*4.3.1. Accuracy significant networks*

In accuracy significant utilization, we define α, β and γ as 2, 1, 1; however if the data accuracy is much more important than other two, we also can define α=3 or much larger. In this research, for simplify, we discuss none other but the case that significant parameter has the significance vector of 2 and the ordinary parameters are 1. According to TOI we can get the best result in fig. 10.

**Figure 10.** Tradeoffs of accuracy significant

From Fig. 10 we find that when the event generation rate is between 0.0001 and 4.0, nonaggregation is the best comparing with full and partial aggregation. When the data generation rate is between 4 and 30, the partial aggregation is the best, and the full aggregation is the best when data generation rate is larger than 30.


**1. Introduction**

The process of covering the surface of an object or substrate with a very thin layer is known as Coating. This layer can be of some sort of a thin polymer sheet, paint, or lacquer, which can be utilized for protective/decorative purposes. Most of industrial products get-up-and-go through the process of coating not only to prevent corrosion but to make them attractive. As the coating involves the development of a thin film layer that can be lacquer or polymeric on a fabric or substrate etc. Therefore, if the substrate starts and ends the process wound up in a roll, the process can be termed "roll-to-roll" or "web-based" coating. Due to multiple usages of coatings, several theoretical and experimental attempts have been devoted and this special issue is one of them. We hope that this issue will not only address the current challenges, but will also provide an overall picture and up-to-date findings to readers of the scientific community that ultimately benefits the industrial sector regarding its specific market niches and end users.

#### **2. Methodologies and Usages**

Two-dimensional, incompressible asymmetric peristaltic propulsion coated with Synovial fluid (non-Newtonian model) with mass transport was investigated in [1]. Because of the coating of the same base-fluid at the surface of the channel, the boundaries become non-porous and exert no slip on the fluid particles. Two illustrative models for the viscosity, namely, shear-thinning (Model 1) and shear-thickening (Model 2), are considered, which reveal the presence and integrity of coating. The perturbation method has been applied to linearize the complicated differential equations. Model 1 predicted higher viscosity values and more significant non-Newtonian behavior than Model 2. It is also observed that the shear-thinning model behaved in quite the opposite manner for the shear thickening model. The converse behavior of Models 1 and 2 occurs due to a curvature of the flow domain. Moreover, Model 1 is not able to capture the correct exponential viscosity dependence on concentration for the whole range of shear rates. On the other hand, the second model shows a strong relationship with accurate power. Solutions are attained for velocity field, concentration profile, and pressure gradient. The novelty of all the essential parameters is analyzed through graphical results. Furthermore, streamlines are also drawn in order to determine the trapping mechanism. The present analysis is beneficial in the study of intrauterine fluid dynamics; furthermore, it is applicable in vivo diagnostic; drug delivery; food diagnostics; protein chips; and, cell chips and packaging, i.e., smart sensors.

Ellahi et al. [2] devoted their efforts to investigate the shiny thin film with a metallic tactile covering of nanoparticles over the surface of a rotating disk. To decorate, glowing silver and gold particles were chosen. Four illustrative base liquids, namely (i) ethanol, (ii) methanol, (iii) ethylene-glycol, and (iv) water were considered with different geometries, which have great importance in industrial use. An emphasis on comparative multi nanofluid analysis was used to make a sound judgment on which one of the fluids best suited the metallic glittering process of spin coating. The film thickness process highly depends on the process of evaporation, which takes some time to settle on the disk's surface. It was found that of the base fluids, the best choices were ethanol alloys with silver. Hence, one can conclude that, from an experimental point of view, if silver alloy is used for coating, then only those liquids can be considered that exhibit ethanol-like properties. The impact of pertinent parameters with different aspects are graphically illustrated in each case

Lu et al. examined the flow of hybrid (nickel–zinc ferrite and ethylene glycol) nanoliquid with entropy optimization and nonlinear thermal radiation coatings past a curved stretching surface [3]. Analysis was carried out in the presence of magnetohydrodynamic, heat generation/absorption, and convective heat and mass flux conditions. A solution of the modeled problem was attained numerically using MATLAB built-in function bvp4c. Impacts of prominent parameters on betrothed distributions were depicted through graphs and they were well supported by requisite discussions. Numerically calculated values of Sherwood number were established in a tabulated form and were scrutinized critically. An excellent concurrence was achieved when the results of the presented model were compared with previously published result; hence, dependable results are being presented. It was observed that concentration field diminished with increasing values of curvature parameter, though the opposite trend was noticed for velocity and temperature distributions. Further, it was detected that Nusselt number decreased with augmented values of radiation and curvature parameters.

Investigation [4] is carried out on the thin film flow of Reiner-Philippoff fluid of boundary-layer type. We have analyzed the flow of thin films of Reiner–Philippoff fluid in the changeable heat transmission and radiation over a time-dependent stretching sheet in two-dimensions (2D). The time-dependent governing equations of Reiner–Philippoff fluid model are simplified with the help of transformation of similarity variables. To investigate the behavior of the Reiner-Philippoff fluid with variable stretching surface for different physical effects, we considered thermophoresis and Brownian motion parameters in the flow. The Homotopy Analysis Method is implemented in the reduced model in order to achieve a solution of the original problem. A numerical convergence of the implemented method is also analyzed. The behavior of temperature, velocity, and concentration profiles have been investigated with the variation of skin friction, Nusselt number, and Sherwood number. A comparative graphical

survey is presented for the velocity gradient, under different parameters. An analytical analysis is presented for the time-dependent parameter over thin film flow. The results that we obtained are better than the previously available results. For the survey, the physical representation of the embedded parameters, are discussed in detail and plotted graphically.

The thin film flow of micropolar fluid in a porous medium under the influence of thermophoresis with the heat effect past a stretching plate is analyzed in [5]. Micropolar fluid is assumed as a base fluid and the plate is considered to move with a linear velocity and subject to the variation of the reference temperature and concentration. The latitude of flow is limited to being two-dimensional and it is steadily affected by sensitive fluid film size with the effect of thermal radiation. The basic equations of fluid flow are changed through the similarity variables into a set of nonlinear coupled differential equations with physical conditions. The suitable transformations for the energy equation is used and the non-dimensional form of the temperature field are different from the published work. The effects of radiation parameter are shown graphically and discussed.

The modern optical fiber required a double-layer resin coating on the glass fiber to provide protection from signal attenuation and mechanical damage. The most important plastics resin used in the coating of fiber optics are plasticized polyvinyle (PVC), low/high density polyethylene (LDPE/HDPE), nylon, and polysulfone. Due to abounded application, the polymer flow during optical fiber coating in a pressure type coating die has been simulated under non-isothermal conditions in [6]. The flow dependent on the wire or fiber velocity, geometry of the die, and the viscosity of the polymer. The wet-on-wet coating process is an efficient process for two-layer coating on the fiber optics. In the present study, the constitutive equation of polymer flow satisfies viscoelastic Phan–Thien–Tanner (PTT) fluid, is used to characterize rheology of the polymer melt. Based on the assumption of the fully developed incompressible and laminar flow, the viscoelastic fluid model of two-immiscible resins-layers modeled for simplified-geometry of capillary-annulus where the glass fiber drawing inside the die at high speed. The equation describing the flow of the polymer melt inside the die was solved, analytically and numerically, by the Runge–Kutta method. The effect of physical characteristics in the problem has been discussed in detail through graphs by assigning numerical values for several parameters of interest. As a first attempt, the model PTT fluid as a coating material for double-layer optical fiber coating using the wet-on-wet coating process is considered. At the end, the present study is compared with the published work as a particular case, and good agreement is found.

The main objective of [7] is to elaborate the characteristics of heat transport and magneto-hydrodynamics finite film flow of human blood with Carbon Nanotubes (CNTs) nanofluids over a stretchable upright cylinder. Two kinds of CNTs nanoparticles, namely (i) SWCNTs (single walled carbon nanotubes) and (ii) MWCNTs (multi walled carbon nanotubes), are used with human blood as a base liquid. In addition, a uniform magnetic field has been perpendicularly conducted to the motion of nanoliquid. The transformation of the partial differential structure into a non-linear ordinary differential structure is made by using appropriate dimensionless quantities. The controlling approach of the Homotopy analysis method has been executed for the result of the velocity and temperature. The thickness of the coating film has been kept variable. The pressure distribution under the variable thickness of the liquid film has been calculated. The impacts of different variables and the rate of spray during coating have been graphically plotted. The coefficient of skin friction and Nusselt number have been presented numerically. In addition, it is noticed that the thermal field of a nanoliquid elevates with rising values of φ and this increase is more in SWCNTs nanofluid than MWCNTs nanofluid.

Lu et al. proposed the unsteady flow and heat transfer analyses of a viscous-based nanofluid over a moving surface emerging from a moving slot [8]. A new form of boundary layer flow resembles with the boundary layer flow over a stretching/shrinking surface depending on the motion of the moving slot. The governing partial differential equations are transformed to correct similar form using the Blasius–Rayleigh–Stokes variable. The transformed equations are numerically solved. The existence of dual solutions is observed for a certain range of moving slot parameter. The range of dual solution is strongly influenced by Brownian and thermophoretic diffusion of nanoparticles.

In [9], the three-dimensional nanofluid thin-film flow of Casson fluid over an inclined steady rotating plane is examined. A thermal radiated nanofluid thin film flow is considered with suction/injection effects. With the help of similarity variables, the partial differential equations are converted into a system of ordinary differential equations. The obtained ODEs are solved by the homotopy analysis method with the association of MATHEMATICA software. The boundary-layer over an inclined steady rotating plane is plotted and explored in detail for the velocity, temperature, and concentration profiles. Additionally, the surface rate of heat transfer and shear stress are described in detail. The impact of numerous embedded parameters, such as the Schmidt number, Brownian motion parameter, thermophoretic parameter, and Casson parameter, etc., were examined on the velocity, temperature, and concentration profiles, respectively. The essential terms of the Nusselt number and Sherwood number were also numerically and physically examined for the temperature and concentration profiles. It was observed that the radiation source improves the energy transport to enhance the flow motion. The smaller values of the Prandtl number, augmented the thermal boundary-layer and decreased the flow field. The increasing values of the rotation parameter decreased the thermal boundary layer thickness. These outputs are examined physically and numerically and are also discussed.

