Achot Khalafian
email: achot.khalafian@ifsttar.frbeatrice.degraeve@univ-catholille.fr

Marie-Axelle Granié

Florent Varet

Béatrice Degraeve

The role of context and perception of road rules in the pedestrian crossing decision: a challenge for the autonomous vehicle

Keywords: Pedestrian, Compliance, Rule perception, Social norms

The role of context and perception of road rules in the

pedestrian crossing decision: a challenge for the autonomous vehicle. In I.

The decisions we make are not always the most rational and prudent. We all know that we do not comply with all existing health, hygiene and safety rules, even if we are fully aware of them. Knowing the traffic rules is not enough to guarantee their application. To fully understand user behaviors, taking into account cognitive mechanisms is not enough. Pedestrian behavior is also based on the construction of social knowledge and representations of the roles of different users, rules and risks.

In this chapter, we propose to review studies that highlights the role of individual and social factors in pedestrian crossing decisions -and how these factors can challenge the implementation of the autonomous vehicle. Specifically, we begin by presenting the results of various studies we have conducted showing that pedestrian compliance with road rules varies according to crossing regulation, the built environment, gender and age. We then address the role of rule perception and internalization. Finally, we emphasize how informal learning, through observation, of road rules can explain the construction of conventional level rules, with contextual application. We end by discussing how knowledge of social norms can be an issue for the autonomous vehicle.

It is a truism to say that user behavior does not always comply with the rules of the road. However, there is a need to better understand the situations in which this lack of compliance occurs and the reasons for it. For example, in a study examining reported and actual behavior of children aged 5-6 years [START_REF] Granié | Gender differences in preschool children's declared and behavioral compliance with pedestrian rules[END_REF], we observed that children are aware of pedestrian rules and report compliance with them, but do not apply the same rules when they are observed on the way home from school with their parents. Knowing a rule does not necessarily mean complying with it. This compliance, which we studied especially among adults, varies in particular according to the physical environment and the perception of road rules.

In a real-life study [START_REF] Tom | Gender Differences in Pedestrian Rule Compliance and Visual Search at Signalized and Unsignalized Crossroads[END_REF], we observed 400 pedestrians (200 men and 200 women) aged 18 to 55 years at 4 intersections, including 2 without traffic lights. The results showed that spatial conformity (use of the pedestrian crossing) varies according to the configuration of the intersection: pedestrians are more compliant with the use of the pedestrian crossing in the presence of a traffic light than in its absence. Crossing regulation is most often implemented when the infrastructure is complex and/or traffic or pedestrian density is high. The presence of regulation not only acts as a reminder of the injunctive norm, but can be interpreted as a sign of a significant dangerousness of the crossing, leading to increased spatial compliance among pedestrians, whose compliance thus varies according to the road layout.

A subsequent study [START_REF] Dommes | Red light violations by adult pedestrians and other safety-related behaviors at signalized crosswalks[END_REF] observed pedestrian behavior at crossings regulated by pedestrian traffic lights by combining in situ observation of the behavior of 422 French adult pedestrians with questionnaires collected after the crossing. The results showed that the size of pedestrian groups and traffic density are associated with safer behaviors, while the presence of vehicles parked near the crossing site are associated with higher risk behaviors. In addition, the driving experience appears to increase safety behaviors and alertness. Pedestrians who report difficulty crossing a road look more towards the traffic light and tend to cross the road in a straight line rather than diagonally. In addition, pedestrians who have already fallen into the street -reported more by older pedestrians -look more closely at other pedestrians before and during the crossing. In terms of temporal compliance (pedestrian green light crossing), pedestrians who cross against the red pedestrian light look less towards the ground and the light, but more towards the traffic before and during the crossing than those who complied with the traffic light. They also run and jaywalk more often. It appears that pedestrians anticipate pedestrian red light violations before they arrive at the intersection to be crossed, depending on traffic conditions and not the color of the pedestrian light, as they look less towards the traffic light before crossing. These behaviors observed before and during the crossing could reveal a deliberate timereduction objective, where the intentional violation of pedestrian red lights goes hand in hand with the vigilance of incoming traffic before and during the crossing, suggesting an awareness of the risk pedestrians take. The role of two gaze targets (traffic light and incoming traffic) should be further examined as possible indicators of an intention to violate the crossing time rule.

Variation according to gender and age

In addition to these variations in spatial and temporal compliance, it appears that pedestrian behavior also varies by sex. The results of Tom & Granié [START_REF] Tom | Gender Differences in Pedestrian Rule Compliance and Visual Search at Signalized and Unsignalized Crossroads[END_REF] showed differences between men and women in temporal compliance and in visual research before and during the crossing. Men's visual focuses are mainly on moving vehicles, while women's visual attention is mainly focused on other pedestrians. In addition, these results show that the visual attention of males does not vary with the crossing configuration (with or without pedestrian light) or the crossing phase and remains focused on the dynamic elements of the environment, such as moving vehicles. On the contrary, women, when crossing with traffic lights, mainly look at the pedestrian light before the crossing and at other pedestrians during the crossing. They seem to be more attentive than men do to the static elements of the scene and -in the absence of external regulation to facilitate decision-making -to the social environment rather than the physical environment. The visual centering of female participants on other pedestrians, particularly in the absence of a time rule for crossing, can be interpreted as the result of a stronger social influence and normativity on decision-making among women than among men.

In another in-situ study [START_REF] Granié | Etude des effets de l'âge et du contexte de traversée de rue sur les comportements observés sur passages piétons régulés[END_REF], we observed, using the same observation grid as in Tom & Granié [START_REF] Tom | Gender Differences in Pedestrian Rule Compliance and Visual Search at Signalized and Unsignalized Crossroads[END_REF], 682 pedestrians (375 women) of two age levels (middle-aged and older adults) at five sites in a major French urban center. The sites all included pedestrian crossings, in mid-block or intersection, regulated or not by traffic and pedestrian lights. The results showed that, in the situation of regulated pedestrian crossings, older people comply with the legal and safety rules more often than middle-aged adults do. They focus on a more diversified number of different visual indicators in preparation for their crossing. Their gazes are more focused on the ground, both before and during the crossing. They also stop more on the sidewalk before crossing, cross when the light is red for traffic, without running. However, the more cautious behavior of older pedestrians is accompanied by less traffic control than among younger adults, who are more likely to violate the pedestrian light rule. Behavioral control and compliance with the rules of caution seem to be accompanied by a delegation of responsibility to the regulatory system in crossing decision-making and a concomitant decrease in traffic condition monitoring.

Variation according to the built environment crossed

This contextual compliance seems to be based, at least among pedestrians, on their perceptions of the crossing environment and, more particularly, on the inferences that pedestrians make from certain clues found in the environment about the pedestriandriver balance of power.

In an exploratory study [START_REF] Granié | Qualitative analysis of pedestrians' perception of the urban environment when crossing streets[END_REF], we analyzed the role of the environment, understood in a broader definition including the built environment, on pedestrians' perception of the situation. We used a focus group methodology with two groups of five and six pedestrians. We had them discuss the walking pleasantness and facility of crossing 20 environments, photographed in order to give a 180° view of the road scenes. The results showed that two types of crossing environments are positively rated by the pedestrians interviewed. Some pedestrians prefer environments that are complex for the driver to deal with (wide sidewalks, lack of parking, markings and pedestrian crossings, context animated by the presence of coffee houses and shops), i.e., environments containing ambiguity and uncertainty [START_REF] Engwicht | Intrigue & Uncertainty[END_REF], requiring increased attention of the drivers towards pedestrians. On the contrary, other pedestrians prefer environments that organize and simplify pedestrian use (presence of pedestrian crossings, well-delimited sidewalks, clear visibility, in a rather calm context, and with moderate traffic). In these environments, legibility and predictability are important, both for the driver and for the pedestrian -thus facilitating decision-making, thanks to the presence of a highly regulated space.

In a second part of the same research project, we set up an experimental study [START_REF] Granié | Influence of built environment on pedestrian's crossing decision[END_REF] whose objective was to identify the variations produced in decisions to cross, depending on different types of urban environments. This experiment also aimed to identify the environmental characteristics that pedestrians take into account and the inferences they develop and use to explain their decision to cross a road. A series of photographs presenting five different types of environments (i.e., city center, inner suburbs, public housing in the outskirts, commercial zone in the outskirts and countryside) were presented to 77 participants divided into three age groups (preadolescents, young and middle-aged adults). Their decision to cross or not to cross, their perception of pleasantness and safety, and the elements they considered when making a decision were collected for each environment presented. The quantitative results showed that pedestrians' perception of the pleasantness and safety of public spaces, in terms of walking, varies widely across urban environments. In addition, the decision to cross varies significantly depending on the environment. Pedestrians were much more likely to make the decision to cross the street in the city center than in the other sites presented. The qualitative analysis of the interviews showed that the presence and function of buildings, the quality of sidewalks and the presence of marked parking spaces are key factors in explaining their decision to cross, allowing them to deduce pedestrian and traffic density and vehicle speed.

