Hugo Rositi
email: hugo.rositi@uca.fr

Owen Kevin Appadoo
email: owen.appadoo@uca.fr

Daniel Mestre
email: daniel.mestre@univ-amu.fr

Sylvie Valarier
email: sylvie.valarier@ch-lepuy.fr

Marie-Claire Ombret
email: marieclaire.ombret@ch-lepuy.fr

Émilie Gadea-Deschamps

Christine Barret-Grimault
email: christine.barret@ch-lepuy.fr

Christophe Lohou
email: christophe.lohou@uca.fr

M.-C Ombret

Presentation of a mixed reality software with a HoloLens headset for a nutrition workshop

Keywords: Mixed reality, HoloLens, therapeutic education, nutrition, clinical study Declarations

Microsoft has recently released a mixed reality headset called Holo-Lens. This semi-transparent visor headset allows the user who wears it to view the projection of 3D virtual objects placed in its real environment. The user can also grasp these 3D objects, which can also interact with each other. The framework of this new technology taking into account this physical approach (interactions, collisions) is called mixed reality. We had the opportunity to digitally transform a conventional nutrition workshop for patients waiting for bariatric surgery by designing a mixed reality software using the HoloLens headset. This software is called HOLO_NUTRI.

In this paper, we present this software and its assessment (acceptance of this technology) by a cohort of thirty patients.

• Funding: This study was funded by a french national grant PEPS INSIS CNRS. • Conflicts of interests/Competing interests: The authors declare that they have no conflict of interest. • Availability of data and material: Data concerning the clinical study are summarized through different tables in this paper. • Code availability: Software is available on demand to C. Lohou.

• Authors' contributions: Hugo Rositi, Owen Kevin Appadoo and Christophe Lohou have contributed to the design, realisation and analysis of the clinical study; they have also contributed to the supervision of the software development and to the writing of this manuscript. Christine Barret-Grimault, Sylvie Valarier, Marie-Claire Ombret and Émilie Gadea-Deschamps have contributed to the design, realisation and analysis of the clinical study. Daniel Mestre has contributed to the design thinking of the feedback forms.

Context

In this paper, we present a software application for patients waiting for bariatric surgery (modification of the anatomy of the digestive system). This application offers them to (virtually) compose their meal and then assess their choices; its educational objective is to inform patients about the modification of their diet (mainly, in terms of optimal quantities) after surgery. This section is structured as follows: the terminology of targeted surgical procedures is presented in section 1.1; we then describe the conventional workshop in section 1.2; in section 1.3, we list three main features expected in the application, one of which will fully justify the use of mixed reality; in section 1.4, we will present the framework of mixed reality; in section 1.5, we will review the state of the art of softwares used in nutritional therapeutic education; in section 1.6, we will present the software implemented for this mixed reality experience.

Terminology

Bariatric surgery aims at patient's weight loss. As part of our research, we are interested in the following two surgical solutions: reduction of the volume of the stomach "Sleeve Surgery" (Figure 1(a)), setting up a deviation from the normal path of progression of foods "Bypass Surgery" (Figure 1(b)).

Conventional workshop of nutritional therapeutic education

The conventional nutritional therapeutic education workshop, later called CONV_WORK, is organized around the nine following steps (also summarized in Table 1 for a more global view): introduction of the session (Step 1); debate around the issue "To feed, eat, when one has been operated on a bariatric surgery, what does it evoke you ?" (Step 2); beginning of the workshop to discuss with patients about the daily diet to adopt after intervention (Step 3); patients are then invited to compose their menu by choosing printed cards representing different foods and in different quantities, the images come from the study SU.VI.MAX [START_REF] Hercberg | The SU.VI.MAX Study: A Randomized, Placebo-Controlled Trial of the Health Effects of Antioxidant Vitamins and Minerals[END_REF][START_REF] Hercberg | Portions alimentaires : Manuel photos pour l'estimation des quantités[END_REF] (Step 4); collection by the clinical team of the menus developed by the patients (Step 5); collegial discussion on the choice made by each patient (Step 6); the dietician then gives to each patient advices according to the food and quantities that he or she chose (Step 7); dissemination of quantitative and qualitative messages (Step 8), qualitative messages focus on the need to vary the number of dishes, the expected duration of the meal, the importance of the chewing time, while quantitative messages focus on reducing the amount of foods compared to a meal before surgery; finally, patients complete forms to evaluate the session (Step 9).

Interests in digitalizing the conventional workshop

We have proposed the digitalization of the classical workshop CONV_WORK, previously described in section 1.2. This new workshop, named DIGIT_WORK afterwards, has the following main objectives to propose:

the digital version of the menu composition using a software that should be rather playful. This software application is called HOLO_NUTRI, a more detailed analysis of the elaborated menu (several quantities associated by food, meal time estimation, taking into account of the chewing time, etc.), the numbers and quantities of foods being more important for this digitalized workshop make it possible to develop more personalized menus, an analog, in the computer application, of the patient's gesture when he or she takes one card representing a food and the associated quantity in the CONV_WORK workshop.

The first two items above can be achieved using the conventional computer/keyboard/screen/mouse set and software development. However, we have searched for the most appropriate solution for the implementation of the gesture to catch the digital model corresponding to the printed card of the food, or which would reinforce this learning [START_REF] Barret-Grimault | Innovons en ETP grâce au casque HoloLens en chirurgie bariatrique ![END_REF]. To do this, we chose a solution setting up a mixed reality framework.

Mixed reality

To justify the framework of our study, we briefly recall the different virtual, augmented and mixed realities and the most commonly used hardware in such context.

In the context of virtual reality, for individual use, opaque headsets are the most used. The best known headsets are the HTC Vive [START_REF]HTC Vive headset[END_REF] (Figure 2(a)) and Oculus Rift [START_REF]Oculus Rift[END_REF]. The main advantage of a virtual reality application is to be able, for example, to simulate an unrealistic environment (large-scale environments, etc.) and/or non real actors (avatars, etc.) and to provide immersive visualization (not related to the real environment). The main drawbacks are the discomfort associated with virtual reality headsets (cybersickness) [START_REF] Laviola | A discussion of cybersickness in virtual environments[END_REF], and a little high latency (time between the triggering of an action and the visual result related to this action). The main interactions by the wearer of this type of headset are carried out using handles (controllers) or gloves (Figure 2(a)). Other rather collective devices exist, such as CAVEs (Cave Automatic Virtual Environment) [START_REF] Cruz-Neira | The CAVE: Audio visual experience automatic virtual environment[END_REF]. In brief, virtual reality headsets are built with opaque visor and prevent users to see their real environment; if taken into consideration, the content of the application can be handled by controllers.

With regard to augmented reality [START_REF] Azuma | A survey of augmented reality[END_REF], the interest is to superimpose the visualization of 3D virtual objects to the one of the real environment. Most applications use markers to superimpose virtual objects (Figure 2(b))). The most common devices are smartphones, tablets for which the real environment is filmed using an embedded camera, or glasses (for example, Epson Moverio [START_REF]Epson Moverio[END_REF], Google Glass [START_REF]Google Glass[END_REF], etc.) and headsets for which the environment is perceived thanks to transparent glasses or to a semi-transparent visor [START_REF]Mira augmented reality[END_REF][START_REF]Meta vision[END_REF]. The main downside of using tablets and smartphones is the need to hold them (Figure 2(c)). If the application does not use markers, then it is necessary to use more sophisticated methods (such as registration for example), not necessarily exploitable or currently in development for light or inexpensive devices (or low quality cameras). This is why most of these devices (tablets, glasses, etc.) also deals mainly with 2D information (Figure 2(d)). Note also that until very recently, occlusion problems were not solved to take into account the real environment on smartphones [START_REF] Watson | Footprints and free space from a single color image[END_REF], in other words, there is no real feeling of depth (Figure 2(e)). Interactions are performed using these same devices, or external cable-connected controllers.

Microsoft recently released the HoloLens headset [20]. In addition to scanning the real environment around the user (Figure 3 The PokemonGO application on a mobile phone, occlusion problems persist and there is no interaction with the user environment. Subfigure (a) is from [START_REF]AS#04 -mixed reality" by artfulspark and is licensed under[END_REF], subfigure (b) is from [START_REF]AR 1" by cubistscarborough and is licensed under CC BY-NC-ND 2[END_REF], subfigure (c) is from [START_REF]Augmented Reality Comics @ Ars Electronica Center[END_REF], subfigure (d) is from [START_REF]The view from Cecilia Abadie's driver's seat[END_REF], subfigure (e) is from [START_REF] Subfigure 2e Reference | Drowzee" by Hackley Public Library and is licensed under CC BY 2[END_REF]. tion of the real environment, and to interact with these same objects thanks to the headset sensors (Figure 3(b)).

It is therefore the consideration of physics in order to manipulate holograms (collisions, etc.) that mainly distinguishes augmented reality from mixed reality. The user can interact with his environment using events from the Gaze, Gesture and Voice triplet (GGV) [START_REF]Gaze, Gesture and Voice[END_REF] by triggering actions programmed in the computer application to respond to these events. Indeed, the integrated gyroscope gives the movements of the head -Gaze, we highlight that this notion of gaze is defined by the orientation of the head (Figure 3(b)), the eyes are not tracked. Depth cameras of the headset allow it to follow some of the user's gestures (fingers pressed, hands opened, etc.) -Gesture and a microphone acquires the words or sounds emitted by the user -Voice.

Let us give more explanations about gaze interactions. When the user looks and keeps its gaze on an object (represented by the blue arrow on Figure 3(b)), if an interaction has been designed in the application with this object, it is therefore possible to interact with it; a blue cursor then appears on one of the 3D object's facet, the closest from the headset and in respect with the user gaze direction (thanks to the environment scanning (Figure 3(a))).

State of the art of softwares in nutritional therapeutic education

We will review some applications in relation to nutritional therapeutic education, according to the three frameworks of virtual, augmented and mixed realities.

Virtual reality experiences

Virtual reality experiences mainly consist of simulating meals or more specific situations (feeling of satiety, choice of sweet foods, etc.).

The simulation of meals may have more or less specific therapeutic aims. In [START_REF]Kokiri Lab. Project Nourished, a gastronomical virtual reality experience[END_REF], a meal simulation is proposed with, in addition to a virtual reality headset, an odour diffuser, a hearing device (sound of chewing) and a gripper (grasping the food), the objectives are to facilitate the management of nutritional disorders or to circumvent food allergies. Another meal simulation [START_REF] Daigneault | Virtual eating : how virtual reality can make you think you're eating pizza when you're not[END_REF] uses electrical probe (salty sensation), thermal probe (spicy or menthol sensation) and electrode (chewing sensation) in addition to a virtual reality headset.