This study of [10] aims to scrutinize the thin film flow of a nanofluid comprising of carbon nanotubes (CNTs), single and multi-walled i.e., (SWCNTs and MWCNTs), with Cattaneo-Christov heat flux and entropy generation. The time-dependent flow is supported by thermal radiation, variable source/sink, and magneto hydrodynamics past a linearly stretched surface. The obtained system of equations is addressed by the numerical approach bvp4c of the MATLAB software. The presented results are validated by comparing them to an already conducted study and an excellent synchronization in both results is achieved. The repercussions of the arising parameters on the involved profiles are portrayed via graphical illustrations and numerically erected tables. It is seen that the axial velocity decreases as the value of film thickness parameter increases. It is further noticed that, for both types of CNTs, the velocity and temperature distributions increase as the solid volume fraction escalates.

The study [11] is about the pressure-driven heated bi-phase flow in two slippery walls. The non-Newtonian couple stress fluid is suspended with spherically homogenous metallic particles. The magnetic susceptibility of Hafnium allures is taken into account. The rough surface of the wall is tackled by lubrication effects. The nonlinear coupled partial differential equations along with the associated boundary conditions are first reduced into a set of ordinary differential equations using appropriate transformations and then numerical results were obtained by engaging the blend of Runge–Kutta and shooting techniques. The sway of physical quantities is graphically examined. Excellent agreement within graphical illustration and numerical results is achieved.

The aim of this research work [12] is to increase our understanding of the exhaustion of energy in engineering and industrial fields. The study of nanofluids provides extraordinary thermal conductivity and an increased heat transmission coefficient as compared to conventional fluids. These specific sorts of nanofluids are important for the succeeding generation of flow and heat transfer fluids. Therefore, the investigation of revolutionary new nanofluids has been taken up by researchers and engineers all over the world. In this article, the study of the thin layer flow of Darcy–Forchheimer nanofluid over a nonlinear radially extending disc is presented. The disc is considered as porous. The impacts of thermal radiation, magnetic field, and heat source/sink are especially focused on. The magnetic field, positive integer, porosity parameter, coefficient of inertia, and fluid layer thickness reduce the velocity profile. The Prandtl number and fluid layer thickness reduce the temperature profile. The heat source/sink, Eckert number, and thermal radiation increase the temperature profile. The suggested model is solved analytically by the homotopy analysis method. The analytical and numerical techniques are compared through graphs and tables, and they have shown good agreement. The influences of embedded parameters on the flow problem are revealed through graphs and tables.

In the analysis [13], peristaltic flow was discussed for magnetohydrodynamic Newtonian fluid through the gap between two coaxial tubes, where the viscosity of the fluid is treated as variable. In addition, the inner tube was considered to be at rest, while the outer tube had the sinusoidal wave traveling down its motion. Further, the assumptions of long wave length and low Reynolds number were taken into account for the formulation of the problem. A closed form solution is presented for general viscosity while using the Adomian decomposition method. Numerical illustrations that show the physical effects and pertinent features were investigated for different physical included phenomenon. It was found that the pressure rise increases with an increase in Hartmann number, and frictional forces for the outer and inner tube decrease with an increase in Hartmann number when the viscosity is constant. It was also observed that the size of the trapping bolus decreases with an increase in Hartmann number, and it increases with an increase in amplitude ratio when the viscosity is parameter.

The magnetohydrodynamic flow of a micropolar nanofluid on an exponential sheet in an Extended–Darcy–Forchheimer porous medium have been considered by Lund et al. [14]. The Buongiorno's model is considered in order to formulate a mathematical model with different boundary conditions. The governing partial differential equations of the nanofluid flow are changed into a third order non-linear quasi-ordinary differential equation, using the pseudo-similarity variable. The resulting equation of the boundary value problems are renewed into initial value problems using a shooting method, and then the initial value problems are solved by a fourth order Runge–Kutta method. The effects of various physical parameters on the profiles of velocity, temperature, microrotation velocity, concentration, skin friction, couple stress coefficients, heat, and concentration transfer are graphically demonstrated. A stability analysis has been performed in order to show the stability of the solutions; only the first solution is stable and physically possible, whereas the remaining two solutions are not stable.

The magnetohydrodynamic flow over a shrinking sheet and heat transfer with viscous dissipation has been studied in [15]. The governing equations of the considered problem are transformed into ordinary differential equations while using similarity transformation. The resultant equations are converted into a system of fractional differential boundary layer equations by employing a Caputo derivative, which is then numerically solved using the Adams-type predictor-corrector method. The results show the existence of two ranges of solutions, namely, dual solutions and no solution. Moreover, the results indicate that dual solutions exist for a certain range of specific parameters which are in line with the results of some previously published work. It is also observed that the velocity boundary layer decreases as the suction and magnetic parameters increase.

The aim of [16] is to present an analytical and numerical treatment of a two-dimensional peristaltic channel along with the coating of laminar layers of nanoparticles with non-Newtonian (Williamson) base liquid. In addition to this, convective heat transfer and magnetic field effects also take into consideration. The geometry is considered as an asymmetric two-dimensional channel experiencing sinusoidal waves propagating across the walls. The walls are supposed to have heat convection at the upper wall and the lower wall is having no temperature gradient. The problem is manufactured under the theory of lubrication approach. The mathematical models are evolved using appropriate transformations. The obtained nonlinear differential equations are solved analytically. Graphical features are presented to find the influence of emerging physical parameters on the stream function, velocity of the nanofluid, heat transfer, nanoparticles concentration, pressure gradient, and pressure increase. It is found that the velocity decreases in the lower part while increasing in the upper side of the channel in the presence of nanoparticles. The temperature is becoming large with increasing amount of nanoparticles and heat convection at the boundaries. It is also observed that nanoparticle concentration is getting higher with Brownian motion parameter, but fluid becomes less thermal against the thermophoresis parameter. The streamlines phenomenon clearly reflects the asymmetry of the channel. The characteristics of viscous fluid can be recovered by switching the Weissenbureg number) to zero.

The impact of second-order slip with thermal and solutal stratification coatings on three-dimensional Williamson nanofluid flow past a bidirectional stretched surface and analytically envisages it by Ramzan et al. [17]. The novelty of the analysis is strengthened by Cattaneo–Christov

heat flux accompanying varying thermal conductivity. The appropriate set of transformations is implemented to get a differential equation system with high nonlinearity. The structure is addressed via the homotopy analysis technique. The authenticity of the presented model is verified by creating a comparison with the limited published results and finding harmony between the two. The impacts of miscellaneous arising parameters are deliberated through graphical structures. Some useful tabulated values of arising parameters versus physical quantities are also discussed here. It is observed that velocity components exhibit an opposite trend with respect to the stretching ratio parameter. Moreover, the Brownian motion parameter shows the opposite behavior versus temperature and concentration distributions.

The peristaltic flow of velocity second slip boundary conditions and inclined magnetic field of Jeffrey fluid by means of heat and mass transfer in asymmetric channel was inspected by Saleem et al. [18]. Leading equations that described the existing flow were then simplified under lubrication approach. Therefore, exact solutions of stream function, concentration and temperature were deduced. Further, the numerical solutions of pressure rise and pressure gradient were computed while using Mathematica software. Furthermore, the effect of the second slip parameter was argued via graphs. It has been depicted that this kind of slip is mandatory and very imperative to foresee the physical model. On the other hand, false results will be obtained.

The importance of Hall current coatings in the establishment of Cattaneo–Christov heat flux model in an unsteady aqueous-based nanofluid flow comprising single (SWCNTs) and multi-walled (MWCNTs) carbon nanotubes (CNTs) amid two parallel rotating stretchable disks in reported in [19]. The novelty of the presented model is strengthened with the presence of homogeneous-heterogeneous reactions and thermal stratification effects. The numerical solution of the system of coupled differential equations with high nonlinearity is obtained by applying the bvp4c function of MATLAB software. To corroborate the authenticity of the present envisioned mathematical model, a comparison table is added to this study in limiting case. An excellent harmony between the two results is obtained. The effects of numerous parameters on involved distributions are displayed graphically and are argued logically in the light of physical laws. Numerical values of coefficient of drag force and Nusselt number are also tabulated for different parameters. It is observed that tangential velocity (function of rotation parameter) is increasing for both CNTs. Further, the incremental values of thermal stratification parameter cause the decrease in fluid temperature parameter.

Alghmdi et al. [20] explored the magnetohydrodynamic stretched flow of viscoelastic nanofluids with heterogeneous–homogeneous reactions. Attention in modeling has been specially focused to constitutive relations of viscoelastic fluids. Thermophoresis and Brownian dispersion are utilized to explore the heat and mass transport process. Resulting nonlinear systems are computed for numerical solutions. Findings for temperature, concentration, concentration rate, skin-friction, local Nusselt, and Sherwood numbers are analyzed for both second grade and elastico-viscous fluids.

The role of mixed convective 3D nanoliquid flow by a rotating disk with activation energy and magnetic field is explored in [21]. Flow was created by a rotating disk. Velocity, concentration, and temperature slips at the surface of a rotating disk were considered. The impacts of Brownian diffusion and thermophoretic were additionally accounted for. The non-linear frameworks are simplified by suitable variables. The shooting method is utilized to develop the numerical solution of resulting problem. Plots were prepared just to explore that how concentration and temperature are impacted by different pertinent flow parameters. The Sherwood and Nusselt numbers were additionally plotted and explored. Furthermore, the concentration and temperature were enhanced for larger values of Hartman number. However, the heat transfer rate (Nusselt number) diminishes when the thermophoresis parameter enlarges.

A liquid coating of bubbly flow with peristaltic motion inside elastic walls was investigated by Ijaz et al. [22]. The proposed model is constructed using the two-fluid approach with the most distinctive collaboration among gas, fluid, pressure, and drag forces. The variation in pressure leads to a change in void fraction. The differential controlling conditions affected by the long wavelength of the peristaltic wave and the slow movement are taken into account. Analytical results of the simplified governing equations are obtained using the homotopy perturbation method (HPM). The features of the significant parameters are shown and examined graphically.

