Perovskite photocatalyst materials are gained special attention due to their exceptional properties because of their flexibility in chemical composition, structure, bandgap, oxidation states, and valence states. The current review is focused on perovskite materials and their applications in photocatalysis. Special attention has been given to the structural, stoichiometric, and compositional flexibility of perovskite photocatalyst materials. The photocatalytic activity of perovskite materials in different photocatalysis applications is also discussed. Various mechanisms involved in photocatalysis application from wastewater treatment to hydrogen production are also provided. The key objective of this review is to encapsulate the role of perovskite materials in photocatalysis along with their fundamental properties to provide valuable insight for addressing future environmental challenges.The massive use of non-renewable energy resources by humankind to fulfill their energy demands is causing severe environmental issues. Photocatalysis is considered one of the potential solutions for a clean and sustainable future because of its cleanliness, inexhaustibility, efficiency, and cost-effectiveness. Significant efforts have been made to design highly proficient photocatalyst materials for various applications such as water pollutant degradation, water splitting, CO 2 reduction in presence of co-catalyst.Photocatalytic reaction for CO The increased concentration of CO2 is causing is global warming and is the leading cause of acid rain, which is dangerous for all living things on the earth. The current decade 2021–2030, is the decade of ecosystem system restoration. Therefore, it is essential to pay attention to clean ways to tackle environmental issues. The new global challenge is the achievement of ecological sustainability. The research focus of this era is oriented toward the way to achieve the eco-friendly system.1–6Rapid industrialization and population growth resulted in increased energy resources utilization and release of harmful pollutants. The increased human activities are depleting the energy resources and polluting our ecosystem. The toxic gasses in the air are causing respiratory diseases, and the direct disposal of industrial and organic waste into the water reservoirs is causing water-borne diseases. Nearly 663 million people in the world have no access to clean water. The amount of CO7,8The blissful gift of Mother Nature, the Sun is an ultimate renewable energy resource that irradiates 3.85 yotta joule (YJ) of energy yearly on the earth's surface. Sunlight is one of the best routes to deal with environmental issues owing to its cleanness and abundant availability. Sunlight can be utilized as an energy medium by photovoltaic, photoelectrochemical catalysis, or photocatalysis in daily activities.2O, CO2, and N2) into fuel . The conversion of water into the fuel (H2) by using light and photocatalyst material is known as photocatalytic water splitting. The conversion of CO2 into the hydrocarbons is known as CO2 reduction, and conversion of nitrogen into NH3 is called nitrogen fixation. All these application deals with the viable way to clean the environment. The main challenge while dealing with photocatalysis is the region of operation of the photocatalyst in the solar spectrum. Most photocatalysts operate under UV radiations, which is only 5% of the sunlight reaching earth. Therefore, suitable photocatalysts are required to respond to the extensive range of the solar spectrum.9–18Photocatalysis is defined as the science of employing a catalyst that uses light to speed up the chemical reaction. In photocatalysis, the photocatalyst material is used, and the energy of the light acts as a source to generate electron–hole pair. The photogenerated electron–hole pair then initiates the redox reaction at the surface of the photocatalyst. This redox reaction can be utilized to degrade water pollutants and convert abundant earth elements  for the decolorization of Prussian blue. Photocatalysis term also appeared simultaneously in the title of the article investigating the degradation of the oxalic acid in the presence of the uranyl salts upon light irradiation. In 1921 Edward Charles Cyril Baly studied the production of formaldehyde using colloidal uranium salts and ferric hydroxides as a catalyst. Soon after, in 1924, the Baur and the Perret at the ETH of Zurich reported the effect of light irradiation on the ZnO suspension and observed that ZnO suspension enhanced reduction of Ag21,23Many researchers from 1940 to 1972 investigated different parameters of the photocatalyst. Photocatalysis gained significant attention when electrochemical photolysis of the water was done by using titanium dioxide under UV irradiation. The oil crisis in 1973 also changed the social and economic status of the western world. The first time, the shortage of fossil fuels became a significant issue, which led to an unprecedented increase in the efforts for alternative energy resources, including photocatalysis. The application of photocatalysis is not limited to energy fuels but can also clean the environment. Much attention has been given to photocatalysis during the last two decades due to its wide range of applications.24,25Photocatalysis has its application in the pollutant degradation and production of fuel. Material can be an excellent photocatalyst if it is responsive to light, especially (UV or visible), biologically and chemically inert, has low cost, and exhibits adsorption and absorption capacity. Normally the metal oxide semiconductors are used in the application of the photocatalyst. Perovskite materials, due to their unique optical properties, also gained importance in the application of photocatalyst during the last decade.26–28 The first discovered perovskite material was CaTiO3.29 The basic chemical formula of the single perovskite is ABX3, where A and B are cations while X is an anion. Normally, A is alkaline earth metals or lanthanides, B is the transition metal, and X is oxygen or any halide. The perovskite is known as perovskite oxide when the anion is oxygen and is known as perovskite halide when the anion is a halide. In single perovskite, the coordination number of A site is 6, the coordination number of B site is 12, and the coordination number of O anion is 6. In single perovskite, the perfect structure of the BO6 octahedral connection results in the cubic lattice.30–33 The structure of single perovskite is shown in Perovskite material was first discovered in 1839 in the Ural Mountains by the German scientist Gustave Rose. The mineral was named perovskite after Russian mineralogist Count Lev Aleksevish von Perovski.34–39 The tolerance factor is a dimensionless quantity given by 38,40The tolerance factor can indicate the distortion and the crystallographic structure of the perovskite material. The tolerance factor of the single perovskites ions was defined by V. M Goldschmidt in 1926. The tolerance factor of the perovskite structure tells the stability of the perovskite structure and the compatibility of the ions in the crystal structure.rA and rB are the ionic radii of the electropositive ions, while rX is the radius of the oxygen or halide ions.41 The perovskite structure can be divided into octahedral hexagonal, tetragonal, and ideal cubic structures on the basis of the tolerance factor. When the tolerance factor is between 0.9 and 1, the structure is the ideal cubic structure with the length of the unit cell (a) as:42In 2O3) and its derivative are considered when the tolerance factor is less than 0.75.43 The stable bixbite polymorph (α-Mn2O3) is favored when the factor decreases. If the tolerance factor is greater than 1, the structure of perovskite is hexagonal close-packed (HCP), and [BO6]-octahedra share faces with the hexagonal c-axis.30,44In non-ideal cases, the tolerance factor deviates from 1, which shows the mismatch in the A–O and B–O bond lengths. The corundum structure  or A2B′B′′O6 (double B-site).47,48 The double perovskite strain energy depends on the charge difference between the two types of B cations (B and B′). There are three ways to arrange the B-type cation based on the charge difference between B and B′. The arrangement is random if the charge difference is one (ΔQ = 1).49 The most common arrangement of the double perovskite is the rock-salt arrangement, also called elpasolite structure, in which the cations change in all three dimensions, and it dominates when the charge difference between the B and B′ is greater than 2 (ΔQ > 2). Due to the difference in the charge size of the B cation in the rock salt order, the crystal symmetries are less than their single perovskite structure. Another arrangement is the layered arrangement of the B and B′, where the cations can change in only one dimension . The distortion in the crystal structure affects the BO3, 264 are experimentally investigated. The ABO3 perovskite structure is divided into five groups depending upon the A and B site charges that are A1+B5+O3, A2+B4+O3, A3+B3+O3, A4+B2+O3, and A5+B1+O3. Theoretically, there are 105 possible material double perovskite, out of which 103 double perovskites are experimentally investigated. For material to be double perovskite, the charge balance of all the charges present in the structure should be equal to 12. Double perovskite allows the elements with high valence shells to accommodate in structure, thus expanding the compositional flexibility.49,68The perovskite structure exhibits a high degree of stoichiometric and compositional flexibility. Theoretically, 346 different kinds of ABO2+ cation is larger compared to the A3+ cation. As the A3+ cation has a smaller size, it limits the size of B-site cations. More than one thousand double perovskite materials are reported in the literature, all of which are prepared at ambient pressure. Some new materials were synthesized at high pressure. More than 720 compounds are reported as the divalent A-site compounds and 200 as trivalent A-site.30,69–71Perovskites' compositional flexibility is often represented by their ease of alloying on A and B sites. Normally, the mixing element in semiconductors is isovalent, while in perovskite, the mixing element is non-isovalent. The simplicity of cation mixing in perovskite materials makes it easy to alter its chemical and physical characteristics. The double perovskite structure also shows interesting properties like half-metallicity, high-temperature ferromagnetism, and many magnetic interactions. The infrastructure of the complicated oxide unit cell depends upon the electronic configuration and the atoms located at the A and B-site. The B-site atoms are considered to be more critical than A-sited cations. The size of the A6 octahedra can be maintained in the presence of vacancies at A, B, and O sites because of the perovskite's excellent structural and compositional flexibility. Applications of the perovskite materials are broad due to these vacancies, and therefore, they carry applications in photocatalysis, photovoltaics, supercapacitors, batteries and fuel cells, etc.30The structural network of BO72 There are two main stability issues; (1) intrinsic stability–stability issues, and (2) extrinsic stability–stability issues of perovskite. Intrinsic stability involves structural stability, electronic band structure, and thermodynamic phase stability. In contrast, extrinsic stability encompasses interaction with water molecules and pathways of degradation, thermochemical stability, light induced ion redistribution, light stability, photochemical degradation, and oxidation/photo-oxidation of charge-transporting layers and perovskites.73The challenge to make use of perovskite photocatalyst materials come from two significant restraints: (1) chemical instability in polar solvents and (2) vulnerability of the surface to transform with chemical species present in solution. Recent progress in this field improved these issues and illustrated different courses of actions to make use of perovskite materials in photocatalytic processes.74,75Perovskite materials, due to their structural, compositional, and stoichiometric flexibility, are considered promising materials for photocatalysis . Perovsk−) and hole (h+) pair. The photogenerated electron–hole pair then initiates the redox reaction at the photocatalyst's surface. There are three steps involved in the mechanism of photocatalysis. The first one is photo-excitation, provided that the energy of the light source should be high enough to overcome the bandgap. The second step is trapping the electron–hole pairs to increase the recombination time. The recombination of photoinduced charges produces heat, which decreases the efficiency. The third and final step is the degradation of the pollutant by the reactive species produced due to a redox reaction or the production of the fuel.76–79 The general reaction mechanism for all the reactions is given by;The light interacts with the surface of the nano photocatalyst, which generates the electron . The doping at these sites can enhance light absorbance in the visible and NIR regions by bandgap alteration. The recombination time can also be addressed by employing the different strategies that includes tuning of bandgap and repression of electron–hole recombination. To solve both light absorption and recombination time problems, a new innovative solution known as defect engineering has been proposed.