The risk of air pollution exposure was higher for the elderly than for other age groups. Within the aging group, a few elderly people had a higher risk of pollution exposure. The high exposure risk communities of the elderly were mainly located in the central and sub-center areas of the city, with a continuous distribution characteristic. No significant difference was found in the exposure risk of children compared to the other populations, but a few children were particularly exposed to pollution. Children’s high-exposure communities were mainly located in suburban areas, with a discrete distribution. Compared with the traditional static PM2.5 exposure assessment, the dynamic assessment method proposed in this paper considers the high mobility of the urban population and air pollution. Thus, it can accurately reveal the actual risk of air pollution and identify areas and populations at high risk of air pollution, which in turn provides a scientific basis for proposing planning policies to reduce urban PM2.5 and improve urban spatial equity.In-depth studies have been conducted on the risk of exposure to air pollution in urban residents, but most of them are static studies based on the population of residential units. Ignoring the real environmental dynamics during daily activity and mobility of individual residents makes it difficult to accurately estimate the level of air pollution exposure among residents and determine populations at higher risk of exposure. This paper uses the example of the Wuhan metropolitan area, high-precision air pollution, and population spatio-temporal dynamic distribution data, and applies geographically weighted regression models, bivariate LISA analysis, and Gini coefficients. The risk of air pollution exposure in elderly, low-age, and working-age communities in Wuhan was measured and the health equity within vulnerable groups such as the elderly and children was studied. We found that ignoring the spatio-temporal behavioral activities of residents underestimated the actual exposure hazard of PM The report of the 19th Congress of the Communist Party of China pointed out that the main social contradiction in China is the contradiction between the people’s growing need for a better life and unbalanced and insufficient development. Health is the most basic condition required for humans to achieve a better life. China confers great importance on the health of its residents. The Outline of Health China 2030 Plan emphasizes the in-depth prevention and control of air pollutants, the establishment of a sound system of environmental and health monitoring, investigation and risk assessment, evaluation of the impact of environmental pollution on the health of the population, and the exploration of the establishment of a health risk assessment system. Therefore, accurate identification of areas and populations at high risk of air pollution exposure is a prerequisite for carrying out environmental optimization and protecting the health of residents and is the basis for achieving a better quality of life.During the past 40 years, China’s rapid urbanization and economic development have made tremendous progress. China’s urbanization rate has reached 65% and its gross domestic product has exceeded 100 trillion CNY. However, rapid urbanization has resulted in strained land resources, severe ecological degradation, and an immense increase in resource consumption, accompanied by various problems related to environmental pollution. Among them, the contradiction between problems of urban air pollution and high-quality urbanization development has become increasingly acute and complex. A large amount of epidemiological evidence has revealed a strong association between long-term exposure to air pollution , urban residents’ spatial and temporal behavioral pollution exposure, and group health disparities and environmental justice. However, most of the current studies on group health disparities and environmental justice in air pollution exposure are mainly focused on western developed countries ,11,12,13Recently, air pollution exposure studies coupled with spatio-temporal behaviors of residents, focusing on the risk of air pollution exposure under individual or group spatio-temporal activities at the micro level, have become a hot research topic. Compared to the static assessment method, this method can reveal the air pollution exposure risk of residents with more accuracy and practicality. The research content includes the study of air pollution exposure under the daily behavioral pattern of the population, exposure based on individual spatial and temporal behavior trajectories, and exposure based on the dynamic distribution of the population.2 and PM2.5 increased when occupational and residential activities were considered, compared to the exposure levels only at residences [Air pollution exposure studies based on the daily behavioral patterns of populations are carried out by categorizing the population based on the behavioral patterns of residents, followed by an investigation of the air pollution exposure levels of residents under different behavioral patterns. For example, Stefan Reis studied the air pollution exposure levels of UK residents at their workplace and residence and found that exposure to both NOsidences . Subsequsidences .2.5 real-time monitors, to measure the air pollution exposure level of individuals in their behavioral trajectories. Kwan Mei-po et al. used the activity trajectories of 117 volunteers in a highly polluted neighborhood in Beijing to accurately measure their exposure and found that residents were able to reduce their exposure risk through daily activities, but those with mobility impairments were always at a higher risk [Air pollution exposure studies based on individual spatio-temporal behavioral trajectories are a type of air pollution exposure level measurement for individuals, usually based on GPS and PMher risk . Using G2.5 observations from stations and introduced the population. The results showed that the risk of PM2.5 pollution exposure based on dynamic population distribution and census-based measurements deviated significantly [2.5 pollution exposure risk measured based on census data was significantly low [The study of air pollution exposure based on dynamic population distribution focuses on the uncertainty of group activity space, integrates the dynamic migration changes of population and air pollutants, and uses the actual space of residents’ activity/travel as the measurement unit to measure the actual pollution exposure level of the group ,14. In cficantly . Claudiontly low .Group differences in air pollution exposure are mainly based on the classification of populations based on socioeconomic attributes to study the levels of air pollution exposure in different populations and their influencing factors. The theoretical basis of such studies is that differences in social attributes affect the spatio-temporal behavior of residents, leading to differences in their air pollution exposure levels. That is, people with different social attributes choose different spatial locations and behavioral patterns due to their economic conditions and thinking patterns, and thus their air pollution exposure levels vary. The level of air pollution exposure of residents varies with age, income, household registration, gender, education level, and ethnicity ,24.Studies on air pollution exposure justice based on age characteristics have shown that the elderly and children bear a greater environmental burden. Pearce et al. reported that in the United Kingdom, the elderly and children produced less air pollution but were exposed to higher concentrations of air pollution, presenting environmental inequities . A study2 pollution exposure levels of community residents in the United Kingdom and found that most community residents in communities with high exposure levels belonged to low-income groups [Studies on the fairness of air pollution exposure based on income disparities have shown that low-income populations tend to be exposed to higher air pollution concentrations. Mitchell et al. studied NOe groups . Pearce e groups . Meir’s e groups . Marshale groups .2.5 pollution exposure was lower among highly educated people on the South Coast of California [Other factors such as education level ,32, the lifornia . By invelifornia . Throughlifornia .In summary, dynamic air pollution exposure studies that couple the spatio-temporal behavior of residents have higher accuracy than static assessment methods. Environmental injustice is widespread worldwide, and residential air pollution exposure levels vary according to age, income, household registration, gender, education level, and race. However, most of the current studies focus on macro-scale such as individual cities or urban agglomerations, and micro-scale such as individual communities or residential blocks, and there is a lack of environmental equity exploration at the scale of urban communities. As a result, most of the research findings are qualitative and broad, and it is difficult to accurately identify urban areas and populations at high risk of air pollution. Over the years, the rapid urbanization and industrialization in China have placed a great burden on the ecological environment, and environmental injustice has intensified. To address the contradiction between people’s growing need for a better life and unbalanced and insufficient development, China emphasizes the establishment of a sound system of environment and health monitoring, investigation, and risk assessment, evaluation of the impact of environmental pollution on the health of the population, and exploration of a health risk assessment system. Therefore, we considered communities in the Wuhan metropolitan area as the research scale, combined residents’ spatial and temporal behaviors, and conducted a study on air pollution exposure patterns and environmental equity in Wuhan, in an attempt to accurately identify areas and populations at high risk of air pollution exposure and provide theoretical basis and policy recommendations for air pollution management in Wuhan.2 and a resident population of 11,212,000. Wuhan city is an important industrial base in China, and PM2.5 pollution in the city has been at a high level for a long time due to industrialization. Relevant studies have pointed out that PM2.5 pollution in Wuhan city has a serious impact on the health of its residents [2 . The aerosol optical thickness data (AOD) were obtained from the Earth Observation Research Center for Data (EORC) and downloaded from the aerosol observation data of the Japan Aerospace Agency Himawari-8 .The data sources used in this study were extensive and contained three main dimensions: air quality, built environment, and socioeconomic data. For air quality data, PMAmong the socioeconomic data, the demographic data consisted of the current population data of Wuhan city in 2017, the third statistical economic unit data of Wuhan city, the original data of social security individual residence and employment, and the original data of the trade union members’ residence and employment . SmartphFor the built environment data, the land data was Wuhan city 2017 land use data, the vegetation index data was NDVI data, and the road traffic data was from OpenStreetMap.2.5 pollution exposure patterns and exposure risk equity in Wuhan, a geographically weighted regression model, and remote sensing inversion techniques were used to measure hourly PM2.5 concentrations and monthly average PM2.5 concentrations in Wuhan metropolitan development area, respectively. Firstly, PM2.5 concentration-related variables were selected, and then a geographically weighted regression model with the monitored PM2.5 concentration as the dependent variable and PM2.5 concentration-related variables as the independent variables was constructed to explore the relationship between the dependent and independent variables and derive the regression equation. Subsequently, a 1000-m-scale fishing net was