In these studies too, it is clear that the rules are used flexibly by pedestrians, depending on the context, and especially on the situation in which the pedestrian and the driver find themselves. This reveals how the pedestrian, a member of a dominated and vulnerable group in the road space, finds himself in need of inferring information about the drivers, a dominant group in terms of density, potential nuisance to the pedestrian and urban development. Thus, pedestrians place great emphasis on environmental analysis, looking for clues to understand their place in the environment in the balance of power with the driver, and driver's current and predictable behavior.

We found further evidence of this estimation of power balance in the study of perceptions of crossing environments among pre-adolescents [START_REF] Granié | Perceptions des environnements de marche connus et inconnus chez des collégiens piétons en France[END_REF]. Three hundred and forty-two secondary school students aged 11 to 16 were asked about their perceptions of the level of pleasantness, safety and convenience of five crossing environments near secondary schools (i.e., the three schools from which the participants came, one from another French region and one from Quebec, Canada). Each environment was represented through five photographs, reproducing a 180° vision from the crossing point. Analysis of the comments showed that the most highly positively rated aspects of the presented environments concern the aesthetic appeal of residential housing, the impression of space emerging from the width of the lanes, as well as the large number of pedestrian crossings and recent road markings.

On the contrary, the relative anarchy of parking also gives pre-adolescents the feeling that pedestrians are not taken into account and respected by drivers. Vehicle parking and the type of built environment therefore seem to be important indicators, allowing both high school students and adult pedestrians to infer the current and future behavior of drivers [START_REF] Granié | Influence of built environment on pedestrian's crossing decision[END_REF]. Similarly, the presence of traffic lights can be negatively perceived by some pre-adolescents who perceive them as an indicator of traffic density (lights are only used when there is a need to regulate vehicle flows) and therefore of more complex and dangerous crossings. On the contrary, according to other pre-adolescents, the presence of traffic lights makes crossing easier, because it allows a delegation of decision-making to the environment, lowering the feeling of danger. This protective effect of traffic lights had already been pointed out in preadolescents who are not very confident about independent travel [START_REF] Granié | Etude qualitative du comportement piéton de collégiens par la méthode de l'autoconfrontation[END_REF] and observations of senior pedestrians seem to show that the regulation of pedestrian-driver interactions by the layout is also sought when perceptual-cognitive abilities decline [START_REF] Dommes | Red light violations by adult pedestrians and other safety-related behaviors at signalized crosswalks[END_REF][START_REF] Granié | Etude des effets de l'âge et du contexte de traversée de rue sur les comportements observés sur passages piétons régulés[END_REF].

Thus, one of the five environments presented is denigrated by all pre-adolescents, including those who frequent it on a daily basis. Participants explained this negative perception by the absence of sidewalks and the degradation of ground markings, including pedestrian crossings, which is perceived as the symbol that pedestrians are not taken into account in the environment. Inversely, the environments perceived positively by pre-adolescents have in common the strong presence of recent road markings, which, like the width of the pavements, not only mark and differentiate everyone's spaces [START_REF] Granié | Qualitative analysis of pedestrians' perception of the urban environment when crossing streets[END_REF] but also reinforce the real but also symbolic visibility of pedestrians.

The role of rule perception

Compliance with traffic rules is not only subject to the local context at the time of decision-making. It also varies according to the perception of road rules themselves. Thus, perception of danger and risk associated with pedestrian behavior, normative beliefs and reported behaviors differ according to whether or not the danger to oneself is intentional.

Differences between errors, transgressions and lapses

As part of a research on the influence of educational practices on risky behavior, we interviewed 258 adult parents (104 males and 154 females), aged 25 to 54 years old, about their perception of danger, self-risk, normative beliefs and reported behaviors in response to injury-risk pedestrian behavior, using the Road User Behavior Perception Scales (EPCUR) [START_REF] Granié | Influence de l'adhésion aux stéréotypes de sexe sur la perception des comportements piétons chez l'adulte[END_REF]. EPCUR is composed of four subscales, each randomly presenting 16 pedestrian behaviors. This list of behaviors, based on Elliott and Baughan's tool [START_REF] Elliott | Developing a self-report method for investigating adolescent road user behavior[END_REF] and previous research on pedestrians [START_REF] Granié | Influence de l'adhésion aux stéréotypes de sexe sur la perception des comportements piétons chez l'adulte[END_REF], differentiate between non-hazardous transgressions (for example, "crossing at a red pedestrian light in a street without a car") and dangerous behaviors without transgression (for example, "crossing while forgetting to look"). The first subscale (Declared Behavior Scale) measures the frequency with which the individual reports manifesting each behavior. In the second subscale (Hazard Perception Scale), the individual must assess the level of danger they perceive about each situation. In the third subscale (Risk Perception Scale), the individual must intuitively assess his or her own probability of having an accident if he or she engages in the above-mentioned behavior. The fourth subscale (Normative Belief Scale) measures individual cognitive norms about the acceptability of behavior [START_REF] Guerra | The role of normative beliefs in children's social behavior[END_REF][START_REF] Huesmann | Children's normative beliefs about aggression and aggressive behavior[END_REF]. The individual must estimate the level of severity of the transgression he or she attributes to each of the behaviors presented.

Analyses of the responses at these different scales showed that individuals differentiate two groups of behaviors for each scale: (1) self-endangerment behaviors, including lack of visual information taking, (2) offence and risk taking behaviors, including absence of temporal or spatial conformity (lights or pedestrian crossings) and errors during pedestrian-vehicle interaction. These analyses of the EPCUR scales thus revealed that pedestrian behavior, whether in terms of perception of danger and risk, normative beliefs or declared behavior, is differentiated by adults on a single dimension: intentionality or not of the endangerment. Moreover, it appears that intentional behaviors include both offences against legal rules and diagnostic errors, concerning the estimation of intervehicular time and the influence of other pedestrians. This lack of differentiation among pedestrians between infringements and errors as shown in this study raises questions that we addressed to other studies. Using the theoretical framework used by Reason in the construction of the Driver Behavior Questionnaire [START_REF] Reason | Errors and violations on the roads: a real distinction?[END_REF] and the scales of aggressive [START_REF] Lawton | Predicting road traffic accidents: the role of social deviance and violations[END_REF] and positive [START_REF] Özkan | A new addition to DBQ: Positive Driver Behaviours Scale[END_REF] driving behaviors towards other users, we developed the Pedestrian Behavior Scale (PBS) [START_REF] Granié | Validation française d'une Echelle de Comportements Piétons[END_REF][START_REF] Granié | Developing a self-reporting method to measure pedestrian behaviors at all ages[END_REF].

The 47 items of this scale are based on existing validated versions of the PBQ [START_REF] Moyano Diaz | Teoria del Comportamiento Planificado e intencion de infringir normas de transito en peatones[END_REF][START_REF] Torquato | Comportamento de Risco do Pedestre ao Atraversaar a Rua: Um Estudo com Universitarios[END_REF][START_REF] Yildirim | Religiousness, conservatism and their relationship with traffic behaviours[END_REF], as well as other reported pedestrian behavior scales [START_REF] Granié | Influence de l'adhésion aux stéréotypes de sexe sur la perception des comportements piétons chez l'adulte[END_REF][START_REF] Elliott | Developing a self-report method for investigating adolescent road user behavior[END_REF], and differentiate between 5 types of pedestrian behavior: 1/ Violations, defined by a deviation from the legal rules of the highway code concerning pedestrian behavior (10 items); 2/ Errors, defined as decision-making that places the pedestrian in danger, but without violations of the legal rules (11 items); 3/ Inattentions, defined by inappropriate behaviors related to a lack of concentration on the task (8 items); 4/ Civic behaviors, defined by behaviors to calm social interactions (5 items); 5/ Aggressive behaviors, defined by conflicting behaviors with other users (6 items). Following Torquato and Bianchi [START_REF] Torquato | Comportamento de Risco do Pedestre ao Atraversaar a Rua: Um Estudo com Universitarios[END_REF], "filter" items were added (7 items).

Study 1 [START_REF] Granié | Validation française d'une Echelle de Comportements Piétons[END_REF] was conducted with 276 students (175 female and 101 male) aged 26 to 40 years. Factor analyses revealed five axes: "violations or errors during the crossing", "inattention", "violations or errors during travel", "aggressive behavior", and "positive behavior". Study 2 [START_REF] Granié | Developing a self-reporting method to measure pedestrian behaviors at all ages[END_REF] was conducted with 343 participants (217 female and 126 male) aged 15 to 78 years. Here, factor analyses revealed four axes. Factor 1 "transgression" includes legal violations and error items. Factor 2 includes "inattention" items. Factor 3 is composed of items of "aggressive behaviors" and factor 4 includes items of "positive behaviors".

