Costanza Santini
email: c.santini@erasmusmc.nl

Alexandra G Arranja

Antonia G Denkova

François Schosseler

Karolina Morawska

Peter Dubruel

Eduardo Mendes

Marion De Jong

Monique R Bernsen

Intravenous and intratumoral injection of Pluronic P94: The effect of administration route on biodistribution and tumor retention

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Introduction

The need for specific and more efficient cancer treatments is well recognized and many efforts are being made towards the development of safe and more effective therapeutic approaches. Specially, the field of nanomedicine has grown considerably in the last decades for this purpose.

Nanomedicines, represented by compounds generally ranging from 1 to 100 nm, are commonly administered through intravenous injection (IV) in order to ensure a complete distribution over the whole body and to reach the primary tumor and metastases. After entry into the circulation, nanoparticles can then accumulate in the tumor tissue profiting from a passive phenomenon known as the Enhanced Permeability and Retention (EPR) effect (1): The leaky tumor vasculature together with reduced lymphatic drainage allow for passive extravasation and specific tumor accumulation of nano-sized medicines. Because of the passive nature of the EPR effect, nanomedicines do not require specific targeting agents and can accumulate in permeable tumors, such as sarcomas [START_REF] Koukourakis | High intratumoral accumulation of stealth liposomal doxorubicin in sarcomas--rationale for combination with radiotherapy[END_REF], head and neck tumors [START_REF] Harrington | Effective targeting of solid tumors in patients with locally advanced cancers by radiolabeled pegylated liposomes[END_REF], and glioblastomas [START_REF] Koukourakis | High intratumoural accumulation of stealth® liposomal doxorubicin (Caelyx®) in glioblastomas and in metastatic brain tumours[END_REF] The extent of accumulation and retention of these nano compounds in the tumors has shown to vary extensively, as it depends on many parameters such as particle's properties (e.g. size, shape and charge) [START_REF] Duan | Physicochemical characteristics of nanoparticles affect circulation, biodistribution, cellular internalization, and trafficking[END_REF] and tumor microenvironment (e.g. tumor internal pressure, blood flow) [START_REF] Song | Effects of tumor microenvironment heterogeneity on nanoparticle disposition and efficacy in breast cancer tumor models[END_REF][START_REF] Miao | Exploring the tumor microenvironment with nanoparticles[END_REF]. When administered IV, high tumor uptake is generally obtained for those particles that can bypass the organism's clearance mechanisms (mainly represented by the kidneys, liver and spleen) [START_REF] Bertrand | The journey of a drug-carrier in the body: an anatomophysiological perspective[END_REF] presenting long circulation times.

In order to improve tumor uptake and to reduce possible side effects, nanomedicines can be applied intratumorally (IT) by injecting the compounds directly at the tumor site. This method can be applied for those solid tumors that are not suitable for surgery, such as brain, head and neck tumors, or inoperable solid tumors [START_REF] Walter | Intratumoral chemotherapy[END_REF][START_REF] Nemunaitis | Head and neck cancer: response to p53-based therapeutics[END_REF][START_REF] Celikoglu | Intratumoral administration of cisplatin through a bronchoscope followed by irradiation for treatment of inoperable nonsmall cell obstructive lung cancer[END_REF]. The injection in loco naturally improves the amount of tracer in the tumor. Moreover, if the compounds together with the radionuclide are retained in the tumor, the IT route avoids the involvement of the systemic circulation evading the main clearance mechanisms and reducing accumulation in off-target tissues. Therefore, using the IT route for the delivery of nanotherapeutics is beneficial for the safety and efficacy of the treatment of localized solid tumors [START_REF] Ray | A phase I study of intratumoral ipilimumab and interleukin-2 in patients with advanced melanoma[END_REF][START_REF] Liang | H-ferritin-nanocaged doxorubicin nanoparticles specifically target and kill tumors with a single-dose injection[END_REF].