As nanofluids have great potential to enhance thermophysical properties and heat transfer performance. Also double diffusion convection plays an important role in natural processes and technical applications. The effect of double convection by diffusion is not limited to oceanography, but is also evident in geology, astrophysics, and metallurgy. For such a vital role of such factors in applications, Alolaiyan et al. [23] have presented the analytical solutions of pumping flow of third-grade nanofluid and described the effects of double diffusion convection through a compliant curved channel. The model used for the third-grade nanofluid includes the presence of Brownian motion and thermophoresis. Additionally, thermal energy expressions suggest regular diffusion and cross-diffusion terms. The governing equations have been constructed for the incompressible laminar flow of the non-Newtonian nanofluid along with the assumption of long wavelength. The obtained analytical expressions for velocity, temperature, and nanoparticle concentration have been sketched for various considerable parameters. The effects of regular buoyancy ratio, buoyancy parameter, modified Dufour parameter, and Dufour-solutal Lewis number have been analyzed along with wall properties and pumping characteristics. This study concludes that fluid becomes hotter with an increase in regular buoyancy ratio and a modified Dufour parameter, but a decrease in temperature is observed for the buoyancy parameter. Moreover, the solutal concentration is behaving inversely against the Defour–Solutal Lewis number.

A mathematical illustration of an application to endoscopy by incorporating hybrid nanoparticles and an induced magnetic field with a rheological fluid model in inspected by Awais et al. [24] for more realistic results. Rheological fluid behavior is characterized by the Ostwald-de-Waele power-law model. A hybrid nanofluid mechanism is considered comprising platelet-shaped nanoparticles, since nanoparticles are potential drug transportation tools in biomedical applications. Moreover, ciliary activity is encountered regarding their extensive applications in performing complex functions along with buoyancy effects. An endoscope is inserted inside a ciliated tube and peristalsis occurred due to ciliary activity in the gap between tube and endoscope. A non-Newtonian model is developed by mathematical formulation, which is analytically tackled using homotopy analysis. The outcomes are interpreted graphically along with the pressure rise and streamlining configuration for the case of negligible inertial forces and long wavelength. A three-dimensional graphical interpretation of axial velocity is also studied. Moreover, tables are prepared and displayed for a more physical insight.

In [25], the authors developed the mathematical model for entropy generation analysis during the peristaltic propulsion of Jeffrey nanofluids passing in a midst of two eccentric asymmetric annuli. The model was structured by implementation of lubrication perspective and dimensionless strategy. Entropy generation caused by the irreversible influence of heat and mass transfer of nanofluid and viscous dissipation of the considered liquid was taken into consideration. A powerful analytical technique handled the governing equations. The comparison of total entropy with the partial entropy was also invoked by discussing Bejan number results. The influence of various associated variables on the profiles of velocity, temperature, nanoparticle concentration, entropy generation, and Bejan number was formulated by portraying the figures. Mainly from graphical observations, they analyzed that, in the matter of thermophoresis parameter and Brownian motion parameter, entropy generation is thoroughly enhanced while inverse readings were reported for the temperature difference parameter and the ratio of temperature to concentration parameters.

Top coating is usually moulded, painted, or sprayed onto the wind blade Leading-Edge surface to prevent rain erosion due to transverse repeated droplet impacts. Wear fatigue failure analysis based on Springer model has been widely referenced and validated to quantitatively predict damage initiation. The model requires liquid, coating, and substrate speed of sound measurements as constant input parameters to analytically define the shockwave progression due to their relative vibro-acoustic properties. The modelling assumes a pure elastic material behavior during the impact event. Recent coating technologies applied to prevent erosion are based on viscoelastic materials and develop high-rate transient pressure build-up and a subsequent relaxation in a range of strain rates. In order to analyze the erosion performance by using Springer model, appropriate impedance characterization for such viscoelastic materials is then required and it represents the main objective of this work to avoid lack of accuracy. In [26], the authors have proposed a modelling methodology that allows one to evaluate the frequency dependent strain-stress behavior of the multilayer coating system under single droplet impingement. The computational tool ponders the operational conditions (impact velocity, droplet size, layer thickness, etc.) with the appropriate variable working frequency range for the speed of sound measurements. Moreover, this research defines in a complementary paper, the ultrasonic testing characterization of different viscoelastic coatings and the methodology validation. The modelling framework is then used to identify suitable coating and substrate combinations due to their acoustic matching optimization and analyze the anti-erosion performance of the coating protection system.

Under droplet impingement, surface leading edge protection (LEP) coating materials for wind turbine blades develop high-rate transient pressure build-up and subsequent relaxation in a range of strain rates. The stress-strain coating LEP behavior at a working frequency range depends on the specific LEP and on the material and operational conditions, as described in this research in a previous work. Wear fatigue failure analysis, based on the Springer model, requires coating and substrate speed of sound measurements as constant input material parameters. It considers a linear elastic response of the polymer subjected to drop impact loads, but it does not account for the frequency dependent viscoelastic effects for the materials involved. The model has been widely used and validated in the literature for different liquid impact erosion problems. In [27], an appropriate definition of the viscoelastic materials properties with ultrasonic techniques is investigated. It is broadly used for developing precise measurements of the speed of sound in thin coatings and laminates. It also allows accurately evaluating elastic moduli and assessing mechanical properties at the high frequencies of interest. In the current work, an investigation into various LEP coating application cases have been undertaken and related with the rain erosion durability factors due to suitable material impedance definition. The proposed numerical procedures to predict wear surface erosion have been evaluated in comparison with the rain erosion testing in order to identify suitable coating and composite substrate combinations. LEP erosion performance at rain erosion testing (RET) technique is used widely in the wind industry as the key metric, in an effort to assess the response of the varying material and operational parameters involved.


**About the Special Issue Editors**

**Teen-Hang Meen**, Dr., was born in Tainan, Taiwan on August 1, 1967. He received his BS degree from Department of Electrical Engineering, National Cheng Kung University (NCKU), Tainan, Taiwan in 1989, his MS degree and PhD from Institute of Electrical Engineering, National Sun Yat-Sen University (NSYSU), Kaohsiung, Taiwan in 1991 and 1994, respectively. He was Chairman of the Department of Electronic Engineering from 2005 to 2011 at National Formosa University, Yunlin, Taiwan. He received prestigious research awards from National Formosa University in both 2008 and 2014. Currently, he is a distinguished professor at the Department of Electronic Engineering, National Formosa University, Yunlin, Taiwan. He is also the president of the International Institute of Knowledge Innovation and Invention (IIKII) and the Chair of the IEEE Tainan Section Sensors Council. He has published more than 100 SCI, SSCI and EI papers in recent years.

**Charles Tijus**, Dr., has worked on visual perception for two years, with Adam Reeves (Vision Lab, Northeastern University, Boston, USA) as a research assistant. Charles Tijus is the Director of the Cognitions Humaine et Artificielle Laboratory, founded with M. Bui and F. Jouen, the CHArt, a cognitive science laboratory (problem solving, understanding, robotics and cognitive ergonomics), and "Laboratoire des Usages des Techniques d'Information Numeriques", with D. Boullier; a ´ new cognitive ergonomics living LAB laboratory, (LUTIN), which is a "USERLAB" (something like the Audience Research Facility, Boston), located at the "Cite des Sciences et de l'Industrie", ´ La Villette. LUTIN is a platform for usability observations and experiments. LUTIN owns most of the analytical equipment required for its work. It provides access to various shared equipment, such as eye-tracking systems, evoked potentials systems, physiological recording systems, video recording and analysi. The advantages of LUTIN are participants for observations, technologies for observation and experimentation, cognitive simulation, interface between disciplines, and links with industries. LUTIN has close relationships with hospitals, industries, and professional teams and users. It offers services and advice for the adequate conception and use of information technologies. Charles Tijus develops a contextual categorization-based approach in order to study the cognitive processes of understanding: thinking, reasoning, decision-making and learning in early child development and adults. How people develop abilities and competencies is the major concern when it comes to adaptive behavior. The methods comprise empirical research, eye-tracking, event-related potentials (ERPs): N400, and computer models for cognitive simulations. The current interdisciplinary and collaborative research (cognitive psychology, neuroscience and computer science) is on problem solving and operative language, as well as figurative language understanding, and on cognitive robotics and other smart cognitive technologies. Charles Tijus was one of the 2014 IBM Faculty Award recipients.

**Jih-Fu Tu**, Dr., received his PhD in Computer Science from Preston University, USA. He is also the professor of the Industrial Engineering and Management Department at St. John's University in Taiwan. He was a technology consultant for WanWell Cop. He is interested in the computer architecture of multiprocessor systems and multithreaded processors, discrete event systems (DES), VLSI design, the I/O devices design of a computer, and AIoT. He was also the peer reviewer of The Journal of Supercomputing, Computers and Electrical Engineering, Microsystem Technologies, and the Committee of International Conference, etc. He has published over 20 journal papers, 5 authored books, 7 edited books, and 30 papers in international conference proceedings.


**5. Conclusion**

We discussed in this brief paper the pros and cons of the standard approach mapping functions + gradients to model the neutral delays of the atmosphere, and more specifically the wet delays caused by the presence of water vapor in the troposphere. If this standard approach is almost perfect for people doing positioning, deformation and VLBI studies, as they see the neutral delays as "noise", it is not so well adapted to people looking at these delays as signals to study atmospheric processes. In particular, the standard definition of gradients is too crude, and does not permit to have access to the horizontal turbulence/convection scales, that are key parameters to model these processes in high resolution NWM models. We therefore propose an alternative way to model the wet tropospheric delays, through a representation of the wet refractivity field as a perturbation over an exponential decay with altitude with a locally adjusted scale height and a time/space series expansion over a suitable basis of orthogonal functions. Our approach is computationally expensive, and maybe not suited for real-time applications, but its

*Beyond Mapping Functions and Gradients DOI: http://dx.doi.org/10.5772/intechopen.96982*

end-product are records of the total and wet refractivity values with high-resolution in time (minute-scale) and distance (sub km-scale), in accordance with the needs of future numerical weather models [38], the emerging field of the modeling of atmospheric rivers [100, 101] and besides does not require the additional step of water vapor tomography, with lower cost, better mobility and simpler operation [102].