The main difference between the factor structure of the DBQ and the Pedestrian Behavior Scale (PBS) is that, contrary to the DBQ for drivers, the transgression axis of the PBS includes both offence and error items. However, this result is consistent with Elliott and Baughan's study [START_REF] Elliott | Developing a self-report method for investigating adolescent road user behavior[END_REF] on the adolescent pedestrian behaviors, whose "unsafe street crossing" dimension is composed of both error and violation behaviors. Similarly, these results can be compared to the previously cited validation study [START_REF] Granié | Influence de l'adhésion aux stéréotypes de sexe sur la perception des comportements piétons chez l'adulte[END_REF], which also differentiated between self-endangerment behaviors (inattention) and intentional risk-taking behaviors (error-transgression). In their perception of pedestrian rules and compliance behavior, it seems that individuals differentiate behavior according to whether or not there is prior intention (present in errors and violations, but not in inattentions) rather than on the basis of objective (consequences of the act for themselves and others) or legal (infringement or not of a traffic rule) criteria.. These risk-taking behaviors (i.e., the deliberate engagement in behaviors in which accidental risk is perceived [START_REF] Saad | Prise de risque ou non perception du danger[END_REF]), are either deviations from legal rules or from informal rules of caution. In other words, French pedestrians do not seem to clearly differentiate legal rules and cautionary rules. In contrast to pedestrians, the studies using the DBQ conducted on drivers (and particularly on French drivers [START_REF] Guého | French validation of a new version of the Driver Behaviour Questionnaire[END_REF]), all showed that drivers differentiate, within intentional risky acts, between behavior that violates legal rules and error behavior. This suggests that legal and social rules are less differentiated among pedestrians than among drivers.

Differences in social value between self-endangerment and risk-taking

This lack of differentiation between transgression and error in pedestrians in favor of a broader differentiation between intentional risk behavior and self-endangerment related to inattention is perceptible from adolescence onwards.

A recent study we conducted analyzed the evolution of relationships with parental and peer behaviors on a sample of 2,473 secondary school students aged 10 to 16 years, equally distributed in terms of gender and age [START_REF] Granié | Effet de l'âge et du sexe sur la relation aux normes des parents et des pairs : étude sur des adolescents piétons. 7èmes entretiens francophones de psychologie[END_REF]. Secondary school students were asked about their perceptions of the frequency -for themselves, their parents and their peers -of intentional or unintentional risky behaviors as pedestrians. The results showed that secondary school students systematically assess their behavior as being more often risky than they reported for their parents, and less often risky than they reported for their peers, regardless of the type of behavior. However, the results showed differences according to age, gender, but also the type of behavior mentioned. Indeed, regarding intentional risky behaviors (and unlike unintentional risky behaviors), the gap between Self and parents is larger for boys and increases with age, while the gap between Self and peers decreases with age and is greater for girls. This suggests that different logics are involved, for each type of behavior (intentional versus unintentional), depending on the way in which relationships with parental and peer norms are constructed. They also show that there is a gap between actual social behavioral norms and adolescents' perceptions of them. By gradually conforming to peer norms on intentional risk behaviors but not on self-endangerment, adolescents, particularly boys, seek to confirm themselves as individuals separated from their family group and to confirm themselves as members of their sex group. Our results among adults [START_REF] Granié | Influence de l'adhésion aux stéréotypes de sexe sur la perception des comportements piétons chez l'adulte[END_REF] seemed to confirm that for men: intentional risk-taking was more acceptable than unintentional endangerment. This shows that intentional risky behavior also seems to be a way of social valuing, at least for adolescent boys.

As we will see, this valuation of intentional risky behavior among men may be the consequence of the socialization process experienced by young pedestrians, who are more confronted during their acculturation on the road with the normative behaviors of their social environment than with the legal rules concerning pedestrian behavior.

Socialization to pedestrian rules and behaviors

Informal learning of traffic rules

To better understand the state of skills and forms of behavioral learning in childhood, we used the auto-and allo-confrontation method [START_REF] Mollo | Auto-and allo-confrontation as tools for reflective activities[END_REF] with nine preadolescents aged 11-12 (5 girls and 4 boys) in the first year of secondary school, observed and then questioned about their behavior during their walk from home to school [START_REF] Granié | Etude qualitative du comportement piéton de collégiens par la méthode de l'autoconfrontation[END_REF]. Our results showed first of all that journeys seem to be affected by a form of routinization based on behavioral and travel habits and a strong knowledge of the specific traffic contexts on the route taken, leading to a strong sense of control of the situation in everyday travel environments. However, children are aware that they cannot use this knowledge in unknown environments, which in turn leads to greater compliance with legal rules when the context changes. In addition, while some important pedestrian skills to handle crossing situations [START_REF] Thomson | Promoting Pedestrian Skill Development in Young Children[END_REF] appear to be fairly well mastered by the children in our sample (i.e., crossing site selection, environmental analysis and time gap estimation), compliance with road rules is far from systematic.

Children comply with the rules according to their perception of the constraints they involve, their knowledge of the road environment and their assessment of traffic conditions. Moreover, their observation of offences committed by pedestrians and motorists leads them to put into perspective both the validity of the rules and the need to comply with them. Their expectations of driver behavior are all the more limited because, in accordance with previous studies [START_REF] Platt | Road safety education for children transferring from primary to secondary school[END_REF], they perceive drivers as not respecting the rules, particularly those governing their interactions with pedestrians. Thus, the road environment is often seen as hostile, insecure, unpredictable, and vehicle-dominated and the relationship with the driver is not perceived as an interaction but as a relationship of driver domination over pedestrians [START_REF] Granié | Les trajets à pied comme temps et objets de socialisation chez les collégiens français[END_REF]. In this respect, these results are in line with the results previously presented [START_REF] Granié | Influence of built environment on pedestrian's crossing decision[END_REF][START_REF] Granié | Perceptions des environnements de marche connus et inconnus chez des collégiens piétons en France[END_REF] in which pedestrians' judgments about environments are based mainly on the estimation of the pedestrian/driver balance of power. The 11-year-old pedestrians interviewed thus use their own rules, which are defensive in nature, to the detriment of legal rules. Moreover, this and other studies [START_REF] Granié | La construction des règles comportementales sur le port de la ceinture chez l'enfant: analyse du contenu d'entretiens auprès d'enfants de 5 et 8 ans[END_REF] show that, in this situation as in others, the child acquires knowledge both through direct confrontation with the situation and through observation of the behavior of others.

During this informal learning, the child can reproduce the behaviors that can be observed in experienced adult pedestrians, but not the skills that lead to their decisions. Traffic knowledge among the young pedestrians interviewed is only based on informal learning of the road space, as part of their usual daily journeys. They identify rules of action, based solely on the behavioral patterns they can observe in other pedestrians, without formal learning of the rules and cognitive skills involved in pedestrian behavior.

Representation of traffic rules and compliance

The studies we have conducted show the contextual variability that children observe in the behavior of others leads them to categorize road rules in the conventional domain, defined in social domain theory as gathering rules of an arbitrary, relative, modifiable and contextual nature [START_REF] Granié | Etude qualitative du comportement piéton de collégiens par la méthode de l'autoconfrontation[END_REF][START_REF] Granié | Socialisation au risque et construction sociale des comportements de l'enfant piéton : éléments de réflexion pour l'éducation routière. Enfances, Familles[END_REF]. This categorization of traffic rules at the conventional level affects the compliance of individuals, because conventional rules remain external and are not internalized (i.e., integrated into the individual's value system) [START_REF] Grolnick | Internalization within the family: the selfdetermination theory perspective[END_REF].

In one of the studies mentioned above, conducted with 162 children (83 boys and 79 girls) in the last year of kindergarten (5-6 years) [START_REF] Granié | Gender differences in preschool children's declared and behavioral compliance with pedestrian rules[END_REF], we observed both the knowledge of road rules, their compliance and internalization and their behavior during the accompanied home-school journey. The rule governing the behavior is considered internalized if the individual avoids to commit this behavior even in the absence of any external disapproval [START_REF] Turiel | The development of morality[END_REF]. The results revealed that children with higher internalization of traffic rules report more cautious and more controlled behaviors on the street.

However, internalization appears to vary with age. Our results on young adolescents aged 11 to 15 years [START_REF] Granié | Sex differences, effects of sex-stereotype conformity, age and internalization on risk-taking among pedestrian adolescents[END_REF] showed that, like the perception of danger, internalization is an important inhibitor of risky pedestrian behavior. However, the reported risky pedestrian behavior increases during adolescence, while the perception of danger and the level of internalization decrease.

The challenges for the autonomous vehicle All the studies presented show that pedestrian behavior at the time of crossing is not based on the traffic rules of the Highway Code. They are based on social norms in which the behaviors to be adopted vary according to the situations, gender and age of individuals, with some intentionally transgressive behaviors being valued by certain social groups. These elements of knowledge about social norms are important to take into account in current thinking about autonomous vehicles for several reasons.

First, the individual has access to these social norms regarding travel in road space before learning the legal rules of the Highway Code. The individual can observe these social norms in action early on, during his or her first travel experiences, as a pedestrian but also as a vehicle passenger. It is therefore important to understand their learning, which will guide the perception of risk and the categorization of legal rules and thus the individual's behavior and compliance with legal rules, throughout life.