(i) Bandgap engineering(ii) Repression of electron–hole pair recombination(iii) Defect engineeringThe bandgap of the various perovskite materials is given in 2 generation, CO2 reduction, contaminants degradation, and nitrogen fixation. The donor and acceptor impurities in the perovskite crystal structure can adjust the bandgap. The donor and acceptor impurities in a perovskite can tune the material's bandgap. The doping at the cations or anions at different sites of perovskite structure enhances the photocatalytic activity by introducing the intra-band energy levels; the shallow and deep intraband energy levels trap the photogenerated electron–hole pair. Doping also reduces the bandgap of the perovskite material  led to the production of donor impurities in the perovskite structure. The high atomic energy of the transition metal ensures better electron mobility without changing conduction band minima of perovskite host material.83,97–99The doping at the different sites of the perovskite structure changes the absorption peak of the material, thus increasing the light absorption. Doping also tailors the material's bandgap and increases the efficiency of the photocatalyst material. Incorporating a new transition energy level in the perovskite structure improves the higher wavelength absorption without changing the intrinsic absorption properties of the material. Hybridization of orbital in perovskite structure and formation heteroatom due to the doing changes the absorption spectra of material. The hybridization decreases the bandgap and therefore increases the absorption of light. Thus, the doping changes the bandgap and increases the recombination time.3 is 3.25 eV, and doping with noble metals like Ru, Rh, Au, etc., introduces the intra-band local states, making the doped SrTiO3 a suitable photocatalyst.100 The Ru doping introduces the intra-band energy state between conduction band (Ti 3d) and valence band . The ionic radius of Ru is greater than Sr and nearly equals Ti; therefore, the Ru substitutes Ti atom in SrTiO3. Ru doping at the B site of the SrTiO3 introduces the local states and increases the photocatalytic performance. The doping of noble metals in SrTiO3 makes it a Vis-IR responsive photocatalyst.86The bandgap of pure SrTiO3 introduces the intra-band energy level and reduces the bandgap. The change in the absorption shift was also reported due to the change in the bandgap with doping.101 It is reported that doping of the sulfur at the O site of the ABO3 introduces the intraband energy level at 1.23 eV. Researchers also noted that doping the nitrogen at the O site of the perovskite material increases photocatalytic activity.102,103Similarly, doping the bismuth (Bi) or copper (Cu) at A site of SrTiO104,105The amount and the quantity of the dopant should also be optimized to impede the recombination of the photogenerated charge carrier. Synthesis methods for preparing the perovskite material also affect the bandgap, recombination of charges, and migration properties of photocatalyst.106–108The movement of photoinduced charge carriers at the photocatalyst's surface initiates the redox reaction at the surface of the photocatalyst. The recombination of charge carriers can occur either at the surface or in bulk. The recombination reduces efficiency, so it must be repressed.109–111 The incident light generates the electron–hole pair below the catalyst surface, known as the absorption length. The development of the space charge region takes place at the phase of the solid–liquid interface, and if the absorption extent exists in the space charge region, the photoinduced electron–hole pair can be easily separated. However, in the case of the bulk material, the recombination rate will be high.112,113 After separating the photo-induced charges, the next step is the migration of the charge carriers. The migration mainly depends on the material's symmetry and diffusion length. Diffusion length is the distance that photogenerated charges travel without recombining or scattering. Diffusion length is the governing factor for controlling the movement of the charges in the absence of the potential gradient. Separation of the photogenerated charges beyond the charge separation region is feasible with materials whose diffusion length is longer. The diffusion length is multiple of the lifetime of the charge carrier and diffusion constant, and it should be of high value to generate the maximum photogenerated charge carriers.114–116 The limiting factor for the range of charge space region is the particle size. The width of the charge space region should not surpass the particle's radius, and therefore the use of nano-size crystals improves the photocatalytic activity.108,117,118The crucial step in photocatalysis is selecting the material, which depends upon the absorption coefficient of the photocatalyst material. The absorption coefficient of photocatalyst depends upon the bandgap and photon energy.79,119–126The perfection in the crystal structures affects the lifetime of the photogenerated charge carrier. The potential gradient also affects the photocatalytic activity because it efficiently separates the photo-induced charges through the support interfaces between the semiconductor–semiconductor, semiconductor–metal, and semiconductor-conductor surfaces, forming the heterojunctions. Therefore, it is necessary to introduce the potential gradient for attaining better efficiency of photocatalytic material. Furthermore, combining the two semiconductors with different bandgaps represses the electron–hole pair recombination. Co-catalyst can also enhance efficiency by introducing active sites and better charge transfer.119,127 The three types of heterojunctions are type I, type II, and type III. The combination of the semiconductor materials forms the Z-scheme, p–n junction, and the Schottky junction based on the type of the semiconductor used  with a highly negative conduction band and highly positive valence band while the bands of the other photocatalyst semiconductor (SP II) lie within it. The drawback of type I heterojunction is that the charges accumulate on the semiconductor with a lower bandgap which is not favorable.131,132In type II, the conduction and valence band of the one semiconductor SPI lies above the conduction and valence band of the SPII. The charge separation of the photogenerated electron–hole pair is efficient in type II because of the electron's downward movement from the conduction band of SPII and an upward movement of the holes from SPI's valence band.120,130In type III heterojunction, the valence and conduction bands difference is more than type II. The photogenerated electrons of the SPI unite with the photogenerated hole of SPII. The SSPII electron and the SPI hole innate the redox reaction in the semiconductor photocatalyst.133–135The understanding of the Z-scheme is necessary to understand the heterojunction. In Z-scheme, two semiconductor photocatalyst materials are linked in a way that they form the letter Z. The electrons from the conduction band of the SPII trans into the valence band of the SPI. SPI acts as a reduction site, and SPII serves as an oxidation site. Due to the redox potential between the two semiconductors, the inbuilt electric field allows efficient charge separation. The direct Z-scheme involves the contact at the interface of two semiconductor materials. In contrast, the indirect Z-scheme involves mediators that allow the charge transfer between the two semiconductor materials. The mediator can be solid or liquid. The iron-based mediators are more commonly used.136,137 Examples of semiconductor–semiconductor photocatalyst are SnO2–TiO2, CdS–TiO2, Bi2O3–Bi2WO6 and Bi2WO6–TiO2, etc.138The heterojunction can also be formed by combining the p and n-type of the semiconductor materials. A free-electron from n-type semi-conductor material migrates toward the p-type semiconductor material upon contact. The migration of free electrons results in the formation of oppositely charged interfaces. This migration also strengthens the inbuilt electric field at the contact of the p–n junction. The built-in electric field facilitates the migration of the photogenerated charge carrier after light irradiation, which increases the photocatalytic performance .136,137 139–141 The prominent examples of metal–semiconductor photocatalysts are Au/TiO2, Au/ZnO, Ag/ZnO, Ag/TiO2, etc.142The increased charge separation of metal–semiconductor photocatalysts arises from the transfer of the electron across the interface of metal–semiconductor. Metal improves photocatalytic performance because it provides active sites and facilitates the separation and transfer of photoinduced charge carriers. When a metal (having a high work function) couples with semiconductor, the electrons flow from the semiconductor to the metal until their Fermi levels get aligned, resulting in upward bending of band edges in semiconductor and accompanied by the band bending, formation of Schottky barrier takes place at the metal–semiconductor interface . The ele143–145Defect engineering is a suitable and easy way to enhance the photocatalytic efficiency of perovskite materials. Substituting the different materials can introduce defects in the perovskite materials in the perovskite structure. These defects act as a site to trap the charge carriers and thus enhance the separation of charge carriers and increase the photocatalytic efficiency. These defects can be a vacancy of an extra atom that results in the formation of the line or screw dislocation.6 octahedra. Furthermore, it is thermodynamically unfavorable to introduce the vacancy at the B site due to the high charge value and the small B site cation size.146,147In perovskite materials, three different alteration sites allow the introduction of the defects or vacancies. The doping at the A and B site cations introduces the dislocation but does not allow structural changes. The structural changes can introduce vacancies at the A site or O site. The vacancy at the B site is challenging to introduce due to the BOx1−AxBO3 and the oxygen-deficient vacancy can be created by doping the sulphur or nitrites. The second approach is doping at the B site, in which the reduction of the B site produces oxygen vacancy. In recent years, many defect-rich perovskite materials with excellent photocatalytic activity have been reported. 