As more specific situations, a serious game has been proposed in [START_REF] Rivas | Students learn the virtual reality of healthy eating and nutrition[END_REF] with a virtual reality headset to take into account the differences in human body reactions depending on sugar consumption (see also [START_REF] Robertson | Atkins made a virtual reality game about sugar, and nothing I say can make sense of it[END_REF]). We can list other works, for example, modifying the visualization, inside the headset, of the volume of food ingested in order to accelerate the impression of being satiated [START_REF] Auriach | Régime façon réalité augmentée[END_REF]; or the simulation with a focus on food selection [START_REF] Ung | Innovations in consumer research: The virtual food buffet[END_REF]; or the modification of the body-shape [START_REF] Ferrer-García | The use of virtual reality in the study, assessment, and treatment of body image in eating disorders and nonclinical samples: A review of the literature[END_REF]. In [START_REF] Persky | Validity of assessing child feeding with virtual reality[END_REF], the validation of the use of virtual reality is questioned for assessing parents' child feeding behavior. Another research work will be noted (neuroscience framework) to decouple the desire to eat from the "intrinsic survival" notion (feeding) [START_REF] Wolcott | Virtual reality, sex and chocolate cake: Desire in a post-virtual world[END_REF]. Other experiments using the term virtual reality are in fact carried out without immersion: for the treatment of obesity in an individual way in front of a screen [START_REF] Perpiñá | Similarities and differences between eating disorders and obese patients in a virtual environment for normalizing eating patterns[END_REF], or even a shopping experience (food) in a 3D supermarket [START_REF] Meißner | Combining virtual reality and mobile eye tracking to provide a naturalistic experimental environment for shopper research[END_REF].

Experiences in augmented reality

Augmented reality experiments mainly consist in providing nutritional information on food products either punctually and individually, or in the form of a medical follow-up.

We can list projects to get information about the composition of meals, with scanners (camera sensors of smartphones and/or tablets) to visualize nutritional information [START_REF] Bayu | Nutritional information visualization using mobile augmented reality technology[END_REF], for the recognition of a fruit and its composition [START_REF] Igue | iOS11ARkit : le nouvel OS des iPhone donne la composition des aliments vus par la caméra, en vidéo[END_REF], to provide a nutritional response according to a predefined diet (preferences or intolerances) [START_REF]Far application[END_REF] or more specifically for sweet drinks [START_REF] Escárcega-Centeno | Augmented-sugar intake: A mobile application to teach population about sugar sweetened beverages[END_REF]. Other hardware also provide access to this information such as connected watches in [START_REF] Campbell | Apple invention uses RFID tags, Apple watch to track food nutrition[END_REF] which use information on food packaging by RFID (Radio Frequency IDentification) tags. In [START_REF] Elsayed | Situated analytics: Demonstrating immersive analytical tools with augmented reality[END_REF], a system of aid for the purchase of food products in a supermarket is proposed based on individual or nutritional criteria. Finally, other projects aimed at nutritional education in general are presented in the reference [START_REF] Menet | Foodtech : la réalité augmentée au service de l'alimentation[END_REF], such as managing children's appetite for vegetables.

Regarding the medical follow-up, work has been proposed for different categories of people (diabetic patients, pregnant women, etc.): analysis of the management of the care for diabetic patients by mobile applications [START_REF] El-Gayar | Mobile applications for diabetes selfmanagement: Status and potential[END_REF][START_REF] Rollo | eHealth technologies to support nutrition and physical activity behaviors indiabetes self-management[END_REF], follow-up of meals taken for several days by pregnant women [START_REF] Ashman | Evaluation of a mobile phone tool for dietary assessment and to guide nutrition counselling among pregnant women[END_REF], estimation of delivered portions [START_REF] Rollo | Servar: An augmented reality tool to guide the serving of food[END_REF][START_REF] Rollo | The effect of an augmented reality aid on error associated with serving food[END_REF].

Mixed reality experiences

The HoloLens headset is used in the medical context mainly for two types of applications: teaching [START_REF] University | HoloAnatomy application[END_REF][START_REF] Gmbh | Insight heart application[END_REF][START_REF]Vimedix AR ultrasound simulator[END_REF] or visual assistance in surgery (arterial network [START_REF] Pratt | Through the HoloLens looking glass: augmented reality for extremity reconstruction surgery using 3D vascular models with perforating vessels[END_REF], prostate [START_REF] Surur | HoloLens used to plan robotic prostate surgery[END_REF], shoulder [START_REF] Bremme | HoloLens : une opération chirurgicale unique au monde en réalité augmentée à Paris[END_REF]). To our knowledge, only the reference [START_REF] O'leary | Back away from the burger ! Microsoft combats overeating with AR patent[END_REF] (nutritional diseases -overweight management) makes a link between HoloLens and the context related to our work.

In the experience described in this paper, as previously stated, the main objective of our application is to compose a meal while strengthening the choice of foods. In order to do that, we have chosen to provide a gesture learning reinforcement which justifies the mixed reality framework. In addition, qualitative and quantitative messages are not focused on the nutritional composition of each food (unlike nutritional scanners) but on the composition of the entire meal. Here, the patient-specific follow-up is limited to verify that the qualitative and quantitative messages have been retained at different times after this experiment.

Technological justification

It is therefore this mixed reality technology using the HoloLens headset that we have chosen for the digital transformation of the CONV_WORK workshop for the following reasons:

patients continue to see the real environment around them through the semi-transparent visor, this avoids any additional stress that can occur with opaque virtual reality headsets (Figure 3(b)), patients must wear this headset about thirty minutes in a one-and-a-half hour session. This seems to us to be too long for uninitiated users of virtual reality. We also have assumed that mixed reality would reinforce learning in the DIGIT_WORK digital workshop. In fact, unlike virtual reality headsets, here, the user acts and sees the movement of his arm, the action of catching a food with the hand. Unlike phones and tablets using at least one hand of the user or the one of an assistant (augmented reality framework), here the user can perform the gesture freely with the HoloLens headset on her/his head (yet the gesture has to be done in an area defined by the headset sensors) (Figure 3(b)).

Plan of the article

In Section 2, we present the developed application and its features. Section 3 presents the framework in which this application was used and the results of the evaluation of its use by a cohort of thirty patients. In Section 4, we will discuss the first conclusions of this study.

Application

List of expected features

The specifications of our application is composed of two parts. The first part must allow the patient to clearly understand the type of operation he or she will undergo (by visualizing the stomach before and after surgery). The second part must provide an interactive composition of a standard menu in two iterations:

the first iteration gives little guidance to the patient and displays at the end a first analysis (qualitative and quantitative messages), the second iteration displays more information (visualization of the stomach in transparency showing its filling as the food is ingested, display of the number of portions), which should allow the patient to better compose his menu.

During both parts of the application, it is requested that the user is the most active possible: for the first part, the user must remove a portion of the stomach depending on the envisaged intervention; for the second part, the patient must compose his meal by choosing foods, one after the other, and their ingestion will then be simulated by placing each food in a "virtual" jaw, which will trigger a chewing animation and prevent the patient from selecting a new food until the chewing time has not elapsed.

We have chosen to treat this type of interaction with mixed reality in order to have a maximum impact on the patient (learning by gesture): it is with the hands that he or she will interact to remove a part of the stomach (first part), and also to choose foods when placing them into the virtual jaw (second part).

Development environment

Whatever the type of reality envisaged, the development of computer applications to use this hardware is done either from proprietary SDKs (Software Development Kit) or by using interactive 3D graphical application development engines such as Unity [START_REF]Unity[END_REF] and Unreal Engine [START_REF]Unreal Engine[END_REF], originally designed for the development of video games but now also addressed to wider professional sectors (automotive, AEC -Architectural, Engineering and Construction-, etc.).

For our application, we first design several 3D resources (3D models and animations made in Blender software [START_REF]Blender software[END_REF], see Section 2.3). The development was carried out with the Unity engine [START_REF]Unity[END_REF] (import of 2D images, 3D models and animations). Several scripts were designed to describe the wanted user interactions on 3D objects; these scripts (integrated in our Unity framework) were written with the C# language from the Microsoft Visual Studio programming environment [START_REF]Microsoft Visual Studio[END_REF] and with the HoloToolKit library [START_REF]Microsoft HoloToolKit[END_REF] specialized for mixed reality with the HoloLens headset. Once all these assets are compiled, it produces our software HOLO_NUTRI (that can be tested on a PC through an emulator). By plugging the HoloLens headset into the PC, the application can then be deployed/embedded into the headset. The user, wearing the headset, can then run HOLO_NUTRI from the HoloLens main menu without the headset being connected to a PC (by either cable or WiFi).

Digital contents

The different 3D models (stomach and jaw) were made with the software Blender [START_REF]Blender software[END_REF], these models must not have too many polygons to maintain the smooth running of the software [START_REF] Lohou | Design of a mixed reality application with a HoloLens headset for a nutrition workshop[END_REF]. For the food selection stage, these are represented by 2D images from SU.VI.MAX study [START_REF] Hercberg | The SU.VI.MAX Study: A Randomized, Placebo-Controlled Trial of the Health Effects of Antioxidant Vitamins and Minerals[END_REF][START_REF] Hercberg | Portions alimentaires : Manuel photos pour l'estimation des quantités[END_REF]. Once the food is selected, it appears as a cube, which will be easier for the patient to handle than a 2D image. The size of this cube is based on the quantity selected for the selected food.

Description of the application HOLO_NUTRI

First part: basic simulation of the surgical intervention

The first part proposes to the user to choose the type of intervention that he or she will undergo (either using buttons in the application (Figure 4(a)), or by scanning a leaflet with the HoloLens (Figure 4(b)), knowing that there is one leaflet for each type of intervention. Fig. 5 User interaction with the two parts (red and gray) of the virtual stomach during the second step of HOLO_NUTRI. The user virtually picks the gray shape of the stomach to take it away from the main red shape that represents the retained stomach part during surgery. From the user point of view, its hand and the object to be moved are aligned (photo taken from the HoloLens camera induces a misalignment).

Then a virtual stomach is displayed and the user will interact with it (Figure 5). For example in the sleeve operation (Section 1.1 and Figure 1(a)) the user, by pinching its thumb and forefinger, will select a part of the stomach that is supposed to be removed by moving it aside from the main part of the stomach. This part was not present in the conventional workshop CONV_WORK. Once this action is done, the second part of HOLO_NUTRI starts.