**Advances in Remote Sensing-Based Disaster Monitoring and Assessment**

Printed Edition of the Special Issue Published in *Remote Sensing* Jungho Im, Haemi Park and Wataru Takeuchi Edited by

www.mdpi.com/journal/remotesensing


*Article* **Snail Overexpression Alters the microRNA Content of Extracellular Vesicles Released from HT29 Colorectal Cancer Cells and Activates Pro-Inflammatory State In Vivo**

**Izabela Papiewska-Paj ˛ak 1,\*, Patrycja Przygodzka 1 , Damian Krzyzanowski ˙ 1 , Kamila Soboska 1,2 , Izabela Szulc-Kiełbik 1 , Olga Stasikowska-Kanicka 3 , Joanna Boncela 1 , Małgorzata W ˛agrowska-Danilewicz 3 and M. Anna Kowalska 1,4,\***

**Simple Summary:** Knowledge of the factors that help migration of carcinoma cells is important for prevention of metastasis. Cancer cells release small particles, extracellular vesicles (EVs) that contain such factors. The aim of this study was to assess if the content of EVs changes through different stages of colorectal cancer (CRC) and evaluate how this process affects cancer progression in vivo in mouse CRC model. We found that EVs released from cells that have migratory properties contain different factors then EVs released from original tumor cells. We also show here that EVs can be incorporated into other cells that facilitate metastasis and change their properties depending on the EVs content. The content of cell-released EVs may also serve as a biomarker that denotes the stage of CRC and may be a target to prevent cancer progression.

**Abstract:** During metastasis, cancer cells undergo phenotype changes in the epithelial-mesenchymal transition (EMT) process. Extracellular vesicles (EVs) released by cancer cells are the mediators of intercellular communication and play a role in metastatic process. Knowledge of factors that influence the modifications of the pre-metastatic niche for the migrating carcinoma cells is important for prevention of metastasis. We focus here on how cancer progression is affected by EVs released from either epithelial-like HT29-cells or from cells that are in early EMT stage triggered by Snail transcription factor (HT29-Snail). We found that EVs released from HT29-Snail, as compared to HT29-pcDNA cells, have a different microRNA profile. We observed the presence of interstitial pneumonias in the lungs of mice injected with HT29-Snail cells and the percent of mice with lung inflammation was higher after injection of HT29-Snail-EVs. Incorporation of EVs released from HT29 pcDNA, but not released from HT29-Snail, leads to the increased secretion of IL-8 from macrophages. We conclude that Snail modifications of CRC cells towards more invasive phenotype also alter the microRNA cargo of released EVs. The content of cell-released EVs may serve as a biomarker that denotes the stage of CRC and EVs-specific microRNAs may be a target to prevent cancer progression.

**Keywords:** Snail transcription factor; extracellular vesicles; colon cancer; pre-metastatic niche

#### **1. Introduction**

Colorectal cancer (CRC) is the third most common cancer in men and second in women worldwide. It is predicted that the number of deaths will rise to 1.1 million per year worldwide by 2030 [1]. Formation of distant metastases are the primary cause of CRC

**Citation:** Papiewska-Paj ˛ak, I.; Przygodzka, P.; Krzyzanowski, D.; ˙ Soboska, K.; Szulc-Kiełbik, I.; Stasikowska-Kanicka, O.; Boncela, J.; W ˛agrowska-Danilewicz, M.; Kowalska, M.A. Snail Overexpression Alters the microRNA Content of Extracellular Vesicles Released from HT29 Colorectal Cancer Cells and Activates Pro-Inflammatory State In Vivo. *Cancers* **2021**, *13*, 172. https:// doi.org/10.3390/cancers13020172

Received: 9 October 2020 Accepted: 29 December 2020 Published: 6 January 2021

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**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/).

treatment failure and patient death [2]. In the metastasis process, to enter the circulation, cancer cells lose their epithelial features, exhibit decreased polarity and intercellular adhesion, undergo cytoskeletal reorganization and become more motile. This phenotypic transformation of epithelial carcinoma into mesenchymal-like cells (EMT) is triggered by several factors including Snail transcription factor that plays crucial role at the beginning of the EMT process [3].

Crucial for cancer progression is also the intercellular cross-talk and subsequent regulation of both local and distant microenvironments. Kaplan et al. [4] introduced the concept termed "pre-metastatic niche" that is defined as the microenvironment that facilitates the formation of metastasis and consists of immune and stromal cells and also the components of the primary tumour. A number of niche-promoting molecules has been identified in various metastasis mouse models [5]. Monocytes and macrophages associated with tumour environment have been widely recognized as immunological effectors [5]. Macrophages display functional plasticity in response to local microenvironment stimuli and participate in cancer-related inflammation, matrix remodelling, immune escape and ultimately in cancer metastasis. Additionally, cancer cell-derived extracellular vesicles (EVs) are recognized as significant contributors to different aspects of modifications of pre-metastatic niches [6].

EVs are defined as a heterogeneous group of a phospholipid bilayer particles that are released to the extracellular environment by most cells in the organism and are present in all body fluids [7]. EVs can be divided into main three subpopulations including exosomes (exo), microvesicles/ectosomes (MVs) and apoptotic bodies (APOs) that differ in size, formation process and content [8]. EVs contain a variety of bioactive molecules, including proteins, lipids and multiple nucleic acid species, including the most extensively studied class of microRNA (miRNA/miR) [6,9]. The cargo of EVs may either reflect the cell of origin or can be actively sorted [10,11]. Recent reviews summarize the importance of EVs as communicators between cells that accelerate cancer progression and metastasis [12,13]. EVs can stimulate angiogenesis, matrix remodelling and modulation of immune response [9,14–16]. Factors, including cytokines, chemokines and EVs that are released from cancer or other cells that are linked to inflammation, influence the environment of pre-metastatic niches in distant organs [17].

We have previously shown that Snail, as an early regulator of EMT, affects the transcriptome and miRNA profile of human HT29 CRC cells and changes HT29 cells phenotype to pro-migratory [18,19]. Here we show that HT29 cells overexpressing Snail, in comparison to epithelial-like HT29 cells, release EVs with different miRNA content and postulate that HT29-Snail-EVs modify the activity of cells constituting the pre-metastatic niche, thus facilitating cancer progression.

#### **2. Results**

## *2.1. Characterization of Extracellular Vesicles (EVs) Released from Control HT29 and HT29-Snail Cells*

#### 2.1.1. Size and Purity

The preparation of EVs free of cellular debris, FBS-derived vesicles or any non-EVs RNAs and proteins is critical for any analyses. We applied a commonly used procedure for isolation of EVs that we used in a previous study [18,19]. Cells release vesicles that according to current nomenclature can be classified as exosomes (exo, 30–120 nm), microvesicles (MVs, 0.1–1.0 µm) and apoptotic bodies (0.8–5.0 µm) [20]. We purified EVs that are the mixture of exo and MVs. EVs sizes were similar for all HT29 clones with sizes of 80 to 240 nm (Figure 1A).

**Figure 1.** Characterization of extracellular vesicles (EVs) released by HT29 clones. EVs were isolated from conditioned media after 24 h of incubation in FBS-free culture media of HT29 clones. (**A**) NTA showed similar numbers and sizes of EVs isolated from HT29 clones. (**B**) Representative Electron microscopic image of EVs derived from HT29-cDNA and HT29-Snail 17. Scale bar, 100 nm. **Figure 1.** Characterization of extracellular vesicles (EVs) released by HT29 clones. EVs were isolated from conditioned media after 24 h of incubation in FBS-free culture media of HT29 clones. (**A**) NTA showed similar numbers and sizes of EVs isolated from HT29 clones. (**B**) Representative Electron microscopic image of EVs derived from HT29-cDNA and HT29-Snail 17. Scale bar, 100 nm.

Similar vesicles number (~5.2 × 1013) for each preparation of EVs were isolated from the same number of cells (~6.8 × 108). The spheroid morphology of EVs as well as their size were confirmed by TEM (Figure 1B). EVs were enriched by markers such as CD63, CD9 and flotillin-1 as compared to lysates of cells of origin (Figure S1). They also contained 70 kDa heat-shock protein (HSP70) and were Annexin V positive; the purity of EVs was confirmed by the absence of cytochrome c and GM130 (Figure S1). Similar vesicles number (~5.2 × 1013) for each preparation of EVs were isolated from the same number of cells (~6.8 × 108 ). The spheroid morphology of EVs as well as their size were confirmed by TEM (Figure 1B). EVs were enriched by markers such as CD63, CD9 and flotillin-1 as compared to lysates of cells of origin (Figure S1). They also contained 70 kDa heat-shock protein (HSP70) and were Annexin V positive; the purity of EVs was confirmed by the absence of cytochrome c and GM130 (Figure S1).

#### 2.1.2. miRNA Content 2.1.2. miRNA Content

The miRNA profiling was performed by Next Generation Sequencing (NGS) analysis of the total mRNA isolated from EVs. There were no significant differences in the number of detectable miRNAs between EVs of control and either of HT29-Snail clones (Figure S2). Further, the two-way hierarchical clustering of miRNA was performed (Figure 2A). As presented in Figure 2B,C, we identified 23 miRNAs differentially expressed in EVs re-The miRNA profiling was performed by Next Generation Sequencing (NGS) analysis of the total mRNA isolated from EVs. There were no significant differences in the number of detectable miRNAs between EVs of control and either of HT29-Snail clones (Figure S2). Further, the two-way hierarchical clustering of miRNA was performed (Figure 2A). As presented in Figure 2B,C, we identified 23 miRNAs differentially expressed in EVs released from HT29-Snail 3, 30 miRNAs from HT29-Snail 8, and 48 miRNAs from HT29-Snail 17.

leased from HT29-Snail 3, 30 miRNAs from HT29-Snail 8, and 48 miRNAs from HT29- Snail 17. Three miRNAs: let-7i, miR-205, and miR-130b were up-regulated while five miRNAs: miR-1246, miR-3131, miR-375, miR-552-3p and miR-552-5p were down-regulated in HT29- Snail-derived EVs (Figure 2C). Additionally, as it is shown in Table S1, among other miRNAs altered more than two times in EVs released from HT29-Snail clones, miR-483-5p (HT29-Snail 17-EVs) was upregulated and miR-142-5p (HT29-Snail 8, -17-EVs) as well as miR-203a and miR-203b-3p (HT29-Snail 17-EVs) were downregulated. Moreover, as shown in Table S2, there were also miRNAs upregulated in EVs released from HT29-Snail cells with a lower fold change. Those differentially expressed miRNAs (marked in red in Tables S1 and S2) were reported to be important in cancer progression as pointed out in the Discussion section.