30,148,149The vacancies at the A site can be induced by substituting the A-site cation with a cation of the high or low valence. The subsisted single perovskite oxide formula is: AEVB) and conduction bands (ECB). Therefore, to investigate the possible applications of semiconductors, it is crucial to have an understanding of the ECB and EVB band edges. In a semiconductor, the Fermi level (EF) determines the electrochemical potential of electrons. At thermodynamic equilibrium, EF tells the occupation of the energy levels, but thermodynamic equilibrium can be perturbed by external bias or irradiation due to injection or photogeneration of holes and electrons. Therefore, the quasi-Fermi levels define the non-equilibrium densities of electrons in conduction band and holes in valence band. Generally, the quasi-Fermi level for the majority carriers approximates the equilibrium EF because of the negligible increase of density of majority carriers. However, the quasi-Fermi level can be shifted because of the small minority carriers at equilibrium.150The potential of semiconductor photocatalyst mainly depends on the electron injection capacity of the material, which is controlled by the energies of valence  in the semiconductor phase, which is associated with the upward bending of the band edges in the n-type semiconductor , and these reactive species then attack the organic pollutants and degrade them into low molecular weight products  within no time.153,157,158The use of the photocatalyst is an eco-friendly way to degrade these water pollutants. The photocatalytic techniques utilize the advanced oxidation process (AOP) for degradation. The interaction of the light with the photocatalyst generates electron–hole pair. Generally, oxygen is employed as an electron scavenger to increase the recombination time for enhanced photocatalytic activity. The redox reaction of the photogenerated electron–hole pair gives reactive species such as . The redox potential of the created reactive ion species depends upon the bandgap of the photocatalyst. The reactive ion species then degrade the contaminant into the smaller fragments, which are then changed into green compounds. The degradation of the contaminants takes place at the surface of the catalyst. Initially, the dye gets adsorbed at the catalyst surface, and after the degradation product gets desorbed, the new pollutant is adsorbed, and this process continues until no contaminant is left. The overall reaction mechanism is shown in  and total organic carbon (TOC). The photocatalytic reaction for degradation of the containments occurs in an aqueous environment. The irradiation of the light on the aqueous system  generates photogenerated electron–hole pair in water. The oxygen or different impurities are added as an electron scavenger to increase the recombination time.shown in .30,76,13165 The first reported perovskite material CaTiO3 exhibited photocatalytic activity and degraded pollutants. The wide bandgap of CaTiO3 (3.0–3.5 eV) responds only to the UV light and efficiently degrade the brilliant green (BG), methyl blue (MG), and rhodamine B (RHb).88,166 The synthesis route and the morphology of the CaTiO3 structure change the bandgap and thus affect the photocatalytic performance for pollutant degradation. CaTiO3 nanocuboid showed the excellent photodegradation of the RhB dye upon visible light irradiation. Zirconium (Zr) doping at the Ti site of CaTiO3 generates oxygen vacancies, producing defects and increasing photocatalytic activity by changing the lattice structure.167,168 The reported photocatalytic activity of the Zr doped CaTiO3 is thirteen times greater than the un-doped CaTiO3. Fe-doped CaTiO3 efficiently degraded the methyl blue using the visible light source.166 The doping of the Fe in the CaTiO3 increased the absorption ability of CaTiO3. The photocatalytic activity also depends upon the calcination temperature and the irradiation time of the light. Fe-doped CaTiO3 showed 100% photodegradation of the methyl blue at optimum condition .169 BaTiO3, SrTiO3, and other titanium-based perovskite materials have also shown better photodegradation of the pollutants.101,102,170Many perovskite materials are used as a photocatalyst, but titanium, bismuth, and ferrite-based single and double perovskite are widely reported due to their excellent photocatalytic properties.3, where A can be La, Bi, Ca, Sr, Gd, etc.) attracted researchers due to their low cost and small bandgap compared to the titanium-based perovskite.171 The widely used ferrite-based perovskite is bismuth ferrite (BiFeO3). The small bandgap (2.0–2.77 eV), excellent stability, and strong photoabsorption of BiFeO3 allow the efficient photodegradation of organic dyes from textile and pharmaceutical industries under visible light.172–174 The pure BiFeO3 in the sunlight showed 69% photodegradation of methyl blue. The doping at different sites of BiFeO3 has shown better results. Sc doped BiFeO3 showed 100% photodegradation of the methyl blue in 3 hours under sunlight. The improved efficiency was attributed to improved ferroelectric properties due to lattice distortion produced by doping.98 Mesh of BiFeO3 showed 98% photodegradation of the methyl blue within 4 hours. The excellent photocatalytic activity of BiFeO3 was due to the high interaction of the dye molecules and photocatalyst. Bi0.90La0.10Fe0.95Mn0.05O3 showed 97% photodegradation of Congo-red within 2 hours under the sunlight.25Ferrites-based perovskite (AFeO3) showed photocatalytic degradation properties. The efficiency of the NaTaO3 based photocatalytic system is quite less due to the wide bandgap of the material. Still, the doping of nonmetals increases its efficiency by creating a local state between the conduction and valence band. The reported photodegradation of methyl blue is 95.21% by nitrogen-based NaTaO3 photocatalyst using sunlight as an irradiation source.175,176 Different perovskite materials used for photocatalytic pollutant degradation are given in Tantalite-based perovskite is also reported for the degradation of water pollutants. Due to excellent photochemical stability, sodium tantalite  in combination with the photocatalyst material produces hydrogen and oxygen, and the reaction is known as hydrogen or oxygen evolution reaction. The process of producing hydrogen and oxygen from water by using semiconductor photocatalyst material and light as an irradiation source to innate the reaction is known as photocatalytic water splitting.218–221One-step photo reaction requires only one semiconductor photocatalyst material, and the overall process is depicted in 2 evolved and the recyclability of the photocatalyst. The reaction rate is not always proportional to the use of photocatalyst mass; therefore, the preferred way is H2 evolved per unit time (μmol h−1) with the mass utilized during reaction rather than writing the amount of H2 produced in a mass normalized from (μmol h−1 g−1).222,223 The intensity of the incident light for the photocatalytic performance is the number of photons collected per particle is directly related to the particle size. The reaction rate for the photocatalyst is proportional to the intensity of the incident light. In some cases, the rate of reaction increases with excitation intensity. In contrast, the increase in intensity in some reactions leads to decreased photocatalytic efficiency due to the second-order recombination of the photo-induced charges.224 At low intensity, the concentration of the photogenerated charge carrier is insignificant compared to the concentration of intrinsic majority carriers. Thus, it can also be assumed that the concentration of minority charges varies with incident light's intensity, whereas the concentration of majority charges stays nearly constant.225–227The performance of the photocatalyst can be evaluated by the quantity of H228–230The recombination of the photogenerated charges can be determined by the quasi-first-order reaction with respect to the concentration of minority carriers induced by the light source. In simple words, the order of a photocatalytic reaction at a specific intensity gives an idea about the photoexcited charge carrier concentration. For comparison, a standard parameter of the apparent quantum efficiency (AQE) and the solar to hydrogen conversion (STH) are reported for measurements of photocatalytic activity.2 under sunlight, the photocatalyst with the bandgap of less than 2.36 must be developed. The perovskites-based photocatalytic materials gained significant attention for the practical application of water splitting under UV light and visible light.231–234Moreover, the stability of photocatalyst for real-world applications is vital as these materials need a life cycle of about ten years with an efficiency of about 5–10% STH to meet the goal price of $ 2.00–4.00 per kg. The absorption wavelength for water splitting should be around 526 nm (2.36 eV) because below this wavelength, the photon's energy is not sufficient to achieve STH efficiency of 5–10%, considering that the reaction takes place for a lower wavelength at the AQE = 1. To meet the industrial demand, the practical need for the evolution of H3, thus are making it a suitable visible light photocatalyst for water splitting reaction. The use of the Pt as a co-catalyst induces the Schottky barrier and can make the photocatalyst responsive to visible light. The presence of the Pt also provides the site for the proton reduction. The quantum yield efficiency by using the visible light as an irradiation source is 0.9% while using the UV light, the quantum yield efficiency of the system is 1.9%. The use of the other metals as Au, Ag, Fe, Ce, and Ni are also reported.235–239Titanium-based perovskite materials are widely reported for photocatalytic water splitting. Doping and co-doping change the morphology and bandgap of SrTiO3 nanocubes is 375 μmol g−1 h−1. The Ag doping at the surface of the KTaO3 increases the hydrogen evolution in UV light. The irradiation of the UV light generates oscillations in the conduction band of absorbed silver and thus allows the easy excitation of the particles to the conduction band of KTaO3. The local electric field of KTaO3 was enhanced with the doping of the silver nanoparticles. The enhanced field promotes the electron–hole pair generation, and therefore, increases the photocatalytic splitting. The reported evolution of the hydrogen in the presence of Ag as a dopant in KTaO3 is 2072 μmol g−1 h−1.240–243 The lanthanum and ferrite-based perovskite material are also reported.244Alkali tantalates are the best perovskite material reported for photocatalytic water splitting when the parameters like the doping of the metal cations and morphology (nanocubes) of tantalates-based material are optimized well. The reported hydrogen evolution by KTaO2 is increasing at an alarming rate due to increased human activities, deforestation, burning of fuels, and industrialization. The increased CO2 emission is causing global warming, and for environmental sustainability, it is essential to convert CO2 into valuable products. Mother Nature blessed humans with photosynthesis, in which chlorophyll act as a natural photocatalyst and, in the presence of sunlight, converts CO2 into water, oxygen, and food for plants. The photocatalyst also