constructed in GIS, and the geographic correlation variables of PM2.5 concentration at the plots of the fishing net were extracted and substituted into the regression equation to measure the PM2.5 concentration at the plots of the fishing net. On this basis, the kriging interpolation method was used to simulate the PM2.5 concentration within the Wuhan metropolitan development area, and the simulated PM2.5 concentration values of the study area were obtained. Finally, the average PM2.5 concentration within the city block was calculated as the PM2.5 concentration in the city block using the city block as the statistical unit. Wuhan city has 10 national monitoring stations, which are evenly distributed in the built-up area of Wuhan. They can theoretically cover an area of 25 square kilometers.To accurately analyze PM2.5 concentration for each hour of a complete week in the Wuhan metropolitan development area, a geographically weighted regression model was constructed in this study, using data averaged for a year and calculated as follows:3) is the average hourly PM2.5 concentration monitored at the monitoring station i at time t; t; t; t; i is located; i; i from the major industrial pollution sources in Wuhan; and To measure the average PM(1)2.5 pollution exposure in urban space based on residents’ spatial and temporal activities.Measurement of PM2.5 pollution exposure intensity was introduced as an indicator to study air pollution exposure. The population exposure risk assessment model proposed by Kousa et al. [2.5 pollution exposure intensity values in urban blocks within the Wuhan metropolitan development area, which was based on the degree of population exposure to PM2.5 concentrations per unit area. Since the cell phone LBS data were collected from 17 March 2017 to 24 March 2017, a complete week of the year containing five working days and two holidays, the population density data for that week could be used instead of the annual data. The calculation formula is as follows:j in μg − person/(m3 − km2) during a day, j at time i (in person/km2), j at time i, j (in hours).Air pollution exposure characterizes the exposure of a population to air pollution over a period of time, and therefore has a more direct link to public health. Identifying areas of high exposure to air pollution plays an important role in reducing the impact of air pollution on the health of the population. In this study, PMa et al.  was used(2)2.5 pollution exposure measurement of urban population based on residents’ spatial and temporal activities.PMIn this study, we set the time for daily travel activities for residents of different age groups. Children and the elderly were designated as vulnerable groups because of health problems and inability to drive motor vehicles due to mobility problems; these groups prefer to spend their time within proximity . Therefo2.5 concentration value in the urban community i during a day, i, ∑2.5 concentration exposure outside of working hours for the population residing in the urban community i in month j, ∑i at the work location in month j, PM2.5 concentration exposure in j urban community i, and ∑i.A walk score is a representative and internationally accepted method for measuring walkability ,41. The The specific steps of the walking index measurement are as follows.(1)Measurement of basic walking index.The basic walking index is obtained by calculating the distance from a certain point to different service facilities within a certain range. The higher the number of service facilities, the richer the category, and the closer the distance, the higher the calculated basic walking index. The different service facilities are weighted according to their importance and substitutability, as shown in the following table .On this basis, the urban development area of Wuhan is divided into 2811 square fishnet grids with a side length of 1000 m as the calculation unit. Using the center of mass point of the fishing net as the starting point, the basic walking index of the starting point is calculated by considering the acceptable walking time and distance for pedestrians and attenuating the weights of different services and facilities. When the distance is within 500 m, no attenuation occurs; between 500 m and 1000 m, the attenuation rate is 25%; between 1000 m and 1500 m, the attenuation rate is 88%; when the service facilities are more than 1500 m away from the starting point, the attenuation rate is 1. After calculating the distance attenuation, the weights of various service facilities around the starting point are summed up to obtain the basic walking index of the starting point.(2)Measurement of the single-point walking index.After obtaining the base walking index of the departure point, the single-point walking index was obtained by further decaying the base walking index according to the intersection density and block scale within the grid cell. Among them, the intersection density and block scale decay rates are divided into five levels each, and the maximum decay rate is 10% .(3)Measurement of the surface area walking index.After calculating the single-point walking index, the face area walking index of urban neighborhoods was calculated using the population weight of the grid as the weight. The population weight of the grid is the ratio of the number of people in the grid to the total population, and the number of people in the grid is obtained by spatially connecting the grid mass to the Wuhan city community demographic data.2.5 concentrations in Wuhan were measured, and the calculation results showed that the R2 were all above 0.6, indicating that the model fit was good. The daily average of 9:00 a.m., 12:00 p.m., and 18:00 p.m. were selected as the displayed results , children (0–6), and working population (19–59) in each community, the communities were classified into five classes: high, medium-high, medium, medium-low, and low, using the natural interruption point grading method, to obtain the distribution maps of high, low, and working age population . In term2.5 concentration and the proportion of the population of different age structures in each community, the communities were classified into high, medium, and low levels using the natural interruption point grading method. By combining the classification results of the two indicators, nine categories of PM2.5 pollution exposure risk communities could be classified from high PM2.5 concentration–high proportion of elderly to low PM2.5 concentration–low proportion of elderly.Based on the weighted PM2.5 concentrations were calculated separately for each decile community, and the age-specific differences in PM2.5 pollution exposure risk were compared The exposure risk of the elderly (65 years old and above) was higher than that of other age groups.2.5 concentration increased steadily with the increase in the proportion of elderly people, and the weighted PM2.5 concentration in the community with the highest proportion of elderly people was 88.77 mg/m3 (D10), whereas the weighted PM2.5 concentration in the community with a higher number of residents of other age groups was 88.01 mg/m3 (D1), which indicates that the elderly people had a higher risk of PM2.5 pollution exposure than other age groups.As shown in (2)Communities with high exposure risk for the elderly were concentrated around the center and sub-center of the city.2.5 concentration for the elderly (65 years old and above) and the population-weighted PM2.5 concentration The exposure risk of children (0–6 years old) was not significantly different from that of the other age groups.2.5 concentration showed a normal distribution with the increase of the proportion of children, that is, it first increased and then decreased and did not differ significantly from those of the other age groups.As shown in (4)Communities with high exposure and high risk of children were scattered in the suburbs of the city, showing a high outer and low inner distribution.2.5 concentration  and weighted PMntration , we foun(5)2.5 pollution exposure was relatively low in the working-age population (19–59 years).The risk of PM2.5 concentration decreased gradually with an increase in the proportion of working-age people, with the highest proportion of working-age people having a weighted PM2.5 concentration of 87.81 mg/m3 (D10) and the communities with more residents of other age groups having a weighted PM2.5 concentration of 88.19 mg/m3 (D1). These results suggest that the risk of PM2.5 exposure was lower for working-age populations than for the other age groups and that this phenomenon was more pronounced as the proportion of working-age populations increased, possibly because communities with high working-age populations were located near large natural ecological spaces where industries were less prevalent and air quality was relatively good. These findings further suggest that vulnerable groups, such as the elderly, were at higher risk of PM2.5 pollution exposure.(6)Cluster distribution of communities with high exposure risk for working-age people outside the first ring.2.5 concentration, the communities with high exposure risk for the working-age population were found to be distributed in clusters outside the first ring road. These areas were mainly the recipients of out-migrating industries, with lower housing prices and more job opportunities, but also faced higher PM2.5 pollution exposure risks.By comparing the spatial distribution of the working-age population (19–59 years) and weighted PM(1)Among vulnerable groups, a few bore higher exposure risks.2.5 concentration and the number of vulnerable people were calculated using Python and then plotted as Lorenz curves. The Gini coefficient was 0.48 for the weighted PM2.5 concentration and the number of elderly people, and 0.58 for the weighted PM2.5 concentration and the number of children, both indicating a “large gap”.To further investigate the equity of the exposure risk for vulnerable people, the Gini coefficients of community-weighted PM2.5 concentration, and 40% of the elderly were exposed to more than 70% of the weighted PM2.5 concentration, with a large exposure risk gap and significant health inequity.Based on the Lorenz curves , 40% of (2)Spatial heterogeneity in areas with high exposure risk.2.5 concentration and number of elderly people, and weighted PM2.5 concentration and number of children, respectively, to further analyze the equity of their spatial distribution. The results showed that the Moran index of weighted PM2.5 concentration and the number of elderly people was 0.179, with a p-value of 0.001 and a z-score of 15.9054, indicating that the clustering of weighted PM2.5 concentration and elderly people was extremely high, that is, the communities with high weighted PM2.5 concentration and the main gathering activities of elderly people were continuously spatially distributed High internal and low external exposure risk for the elderly and high internal and low external exposure risk for children.2.5 concentration and the number of vulnerable people, the risk of PM2.5 pollution exposure in the elderly showed a spatial differentiation of activities with high internal and low external exposure. Among 1819 communities in the metropolitan area of Wuhan, 163 had a high weighted PM2.5 concentration–high number of elderly people; these communities were concentrated between the first and third rings. Among these, the Wuchang and Jiang’an districts were concentrated and distributed in a large number. There were 112 low-weighted PM2.5 concentration–high elderly population communities located near the ecological environment along the third ring road. This was mainly because elderly people tend to gather in and around the inner-city center of the third ring road, where the high-density production, living, and high volume of automobile traffic are likely to cause air pollution problems, thus threatening the elderly population in the area. The lack of ecological green space further exacerbates this risk. On the other hand, the suburban area attracts