Secondly, it must be taken into account that these social norms govern the actual functioning of road space, more than legal rules. These social norms (what the subject may define as "normal" behavior to adopt when travelling in an urban environment) are not necessarily in line with legal norms. The more the social norms to which users refer and comply differ from legal norms, the more difficult their behavior may be to anticipate, if these social norms are not known by the users with whom they interact. Individuals must therefore acquire knowledge both of the social norms to which they must comply, but also of the social norms to which other types of users comply.

Making decisions in road space -whether as pedestrian or driver -requires anticipating the behavior of others according to the role they play in that space (for example, the use of another mode of transport) and the social rules that are linked to that role [START_REF] Foot | Children's understanding of drivers' intentions[END_REF]. Thus, knowledge of legal norms is not sufficient to anticipate correctly the behavior of others, it is also essential to know and understand the different social norms by 1/ identifying them through the behavior patterns of different types of users [START_REF] Granié | La construction des règles comportementales sur le port de la ceinture chez l'enfant: analyse du contenu d'entretiens auprès d'enfants de 5 et 8 ans[END_REF] ; 2/ within the same type of users, differentiating them according to certain criteria of belonging to social groups: women, men, young people, the elderly, motorcyclists, truck drivers, drivers of powerful cars, etc. [START_REF] Granié | Etude qualitative du comportement piéton de collégiens par la méthode de l'autoconfrontation[END_REF]. These social categorizations will allow a classification of observed behaviors that will facilitate the anticipation of other users' behaviors, depending on the situation, their roles in the urban space, and refined by taking into account their membership of a certain social group [START_REF] Mundutéguy | Perception et anticipation du comportement d'autrui en situation de conduite automobile[END_REF]. As a result, expectations of others are not based on legal norms, but on the social norms corresponding to their social group and mode of travel. Knowledge of social norms is thus essential to understand and anticipate the behavior of others during interactions between users in the road space. These elements should be considered in the ongoing discussion on the autonomous vehicle in all industrialized countries.

This knowledge can also be taken into account in the necessary discussions on the pedestrian behavior models that will have to be implemented in the autonomous vehicle, so that it correctly anticipates the behavior of the pedestrians with whom it is likely to interact. The research presented in this chapter showed that pedestrian behavior is strongly related to context (presence or not of regulated crossings, built environments of crossing locations) and individual variables (gender, age, but also time pressure). They also show that pedestrians modify their behavior according to the balance of power with the driver, which they consider more or less favorable depending on the situation. The autonomous vehicle must therefore take into account the fact that the pedestrian's behavior towards it may vary according to the spaces crossed but also according to individual characteristics.

They can be taken into account in the discussions on the behavior models that must be implemented in the autonomous vehicle so that its behavior is understandable and predictable by users of the different transport modes. Using social norms in the model of driver behavior would make the behavior of the autonomous vehicle more understandable and easier to anticipate by other drivers but also by pedestrians. For interactions with other vehicles, this consideration of social standards should at least be necessary on a transitional basis, pending a complete homogenization of the fleet of vehicles on the road. It will also be necessary to secure interactions with users of other modes of travel that will remain un-automated. This requires a correct understanding of the content of the social norms used by drivers (which may vary according to culture), interacting with other drivers but also with other types of users. This could be studied through observations in real or virtual situations, but also

  Granié, M.-A., Varet, F., Degraeve, B., & Khalafian, A. (2019). The role of context and perception of road rules in the pedestrian crossing decision: a challenge for the autonomous vehicle. In I. Nunes (Ed.), Advances in Human Factors and Systems Interaction. AHFE 2019. Advances in Intelligent Systems and Computing (Vol. 959, pp. 221-233): Springer, Cham.

  Granié, M.-A., Varet, F., Degraeve, B., & Khalafian, A. (2019). The role of context and perception of road rules in the pedestrian crossing decision: a challenge for the autonomous vehicle. In I. Nunes (Ed.), Advances in Human Factors and Systems Interaction. AHFE 2019. Advances in Intelligent Systems and Computing (Vol. 959, pp. 221-233): Springer, Cham.

  Granié, M.-A., Varet, F., Degraeve, B., & Khalafian, A. (2019). The role of context and perception of road rules in the pedestrian crossing decision: a challenge for the autonomous vehicle. In I. Nunes (Ed.), Advances in Human Factors and Systems Interaction. AHFE 2019. Advances in Intelligent Systems and Computing (Vol. 959, pp. 221-233): Springer, Cham.

  Granié, M.-A., Varet, F., Degraeve, B., & Khalafian, A. (2019). The role of context and perception of road rules in the pedestrian crossing decision: a challenge for the autonomous vehicle. In I. Nunes (Ed.), Advances in Human Factors and Systems Interaction. AHFE 2019. Advances in Intelligent Systems and Computing (Vol. 959, pp. 221-233): Springer, Cham.

  Granié, M.-A., Varet, F., Degraeve, B., & Khalafian, A. (2019). The role of context and perception of road rules in the pedestrian crossing decision: a challenge for the autonomous vehicle. In I. Nunes (Ed.), Advances in Human Factors and Systems Interaction. AHFE 2019. Advances in Intelligent Systems and Computing (Vol. 959, pp. 221-233): Springer, Cham.

  Granié, M.-A., Varet, F., Degraeve, B., & Khalafian, A. (2019). The role of context and perception of road rules in the pedestrian crossing decision: a challenge for the autonomous vehicle. In I. Nunes (Ed.), Advances in Human Factors and Systems Interaction. AHFE 2019. Advances in Intelligent Systems and Computing (Vol. 959, pp. 221-233): Springer, Cham.

  Granié, M.-A., Varet, F., Degraeve, B., & Khalafian, A. (2019). The role of context and perception of road rules in the pedestrian crossing decision: a challenge for the autonomous vehicle. In I. Nunes (Ed.), Advances in Human Factors and Systems Interaction. AHFE 2019. Advances in Intelligent Systems and Computing (Vol. 959, pp. 221-233): Springer, Cham.


I Tsapis

A Andreana

C Chamarat

Camilla Foged

M Delomenie

Nabil Noiray

El

Jean-Pierre Pierre Majoral

Serge Mignani

Adam Bohr

Nicolas Tsapis

Ilaria Andreana

Anais Chamarat

Claudine Delomenie

Magali Noiray

Nabil El Brahmi

Ph.D Elias ; Fattal
email: elias.fattal@u-psud.fr

ANTI-INFLAMMATORY EFFECT OF ANTI-TNF-siRNA CATIONIC PHOSPHOROUS DENDRIMERS NANOCOMPLEXES ADMINISTERED INTRANASALLY IN A MURINE ACUTE LUNG INJURY MODEL

Keywords: siRNA, TNF-α, lung inflammation, dendriplex, phosphorous dendrimer, transfection efficiency, LPS

HAL is

Introduction

Inflammation is an essential component of many lung diseases such as asthma, chronic obstructive pulmonary disease (COPD) or acute lung injury (ALI) 1,2 . ALI and the acute respiratory distress syndrome (ARDS) are common causes of intensive care mortality 3 . In ALI, resident and recruited alveolar macrophages play a key role in initiating and maintaining pulmonary inflammation 4 .

Alveolar macrophages from ALI patients have increased baseline and stimulated secretory levels of proinflammatory proteins, including certain cytokines and chemokines 5 . Of the numerous cytokines, tumor necrosis factor (TNF-α) is believed to play a key role in the inflammation process 6 . Current medicinal treatment of the inflammation involves anti-inflammatory drugs, such as glucocorticoids, which quite often result in systemic drug exposure and undesired side effects 7 .

Treatment based on small interfering RNA (siRNA) may therefore provide a more specific and less toxic therapy in which the inflammatory pathways leading to TNF- secretion can be targeted. One of the major obstacles limiting the use of siRNA as a drug is their low permeation across cell membranes due to their unfavorable physicochemical properties (high molecular weight and positive charge) and their poor stability in biological fluids. Thus, synthetic carriers, including nanocarriers using cationic lipids or cationic polymers e.g., polyethylenimine (PEI) or chitosan, have been designed for lung delivery of siRNA 8,9 as they protect them from enzymatic degradation and further facilitate their transmembrane delivery and release into the cytoplasm 10 . In this work, we have applied a new family of dendrimers as siRNA carriers. Dendrimers are synthetic polymers characterized by a branched structure and a globular shape with high monodispersity and vast opportunities for surface tailoring as well as their ability to condense nucleic acids into nanoparticles 11,12 . Indeed, dendrimers have been shown to be efficient siRNA delivery systems in vitro and in vivo in several studies, where mostly the commercially available polyamidoamine (PAMAM) dendrimers have been used 13,14 . However, in very preliminary in vitro experiments, phosphorous dendrimers have been shown to be more efficient in cellular delivery of siRNA compared to PAMAM dendrimers 15 . Phosphorous dendrimers have a backbone consisting of aminothiophosphates at every branching point. In addition, they possess intrinsic properties that are the easy regioselective functionalization of the core, the possibility to play with the hydrophilicity/hydrophobicity of the surface and of the core, the interior being hydrophobic, their biocompatibility and biodegradability as well as high thermal stability [16][17][18] . Further, it has been shown that phosphorous dendrimers may exhibit anti-inflammatory properties 19,20 . Indeed, incubation of a polyanionic phosphorous dendrimer with activated monocytes resulted in the upregulation of the expression of several anti-inflammatory cytokines, including interleukin (IL)-10 considered to exert its effect directly on the inflamed tissue. Interactions with serum proteins that degrade siRNA after intravenous administration is mitigated as serum is absent on the air-side of the lung and nuclease activity is comparably low 9,[START_REF] Zheng | [END_REF]23 .