converts CO2 into valuable products in sunlight.16,277,284,285The atmospheric concentration of CO2 reduction requires more electrons than hydrogen evolution reaction. The end product from depending upon the number of available electrons. One of the limiting factors is the low water solubility of CO2. The complete reduction of the CO2 in water also produces H2. The Z-scheme or heterojunction process for CO2 reduction is reported frequently.30,122Photocatalytic CO2 reduction involves the radical anion formation at −1.9 eV, which is impossible for most perovskite material due to less negative CB potentials of the perovskite material; therefore, high activation energy is required. The surface adsorption of CO2 by catalyst generates the charge CO2δ− which facilities the reaction to proceed. Surface engineering can enhance the CO2 adsorption to the photocatalyst's surface and thus, increases the conversion efficiency. The low conversion efficiency of the CO2 is considered a significant concern in dealing with photocatalytic CO2 reduction.286,287 The co-catalyst can lower the activation energy resulting in enhanced photocatalytic activity. The reaction mechanism is shown in 2 reduction, the CO2 gets adsorbed on the photocatalyst's surface, and electron–hole pair is produced upon irradiation. The photo-generated charges then move to the surface of the photocatalyst, and CO2 is reduced into the valuable fuel. After the reduction of CO2 by the photogenerated electrons and the formation of O2 by photogenerated holes, the reduced and the oxidized product is formed by the desorbed hole and electron, and the cycle continues. The end product depends upon the total number of electrons used in the procedure. For a better photocatalytic activity, a significant number of electrons should migrate towards the surface of the photocatalyst, and the bottom level of the photocatalyst's conduction band should be more negative than the redox potential of CO2. The water or additional sacrificial reagents must consume the photogenerated holes; otherwise, holes recombine with the oxygen. The photocatalytic CO2 reduction can be enhanced by optimizing CO2 adsorption, charge separation, and desorption of the products. The process of CO2 reduction in the presence of co-catalyst is shown in 288,289The first step in CO2 reduction by photocatalyst takes place in the presence of a water molecule, while the reaction in the gaseous or aqueous phase. The reduction potential of CO2 should be small compared to the reduction potential of conduction band minimum and water to obtain valuable solar fuel. During the oxidation, the oxygen molecule is formed by the reaction of the water with a valence band hole.The CO2 reduction.290–292Calculation of the AQE is used to access the performance of the perovskite material for photocatalytic COThe reactions are shown as:2 compared to the tantalates and niobates. SrTiO3 perovskite material is widely reported due to better charge transport properties and bandgap equivalent to TiO2 (3.2 eV), but the poor CO2 surface adsorption of photocatalyst (SrTiO3) creates a problem. This problem can be solved by using the metal as a co-catalyst and introducing the oxygen vacancies to improve the adsorption. In the gas phase reactor using visible light, Fe doped SrTiO3 photocatalyst with Pd (0.5 wt%) as a co-catalyst reduces CO2 to CH4. The yield of the product is 421, and the CO selectivity is 84%.16,293–295Titanium-based perovskite oxides exhibit excellent photo-stability for CO2 reduction because of their wide bandgap. NaTaO3 photocatalyst in combination with Au (0.5 wt%) as a co-catalyst reduces CO to CH4 fuel in the gas phase reactor and uses UV-vis light as the irradiation source. The selectivity of CO is 96%, but the yield is small.2962 reduction.Tantalum-based perovskites are not widely reported for CO2). Dinitrogen air converts into ammonia by the process known as nitrogen fixation. Plants and soil then use the ammonia formed by atmospheric nitrogen to build several processes. The mechanism to create ammonia by nitrogen is the Haber cycle.314 At standard conditions, the ammonia formation is a thermodynamically spontaneous process (ΔH = −92.2 kJ mol−1). Due to its high ionization energy of 15.58 eV and low electron affinity, the dinitrogen in the air is highly stable; therefore, the atmospheric dinitrogen in the normal conditions cannot be used to prepare the ammonia unless some catalyst or some industrial fixation is used. In industry, by using iron or ruthenium as a base catalyst at high temperatures, the dinitrogen is converted into ammonia by the Haber–Borsch process. But even after the century's advancement, the production of the indusial fixation is still quite low. The NH3 is one of the essential components in manufacturing fertilizers and hydrogen storage. Therefore, it is essential to synthesize an alternative green route to deal with energy crises and global warming.77,315Nitrogen plays a vital part in building biomolecules such as amino acids, proteins, and other molecules. Earth's atmosphere has 78% of the dinitrogen  scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>N bond. Light irradiation generates the electron–hole pair. These photogenerated charge carriers then move to the photocatalyst's surface, and at the conduction band, the dinitrogen is reduced to the ammonia by multi-step transfer of the electron and proton available from the water. The hole in the valence band oxidizes the water molecule and gives O2. The chemisorption of the N2 and H+ ions occurs on the surface of the conduction band accompanied by the association or dissociation by H2 incorporation of N2 molecules absorption and NH3 formation. Lastly, desorption of NH3 from the photocatalyst's surface after the ammonia formation occurs.85,319–321Recently the photocatalyst material for nitrogen fixation has gained much attention from researchers. The nitrogen fixation by the photocatalyst utilizes only sunlight and water in the presence of the dinitrogen. The six electrons do the complete conversion of NN bond occurs in the dissociative pathway before adding the hydrogen atom as a reducing agent, which is impossible in an ambient environment.322 In an associative pathway, breaking the NN bond is unnecessary, so nitrogen fixation is possible at ambient conditions. The nitrogen is adsorbed at the surface of the photocatalyst and then converted into the NH3, which is then desorbed after the breakage of the N–N bond. In the associative pathway, the hydrogenation takes place with the sideway nitrogen, which is not directly attached to the surface of the photocatalyst, which generates the NH3.323,324 The remaining nitrogen attached photocatalyst's surface undergoes hydrogenation and generates NH3. But this route is challenging because the chemisorption of nitrogen molecules on the surface of the catalyst is not easy; therefore, surface engineering is an important parameter to enhance the photocatalyst activity for nitrogen fixation.8,321,325 The yield and the efficiency of the reaction are given by the apparent quantum efficiency (AQE) and can be calculated as;326The nitrogen fixation in the presence of the photocatalyst takes place in two ways, either by the associative way or by the dissociative way. The breakage of the N3, KNbO3, LaCoO3, and layered double perovskite Bi2WO6.129,327–329 The efficiency of the perovskite material is less due to the selectivity of N2 and adsorption of N2 at the surface of the photocatalyst material; however, more research is needed in the field of photocatalytic nitrogen fixation to compete with the available industrial method of nitrogen fixation (Haber cycle).319,330 Some of the reported perovskite photocatalyst materials for nitrogen fixation are listed in Limited numbers of the perovskite materials are reported due to the less AQE and yield. The reported materials are SrTiO6  were synthesized and degraded MB. Researchers reported that rare-earth cation-dependent compounds  showed significant photocatalytic activity. CaCu3Ti4O12 possessed an indirect bandgap of 1.27 eV, and Pt loaded CaCu3Ti4O12 showed the degradation of MO under visible irradiation. Double perovskite materials such as Sr2CoWO6, Ba2CoWO6, and Sr2NiWO6, Ba2NiWO6, are reported to be stable for O2 evolution with sacrificial agents.124,125 Although the photophysical properties of certain double perovskites also have been investigated and reported, still a lot of efforts are required before utilizing these materials on an industrial scale.The researchers have investigated photocatalytic activity of rare earth and bismuth-based double perovskites under visible light. Double perovskites Ba2 reduction, and nitrogen fixation. The critical perovskite materials for each photocatalytic application are also listed with their properties in this study. Overall, it can be inferred that although perovskite oxide-based materials have exhibited significant photocatalytic performance still, extensive challenges are ahead of their design, fabrication, cost, and efficiency for industrial and large-scale production. The biggest challenge in the industrialization of photocatalyst technology is the development of an ideal photocatalyst, which should possess four features, including high photocatalytic efficiency, a large specific surface area, full utilization of sunlight, and recyclability. Nonetheless, there is no doubt that perovskite-based oxide materials will be investigated more intensively in the coming years due to their exceptional properties and applications for a sustainable future.In summary, the abundantly available sun energy can be harvested through photocatalysis to deal with the concerns pertaining to the environment and humankind. This article aimed to discuss the imperative properties of perovskite materials which play vital role in photocatalysis, and will assist in understanding the fundamentals of photocatalytic mechanisms involved in designing highly efficient photocatalysts. The three altercation sites  of perovskite materials ma them suitable for numerous applications, particularly photocatalysis. These sites help in tailoring chemical, physical, optical, and photocatalytic properties for desired photocatalytic reactions. The structural and compositional suppleness in perovskite photocatalyst strongly affects photo-generated carriers' mobility, separation, and recombination. The current article also describes the defect engineering in perovskite materials for enhanced photocatalytic performance. Recently, defect engineering gained much attention among researchers because it resulted in visible light photocatalytic activity without doping. Furthermore, surface defects provide reactive sites beneficial for a process like nitrogen fixation at ambient conditions. The review also provides an insight into the different applications of photocatalysis, including wastewater treatment, water splitting, COAll authors verify their contribution to the current review article as follows: design, study conception, and supervision of the whole article is done by Muneeb Irshad and Quar tul Ain; data collection is done by; Asif Nadeem Tabish, Muhammad Usman, Masood ul Hassan Farooq, Mohammed A. Assiri, Muhammad Imran; analysis and interpretation of results by; Muneeb Irshad, Quar tul Ain, Muhammad Zaman, Muhammad Zeeshan Aslam, Naila Kousar, Muhammad Asim, Muhammad Rafique, and Khurram Siraj; draft manuscript preparation and proofreading by; Muneeb Irshad, Quar tul Ain, and Muhammad Zaman. Moreover, Mohammed A. Assiri and Muhammad Imran provided the necessary funding to complete the article. All authors reviewed the results and approved the final version of the manuscript.There are no conflicts to declare.