the elderly population due to the convenience brought by the proximity to the central area. There are large-scale ecological spaces such as East Lake, Moon Lake, and South Lake, which have significant effects on reducing air pollution and play a positive role in the health of the elderly. There were 120 communities with high weighted PM2.5 concentration–low elderly population and 268 communities with low weighted PM2.5 concentration–low elderly population, all of which were located in the periphery of metropolitan areas, and the risk of PM2.5 pollution exposure in these spaces was relatively low due to the sparse population distribution. Overall, the PM2.5 pollution exposure risk of elderly people showed a spatial variation of high on the inside and low on the outside, reflecting the unevenness of spatial risk, that is, the existence of inequity.According to the clustering results of LISA analysis of weighted PM2.5 pollution exposure risk showed a spatial differentiation of activities with low internal and high external exposure. There were 49 communities with high-weighted PM2.5 concentration–high number of children among 1819 communities in the metropolitan area of Wuhan and that were discrete in the suburban areas along the third ring road. These areas, as catchment areas for heavy industries, offer many employment opportunities and have low-cost housing that attracts young people, most of whom are of childbearing age, thus increasing the number of children in the area. The 141 communities with low weighted PM2.5 concentration–high number of children were located near the ecological space outside the third ring road, and these areas were also the employment centers of the district, where the spatial differentiation of the young generation lived as high housing prices forced them to work nearby and live together with their children. There were 131 communities with high-weighted PM2.5 concentration–low number of children and 228 communities with low-weighted PM2.5 concentration–low number of children, all of which were located in the periphery of the metropolitan area, and these areas were at low risk. Overall, the risk of children’s PM2.5 pollution exposure showed a spatial differentiation of activities with low internal and high external exposure, and the presence of inequity.In contrast to the elderly, children’s PM2.5 concentration. Under the law of land rent, large ecological spaces are mostly located at the periphery of the city, and the urban blocks around them enjoy the convenience of the urban environment as well as clean air brought by the natural environment. Thus, the spatial pattern of PM2.5 exposure risk in urban blocks shows the circle differentiation of “high on the inside and low on the outside, decreasing circles” and the distribution characteristics of “central concentration, axis extension” in high-risk urban blocks.In general, urban centers are built with a mature environment and are rich in services and pedestrian systems, which attracts people to gather and move, thus generating a large amount of motorized traffic and increasing the PM2.5 pollution and their spatial distribution varies significantly, with the risk of PM2.5 pollution exposure being higher on the inside and lower on the outside for the elderly and higher outside and lower inside for children. The main reason for this phenomenon is the urban governance corporatism and the capital-led urbanization model that accompany the urbanization process in China. Capital-led urban space production neglects the survival and development of human beings, resulting in the alienation of space and eventually the over-capitalization of public space. The high-quality, healthy spaces in the city are inevitably developed for commercial purposes, increasing the economic value of the land. Therefore, industrial spaces and reserved ecological spaces are mostly located in the low-priced areas in the suburbs of the city. The aging phenomenon in China is becoming more pronounced, with older people living mostly in older neighborhoods in urban centers, enjoying the benefits of a built environment but also suffering from the threat of air pollution due to widespread and massive motor vehicle emissions and a lack of ecological space. China’s fertility rate has been declining in recent years, and despite the government’s “three-child policy,” the willingness of young people to marry and have children remains low due to China’s rising housing prices. With the recent urban sprawl and relocation of downtown industries, many large corporations have moved to the suburbs, and young people tend to move there as well, as jobs are available and housing costs are lower, and then start a family there until they are middle-aged and purchase a second, improved home; therefore, their children are often exposed to severe industrial air pollution during their childhood.Vulnerable groups such as the elderly and children are disproportionately exposed to PM2.5 air pollution and address the injustice of air pollution exposure to the elderly and children, the following urban planning policies and recommendations are proposed:(1)2.5 pollution among the elderly are mainly located in high-intensity built-up areas in the city center, for which, in conjunction with China’s ongoing urban renewal initiatives, this study proposes a morphology-guided, functionally mixed strategy. We propose that the guidance of block texture and morphology should be used, an urban ventilation system should be constructed, the internal ventilation corridor of the block should be built, and the internal ventilation corridor should be linked with the urban ventilation corridor to form a “sewage system”. Improving the mix of functions can reduce the travel distance and the probability of motor vehicle travel for urban residents, and reduce the total carbon emission and path exposure of residents’ travel;Neighborhoods at high risk of PM(2)2.5 pollution exposure for the elderly and children is the insufficient construction of green and open spaces. The study proposes the strategy of increasing their extent, improving planning, and optimizing the layout. A scattered layout should be avoided in the process of configuring green space, and the centralization and scale of green space should be promoted. Regarding the layout of green and open space, the layout should be optimized according to the functional characteristics;The common feature of typical high-risk areas for PM(3)2.5 pollution exposure;As a road connecting urban areas, the trunk road is characterized by high traffic volume and high pollution. In the dense area of the trunk road network, the study proposes the protection retreat line restriction function strategy. During the construction process, the threshold distance of significant reduction of pollutants on both sides of the road should be calculated as the basis of the protection retreat line. Strict functional construction restrictions should be made in the high concentration range to avoid crowd-gathering spaces such as schools, residences, and open spaces from being too close to the main roads to reduce the risk of PM(4)During the construction of new cities, emphasis should be placed on the integration of industries and cities, and the location of large industrial enterprises should not only focus on industrial benefits but should also consider ecological benefits. The residential land of large industrial enterprises should be close to large ecological spaces to reduce industrial waste gas pollution to the residents. At the same time, adequate protective measures should be set up outside the industrial land of concentrated scale to guide the pollutants to stop their diffusion to the isolation and absorption green belt and avoid their flow to the crowd-gathering space.To mitigate urban PM2.5 concentration built environment influencing factors, namely AOD (aerosol data), road traffic, population density, land use, and industrial pollution impact, were integrated to simulate high precision hourly PM2.5 concentrations and measure PM2.5 pollution exposure intensity in urban neighborhoods by combining the dynamic population distribution characterized by hourly precision LBS data. On this basis, the spatial pattern of PM2.5 pollution exposure risk is further explored in conjunction with the built environment walkability, revealing the actual PM2.5 pollution exposure risk in the built environment and enriching the understanding of the built environment air pollution exposure risk pattern based on the dynamic distribution of population and PM2.5. This study provides a feasible method and scientific basis for studying the differences in air pollution exposure among different groups and the derived environmental justice issues, and for accurately identifying the high-risk areas and populations of air pollution exposure in urban areas.In this study, five types of PM2.5. In future studies, the use of data with longer time spans could be considered for the study. (2) In the study of community population differentiation of PM2.5 pollution exposure, the pathway exposure generated by different groups of traffic travel processes was not calculated, which could lead to some deviation between the calculated results of PM2.5 pollution exposure and the actual exposure of the population. Future studies should divide the population according to their daily behavioral patterns, simulate their traffic travel paths according to their behavioral characteristics, calculate their traffic travel exposure levels, and more comprehensively assess the differences in PM2.5 pollution exposure among different populations. (3) There are currently four main approaches to air pollution exposure measurement, including proximity models, atmospheric dispersion models, interpolation models, and regression models [2.5 distribution in Wuhan City using the technique with an accuracy of 1 KM×1 KM. The accuracy and fit are high enough to support the conclusion. However, the multi-variate models and kriging techniques may not necessarily capture the complex urban PM2.5 distribution. Nowadays, various intelligent methods such as neural network algorithms and their improvement algorithms have emerged and have initially been applied to the measurement of urban PM2.5 and PM10 pollutant concentrations. For example, Perez et al. [2.5 hourly concentration using multilayer neural network and linear regression method, Grivas et al. [2.5 based on BP artificial neural network, and all of them concluded that the neural network method is better than the linear regression method and kriging interpolation method. In the following research, we will improve the method.This study has a few shortcomings. (1) Due to data limitations, the time period studied in this research is one week, and although that week is a full week with five working days and two rest days, it is still contingent in the calculation in the measurement of PMn models ,43. In gz et al.  predictes et al.  combineds et al.  measured2.5 pollution and its variability based on the dynamic distribution of population activities and PM2.5 concentrations, as well as the health equity of PM2.5 pollution exposure risk based on the spatial and temporal activities of the residents. Compared with the traditional static PM2.5 pollution exposure assessment, the dynamic assessment method also considers the urban population and the high mobility of air pollution, thus accurately revealing the actual risk of air pollution. This study enriches our understanding of PM2.5 pollution exposure risk patterns in the built environment, expands the understanding of population differentiation of PM2.5 pollution exposure risk for the spatial and temporal activities of residents, and provides effective references and suggestions for urban planning decision makers and program designers to improve air quality, reduce PM2.5 pollution exposure risk, and promote residents’ health equality in a targeted manner. [This study systematically analyzes the exposure risk of the spatial pattern of urban PM manner.