Second part: Meal composition

The second part displays a virtual buffet of about thirty different foods. Due to the quantity of informations and in order to render those informations in the most visible way, this buffet is provided in a semi vertical cylinder manner, with for the same column, three different portion sizes (small, medium, large) of the same food (Figures 6 and7). The user chooses the food by looking at it through the transparent visor (HoloLens' gaze) then the user can grab it by pinching his/her fingers together and by releasing them afterwards (HoloLens' gesture). The selected food appears as a cube in front of the 3D jaw (Figure 8(a)). The user grabs once again the food and brings it to his/her own mouth, an animation is then triggered (opening and closing cycle of the jaw, Figures 8(a) and (b)), forbidding any other action from the user, this essential step is designed to draw attention on the importance of the chewing time. The user repeats this procedure as long as he or she believes it is necessary to add foods to fulfill her/his meal. When the user is done, a first recap is shown to inform about the number of food selected and the portion size associated (Figure 9). In a next step, qualitiative (Figure 10(a)) and quantitative (Figure 10(b)) messages are displayed (according to the ones delivered in the conventional workshop). The qualitative messages inform on the necessity to have a diet as various as possible and to observe the chewing time (Figure 10(a)). The quantitative messages point the significant stomach volume reduction out (Figure 10(b)). The user is then allowed to perform a second iteration of this part of the workshop. For this second iteration, supplementary indicators (number of swallowed foods (Figure 11), transparent stomach (Figure 12), etc.) may help her or him to better compose their meals.

Results

Digital workshop presentation

HOLO_NUTRI software has been used by patients during the DIGIT_WORK workshop which goal was to make patients aware of the post-surgery diet modification they will endure due to the bariatric surgery they will undergo. This workshop is composed of nine steps (also depicted in Table 2 for a more global understanding): session introduction (step 1), identic to step 1 of CONV_WORK workshop; debate around the central question "To feed, eat, when one has been operated on a bariatric surgery, what does it evoke you ?" (step 2), identic to step 2 of CONV_WORK workshop; computer scientists team presentation (step 3); hardware and its using presentation (step 4) by showing a video. A dedicated software named LEARN_EX has been developped by us to introduce basic gestures to control the HoloLens and essential to the good use of HOLO_NUTRI (Figure 13). The computer scientist team gives information about this LEARN_EX learning exercise (step 5). This workshop starts with a discussion between all patients about the daily diet post-surgery (step 6), identic to step 5 of CONV_WORK workshop; patients are then invited to compose their own meal with HOLO_NUTRI software (step 7); the nutritionist delivers several advices depending on the food and quantities selected by the patient (step 8); feedback forms are then filled by patients in order to evaluate the session (step 9), identic to step 9 of CONV_WORK workshop. We have set up a specific communication to make patients able to use this technology and application as fast as possible: this communication is composed of laminated leaflets (surgery details, gestures to be used with the headset), several video sequences (basic gestures description to use both the headset and the application), and also a custom-made learning exercise LEARN_EX (to address specific gestures). More details about this dedicated communication is available in [START_REF] Appadoo | Multimedia contents design for a mixed reality experience with a HoloLens headset, on the occasion of the digitalization of human nutrition sessions[END_REF].

Experimental procedure

The software presented in this article has been used in a hospital hosted workshop that aimed to help patient prepare for a bariatric surgery (Centre Hospi- talier Émile Roux, Le Puy-en-Velay, France), several sessions of one hour and a half had been organized. For each one of them, a group of three patients were supposed to simultaneously handle first the headset HoloLens then to use the application HOLO_NUTRI. We, on purpose, limited the number of participants to three for the following reasons: size of the experimental room (Figure 14), only three HoloLens at our disposal, involvement of three researchers to help patients in the use of both the equipment and the software.

To allow this simultaneous participation of three patients, we have used the HoloLens App application [START_REF]Microsoft HoloLens application[END_REF] from the Microsoft Store in order to connect (using WiFi) the three HoloLens to three different computers to get access to a video feedback of each patient to better help them in their use of both this hardware and the software (Figure 14).

Application evaluation by the patients

HOLO_NUTRI has been used and assessed for 10 sessions by a total of 30 patients (27 women and 3 men) evenly distributed among those 10 sessions. These questions (12 in number) have been gathered into five different categories; the answers to these questions are shown in Tables 3 to 7. A number of these questions can be answered by a simple yes or no, while others expect an answer by scale (e.g. not at all ; slightly; moderately; severely). We write in each table the number of patients answering a given answer (by column) in the first line and the corresponding percentage in the second line in regard to the whole cohort of 30 patients. The average age of this group is 46 years old (standard deviation of 11.57 years, with a minimum age of 21 and a maximum age of 68). The average duration of the workshop introduction (explanations and video presentation of the headset) and of both the presentation and the completion of LEARN_EX is 13:07 minutes (standard deviation of 2:32 minutes, minimal duration of 8 minutes and maximal of 22 minutes). The mean duration use of HOLO_NUTRI simulation (first part) and two iterations of the meal composition (second part) is 17:53 minutes (standard deviation of 4:05 minutes, minimal duration of 12 minutes and maximal duration of 30

Yes

No Q1. Have you ever used an augmented reality material or application before (e.g. Pokemon Go, Snapchat, etc.) ? 6 24 20% 80% minutes). For five of those patients, the duration of the different workshop phases could not have been recorded. We have provided a series of questions to patients in order to get their feedback about this mixed reality experience. Those questions were focused on the patient technological knowledge (Table 3, Q1), on the physical feeling during the experience (Table 4, Q2), on the assessment of the software use (Table 5, Q3-Q7), on the assessment of this mixed reality technology compared to a more conventional one with the set computer/keyboard/screen/mouse (Table 6, Q8-Q11) and on the expected educational benefit (Table 7, Q12).

We present these five groups of results in the following tables from 3 to 7.

• (Group 1, Table 3). 80% of patients state that they have no technological skill at all in the use of augmented reality material or application. We can then conclude that this experience is the first with this kind of mixed reality material for most of them.

• (Group 2, Table 4). Half of our patient cohort does not seem to feel any general discomfort (Q2a). Only 2 patients among 29 (1 missing data) state feeling general tiredness following the experience (Q2b). 16 patients declare feeling a slight to severe ocular tiredness (Q2c). Only one patient has felt nauseous (Q2d). Over the 30 patients, 3 have found the headset to be heavy, 2 showed headaches with 1 who has declared feeling a severe ocular tiredness (Q2c). We can conclude that the physical feeling of patients is rather satisfying considering that this experience is the first with mixed reality for most of them.

• (Group 3, Table 5). 28 patients report not feeling the workload to be too much tiring (Q3). 24 patients have felt rather relaxed, satisfied and selfconfident while using the HOLO_NUTRI software (Q4). The duration of this experience has been perceived as rather satisfying by 28 patients (as a reminder, the session was planned to last one hour and a half with an effective use of HOLO_NUTRI of 18 minutes) (Q5), the two patients who have not 4, Q2b). Three patients only have not perceived this experience as satisfying (Q7): two of them for the reason previously stated in the question about recommendation to another person (Q6), and the last patient has not given us any feedback. In a summary, this experience has been rather tough for three patients (difficulties in performing the required gestures willing to a certain annoyance and preventing them from fulfilling the workshop goals, i.e. the total completion of two iterations of the second part of HOLO_NUTRI). For the rest of the cohort, this experience has been perceived as satisfying to very satisfying, which is rather positive to repeat this kind of mixed reality experience in a medical context, despite technologies that could be seen as too modern and not user-friendly. The same bias seem to be refuted by the results we present on the assessment of this new technology over a more conventional approach (Table 4).

• (Group 4, Table 6). 28 patients have not been too much disturbed by the superimposition of virtual elements onto their real environment (Q8). To our great surprise, a majority of patients has believed to handle real objects (14 "yes absolutely" and 4 "yes rather") during the use of HOLO_NUTRI (Q9). About the patient's focus on the workshop goal, more than 75% of them stated not being troubled by the use of this technology (17 "not at all" and 8 "not really"); 5 patients have stated to feel a focus trouble due to the use of this material (Q10). Finally, 60% of the patient cohort declared to be rather interested in having a workshop with this kind of material and set up rather than with a more conventional approach (the set computer/keyboard/screen/mouse or tablet) (Q11). The summary of the use of mixed reality in this awareness workshop on post bariatric surgery nutrition is, in accordance with the feedback we get from the patient themselves, rather positive and quite well perceived; this experience has been rather well received.

• (Group 5, Table 7). A question relative to the digitalization of this workshop is to understand if we can expect a better educational benefit with a mixed reality headset and an associated application compared to a classic information technology approach (computer/keyboard/screen/mouse set). We briefly speak about this question in this communication but it is presented with more detailed information in [START_REF] Barret-Grimault | Innovons en ETP grâce au casque HoloLens en chirurgie bariatrique ![END_REF]. Thus, the majority of the patient cohort that represents 93% (excepting one patient and one missing data to this question) has found a real motivation in learning from the nutritional messages delivered through the handling of virtual objects (i.e. meal composition with 3D foods).

Discussion and conclusion

We will start by listing the positive and negative aspects we have noted during this mixed reality experience in a medical context. We will consider five criteria: 1. material, 2. software, 3. developer, 4. clinical, 5. patient. We will conclude this section by a discussion and by giving perspectives to this application and its framework.

Positive aspects: an only educative use (e.g. the headset may work as a camera to broadcast a surgeon point of view to an audience of students in order to provide a better understanding of the surgical gestures to perform), during a surgical operation, a visualization of the pre-operative data is given to the surgeon (e.g. patient clinical file, patient 3D organ models, etc.); this may represent a real help for the surgeon in its practice [START_REF] Pratt | Through the HoloLens looking glass: augmented reality for extremity reconstruction surgery using 3D vascular models with perforating vessels[END_REF][START_REF] Jang | Threedimensional holographic visualization of high-resolution myocardial scar on hololens[END_REF][START_REF] Kuhlemann | Towards X-ray free endovascular interventions -using HoloLens for on-line holographic visualisation[END_REF][START_REF] Garcia-Vázquez | Navigation and visualisation with HoloLens in endovascular aortic repair[END_REF][START_REF] Liu | Augmented reality based navigation for computer assisted hip resurfacing: A proof of concept study[END_REF]. This visualization is mainly provided without any registration. 5. This new technology allows researchers to address new problems, by bringing efficient and innovative answer like in our experience we can note a reinforced learning from the gesture [START_REF] Barret-Grimault | Innovons en ETP grâce au casque HoloLens en chirurgie bariatrique ![END_REF] and therefore bring a real benefit to the patient in the end.

