating AMD wastewater. So various industrial by-products are examined by researchers. For example, the by-products generated from the Calcium oxide production process are used to treat sulfate and metals like cobalt, nickel, zing copper cadmium with better efficiency. Some other industrial outcomes like fly ash, steel slag, cement kiln dust, and bayer residue have possible calcium oxide and calcium hydroxide alternatives to treat AMD. The availability of these materials is generally high, which offers costeffective neutralizing materials for the treatment process.


*Review* **Protein-Based Nanoparticles for the Imaging and Treatment of Solid Tumors: The Case of Ferritin Nanocages, a Narrative Review**

**Francesco Mainini 1, Arianna Bonizzi 1, Marta Sevieri 1, Leopoldo Sitia 1, Marta Truffi 2, Fabio Corsi 1,2,\* and Serena Mazzucchelli 1,\***

**Abstract:** Protein nanocages have been studied extensively, due to their unique architecture, exceptional biocompatibility and highly customization capabilities. In particular, ferritin nanocages (FNs) have been employed for the delivery of a vast array of molecules, ranging from chemotherapeutics to imaging agents, among others. One of the main favorable characteristics of FNs is their intrinsic targeting efficiency toward the Transferrin Receptor 1, which is overexpressed in many tumors. Furthermore, genetic manipulation can be employed to introduce novel variants that are able to improve the loading capacity, targeting capabilities and bio-availability of this versatile drug delivery system. In this review, we discuss the main characteristics of FN and the most recent applications of this promising nanotechnology in the field of oncology with a particular emphasis on the imaging and treatment of solid tumors.

**Keywords:** ferritin; cancer; tumor targeting; drug delivery; imaging

#### **1. Introduction**

Nanoparticle-based drug delivery systems have the capacity to enhance the physicochemical properties of a wide variety of drugs used in oncology to limit off-site side effects and improve their therapeutic efficacy [1–3]. The ideal nanocarrier should be bio-compatible and be able to avoid recognition by the reticuloendothelial system (RES), composed of tissue-resident macrophages and phagocytes in the bloodstream, capable of efficiently clearing exogenous nanoparticles (NP) from the circulation [4]. Natural proteins nanocages have a distinctive advantage in this regard, in comparison to synthetic NP (liposomes, polymeric NP, micelles and dendrimers), since they are virtually invisible to the immune system and display great biocompatibility coupled with minimal toxicity. An exception is represented by virus-like particles (VLPs), which are also composed of self-assembled proteins that are, in some cases, highly immunogenic [5].

Endogenous self-assembled NPs can be synthesized by many cell types and are primarily used to store and/or distribute to different tissues a wide variety of molecules, such as nutrients and biochemical signals. NPs of this kind are quite diverse in terms of size and physiological activity. Some examples are ferritin nanocages (FNs), heat-shock protein cages, vault ribonucleoparticles, the E2 protein of the pyruvate dehydrogenase multienzyme complex, chaperones, carboxysomes and other enzyme complexes [6]. Unfortunately, most of these protein-based NP are understudied and have, so far, limited applications in the field of oncology. However, FNs have been studied extensively due to their intrinsic targeting capabilities toward the Transferrin Receptor 1 (TfR1), which is highly expressed in many tumors, making them very appealing for drug-delivery applications in oncology. Furthermore, the small size and high customization potential make them ideal candidates

**Citation:** Mainini, F.; Bonizzi, A.; Sevieri, M.; Sitia, L.; Truffi, M.; Corsi, F.; Mazzucchelli, S. Protein-Based Nanoparticles for the Imaging and Treatment of Solid Tumors: The Case of Ferritin Nanocages, a Narrative Review. *Pharmaceutics* **2021**, *13*, 2000. https://doi.org/10.3390/ pharmaceutics13122000

Academic Editor: Xiangyang Shi

Received: 2 November 2021 Accepted: 19 November 2021 Published: 25 November 2021

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

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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for the development of novel nanomedicines able to deliver a wide variety of drugs to the tumor microenvironment (TME). This review describes the structure and function of FN, modifications of the nanocages by chemical or genetic manipulation (Figure 1) and novel applications of this nanotechnology for the imaging and treatment of solid tumors (Figure 2).

**Figure 1.** FN as a protein-based delivery system for oncological therapeutics and imaging agents. FNs are composed of 24 Hc subunits that can be chemically or genetically modified to couple a large variety of molecules (antibodies, peptides, fluorophores, polyethylene glycol (PEG) and others) to their surface (N-terminus) or internal cavity (C-terminus). Furthermore, FNs can be loaded with different drugs and imaging agents and have intrinsic targeting capabilities toward the receptor TfR1, which is overexpressed in many tumors. This cartoon was created by using BioRender (https://biorender.com/, accessed on 2 November 2021).

**Figure 2.** Applications of FNs in the field of oncology. FNs are versatile drug delivery systems. They can be loaded simultaneously with anticancer and imaging agents to provide effective antitumor therapy that can be monitored by different imaging modalities. In addition, FNs can be loaded with immunomodulatory drugs to remodel the TME or can be developed to incorporate tumor associated antigens to induce specific adaptive immune responses against cancer cells, in the case of nanovaccines. This cartoon was created by using BioRender (https://biorender.com/, accessed on 2 November 2021).