At the moment, the field of biomedical materials science is actively developing, which aims at creating new functional materials. A developing direction in biomedical materials science is that towards the treatment of diseases associated with bone tissue disorders, using biodegradable composite materials based on polymer and calcium phosphate materials. We developed a material based on polyvinyl alcohol cryogel, mineralized with calcium phosphate. A material based on cryogel of polyvinyl alcohol mineralized with calcium phosphate was developed. The composites were obtained by the method of cyclic freezing–thawing, and the synthesis of calcium phosphates was carried out in situ with heating, stirring, and exposure to microwave radiation. The phase composition, as well as the composition of functional groups, was determined by IR spectroscopy and X-ray phase analysis. Monocytes isolated from human blood showed higher viability compared to the controls. Bone defects caused by various infectious diseases, trauma, and genetic factors can lead to dysfunction and problems with the musculoskeletal system. Traditional methods of treatment, such as autologous bone grafting, face such problems as high trauma and the risk of infection, which limit their clinical use . HoweverBone tissue is a solid and dense substance, consisting mainly of two parts: cortical bone and spongy bone . Accordi10(PO4)6(OH)2 is the main inorganic component of bone tissue [Hydroxyapatite with the general formula Cae tissue . This mae tissue . The bioe tissue ,13, it ce tissue .However, despite all the advantages of hydroxyapatite, its use in bone implants is limited . This isPolyvinyl alcohol (PVA) is a synthetic biocompatible and non-toxic polymer whose cryogels have a porous structure ,23. The Cryogels have important advantages in bone tissue repair. Due to the fact that cryogels consist of three-dimensional hydrophilic polymer chains, which have high mechanical strength, they are able to create a nutrient medium for the growth of endogenous cells. They are also able to imitate the extracellular matrix of bone, which makes it possible to encapsulate bioactive cells and molecules. Due to their network structure, cryogels can control the release of materials as needed . In addiTo obtain materials K1 and K2, a suspension of calcium hydroxide in water (3.68 g in 20 mL) was added to a 30% polyvinyl alcohol gel in water (15 mL) and stirred for 2 h. After two hours of stirring, to obtain material K1, a solution of ammonium hydrogen phosphate (3.95 g in 10 mL) was added and stirred for 6 h, and to obtain K2, an 87% solution of phosphoric acid was added and stirred for 4 h. Further, the resulting suspensions were treated with microwave radiation (power 100 W), kept for two days to preserve the precipitate, and frozen for 12 h at −20 °C. Using the same methods, but without PVA, pure HA(K1) and HA(K2) hydroxyapatite was synthesized to compare the viability of monocytes in the presence of materials .−1 in the range 400–4000 cm−1 using an ATR attachment.The phase composition of the resulting composite materials based on cryogels of polyvinyl alcohol and calcium phosphates was determined on a Rigaki diffractometer (MiniFlex 600) at 40 kV and speed 3°/min. Phase interpretation and identification was carried out using the ICDD diffraction database (PDF-2/Release 2012 RDB). The spectra of the surface layer of samples of various materials were recorded on a Nicolet 6700 IR Fourier spectrometer (Thermo Scientific). The spectra were recorded with a resolution of 4 cmThe surface morphology of composite materials based on cryogels of polyvinyl alcohol and calcium phosphates was studied using a HITACHI TM-3000 electron microscope at an accelerating voltage of 5 kV, without applying metals to the surface. The particle size was calculated by the linear-intercept method, in which the size measurement is made using a straight line at one selected angle for all particles, followed by plotting a diagram from the resulting data array.2+ calcium ions in physiological saline at 37 °C, in which the samples were kept for 20 days to achieve saturation with respect to the solid phase, was determined by complexometric titration in the presence of Eriochrome Black T with an ammonia buffer solution, pH = 10. Titration was carried out every 7 days.To assess the solubility of HA in the composition of the composites, the total concentration of CaThe compressive strength was measured, and the modulus of elasticity was calculated on a setup assembled on the basis of an Arduino Board Mega 2560 and a strain gauge with a sample deformation of 20%.To determine the assessment of the effect of materials on the viability of cells of the immune system, a cytotoxicity test with an Alamar Blue indicator was used. The signal intensity was measured using a Tecan Infinite 200 microreader at a wavelength of 540 nm.−1, which indicates the stretching vibrations of the O-H bond, which is a characteristic band for alcohols. The absorption at 2900 cm−1 refers to the stretching vibrations of the CH2 bond. Absorption is observed in the region of 1600 cm−1, which is characteristic of the stretching vibrations of the C=O bond: this band could appear namely as a result of the production of polyvinyl alcohol, during the saponification of polyvinyl acetate. An absorption band is also observed in the region of 1385 cm−1, which is also characteristic of the bending vibrations of O-H groups, but for the K2 sample, an increase in the intensity of this band can be observed. This is due to the formation of a larger number of hydrogen bonds. In the spectrum of hydroxyapatite, an intense absorption band is observed in the region of 1000 cm−1, which refers to the stretching vibrations of phosphate P-O groups, and the bands at 570 cm−1 refer to their deformation vibrations.Analysis of the resulting composites by IR spectroscopy showed that the spectra  have banWhen examining samples by X-ray phase analysis , we can The results of surface morphology studies by SEM showed that the K1 composite is more homogeneous compared to the K2 sample. On the histograms, we can observe that the average particle size differs slightly and is 3 µm . ParticlComposites K1 and K2, obtained by mineralization of the polymer matrix in situ, were divided into several parts and immersed in saline, after which they were placed in a thermostat at a temperature of 37 °C and kept for four weeks. In total, four series of tests were made, and after each week, the physiological solution in which the composites were kept was titrated for the calcium content in the solution. The physiological solution was titrated with a solution of 0.01 M Trilon-B, with the indicator Eriochrome Black. From the results of the titration, we can observe that the release of calcium in the K1 composite is higher in all test series, relative to the K2 composite. For composite K1, complete diffusion occurs as early as the third week, and does not change further. The reason for the low release of calcium in the K2 composite may be the presence of only hydroxyapatite, which is a poorly soluble compound and entails low diffusion .The mechanical compressive resilience of composite materials was determined by measuring the relative stress and strain of the composites. Based on the measurement results, dependency graphs were obtained .0.02 Based on the results of these measurements, the graphs were processed, and the average modulus of elasticity was calculated . Based oWhen analyzing the viability of monocytes in the presence of materials, in addition to composite materials, pure hydroxyapatite was also synthesized according to two methods . First, 10(PO4)6(OH)2; however, in K1 there is also a phase of calcium hydroxide Ca(OH)2, and in K2 there is a phase of calcium hydrophosphate CaHPO4. The release of calcium in composite K2 occurs much faster due to the more soluble phase of calcium hydrogen phosphate CaHPO4. For composite K2, the average modulus of elasticity is 157 kPa, which is significantly higher than that of K1, whose average modulus of elasticity is 101 kPa. The study of the surface morphology of HA/PVA showed the presence of a homogeneous structure of the K1 composite and an inhomogeneous structure of the K2 composite; the average particle size is 3 µm. The study of the viability of monocytes showed that about 60–80% of cells survive in the presence of pure HA, and in the presence of pure PVA and composites, it is comparable with the control sample. As we can see, the resulting materials do not have outstanding mechanical properties, but can be considered biocompatible since the cells of the immune response monocytes show high viability in the presence of materials. Such a material can be used to fill the voids that have formed due to pathological processes in the bone, between the bone, and the implant bearing a mechanical load.Composite materials based on a 10% solution of polyvinyl alcohol mineralized with hydroxyapatite, synthesized from a solution of calcium hydroxide, ammonium hydrogen phosphate, and phosphoric acid were obtained. It was established by X-ray phase analysis that the main phase of the mineral filler of the composite is hydroxyapatite Ca