Negative aspects: 1. This material carries some limitations such as its high financial cost, a low screen resolution, a narrow field of view and a quite heavy weight. 2. The release of HoloLens 2 less than two years and a half after the first version in France, asks us the necessity to update the HOLO_NUTRI application. 3. For the developer, the learning curve is steep because it needs Unity knowledge, several in-development libraries and the dedicated documentation, which is not keep up-to-date on a regular basis. A specific development is required to take in consideration the specificity of each environment where the application will be used. For us, a specific distance was tuned to allow patients to easily grab the virtual 3D food, size of the semi-cylinder was also tuned to fit with the HoloLens field-of-view [START_REF] Lohou | Design of a mixed reality application with a HoloLens headset for a nutrition workshop[END_REF]. 4. Non computer scientist members of this experience would not feel comfortable enough with this kind of material to perform this whole experience in an autonomous way.

5. From our side, it was not possible to let this kind of material at the free disposal of the patients, therefore, we could not consider to perform this experience at home (technical difficulties to handle this material without assistance) and even less a long term follow-up; unlike augmented reality based on the use of a sole smartphone (as described in section 1.5), which would give more autonomy to the patients.

To conclude, we realised a translational research study (from the clinical needs towards the patient) with limited means (one student, three months of development, one Unity licence and one computer for programming), not forgetting the purchase of the HoloLens. In the frame of this nutritional workshop, we remind that 60% of patients who participated in this experience have assessed that this mixed reality approach was preferable to a more conventional one (computer/keyboard/screen/mouse set). Thus, we can easily extend this kind of work to other pathologies or to similar prevention workshops. We would like to insist on the fact that it would have been really hard to consider the same application without the HoloLens material. The use of 3D models in our application does not limit to the sole visualization, to a spatial positioning in a virtual environment or to a scaling of those latest but it tends to make the patient the more active as possible by giving them the opportunity to handle them directly. This gesture reinforced learning is reviewed in [START_REF] Barret-Grimault | Innovons en ETP grâce au casque HoloLens en chirurgie bariatrique ![END_REF]. Our future work would be to add nutritional informations per food in order to give a more detailed analysis than the one currently proposed on the meal composed by the patient (only the number and the size of the portions selected are given). Finally, our experience on this kind of application development for HoloLens in a medical context has also allowed us to develop a prototype of needle inserting assistance during hemodialysis sessions [START_REF] Lohou | Mixed reality experiment for hemodialysis treatment[END_REF] or another one to assist surgeons during trocar placement [START_REF] Lohou | Preliminary experiment of the interactive registration of a trocar for thoracoscopy with HoloLens headset[END_REF].

Fig. 1

 1 Fig. 1 Both types of considered interventions: (a) Sleeve Surgery -the dark part is removed, (b) Bypass surgery -the dark part represents the medium part of the small intestine that bypasses the normal food path. Figure from [1].

Fig. 2

 2 Fig.2(a) A user of the HTC Vive virtual reality headset, holding one controller in each hand. (b) Superimposition of a 3D virtual object on a marker. (c) Example of an augmented reality application using a tablet to scan a painting; there is a necessity to hold the device with one or two hands. (d) This view is from one user of the Google Glass device; the projected information are mainly in 2D and the user cannot interact with its environment. (e) The PokemonGO application on a mobile phone, occlusion problems persist and there is no interaction with the user environment. Subfigure (a) is from[START_REF]AS#04 -mixed reality" by artfulspark and is licensed under[END_REF], subfigure (b) is from[START_REF]AR 1" by cubistscarborough and is licensed under CC BY-NC-ND 2[END_REF], subfigure (c) is from[START_REF]Augmented Reality Comics @ Ars Electronica Center[END_REF], subfigure (d) is from[START_REF]The view from Cecilia Abadie's driver's seat[END_REF], subfigure (e) is from[START_REF] Subfigure 2e Reference | Drowzee" by Hackley Public Library and is licensed under CC BY 2[END_REF].

Fig. 3

 3 Fig. 3 (a) Example of the environment scanning performed by the HoloLens; it is decomposed in multiple triangles in order to create a 3D model of the scene where possible interactions will be allowed. (b) Photo montage illustrating the user of a HoloLens mixed reality headset and the content as seen by him in his own environment (boxed). The blue circle indicates the HoloLens cursor as seen by the user in the scene. The blue arrow indicates the gaze direction of the user. Subfigure (a) is from [21].

Fig. 4

 4 Fig.4(a) "Sleeve Surgery" button (first button on the left in the upper menu) and "Bypass" button (second bouton on the left in the upper menu). (b) Scanned leaflet with 3D model (Sleeve surgery).

Fig. 6

 6 Fig. 6 Buffet screenshot (part of the semi cylinder).

Fig. 7

 7 Fig. 7 Different portions for the same food.

Fig. 8

 8 Fig. 8 (a) 3D representation of foods (red delineated cube) and of the jawbone. (b) Jawbone animation (opening-closing cycle) and display of the remaining chewing time (red arrow).

Fig. 9

 9 Fig. 9 Recap panel of the meal; on the left are shown the meal duration and the number of selected foods, on the right a message about the number of portions and foods composing the meal and a logo corresponding to that message.

Fig. 10

 10 Fig. 10 Informative panels on the qualitative (a) and quantitative (b) aspects of the meal.

Fig. 11

 11 Fig. 11 Supplementary indicators for the second iteration: on the left, portions and foods numbers along with the duration of the meal are displayed; on the right, swallowed foods are shown.

Fig. 12

 12 Fig. 12 Transparency of the 3D stomachs (on the right, before surgery and on the left, after surgery) to enhance visualization of the occupied volume into them by the swallowed foods.

Fig. 13

 13 Fig. 13 The consecutive three steps of LEARN_EX learning exercise : (a) Menu validation, (b) 3D object selection, (c) 3D object movement.

Fig. 14

 14 Fig. 14 DIGIT_WORK workshop layout into the experimental room. In the box, video feedback of HOLO_NUTRI on a laptop.

Table 1

 1 Description of the main constituent steps of the conventional workshop CONV_WORK.

	1. Session introduction
	1.1. Presentation of the protocol and signature of the consent forms
	1.2. Signature by patient
	1.3. Group presentation (clinicians / patients)
	1.4. Presentation of the objectives of the session
	2. Debate around the question "To feed, eat, when one has been operated on a bariatric
	surgery, what does it evoke you ?"
	3. Beginning of the workshop: Initial question "After the surgery, how to eat daily ?"
	4. Carrying out the workshop experience with cards
	4.1. Presentation of the workshop with cards
	4.2. Food choices via cards
	4.3. Register quantities in a table
	5. Collection of the patients choices regarding the menus
	6. Collegial discussion on the choice of each patient
	7. Advices are given to the patient according to their food and quantities choices
	8. Informative messages (quantitative and qualitative)
	9. Forms (assessment knowledge + satisfaction)

Table 2

 2 Description of the main constituent steps of the digital workshop DIGIT_WORK.

Table 3

 3 Evaluation of patient knowledge on augmented reality devices and related applications.

	Q2. Have you felt any discom-	Not	Slightly	Moder-	Sever-	Missing
	fort due to wearing
	the headset:	at all		ately	ely	data
	a. General discomfort ?	15 50%	11 36.67%	3 10%	1 3.33%
	b. General tiredness ?	27 90%	1 3.33%	1 3.33%	0 0%	1 3.33%
	c. Ocular tiredness ?	14 46.67%	11 36.67%	4 13.33%	1 3.33%
	d. Nauseas ?	29 96.67%	0 0%	1 3.33%	0 0%

Table 4

 4 Evaluation of patient physical feeling during the experiment.

Table 5

 5 Assessment of HOLO_NUTRI application use by the patients. appreciated the duration have also declared feeling ocular tiredness (Table4, Q2c). 28 patients would recommend this experience and 15 of them with no restriction (Q6); 2 patients would not really recommend this experience, one finds no interest in this workshop compared to the classic CONV_WORK and the other finds this technology too modern, requiring a very high level of concentration to use this device which deviates attention from the main goal of the workshop, this patient has also declared feeling general tiredness following this experience (Table

Table 6

 6 Assessment by the patients of this mixed reality technology compared to a more conventional information technology approach (computer/keyboard/screen/mouse).

	Not	Not	Yes	Yes
	at all	really	rather	absolutely

Table 7

 7 Expected educational benefit evaluation from the patient's point of view.

  1. The material we used is working in an autonomous way, an embedded computer is stored in the headset itself. No other equipment is needed to work, all sensors are present in or on the headset; no cable connexion is required. 2. With an appropriate communication (e.g. video tutorial), 20 minutes are enough to handle this equipment and the application [1]. 3. Application development for the HoloLens may benefit from several frameworks dedicated to development of 3D interactive graphical applications such as Unity and Vuforia. Therefore, there is no need to program with a low level library like DirectX [58] 4. More and more medical applications use the HoloLens headset. We can note two main uses:

Acknowledgements Authors would like to thank Arthur Jacquin, student in DUT Informatique Graphique at IUT de Clermont-Ferrand, France (site Le Puy-en-Velay) for the software design. This project is funded by a french national grant: PEPS INSIS CNRS "Sciences de l'Ingénierie pour la Santé pour accompagner des projets translationnels", 2017, project AVACM (Assistance Visuelle Augmentée lors de Consultations Médicales).


Rahmet Parilti

Alba Castañon

Muriel Lansalot

Franck D ' Agosto

Christine Jérôme

Steven M Howdle

Hydrocarbon based stabilisers for the synthesis of cross-linked poly(2-hydroxyethyl methacrylate) particles in supercritical carbon dioxide

An environmentally-friendly process to prepare well-defined cross-linked particles of poly(2-hydroxyethyl methacrylate) (PHEMA) was investigated. The dispersion polymerisation of 2-hydroxyethyl methacrylate (HEMA) with a cross-linker ethylene glycol dimethacrylate (EGDMA) was performed in supercritical carbon dioxide (scCO 2 ). Stabilisers based only on vinyl acetate (VAc) and vinyl pivalate (VPi) random copolymers P(VAc-stat-VPi) were employed thus avoiding the use of silicone or fluorine based stabilisers which are normally required in scCO2. The effect of molecular weight and composition of the P(VAc-stat-VPi) stabilisers on the size and shape of the produced microparticles was investigated. A copolymer stabiliser with 56:44 (VAc:VPi) molar composition (Mn = 12.8 kg mol -1 ) was found to be the most efficient, resulting in welldefined spherical cross-linked PHEMA particles with a diameter of 1.2 µm. This particular stabiliser was found to provide good steric stabilisation for a range of cross-linker ratios and the addition of a co-monomer such as N-vinylpyrrolidone (NVP) allowed tuning of the properties of the microparticulate products. Finally, the stabiliser plays a second very important role.