**Figure 2.** Changes in the expression of miRNAs in EVs released by HT29-Snail cells as compared to EVs from HT29-pcDNA cells. (**A**) Heatmap and unsupervised hierarchical clustering by sample and miRNA. (**B**) Number of differentially expressed miRNAs detected by NGS analysis, that were either significantly upregulated (red) or downregulated (green) in EVs derived from HT29-Snail-3, -8 and -17 versus EVs from control cells. (**C**) Venn diagrams show differentially expressed miR-NAs. The most regulated miRNAs in all three or two clone-EVs are marked. (corrected FDR *p* < 0.05 and fold change > 2). miRs marked in red are discussed in the text. **Figure 2.** Changes in the expression of miRNAs in EVs released by HT29-Snail cells as compared to EVs from HT29-pcDNA cells. (**A**) Heatmap and unsupervised hierarchical clustering by sample and miRNA. (**B**) Number of differentially expressed miRNAs detected by NGS analysis, that were either significantly upregulated (red) or downregulated (green) in EVs derived from HT29-Snail-3, -8 and -17 versus EVs from control cells. (**C**) Venn diagrams show differentially expressed miRNAs. The most regulated miRNAs in all three or two clone-EVs are marked. (corrected FDR *p* < 0.05 and fold change > 2). miRs marked in red are discussed in the text.

#### Three miRNAs: let-7i, miR-205, and miR-130b were up-regulated while five miRNAs: 2.1.3. Gene Ontology (GO) Enrichment Analysis

miR-1246, miR-3131, miR-375, miR-552-3p and miR-552-5p were down-regulated in HT29-Snail-derived EVs (Figure 2C). Additionally, as it is shown in Table S1, among other miRNAs altered more than two times in EVs released from HT29-Snail clones, miR-483- 5p (HT29-Snail 17-EVs) was upregulated and miR-142-5p (HT29-Snail 8, -17-EVs) as well as miR-203a and miR-203b-3p (HT29-Snail 17-EVs) were downregulated. Moreover, as shown in Table S2, there were also miRNAs upregulated in EVs released from HT29-Snail cells with a lower fold change. Those differentially expressed miRNAs (marked in red in To identify the biological processes that might be triggered in the response to elevated Snail levels in the HT29 clones that are shedding EVs, Gene Ontology (GO) enrichment analysis was performed to identify GO terms that are significantly associated with differentially expressed microRNAs in EVs released by the clones. Positive regulation of thyrosine phosphorylation of STAT-3 and -1 was at the top of Biological Process in the GO analysis (Figure S3).

#### Tables S1 and S2) were reported to be important in cancer progression as pointed out in *2.2. In Vivo Studies*

the Discussion section. 2.1.3. Gene Ontology (GO) Enrichment Analysis To look for the in vivo effect of Snail on the cancer progression we injected athymic mice subcutaneously (s.c.) with the same number (1.5 × 106/mouse) of control (HT29 pcDNA) and HT29-Snail 17 cells. Clone 17 contains the most elevated Snail expression ([18] and Figure S5), and their EVs have the highest number of differentially expressed miRNA (Figure 2 and Tables S1 and S2). Further, animals of both groups were injected intravenously

(i.v. day 8, 12, 15 and 18) with the same amount (10 µg/mouse) of HT29-pcDNA-EVs, and observed for 28 days. Administration of HT29-pcDNA-EVs did not have statistical impact on neither tumour growth nor plasma MCP-1/CCL2 levels (Figure 3A,C, Table S3) within the groups of mice injected with either HT29-pcDNA cells or HT29-Snail cells. However, there was a ~20% difference in the growth rate of tumour (day 12 to 28) between the control and HT29-Snail mice, regardless of the injection of EVs (Figure 3A). Growth of tumours in mice administered s.c. with HT29-Snail cells was slower in comparison to administration of control cells, which is not surprising since HT29-Snail cells are already in the early EMT stage [18,19]. We have also observed an increase in the concentration of monocyte chemotactic protein-1 (MCP-1/CCL2) at day -7 (Figure 3B) and -28 (Figure 3C) in the plasma of mice that bore a tumour induced by injection of HT29-Snail17 cells as compared to control mice injected with HT29-pcDNA cells. We did not observe any metastatic sites of HT29 cells in the lungs, livers or intestines after 28 days of tumour growth. However, early interstitial pneumonias were observed after 28 days in animals injected with HT29-Snail17 cells as compared to animals injected with HT29-pcDNA (Table 1, group IV and II, respectively). Injection of EVs released from control HT29-pcDNA cells slightly increased the number of mice with lung infection in control group of mice that bore a tumor induced by injection of HT29-pcDNA cells (Table 1, group II and III). Based on the fact that HT29-Snail cells injected s.c. into mice may secrete additional HT29-Snail-EVs, we have additionally injected such EVs into the group of mice bearing tumors induced by HT-29- Snail cells. We did not observe any changes in either tumor growth or MCP-1/CCL2 levels (Table S3). However, in this group (Table 1, group VI) we observed the highest number of animals presenting lung inflammation (80%) As presented on Figure 3D, inflammatory infiltration from lymphocytes and thickened interalveolar septum were observed in lungs of those mice.

**Table 1.** Incidence of lung infection in mice with tumours inflicted by injection of control HT29 pcDNA or HT29-Snail cells followed by injections of various EVs.

**Figure 3.** In vivo mouse studies in animals injected s.c. with HT29-pcDNA- (small dot) or HT29-Snail17- (small cube) cells. (**A**) The growth rate of tumours (day 12–28) in animals with HT29-pcDNA or HT29-Snail administered s.c. and additionally injected i.v. with EVs released by HT29-pcDNA, mean value ± SEM: 0.052 ± 0.04 and 0.039 ± 0.03, respectively (**B**) Levels of monocyte chemoattractant protein-1 (MCP-1/CCL2) measured in the plasma of mice at day 7 (before first injection of EVs). (**C**) Levels of monocyte chemoattractant protein-1 (MCP-1/CCL2) measured in the plasma of mice at day 28 (end of experiment). Mean value ± SEM for animals injected s.c. with HT29-pcDNA and HT29-Snail were 24.8 ± 2.7 and 41.5 ± 4.1, respectively. (**D**) Representative H&E staining of murine lungs. 100× magnification. The images of groups IV and VI (see Table 1) show interstitial pneumonia pictures. *n* = 10–15, \* *p* < 0.02; \*\* *p* < 0.01. **Figure 3.** In vivo mouse studies in animals injected s.c. with HT29-pcDNA- (small dot) or HT29-Snail17- (small cube) cells. (**A**) The growth rate of tumours (day 12–28) in animals with HT29-pcDNA or HT29-Snail administered s.c. and additionally injected i.v. with EVs released by HT29-pcDNA, mean value ± SEM: 0.052 ± 0.04 and 0.039 ± 0.03, respectively (**B**) Levels of monocyte chemoattractant protein-1 (MCP-1/CCL2) measured in the plasma of mice at day 7 (before first injection of EVs). (**C**) Levels of monocyte chemoattractant protein-1 (MCP-1/CCL2) measured in the plasma of mice at day 28 (end of experiment). Mean value ± SEM for animals injected s.c. with HT29-pcDNA and HT29-Snail were 24.8 ± 2.7 and 41.5 ± 4.1, respectively. (**D**) Representative H&E staining of murine lungs. 100× magnification. The images of groups IV and VI (see Table 1) show interstitial pneumonia pictures. *n* = 10–15, \* *p* < 0.02; \*\* *p* < 0.01.

#### **Table 1.** Incidence of lung infection in mice with tumours inflicted by injection of control HT29 pcDNA or HT29-Snail cells followed by injections of various EVs. *2.3. In Vitro Studies*

*2.3. In Vitro Studies*

#### 2.3.1. Uptake of EVs by THP-1 Derived Macrophages (TDM)

**Group Cell Injection Day 0 EV Injection Day 8, 12, 15, 18 Lung Infection Day 28**  I None None 0/5 (0%) II HT29-pcDNA None 0/5 (0%) The uptake of EVs by TDM was visualized by confocal imaging (Figure 4A,B). EVs obtained from either control HT29-pcDNA or HT29-Snail clones -3, -8 and -17 were incorporated into TDM in FBS-free media for 4 h. The viability of TDM cells was not changed by incorporation of any EVs (Figure S4).

III HT29-pcDNA HT29-pcDNA 1/5 (20%)

#### IV HT29-Snail17 None 2/5 (40%) 2.3.2. Effect of EVs Released from HT29 Clones on Macrophage Activity

V HT29-Snail17 HT29-pcDNA 2/5 (40%) VI HT29-Snail17 HT29-Snail17 4/5 (80%) We examined whether the incorporation of EVs released by HT29-Snail cells that represent the intermediate state of EMT, affects the activity of macrophages differently than incorporation of EVs released from epithelial-like HT29 cells (Figure 5).

2.3.1. Uptake of EVs by THP-1 Derived Macrophages (TDM)

by incorporation of any EVs (Figure S4).

The uptake of EVs by TDM was visualized by confocal imaging (Figure 4A,B). EVs obtained from either control HT29-pcDNA or HT29-Snail clones -3, -8 and -17 were incorporated into TDM in FBS-free media for 4 h. The viability of TDM cells was not changed

**Figure 4.** Uptake of EVs derived from Snail-HT29 cells (clone 17) by TDM. HT29-Snail EVs were labeled with PKH67 dye (green). Nuclei were stained with Hoechst (blue), actin was stained with Phalloidin Texas Red (red). (**A**) Representative confocal laser scanning microscope. (**B**) Z-stack analysis of cells and incorporated EVs. Note: Viability of TDM cells was not affected by the incorporation of EVs (Figure S4). **Figure 4.** Uptake of EVs derived from Snail-HT29 cells (clone 17) by TDM. HT29-Snail EVs were labeled with PKH67 dye (green). Nuclei were stained with Hoechst (blue), actin was stained with Phalloidin Texas Red (red). (**A**) Representative confocal laser scanning microscope. (**B**) Z-stack analysis of cells and incorporated EVs. Note: Viability of TDM cells was not affected by the incorporation of EVs (Figure S4). *Cancers* **2021**, *13*, x 8 of 15

**Figure 5.** In vitro effect of EVs on macrophages. (**A**) Motility of TDM cells before (small dot) after the uptake of EVs isolated from control HT29-pcDNA (small cube) or HT29-Snail cells (clone 3- small triangle, clone 8- small upside triangle and clone 17- diamond). (**B**) IL-8 release from TDM after treatment with EVs indicated. (**C**) IL-8 release from macrophages isolated from healthy donors blood (MDM) and incubated with EVs. *n* = 3–7, \* *p* < 0.05; \*\* *p* < 0.01. **Figure 5.** In vitro effect of EVs on macrophages. (**A**) Motility of TDM cells before (small dot) after the uptake of EVs isolated from control HT29-pcDNA (small cube) or HT29-Snail cells (clone 3- small triangle, clone 8- small upside triangle and clone 17- diamond). (**B**) IL-8 release from TDM after treatment with EVs indicated. (**C**) IL-8 release from macrophages isolated from healthy donors blood (MDM) and incubated with EVs. *n* = 3–7, \* *p* < 0.05; \*\* *p* < 0.01.