A large amount of coal fly ash produced in thermal power plants is disposed of in landfills which causes many environmental problems. The utilization of fly ash can be encouraged in geotechnical engineering projects. In this paper, the effects of class C and class F fly ash on the mechanical and microstructural behavior and stabilization of clay soil were evaluated through a program of laboratory experiments. The experiments included compaction, unconfined compressive strength, consolidated-undrained triaxial, one-dimensional consolidation tests, and scanning electron microscopy analysis on samples of fly ash-stabilized clay soil after 1, 7, and 28 days of curing. The tests were conducted on mixtures of clay with class C or class F fly ash, ranging from 0% to 30% of the soil. Experimental results showed that the strength parameters and permeability of the stabilized soil improved while the compression and swelling indices decreased by the addition of fly ash and by the increase of curing days. The results obtained from the mechanical tests agreed with the results from the SEM analysis. Based on the results, the soil could be successfully stabilized by using class C fly ash. The improvements in strength, swelling, and permeability parameters of the stabilized soil were higher with the class C fly ash compared with class F fly ash. Fly ash is an industrial by-product generated during the combustion of coal in thermal power plants ,2. It isTherefore, the stabilization of different types of soil with fly ash has encouraged various researchers to carry out experimental or/and field studies. The available literature on clay soils stabilized with fly ash is summarized in Seyrek  conductePrabakar et al.  carried r) tests. They reported, on the basis of the test results, that the inclusion of fly ash (10–30%) provides a significant increase in the CBR and Mr parameters of the inorganic soils. The CBR values increased four and eight times with the addition of 10% and 18% fly ash, respectively. On the other hand, the effect of fly ash on organic soil was insignificant. Tastan et al. [r) and UCS tests were carried out on soil stabilized with 10%, 20%, and 30% fly ash. The results indicated that the different fly ash contents and soil types significantly affected the effectiveness of the stabilization. For example, the UCS increased about 400 kPa with addition of fly ash to two types of clayey soil with organic content less than 10%. However, by adding the same amount of fly ash, the UCS of an organic sandy silty peat with 27% organic content only increased 100 kPa. Edil et al.  analyzedn et al.  investigThe review of the literature suggests that fly ash stabilization of soil has great potential for improving the mechanical and physical properties of geomaterials. Common tests used for the study of fly ash stabilized soils are UCS, free swell index, consistency limits, and CBR tests. However, little information is available on shear and consolidation behavior of fly ash stabilized clay soils. Furthermore, previous studies did not fully clarify the different effects of using class C fly ash and class F fly ash on shear, consolidation, and microstructural behavior of stabilized soils. Therefore, there is a need to improve the fundamental understanding of how class C and class F fly ash affect the overall shear, consolidation, and microstructural behavior on soil samples. Shear strength parameters play an important role in estimating the bearing capacity of soils and in the assessment of the stability of geotechnical structures, while consolidation parameters allow the analysis of settlement behavior of soils.This study presents a comparison of class C and class F fly ash on the stabilization of a clay soil (kaolinite). Their effects are evaluated through a program of laboratory tests including compaction, UCS, consolidated-undrained (CU) triaxial, one-dimensional consolidation (oedometer) tests, and scanning electron microscopy (SEM) analysis. The outcome of the study will improve the current understanding of the effects of class C and class F fly ash on the mechanical behavior and microstructural characteristics of stabilized soils, help determine and compare the suitability of class C and class F fly ash as alternative soil stabilizing agents and encourage the utilization of fly ash to reduce the environmental impacts of fly ash disposal. Another focus will be to identify the optimal amounts of the two types of fly ash for the stabilization of clay soil.dmax) of 15.2 kN/m3 and optimum moisture content (wopt) of 21%. The specific gravity of the soil was found to be 2.6 from the small pycnometer test.A kaolinite powder (China clay) was used in this study. Atterberg limit, specific gravity, and standard proctor tests were conducted to characterize the soil, according to the British Standards (BS) ,20. The X-ray diffraction (XRD) analysis was carried out to obtain mineralogical characterization of the soil using a Bruker D8 advanced XRD equipment . The recorded angular range was 5 to 65° (2θ) from the X-Ray generator. SEM analysis was conducted with 2.00 kx magnification factor on the soil, with the use of TESCAN VEGA3 SEM detector . Based on the XRD results, the dominant minerals of the soil were found to be kaolinite, quartz, and illite . Also, aClass C fly ash was sourced from the MUEG company in Germany (Braunsbedra), and class F fly ash was supplied from Power Minerals Ltd. in the UK (Birmingham). Both fly ashes were silt sized. Thus, the parameters of the plasticity index could not be evaluated, and it was recorded as nonplastic (NP). The values of specific gravity of the class C and the class F fly ash were found to be 2.4 and 2.32, respectively.The average chemical compositions of the class C and the class F fly ash are shown in 3 based on the different fly ash contents of the stabilized soil samples. The relevant optimum moisture contents of the control sample and fly ash-stabilized soil samples obtained from standard proctor tests were used. The prepared samples were 50 mm in diameter and 100 mm in height and were compacted in 3 layers for UCS and CU triaxial tests. The samples, 50 mm in diameter and 20 mm in height, were prepared in one layer for one-dimensional consolidation tests. After compaction, the samples were sealed and stored in laboratory conditions (23–26 °C) and cured in vacuum desiccators for 1, 7, and 28 days before testing.Control samples and fly ash stabilized soil samples were prepared by static compaction using an Instron 3382 Floor Model Universal Loading System  for the UCS, triaxial, and one-dimensional consolidation tests. Each layer was compacted by increasing the load at a displacement rate of 2.5 mm/min up to a constant maximum load. The maximum dry unit weight of the compacted samples was recorded between 13.9–15.2 kN/mThe experiments conducted in this study focused on the effects of the type and amount of fly ash on the mechanical and microstructural behavior of soil. A series of laboratory tests including compaction, UCS, CU triaxial, one-dimensional consolidation tests, and SEM were conducted on the control (neat clay) and stabilized soil samples. dmax might be affected with compaction delays. Mahvash et al. [To finds the optimum moisture content and maximum the dry unit weight of the control sample and the soil samples stabilized with 5%, 10%, 15%, 20%, 25%, and 30% fly ash (based on weight of the dry soil), standard proctor compaction tests were conducted according to BS ,14,15,17h et al.  stated tUCS tests were conducted on the control samples and samples of soil stabilized with 5%, 10%, 15%, 20%, 25%, and 30% fly ash with 1-, 7-, and 28-day curing times. The samples were loaded between two metal plates at a displacement rate of 1 mm/min. All UCS tests were conducted on duplicate samples and average results were used. Based on the results obtained from the UCS tests, the fly ash contents of 15% and 25% were chosen for the triaxial tests. According to the UCS tests, the strength parameters generally decreased after 25% of fly ash content. Therefore, 25% fly ash was considered as optimal. A sample of 15% fly ash was selected to compare and determine the effects of changes in fly ash content from 15% to 25%. Cylindrical samples were prepared by static compaction. Isotropically consolidated undrained triaxial tests were conducted according to the BS . The maiA series of standard one-dimensional consolidation tests were carried out to determine the consolidation properties of the control sample and the fly ash-stabilized soil with 15% and 25% fly ash. The tests were conducted in accordance with the British Standard, BS [The microstructural investigation of the control sample and the soil samples stabilized with class C and class F fly ash was carried out using TESCAN Vega 3 SEM. SEM tests were applied on the stabilized soil samples with 1, 7, and 28 days of curing time. The accelerating voltage of 10 kV and beam intensity of 10 were used in this study. The dried samples were mounted on the stubs using carbon tape and coated by Quorum Q150 TES Sputter  coater to improve their conductivity prior to the SEM analysis. dmax decreased and wopt increased with the increase of the class C or class F fly ash content. This trend is similar to that found by other researchers [dmax with the addition of fly ash is due to the lower specific gravity of fly ash than kaolinite [dmax was smaller for the soil samples stabilized with class C fly ash compared to class F fly ash. Agglomeration and flocculation occurred due to the cation exchange reaction which resulted in a change of gradation in the stabilized soil samples. This could be another reason for the decrease in γdmax [dmax in