#### **2. FN Structure and Properties**

Ferritin self-assembles in hollow icosahedral-shaped nanocages with inner and outer dimensions of 8 and 12 nm, respectively [7]. In mammalian cells, ferritin is composed of heavy-chain (Hc, 21 kDa) and light-chain (Lc, 19 kDa) subunits (24 in total between the two) which are structurally similar. FN employed as a delivery device in cancer application are mostly constituted only by Hc subunits of human ferritin. Ferritin and FNs are remarkably stable in biological fluids and are resistant to denaturants, including high temperatures (>80 ◦C) [8]. Each subunit is composed of four long helixes, a short helix and a long loop [9]. The C-terminal of each subunit folds into the inner cavity, while the N-terminal is exposed on the outer surface of the nanocage. The ratio between Hc and Lc subunits is determined by ferritin's primary role in tissues. For example, in the hearth and brain, the Hc is more abundant, while, in the liver and spleen, the Lc is predominant. The Hc subunit contains a dinuclear ferroxidase site that is located within the four-helix bundle, while the Lc provides efficient sites for iron nucleation and mineralization [10]. Ferritin and FN carry six C4 channels and eight C3 channels. The C3 channels have hydrophilic properties and allow the passage of Fe(II) ions and water molecules in and out of the protein cage. On the other hand, the C4 channels allow the passage of small hydrophobic molecules [8].

Ferritin in the bloodstream is mainly composed of Lc subunits [8], which seem to be secreted primarily by macrophages [11]; however, their role in the serum is still highly debated. Nonetheless, high ferritin levels have been linked with ongoing infections and chronic inflammation, while its reduced levels have been correlated with iron

deficiency [12–14]. Interestingly, it can be localized in cells both in the cytoplasm and in the nucleus. Iron stored in ferritin can be utilized by the cell in a process mediated by autophagy, where it is transported to the lysosomes and iron is released in a pH-dependent manner [15]. On the other hand, in conditions of oxidative stress, ferritin can convert DNA damaging Fe(II) to harmless Fe(III), thus limiting DNA damage mediated by the formation of hydroxyl radicals through the Fenton reaction [16–18]. Two proteins, poly(rC)– binding protein 1 (PCBP1) and nuclear receptor coactivator 4 (NCOA4), are involved in the transport of iron inside and outside ferritin [19]. Furthermore, it has been proposed that O-glycosylation of the Hc could be involved in the nuclear translocation of Ferritin, which maintains its intact structure during this process [20,21]. FNs share all structural features and properties with their physiological form, and they often have been demonstrated to be managed by the cells and the tissue as natural ferritin [21,22].

#### *Strategies for Loading FN*

Molecular cargoes can be loaded into the inner core of FN by different methodologies (Figure 3). Extreme pH (2 or 13) is used to transiently disassemble the protein nanocage into monomers that can reassemble by adjusting the pH toward neutrality. By employing this methodology, FN can be loaded with different chemotherapeutic drugs. Interestingly, only minor differences in the loading efficiency between doxorubicin (DOX), epirubicin (EPI), daunorubicin (DAU) and idarubicin (IDA) were seen, despite their differences in terms of hydrophobicity [23]. In addition, high concentrations of guanidine hydrochloride (GuHCl) or urea are able to disrupt the non-covalent forces which support FNs' structure, leading to their disassembly. This process can be reversed by dialysis to remove the excess of chaotropic agents, leading to the recovery of the original nanostructure with consequent loading of molecular cargoes in the inner cavity [24]. More recently, atmospheric cold plasma (ACP) technology was implemented to reduce the α-helix/β-sheet contents and thermal stability of FN to allow disassembly at a pH of 4. This technique can be utilized to load molecules which are susceptible to extreme pH conditions and could be degraded during the loading procedure [25]. In addition, our group has showed that the loading of molecules sensitive to low pH can be achieved during the reassembly phase by adding the molecule of interest to the ferritin-containing solution after the adjustment of the pH toward neutrality [26]. In another report, Jiang and colleagues developed a methodology that is able to provide high loading of DOX and high recovery of FN by incubating DOX with FN at 60 ◦C for 4 h [27]. This loading methodology enables the opening of FN's channel to introduce DOX without disrupting FN's structure. Lastly, by taking advantage of the natural capacity of FNs to encapsulate iron in their cavity, several metal ions can be coupled to molecules of interest that can then be loaded into FNs. In this case, the final loading efficiency of the chosen drug depends on its binding affinity for the metal ion, the FN species used and preparation conditions. For example, Zhen and colleagues suggested that, between Cu(II), Mn(II), Zn(II) and Fe(III) metal ions, the use of copper resulted in the highest loading rate of DOX into FN [28]. A more detailed comparison of loading methodologies for DOX into FN has been reviewed by He and colleagues [29], while Zhang and colleagues provide specific protocols regarding the loading of various drugs into FNs by using different methodologies [30].