**3. Discussion**

The motility of TDM was evaluated by measuring the cell speed changes in time.

Our group has recently reported that overexpression of Snail drives HT29 colon cancer cell to a partial-EMT and modulates the expression of specific protein transcripts and miRNAs [18,19,21]. We reasoned that cancer cells at various EMT stages will release extracellular vesicles of different content and thus differently affect recipient cells. Changes in the protein levels in EVs released from cells stimulated by Snail were already observed

However, the motility of TDM cells with incorporated EVs released from HT29-Snail cells was inhibited in comparison to cells containing HT29-pcDNA-EVs. (Figure 5A). We have also quantified the level of cytokines secreted by TDM. We observed a significantly increased level of IL-8 released by TDM (Figure 5B) treated by EVs released from HT29 pcDNA cells, as compared to non-treated TDMs (no EVs). However, incorporation of HT29-Snail-EVs resulted in decreased IL-8 levels (Figure 5B). Next, we performed the analysis of cytokines released by macrophages derived from monocytes isolated from healthy donors and differentiated with GM-CSF (MDM). Consistent with the previous results, MDM cells incubated with EVs released from HT29-Snail clones secreted less IL-

8 as compared to cells incubated with control HT29-pcDNA-EVs (Figure 5C).

The motility of TDM was evaluated by measuring the cell speed changes in time. There was a slight, but not significant, increase in the motility of TDM that had incorporated EVs released from HT29-pcDNA as compared to control TDM cells (Figure 5A). However, the motility of TDM cells with incorporated EVs released from HT29-Snail cells was inhibited in comparison to cells containing HT29-pcDNA-EVs. (Figure 5A). We have also quantified the level of cytokines secreted by TDM. We observed a significantly increased level of IL-8 released by TDM (Figure 5B) treated by EVs released from HT29-pcDNA cells, as compared to non-treated TDMs (no EVs). However, incorporation of HT29-Snail-EVs resulted in decreased IL-8 levels (Figure 5B). Next, we performed the analysis of cytokines released by macrophages derived from monocytes isolated from healthy donors and differentiated with GM-CSF (MDM). Consistent with the previous results, MDM cells incubated with EVs released from HT29-Snail clones secreted less IL-8 as compared to cells incubated with control HT29-pcDNA-EVs (Figure 5C).

#### **3. Discussion**

Our group has recently reported that overexpression of Snail drives HT29 colon cancer cell to a partial-EMT and modulates the expression of specific protein transcripts and miRNAs [18,19,21]. We reasoned that cancer cells at various EMT stages will release extracellular vesicles of different content and thus differently affect recipient cells. Changes in the protein levels in EVs released from cells stimulated by Snail were already observed [22]. As a rule, the cargo loaded into EVs reflects the status of cancer cells [23], but potential preferential packaging into EVs has also been suggested [11]. The most extensively studied class of factors transported between cells by EVs are miRNAs that regulate the translation of target mRNAs in recipient cells [10,24]. We show here that overexpression of Snail in HT29 cells significantly triggers changes on individual miRNAs levels.

We are aware of the fact that our HT29-Snail clones differ in the amount of Snail overexpressed in their cells. In this study, we used several HT29-Snail clones with clone 3 representing the lower and clone 17 the highest Snail level [18]. Thus, each of the clones may reflect a slightly different EMT stage.

In EVs released from all three HT29-Snail clones let-7i, miR-205 and miR-130b-5p miRNAs were highly upregulated. There are conflicting reports concerning the role of miR-205 and let-7i in cancer [25–27]. Whether particular miRNA is considered as tumor suppressor or onco-miRNA appears to be dependent on the specific cancer and tumorenvironment [28,29]. Increased migratory properties of HT29-Snail cells with elevated miR-205 and let-7i expression was shown previously by us, pointing on their role in CRC progression [19]. The mRNA targets for miR-205-5p and let7i-5p were also shown in our previous study. Potentiated miR-205 expression was correlated with Dynamin 3 (DNM3) mRNA decrease. DNM3 is considered as a cancer suppressor. Thus, from the tumour point of view redundant during progression of the disease and decreased in HT29-Snail cells [19].

We observed miR-130b-5p enrichment in EVs released from all our HT29-Snail clones. In contrast to miR-205 and let-7i up-regulation in EVs, which mirrored intracellular changes in miRNAs expression, miR-130b-5p was not up-regulated in HT29-Snail cells [19]. This suggests that miR-130b-5p could be actively sorted into vesicles released from cells that overexpress Snail and thus undergo EMT. MiR-130b was identified in the microvesicles of leukemia K562 cells, but, unlike in CRC cells in this study, at an equal expression level as in cells of origin [30]. In various human tumor types altered miR-130b expression has been implicated as either promoting or suppressing tumorigenesis; miR-130b is significantly downregulated in pituitary adenomas and endometrial cancer [31,32] whereas it is upregulated in bladder cancer, melanoma, metastatic renal carcinoma [33–35]. The contribution of miR-130b to CRC progression is also the subject of the debate. In cell lines SW-480 and SW-620 over-expression of miR-130b downregulates integrin β1, leading to the impaired migration and invasion of CRC cells [36] whereas another analysis of a series of CRC cell lines showed that miR-130b acts as an efficient inducer of EMT in vivo and in vitro, likely

through up-regulation of Snail and ZEB1 transcription factors. The effects of miR-130b on promoting cell migration and invasion of CRC cells with poor prognosis for colorectal cancer was also indicated [37]. Whether the miR-130b that is packed into EVs released from HT29-Snail clones affects positively colorectal cancer progression is not definitely comprehensible from our studies. However, our in vivo studies (Table 1) suggest that the presence of EVs released from CRC cells that undergo EMT and contain packed miR-130b may lead to increased lung inflammation that facilitates cancer progression. This is in agreement with the previously identified effects [37] and suggests that miR-130b may be a target to attempt to slow the CRC progression. Additionally, the presence of miR-130b on EVs released from CRC cells can serve as a biomarker of an advanced stage of CRC.

We found increased amount of miR-483-5p in EVs released from HT29-Snail clone 17 that express the highest amount of Snail and was used in our in vivo studies. Increased content of miR-483-5p was observed in exosomes isolated from plasma of a CRC-patient in various stages and was found in EVs from SW480 CRC line suggesting its diagnostic potential [38]. We have also observed that miR-221, miR-222, and miR-125a were overexpressed in HT29-Snail-EVs, although with lower fold change. MiR-221 is among commonly upregulated miRNAs in CRC in tumors [39] and in patient's serum [40] while miR-222- 3p promotes macrophage polarization and differentiation to M2 phenotype in vitro and in vivo, which enhance the progression of epithelial ovarian cancer [41]. Additionally, the decrease in miRNA-34a and miRNA-203b in EVs released from HT29-Snail confirms the previously described reports about repression of miRNA-34 and miRNA-203 by Snail as a part of the EMT program in cells [42,43] that leads to cancer progression. Thus, our analysis of the differences in individual miRNA expression shows that EVs released from cells in early EMT stage can carry miRNAs that promote cell migration and invasion of CRC cells and are associated with poor prognosis for colorectal cancer. Further, the positive regulation of STAT3 is the most significant GO term for Biological Process (Figure S3). Cytokine-driven JAK/STAT3 pathway plays an important role in the processes of signal transduction and is associated with the hyperproliferative and invasive phenotype of CRC cells [44].

Tumors induced by injection of HT29-Snail, as compared to control HT20-pcDNA cells, tend to have a lower rate of growth (Figure 3A). This observation is in an agreement with the inhibition of cell proliferation within the growing tumor due to the cell EMT progression caused by Snail [18]. At the same time we have observed the increased amount of MCP-1/CCL2 in the plasma of mice bearing the tumours induced by HT29-Snail. These data are in agreement with the earlier findings suggesting that Snail, as EMT inducer, can induce MCP-1/CCL2 production [45]. MCP-1/CCL2 is a potent chemoattractant for circulating blood monocytes via binding to its receptor CCR2 but is not an effective chemoattractant for differentiated monocyte-derived macrophages [46,47]. In parallel with the results of inhibited tumour growth and increased production of MCP-1/CCL2 we have observed the increased number of early interstitial pneumonias in mice injected with HT29-Snail cells, as compared to HT29-pcDNA cells (Table 1, group IV and II). Additionally, almost all mice bearing tumour induced by HT29-Snail cells that were also injected with EVs released from HT29-Snail cells, presented the appearance of early interstitial pneumonias (Table 1, group VI), while injection of HT29-pcDNA-EV had no effect (Table 1, group V and IV, respectively). As HT29-Snail-EVs were injected only into the group that was bearing tumours induced by injection of HT29-Snail cells, we cannot exclude the possibility that HT29-Snail-EVs would have an effect on control (HT29-pcDNA) mice and that remains to be elucidated.

Our in vitro findings (Figure 5) suggest that EVs released from HT29-Snail cells that are in an early EMT stage affect macrophages differently than the EVs released from epithelial-like HT29 cells. We observed the inhibition of random motility of macrophages that were treated with HT29-Snail-EVs cells as compared to control EVs released from HT29 pcDNA cells. Thus, the macrophages that are affected by HT29-Snail-EVs may become retained in pre-metastatic niche. We have also found the differences in the secretion

of IL-8 by macrophages (Figure 5B,C) that may be attributed to the variability in EVs miRNA profiles. We found for example that miR-142-5p is downregulated in EVs released from HT29-Snail 8 and -17, so relatively there is higher amount of miR-142-5p in control EVs. High concentrations of miR-142-5p were observed in ulcerative colitis (UC), the inflammatory disease that frequently leads to development of colorectal cancer. MiR-142-5p levels were negatively correlated with the expression of suppressor of cytokine signalling 1 (SOCS1) in UC patients [48]. Further, miR-142-5p increased the secretion of IL-6 and IL-8 in TNFα-treated-HT29 [48]. Thus, we postulate that downregulation of IL-8 secretion macrophages treated with HT29-Snail-EVs might be associated to some extend with lower levels of miR-142-5p.