stabilized soil. According to Mir and Sridharan [opt with the addition of fly ash is due to the presence of some large and hollow spheres in fly ash situated in stabilized soil.The compaction curves of the control sample and the soil samples stabilized with class C and class F fly ash are shown in earchers ,27,28,29aolinite ,31. The in γdmax . Seyrek in γdmax  also argopt and γdmax changed from 21% and 15.2 kN/m3 for kaolinite to 25.8% and 14.2 kN/m3 with 30% of class C fly ash, and to 28.2% and 13.9 kN/m3 with 30% of class F fly ash addition. Also, the compaction curves for the stabilized soil with 15%, 20%, and 25% class C fly ash showed shallower peak in comparison with kaolinite. The shallow curve indicates that the soil samples stabilized with 15%, 20%, and 25% class C fly ash did not show significant change over a wide range of water content. However, this trend  was not observed for the soil stabilized with class F fly ash. Hence, it can be said that soils stabilized with class C fly ash are more adaptable in field applications compared to class F fly ash stabilized soils.It is seen from u) and the fly ash content for different curing periods is presented in 2 ratio in fly ash, the greater the unconfined compressive strength and resilient modulus [2 ratio (32.4%/28.3%) and hence gave a higher compressive strength than the class F fly ash with ratio of 2.2%/46.8%. Furthermore, mineralogical analysis confirmed a non-negligible presence of lime, which has self-cementing properties, in the class C fly ash. Also, anhydrite in class C fly ash reacts with water and produces gypsum which has binding effects. On the other hand, class F fly ash does not have enough cementitious properties because of the low calcium content, and thus, it does not give a strong reaction with soil [The relationship between the unconfined compressive strength  for the soils stabilized with class C and class F fly ash, obtained from the results of the UCS tests at 1, 7 and 28 days of curing are presented in a) behavior of the control sample and the soil samples stabilized with 15% and 25% class C and class F fly ash, cured for 1, 7, and 28 days, at 600 kPa effective confining pressure (σc’). It is seen that at a given axial strain, the deviator stress of the soil stabilized with both types of fly ash increased with increasing fly ash content. This trend is consistent with the findings from Prabakar et al. [max) reached 359 kPa at around 11% axial strain, while in the soil stabilized with 25% class C fly ash, the sample reached the qmax of 889 kPa at around 2% axial strain. This could be that the cementitious properties of class C fly ash produced stiff bridges in the soil structure, and lower strains are enough to break such bridges.The shear strength parameters of the soil were obtained from the Mohr–Coulomb failure criterion. Mohr circles were plotted for the control and stabilized soil samples at three different confining pressures.The values of the effective angle of shearing resistance (ϕ’) and effective cohesion (c’) are shown in The values of the effective angle of shearing resistance improved with the addition of both types of fly ash. However, class C fly ash was more pronounced in comparison with class F fly ash in improving the effective angle of shearing resistance due to its chemical nature. According to Bryson et al. , the incThe results also indicate that the y-intercept of the critical state lines in the space of deviator stress versus mean effective stress, increased with the addition of class C fly ash for all curing times (except the soil sample stabilized with 15% class C fly ash at one day curing). The y-intercept of the CSL is related to cohesion and is a result of cementation . It coulcC), swelling index (sC), volume compressibility (vm), coefficient of consolidation (cv), and permeability (k).One-dimensional consolidation tests were carried out to evaluate the effects of fly ash on the consolidation characteristics of the soil including compression index  represents the amount of change in unit volume due to a unit change in effective stress:e/Δσ′ is the slope of the e/σ′ curve [The coefficient of volume compressibility , the coefficient of volume compressibility (mv), and the unit weight of water (γw) [The coefficient of permeability (k) of the samples was also estimated based on the results of the coefficient of consolidation (cter (γw) :K = cvmvv and k for different fly ash contents and curing times for an effective stress increment of 40–80 kPa. The results show that the value of cv of the soil increased with the inclusion of fly ash. A similar trend was observed from the analysis of permeability results. According to Wang and Tanttu [v leads to an increase in permeability. The permeability of the soil increased with the increase of fly ash content. This trend is comparable with the finding of Phanikumar [2+ ions from the fly ash lead to the formation of a flocculated structure in the clay. The flocculation leads to increase of permeability [v and k for the fly ash stabilized soil. The soil stabilized with class C fly ash showed a higher permeability than the control samples with one day and seven days of curing. However, the stabilized soil became less permeable in comparison with the control sample at 28 days of curing. On the other hand, the class F fly ash stabilized soils showed a higher k value than the control samples with 1, 7, and 28 days of curing, although the k value of the stabilized samples decreased with the curing time. The long-term decrease of permeability is due to the reaction of calcium aluminium silicate hydrate (CASH) and/or calcium silicate hydrate (CSH) [d Tanttu , the incanikumar . Accordianikumar , the Ca2eability . Mir andeability  also indeability  reportedte (CSH) . This cete (CSH) . Chew ette (CSH)  analyzedte (CSH)  reportedThe SEM analysis was carried out to evaluate the microstructural changes in the soil with the increase of curing days and fly ash percentages. The microstructures of the soil stabilized with class C and class F fly ash are shown in This paper investigated the influence of class C and class F fly ash on the strength, consolidation, and microstructural characteristics of a clay soil. The following conclusions can be drawn from the results presented in this paper.dmax and lower wopt in comparison with the soil stabilized with class F fly ash.The maximum dry unit weight of the soil decreased, and the optimum moisture content increased with increasing percentages of both types of fly ash. The soil stabilized with class C fly ash had higher γThe compressive strength of the soil increased with the addition of both types of fly ash and with the curing time. However, when the fly ash content increased from 25% to 30%, the compressive strength of the stabilized soil slightly decreased for both types of fly ash and for different curing times. Therefore, the optimal fly ash content appears to be 25% for both types of fly ash. Also, class C fly ash was found to be much more effective in improving the compressive strength of the soil than class F fly ash. The elastic modulus of the soil increased with the addition of both types of fly ash up to 25% and with increasing the curing time.The results of the CU triaxial tests indicated an improvement in the angle of shearing resistance and cohesion intercept with the addition of class C fly ash, whereas the cohesion intercept of the soil stabilized with class F fly ash was lower than the control sample. The curing time was effective in improving the values of c’ and ϕ’ for both types of fly ash stabilized soil. The gradient of the critical state line increased with increasing the class C and class F fly ash contents and with increasing the curing time.c of the soil stabilized with class C fly ash compared to the control sample. Furthermore, the Cc decreased with curing time for both types of fly ash. However, with class F fly ash, the Cc initially increased up to the particular fly ash content and thereafter decreased at one day of curing. The mv value showed a similar trend to the compression index. Cs decreased by the addition of the class C or class F fly ash. In addition, curing time was found to be an effective parameter in decreasing the swelling index.The results from the one-dimensional consolidation tests indicated a decrease in the CThe values of cv and k increased with the addition of class C or class F fly ash. However, both cv and k decreased with increasing the curing time for fly ash stabilized soils.The SEM analysis conducted on the soil stabilized with both class C and class F fly ash confirmed the gradual improvement in the soil properties and strength due to the formation of reaction products in the soil with the increase of curing time. However, the soil stabilized with class C fly ash had more reaction products and denser fabric than the soil stabilized with class F fly ash due to the better cementitious properties of class C fly ash. The results from the UCS, triaxial, and consolidation tests were found in agreement with the results from the SEM analysis.In general, it was observed that class C fly ash is more effective in improving the mechanical properties of the soil compared to class F fly ash. The findings have proven that class C fly ash can be used effectively in the stabilization of clay soils. Class F fly ash can be used with the other additives such as lime or alkali activators to achieve higher mechanical properties in clay soils.