For this study, two different cationic, phosphorous dendrimers of generation 3 (G3) were synthesized, functionalized with either cationic pyrrolidinium or morpholinium surface groups.

These surface groups are secondary amines, which have both been considered for their excellent binding properties versus nucleic acids 24,25 . The synthesized dendrimers were investigated as transfection agents for the delivery of siRNA directed against TNF-α, and the resulting dendriplexes were characterized and their performance assessed. An activated mouse macrophage cell line and a murine acute lung injury model were used for biological assessment of the prepared siRNA formulations.

Experimental Section

Materials

Phosphorous dendrimers were synthesized and characterized by 1 H, 13 C, and 31 P NMR. The phosphorus dendrimer of generation 3 (1G3) bearing 24 terminal P(S)Cl 2 was synthetized as previously described 26 . Grafting of amino pyrrolidine or aminomorpholine was conducted as reported for the grafting of other diamines 27 and leads to dendrimers bearing 48 terminal cationic pyrrolidinium groups (DP) or 48 morpholinium groups (DM) (figure 1). Dicer substrate asymmetric duplex siRNA directed against TNF-α 14 was provided by Integrated DNA Technologies Inc. (IDT, Coralville, IA, USA) as dried, purified and desalted duplexes. The TNF-α siRNA consisted of the following sequences: Sense 5'-pGUCUCAGCCUCUUCUCAUUCCUGct-3', Antisense 5'-AGCAGGAAUGAGAAGAGGCUGAGACAU-3', where the lower case letters indicate 2'-deoxyribonucleotides, underlined capital letters represent 2'-O-methylribonucleotides and p is a phosphate residue (Mw 17,047 Da). Negative control sequences of siRNA (scrambled) were acquired from Eurogentec (Eurogentec, Angers, France). The exact sequence of the negative control siRNA was not disclosed by the supplier. Fluorescent dicer substrate siRNA labeled with the dye TYE™ 665 was also provided by IDT with a sequence directed against luciferase: Sense 5'-pGGUUCCUGGAACAAUUGCUUUUAca-3', Antisense 5'-UGUAAAAGCAAUUGUUCCAGGAACCAG-3'. RNAse-free diethyl pyrocarbonate (DEPC)-treated water was used for dilutions, buffers and all solutions containing siRNA and dendrimers. The duplexes were re-annealed in duplex buffer consisting of 30 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES) and 100 mM potassium acetate prior to any formulation, as recommended by the supplier. All experiments were performed at 25 ºC in 10 mM HEPES buffer, pH 7.4 unless otherwise stated. Primers for PCR (Supplementary material, Table S1) were provided by Eurofins MWG Operon (Ebersberg, DE). Lipopolysaccharide (LPS) (γ-irradiated, <1% protein) was obtained from Sigma-Aldrich (St Quentin Fallavier, France), All additional chemicals used were obtained commercially and were of analytical grade.

Acid-base titration

The pKa value of each dendrimer was determined after titration. Each dendrimer was dissolved in a 30 mL aqueous solution at a 0.2 mg/mL concentration. The solution was then adjusted to a pH value of 10 using 1 M NaOH, and the pH was measured using a pH meter (Mettler Toledo, Viroflay, France). The solution was then titrated using 1 M HCl in 2 µl volume increments down to a pH value of 2 according to the volume of HCl added. From these curves the pKa value was estimated.

Size and zeta potential measurements

The average particle size (z-average) and polydispersity index (PDI) of the dendrimers were measured by dynamic light scattering (DLS), and the surface charge was assessed by zeta potential analysis (laser-Doppler electrophoresis). Measurements were performed using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, United-Kingdom) equipped with a 633 nm laser and 173° detection optics. Dendrimer solutions were prepared at 5.6 µM in 10 mM HEPES buffer and a volume of 1 ml was added to the cuvette for measurements. The Malvern DTS v.6.10 software (Malvern Instruments) was used for data acquisition and analysis, and a zeta potential transfer standard (-50 ± 5 mV, Malvern Instruments) was used to verify the performance of the instrument.

Preparation of dendriplexes

All dendriplexes were prepared at a nitrogen-to-phosphate (N/P) ratio (the molar charge ratio between the cationic dendrimer and the anionic siRNA) of 5, 10 or 20, respectively in 10 mM HEPES buffer. In brief, the dendrimer and siRNA solutions were prepared separately in a total volume of 100 µl, and 20 µl dendrimer solution was added to 80 µl of siRNA solution, followed by vortex mixing. The mixture was left at room temperature for 10 min to allow the complexes to form. The final concentrations of the mixtures were 5 µM for siRNA and 28, 56 112 µM for the dendrimers at N/P ratios of 5, 10, and 20, respectively. Further adjustment of the concentration was obtained by subsequently diluting in 10 mM HEPES buffer to obtain lower concentrations and by using filter centrifugation for up-concentrating the samples. Amicon Ultra-0.5 mL centrifuge tubes (Thermo-Fisher, Villebon, FR) were used to centrifuge dendriplex dispersions at 11,740 g for 20 min.

Characterization of dendriplexes

The z-average, polydispersity index (PDI) and the zeta potential of the dendriplexes were measured as described above for dendrimers. Measurements were performed on diluted samples at 500 nM and 2.8 µM siRNA and dendrimer, respectively. All samples were prepared in triplicate (n=3) and for each sample, a minimum of three measurements were performed. Gel electrophoresis was performed to assess the binding of siRNA to the dendrimers as dendriplexes. A total amount of 0.1 nmol siRNA was used per well and the electrophoresis was carried out using 1% (w/v) agarose gels (Promega, Madison, WI, USA). The gels were run at 100 V for 20 min in Tris-borate-EDTA (TBE) buffer (pH 8.2) supplemented with 5 µL of 2.5 mg/mL ethidium bromide, and the siRNA bands were visualized using a MF-ChemiBIS gel imaging system (DNR Bio-Imaging Systems, Jerusalem, Israel).

Cell culture

The mouse macrophage cell line RAW264.7 was acquired from American Type Culture Collection (ATCC), and was used at an unspecified passage number in all cell-based experiments. The cells were cultured using Dulbecco's Modified Eagle's Medium (DMEM) (Sigma Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) fetal bovine serum (PAA Laboratories, Pasching, Austria), 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were incubated in an atmosphere of 5% CO 2 and 95% O 2 at 37 °C and sub-cultured twice a week by scraping the cells from the culture flask and re-suspending them in fresh culture medium. The cells were used at 80% confluence between passages 5-10.

Cell viability

The viability of the RAW264.7 cellswas measured using the 3-(4,5-dimethylthiazol-2-Yl)-2,5diphenyltetrazolium bromide (MTT) assay. Cells were detached from the surface of culture flasks, pooled, counted, adjusted to the right concentration, and seeded in 96-well plates at a density of 8 × 10 3 cells per well and left overnight. Subsequently, they were incubated with dendriplexes (N/P ratio of 5) at different concentrations for 24 h and the medium was replaced with fresh medium. For each well, 10 µl of MTT-solution [5mg/ml in phosphate-buffered saline (PBS), pH 7.4] was added to 100 μl of cell culture medium. The plate was incubated for 2 h at 37 °C to allow the transformation of MTT to formazan crystals. The medium was then removed, and 200 µl of dimethylsulfoxide was added to each well to dissolve the MTT formazan crystals. Absorbance was spectrophotometrically detected at a wavelength of 570 nm using a microplate reader (FLUOstar OPTIMA, BMG labtech, Champigny-sur-Marne, France). The viability of the cells was calculated as percentage by comparing the absorbance from cell suspensions exposed to dendriplex samples with those from corresponding untreated control cell suspensions. The half-maximal inhibitory concentration (IC50) was determined based on the dose-response curve using linear regression and estimated from the fitted line.