Among nanoparticles, polymers and Pluronic copolymers in particular have raised considerable interest due to their versatility and valuable properties. These polymer chains can be easily modified with different agents for imaging applications, but they can also be combined with drugs becoming especially suitable for theranostic purposes. Moreover, certain compositions of Pluronic copolymers have shown a typical biological activity such as the capacity to sensitize multi-drug resistant cancer cells increasing therefore the cytotoxic potential of chemotherapeutic drugs [START_REF] Kabanov | Pluronic block copolymers as novel polymer therapeutics for drug and gene delivery[END_REF][START_REF] Batrakova | Pluronic block copolymers: evolution of drug delivery concept from inert nanocarriers to biological response modifiers[END_REF]. Pluronic P94 is an amphiphilic linear triblock copolymer of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) and has shown interesting properties in vitro [START_REF] Arranja | Cytotoxicity and internalization of Pluronic micelles stabilized by core cross-linking[END_REF][START_REF] Arranja | Interactions of Pluronic nanocarriers with 2D and 3D cell cultures: Effects of PEO block length and aggregation state[END_REF] as well as in vivo [START_REF] Arranja | SPECT/CT Imaging of Pluronic Nanocarriers with Varying Poly(ethylene oxide) Block Length and Aggregation State[END_REF] for imaging and therapeutic applications. In preclinical settings, Pluronic P94 has shown to be internalized efficiently by tumor cells, with reduced exocytosis when compared with other formulations, such as Pluronic F127. It has also demonstrated to be able to disaggregate tumor spheroids in vitro [START_REF] Arranja | Interactions of Pluronic nanocarriers with 2D and 3D cell cultures: Effects of PEO block length and aggregation state[END_REF], suggesting potentially relevant therapeutic effects. In vivo studies performed with 111 In radiolabeled P94 copolymers in healthy mice have shown high in vivo stability and relatively long circulation time, while acute whole body toxicity was not reported [START_REF] Arranja | SPECT/CT Imaging of Pluronic Nanocarriers with Varying Poly(ethylene oxide) Block Length and Aggregation State[END_REF]. These results suggest that Pluronic P94 copolymer might be suitable for imaging and/or therapeutic applications.

In this study, we applied 111 In-radiolabeled P94 copolymer in tumor-bearing mice via IV and IT injection routes to evaluate for the first time the in vivo potential of Pluronic P94 triblock copolymer regarding tumor accumulation and retention.

Materials and Methods

Functionalization of Pluronic P94 copolymer with DTPA chelator (P94-DTPA)

Pluronic P94 (PEO 26 PPO 48 PEO 26 ; M n PEO blocks: 2200 Da; M n PPO block: 2800 Da) were supplied by BASF and end-functionalized with the chelator DTPA following procedures described previously [START_REF] Arranja | SPECT/CT Imaging of Pluronic Nanocarriers with Varying Poly(ethylene oxide) Block Length and Aggregation State[END_REF][START_REF] Lu | Galactosylated PVDF membrane promotes hepatocyte attachment and functional maintenance[END_REF][START_REF] Hoang | Noninvasive monitoring of the fate of 111Inlabeled block copolymer micelles by high resolution and high sensitivity microSPECT/CT imaging[END_REF].

Briefly, P94 unimers were purified from low molecular weight contaminants [START_REF] Arranja | Lack of a unique kinetic pathway in the growth and decay of Pluronic micelles[END_REF] and functionalized with 1,1-carbodiimidazole (CDI-Pluronic) and primary amines (Pluronic-NH 2 ) [START_REF] Arranja | SPECT/CT Imaging of Pluronic Nanocarriers with Varying Poly(ethylene oxide) Block Length and Aggregation State[END_REF]. The modified chelator p-SCN-Bn-DTPA was then conjugated to the amine-terminated copolymers. The yield of the different chemical intermediates was determined by 1 H-NMR (Bruker WH 300 MHz instrument) and is reported elsewhere [START_REF] Arranja | SPECT/CT Imaging of Pluronic Nanocarriers with Varying Poly(ethylene oxide) Block Length and Aggregation State[END_REF].