The effect of the replacement rate of silica fume (SF) on the correlation between the compressive strength and ultrasonic pulse velocity (UPV) of cement mortar was experimentally analyzed. Specimens were fabricated with different replacement rates of SF, the compressive strength and UPV were measured, and isothermal calorimetry and mercury intrusion porosimetry tests were conducted to analyze the effects of replacement on the hydration kinetics and microstructures on these properties. Field emission scanning electron microscopy analysis was performed to observe SF particles and microstructure. The substitution of SF changed the cement mortar’s hydration kinetics and microstructures, resulting in different strengths and UPVs depending on the replacement rate. The compressive strength and UPV for cement mortars blended with SF also showed a different exponential relationship depending on the SF replacement rate. Dotto e studies . Althoug studies ,10,11,12 studies ,14,15.The mechanical properties of cementitious materials blended with SF are sensitive to environmental factors, mixture proportions, raw-material properties, and age, compared with those of normal cementitious materials ,17. TherThis study aimed to comprehensively investigate various factors related to the compressive strength and UPV of cement mortars blended with SF and to analyze their correlation. To this end, cement mortar specimens with SF replacing other binder materials at replacement rates of 0, 10, 20, and 30% were fabricated. The compressive strength and UPV of each specimen were measured at 1, 3, 7, 14, and 28 days of age. The microstructure and hydration kinetics were analyzed by mercury intrusion porosimetry (MIP) and isothermal calorimetry experiments, respectively. The variation in the compressive strength and UPV of each specimen and their correlation were determined. Field emission scanning electron microscopy (FE-SEM) analysis was also performed to evaluate the effect of age and SF addition on the microstructural change of the cement matrix.2 content of SF was 93.1%. The surface areas of the cement and SF were 3300 and 217,000 cm2/g, respectively. 2 by mass. A polycarboxylic acid chemical admixture (Rheobuild SP8HU (SR), BASF Japan Ltd., Tokyo, Japan) was used to enhance the workability.Type-I Portland cement  and SF  were used as binder materials. The SiODry cement, SF, and sand were mixed for 5 min to ensure a homogenous distribution. Next, water and chemical admixture were added to the mixture and mixed at a low speed (140 ± 5 rev/min) for 3 min. The wall of the mixer bowl was scraped to prevent heterogeneous mixing by the mixture attached to the wall, and the fresh cement mortar mixtures were mixed at an intermediate speed (285 ± 10 rev/min) for 5 min. The mixtures were then cast into molds.3 beam-shaped mold, they were cured at room temperature (23 ± 1 °C) under sealed conditions, using aluminum foil tape, to prevent moisture escaping. Upon completion of curing, the specimens were split in half in the transverse direction and the compressive strength was measured. The arrival time of the ultrasonic pulse was measured with a cylindrical specimen having a size of 100 × 200 mm2 using the Ultracon-170 ultrasonic detector  for UPV analysis. The transducer has a frequency of 52 kHz. The specimen was cured in a sealed state in the same environment as the compressive strength measurement specimen.The compressive strengths of the cement mortar specimens were measured at different ages  in accordance with the ISO 679 standard. After casting the fresh cement mortar mixtures into a 40 × 40 × 160 mm3 cuboid shapes. Subsequently, the specimens were immersed in an acetone solution for 24 h at room temperature in order to stop the hydration reaction. Thereafter, the specimens were placed in an oven at 60 °C for 24 h to remove acetone and moisture from the specimens. Finally, measurements of total porosity and pore-size distributions of the specimens at 1, 3, 7, 14, and 28 days of ages were performed using Micromeritics’s AutoPore IV9500 . In addition to the MIP test, FE-SEM  analysis was also performed on a fractured specimen to confirm the microstructure of the cement matrix after measuring the compressive strength in the same manner as the MIP test. The FE-SEM used in this study has the advantage of less radiation damage to the sample because it can observe high magnification at a low-speed voltage. The specimens were cut into 1 × 1 mm2 thin film forms, which were immersed in acetone solution and dried in an oven. Subsequently, their surfaces were coated with Pt prior to the SEM analysis.To measure the heat evolution rate of the specimens with age, an isothermal calorimetry test was conducted using a TAM-AIR isothermal calorimeter  in accordance with ASTM C1702 standards. After mixing fresh cement mortar, approximately 4 g of the mixture was put into a glass ampoule, and the isothermal calorimetry test was conducted at room temperature for 28 days. The MIP test for microstructure analysis was performed using the specimens fractured after measuring the compressive strength. The specimens were cut into 10 × 10 × 10 mm2+ ions are rapidly released from the cement particles. SF particles adsorb calcium ions on the surface and reduce the concentration of Ca2+ in the pore solution, boosting the dissolution of C3S and accelerating the hydration reaction [reaction . After tTwo transducers were placed on both ends of each cylindrical cement mortar specimen, and the UPV (V) was determined by:Since the ultrasonic pulse is transmitted through the solid phases, the UPV of cementitious materials having porous characteristics has a proportional relationship with the volume fraction of the solid phase and an inverse relationship with porosity ,38. TherAs seen in As SF replacement rates increased, the hydration kinetics decreased at early ages, but it increased due to SF’s pozzolanic reaction at a later age. In addition, SF reduced the porosity of the cement mortar by acting as a filler in the pores. The number of mesopores increased and capillary pores decreased, as the content of SF increased.As the SF replacement rate increased, the compressive strength of cement mortar increased. The strength increased because of the filler effect of SF at an early age, and the strength increased through the pozzolanic reaction in combination with the filler effect at later ages.The volume fraction of the solid phase increased as the pores filled with moisture were filled with hydration products with age. The UPV of the specimen gradually increased as the solid phases were connected. The development of UPV showed a nonlinear relationship with the replacement rate of SF, which is closely related to the density and microstructure of the specimen.The UPV and compressive strength of cement mortar have an exponential relationship, which differs by the replacement rate of SF. In general, the substitution of SF had a significant effect on the relationship of the exponential function over the reference specimen; however, variation in the replacement rate of SF was found to have a relatively minor effect.In this study, the correlation between the compressive strength and UPV of cement mortars blended with SF was analyzed for up to 28 days. For this purpose, cement mortars were prepared for varied SF replacement rates, and their compressive strengths and UPVs were measured. Additionally, isothermal calorimetry and MIP experiments were conducted to analyze the hydration kinetics and microstructure, to assess how the replacement rate of SF affects the correlation between compressive strength and UPV. The test results can be summed up as follows:In previous studies on the relationship between compressive strength and UPV, a correlation analysis was performed for all specimens regardless of the mixture proportion of cementitious materials. The present work evaluated the effect of hydration kinetics and microstructure changes on UPV and compressive strength due to the difference in mixture proportions and examined the correlation between comprehensive strength and UPV for each mixture. The findings from this study can be used to estimate the compressive strength of cementitious materials blended with SF and provide preliminary comparison data for studies that aim to analyze the correlation between the compressive strength and UPV of cementitious materials containing SF and other SCMs.


The results of experimental and clinical trials of the agents based on oncolytic Newcastle disease virus (NDV) strains provided hope for the development of virotherapy as a promising method for treating human tumors. However, the mechanism of the antitumor effect of NDV and realization of its cytotoxic potential in a cancer cell remains to be elucidated. In the current work, we have studied the antitumor effect of NDV in a syngeneic model of mouse Krebs-2 carcinoma treated with intratumoral injections of a wild-type strain NDV/Altai/pigeon/770/2011. Virological methods were used for preparation of a virus-containing sample. Colorimetric MTS assay was used to assess the viability of Krebs-2 tumor cells infected with a viral strain in vitro. In vivo virotherapy was performed in eight-week-old male BALB/c mice treated with serial intratumoral injections of NDV in an experimental model of Krebs-2 solid carcinoma. Changes in the tumor nodes of Krebs-2 carcinoma after virotherapy were visualized by MRI and immunohistological staining. Light microscopy examination, immunohistochemical and morphometric analyses have shown that intratumoral viral injections contribute to the inhibition of tumor growth, appearance of necrosis-like changes in the tumor tissue and the antiangiogenic effect of the virus. It has been established that a course of intratumoral virotherapy with NDV/Altai/pigeon/770/2011 strain in a mouse Krebs-2 carcinoma resulted in increased destructive changes in the tumor tissue, in the volume density of necrotic foci and numerical density of endothelial cells expressing CD34 and VEGFR. These results indicate that intratumoral NDV injection reduces tumor progression of an aggressive tumor. The widespread acceptance of the idea of using viruses as antitumor agents and encouraging results of successful experimental treatment of tumors obtained in numerous preclinical studies have led to the establishment and further development of targeted cancer therapy (virotherapy), which is based on the use of infectious oncolytic agents for the selective destruction of tumor cells. A wide range of wild-type and genetically modified viruses have been proposed as oncolytic agents ,2. One sAn important trend in the study of viruses with oncolytic potential is the identification of principally new strains allowing the use of wild-type non-pathogenic isolates as an antitumor agent. The use of animal viruses  that are non-pathogenic for humans in virotherapy can significantly reduce the risks of infectious diseases. Since NDV is an avian virus, it is not eliminated by the mammalian immune system due to the presence of the antibodies to the viral agent in the blood, while the existence of natural immunological response to the adenoviruses most commonly used in virotherapy, measles, herpes simplex, and mumps viruses limits their clinical use ,6,7,8,9.NDV requires neither additional development of the strategies for the effective viral delivery  nor overcoming the host antiviral immune response (since it belongs to the avian viruses).NDV strains are divided into two groups based on their ability to infect human cells and replicate in them: lytic and non-lytic viruses. When studying both groups of NDV strains, several possible concepts for the use of their antitumor potential for the effective treatment of tumors have been proposed, which has led to the emergence of different virotherapy approaches. One of them is the use of lytic NDV strains for the selective destruction of malignant cells via the rapidly spreading cytotoxic effect on tumor cells due to effective viral replication and release of numerous infectious viral particles ,11.Most scientific papers that suggest possible mechanisms of virus-mediated tumor cell death mention apoptosis as the main specific pathway for manifestation of the antitumor cytotoxic potential in NDV-infected cells. While apoptotic cell death is rarely detected in a tumor of epithelial origin in in vivo experiments on antitumor therapy, in vitro apoptosis of cancer cells can be only caused by relatively high drug doses, which are high in terms of therapeutic doses for humans . In receIn addition to the cytotoxic effect causing direct destruction of tumor cells, oncolytic viruses have also attracted researchers’ attention due to their antiangiogenic properties ,17,18. NThere are few experimental examples of the antiangiogenic properties of NDV. Nevertheless, a number