**Figure 3.** Biochemical strategies used to load different cargoes into FNs: (**A**) pH or urea-mediated disassembly–reassembly methodology; (**B**) modified ferritins or ACP can be utilized to disassemble FN at pH 4, and then pH 7.5 is used to reassemble FN; (**C**) high temperatures partially destabilize FN to allow channel openings with consequent drug loading, and then lowering the temperature can slowly reconstitute the natural conformation of FN; (**D**) molecular cargoes can be complexed with Cu(II) or other metal ions which have a high affinity for the internal cavity of FN. This methodology permits the loading of hydrophobic molecules with some limitations.

Overall, FNs' physicochemical properties (small size and negative Z potential), together with their intrinsic capacity to avoid recognition by RES and targeting capability toward TfR1-expressing tumor cells, make them an ideal candidate for the development of drug delivery systems for nanobiotechnological applications in the field of oncology (Table 1). Moreover, drug loading and targeting efficiency could be enhanced by chemical and genetic manipulations of FNs, as is discussed in the following section.

**Table 1.** *Cont.*

#### **3. Production and Modifications of FN**

FNs utilized in preclinical studies are usually produced as recombinant protein in *E. coli* strains engineered to express only the human Hc subunit. This procedure involves the transformation of bacteria with a plasmid containing the Hc sequence of interest, which is then purified by anion-exchanger columns after treatment at 70 ◦C. The resulting FNs are composed, in this case, of 100% Hc subunits [66]. Otherwise, FNs can be purified from the horse spleen, where the ratio between Hc and Lc subunits was found to be ~1/10 [67]. To ensure that purified FNs are not contaminated by endotoxins that could impact both in vitro and in vivo experiments, additional procedures to remove endotoxins might be required [68].

The genetic manipulation of the Hc-FN DNA sequence led to the development of more than one hundred variants to introduce novel functionalities that are able to improve the drug loading, biodistribution and targeting properties of FNs [69]. For example, the self-assembly properties of FNs can be altered to produce novel nanostructures comprising 8 or 48 subunits instead of 24 [70,71]. In addition, ferritin can be modified to produce nanocages that can disassemble at a pH of 4 or 6, instead of 2 [72–74]. Intriguingly, Gu and colleagues developed His-modified ferritins that do not self-assemble at neutral pH. However, metal ions or a pH of 10 induce self-assembly with consequent increases of the drug-loading efficiency, as compared to the standard pH methodology discussed previously [75]. Unfortunately, the stability in serum of His-modified ferritins was not evaluated, and it is unclear if these nanoconstructs are suitable for in vivo studies.

Different strategies have been recently employed to enhance the half-life of FNs in the circulation to provide higher tumor accumulation and reduce clearance by RES. For example, Wang and colleagues developed a novel FN that includes an albumin-binding domain that is able to increase FNs' half-life by 17 times, as compared to the standard FN [76]. In another report, an amino acid sequence rich in proline (P), serine (S) and alanine (A) residues (PAS polypeptide) was inserted by genetic manipulation into FN to increase blood half-life and DOX encapsulation efficiency [77,78]. Interestingly, the insertion of two glutamate residues in the PAS sequence (PASE) further improved FNs' accumulation to the tumor site [79]. In another report, Jin and colleagues introduced in the ferritin construct a

blood circulation prolonging (BCP) peptide derived from the phage M13. The generated FN (BCP1–FN) showed improved circulation time compared to standard FN (20 h compared to 2 h). In addition, when loaded with DOX, BCP1–FN–DOX showed superior therapeutic efficacy in a mouse model of melanoma compared to FN–DOX and free DOX. Intriguingly, the authors suggested that the RGD portion of the BCP1 peptide could be responsible for the binding of BCP1–FN to peripheral blood cells, particularly platelets, which are able to protect the nanocages from RES recognition [31]. Nonetheless, blood cells' "hitchhiking" has recently emerged as one of the strategies that can be employed to enhance the delivery of NP to the tumor site [80,81].

Since FNs are composed of different subunits, novel FN-based nanostructures have been developed with combinations of different ferritins resulting in hybrid nanocages with interesting physicochemical properties. Ahn and colleague developed a hybrid FN composed of modified subunits (F160) and standard Hc in a ratio 1:1. F160 was devised to provide large pores to FN and was produced by removing the C-terminal channel forming E-helix from the Hc sequence. The resulting hybrid FN (nicked–FN) allows for the encapsulation of DOX by simple incubation, improving the loading of DOX and the recovery of the nicked–FN–DOX in comparison to encapsulation in unmodified FN or with the pH-mediated disassembly and reassembly methodology [82]. Another strategy to enhance DOX loading into FN was developed by producing a mutant FN that displays an enhanced affinity for copper ions [32]. In another report, FNs were modified with the addition of biotin accepted peptide, which resulted in biotinylated FN that can be more easily modified by the addition of streptavidin-tagged molecules [83]. These modified FN could be used in a variety of immunoassays based on streptavidin-tagged antibodies to increase the sensitivity. It is unsure if they can be employed for in vivo studies.

The delivery of nucleic acids by NP-based delivery systems has always been a primary goal of the research effort in the field of nanotechnology. Interestingly, modified FNs with the addition of a cationic polypeptide were developed to facilitate the incorporation of siRNAs in FNs' nanostructure [33,84]. However, it has also recently been shown that unmodified FNs could incorporate siRNAs by pH-mediated disassembly and reassembly methodology [85].