This study represents a pioneering work on the extensional magnetorheological properties of human blood analogue fluids loaded with magnetic microparticles. Dynabeads M-270 particles were dispersed in Newtonian and viscoelastic blood analogue fluids at 5% wt. Capillary breakup experiments were performed, with and without the influence of an external magnetic field aligned with the flow direction. The presence of the particles increased the viscosity of the fluid, and that increment was larger when embedded within a polymeric matrix. The application of an external magnetic field led to an even larger increment of the viscosity of the working fluids, as the formation of small aggregates induced an increment in the effective volume fraction of particles. Regarding the liquid bridge stability, the Newtonian blood analogue fluid remained as a Newtonian liquid exhibiting a pinch-off at the breakup time in any circumstance. However, in the case of the viscoelastic blood analogue fluid, the presence of the particles and the simultaneous application of the magnetic field enhanced the formation of the beads-on-a-string structure, as the Ohnesorge number remained basically unaltered, whereas the time of the experiment increased due to its larger viscosity, which resulted in a decrease in the Deborah Number. This result was confirmed with fluids containing larger concentrations of xanthan gum. Human blood is a complex fluid, composed of cellular elements like red blood cells (RBCs), white blood cells (WBCs) and platelets suspended in plasma, an aqueous solution (approximately 90–92 wt.% water) containing organic molecules, proteins and salts ,2,3. It A complete rheological characterization of the whole human blood is of great importance for the diagnosis and treatment of main cardiovascular diseases to understand and to plan drug delivery through the circulatory system, and for the design and development of medical equipment such as blood pumps, heart valves or stents, among others ,8,9. TheBlood analogue solutions have been developed to replace real human blood for in vitro experiments. They possess characteristics such as nontoxicity, low cost and transparency, and avoid several complications regarding ethics, safety and costs ,17,18. TNewtonian and non-Newtonian (viscoelastic non-particulate) blood analogues were prepared and used for the extensional tests. An aqueous solution of 52 wt.% of dimethyl sulfoxide (DMSO) was prepared as a Newtonian blood analogue . To addr3  with a particle diameter of 2.75 µm and a density of 1.6 g/cm3 , were co3 . They we3O4 or γ-Fe2O3) in a porous matrix, in a 14% wt. Regarding the magnetization curve, three regions may be distinguished: (a) A first region (H ≤ 5 kA/m), corresponding to weak external magnetic fields, where the microparticle magnetization is proportional to the applied magnetic flux density . The initial aspect ratio is given by The rheological characterization under extensional flow was performed in the Capillary Breakup Extensional Rheometer , equipped with 4 mm diameter plates (2R0.5<Λ0<1 . Conside0.5<Λ0<1 .b). In order to capture key information about the elastocapillary regime, the experiments were repeated, and the last part of the thinning process was recorded with higher magnification. Each set of images acquired during the course of each experiment was postprocessed in Matlab to detect the filament interface and determine the minimum diameter (2Rmin) along its axis of symmetry. Previously, the ratio micron/pixel was determined by means of recording a series of standard filament diameters  with the optical setup at the same experimental conditions, providing resolutions of 3.6 µm/pix and 1.0 µm/pix , equipped with a set of optical lenses . A 52 mm Telecentric Backlight Illuminator provided a correct illumination and high contrast images of the liquid bridge profile, which was connected to a metal halide light source  by means of an optical fiber cable, as detailed in recent works ,31,32. H0 µm/pix . For eacThe application of a constant magnetic field parallel to the extensional flow during the CaBER experiments was carried out by means of an apparatus developed by Galindo-Rosales et al. . The fix3) and the carrier liquid (3), once the sample was loaded in the CaBER the magnetic field was immediately activated, then the plates were separated and the filament thinning process recorded. All this procedure, including the duration of the filament thinning process, lasted less than 5 s, which is significantly smaller than the characteristic sedimentation time (~50 s).To avoid particle sedimentation due to density mismatch between the particles  along the abscissa axis so that the initial potential flow regimes collapse onto the XG0 curve (which corresponds to the pure solvent). This behavior is normally noticed in low viscous fluids where the capillary pressure is only resisted by the inertia of the accelerating fluid molecules. On the contrary, for high enough extension rates , the minimum filament radius is initially moving towards the end-drops and the thin filament formed (stabilized by the unravelling polymer) is located on both sides between a large satellite drop in the middle and the two end-drops [The results from CaBER experiments for formulations not loaded with magnetic particles are shown in nd-drops .Then, it was confirmed from In the case of the Newtonian analogue fluid a, the prue fluid :(1)η=ηs(The application of the magnetic field (11.9 kA/m) slightly increases further the viscosity of the suspension in the Newtonian analogue fluid. While the magnetic field is not strong enough to induce a full magnetization of the particles and, consequently, a percolated network of particles cannot be generated, a mild magnetization is induced, resulting in magnetostatic forces that are strong enough to allow the formation of small clusters or aggregates of magnetic particles. These small clusters of particles are responsible for an effective volume fraction larger than the volume fraction calculated from the concentration  interconnected by slender threads, and they are characteristic of viscoelastic liquids that have undergone a filament thinning process . However diagram  reportedThe breakup time is the time it takes for a liquid bridge to break under a step-strain extensional experiment in the CaBER-1 device and it is shown in This study represents a pioneering work within the frame of elucidating the effect of the dispersing magnetic microparticles when a simultaneous external magnetic field is applied on the rheological properties of blood. Rheological blood analogue fluids have been considered in this work.The dispersion of Dynabeads M-270 particles in the working fluids resulted in an increment of the viscosity, which was larger when embedded within a polymeric matrix. That increment was even larger under the influence of the magnetic field, as the formation of small aggregates induced an increment of the effective volume fraction of particles.In the case of the Newtonian blood analogue, it remained as a Newtonian liquid exhibiting a pinch-off at the breakup time in any circumstance. However, in the case of the viscoelastic blood analogue fluid (XG100), the presence of the particle and the application of an external magnetic field induced the formation of the beads-on-a-string structure. The same behavior was observed for larger concentrations of XG. These results are of paramount importance as they constitute clear evidence of the variation of the rheological properties of blood, especially in the cases of a high degree of elasticity that can be related to some blood diseases. This fact would help to find more precise diagnostic techniques. In future works, we will explore the influence of larger magnetic fields, different particle sizes and concentrations in the magnetorheological properties of blood.


The affinity of an antibody for its antigen is primarily determined by the specific sequence and structural arrangement of the complementarity-determining regions (CDRs). Recent evidence, however, points toward a nontrivial relation between the CDR and distal sites: variations in the binding strengths have been observed upon mutating residues separated from the paratope by several nanometers, thus suggesting the existence of a communication network within antibodies, whose extension and relevance might be deeper than insofar expected. In this work, we test this hypothesis by means of molecular dynamics (MD) simulations of the IgG4 monoclonal antibody pembrolizumab, an approved drug that targets the programmed cell death protein 1 (PD-1). The molecule is simulated in both the apo and holo states, totalling 4 μs of MD trajectory. The analysis of these simulations shows that the bound antibody explores a restricted range of conformations with respect to the apo one, and that the global conformation of the molecule correlates with that of the CDR. These results support the hypothesis that pembrolizumab featues a multi-scale hierarchy of intertwined global and local conformational changes. The analysis pipeline developed in this work is general, and it can help shed further light on the mechanistic aspects of antibody function. On the one side, the selection of the isotype, and therefore those structural/dynamical features of the constant region that come with it, leads to different immune responses, and is thus performed on the basis of the planned application8; on the other side, modifications of single residues can determine a higher therapeutic efficacy, as in the case of those mutations introduced in the Fc domain to enhance effector function and recruitment of additional proteins11. A remarkable example is the single-residue mutation that, in the hinge of IgG4 antibodies, prevents Fab-arm exchange14.Even in the case of naked immunoglobuline drugs, which do not involve conjugation with radionuclides or small molecules15. However, the CDR loops are not the only possible loci of intervention; it is experimentally shown that both mutations near and far from the antigen-binding site can affect affinity16. Such mutations act by modulating the interdomain conformational dynamics of the antigen-binding fragment, which eventually reflects on the paratope, namely the antigen binding site. On a similar note, NMR relaxation dispersion experiments allowed researchers to detect important fluctuating residues that are not located in the CDR, whose point mutation can nonetheless increase antigen-antibody affinity17.Modifications of this type, which may be distributed throughout the whole antibody sequence, are usually introduced for reasons that are not directly linked to antigen affinity modulation. Engineering efforts intended to increase specificity and affinity are in fact mostly focused on the residues of the six loops comprising the complementarity-determining region (CDR), because of their preeminent role in antigen binding18. Molecular dynamics (MD) simulations, on the other hand, offer a valuable tool for the investigation of the interplay between stabilizing interactions, fluctuation correlations, and conformational variability at different levels of resolution and experimental conditions22. In silico structural investigation of immunoglobulins and antigen-antibody complexes has been successfully employed with different objectives, which include the detailed description of the dynamics of CDR loops and the transitions between their conformational states25, as well as the comprehension of structural rearrangements and allosteric modifications following antigen binding28.Empirical experimental optimization of binding affinity can be laborious and costly, both in terms of time and resources29 , a 288-residues long receptor located on the membrane of T cells, B cells, and natural killer cells34. PD-1 promotes apoptosis of the lymphocyte when activated by the programmed cell death receptor ligands PD-L1 and PD-L2, whose expression is upregulated in malignant cells35. The large contact area between