of studies have shown that NDV strains can inhibit angiogenesis. An in vitro experiment in glioblastoma tumor cells demonstrated a significant antiangiogenic effect 18 h after infection of the cells with the Iraqi NDV strain as compared to the untreated control cells. Suppression of angiogenesis is observed upon inhibition of the expression of angiogenic factors such as fibroblast growth factor, keratinocyte growth factor, vascular endothelial growth factor, and inter-cellular adhesion volecule-1 ,22. ThusSui  and the In addition, it has been shown that hypoxia in NDV-infected tumor tissue, which often occurs during tumor development, enhances NDV-mediated oncolysis of human kidney cancer cells by a velogenic strain AF2240 isolated in Malaysia [Another approach to virotherapy with NDV is based on the idea of a therapeutic antitumor effect via stimulation of non-specific immunity through the induction of cytokines and interferons, better recognition of tumor antigens by the immune system and stimulation of cytotoxic cells: natural killers and cytotoxic T lymphocytes ,26.The encouraging results obtained in preclinical and clinical trials on the antitumor effect of viruses dictate the need for further research on the virus-cell interaction mechanisms leading to the death of tumor cells.In previously published studies, the NDV/Altai/pigeon/770/2011 strain  demonstrNewcastle disease virus NDV/Altai/pigeon/770/2011 strain  with an The presence of NDV in the allantoic fluid was determined by hemagglutination assay and real-time RT-PCR with specific primers. Viral RNA was isolated from the virus-containing liquid (VCL). Primary identification of the isolated Newcastle disease virus was performed by RT-PCR with real-time detection in a closed tube using primers and a probe specific to the conserved regions of the Newcastle disease virus M gene according to .Experimental studies with Newcastle disease virus were carried out in accordance with the sanitary safety requirements for working with microorganisms of pathogenicity groups III–IV and helminths .An implantable strain of Krebs-2 ascites carcinoma was kindly provided by a senior scientist at the Laboratory of Gene Expression Regulation of the Institute of Cytology and Genetics, Popova N.A, PhD. Krebs-2 tumor cells were maintained through regular passages in the mouse. Having first occurred as a spontaneous breast tumor in a hybrid mouse, Krebs-2 was further converted to the ascitic form and, to date, Krebs-2 cells are presented with poorly differentiated forms. Krebs-2 cells do not contain genes of the major histocompatibility complex. Therefore, they are effectively transplanted into inbred mouse lines. A solid node is formed in the thigh region upon intramuscular implantation. The tumor does not give rise to metastases.2 incubator. Next, the cells were concentrated by centrifugation at 1500 g for 5 min, virus-containing supernatant was removed, and the cell pellet was resuspended in fresh growth medium and seeded in a 96-well plate at a concentration of 10,000 cells per well in a volume of 100 μL. Control untreated cells were incubated in growth medium under the same conditions.To evaluate the cell viability after infection with Newcastle disease virus, suspension culture of mouse Krebs-2 ascites carcinoma was infected with a NDV strain. For this, a suspension of Krebs-2 cells in DMEM medium was aliquoted into Eppendorf microtubes. Cells were concentrated by centrifugation at 1500 g for 5 min, the supernatant was removed, and the cells were incubated with different dilutions of the virus: 2, 8 and 16 HAU per 10,000 cells. Virus dilutions were prepared in DMEM medium (Gibco Inc.) containing 10% FBS (Gibco Inc.) and 60 μg/mL gentamicin sulfate. Cells were incubated with the virus for 1 h 30 min at 37 °C in a 5% CO2. The optical density was measured at a wavelength of 490 nm on an Elx808 Absorbance Microplate Reader (Biotek).Cell viability was measured by colorimetric assay using CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega) according to the manufacturer’s protocol four days after infection with NDV strains. The reagent contains a tetrazolium compound, MTS, which is reduced to formazan and stains intact cells with enzymatic activity. A total of 20 μL of the reagent was added to each well containing 100 μL of culture medium; cells were incubated for 2 h at 37 °C in the presence of 5% COViability of the control Krebs-2 carcinoma cells was evaluated 4 days after infection with NDV strains based on the percentage of living cells versus the untreated control.Animals were fed a standard diet with free access to food and water. The adaptation period was one week prior to the onset of experiments. The study was carried out in compliance with the principles of humanity described in the Guidelines of the European Community (86/609/EEC), Helsinki Declaration and in accordance with the Order No. 755 of the USSR Ministry of Health on 12.08.1977 “On measures for further improvement of organizational forms of work using experimental animals”. Viral experiment consisted of two stages.At the first stage, a pilot experiment was performed using 4 eight-week-old male -BALB/c mice. Krebs-2 ascites carcinoma was implanted intramuscularly into the thigh of all mice at a concentration of 500,000 cells in 100 μL of PBS. On the third day after implantation, two experimental mice were injected intratumorally in the thigh with 100 μL of the NDV/Altai/pigeon/770/2011 strain at the maximum concentration (256 HAU per 100 μL), and two control mice received physiological saline. Single injections were administered daily for 5 days. On day 28 after tumor implantation, the spread of the intramuscular tumor process was assessed by magnetic resonance imaging (MRI) at the Center for Genetic Resources of Laboratory Animals of the SPF Vivarium at the Institute of Cytology and Genetics. After MRI, animals were sacrificed by dislocation of the cervical vertebrae, solid tumor node with the surrounding muscle tissues were collected for further morphological examination by light microscopy.To study the dynamics of tumor progression and pathomorphological changes in Krebs-2 carcinoma tissue treated with intratumoral injections of the NDV/Altai/pigeon/770/2011 strain, the second stage of the in vivo experiment was carried out. The study was conducted in 60 7-to-8-week-old white male BALB/c mice obtained from the mouse bank of the State Research Center of Virology and Biotechnology Vector . A total of three groups of animals, each consisting of 20 mice, were formed. Krebs-2 ascites carcinoma was implanted intramuscularly into the thigh of all mice at a concentration of 500,000 cells in 100 μL of PBS. The first control group was comprised of animals not receiving injections after tumor implantation. The second, control , group consisted of animals receiving injections of the uninfected (virus-free) allantoic fluid of the chick embryo. The third, treated, group included animals that received a five-day course of intratumoral virotherapy starting from the third day after tumor implantation at a dose of 100 μL of the NDV/Altai/pigeon/770/2011 strain containing 256 HAU per 100 µL.Viral injection dose, the scheme of virotherapy and allantoic fluid administration, the volume of injections, and housing conditions were completely similar to those of the first experiment. Animals were weighed on a daily basis. Tumor growth was daily assessed by measuring the affected limb using a caliper. The tumor volume (mm3) value was obtained by multiplying three orthogonal measurements. On day 5, 10, 15, and 20 after completion of the injection course , 5 animals of each group were sacrificed at a time by dislocation of the cervical vertebrae; tissue samples were collected similarly to the first experiment.For light-optical examination, a Krebs-2 tumor tissue sample isolated from the mouse thigh muscle was fixed in 10% formalin at +4 °C for three days. Next, according to a standard protocol, the samples were dehydrated in a series of graded concentrations of alcohol, then butanol and xylene and embedded in HISTAMIX paraffin. The obtained paraffin blocks were divided into 4.5–5-μm-thick sections on a Microm HM355S rotary microtome (Thermo Fisher Scientific). Deparaffined sections were subjected to hematoxylin-eosin overview staining according to the standard procedure and embedded in Vitrogel mounting medium. The stained samples were examined on an Axio Imager A1 microscope with an AxioCamMRc camera using AxioVision software (rel. 4.12) (ZEISS) at the “Modern Optical Systems” core facility of the Federal Research Center of Fundamental and Translational Medicine.Polyclonal antibodies against the whole virion of Newcastle disease virus were generated by twice immunization of rabbits with the NDV/Altai/pigeon/770/2011 strain in complete Freund’s adjuvant with a 23-day interval. On day 46, blood serum samples treated with receptor-destroying enzyme (RDE) were obtained and analyzed for the presence of specific antibodies. The IgG fraction of rabbit antiserum samples was purified by affinity chromatography on a protein A-Sepharose 4B column in 0.02 M potassium phosphate buffer (pH 7.3), 0.15 M NaCl, 0.02% sodium azide at the laboratory of Biotechnology of the Institute of Chemical Biology and Fundamental Medicine .For virus detection, tumor node sections were dewaxed and rehydrated, antigens were unmasked in citrate buffer (pH 9.0) in a microwave oven at 700 W. Sections were incubated with primary antibodies at 37 °C for 30 min, then with streptavidin–peroxidase complex, NovoLink DAB substrate buffer (Novocastra) and additionally stained with Mayer’s hematoxylin.Krebs-2 tumor samples were also stained by immunohistochemistry  with pri5 μm2; 50–100 visual fields were counted. Morphometric study included evaluation of the degree of structural changes in the tumor tissue taking into account the volume density of necrosis (Vv), numerical density of the microvasculature vessels (Nai) and endothelial cells expressing the selected IHC markers.Histological samples were analyzed on an Axio Imager A1 microscope with an AxioCamMRc camera using AxioVision software (rel. 4.12) (ZEISS). Morphometric analysis of the tissue structural elements was conducted using a closed test system consisting of 100 testing points in a testing area equal to 3.64 × 10p < 0.05. Cell viability assessment results are presented on a histogram as relative mean values of the percentage of living cells on the fourth day after treatment with virus to the percentage of control untreated cells plus standard deviation .Mean values of the parameters for the assessment of Krebs-2 cell viability were determined using the standard STATISTICA V.6.0 software package; Student’s t-test was used. The differences were considered significant at The results of the colorimetric MTS assay presented as a histogram in In a pilot experiment, Krebs-2 tumor cells were implanted intramuscularly in the right thigh of all four mice. On the day 3 after implantation, a palpable mass was observed, which is a characteristic of tumor formation. NDV/Altai/pigeon/770/2011 virus strain was injected intratumorally in the experimental group of mice starting from the onset of tumor formation (day 4 after tumor implantation). For this purpose, 100 μL of VCL was injected into the tumor node site in the experimental animals at a concentration of 256 HAU per 100 μL. No specific conclusions regarding changes in the tumor size, its progression or the onset of regression can be drawn during the next five days of the experiment, in which intramuscular injections of the virus were performed, since the inflammatory process develops in the tumor area in the first stages of virus introduction, which causes swelling and hinders visual assessment of possible changes.Tumors implanted in control animals were treated with saline injections according to a similar scheme and at the same doses as for the experimental group. Similar processes  were observed in the control group receiving intratumoral injections of saline.On day 10 after tumor cell implantation, the tumor-bearing thigh of the control mice was visually larger in size due to tumor growth than in experimental mice receiving virotherapy with a NDV strain. During the experiment, mice of the both groups actively consumed food and water. However, control animals not receiving virotherapy became less active by day 14 of the experiment.In order to visualize the tumor node growth in detail in the control group and compare the differences in the size of the intramuscular tumor with those of the experimental group animals, MRI was performed on day 28 of the experiment. The tumor is clearly visualized next to the normal tissues in a series of frontal sections on MRI. The obtained MRI data revealed almost complete tumor regression in one experimental