FNs can also be modified to include immunogenic peptides that are able to induce immune responses against specific antigens. As proof of principle, Kanekiyo and colleagues developed a nanovaccine against the H1N1 virus based of FNs that were modified to include the viral hemagglutinin sequence. Preclinical testing of the developed nanoformulation showed induced protection of animal models to H1N1 infection [86]. More recently, FN-based anticancer nanovaccines have been developed and were tested successfully in preclinical models [56,87,88].

Interestingly, many FN variants have been developed to include novel targeting ligands. For example, Jiang and colleagues introduced, by genetic manipulation, a hepatocellular carcinoma (HCC)-targeting peptide to the FN's structure that was then loaded with DOX. This novel formulation showed superior activity compared to free DOX in reducing HCC tumor growth and metastases in preclinical models [48,89]. In another report, the PD-L1 binding peptide 1 (PD-L1pep1, CLQKTPKQC) was introduced into ferritin's sequence to generate an FN targeted to PD-L1 [34]. Another well-studied tumor-targeting ligand is the tLyp-1 peptide, which binds the receptor Neuropilin 1 expressed in the stroma of many types of tumors [90]. Modified FNs were developed to include the tLyp-1 peptide in the external structure of the nanocage and were subsequently loaded with Paclitaxel (PTX). The resulting FNs (tLyp–FN–PTXs) showed enhanced uptake by tumor cells and were able to control tumor growth in vivo compared to free-PTX or FN, where the sequence of tLyp was mutated (m-tLyp–FN–PTX) [35].

Beyond genetic manipulation, FNs have available primary amines on their surface that can be exploited for chemical conjugation purposes. N-hydroxysuccinimide (NHS) ester or maleimide groups, in combination with 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (EDC), are often used to couple peptides, PEG, fluorophores or antibodies to FN in a

buffered solution, without the use of organic solvents [42,91]. This coupling methodology is often employed to develop fluorescent versions of FNs that can be utilized in a variety of in vitro and in vivo assays, including flow cytometry, fluorescence microscopy and live imaging, which are critical techniques for NP characterization and for the evaluation of biodistribution and targeting capacity of novel formulations of FNs. In a recent report, FNs were modified with the addition of positively charged polyamine dendrimers (PAMAM) to allow efficient loading of nucleic acids. MiRNA-loaded FNs were successfully used to target leukemia cells and showed promising in vitro results [92].

Overall, both genetic and chemical manipulations can enhance multiple aspects of the intrinsic properties of FNs, such as targeting, loading and half-life. However, it is unknown if these modifications could induce the production of specific anti-ferritin antibodies when administered in humans. Interestingly, it has been shown that modifications such as PEGylation could result in the generation of anti-PEG antibodies [93]. Therefore, it is plausible that some of the developed modifications of the native human Hc subunit, by both genetic and chemical manipulation, could potentially reduce the effectiveness of FN after multiple administrations, limit their targeting capabilities and induce undesirable immunogenic reactions [93]. Furthermore, the FN's origin could be an important factor contributing to immunological side effect. These potential complications should be carefully taken in consideration to ensure the success of modified FNs in the prospect of clinical translation [94].

#### **4. FN-Based NPs for Cancer Treatment in Preclinical Models**

One of the main issues in the delivery of chemotherapeutics for cancer treatment is the onset of off-site side effects, which can cause a wide spectrum of complications, such as infections, neuropathies, cytopenias, nephrotoxicity, cardiotoxicity and hepatotoxicity [95–98]. NP-based delivery systems are utilized in oncology primarily to reduce the severity of these side effects, improving drug accumulation at the tumor site. Examples of nanotherapeutics currently used in the clinical practice are Doxil™, Abraxane™, Marqibo™ and DaunoXome™, which are NP-based platforms for the delivery of DOX, PTX, Vincristine and DAU, respectively [1].

Interestingly, FNs have been extensively studied as nanocarriers for DOX, since this hydrophilic drug can be encapsulated efficiently into FNs and can be delivered to tumor cells by the TfR1-mediated intrinsic targeting capabilities of FNs. Our group and others have shown that not only are FN–DOX formulations superior to free DOX or Doxil™ in controlling tumor burden in preclinical models of cancer, but they also dramatically reduced drug cardiotoxic effects, as compared to the free DOX [48,99–101]. In another report, Huang and colleagues developed a hybrid FN–DOX formulation for the treatment of lung cancer. It is composed of PEGylated Hc subunits to provide stealth capabilities and non-PEGylated Hc subunits to allow the binding of the nanocage to TfR1. In vivo results showed that, after intratracheal administration of hybrid FN–DOX, the tumor burden in a orthotopic murine model of lung cancer (3LL) was dramatically reduced when compared to free DOX [37]. Apart from DOX, platinum-based chemotherapeutics (cisplatin, oxaliplatin, Pt(II) terpyridine and carboplatin) have been successfully encapsulated in FNs and have shown encouraging antitumor activity in preclinical models of cancer [53,102,103]. Recently, Ferraro and colleagues developed a novel FN loaded with Arsenoplatin-1 (Pt(μ-NHC(CH3)O)2ClAs(OH)2), which combines the cytotoxic effects of both cisplatin and arsenic trioxide. Preliminary in vitro results showed that this novel formulation provides selectivity toward cancer cells, but, unfortunately, it was not tested in vivo [50].