Cell uptake

Flow cytometry and fluorescence microscopy were employed to assess the cellular siRNA uptake and intracellular distribution in RAW264.7 cells. For flow cytometry studies, the cells were cultured in 12-well plates in full culture medium at a density of 5 × 10 5 cells per well. The cells were treated with fluorescent siRNA, either non-complexed or in dendriplexes (N/P ratio of 5) at a concentration of 100 nM fluorescent siRNA for 0.5-4 h before resuspension and analysis by flow cytometry using an Accuri C6 (BD biosciences, Franklin Lakes, NJ, USA). These measurements were used to determine the relative uptake, the ratio between the mean fluorescence intensity (MFI) of the treated samples, and the non-treated negative control, expressed in arbitrary units. For fluorescence microscopy, cells were cultured in chambered coverslips, µ-Slide 8 well (Ibidi, Planegg, Germany), at a density of 5 × 10 4 cells per well. The cells were treated with fluorescent siRNA, either noncomplexed or as dendriplexes (N/P ratio of 5) at a concentration of 100 nM fluorescent siRNA for 4 h and subsequently washed with PBS and fixed using 4% (v/v) paraformaldehyde. Microscopy images were captured at a 63x magnification with a Zeiss LSM 510 (Carl Zeiss, Jena, DE) fluorescence microscope equipped with a 1 mW helium-neon laser and a plan-apochromat 63X objective lens (numerical aperture 1.40, oil immersion). Differential interferential contrast (DIC) images were collected simultaneously. The TYE™ 665 dye was excited using a 633 nm laser illumination and collected with a 650 nm long pass emission filter. The pinhole was set at 1.0 Airy unit (0.8 µm optical slice thickness). Twelve bit numerical images were acquired with LSM 510 software version 3.2 (Carl Zeiss).

Cell transfection

Transfection experiments were performed essentially as previously described 14 . In brief, cells were seeded in 6-well culture plates at a density of 1 × 10 6 cells per well in full culture medium and left overnight. The dendriplexes were prepared as described above (N/P ratio of 5) and 100 µl dendriplex suspension was added to each well at different dilutions to achieve final siRNA concentrations of 6, 12, 25, 50 and 100 nM, respectively, in the wells, and incubated for 24 h. Three hours prior to harvesting, 100 µl PBS containing LPS was added to each well to obtain a final LPS concentration of 5 ng/ml. Positive controls for inflammation response were not treated with siRNA or dendrimer but were treated with LPS while negative controls were incubated with PBS. At harvesting, the culture medium was removed from the wells and the cells were washed with PBS and subsequently 1 ml cold TRIzol (Thermo Fisher, Villebon-sur-Yvette, France) was added to each well, and the cells were loosened by scraping. The contents of each well was mixed by pipetting, transferred to centrifuge tubes and kept on ice.

RNA extraction was performed from the TRIzol-suspended cell homogenates following the instructions of the protocol provided with TRIzol reagent. The total RNA content of the extract samples was quantified using a UV spectrophotometer, Biomate 3 (Thermo Fisher) with a TrayCell fiber-optic ultra-micro cell (Hellma Analytics, Paris, France) and quality control of the RNA samples was performed with a RNA LabChip, using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Reverse transcription of 1 µg RNA samples was done using a mixture of primers applying an iScript cDNA Synthesis Kit (Biorad, Hercules, CA, USA). The cDNA samples were diluted 1:10 in RNAse-free water and stored at -80°C. All RNA samples showed a 260/280 ratio between 2.0-2.2 and an RNA integrity number (RIN) number between 9 and 10.

The knockdown of TNF-α mRNA was assessed by real time reverse transcription polymerase chain reaction (RT-PCR) essentially as previously described 14 . All PCR reactions were performed in duplicate in a total reaction volume of 20 µl using 10 µl of SsoAdvanced Universal SYBR Green Supermix (Biorad, Hercules, CA, USA), 5 µl cDNA working solution (1:10 dilution) and 0.5 µM of forward and reverse primers (Table ST1, supplementary material). Real-time PCR was performed using a C1000 Thermal Cycler instrument with a CFX96 Real-Time System (Biorad) with the following cycling conditions: An initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and elongation at 72 °C for 10 s. The CFX manager software 3.0 (Biorad) was used for crossing point (CP) analysis, and the values were normalized against the average of the two reference genes, glucoronidase b (GUSb) and ribosomal protein, large P2 (RPLP2). These reference genes were chosen from a screening test of eight reference candidates, which showed that the optimal number of reference targets is two or three (geNorm V < 0.15) where good reference target stability is achieved. Gene knockdown data is presented as the relative gene expression compared to the positive control (LPS-treated cells).

In vivo studies

Mice

Experiments were conducted according to the European rules (86/609/EEC and 2010/63/EU) and the Principles of Laboratory Animal Care and national French legislation (Decree No. 2013-118 of February 1, 2013). All techniques/procedures were refined to provide for maximal comfort and minimal stress to the animals. Mice were housed in groups of four with access to water and food ad libitum and kept at constant temperature (19-22°C) and relative humidity (45-65%). Female CD-1 mice were purchased from Envigo (Gannat, France), and all experiments were performed using mice at the age of 6 to 8 weeks.

Lung inflammation model

A widely used protocol for acute LPS-induced lung inflammation [28][29][30] was adjusted for CD-1 mice.

The mice were anesthetized by intraperitoneal (i.p.) injection of 200 µl Ketamine (3.5 mg/ml)/Xylazine solution (5 mg/ml), and the animals were monitored until disappearance of the plantar reflex. Using a pipette, the LPS solution (3.2 mg/kg) was administered to the mice by nasal administration (1.2 µl LPS solution/g of mouse weight) by placing the mice on their backs and allowing them to inhale the solution through the nostrils. Subsequently, the mice were allowed to wake up and were monitored over the next days. For bronchoalveolar lavage (BAL), the mice were euthanized via an overdose of pentobarbital (i.p. injection of 200 µl 20 mg/ml pentobarbital solution), and the trachea was exposed from an incision and cannulated with a 22-gauge catheter (BD biosciences). Subsequently, BAL was performed by flushing the lungs with seven times 0.3 ml PBS, and the first two lavages (collected separately) were used for protein quantification and cell counting/differentiation, whereas the last five lavages were used only for cell counting/differentiation. The BAL was centrifuged at 400g for 10 min, the supernatant was stored at -20 °C for protein quantification, and the pellets were pooled and resuspended in fresh PBS.

10.3 Experimental design

Mice were dosed with the siRNA as a prophylactic treatment prior to inducing inflammation in their lungs (Figure 2). Dendriplexes (N/P ratio of 5) were administered via nasal administration as described for the LPS administration. They were given at an siRNA concentration of 2.0 mg/kg in an average volume of 30 µl 24 h prior to administration of LPS. The mice were assessed 4 and 72 h after the LPS challenge, and a minimum of five mice were included in each treatment group. They were dosed either with siRNA targeting TNF-α or non-coding scrambled siRNA (SCR). Positive controls were pretreated with PBS, and were subsequently dosed with LPS. Negative controls were pretreated with PBS and were subsequently given PBS.

Cell counting and differentiation

The total cell number in each lavage was counted using KOVA Glasstic hemocytometer slides, (KOVA international, Amsterdam, The Netherlands). For each animal, the ratio between neutrophils and macrophages (N/M ratio) was calculated based on counting a total of 300 cells.

Protein quantification

Protein levels in the BAL were quantified using the Cytometric Bead Array -Mouse inflammation kit (BD biosciences), and the IL-6, IL-10, Interferon-γ (IFN-γ) and TNF-α protein levels in the BAL were measured. BAL supernatants were stored at -20 °C and were used directly or diluted (1:3, v/v) in assay diluent from the kit, and the samples were prepared following the instructions in the protocol of the kit. The BAL protein levels were finally quantified by flow cytometry using an Accuri C6) with 2000 ROI counts measured for each sample. All samples were prepared in duplicates and analyzed using the BD Accuri C6 software.

Statistics

Data reported for the cell studies are given as mean values ± SD. Data reported for the in vivo studies are given as mean values ± SEM. The in vitro and in vivo data were all based on independent replicates with each sample set consisting of samples prepared in different days.

Statistical significance was confirmed by using the two-tailed and unpaired Student's t-test.

Statistical analyses were performed by using the student's t-test. The level of significance was set at p < 0.05.

Results and discussion

Physicochemical characterization of dendrimers and dendriplexes

All dendriplexes have a mean size between 120-190 nm and a PDI between 0.27-0.44 (Figure 3, A andB) whereas the hydrodynamic diameter of both dendrimers was around 3 nm (Table 1). Their polydispersity is consistent with what is reported in the literature for siRNA complexes selfassemble into larger aggregates 31 . No clear correlation between the N/P ratio and the resulting hydrodynamic diameter and PDI of the dendriplexes was observed. Similar size and PDI of DP and DM dendriplexes were measured. A positive zeta potential was observed for all dendriplexes, indicating an overall positive surface charge (Figure 3C). The zeta potential increased as a function of N/P ratio due to the higher charge ratio resulting in better siRNA condensation and higher surface charge. Higher zeta potential values were generally observed for DP dendriplexes than for DM dendriplexes at all N/P ratios. This is related to the difference in pKa value of the two Therefore at a pH of 7.4 in HEPES buffer, DP dendrimers are more ionized than DM dendrimers resulting in higher positive zeta potential values. Gel electrophoresis studies give an indication of the binding affinity between siRNA and the dendrimers. These studies show stronger binding between siRNA and dendrimers at higher N/P ratios for both DP and DM dendrimers confirming the zeta potential observations (Figure 3D).