mouse and a significant tumor reduction in another mouse compared to the control animals ; intramuStudy of the paraffin sections of tumor nodes obtained from experimental animals receiving intratumoral injections of the NDV/Altai/pigeon/770/2011 strain confirmed the almost complete absence of a tumor node  in one mouse . NecrotiCells of the immune system, such as neutrophils, lymphocytes, and macrophages, were detected in the muscle tissue adjacent to the tumor node , which iA solid node was visually noticeable at the site of tumor cell implantation on day 7 of the experiment. Tumor growth and progression were observed in the control groups, while a decrease in the tumor growth rate was noted in the group receiving virotherapy.Macroscopic examination showed that the tumor node in the thigh has a round-oval shape with clear boundaries with the surrounding tissue, hemorrhages and necrosis foci appear with an increase in the tumor volume. Light optical microscopy revealed that the central tumor region is represented by broad areas of necrosis containing islets of large tumor cells of various sizes. The subcapsular tumor area presents dense tissue consisting of poorly differentiated cells with large nuclei, in which numerous impaired mitotic events are observed.On day 20 after the end of the series of injections, the average size of the tumor-bearing thigh in the group of mice receiving live virus injections was 1779.1 ± 79.98 mm3, which is 2.6 times lower than that of the control group and the group receiving injections of uninfected chick allantois: 4659.9 ± 704.75 mm3 and 4899.1 ± 807.69 mm3, respectively . Tumor dThe microscopic specimen was tumor tissue consisting of atypical round tumor cells; a large amount of necrotic cells were noted. Muscle fibers destroyed due to invasive tumor growth were observed on the microscopic slides. No metastasis was observed.Spread of the necrotic area was observed in the tumor tissue of the control untreated and experimental animals receiving live virus . A totalLive virus injections triggered necrotic processes on day 5, which occupied 16.7 ± 2.35% of the tissue volume density. This parameter slightly changed by the day 10 and 15 and estimated 15.5 ± 1.51% and 13.6 ± 1.95%, respectively. On the day 10 and 15, the value of the volume density of necrosis corresponded to that of the control group. However, by the 20th day, the value increased to 25.7 ± 2.68%.As a result, an increase in the volume density of necrosis is observed in the tumor node in all groups of animals. However, in the control group, the growth rate was the slowest with a maximum of 19.8 ± 3.55%, while intratumoral injection of the wild-type NDV/Altai/pigeon/770/2011 strain by the day 5 contributes to the high necrosis density value, which increases significantly (to 25.7 ± 2.68%) by the day 20.Numerical density of blood vessels in the tumor tissue was found to be approximately 2 or more times lower after virus injection than in the control group. Morphometric analysis of large blood vessels  in the tIn order to more accurately estimate the number of vessels and study the mechanism of neoangiogenesis inhibition, the numerical density of endothelial cells expressing hematopoietic marker CD34 and marker VEGFR was determined. Expression of these markers was observed in the tumor cells of control and treated mice, with a tendency of the number of cells immunopositive for CD34 and VEGFR to decrease in the treated group.The numerical density of CD34+ cells in the tumor of the treated group was lower by 72.69% and by 57.57% than in untreated mice by the day 5 and 20 after virotherapy, respectively. By day 20 after virotherapy, the number of CD34+ cells was 2.36 times lower in the tumor of mice treated with the virus than in untreated animals, with a 62.3% difference in the parameter from that of the treated animals by day 5 .An IHC study of VEGFR expression in the tumor of mice inoculated with NDV/Altai/pigeon/770/2011 showed a significant decrease in the numerical density of VEGFR-expressing cells (by 37.45%) compared to that for the non-injected group on days 10 and 20 of observation .Oncolytic viruses as agents for antitumor therapy offer several important advantages over traditional cancer treatment approaches and other targeted therapies: (1) tumor-selective infection and viral reproduction in the tumor cell, leading to death, without cytotoxic effects on healthy cells; (2) involvement of infected tumor cells in cytolytic death by direct membrane lysis, in addition to activation of cell death mechanisms; (3) viral reproduction by replication in cancer cells, ensuring the spread of infectious particles in the tumor tissue after the release of the viral offspring, which increases the number of active agent and the number of infected cells and, as a result, increases the therapeutic effect; (4) the death of tumor cells (by direct oncolysis or programmed cell death) increases the pro-inflammatory microenvironment of the tumor and the involvement of immune system cells.With all of these broad therapeutic options, tumor NDV-therapy has great potential for cancer treatment , but forStudies conducted in an animal model of experimental oncogenesis were aimed at investigating the effect of the course of virotherapy injections of the wild-type strain NDV/Altai/pigeon/770/2011 on the progression of mouse Krebs-2 carcinoma in vivo. The animal model and the method of virus introduction were selected in a pilot experiment on virotherapy of mouse Krebs-2 carcinoma. To confirm the oncolytic potential of Newcastle disease virus, the viability of mouse Krebs-2 tumor cells was first assessed in an in vitro system using the selected mesogenic strain NDV/Altai/pigeon/770/2011.The in vitro study result demonstrates that the NDV/Altai/pigeon/770/2011 strain can be used for further works on experimental virotherapy in an animal model despite the fact that the oncolytic potential in relation to the mouse Krebs-2 carcinoma cells is significantly lower than that obtained in a similar experiment using human tumor cell lines .Pilot experiment was performed on a small number of animals. The aim of the study was to assess intramuscular oncogenesis of the Krebs-2 carcinoma with the formation of a solid tumor node in an in vivo model, to monitor the condition of animals during tumor progression and to preliminary estimate the antitumor effect of a series of intratumoral injections of the wild-type strain NDV/Altai/pigeon/770/2011 of the Newcastle disease virus.The second stage of the experiment on the model of mouse Krebs-2 carcinoma was aimed at the study and assessment of changes in the development of a solid tumor node at different time points after a course of intratumoral virotherapy and evaluation of morphological changes in the tumor tissue after virus injection in comparison with untreated animal tumors.The obtained data on the tumor growth inhibition after serial intratumoral injections of Newcastle disease virus required clarification of the morphological changes and possible mechanisms of tumor growth suppression.Necrotic foci are observed in the experimental group at different time points as compared to the tumor tissue of control animals and animals receiving a course of virotherapy on day 5 and day 20 . IntratuAssessment of the immune status after a course of virotherapy is complicated, since no cells involved in immune response activation  were found in the tumor tissue of experimental animals. Presumably, this may indicate a predominantly direct effect of the virus on the tumor tissue via direct oncolysis of tumor cells with the appearance of extensive necrotic foci.It is possible that the appearance of extensive necrotic lesions in the control group may reflect the formation of ischemic foci in the tumor tissue resulting from the rapid tumor node growth and delayed neoangiogenesis.We suggest that extensive necrotic lesions in the control group may reflect the formation of ischemic foci in the tumor tissue resulting from the rapid tumor node growth.There is a correlation between an increase in the tumor node volume and an increase in the volume density of necrosis in the tumor tissue, which is due to the rapid tumor growth and the lack of feeding vessels. However, it should be noted that virus injections contribute to necrotic changes in the earlier stages of tumor growth and the appearance of a significant proportion of necrosis at a later date.A sharp decrease in the number of large visually distinguishable vessels in the tumor tissue of the control animals on day 10 is, apparently, due to the high tumor growth rates, when new vessels do not have time to form and, as a result, nutrient supply of the tumor tissue is disturbed. On the contrary, an increase in the number of vessels by day 20 indicates that neoangiogenesis takes place in the tumor tissue.There is almost a 5-fold difference in the number of large vessels between the control and experimental groups on day 20 of the experiment. Apparently, a small number of vessels in the experimental group on the 20th day indicates that virus is able to directly or indirectly affect neoangiogenesis of the growing tumor by regulating nutrient supply to the tissue.Based on the estimation of numerical density of endothelial cells expressing CD34 in Krebs-2 carcinoma tissue, we suggest that intratumoral injection of the strain NDV/Altai/pigeon/770/2011 indicates that virus is able to directly or indirectly affect neoangiogenesis of the developing tumor, interfere with normal regulation of the nutrient supply to the tumor tissue and result in extensive necrotic lesions.In order to explore the mechanisms of neoangiogenesis inhibition, an IHC study was carried out using a marker to the VEGF receptor, which is involved in the signal pathways that serve as a target for the majority of drugs inhibiting angiogenesis.It is possible that the virus facilitates suppression of the angiogenic signal by decreasing the expression of endothelial factors and suppressing vascularization after viral therapy. We suggest that one of the mechanisms responsible for the delayed processes of neoangiogenesis in the tumor tissue may be the suppression of VEGF receptors.Thus, the conducted study demonstrated that intratumoral injections of the wild-type NDV/Altai/pigeon/770/2011 strain in BALB/c mice with intramuscular Krebs-2 carcinoma had a restraining effect on the tumor node growth in comparison with the untreated animals. There is no doubt that virus-induced necrotic changes in the tumor serve as the main mechanism for the Krebs-2 carcinoma cell death. Apparently, the effect of intratumoral injections of the NDV/Altai/pigeon/770/2011 strain on tumor angiogenesis is an additional mechanism suppressing tumor growth, which affects regulation of the nutrient supply to the tumor tissue and, as a result, leads to the spread of extensive necrotic foci.Apparently, the Newcastle disease virus circulating in a population of wild birds is a promising object for the analysis on the presence of antitumor properties. NDV isolated from a natural reservoir has been shown to be able to successfully kill tumor cells with various etiology and histogenesis. Modern research methods have made it possible not only to determine the effectiveness of the oncolytic effect of NDV strains but also study the mechanism of virus-mediated cell death, explore the interaction between the virus and the tumor cell and analyze the effect of the virus on the features of the immune response activation.Furthermore, the obtained results indicate that wild-type NDV strains can serve as the basis for creation of an effective antitumor drug. The cytotoxic action of the NDV strains on the tumor cell lines and safety for the healthy cells demonstrates their effectiveness in the fight against cancer. Study of the antitumor activity of NDV in laboratory animals with implanted tumors provides data on the antitumor potential and the possibility of conducting clinical trials on oncotherapy in humans. Understanding of the processes of selective cytotoxic action, the mechanisms of the spread and cell tropism of the virus, its ability to affect angiogenesis in the tumor tissue as well as the immune system is important for the development of a prototype drug based on a wild-type NDV strain.In conclusion, the intratumoral injections of the wild-type NDV/Altai/pigeon/770/2011 strain in BALB/c mice with intramuscular Krebs-2 carcinoma have a restraining effect on the tumor node growth. Furthermore, an additional mechanism suppressing tumor growth with NDV treatment is possibly regulation of the angiogenesis in tumor that leads to the spread of extensive necrotic foci. These results suggest that wild-type NDV strain has the antitumor properties to be able to successfully kill tumor and strains can serve as the perspective effective antitumor drug.