The development of drug resistance often occurs after treatment with standard chemotherapeutics, and it can be mediated by the activity of the transporter multidrug resistance protein 1 (MDR1), which is upregulated in the hypoxic areas of tumors and facilitates the excretion of chemotherapeutics outside the tumor cell membrane [104]. Interestingly, hypoxia in the TME can induce the expression of TfR1 mediated by hypoxia-inducible factor-1α (HIF-1α) in tumor cells [105]. Hence, FN-based NPs could be employed to specifically target hypoxic areas in tumors. For this purpose, Huang and colleagues developed a hybrid FN (composed of 75% PEGylated subunits) (Figure 4) for the delivery of the HIF-1α inhibitor Acriflavine(AF). This nanoformulation was particularly effective when used in combination with cisplatin, since the delivery of AF to the TME was able to reduce the expression of MDR1 on tumor cells, thus reducing the development of resistance to cisplatin, which was not effective as standalone treatment in the 3LL lung cancer xenograft model [38].

**Figure 4.** Structure and loading procedure for AF-loaded hybrid PEGylated FN. Hybrid FNs can be developed by utilizing the pH disassembly/reassembly methodology, starting from two different FNs (in this case, a PEGylated FN and non-PEGylated FN). In addition, prior to the reassembly phase, anticancer drugs can be added, resulting in their inclusion inside the FN nanostructure after reassembly. Adapted from [39], American Chemical Society, 2019.

Ferritin was also recently employed to develop a novel nanoformulation containing both Rapamycin, an mTOR inhibitor, and Erastin, a ferroptosis inducer. NPs were produced by the emulsification technique, which was shown to be superior compared to the standard pH disassembly–reassembly methodology in regards to drug loading. Interestingly, the size of the NP formed was 7-fold larger than standard FN (78 compared to 12 nm). Nonetheless, this novel formulation achieved impressive results in controlling the tumor growth in a murine model of breast cancer which recapitulates tumor relapse and metastases formation. Briefly, the primary tumor was allowed to grow, and it was excised to simulate surgery. Subsequently, NPs or the free drugs were included in a thermo-responsive F-127 hydrogel and injected into the tumor resection cavity to test the ability of the nanoformulation to prevent tumor recurrence [51]. Unfortunately, the authors did not evaluate the differences in uptake between standard a FN and the modified version developed. Furthermore, since NPs were not administered intravenously, their biodistribution was not evaluated.

FN has also been explored as a nanocarrier for a large variety of drugs for cancer therapy, such as Olaparib [39], Everolimus [40], Curcumin [41], Oxaliplatin + Panitumumab [53], Mertansine [49], Resveratrol [54] and Navitoclax [42].

#### *4.1. FN-Based NPs for Immunomodulation and Immunotherapy*

Another therapeutic strategy which has recently emerged in cancer therapy is the immunomodulation of the TME, in particular, the reprogramming of tumor associated macrophages (TAMs). In solid tumors, TAMs constitute up to 50% of the tumor mass and have been shown to support local immunosuppression and metastases formation [106]. They are recruited from the bloodstream and surrounding tissues by growth factors and chemokines, including colony-stimulating factor 1, C-C motif ligand 2 and vascular endothelial growth factor [107,108]. Interestingly, TAMs are conventionally categorized as anti-inflammatory M2-like macrophages and express high levels of TfR1 compared to proinflammatory M1-like macrophages [109,110]. For this reason, TAMs could be effectively targeted by FN-based therapeutics. Of note, macrophages, in general, can be considered as "gate-keepers" of iron metabolism due to their involvement in the recycling of iron from dying erythrocytes [111]. In addition, TAMs-derived FNs have been shown to function as

growth factors on malignant mammary epithelium in a process independent of iron [112]. In order to re-educate TAMs and promote a phenotype switch from M2-like to antitumoral M1-like, FNs have been developed to deliver the toll-like receptor 9 agonist CpG, a nucleic acid with M1-polarizing properties [57]. FNs were functionalized with the TAMs-targeting peptide M2pep (YEQDPWGVKWWY), which was combined with a cationic peptide to allow the attachment of the negatively charged CpG. Interestingly, this novel formulation was able to achieve reduction in tumor growth in a murine model of breast cancer. However, a similar effect was seen even when CpG was absent. The authors hypothesized that the antitumoral effect mediated by the M2pep-modifed FN could be mediated by the intrinsic M1-polarizing activity of the cationic peptide included in the modified FN, since the unmodified FN showed only minor antitumor activity [57].