Partial hydrolysis of the ester groups of the stabiliser located on the surface of the microparticles leads to a hydrophilic surface with alcohol moieties and hence facilitates dispersion of the microparticles into water leading to valuable microgels.

Introduction

Green chemistry is defined as the design of products and processes that eliminate the use and/or the generation of hazardous substances. [START_REF] Anastas | Green Chemical Syntheses and Processes[END_REF] Green chemistry is described by 12 principles and across the world research groups and companies are trying to design synthetic methods that utilise these principles; for example to replace volatile organic conventional solvents. [START_REF] Anastas | [END_REF] In that framework, supercritical carbon dioxide (scCO2) is an excellent candidate with accessible critical temperature (31.1 °C) and pressure (73.8 bar), gas-like diffusivity and liquid-like densities. Moreover, scCO2 is a naturally highly abundant, non-toxic, non-flammable solvent which makes it a good candidate for VOC replacement. Most importantly, scCO2 can be removed by simple depressurisation, this can also help significantly in removing unreacted monomer and hence eliminating post-reaction purification processes. 3 The use of scCO2 as a solvent has witnessed tremendous growth 4 in many areas; from commercialized extractions in the food industry 5 to intricate pharmaceutical formulations for drug delivery 6 or as a solvent or non-solvent to produce polymer particles with controlled size and shape. 7 One of the most versatile routes to polymer particles is heterogeneous freeradical polymerisation. 8 Most common monomers are soluble in scCO2, but their corresponding high molecular weight polymers are usually insoluble making scCO2 an ideal candidate for dispersion polymerisation. 8 A key factor for the successful dispersion polymerization and control of the particle size is the selection of a relevant stabiliser. Stabilisers can either adsorb or chemically graft onto the growing polymer microparticles limiting growth and preventing aggregation by steric stabilisation. 9 Efficient stabilisers for dispersion polymerization in scCO2 require macromolecules exhibiting a "CO2-philic" part to ensure solubility in scCO 2 and provide steric stabilisation to prevent particle aggregation. 10 Up till now only fluorinated and silicone based polymers have shown sufficient scCO2 solubility. However, these are expensive and this might limit large scale application. 11 Consequently, an area of interest has been the synthesis of alternative hydrocarbon based stabilisers. Kazarian et al. have shown that polymers containing electron donating functional groups such as carbonyls, interact with CO2 molecules via Lewis acid-base type interactions. 12 Sarbu et al. proposed that a CO 2 -philic block could be based on a combination of a monomer (A) which gives flexibility and free Please do not adjust margins Please do not adjust margins volume to the polymer chain and enhances the entropy of mixing, and a co-monomer (B) containing a Lewis base group, such as a carbonyl group, to facilitate interaction with CO2. Poly(ether carbonate)s (PEC)s were reported to be scCO2 soluble at reasonable temperatures and pressures. 13 Similarly, Tan et al. reported the facile synthesis of scCO2 soluble poly(ether carbonate)s and poly(ether ester)s by step-growth polymerisation. 14 In addition they showed that block copolymers composed of poly(ethylene glycol) (PEG) and oligo(vinyl acetate) (OVAc), PEG-b-OVAc diblock and OVAc-b-PEG-b-OVAc triblock copolymers could be efficient emulsifiers for water in scCO2. These diblock and triblock copolymers were able to emulsify significant levels of water in CO2 and polymerisation in this medium resulted in highly porous poly(acrylamide) (PAM) networks. 15 The porosities of the PAM networks were found to be higher than those of networks synthesized with a perfluoropolyether (PFPE) showing that OVAc-based block copolymers might advantageously replace fluorinated materials in scCO2. 15,16 Poly(vinyl acetate-alt-dibutyl maleate) P(VAc-alt-DBM) copolymers also showed good CO2 solubility. 17 Cloud point measurements demonstrated that random copolymers of vinyl acetate and vinyl butyrate P(VAcstat-VBu), 50:50 molar composition, have a higher solubility than PVAc and PVBu homopolymers. These materials were then exploited to perform the first successful dispersion polymerisations in scCO2 using hydrocarbon based stabilisers. 18 The dispersion polymerisation of N-vinylpyrrolidone (NVP) led to monodisperse poly(N-vinylpyrrolidone) (PNVP) microparticles. 18 This work was then extended by using stabilisers from copolymers of vinyl pivalate (VPi) and vinyl acetate (VAc) (P(VAc-stat-VPi)). Again, micron sized spherical PNVP particles were produced successfully. 19 Park et al. extended this study to the synthesis of copolymers based on VAc, vinyl butyrate (VBu) and vinyl octanoate (VOc). Their cloud point data indicate that the solubility of the P(VAc-stat-VBu) stabiliser increased with increasing VBu content in the copolymer, which also lead to better stabilisation of PNVP particles, while in the case of P(VAc-stat-VOc) stabilisers the opposite behaviour was observed. 20 Girard et al. reported the synthesis of poly(N,N-dimethyl acrylamide)-b-poly(vinyl acetate) (PDMA-b-PVAc) diblock copolymers and showed that their solubility in scCO2 decreased at higher PDMA content. 3 Cross-linked PHEMA 21 microparticles have shown utility as drug carriers, in dental, ophthalmic and tissue engineering applications. 22 A key challenge is to find routes to their synthesis that are clean, but do not require perfluorinated or silicone based stabilisers. Moreover, the materials produced should be easily dispersible in water. Hence, in this paper we focus on the dispersion polymerisation of 2-hydroxyethyl methacrylate (HEMA) in the presence of ethylene glycol dimethacrylate (EGDMA) as a cross-linker using P(VAc-stat-VPi) copolymer stabilisers to obtain microparticulate PHEMA networks directly from scCO2. Block copolymer architectures have been shown to be less soluble than comparable statistical copolymer stabilisers. 23 This was attributed to the segregated arrangement of the VAc and VPi components. To ensure a high enough solubility it was essential that the block copolymers had a lower overall molecular weight. This led to spherical microparticles, albeit with larger particle size diameter and broader particle size distributions than those obtained from the statistical copolymers of the same chain length. After successful synthesis of microparticles, P(VAc-stat-VPi) stabiliser must be present on the microparticle surface. One idea that we intend to explore is to determine whether hydrolysis could provide a facile route to surface bound hydrophilic vinyl alcohol units to allow dispersion of the microparticles into water.

Experimental Materials

All chemicals were used as received unless otherwise noted.

Methods

Synthesis of O-ethyl-S-(1-methoxycarbonyl) ethyl dithiocarbonate

Methyl bromoacetate (16 g, 104.6 mmol) was dissolved in methanol (75 mL) in a 250 mL round bottom flask with a stirring bar and in an ice bath. Potassium ethyl xanthogenate (19.515 g, 121.7 mmol) was dissolved in ethanol (100 mL) in a beaker for 30 minutes and added dropwise to the flask using an addition funnel. The mixture was stirred at room temperature for 24 hours and filtered to remove KBr salts. The filtrate was concentrated under reduced pressure, dissolved in dichloromethane (50 mL) and washed with water (3x15 mL). The organic phase was dried with magnesium sulfate and dried under vacuum to yield a yellow liquid. Yield = 79%. 1 H-NMR (CDCl3): δ = 4.5 ppm (q, 2 H), 3.8 ppm (s, 2H), 3.6 ppm (s, 3H), 1.3 ppm (t, 3H). Synthesis of P(VAc-stat-VPi) copolymers Typical copolymer synthesis in bulk: vinyl acetate (17.99 g, 209 mmol), vinyl pivalate (2.97 g, 23.23 mmol), O-ethyl-S-(1-methoxycarbonyl) ethyl dithiocarbonate (0.684 g, 3.6 mmol) and AIBN (0.06 g, 0.36 mmol) (RAFT:AIBN molar ratio = 10:1) were placed in a sealed round bottom flask with a stirring bar and degassed with argon for 30 minutes in an ice bath. The mixture was stirred at 60 °C for 5 hours to a conversion of around 60% as determined by NMR. The polymer product was precipitated in a mixture of ice cold methanol:water (4:1 v:v) and dried under vacuum for 24 Please do not adjust margins Please do not adjust margins hours to remove both the solvent and unreacted monomer. [START_REF] Anastas | Green Chemical Syntheses and Processes[END_REF] H-NMR (CDCl3) is shown in Figure 1.

Dispersion polymerisation in scCO2

Dispersion polymerisations were performed in a 60 mL high pressure autoclave. A typical one-pot procedure was followed for the free radical dispersion polymerisation of HEMA and/or copolymerisation with NVP in scCO2 unless otherwise noted. The autoclave body was charged with 6 mL of monomer (10% vmonomer/vCO2), 0.1368 g of V-70 (1% wV-70/wmonomer), 60 µL of EGDMA (1% vcross-linker/vmonomer), and 0.6 g of stabiliser (10% wstabiliser/vmonomer). Initially the autoclave was filled with 800 psi with CO2 (55 bar), then stirring was started at 200 rpm. The cell was heated up to 35 °C. When the final temperature was reached and stabilised for a few minutes, the cell was pressurised progressively to 3500 psi (240 bar) or the desired final pressure. The reaction was left stirring overnight at 35 °C and 3500 psi. To stop the reaction the heating jacket was disconnected, and the autoclave was allowed to cool down to room temperature and depressurised very slowly by the outlet tap into the fume hood. Stirring was left on at all times until a low pressure (700-600 psi) was reached. The autoclave was opened once atmospheric pressure was reached (14-18 psi). Polymer products were then collected for analysis.