#### **4. Materials and Methods**

#### *4.1. Cell Culture and Differentiation*

The HT29 cell line (cells: colon, disease: colorectal adenocarcinoma) was obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in McCoy's 5A medium (LifeTechnologies, Waltham, MA, USA), supplemented with 10% FBS (LifeTechnologies) and antibiotics—streptomycin and penicillin (P/S) (Sigma-Aldrich, St. Louis, MO, USA), primocin (Invivogen, San Diego, CA, USA). The cells were periodically tested for mycoplasma using the PlasmoTest (Invivogen). For isolation of EVs released by HT29, serum-free medium with P/S was used to rule out the effect of exosomes of foetal bovine serum.

THP-1 monocyte/macrophage-like cell line from American Type Culture Collection (Manassas, VA, USA) was cultured in RPMI-1640 culture medium supplemented with 1 mM sodium pyruvate, 10% FBS, 0.05 mM 2-ME, P/S and primocin. For differentiation of THP-1 monocytes into THP-1-derived macrophages (TDM), cells were cultured with 20 ng/mL of phorbol ester (PMA) in culture medium for 48 h. Isolations of monocytes from healthy donors were performed as described previously [49]. To generate monocytederived macrophages (MDM) human monocytes were cultured 7 days with 10 ng/mL of GM-CSF (Thermo Scientific, Waltham, MA, USA) in RPMI-1640 supplemented with 10% human serum type AB from Sigma-Aldrich (St. Louis, MO, USA). All cell cultures were performed in a 90–95% humidified atmosphere of 5% CO2.

#### *4.2. HT29 Stable Clone Generation and Isolation of EVs from Culture Supernatants*

The pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) and pcDNA3.1 vector expressing Snail was obtained from Prof. Muh-Hwa Yang (Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan). HT29 nucleofection and clone generation was performed as previously described [18]. Western blot showing the levels of Snail in clones -3, -8, and -17 is shown in Supplemental Figure S5. The HT29 cell line and HT29-pcDNA control clone were authenticated by ATCC using Short Tandem Repeat (STR) analysis. Extracellular vesicles were isolated by differential centrifugations and subsequent ultra-centrifugations as described previously [19]. HT29-Snail 3, 8, 17 and HT29-pcDNA clones were grown to 70–80% confluence on 15-cm dishes, washed three times with empty medium to remove vesicles present in FBS of culture medium and then cultured for 24 h to obtain conditioned medium. Next the medium was collected and centrifuged (350× *g* for 10 min) to remove floating cells. The supernatants were then collected and centrifuged at 2000× *g* for 20 min to remove APOs. Finally, EVs pellets were obtained after ultra-centrifugation for 1.5 h at 100,000× *g* using OPTIMA L-80 Ultracentrifuge and Type 45 Ti Rotor, Fixed Angle (Beckman Coulter, Inc., Brea, CA, USA). The pellets were next washed by diluting in PBS and centrifuging for 1.5 h at 100,000× *g*. All centrifugations were performed at 4 ◦C. Finally, EVs pellets were resuspend in PBS for Western blots or in appropriate media for functional experiments.

#### *4.3. Nanoparticle Tracking Analysis (NTA)*

EV size distribution and quantification of vesicles were analyzed by NTA using a NanoSight NS300 System (Malvern Panalytical Ltd., Malvern, UK) by a courtesy of the representative of company (A.P. Instruments, Warsaw, Poland).

#### *4.4. Transmission Electron Microscopy (TEM)*

TEM assay was used to evaluate the shape and size of EVs. Ten microliters of the sample were placed on 200-mesh copper grids with a carbon surface. The samples were negatively stained with 2% uranyl acetate for 1 min. and dried at room temperature. The transmission electron microscopy images were obtained using JEOL-1010 (Akishima, Japan).

#### *4.5. miRNA Isolation from Extracellular Vesicles and miRNA Content Analysis*

EVs pellets were treated with RNAze A (20 µg/mL in PBS). Total RNA was isolated and quality control of RNA was performed as described earlier [19]. Next-generation sequencing (NGS) analysis of the miRNAs was performed by Exiqon (https://www. exiqon.com/small-rna-ngs). For the comparisons between control EVs released from HT29 pcDNA and EVs released from clones HT29-Snail 3, -8, -17, the Benjamini-Hochberg FDR corrected *p*-values were calculated.

#### *4.6. Animal Studies*

The animal experiments were performed in the Center for Experimental Medicine Medical University of Bialystok (PL), in compliance with the Local Ethical Committee for Experiments on Animals in Olsztyn. Female CByJ.Cg- Foxn1<nu>/ccmdb mice (~20 g, 6–8 weeks old) were obtained from the same Center. Animals in group I were left as controls. The primary tumours were established by inoculating HT29-pcDNA or HT29- Snail 17 cells (1.5 × 106/100 µL PBS) subcutaneously into the flank of the mice (group II and IV respectively). Tumour volumes were calculated: (length x width2 )/2. Animals from group II and IV were additionally injected intravenously (tail vein) with 10 µg/mouse of indicated EVs (groups III, V and VI). Blood was collected from the retroorbital sinus (~50µL at day 7) and during the section of animals at the end of the procedure (day 28). Collected organs (liver, lungs, large intestine) were divided and fixed and stained with haematoxylin and eosin (H & E). Tumour growth was estimated by fitting each animal's tumour growth to an exponential model. To fit data to this model, at first low volumes were truncated (up to 12 day) and log10 tumour volume versus time for each animal was plotted. The slopes and R2 values for the fits were calculated using linear regression [50].

#### *4.7. PKH67 Labelling of Extracellular Vesicles and Their Uptake into TDM Cells*

Extracellular vesicles were labelled using PKH67 Fluorescent Cell Linker kit (Sigma-Aldrich) according to the manufacturer's instructions, with minor modifications [51]. After 4 h of PKH67-labelled EV incorporation into TDM, cells were fixed, treated with 0.1% Triton X-100 and incubated in sequence with Texas Red®-X phalloidin (F-actin marker) and Hoechst 33,342 (cell-permeant nuclear dye). The uptake was visualized using a confocal microscope (Nikon D-Eclipse C1) and analysed with EZ-C1 software.

#### *4.8. Cell Motility Measurements*

Measurements of cell motility were performed between the 7th and 25th hour after beginning of incubation of TDM with EVs using HoloMonitor M4 (Phase Holographic Imaging PHI AB, Lund, Sweden—Courtesy of the representative of company). Cell speed was calculated using the App Suite software of the same company. A plot of cell speed versus time was generated and the area under curve (AUC) was computed using GraphPad Prism 7.05 software.

#### *4.9. Measurements of Cytokine Release*

Macrophages were incubated with EVs in FBS-free RPMI-1640 culture medium for 24 h, the medium collected and centrifuged for 20 min at 18,000× *g*. IL-8 levels in the cells supernatants were analysed by flow cytometry according to the manufacturer's procedure using the BD Cytometric Bead Array (CBA) Human Inflammatory Cytokines Kit. The BD Cytometric Bead Array (CBA) Mouse Inflammation Kit was used to measure MCP-1/CCL2 in the plasma of mice. Data were analysed using FCAP ArrayTM Software Version 3.0 (BD Life Sciences—Biosciences, San Jose, CA, USA).

#### *4.10. Statistical Analyses*

Data are presented as mean ± SEM. All experiments were performed at least in triplicate. The Shapiro-Wilk test was used to confirm the Gaussian distributions of raw data. Analysis of variance (ANOVA) was used for multiple comparisons. The Kruskal-Wallis analysis was performed to test the differences between groups of data with nonnormal distributions. For analyses of two groups, the appropriate Student's *t* test (or the Welch's test for unequal variances) was performed to test the differences between groups for normally distributed data. p value less than 0.05 was considered statistically significant. All statistical analysis was performed using GraphPad Prism 7.05 software.

#### **5. Conclusions**

We conclude that Snail that modifies CRC cells towards a more invasive phenotype, can also alter microRNA cargo of cell-released EVs. Thus, the content of cell-released EVs may serve as a biomarker that defines the stage of CRC and either Snail, or the different microRNAs that is carried by EVs to the destination sites, which serves as a pre-metastatic niche, may be a target to prevent cancer progression. We also point to the macrophages that may reside in the tumour pre-metastatic niche, as one of the possible EVs recipient cells that is modified during growing CRC invasiveness.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2072-6 694/13/2/172/s1, Figure S1: Western blot of EVs markers, Figure S2: Global efficiency of miRNA processing, Figure S3: Gene Ontology Enrichment Analysis, Figure S4: Cell viability assay, Figure S5: Snail transcription factor expression in HT29 clones, Table S1: Clone-specific miRNA evaluated during analysis of differentially expressed extracellular vesicles miRNA (fold change ≤ −2.0 for down-regulation and ≥ 2.0 for up-regulation) between each clone overexpressing Snail and control clone, Table S2: Differentially expressed miRNA evaluated during analysis of extracellular vesicles miRNA between each clone overexpressing Snail and control clone, Table S3: The growth rate of tumours and MCP-1/CCL2 levels in plasma of mice injected s.c. with of control HT29-pcDNA or HT29-Snail cells followed by i.v. injections of various EVs.

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

**Funding:** This research was funded by the National Science Centre, Cracow, Poland, grant number 2011/02/A/NZ3/00068 and 2018/29/B/NZ5/01756 (M.A.K), 2016/22/E/NZ3/00341 (P.P) and IBM PAS Statutory Funds (J.B.). The APC was funded by IBM PAS.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Local Ethical Committee for Experiments on Animals in Olsztyn (approval number 55/2017, date: 2017/07/25).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Publicly available datasets were analyzed in this study. This data can be found here: https://www.ncbi.nlm.nih.gov/sra/PRJNA674779.