The discovery of the molecular mechanisms underpinning the immunosuppressive state in the TME led to the FDA approval of immune checkpoint inhibitors (ICIs) for cancer therapy, giving rise to novel immunotherapeutic options that are able to induce a strong infiltration of active immune cells in the TME, with consequent control of tumor growth [113]. ICIs currently used in the clinical setting are monoclonal antibodies (mAbs) that are able to block the activity of the programmed cell death protein 1 (PD-1)/PD-L1 interaction or cytotoxic T-lymphocyte antigen-4 expressed by T cells. Recently, a DOX-loaded engineered FN displaying the PD-L1 binding peptide (PpNF) was developed by Seon and colleagues [34]. Interestingly, this novel nanoformulation was able to achieve enhanced tumor-growth reduction in the colon carcinoma CT26 xenograft model, as compared to anti-PD-L1 mAb and free DOX (Figure 5). In addition, the engineered FNs without DOX were shown to be superior to anti-PD-L1 mAb in enhancing the activity of T cells in vitro.

**Figure 5.** Antitumor activity of FNs displaying the PD-L1 binding peptide (PpNF). (**A**) Experimental schemes for antitumor treatments. Mice bearing s.c. CT26 syngeneic colon tumors were treated with DOX-loaded PpNF (PpNF(Dox)) or PpNF or Dox administered by i.v. injection three times per week. Anti-PD-L1 antibody was administered by i.p. injection twice per week. (**B**) Tumor volumes after treatment. (**C**) The weights of excised tumors from each group at the 19-day post-injection. (**D**) Body weights. The data represent means ± SEM (\* *p* < 0.05 and \*\* *p* < 0.01; *t*-test). Reproduced with permission from [35], Elsevier, 2021.

We hypothesize that, in future years, novel FN-based delivery system will be employed to modulate the activity of immune cells, since a new paradigm for NP-based anticancer therapeutics is emerging [114]. In fact, the expanding arsenal of nanomedicines able to modulate the activity of TME-infiltrating immune cells could be utilized to support standard chemotherapeutics or immunotherapies in order to reactivate the antitumor immunity [115].

#### *4.2. FN-Based NPs for the Treatment of Brain Tumors*

The blood–brain barrier (BBB) is a diffusion barrier that impedes the influx of most compounds from the bloodstream to the brain parenchyma and represents a protective interface between the central nervous system and peripheral blood circulation [116]. Interestingly, brain cells require iron for metabolic processes; thus, transferrin and ferritin have to bypass the BBB in a process mediated by ligand–receptor recognition. Within the brain, TfR1 was shown to be expressed by capillary endothelial cells, choroid plexus epithelial cells and neurons, which increase the expression of TfR1 in condition of iron deficiency [117]. In addition, TfR1 has been shown to be overexpressed in brain tumors, particularly in glioblastomas, and its overexpression is associated with worse prognosis [118]. Taking into consideration these experimental evidences, we see that FNs are promising candidates for effective brain tumor therapy, due to their intrinsic targeting capability toward TfR1.

Fan and colleagues demonstrated that DOX-loaded FNs are able to bypass the BBB and deliver DOX to brain tumors in mice, dramatically increasing their survival compared to mice treated with control treatments (free DOX and Doxil™) [43]. Interestingly, FNs maintain their intact structure after crossing the BBB by transcytosis. This process is mediated by endothelial cells and allows the accumulation of FNs in the brain parenchyma in healthy mice. However, FNs co-localize with lysosomes after internalization in glioma cells. These results corroborate the idea that FNs traverse the BBB and effectively deliver therapeutics to brain tumors without affecting the surrounding tissues. The authors speculate that the different fate of FNs between endothelial and tumor cells could be due to the differences in expression of TfR1 [43]. More recently, α2β1-targeted Dox-loaded FN (αβ-FN-DOX) was shown to have enhanced activity compared to FN–DOX in controlling the tumor growth of glioblastoma in an orthotopic model of brain tumor (U-87MG) [44]. Interestingly, αβ-FN–DOX had a higher drug-loading capacity compared to FN–DOX (60 vs. 15%, respectively). The authors speculate that this could be due to the modified integrin α2β1 targeting sequence, which possesses multiple carboxyl groups that could have an impact in ionic interactions between DOX and αβ-FN.

In another report, PTX-loaded FNs were successfully developed by the disassembly and reassembly methodology and were used to treat C6 glioma bearing mice. The results showed enhanced activity of FN–PTX compared to the free drug in controlling tumor growth. Furthermore, treatment with FN–PTX showed no apparent signs of toxicity in the heart, liver, spleen, lung and kidneys of treated animals [45].

Other anticancer compounds, such as Au(III) thiosemicarbazone [55] and vincristine [52], have been successfully loaded into FNs and used to treat brain tumors in various murine models of cancer, achieving impressive results in controlling tumor growth. Interestingly, FNs can also facilitate the delivery of therapeutic mAb through the BBB. Our group has developed FNs coupled with Trastuzumab or Cetuximab, two FDA-approved mABs that are able to target the human epidermal growth factor receptor 2 and the epidermal growth factor receptor, respectively [46]. In addition, our group has developed a methodology to specifically study the translocation of FNs (or potentially other nanocarriers) through the BBB. This ex vivo model is based on layers of primary rat brain microvascular endothelial cells and astrocytes, which are used as a surrogate of the BBB [119].