Hydrolysis of the stabiliser and stabilised particles

Two conditions have been tested to hydrolyse the P(VAc-stat-VPi) copolymer into PVOH based on previously reported conditions for PVAc homopolymer (condition 1) and for PVPi homopolymer (condition 2) by Bernard et al. 24 The following procedures were applied first on the stabiliser only and then on the PHEMA stabilised particles. Condition 1 "PVAc conditions": 100 mg of statistical copolymer was dissolved (or 100 mg of particles were suspended) in 10 mL of methanol and a solution of 200 mg of KOH in 10 mL methanol was added. The mixture was stirred for 24 hours at room temperature then dialysed against methanol for 24 hours with Spectropore (cut-off 3.5kDa) dialysis membranes from Spectrum. The solvent was removed under reduced pressure (particles were freeze-dried in order to eliminate the small amount of residual solvent). Condition 2 "PVPi conditions": 90 mg of statistical copolymer was dissolved (or 90 mg of particles were suspended) in 1 mL ethanol and a 1 mL solution of NaOH (1 wt% in methanol) was added on the mixture. The mixture was stirred for 24 hours at 60 °C then dialysed against methanol for 24 hours. The solvent was removed under reduced pressure (particles were freezedried in order to eliminate the small amount of residual solvent).

Characterisation

Cloud point measurements of the stabilisers in scCO2 / monomer mixture

The solubility of the statistical copolymers in scCO2 indicates their potential to be used as stabiliser for dispersion polymerisation in this solvent/medium. Cloud point measurements were recorded for a range of copolymers of different compositions and molecular weights. The cloud point in scCO2 can be defined as the pressure at which the polymer starts to precipitate out from the solution at a fixed temperature. 14 The cloud points of synthesised statistical copolymers were determined in the CO2/monomer mixture by using a high pressure variable volume view cell. 25 For a typical cloud point measurement 3 g (15 wt% monomer/CO2) of HEMA, 150 mg of stabiliser (5 wt% stabiliser/HEMA) and 20 g of CO2 were used. The stabiliser was dissolved in a given amount of HEMA and injected into the view cell. CO2 was then transferred to the view cell by using a small stainless-steel CO2 sample cylinder, the weight of which was recorded before and after addition to calculate the exact amount of CO2 introduced in the cell. The cell was heated up to the desired temperature and allowed to stabilise. Thereafter, the hydraulic piston was slowly moved forward to increase the pressure in the cell until a single phase was observed through the sapphire window. At this pressure, the light placed at the end of the view cell was visible. The piston was moved back again slowly to decrease the pressure in a controlled manner to observe the pressure at which the solution was no longer a single phase. A cloudy mixture was observed due to precipitation of the polymer from the solution. Measurements were repeated in triplicate. Cloud points were taken at 35, 45, 55, 65 and 75 °C. The cloud points are reported as an average value of the three repetitions. 19 The glass transition temperatures (Tgs) of the collected particles were analysed by using a Triton Technology TT DMA using powder pockets which allow analysis of samples in powder form. The analysis pocket was filled with the particles as collected from the high-pressure reactor. Dynamic mechanical analysis (DMA) was performed in single cantilever bending mode with a temperature ramp from room temperature to 120° C or 150 °C at a heating rate of 5 °C min -1 . 1 H-NMR was used to determine the structure, composition, conversion and molecular weights of the polymers. Analyses were performed on a Bruker AV3400 spectrometer equipped with a 400 MHz magnet, using deuterated chloroform (CDCl3) as solvent. End group analysis was performed using relaxation times of 1 second and after recording 256 scans. All data were processed using the MestreNova software. Size exclusion chromatography (SEC) was performed using an Agilent 1260 Infinity system equipped with two polystyrene columns of 8x300 mm and a Wyatt OptilabrEX refractive index detector. THF was used as eluent at 0.5 mL min -1 flow rate. PMMA standards were used for calibration. The morphology of the obtained polymer particles was analysed by scanning electron microscopy (SEM) using a JEOL 6490LV SEM and JEOL JSM 840-A in secondary electron image (SEI) mode under high vacuum (HV). Samples were prepared on aluminium stubs supplied by Agar. Two-sided carbon discs were placed on the aluminium stub and sufficient amounts of samples were spread on the discs. Average diameter of the particles (Dn) is determined from a conventional SEM images by measuring of randomly selected individual particles with the aid This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins of software (VistaMetrix, V.1.33.0). Approximately 25-30 particles were counted per image and several images of the same sample were analysed to get statistical measurement. Coefficient of variance (Cv) is calculated according to the following equation: Cv = (σ/D n )*100. Hydrodynamic diameters of polymer particles in diluted solutions were recorded using DelsaNano C by Beckman Coulter. The size distribution of the diluted water dispersions was estimated by photon correlation spectroscopy (PCS) using a particle-size analyser at 25 °C. The intensity of the scattered light was detected at an angle of 165° to the incident beam.

Results and discussions

Synthesis of the stabilisers via RAFT polymerisation

Stabilisers composed of VAc and VPi of different composition and molecular weight were synthesized via RAFT/MADIX polymerisation (Figure 1) because of its ease of implementation and good control over the polymerisation of vinyl ester monomers. 26,27 Conversions around 60% were achieved after 5 hours in all cases. Reactions were typically stopped at 60% conversion to optimize the control of chain growth. The macromolecular characteristics of the obtained copolymers were determined by [START_REF] Anastas | Green Chemical Syntheses and Processes[END_REF] H-NMR and SEC after purification and all results are summarized in Table 1. A representative 1 H-NMR spectrum is shown in Figure S1 as an example. The VAc:VPi ratio was calculated by comparison of the integrations of the resonances b (VPi, 9H from t Bu), c and d [5VAc (3H from CH3 + 2H from CH2)+ 2VPi (2H from CH2)] (Figure 1). Molecular weight was calculated assuming 100% of the chains were initiated with the reinitiating fragment of the initial RAFT agent and using the integral of the resonance for the corresponding methoxy end group (f in Figure 1) as a reference. The degree of polymerisation was then obtained considering the integration of the signal h (Figure 1). The data provided in Table 1 show that P(VAc-stat-VPi) with Mn between 4.6 and 22 kg mol -1 and VAc:VPi molar ratio ranging between 9:91 to 75:25 were successfully obtained with rather low dispersities (<1.5). There is a good agreement between theoretical Mn values (estimated taking into account the conversion) and those calculated by NMR from the integration of the chain end protons (Table 1). Table 1 Macromolecular characteristics of the synthesized stabilisers and the size and size distribution of the PHEMA particles obtained using those stabilisers in dispersion polymerisation in scCO2. a: The average size of the particles determined from individual SEM images (Dn), b: Cv = (σ/Dn)*100 where σ indicates the standard deviation. *: In these samples a particle size is reported for individual particles but also significant aggregation was observed. For entry 3 the RAFT ends could not be determined.

Cloud point measurements

The cloud point in scCO2 is defined as the pressure where the polymer starts to precipitate out from the medium at a given temperature and is measured at the point where the LED lights in the system are no longer visible. Therefore, by measuring cloud points one is able to estimate the reaction conditions for the copolymer to be used efficiently as a stabiliser in dispersion polymerisation in scCO2. The cloud points of the two synthesized P(VAc-stat-VPi) copolymers shown in Table 2 were measured in the dispersing medium (scCO2) containing the HEMA monomer which mimics the initial phase of dispersion polymerisation. Moreover, in this mixture the monomer also behaves as a co-solvent enhancing the solubility of the copolymers in scCO2. Photographic images (Figure 2) were taken at discrete phase changes during cloud point measurements, which represent the different stages of a cloud point experiment (Figure 2). A clear trend is observed; an increase in temperature (35 °C to 75 °C) led to higher cloud point pressures for both samples. This behaviour is strongly related to the lower CO2 density at higher temperatures, which necessitates an increase in the pressure in order to have the same solvent strength. Nevertheless, the copolymers show similar cloud points at 35 °C (175 bar) to the conditions under which our dispersion polymerisations in scCO2 will operate. In addition, the cloud point of a stabiliser with roughly similar molecular characteristics (VAc:VPi = 44:56/Mn = 9.4 kg mol -1 /Ð = 1.5/Tg = 47.4 °C) was measured in scCO2 in the presence of a different monomer, NVP (Table 2-Entry 3). The cloud point data at 35 °C show very similar values to the HEMA system, i.e. 154 bar (2236 psi) thus indicating that perhaps the syntheses with HEMA may proceed just as effectively as those previously demonstrated for NVP. 19 In light of these measurements, it would be expected that in terms of copolymer composition when the VPi content increases the solubility in CO2 increases. And all the synthesized stabilisers should be soluble for HEMA dispersion polymerisations carried out 35 °C and 240 bar.

Dispersion polymerisation of HEMA in scCO2

Dispersion polymerisation of HEMA was performed in scCO2 as described above. Cross-linked PHEMA particles have been widely investigated for biomedical applications especially for sustained release. The cross-linking degree defines the swelling of the networks thus affect the release behaviour of the entrapped molecules within the network. Synthesis conditions and feed ratios for producing cross-linked PHEMA particles have been selected on the basis of cloud point measurements along with our previous work using fluorinated stabilisers, 9 i.e. 35 °C and 240 bar which are above the critical conditions for CO2.

In the course of the dispersion polymerisations in scCO2 we observed an increase of the final pressure of around 14 bar (200 psi). This phenomenon was also reported by other researchers for polymerisation of styrene (Sty) 28 , methyl methacrylate (MMA) 29 and HEMA 30 in scCO2 and is attributed to the thermodynamics of mixing leading to expulsion of both CO2 and monomer from the polymer phase.

After overnight polymerisation at 35 °C and 240 bar the heating was turned off and the autoclave was left to cool to room temperature and remaining CO2 was slowly vented. After complete depressurisation, the polymer was collected from within the autoclave as either a free-flowing powder or as a brittle disc from the bottom of the reactor (See supporting info-S2). Further inspection by SEM of polymer obtained in two of the experiments (Figure 3) showed that only aggregated polymer products are formed by coalescence of well-defined spherical particles. Similarly, large aggregates of polystyrene particles have been observed before by Canelas et al. using poly(styrene)-block-poly(dimethylsiloxane) (PS-b-PDMS) block copolymers as stabiliser. 28 They explained the formation of polystyrene aggregates by the collapsing of PDMS chains as solubility in CO2 decreased during venting process.

Effect of stabiliser composition and molecular weight i. Composition

A series of P(VAc-stat-VPi) copolymers having roughly similar molecular weights (~ 10 kg mol -1 according to NMR) with different compositions (Table 1, entries 3/4/5/7/8/9/11/15/16) were tested to find the most suitable VAc:VPi ratio for stabilising PHEMA particles in scCO2. The initial feed ratios were kept constant for all the reactants; HEMA (monomer, 10% v/vCO2), EGDMA (cross-linker, 1% v/vmonomer), stabiliser (10% w/vmonomer) and low temperature initiator (V-70, 1% w/vmonomer). After overnight polymerisation, for all copolymer stabilisers, high HEMA conversions were achieved typically >90% in each case. However, depending on the molecular characteristics of the stabilisers, different polymer product morphologies were observed by SEM (Figure 4). The stabilisers and the characteristics of the corresponding particles are listed in Table 1.