N,N-diisopropylamino)ethyl methacrylate and 2-methacryloyloxyethyl phosphorylcholine was investigated in aqueous media. Their pH-responsive behaviors were investigated in aqueous media by dynamic light scattering (DLS) and fluorescence measurements using a pyrene hydrophobic fluorescence probe. In an acidic environment, these copolymers existed as single polymer chains that did not interact with each other. In contrast, upon increasing the pH of the solution above the critical value of ~8, separated micelles were formed in the mixture, which was indicated by bimodal distribution in DLS results with radius of 4.5 and 10.4 nm, corresponding to the random and block copolymer micelles, respectively. Fluorescence resonance energy transfer efficiencies were near to zero in the mixture of the donor labeled block and acceptor labeled random copolymers under both acidic and basic pH. These results demonstrated the coexistence of two distinct micelles.The self-assembly of pH-responsive random and block copolymers composed of 2-( N,N-Diisopropylamino)ethyl methacrylate (DPAEMA) is one of the most widely used compounds for producing pH-responsive polymers with an acid constant (pKa) of 6.8 0 are the initial concentrations of the monomers and CTA, respectively, p is the percentage of conversion, and Mm and MCTA are the molecular weights of the monomer and CTA, respectively.Random and block copolymers were prepared via RAFT. After collecting the samples by freeze-drying, the polymer powders were dissolved in methanol-polymers . The molMn(GPC) and Mw/Mn of the copolymers, GPC measurements were performed using a 0.3 M Na2SO4 aqueous solution containing 0.5 M acetic acid as an eluent  estimated using the NMR spectra. However, the Mn(GPC) values were about twofold lower due to the different principles of these methods. Thus, Mn(NMR) is an absolute value calculated from the intensity ratios of resonances corresponding to protons of the polymers, whereas Mn(GPC) is estimated using a standard polymer sample having a different chemical structure from that of our polymer. In addition, it should be noted that although the random and block copolymers were designed to exhibit similar DPs and their Mn(NMR) and Mn(theory) were also similar, the Mn(GPC) of the random copolymer was lower than that of the block copolymer (Mn(GPC) values.To determine the n eluent . Narrow opolymer . This caI3/I1) increase. Therefore, aqueous solutions of the random and block copolymers and their equimolar mixture at different pH values from 3 to 11 were subjected to fluorescence measurements. The obtained I3/I1 values were plotted as a function of the solution pH to determine the critical pH values, which were found to be close to 8 for all the samples  close to 0.2 Mcps, suggesting that both copolymers were totally dissolved in water, and no attractive interaction between the random and block copolymers occurred at acidic pH. At pH 10, both Rh values  for the random copolymer was close to unity (0.83), which means that each single polymer chain formed unimolecular micelles in the aqueous solution. In contrast, about seven polymer chains aggregated in the polymer micelles of the block copolymer solution. These different behaviors were caused by the difference in the sequence of the hydrophobic and hydrophilic units in the polymer chains and in the balance between the hydrophilic and hydrophobic interactions. In the mixture of the random and block copolymers, the Rg value was close to that of the block copolymer micelles, whereas intermediate values between those of the random and block copolymer single solutions were obtained for Mw and Nagg, which was probably caused by the coexistence of two distinct random and block copolymer micelles.The polymers were further characterized by performing SLS measurements . Accordil shapes ,33,34. Id) of the copolymer micelles was estimated using Equation (2):V is the volume of the micelle calculated according to the expression 4/3πRh3, Mw(SLS) is the molecular weight of the micelle estimated from the SLS measurement, and NA is Avogadro’s number. The results are listed in d value of the aggregates produced from the block copolymers was smaller than that of the random copolymers. This implies that the block copolymer micelles were more hydrated than the random copolymers micelles, which is presumably due to the presence of the stabilizing hydrophilic PMPC blocks with a DP of 52 in the copolymers. This result is also consistent with the Rg/Rh of 1.12 obtained for the block copolymer micelles. Besides, the random copolymers self-folded to form compact unimer micelles, which contributes to the higher density of this sample.The density (1H NMR spectra recorded in D2O at a Cp of 10 g/L and pH 3 (Mn and Mw/Mn of the labeled copolymers were similar to those of the copolymers before labeling (1H NMR and GPC results prove that the structures of the copolymers were well maintained after labeling. Using the absorbance and molar extinction coefficient of A555 at 556 nm and A488 at 495 nm (R0), which is the distance at which the fluorescence resonance energy transfer from the donor dye to the acceptor dye is 50% efficient. The R0 value of the A488 and A555 pair has been reported to be 7 nm [R0 value, and FRET would not occur. In addition, if the labeled random and block copolymers are dissolved in a poor solvent at a sufficiently high concentration of the fluorophore for donor and acceptor to be close enough to ensure the energy transfer, highly efficient FRET could be achieved . Fluorescence spectra were recorded for these samples at pH 2 and 11 (E) of the energy transfer from the donor to the acceptor can be deduced from the quenching of the donor using Equation (3):E values calculated for the mixture at pH 2 and 11 were close to 0 in both cases, suggesting that there was no energy transfer from the donor to the acceptor. This suggests that separated polymer micelles were formed in the mixed aqueous solution of random and block polymers at pH 11.For these experiments, equimolar mixtures of labeled and non-labeled random and block polymers were prepared at a total 2 and 11 a. The re2 and 11 b,c. The Cp of 10.0 g/L for each polymer. However, the copolymers could not dissolve in DMSO, as seen with the naked eye. Thus, about 10 µL of 6 M HCl was added in 2 mL of the solutions to ensure complete dissolution of the copolymers, and the fluorescence measurements were then conducted  values of these copolymers. Py is an indicator for the hydrophobicity of its surrounding environment [I3/I1) between the third to the first peak in the Py emission spectra is low, and this value increases when the copolymer micelles formed at a Cp higher than CMC. Using this, I3/I1 values were plotted as a function of the copolymer concentration to estimate the CMC (Cp increased to be around or higher than CMC, I3/I1 gradually increased because Py was entrapped in the hydrophobic core of the micelles. Whereas, in the mixture of Py and the random copolymer, I3/I1 values were nearly the same when Cp varied from 0.0005 to 0.05 g/L, indicating that unimer micelles always formed regardless of the Cp. Hydrophobicity of the hydrophobic domain of the unimer micelle is lower than that of the block copolymer micelles.The FRET experiments clearly demonstrate that separated micelles were formed in the mixture of the random and block copolymers composed of MPC and DPAEMA. This phenomenon might be due to the incompatibility of these copolymers caused by the different arrangement of the monomer units along the polymer chains. Moreover, Py fluorescence spectra were recorded in the presence and absence of the random or block copolymers in 0.1 M NaCl at different ironment ,31. Belo the CMC . CMC valRandom and block copolymers were successfully prepared from MPC and DPAEMA via RAFT polymerization. In aqueous environment, the critical pH values of the random and block copolymers and their equimolar mixture were close to 8. These copolymers self-assembled to form polymer micelles in aqueous solutions at pH values above the critical pH. Separated micelles were formed in the mixture of these copolymers, which indicated that the compatibility in the structure of the copolymers is important for their assembly. The formation of separated micelles was confirmed by the appearance of a bimodal size distribution in their mixed aqueous solution and by the absence of FRET between the acceptor and donor in the random and block copolymer chains. The sizes of the polymer aggregates were less than 100 nm, which renders them as promising carriers in drug delivery systems.