Binding with siRNA was substantially different for DP and DM dendrimers with DP dendriplexes retaining their siRNA, while DM dendriplexes mostly disassociated upon electrophoresis, indicating stronger binding for DP dendriplexes. This result is again related to the difference in ionization of the dendrimers due to their different pKa values. As complexation is performed at a pH value slightly above the pKa value of DM dendrimers, only a few amine groups are charged and the binding is weaker than for DP dendrimers. Given the results obtained at different N/P ratios, biological studies were carried out at an N/P ratio of 5 to maximize the amount of siRNA associated to the dendriplexes while providing dendriplexes with comparable hydrodynamic size and siRNA binding affinity.

Cytotoxicity and cell uptake of dendriplexes

The cell viability was evaluated by measurement of the mitochondrial activity using the MTT assay on RAW 264.7 cells to determine the maximal tolerated concentration for further transfection experiments. Viability studies showed that dendriplexes prepared using the two different types of dendrimers possess significantly different toxicity profiles (Figure 4A). DM dendriplexes did not show any notable reduction in cell viability in the studied concentration range (IC 50 > 15 µM siRNA). In contrast, DP dendriplexes exhibited a reduction in cellular viability as a function of siRNA concentration, with a calculated IC 50 siRNA concentration of 470 nM, corresponding to a dendrimer concentration of 41 µg/ml. A similar toxicity profile was observed for the noncomplexed dendrimers (Figure S1). Also here, the DP dendrimers showed significantly higher toxicity compared with the DM dendrimers, which did not have any measurable toxicity. The higher toxicity of DP dendriplexes measured in this study could partly be related to their higher surface charge, or perhaps due to higher cell permeation and a higher degree of interaction with cellular components. The assessment of cytotoxicity again highlights the pronounced difference between the two types of dendrimers with only slightly different surface functional groups. The cytoxicity measured for the DP dendriplexes (IC 50 = 41 µg/ml) is comparable to that observed for other cationic polymer-based transfection agents, e.g., PEI (IC 50 = 40 µg/ml) and poly-L-lysine (PLL) (IC 50 = 45 µg/ml) 32 . For all subsequent cell studies, siRNA concentrations lower than 100 nM, corresponding to dendrimer concentrations of 15 µg/ml, were used in order to remain below the minimal toxic concentration for the DP dendriplexes (80%).

The cell uptake kinetics of non-complexed siRNA and dendriplexes was measured using flow cytometry (Figure 4B). For non-complexed siRNA, as expected, the relative uptake remained very low over time. For the DM dendriplexes, a very modest yet statistically significant uptake of siRNA was observed. For the DP dendriplexes, a significantly higher uptake of siRNA was demonstrated.

The MFI of cells incubated with DP dendriplexes was five times higher than the MFI of cells incubated with non-complexed siRNA after 4 h incubation, while only a two-fold increase was observed for cells incubated with DM dendriplexes. To further investigate siRNA uptake, cells incubated with non-complexed siRNA or dendriplexes for 4 h were observed by confocal/DIC microscopy. Observations corroborated the flow cytometry measurements (Figure 5), and were verified by z-stack images (not shown). Cells incubated with non-complexed siRNA did not show any detectable fluorescence above background levels, proving that no cell internalization of the siRNA occurred (Figure 5B). Cells incubated with DM dendriplexes showed a small amount of the siRNA internalization, while the majority of the siRNA was adsorbed on the surface of the cells (Figure 5C). However, cells incubated with DP dendriplexes showed internalization of siRNA in the cells with siRNA distributed within the cytoplasm (Figure 5D).

The images indicate that DP dendriplexes are mainly taken up by cells whereas DM dendriplexes mainly tend to adhere to the outer surface of the cell membrane as aggregates. This indicates better internalization of DP dendriplexes compared with DM dendriplexes, which also confirms the observations from the flow cytometry measurements. The higher uptake observed for the DP dendriplexes could be explained by their stronger positive charge and better stability. DM dendriplexes most probably dissociate in the cell culture medium before aggregating on the cell surface. These results are in agreement with the stronger binding observed for DP dendriplexes than for DM dendriplexes and possibly also their higher net surface charge, which has been shown in several studies to result in higher cellular uptake [33][34][35] . The cell uptake results could also explain the difference of toxicity observed between the two dendriplexes. Indeed constructs with high positive charge have been reported to results in some cytotoxicity 34 . As DM dendrimers lead to a lower internalization, the mitochondrial activity is less affected and the resulting viability measured by MTT-assay remains higher.

In vitro inhibition of TNF- by dendriplexes in LPS treated macrophages

Cells treated with the dendriplexes containing TNF-α siRNA and subsequently activated with LPS were assessed at the mRNA level using RT-PCR (Figure 6). The relative TNF-α expression levels were lower for all TNF-α based samples compared with the corresponding scrambled siRNA samples. Non-complexed TNF-α siRNA showed 28 % inhibition, compared with the control, and a 26 % inhibition compared with scrambled siRNA, both statistically significant (p < 0.05). The DM dendriplexes with TNF-α siRNA showed 34 % inhibition, compared with the control, and 33% inhibition, compared with scrambled siRNA, which were statistically significant (p < 0.05). The DP dendriplexes with TNF-α siRNA showed a rather substantial inhibition of 74 %, compared with the control, and a 50% inhibition, compared with scrambled siRNA, which were also statistically significant (p < 0.01). Dose-response studies were performed for DP (Figure 6B) and DM (Figure 6C) dendriplexes in the 6-100 nM siRNA concentration range to assess the influence of the siRNA concentration on the gene inhibition, and ascertain that the observed effects were specific. For both DM and DP dendriplexes, the gene silencing was concentration-dependent. DM dendriplexes were shown to provide significant TNF-α knockdown from 50 nM siRNA and up while DP dendriplexes were shown to provide significant TNF-α knockdown from 25 nM siRNA and up. For both dendriplexes the highest dose (100 nM siRNA) was below the toxic level of dendriplexes for the cells. The transfection efficiency data correlates well with the observations from the cell uptake study, where it was demonstrated that DP dendriplexes result in higher cell internalization compared with DM dendriplexes and non-complexed siRNA. The higher silencing obtained with DP dendriplexes can thus be explained by their higher cell uptake and their higher stability and surface charge. Although DM dendriplexes did not indicate convincing cell uptake and structural stability, the nevertheless demonstrated a significant silencing of TNF-α expression. It was similar for the non-complexed siRNA, which showed a modest but significant silencing of TNF-α expression at the highest concentration, despite low cellular 435 uptake. This could indicate that only a small fraction of cellular siRNA uptake is sufficient to provide a silencing effect.

Modulation of the inflammation by dendriplexes in a murine acute lung injury model

An ALI model was used to assess the efficacy of the dendriplex formulations in vivo. LPS-induced ALI is considered one of the most important and applicable in vivo models to study molecular mechanisms and potential therapies for inflammation-associated lung injury as described 36 . In humans, once a patient develops lung injury, the best known strategy is supportive care 37 . However, many therapeutic agents have demonstrated a reduced efficacy, in part due the delay in initiating the therapy several days after the onset of lung injury 37,38 . This is why we intentionally decided to pretreat animals prior to administration of LPS, as a prophylactic treatment, and this is believed to represent the optimal therapeutic scheme.

The method for nasal instillation of the liquid suspensions was validated by administering a trypan blue solution to the mice and subsequently evaluating the deposition in the lungs and stomach. The lungs were entirely stained with trypan blue while the stomach did not show any dye, which indicates that most of the dye was delivered to the lungs (data not shown). The total cell count of the BAL indicates strong cell recruitment within 24 h after LPS challenge and is subsequently reduced as seen in the following time points (Figure S2A). The same trend was observed for the neutrophil-to-macrophage (N/M) ratio where neutrophil recruitment could be measured already after 4h. This also highlights that the majority of the subsequent cell recruitment observed occurs from the neutrophils (Figure S2B). Similar observations were made in other studies using intranasal instillation and LPS to assess therapeutic efficacy 39,40 The cytokine profiles measured in the BAL after LPS challenge showed that the TNF-α levels (Figure S3) are very high 4 h after the animals are challenged with LPS and subsequently decrease over the next 3 days until they return to normal levels (32 pg/ml) after 7 days. A large increase was also observed for IL-6, IFN-ɣ and IL-10, for which the cytokine levels in the lungs were either sustained (IL-6, IL-10) or increased progressively (IFN-ɣ), and for the latter maximum levels were reached at day 3 after LPS challenge, followed by a decrease to normal levels at day 7. Additional inspection of the mice showed a significant reduction in weight (approx. 10%) and lack of grooming in the first three days following LPS challenge (data not shown).