**Acknowledgments:** The Authors gratefully acknowledge Łucja Balcerzak and Sylwia Michlewska from Laboratory of Microscopic Imaging and Specialized Biological Techniques, Faculty of Biology and Environmental Protection, University of Lodz, Poland, for transmission electron microscopy (TEM) imaging of the EVs samples.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


*2.3.5 Application of nanotechnology in electrochemical biosensors*

In electrochemical biosensors, nanomaterials are mainly applied for modifying the sensitive interface of the biosensors or immobilizing biomolecules. Because of its large specific surface area and high surface energy, nanomaterials can become an

#### **Figure 4.**

*Schematic diagram of rapid direct identification of SARS-CoV-2 using the PMO-functionalized G-FET nano-sensors.*

#### *Perspective Chapter: Novel Diagnostics Methods for SARS-CoV-2 DOI: http://dx.doi.org/10.5772/intechopen.105912*

active electron acceptor or electron donor. The electrode modified by nanomaterials can significantly improve the specific surface area of the electrode, improve the conductivity of the electrode, load more biomolecules, improve the sensitivity and stability of the biosensors, and speed up the response of the biosensor [49].

Nanomaterials commonly used in electrochemical biosensors include noble metal nanomaterials, carbon nanotubes, graphene, magnetic oxide nanoparticles, and so on. Noble metal nanoparticles represented by gold nanoparticles not only have large specific surface area and high surface energy, but also have high catalytic efficiency, strong adsorption, and excellent biocompatibility, which can effectively load and label target biomolecules. In addition, gold nanoparticles have excellent electrochemical activity and can effectively improve the electron transmission efficiency. Au nanoparticles can also form strong covalent binding through Au-S bond and Au-N bond, which is conducive to the coordination of biomolecules containing -SH and -NH2. Carbon-based materials such as carbon nanotubes and graphene have large specific surface area and strong electron conduction ability. The modification of this kind of carbon-based nanomaterials can greatly improve the electrochemical activity of the electrode, improve the detection sensitivity, increase the current signal, and improve the response time of the electrochemical biosensors [50]. Magnetic nanoparticles can be modified on the surface of the electrode to improve the specific surface area of the electrode, which can also be applied for immobilizing biomolecules to improve the selectivity and specificity of electrochemical detection and avoid the interference of other impurities in the biological environment to the target molecules.

#### **2.4 Magnetic biosensors**

Magnetic biosensors have attracted extensive attention of researchers in the past two decades. The biosensors can be divided into surface-based and volume-based magnetic biosensors, which are widely used in the detection of viruses, pathogens, and cancer biomarkers [51–53]. In magnetic biosensors, magnetic nanoparticles modified by suitable antibodies or DNA/RNA probes are usually used as magnetic nanotags, which can skillfully convert the concentration of analytes into magnetic signals [54]. Compared with optical, plasma, and electrochemical biosensors, magnetic biosensors have lower background noise. Because the biological environment of most biomolecules is non-magnetic, the magnetic biosensors will not be disturbed by the biological environment, so as to produce more accurate and reliable detection results [55]. Magnetic biosensors can be roughly divided into three categories: magnetoresistive (MR) biosensors, magnetic particle spectroscopy (MPS) platforms, and nuclear magnetic resonance (NMR) platforms.

#### *2.4.1 Magnetoresistive biosensor*

Magnetoresistive biosensor is a surface-based sensing technology, which is very sensitive to the stray field from generated by magnetic nanoparticles close to the sensor surface, so as to convert the binding of magnetic nanoparticles with analytes into readable electrical signals [56, 57]. Magnetic nanotags in magnetoresistive biosensors need to produce high magnetic moment without losing paramagnetic properties.

At present, there are few studies on magnetoresistive biosensors for the detection of SARS-CoV-2, and we infer main reason is that magnetic nanoparticles would inevitably reduce the magnetic moment when their size decreases. Jinhong Guo's team at the University of Electronic Science and technology of China [58] constructed

an LFIA detection platform based on superparamagnetic nanoparticles and giant magnetoresistive sensing system to detect the immunoglobulin IgM and IgG of SARS-CoV-2 at the same time. Among them, the giant magnetoresistive sensing platform can transmit medical data to smart phones through Bluetooth, which is convenient for medical personnel to obtain patient information. Superparamagnetic nanoparticles with an average size of 68 nm were synthesized by a simple and rapid coprecipitation method with excellent dispersion and magnetic properties. This sensing technique has the advantages of low cost, rapidity, easy operation, and high sensitivity, which can simultaneously detect two antibodies of SARS-CoV-2 within 10 min with the LOD of 10 ng/mL for IgM and the LOD of 5 ng/mL for IgG.

#### *2.4.2 Nuclear magnetic resonance (NMR) platform*

The nuclear magnetic resonance (NMR) platform uses magnetic nanoparticles as contrast enhancer, which causes the nonuniformity of local magnetic field and disturbs the precession frequency variations of surrounding water protons [59]. Therefore, the development of high-sensitivity NMR platform essentially depends on the application of appropriate magnetic nanoparticles with high transverse relaxation.

Siwei Yang's team of Shanghai Institute of Microsystems and Information Technology, Chinese Academy of Sciences [60] reported a rapid and highly sensitive detection of SARS-CoV-2 pathogens based on ultralow-field NMR relaxometry (**Figure 5a**). This method utilizes magnetic graphene quantum dots modified by SARS-CoV-2 antibody as a probe to construct a magnetic relaxation switch to specifically detect novel coronavirus. It is worth noting that closed-tube one-step strategy is safer for experimenters without samples preparation. This one-step detection method has the characteristics of excellent sensitivity and rapid detection with 248 particles/ mL within 2 min.

NMR spectroscopy, like infrared spectroscopy and Raman spectroscopy, can analyze the structure of the molecules to be measured. Different from infrared spectroscopy and Raman spectroscopy, which can directly reflect the molecular structure information, NMR spectroscopy obtains the skeleton structure of the molecules to be tested by analyzing the 1 H, 13C, and 15N NMR spectra. Nuclear magnetic resonance spectroscopy is also widely applied to the screening of antibodies of SARS-CoV-2 and the characterization of protein and nucleic acid structures. Magnetic nanoparticles can be used as a signal amplification tags in this technology to improve the sensitivity of detection. Schoenle et al. [61] reported the sequence-specific backbone assignment of the SARS-CoV-2 RBD and proved that biomolecular NMR spectroscopy chemical shift perturbation (CSP) mapping can quickly and successfully identify the molecular epitopes of RBD-specific antibodies. CSP mapping combined with other detection technology of biomolecules could help us accurately recognize the interaction between RBD and antibody, which is of great significance for antibody screening and further vaccine development.

#### *2.4.3 Magnetic particle spectroscopy platform*

Magnetic particle spectroscopy platform is a volume-based detection technology, which directly detects the dynamic magnetic responses of magnetic nanoparticles [62, 63]. Therefore, for this kind of biosensor, the properties of excitation magnetic field, saturation magnetization, and anisotropy should be considered. Rosch et al. [64] reported a novel SARS-CoV-2 nucleic acid detection platform based on

*Perspective Chapter: Novel Diagnostics Methods for SARS-CoV-2 DOI: http://dx.doi.org/10.5772/intechopen.105912*

#### **Figure 5.**

*a: The detection process of SARS-CoV-2 of the magnetic relaxation switches assay with ULF NMR. b: The schematic diagram of the detection of SARS-CoV-2 RNA based on magnetic particle spectroscopy biosensors.*

magnetic response changes of magnetic nanoparticles (**Figure 5b**). The specific modified magnetic nanoparticles and target molecules mediated assembling will lead to the increase of hydrodynamic radius, which can be measured by the magnetic particles spectrum in alternating magnetic field. This sensing technology has high sensitivity for the detection of SARS-CoV-2 RNA, with LOD of 0.28 nmol/L, and the biological environment such as saliva will not affect the performance of the detection platform.

Compared with optical biosensors, magnetic biosensors have simpler sample processing steps in the detection process, and magnetic tags are safer than electrochemical biosensors. Because of its high sensitivity, accuracy, and specificity, magnetic sensing technology is expected to be employed for on-site detection tools to restrain the spread of SARS-CoV-2.

#### *2.4.4 Application of nanotechnology in magnetic biosensors*

The unique magnetic relaxation properties and good biocompatibility of magnetic nanomaterials give many biosensors high sensitivity and selectivity. In the process of designing magnetic biosensors, which rely on magnetic nanoparticles tags, the size of magnetic nanotags is required to match that of analytes [65]. However, with the decrease of the size of magnetic nanoparticles, these magnetic nanotags often have low magnetic moment and uneven particle size distribution. In addition, the serious surface defects on the surface of nanoparticles and the unavoidable magnetocaloric effect will cause the fluctuation of magnetic signal in the detection of low-concentration analytes. Therefore, the development of magnetic biosensors should focus on how to prepare magnetic nanoparticles with uniform size and good dispersion and the point-of-care detection of magnetic biosensors.

## **3. Conclusions**

Up to now, SARS-CoV-2 has continued to diffuse and spread all over the world, and the epidemic of COVID-19 is facing the dilemma of globalization and time sustainability. Especially in view of the continuous variants of SARS-CoV-2 with

increasing transmission speed, concealment, and the proportion of immune escape, even new variants still pose a serious and death threat to people with low immunity and the elderly. Therefore, it is necessary to further improve the conventional detection methods and break through the limitations of the original detection methods and develop new methods as a supplement or substitute for future monitoring and detection tools. Especially in some developing countries with a shortage of medical resources, it is particularly important to develop rapid, simple, high-throughput, and intelligent detection methods.

The nanotechnology attached to the novel nano-biosensing technology has developed relatively mature, but the application of biomacromolecules detection still needs to be further improved, such as the biological toxicity of nanomaterials, the modification of biomolecules on the surface of nanomaterials, the large-scale manufacturing of nanomaterials, and so on. For the novel diagnostics methods, the following aspects need to be considered:

*Perspective Chapter: Novel Diagnostics Methods for SARS-CoV-2 DOI: http://dx.doi.org/10.5772/intechopen.105912*

with the existing nucleic acid, antibody, and pathogen detection methods and complementing their advantages, the combined use of them is expected to achieve the accurate and rapid detection of SARS-CoV-2. This method not only provides a powerful means for the current outbreak of COVID-19 and the detection of unknown pathogens in the future, but also has important practical significance for the future application in the fields of respiratory disease differential diagnosis, environmental monitoring, food safety assessment, etc.

7.Intelligent detection. Standardize the high-throughput screening results, instrument interpretation results, and analysis results for large population to avoid the lag and subjectivity of manual interpretation. The interpretation results are not only comparable, but also can be output in time for on-site or remote research and judgment.