We speculate that the development of novel FN therapeutics for the treatment of brain tumors will be particularly prominent in the coming years, since FNs have shown to effectively bypass the BBB without disassembly, leading to the release of FN-loaded therapeutics directly to tumor cells and avoiding off-site side effects.

#### **5. Preclinical Exploitation of FN-Based NPs for Tumor Imaging**

There is an ever-growing need for novel imaging agents that are able to effectively identify the presence of very small tumors in the early stage of the pathology, when they can be successfully treated by surgery or anticancer therapies. In addition, after successful surgery, patients undergo routine diagnostic tests to reveal the insurgence of metastatic events that could occur even several years after the original diagnosis [120]. Unfortunately, metastatic tumors are often incurable, since they usually become resistant to standard therapies and account for 90% of total cancer death worldwide [121]. Hence, it is critical to identify the presence of metastases with the current imaging modalities (computerized tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET)) which are often utilized together with contrast agents that are able to accumulate specifically in the TME.

In regards to MRI, gadolinium-based contrast agents are widely used, since they are able to identify highly vascularized tissues, such as tumors [122]. However, these types of agents are not cancer-specific and can result in a high rate of false positives. In addition, standard MRI does not have the sufficient spatial resolution to detect micro-metastases, thus leading to possible misdiagnoses of oligometastatic disease [123]. On the other hand, the glucose analog 18F-Fluorodeoxyglucose is a contrast agent utilized in PET/CT imaging to detect tumors, allowing for the visualization of areas with high metabolic activity [124]. Unfortunately, other areas characterized by active metabolic activity (benign tumors, inflammation sites and areas of ongoing infections) can give rise to false-positive results. Lastly, PET scans are quite expensive and require the use of radioactive contrast agents based on glucose that cannot be utilized in pregnant women or diabetic patients, due to off-target side effects [125].

NP-based delivery systems have been employed to enhance the specificity of contrast agents for tumor cells [126–128]. FNs have also been explored as a delivery platform for imaging probes, particularly for metal-based contrast agents. For example, multimodal FNs loaded with superparamagnetic iron oxide (Magnetoferritins) and a near-infrared fluorescence dyes were developed to efficiently detect tumors by multiple imaging modalities [58,59,65]. Interestingly, Magnetoferritins can efficiently identify very small tumors (~1 mm) by MRI in murine models of cancer, thus dramatically increasing the limit of detection of current contrast agents for MRI. Iron-loaded FNs derived from equine spleen (HoS–FN, composed of 85% of Lc and 15% of Hc subunits) were also utilized for MRI visualization of tumors [61]. Interestingly, HoS–FN showed enhanced uptake by SCAR5-positive cells, due to the specific targeting of this receptor mediated by Lc subunits. Furthermore, HoS–FNs were also able to reduce tumor growth, as compared to apoferritin HoS–FN in a murine model of breast cancer.

Imaging agents are also used in fluorescence-guided oncological surgery to assist the surgeon in the identification of metastatic foci, particularly in lymph nodes [129]. Indocyanine-green (ICG) is one of the most used FDA-approved fluorescent dyes for this purpose, since it can be visualized avoiding background autofluorescence (mainly due to hemoglobin) and has a low risk of adverse events [130,131]. Our group has developed ICG-loaded FNs with improved fluorescence accumulation in tumors in comparison to free ICG in a murine model of breast cancer [26,63]. Since specific accumulation of FN–ICG in tumors can be detected up to 24 h after intravenous injection in mice, we speculate that FN–ICG could be administered prior to surgery, and it could be visualized during surgery by fluorescence-guided endoscopy. This methodology could potentially reduce surgery time and improve the detection of small metastases, particularly in lymph nodes.

FNs were also developed to specifically visualize bone metastases by genetic manipulation of ferritin to include osteoblast and hydroxyapatite-binding peptides [64]. In another report, folic acid–functionalized FNs were developed to target tumor cells and deliver

perfluoropentane, a compound used for low-intensity focused ultrasound imaging and therapy [62]. Lastly, gold NPs were efficiently encapsulated into 2-amino-2-deoxy-glucosefunctionalized FNs to develop a tumor-targeted FN for CT imaging [60].

Overall, FN-based nanostructures can be utilized for the tumor-specific delivery of numerous contrast agents to improve their pharmacokinetic characteristics and enhance tumor accumulation.

#### **6. Drawbacks and Future Perspective of FN**

FNs are a versatile drug delivery system for chemotherapeutics and imaging agents. However, one of the major limitations of FNs in regards to drug loading is the low encapsulation capacity for hydrophobic compounds. This is primarily due to the leakage of the loaded hydrophobic drug from FN soon after encapsulation. Nonetheless, hydrophobic drugs can still be loaded inside FN by utilizing methodologies such as the pre-complexation with copper ions or the modification of native ferritin with hydrophobic amino acid sequences, that are able to enhance the affinity of hydrophobic compounds for ferritin. For this purpose, Wang and colleagues designed a novel FN construct (Am-PNCage) by linking the sequence of the Pout peptide (GRGDSKKHHHHHHAFAFAFAFVVVAA) to the C terminus of Hc ferritin through a flexible amino acid sequence GGSG, which replaced the E helix amino acids of Hc. This novel FN was employed to achieve the co-loading of the hydrophilic anthracycline EPI and the hydrophobic topoisomerase inhibitor Camptothecin and showed impressive antitumor activity in different murine models of cancer [47]. This novel FN construct could pave the way for the development of sophisticated FNbased nanostructures that are able to integrate multiple drugs with different mechanisms of action. This combinatorial nanotherapy could synergistically strike solid tumors by taking advantage of specific chemosensitivities to limit the insurgence of resistance to single chemotherapeutics.