pembrolizumab and PD-1 hinders the binding of PD-L1 and PD-L2, thus preventing down-regulation of the anti-tumor activity of T cells38.Here, we employ atomistic MD simulations to investigate the internal dynamics of full-length pembrolizumab, a humanized IgG4 antibody used in immunotherapy, whose full structure has been experimentally solved29 Fig. . PembrolWe performed a total of 4 μs of dynamics of the deglycosylated antibody, both in presence and in absence of the antigen PD-1. The analysis pipeline, which combines structural analysis, investigation of chemical interactions, and information theory-based measures of correlation, allowed us to rationalize at a residue-level the observed conformational dynamics. Our simulations highlighted the particular role of key residues of the hinge, resulting in an asymmetric behavior of the two hinge segments in pembrolizumab; moreover, an interplay between large-scale conformation and binding state is observed, and the residues allowing such information routing from the paratope throughout the antibody structure are identified. We believe that the residue-level elucidation of the complex dynamics of potent and highly selective mAbs—of which pembrolizumab is a notable example—in relation to their binding state may assist in the process of antibody engineering, for the rational design of novel, optimized therapeutic agents.14. A schematic representation of the protein is given in Fig. 29. We label Fab1 the Fab domain formed by chains A and B, and Fab2 the one including chains C and D; similarly, Fc1 includes domains CH2 and CH3 from chain B, while Fc2 includes those from chain D. Throughout the manuscript, all references to residue indices follow the EU numbering scheme39.Pembrolizumab is a 1324-residues long therapeutic antibody, belonging to the IgG4 class. On both hinge segments, it presents the typical S228P mutation, introduced to prevent fab-arm exchange in IgG4 antibodiesapo form) and bound to its antigen, the ectodomain of the protein PD-1 (holo form). The latter, comprising 114 residues, corresponds to the region of PD-1 whose structure has been experimentally solved in complex with pembrolizumab Fab40. To allow for an unbiased comparison, the starting structure of the holo system was built on the same initial antibody conformation of the apo case; specifically, Fab2 of pembrolizumab (formed by chains C and D) was replaced with the Fab-PD1 complex (PDB ID: 5GGS), after structural alignment on the antibody domain.We focused on the analysis of conformations and the rationale behind domain motion in dependence of the binding state; to this aim, the results presented here are based on a comparison between the deglycosylated antibody alone  of Cdy Figs.  and S5; re Figs.  and S7.27.To facilitate the analysis of antibody flexibility, the conformations sampled from MD simulations are collected in clusters on the basis of their structural similarity Fig. , as measentclass1pt{minimaentclass1pt{minimaFor each system (apo or holo), clusters are indexed according to increasing radii of gyration; in this way, clusters ter Fig. .41. This conformational preference has been explained on the basis of the short hinge of IgG4 antibodies with respect to other IgG subclasses; IgG4 hinge has indeed a three amino acid deletion, compared to the one of IgG142. Another factor that can favour the compact arrangement is the tilted conformation of the CH2 domain in the Fc29, which displays a rigid-body rotation of Streptococcus pyogenes, which binds the CH2–CH3 interface43. The asymmetric orientation of the two CH2 domains can also explain the contact area between Fabs and Fc, reported in Fig. 13.The relative cluster population  of CThe most open conformations  with dense internal correlations, but sparse inter-community connections  for each residue i as the sum of GCC values with all the other antibody residues. This allowed us to distinguish intra-domain scores  Section , ranging Section , particures Fig. , where tres Fig. , which ters Fig. . This reMutual information shows the highest correlation between the antigen molecule and all the antibody domains in cluster }0H Fig. , especia}1A Fig. ; in addi}1A Fig. . A simil58.While impressive progress has been made in the experimental characterisation and manipulation of antibodies, a detailed, atomistic investigation of their properties is still incomplete. This is particularly true for the interplay between the molecule structure and its dynamics, which is extremely rich and varied, as several studies have recently shown17. This is especially the case for IgG4 antibodies such as pembrolizumab, whose short hinge reduces the degree of flexibility and tightens the interactions among the various domains.If, on the one hand, the specific sequence of the CDR plays the most prominent role in the selectivity and binding affinity of the antibody, on the other hand the observation that a modulation of the binding strength can be effected though mutations in distal sites shows that the internal mechanics of these molecules can be extremely complexIn this work, we have made use of atomistic MD simulations and information-theoretical analysis methods to elucidate the relation between the large-scale arrangement of pembrolizumab and the stability of the antigen binding. The analysis pipeline employed has allowed us to highlight a substantial conformational variability, a quality rather different from that one might observe in the case of loosely-connected rigid bodies. On the contrary, a complex pattern of structural arrangements, intramolecular communication pathways, and binding strength has emerged.In accordance with experimental results, we observed that the antibody is prevalently found in a compact, asymmetric shape. This particular arrangement can be explained on the basis of the short hinge, which limits the conformational space accessible to Fab arms, as well as the rotated CH2 domain of chain B. Furthermore, the spectrum of structures sampled by the molecule is modulated by the binding state: the large conformational variability of the apo case is substantially restricted when the antibody is complexed with the antigen. Similarly, the flexibility of the hinge (hinge 1 in particular) is reduced in this latter case, with a high degree of correlation emerging between the binding site on Fab2 and hinge 1. The binding site was shown to bear strict ties with the rest of the molecule in general. The analysis of the distributions of the apo binding site RMSD, computed taking the experimentally resolved holo structure as a reference, highlighted a correlation between the binding site and the large-scale arrangement of the molecule as a whole.The binding of the PD1 antigen to Fab2 is, in general, rather stable in all holo state simulations; however, the strength of the binding is different across the various conformational clusters. The trend follows the intensity of the inter-domain correlations not only within the antibody, but also between the antigen and the antibody itself; a particularly strong affinity is observed in a specific configuration in which Fab1 is bent toward Fab2 and Fc. The tight binding and high correlations in this cluster suggest an interplay between the binding mode and the intensity of correlations, which manifest themselves in an extended network of interactions.The results reported in this work return a picture of antibodies as extremely complex molecules, with a rich pattern of structural and dynamical features. The analysis protocol here applied to pembrolizumab is completely general, which enables its widespread application to other antibodies, with the objective of acquiring an ever deeper understanding of their inner life and, in turn, providing sharper tools for the manipulation of these molecules for medical and technological purposes.29 was used as a starting structure for the MD simulations. Missing residues  were modelled with Chimera59, using an IgG2a antibody (PDB ID: 1IGT60) as a template. Glycans were removed from the crystal structure, so as to isolate the role of structural features of the antibody itself in the emerging dynamics. The system in the holo form (Ab2) was obtained by replacing one Fab in Ab1 with the crystallized structure of Fab-PD1 complex (PDB ID: 5GGS)40, after structural alignment. The Fab-fragment alone bound to the PD-1 (Ab3) was also simulated, in order to assess possible differences in the interaction with the antigen with respect to the full-length antibody.The crystallographic structure of pembrolizumab in the apo form 62. The amber99SB-ILDN force field63 was used to define the topology of the systems. The protein was solvated in a box of TIP3P water molecules64 and the charge was neutralized with Cl65 and the Parrinello-Rahman barostat66. Temperature was set at 300 K and pressure at 1 bar, and the time constants were set to 67. A cutoff of 10 Å was used for Van der Waals and short-range Coulomb interactions; long range electrostatics was treated with Particle Mesh Ewald. Ab1 and Ab2 were simulated for a total of 2 μs each, in 4 independent replicas of 500 ns. Ab3 was simulated for 500 ns. Additional simulations of the apo state were started from the representative holo conformations, after removal of the antigen; the structures were solvated and equilibrated using the above-mentioned procedure, and three 100ns-long replicas were simulated for each system.MD simulations were performed with the Gromacs 2018 software68. The distance used in the cluster definition is based on the root-mean-square deviation between C69. Calculation of atomic displacement, residue fluctuations, interatomic distances and contact surface areas, were performed using Gromacs 2018 utilities, while calculation of MM/PBSA energy was performed with the software g_mmpbsa52. Additional details on the MM/PBSA method and on the PAD70, setting an angle cutoff of 25°  and a maximum donor-acceptor distance of 3 Å.Clustering of the simulation frames according to structural similarity was performed with an in-house script using hierarchical clustering (UPGMA)gmx covar and gmx anaeig62. In order to visualize the direction of movements captured by the eigenvectors, porcupine plots were generated using the extreme projections on the first three principal component, and visualized with VMD; here, the direction of the arrow on each CPrincipal component analysis (PCA) was performed for each cluster using CMI) was calculated as a measure of inter-residue communications. MI is defined as follows:i-th or j-th atoms with a displacement equal to GCC) is computed as:Mutual information  to 1 (perfect correlation)54. Calculations were performed through in-house scripts.GCC was in turn employed to calculate the correlation score (CS). For each residue i, i and the other protein residues:intra-domain CS, the sum extends to the residues belonging to the same structural domain as residue i; in the case of the inter-domain CS, the summation takes into account only residues belonging to all the other structural domains, excluding that of residue i.In the case of the Networks were defined as sets of interconnected nodes centered on the Ci and j is defined as:Each edge is weighted according to the generalized correlation coefficient measure; specifically, the weight of the edge between nodes entclass1pt{minima71, which makes use of the NetworkX package72 and is optimized using Cython73 and Numba74. In the weighted networks, the sets of communities were identified using the Girvan-Newman algorithm76. The importance of the edge for communication within the network was measured in terms of edge betweenness, which is defined as the number of shortest pathways that cross the edge; this quantity was calculated with the commonly used Floyd–Warshall algorithm78. For a detailed description of the methods used for network analysis, the reader is referred to the Supplementary Information.The network analysis was performed with the Python implementation found in Melo et al.Supplementary Information 1.