Cytokine levels were compared for mice treated with LPS (Control +) and mice treated with noncomplexed dendrimers in solution, as well as untreated mice (Control -) and were evaluated in order to assess the potential inflammatory response of the non-complexed dendrimers. Both dendrimers showed a small response for TNF-α, which was minor compared with the effect of LPS, yet significantly higher than the negative control, meaning that the dendrimers resulted in a small inflammatory up-regulation (Figure S4A). No visible effect was observed for IL-6 and IFN-ɣ relative to the negative control (Figure S4A and S4B). However, both dendrimers experienced a slight increase in their IL-10 levels compared with the negative control indicating a small antiinflammatory response by the dendrimers (Figure S4D). The total BAL cell count was assessed for the different treatment conditions at 4 and 72 hours after LPS challenge. The cell population was not significantly different after 72 h (Figure S5A). The N/M ratio of the BAL for the different treatment conditions at 72 after LPS challenge was also assessed (Figure S5B). It was shown that the N/M ratio was lower for animals receiving a treatment containing anti-TNF-α treatment, although the differences in N/M ratio were not statistically significant. Yet, this could indicate a higher relative neutrophil recruitment for the positive control than for treated animals, which could also indicate a lower level of inflammation in the treated animals. Cytokine levels in the BAL were measured at different time points (4 and 72 h) after siRNA pre-treatment followed by LPS challenge. The siRNA dose was administered, based on previous pulmonary siRNA delivery studies (0.6-3 mg/kg siRNA administered) [41][42][43] , to ensure a sufficient level to observe a potential therapeutic effect. An siRNA dose of 2 mg/kg was chosen.

The TNF-α protein levels measured 4 h after LPS challenge (Figure 7A) show that all treatments containing TNF-α siRNA resulted in a reduction in the TNF-α expression, compared with the corresponding treatments containing scrambled siRNA. The strongest inhibition was observed for DP TNF-α (55 %) compared to non-complexed TNF-α siRNA (17 %). The specificity of this inhibition was demonstrated by the weakest effect of the scrambled siRNA delivered either noncomplexed or by dendriplexes. DM TNF-α showed a slight increase in the TNF-α protein expression level, compared with the positive control confirming the lack of efficiency of these dendriplexes as shown in vitro.

Measurements of other cytokine levels show that the TNF-α based siRNA also has an influence on these, which is expected, as their regulation is interdependent. Significantly increased IL-10 protein levels were observed for DP TNF-α (18 fold increase, Figure 7D) compared with the positive control at 4 h after LPS administration. In contrast, the DM dendriplexes did not affect the BAL IL-10 level. This indicates that the DP TNF-α results in an anti-inflammatory response immediately after administering LPS. A substantial increase in the IL-6 protein secretion was observed for DP TNF-α (four-fold increase) compared with the positive control at 4 h after LPS administration (Figure 7C). The influence of non-complexed DP dendrimer on the cytokine expression was investigated by administering non-complexed DP dendrimer with a subsequent LPS challenge (Figure S6). It was observed, 4 h after LPS administration, that the non-complexed DP dendrimer resulted in an increase in IL-6 and IL-10, much lower than the secretion due to DP TNF-α and without any change in the production of TNF-α. The same trend is observed with scrambledassociated dendriplexes. If an increase in IL-10 results from anti-inflammatory effects, IL-6 should be downregulated as this is the case for TNF-α. We believe that IL-6 upregulation is due either to DP or to the specific sequence of siRNA, or both. Many authors have demonstrated that IL-6 in the murine LPS-induced lung injury model displays anti-inflammatory properties 44,45 and could contribute to reducing TNF-α to a certain extent as this was demonstrated on the same model after intratracheal administration of IL-6 46 . Although TNF-α and IL 6 return to very low levels 72 h after LPS challenge (Figure 8A andC), DP TNF-α siRNA still induces a strong inhibition of 44% for TNF-α compared with the positive control, much higher than non-complexed TNF-α siRNA or DM TNF-α. This sustained efficiency, three days following the onset of lung inflammation, suggests a clear and efficient silencing effect provided by the siRNA. At 72 h, IL-6 levels had decreased for both DP-TNF-α and scrambled associated dendriplexes (Figure 8C). Finally, IFN-ɣ, a pro-inflammatory cytokine, was slightly reduced for mice treated with DP dendriplexes and non-complexed siRNA compared with the positive control at 4-h after LPS administration but not in a significant manner, which is normal since the kinetics of secretion of IFN-ɣ is slowed down compared to the other cytokines (Figure 7 D). The overall trend of the cytokine levels indicates an anti-inflammatory effect of DP TNF-α compared with the positive control, induced by the effective transfection with anti-TNF-α siRNA. Unlike observations made at 4 h, a considerable inhibition of IFN-ɣ levels was observed for dendriplexes compared with the positive control and compared with non-complexed siRNA (Figure 8B). Also a ten-fold increase in the INF-ɣ levels was observed from 4 h to 72 h following LPS challenge whereas all other cytokines studied decreased in their levels over the same period.

From the in vivo assessment of the performance of the siRNA-loaded dendriplexes it can be established that the DP dendriplexes provided a more efficient silencing of TNF-α as well as a better anti-inflammatory regulation of other cytokines involved, compared with DM dendriplexes and non-complexed siRNA. This correlates well with the findings from in vitro transfection studies where the same trend was observed. The superior performance of DP dendriplexes is explained by the better ability of DP dendrimers to form stable complexes with siRNA therefore leading to a higher internalization in macrophages, which plays a key role in the production of TNF-α, a strong proinflammatory cytokine.

Conclusion

In this study, siRNA therapeutics was assessed for the treatment of acute inflammation, which was achieved both in vitro and in vivo. Major inhibition of TNF-α was obtained by means of a cationic 550 phosphorous dendrimer containing pyrrolidinium and binding efficiently anti-TNF-α siRNA. The obtained dendriplexes are highly efficient in delivering siRNA to LPS-activated macrophages. After intranasal administration, TNF- inhibition resulted in a strong anti-inflammatory effect as demonstrated by modulation of all other cytokines involved in the inflammation process.

Figure 1 .

 1 Figure 1. Synthesis and chemical structures of cationic phosphorus dendrimers of generation 3: DP (pyrrolidinium end groups) and DM (morpholinium end groups).

Figure 2 .

 2 Figure 2. Schematic overview of the timeline in the animal experimental model and the subsequent

Figure 3 .

 3 Figure 3. Z-average (A), polydispersity index (PDI) (B) and zeta potential (C) of DP and DM

Figure 4 .

 4 Figure 4. Cell viability of RAW 264.7 cells as a function of siRNA concentration, measured after

Figure 5 .

 5 Figure 5. Representative image of the cellular uptake of dendriplexes in RAW 264.7 cells after 4 h

Figure 6 .

 6 Figure 6. Dendriplex-mediated TNF-α mRNA silencing in RAW 264.7 cells. Normalization to LPS-treated

Figure 7 .

 7 Figure 7. Cytokine concentration in BAL of mice challenged with LPS 24 h after administration of siRNA

Figure 8 .

 8 Figure 8. Cytokine concentration in bronchoalveolar lavage of mice challenged with LPS 24h after

Table 1 .

 1 Physicochemical properties of cationic phosphorous dendrimers

	Dendrimer Surface group Primary	MW	pKa	Hydrodynamic
		amines	(g/mol)		diameter (nm)
	DP	Pyrrolidinium 48	16188.11 8.1	2.7
	DM	Morpholinium 48	16956.06 7.1	2.9

Acknowledgements

The authors would like to thank the Danish Council for Independent Research (Grant No. DFF-12-131927) for financial support of this project. Further, Valérie Nicolas and Stéphanie Denis are acknowledged for their help with confocal microscopy and cell culture respectively. Authors would like to thank N. Szely, M. Beck-Broichsitter, J. Vergnaud and A. Biola-Vidamment for fruitful discussion. This study has been carried out with financial support from the Commission of the European Communities, Priority 3 "Nanotechnologies and Nanosciences, Knowledge Based Multifunctional Materials, New Production Processes and Devices" of the Sixth Framework Programme for Research and Technological Development (Targeted Delivery of Nanomedicine: NMP4-CT-2006-026668). The authors would also like to acknowledge the financial support provided by COST-European Cooperation in Science and Technology, to the COST Action MP1404: Simulation and pharmaceutical technologies for advanced patient-tailored inhaled medicines (Siminhale).

Supporting Information

-Procedure for dendrimer synthesis -Primer sequences used for the RT-qPCR analyses -Cell viability of RAW264.7 cells versus non-complexed dendrimers -Bronchoalveolar lavage cell count and neutrophil-to-macrophage ratio in mice and following treatment and 72 hours after lipopolysaccharide chalenge -Cytokine concentrations in bronchoalveolar lavage of untreated mice or mice treated with non complexed dendrimers vesus dendriplexes, 4h afer intranasal challenge with phosphate buffer or lipopolysaccharide

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