Figure 4: SEM images of the particles obtained after dispersion polymerization of HEMA (10% v/vCO2) and EGDMA (cross-linker, 1% v/vmonomer) with stabiliser (10% w/vmonomer) of different compositions (see entries of Table 1) and initiator (V-70, 1% w/vmonomer).

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The copolymer composition was shown to have a major impact on particle stabilisation, morphology and size. It was observed that the best defined and smallest particles were obtained when the copolymer compositions (VAc:VPi) were close to 50:50 (especially 56:44, Entry 15). Increased VAc content in the copolymers resulted in better stabilisation of the PHEMA crosslinked particles (Entry 5 to Entry 15) affording diameters around 2 microns. This behaviour of the P(VAc-stat-VPi) copolymer was in line with already reported observations for the dispersion polymerisation of NVP in scCO2. 19 There appears to be a morphology change of the PHEMA product for stabiliser compositions that were clearly rich in one of the co-monomers (23:77 and 75:25 VAc/VPi ratio). These copolymers led to formation of porous PHEMA monoliths instead of particles (Figure 4, Entry 4 and 16). For these extreme compositions the copolymers were not able to stabilize the growing PHEMA particles, and the breakdown of steric stabilisation leads to a porous PHEMA network.

It is worth mentioning that anchoring to the particles via RAFT end-groups cannot be considered since xanthates are indeed known to show no reactivity towards methacrylic radicals. 18 Therefore, the origin of stabilisation appears to be mainly by adsorption of the stabiliser on the particle surface which imparts long-range repulsion.

ii. Molecular weight P(VAc-stat-VPi) stabilisers with different molecular weight were tested for their effect on particle formation, morphology and size. Surprisingly, quite well-defined particles are formed for stabilisers with very low VAc content and low molecular weights (Table 1 -Entry 1/2/3). This is attributed to the higher solubility of these lower molecular weight stabilisers. However, the particles produced do remain quite large in diameter (Table 1 -Entry 1/2/3). The best-defined and smallest particles are produced with a composition of 56/44 (Figure 4 and Table 1: Entry 14/15). In general, the composition appears to be the dominant parameter for efficient stabilisation of the particles. At this composition (56/44) an increase of molecular weight from 8.6 kg mol -1 to 12.8 kg mol -1 shows a significant and controllable decrease of the particle size from 2.2 µm to 1.3 µm, although the size distribution was slightly broadened.

In previous work we produced PHEMA nanoparticles in scCO2 aided by a perfluorinated diblock stabiliser composed of polyethylene oxide (PEO) and poly(heptadecafluorodecyl acrylate) (PFDA) (PEO-b-PFDA). The particles were obtained with diameter as small as 200 nm. 9 Others have shown that dispersion polymerisation in scCO2 using a silicone based stabiliser can yield controlled particle sizes from 300 nm to 7 m for PMMA. 31 Although the particles obtained in this work are of a larger size (micrometres) they are still very well defined and of practical interest. In conclusion, the most efficient stabiliser was found to be the copolymer with 56:44 composition and 12.8 kg mol -1 (Figure 4 and Table 1, Entry 15) and was therefore selected for further investigation.

Effect of cross-linker content

It has been reported that mechanical properties such as stiffness and surface hardness of bulk or particle hydrogel networks can be tuned by tailoring the composition of the network by incorporating co-monomers or varying cross-linking density. 32,33 Thus, dispersion polymerisation of HEMA was carried out with three different initial concentrations of EGDMA whilst keeping the other parameters constant (i.e. stabiliser composition, ratio). The effects of initial cross-linking content on the particle size and morphology were demonstrated by SEM images (Figure 5). Surprisingly we found that in our control experiments with no added EGDMA the PHEMA particles were found to be slightly cross-linked as evidenced by the particles swelling but not dissolving in DMF, a good solvent for PHEMA.

We believe this can be explained by small levels of EGDMA that are present as an impurity in the monomer. 34 A clear trend (Figure 5) is seen as the cross-linker initial concentration increases (0 to 3 wt%); more uniform and discrete particles were formed. The likely reason for the apparently better stabilisation is that the particle surfaces have become rigid because of the action of the cross-linker and this helps in preventing coalescence of the particles when they collide in the dispersion process or after depressurisation. 35 In addition to better stabilisation, the particle size also decreased from 1.3 to 1.2 µm as EGDMA cross-linker concentration was increased from 1 wt% to 3 wt%. Dynamic mechanical analysis (DMA) was conducted in order to see the effect of the cross-linking density on the particle properties. DMA plots (Figure 6) indicate two distinctive peaks. This journal is © The Royal Society of Chemistry 20xx

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The first peak at around 50 °C in the DMA curves is ascribed to the stabiliser present on the particle surface, a conclusion drawn from comparison with DSC analyses of the stabiliser alone which gives a peak at around 50 °C (Table 1). The second peak (80 °C -140 °C) is clearly assigned to the PHEMA network (Figure 6) based on literature data 36 and this peak increases with cross-linking density. A clear trend is seen, with high concentration of cross-linker leading to higher Tgs and indicating that higher cross-linking density was achieved in the resultant networks (Table S3 -Entry 3).

Copolymerisation with NVP

As mentioned earlier, another approach to vary the properties of the PHEMA particles is to introduce a co-monomer. To test this approach, copolymerisation of HEMA with NVP has been performed using 50% v/vHEMA initial feed ratio in the presence of 1 wt% EGDMA crosslinker. The incorporation of NVP as comonomer was evidenced using ATR-IR (Figure S4). Successful copolymerisation of HEMA with NVP was confirmed by the appearance of the characteristic peak of a carbonyl group (C=O) assigned to the NVP moieties at 1650 cm -1 . In addition, as shown by the SEM image (Figure 7), the copolymerisation of HEMA with NVP results in largely spherical particles (1.8 µm) with a very small amount of aggregation, essentially very similar to those obtained for PHEMA (1.3 µm) with the same stabiliser. While the implication of the xanthate chain end was previously ruled out in the case of HEMA polymerization, the addition of NVP as a comonomer requires to reconsider this assumption. Indeed, previous studies have investigated the effect of the xanthate end groups in dispersion polymerisations in scCO 2 . Poly(vinylalkylate) stabilisers bearing xanthate end groups were found to easily and reproducibly form micron-sized PNVP particles. 18,19 Lee et al. demonstrated that the RAFT mechanism was essential for that stabiliser P(VAc-co-VBu) to anchor onto the PNVP growing particles in scCO2. This was proven by an experiment in which the xanthate end-group was converted to a thiol. Even though the thiol terminated copolymer had roughly similar solubility (similar cloud points) in scCO2 compared to xanthate bearing copolymer it was found to be ineffective for stabilisation of PVP particles in dispersion polymerisation.

Therefore, we hypothesised that the observed stabilisation of the particles in the presence of NVP might be slightly different from that obtained with HEMA alone as the presence of the xanthate chain end of the stabiliser will not be neutral. The literature offers a bunch of reactivity ratios for NVP/HEMA copolymerization systems 37,38 that, although different, are all pointing to a faster incorporation of HEMA in the chains compared to NVP. Although HEMA terminated growing radicals will not add to the xanthate moiety of the stabilizer as mentioned above, PNVP oligoradicals can add onto the xanthate chain end, and release P(VAc-stat-VPi) macroradicals to form block copolymers that will be involved in particle stabilization.

The introduction of NVP units in the PHEMA network also impacts the properties of the particles (Figure 6). DMA measurements demonstrate that the Tg of the network containing NVP is shifted from 101 °C to 113 °C as would be expected from the higher Tg of PNVP.

All of these data clearly show that we have developed a robust and fluorine-free dispersion process in scCO2 that allows production of well-defined hydrophilic networks of PHEMA and P(HEMA-co-NVP) with adjustable Tg in both cases and variable cross-link density; both of which could be used to potentially modulate their swelling in water.

Hydrolysis of the stabilizer towards hydrogel microparticles

Finally, the dispersion process results in particles that have stabiliser located at the surface; in this case P(VAc-stat-VPi), which is hydrophobic and does not allow the cross-linked particles to be dispersed in aqueous systems. To make these materials dispersible in water, we began to look closely at the possibility of hydrolysing the stabiliser attached to the particle surface. Poly(vinyl esters) (PVAc and PVPi) are used commercially to obtain water soluble systems by conversion through hydrolysis to introduce some level of poly(vinyl alcohol) (PVOH). Therefore, we hypothesized that subjecting the synthesized particles to hydrolysis would convert the surface located P(VAc-stat-VPi) to PVOH which would enable us to redisperse the particles in water. However, the challenge is to check that this can occur successfully on the microparticle surfaces. Two different Please do not adjust margins Please do not adjust margins hydrolysis procedures were initially tested on the stabiliser itself and were analysed by FTIR-ATR (Figure S5). The results clearly demonstrate that hydrolysis occurs since broad -OH peaks have clearly appeared at around 3500 cm -1 . As expected, the carbonyl peak at around 1700 cm -1 decreases, but it does not completely disappear. Most importantly, when applied to our crosslinked particles, this very simple hydrolysis system must provide sufficient hydrolysis of the surface located stabiliser molecules to allow the microparticles to become dispersible in water. This would represent a significant improvement upon the previously published fluorinated systems 9 that are hydrophobic and required a photochemical cleavage process.

Having established the two sets of hydrolysis parameters, we repeated these experiments on PHEMA particles prepared with the selected P(VAc-stat-VPi) stabiliser in presence of 1% or 3% of cross-linker. Hydrolysed particles were isolated from the hydrolysis process by removing the solvent under reduced pressure and subsequent freeze-drying. Particle morphologies were further inspected by SEM and compared to the nontreated particles. SEM images (Figure 8 -left) clearly show that hydrolysis was non-destructive for the PHEMA particle morphology, especially for the 3% cross-linker content. The hydrolysed particles were redispersed in water at a concentration of 5 mg mL -1 and treated for 2 minutes using a horn sonicator at high amplitude and frequency. The solution behaviour (Figure 8 -right) clearly demonstrates hydrolysis enables dispersion in water. Before hydrolysis, most of the particles settle to the bottom of the vial (Figure 8 -Vials 1 and 4) whereas after hydrolysis a much larger portion of the particles are clearly dispersed in water (Figure 8 -Vials 2, 3, 5 and 6) and the dispersion remain stable for a significant period of time (ca. 45 minutes). This shows clearly the increased hydrophilicity of the microparticle surfaces through hydrolysis of the ester groups of the stabiliser. Having established these hydrolysis parameters we repeated the same experiment for PHEMA cross-linked particles that had been prepared in the presence of the PEO-b-PFDA diblock copolymer stabiliser. 9 Here, the hydrolysis procedure had a minimal effect (Figure S6) with just a very slight improvement in the dispersion of the particles in aqueous media, and the resettling of the PEO-b-PFDA stabilised particles was found to be rapid. The fact that there was any improvement at all is somewhat surprising but could perhaps be a result of some modest hydrolysis of PFDA polymer at or near the particle surface. Nevertheless, the hydrolysis procedure very strongly enhances the redispersibility of the particles synthesised in the presence of the P(VAc-stat-VPi) stabiliser.