An important area that is currently understudied is the relevance of the various modifications of FNs in regards to uptake and toxicity, particularly toward immune cells and erythrocytes in the bloodstream. For example, RGD-modified NP (nano-emulsions and liposomes) have been shown to be taken up by phagocytes in the bloodstream that are then able to transport NPs to specific sites in the body where there is ongoing inflammation and/or angiogenesis, such as the TME [132,133]. In fact, a majority of research efforts have been focused on showing that FN-loaded drugs can induce fewer side effects as compared to the free drug. This has been shown extensively for DOX, since FN–DOXs have an encouraging minimal effect on cardiomyocytes when compared to DOX [99,100]. However, it remains unclear if FN modifications can impact their uptake on different cell types present in the bloodstream. This area of study could be particularly relevant to pursue since it has been recently speculated that the enhanced accumulation of nanotherapeutics in the TME could be mediated not only by the EPR effect but also by the phenomenon of NP hitchhiking [81,114,133,134].

Indeed, FNs have favorable and interesting characteristics as an NP-based delivery system. Their efficient loading capacity for different drugs used in oncology, intrinsic targeting toward TfR1 and biocompatibility make them an ideal nano-platform for the treatment and imaging of tumors. Unfortunately, to date, FNs have not yet reached the clinical stage. In fact, the current high cost of production somewhat limits their translational potential. However, we speculate that the recent advancement concerning drug-loading efficiency and customization capabilities could facilitate the interest of pharmaceutical industries in developing novel production protocols for FNs that are aimed at enhancing purity, while, at the same time, reducing the costs of production. Collectively, the number of experimental evidences in support of the use of FNs as nano-delivery systems are everincreasing, making their translation from bench to bedside a reasonable possibility. Lastly, the opportunity of co-encapsulating different drugs into FNs allows for the development of novel FN-based theranostic agents that are able to combine both imaging and therapeutic functionality in a fully biocompatible nanosystem. For these reasons, we believe that, in

the near future, the clinical application of FNs could play a pivotal role in the diagnosis and treatment of solid tumors.

**Author Contributions:** F.C., F.M. and S.M. conceived of and designed the work; F.M., A.B., M.S., L.S., M.T., F.C. and S.M. wrote the manuscript; M.T., S.M., F.M. and F.C. revised the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research leading to these results has received funding from AIRC under IG 2017—ID. 20172 project—P.I. Corsi Fabio.

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

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors acknowledge AIRC for F.M. postdoctoral fellowship and University of Milan for A.B., M.S. and L.S. doctoral and postdoctoral fellowship, respectively. Figures 1 and 2 were created with BioRender.com (accessed on 2 November 2021).

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

#### **References**


Meet the editor

Sara Savić, PhD, DVM, is a researcher working in a diagnostic laboratory within the Scientific Veterinary Institute "Novi Sad" in Novi Sad, Serbia. Her main work is based on the diagnostic procedures for zoonotic diseases and vector-borne zoonoses. Dr. Savić completed her PhD degree on Diagnostics of Lyme disease in dogs and ticks, and then turned her interests and career towards other vector-borne diseases important for public health

and One Health issues. The significance of multidisciplinary and transdisciplinary work has become most interesting during the past decade. Her expertise is in bacterial and parasitic vector-borne zoonoses, especially in blood parasites. Dr. Savić has published over 100 publications so far as a leading author or as a coauthor in different scientific journals or at conferences.

**Preface III**

Introduction **1**

**Chapter 1 3**

Vectors **9**

**Chapter 2 11**

**Chapter 3 33**

Vector Borne Zoonotic Diseases and Pathogens **49**

**Chapter 4 51**

**Chapter 5 73**

**Chapter 6 87**

Vector Control **105**

**Chapter 7 107**

Use of Artificial Intelligence on the Control of Vector-Borne Diseases *by Daniel da Silva Motta, Roberto Badaró, Alex Santos and Frank Kirchner*

Introductory Chapter: Vectors and Vector-Borne Pathogens around Us

Aedes: What Do We Know about Them and What Can They Transmit?

Sandfly-Borne Viruses of Demonstrated/Relevant Medical Importance

An Overview of Leishmaniasis: Historic to Future Perspectives

*by Biswadeep Das, Sayam Ghosal and Swabhiman Mohanty*

*by Ana Sofia Santos and Maria Margarida Santos-Silva*

*by Nazli Ayhan and Remi N. Charrel*

Vector-Borne Infections in Bulgaria

*Ixodes ventalloi* Gil Collado, 1936: A Vector Role to be Explored

**Section 1**

Contents

*by Sara Savić*

**Section 2**

**Section 3**

*by Mümtaz Güran*

*by Iva Christova*

**Section 4**