Swelling of the homo/copolymer networks in water

Since PHEMA is highly hydrophilic, the swelling of the networks after hydrolysis can be gauged by DLS. The size of the particles was measured by DLS directly after suspension in water. DLS measurements were done in triplicates and the values are reported as an average in Table 3. The obtained values were compared to the size measured in the dry state from SEM micrographs. It is clear that all the particles exhibit a significant size increase in water compared to the dry state confirming their capability to swell rapidly. Please do not adjust margins Please do not adjust margins

In the case of lightly cross-linked (1%) PHEMA, a bimodal distribution is observed with two peaks (at around 350 nm and 3.7 µm). This bimodal distribution suggests that not all the particles were evenly cross-linked leading to size differences throughout the sample. By contrast a monomodal distribution was observed for PHEMA particles with higher cross-linker content (3%). In this case, particle size increases from ~ 1 µm in the dry state to ~3 µm in water. As anticipated, the more highly cross-linked particles are more constrained and hence swell less. The P(HEMA-co-NVP) particles showed a rapid swelling from ~2 µm to ~5 µm. This higher swelling capacity is likely due to increased hydrophilicity of the network owing to the presence of lactam groups of NVP units. 32 Thus, DMA and DLS analyses demonstrate that the mechanical and swelling properties of the polymer networks can be altered either by copolymerisation or by varying the cross-linking density. More importantly, the swelling behaviour of the homo/copolymer networks in the aqueous suspensions provides a clean route to PHEMA microgels that is highly versatile and can be adjusted by varying various parameters to obtain particles with tailored properties.

Conclusions

PHEMA particles were successfully obtained in scCO2 using a fluorine-free copolymer composed of VAc and VPi as the stabiliser system. After studying various stabilisers of different molecular weight and composition, the optimised stabiliser was based upon P(VAc-stat-VPi) with a molecular weight of 12.8 kg mol -1 and a molar composition of 56:44. Indeed, this stabiliser is the most efficient for the dispersion copolymerisation of HEMA and EGDMA in scCO2, leading to well-defined particles of ca. 1.2 µm (for 3% EGDMA). The stabiliser was sufficiently robust to be applied to HEMA copolymerisations with various amounts of cross-linker and addition of other monomers, e.g. NVP allowing control of the physico-mechanical properties of the obtained particles. The glass transition temperatures (Tg) of the networks could also be modified by varying the degree of cross-linking or by introducing a comonomer (i.e. NVP). In addition, the P(VAc-stat-VPi) stabiliser located on the surface of the particles could be hydrolysed to PVOH to facilitate the redispersion of the particles in water leading to hydrogels of a few micron-size. This facile and green route to PHEMA based microgels opens a pathway to a wide range of formulations for biomedical applications and could be used to encapsulate solvent and temperature sensitive bioactive ingredients, such as protein/peptide-based drugs.

Figure 1 :

 1 Figure 1: Chemical structure of the P(VAc-stat-VPi) stabilizers employed and protons used to determine the composition and the molecular weight by 1 H NMR. See Figure S1 for complete 1 H NMR assignments.

Figure 2 :

 2 Figure 2: Illustrative pictures of the cloud point measurements for the typical P(VAc-co-VPi) stabiliser. 1: Initial mixture of HEMA (3 g), stabiliser (150 mg) and carbon dioxide (20 g) at initial pressure (around 50 bar) and room temperature; 2:The view cell is heated to the desired temperature (i.e. 35 °C) and pressure is increased until there is complete dissolution of the copolymer in the monomer and carbon dioxide mixture; 3: Keeping the temperature constant the rear sapphire piston is withdrawn to lower the pressure (and density) and the copolymer starts to precipitate out from the mixture forming the cloud and obscuring the LED back-lights.

6 |

 6 J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins

Figure 3 :

 3 Figure 3: Representative SEM images of the solid disc polymer product obtained by dispersion polymerisation in scCO2; (Table 1: Entry 14) i. 100X magnification ii. 1000x magnification; (Table 1: Entry 15) iii. 100X magnification iv. 1000X magnification.

Figure 5 :

 5 Figure 5: Effect of cross-linker concentration on particle morphology. A: 0% EGDMA, B: 1% EGDMA, C: 3% EGDMA added (v/vmonomer). The feed ratios for stabiliser (10% w/vmonomer) and initiator (1% wV-70/vmonomer) were kept constant as well as the reaction conditions (35 °C and 240 bar). Stabiliser characteristics are listed inTable 1 Entry 15.

Figure 6 :

 6 Figure 6: Dynamic mechanical analysis of the PHEMA particles obtained with various cross-linker concentrations as well as copolymerisation of HEMA with NVP using 1% cross-linker.

Figure 7 :

 7 Figure 7: Copolymerisation with 50% v/v NVP with respect to HEMA. Stabiliser: 10% w/vmonomer (VAc:VPi 56/44, 12.8 kg mol -1 , Table 1 -Entry 15); cross-linker EGDMA: 1% v/vmonomer ; initiator V-70: 1% w/vmonomer; 35 °C and 240 bar.

Figure 8 :

 8 Figure 8: Left: SEM images for PHEMA particles obtained with either 1% (Samples 1 to 3) or 3% cross-linker (samples 4 to 6), before and after hydrolysis. Right: pictures of the same particles re-dispersed in water, clearly showing that only the hydrolysed samples (2,3 & 5,6) are dispersed. The others (1,4) are clumped on the bottom of the vial.

  Electronic Supplementary Information (ESI) available: Figures and tables from S1 to S6 are available in the supplementary information See DOI: 10.1039/x0xx00000x

	a. School of Chemistry, University of Nottingham, University Park, Nottingham, NG7
	2RD, UK. E-mail: steve.howdle@nottingham.ac.uk; Fax: +44 (0) 115 846 8459; Tel:
	+44 (0) 115 9513486

b. CERM, CESAM, University of Liege, 11, Allée du Six Août, B-4000 Liege, Belgium c. Univ Lyon, Université Claude Bernard Lyon 1, CPE Lyon, CNRS UMR 5265, Chemistry, Catalysis, Polymers & Processes (C2P2), 43 Bd du 11 Novembre 1918, 69616 Villeurbanne, France.

Table 2

 2 Cloud points of two representative copolymers in HEMA/CO2 (Entries 1 and 2) or NVP/CO2 (Entry 3).

	Entry		Copolymer				Cloud Points [T (°C) vs. P(bar)]
		[VAc: VPi] ( 1 H-NMR) Mn total (kg mol -1 , 1 H-NMR)	Ð (SEC)	Tg (°C, DSC)	35 °C	45 °C	55 °C	65 °C	75 °C
	1	47:53	7.8	1.13	51.5	174	207	237	263	286
	2	57:43	9.3	1.02	48.3	175	217	250	278	305
	3	44:56	9.4	1.50	47.4	154*

*Data was taken from the reference 19.

VAc:VPİ], 1 H-NMR) Mn (stabiliser) (kg mol -1 , theo) Mn (stabiliser) (kg mol -1 , 1 H-NMR) Mn (stabiliser) (kg mol -1 , SEC)

						Ð	Particle Size	Cv
						(SEC)	(µm, SEM) a	(%) b
	1	9:91	5.0	4.6	4.5	1.22	3.7	11.2
	2	12:88	6.0	7.5	7.4	1.42	2.3	8.3
	3	17:83	12.0	9.8	10.6	1.49	4.4	9.6
	4	23:77	11.0	9.8	11.9	1.42	Porous
	5	29:71	9.3	10.6	9.0	1.20	Aggregate
	6	36:64	8.8	7.8	10.0	1.02	1.2*	10.8
	7	36:64	8.0	9.9	11.5	1.08	2.1	11.6
	8	42:58	8.5	10.3	10.0	1.20	Aggregate
	9	44:56	12.8	10.7	12.0	1.08	2.3	10.5
	10	47:53	7.8	6.6	8.2	1.13	Irregular shaped
	11	50:50	14.0	12.0	13.8	1.47	1.9	9.0
	12	49:51	17.0	14.5	21.8	1.50	Aggregate
	13	50:50	25.0	22.0	29.4	1.42	0.9*	12.5
	14	56:44	8.4	8.6	10.0	1.05	2.2	7.5
	15	56:44	9.8	12.8	14.0	1.07	1.3	13.2
	16	75:25	12.0	9.5	13.5	1.38	Porous

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Table 1 :

 1 Entry 14) i. 100X magnification ii. 1000x magnification; (Table 1: Entry 15) iii. 100X magnification iv. 1000X magnification.

Table 1

 1 Entry 15.

Table 3

 3 Particle size for the homo-and copolymer networks determined by SEM and DLS This journal is © The Royal Society of Chemistry 20xx

	Entry Monomer(s) Cross-linker SEM size	DLS size (µm)
			(%v)	(Dry, µm)	Peak 1	Peak 2
	1	HEMA	1	1.3	0.35	3.7
	2	HEMA	3	1.2	3.0
	3	HEMA+NVP	1	1.8	4.8

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Acknowledgements

RP, CJ and SMH thank for the European financial support in the frame of the NanoFar program, an Erasmus Mundus Joint Doctorate (EMJD) program in nanomedicine and pharmaceutical innovation. AC is grateful for the financial support of the SINCHEM program, an Erasmus Mundus Joint Doctorate (EMJD) program in Sustainable and industrial chemistry. CERM is indebted to Interuniversity Attraction Poles (IAP-7/5-FS2). RP, AC and SMH are grateful for invaluable general laboratory support of Mr. Mark Guyler, and highpressure equipment technical and safety support from Mr. Peter Fields and Mr. Richard Wilson at the University of Nottingham.