Radiolabeling of P94-DTPA with 111 In (P94-DTPA-111 In) and radiolabeling efficiency

The radiolabeling of P94-DTPA with 111 In was performed at neutral pH and at room temperature for 30 minutes. A stock solution of P94-DTPA (8 μL) was added to a solution containing 1800 μL of 1X PBS and 200 μL of 111 InCl 3 (~150 MBq). 111 InCl 3 was obtained from Mallinckrodt Pharmaceuticals (Petten, The Netherlands). The final concentration of P94-DTPA was 1 mg/mL, with a specific activity of 75 MBq/mL. The efficiency of the labeling reaction was analyzed by instant thin layer chromatography (iTLC) in aluminum sheets of Silica gel 60 (Merck Millipore) and EDTA/NH 4 Ac (1:1) buffer (R f P94-DTPA-111 In = 0; R f free 111 In = 1; R f p-SCN-Bn-DTPA-111 In = 0.5-1) as described previously [START_REF] Arranja | SPECT/CT Imaging of Pluronic Nanocarriers with Varying Poly(ethylene oxide) Block Length and Aggregation State[END_REF]. For negative control studies, free DTPA was labeled with 111 In (200 pmol, 3 MBq) ( 111 In-DTPA) following the same procedure already used for labeling the DTPA-P94 polymer. Labeling efficiency was assessed with iTLC chromatography [START_REF] Chen | A simple two-strip method to determine the radiochemical purity of technetium-99m mercaptoacetyltriglycine[END_REF] on aluminum paper (TLC Aluminum Sheets, EMD/Merck KGaA).

Tumor and animal model

Human small cell lung cancer, H69 (ECACC, Salisbury, UK), were cultured under standard conditions in Gibco's RPMI medium (Invitrogen Corp., Breda, The Netherlands) supplemented with 10% v/v fetal bovine serum and 1% v/v penicillin/streptomycin. Cells were suspended in a 1:2 v/v Seligmann's buffered salt solution (SBSS) medium and Matrigel (BD Biosciences, Mississauga, Canada). The suspension was diluted at a dose of 100 µL/mouse (~3×10 6 cell/mouse) and inoculated subcutaneously in 20 young adult male Balb/c-nu mice (Janvier, France). Tumor growth was monitored and measured with a caliper ruler three times per week. All animal experiments were performed in accordance with Dutch animal welfare regulations and approved by the local ethics committee

In vivo studies

When the average tumor volume reached 300 mm 3 , the mice were randomly subdivided in three groups: one for intravenous injection (IV) (N=8) studies, one for intratumoral injection (IT) (N=8) studies and third one for IT negative studies (N=4). Mice from the IV group (N=8) received an injection of ~200 μL (~0.2 mg and ~15 MBq/animal) in the tail vein.Mice from the IT group (N=8) received ~100 μL (~0.1 mg and ~7.5 MBq/animal) in one single injection in the center of the tumor mass. The 4 mice of the IT negative control group were injected IT with 100 μL of 111 In-DTPA (200 pmol and ~3 MBq).

In vivo imaging SPECT/CT and biodistribution

After the injection, mice were anesthetized with a gas mixture of isoflurane (Pharmachemie, Haarlem, The Netherlands) (4% induction, 2% maintenance) and oxygen and subjected to whole body and focus SPECT/CT static scan at different time points. Six mice from the IV group and six mice from the IT group were randomly chosen and further subjected to SPECT/CT scan.

Nuclear images were acquired using the U-SPECT-II system (MiLabs B.V., Utrecht, The Netherlands) equipped with the new ultra-high-sensitivity collimator Mouse 2.0, having 54 conical pinholes of 2.0 mm diameter [START_REF] Ivashchenko | Ultrahigh-sensitivity submillimeter mouse SPECT[END_REF]. The U-SPECT-II system has a heating system to prevent the hypothermia of the animals during the scan. The acquisition time of each scan was approximately 36 minutes for the whole body (40-42 positions, 52 sec/position) and 15 min (14 positions, 66 sec/position) for the focus scan. A CT scan (240 projections, 500 ms exposure time 55 kVp tube voltage) was also performed for anatomical reference. The focus SPECT/CT scan was performed at 0.5 h post injection (pi) in IT injected mice. Whole body SPECT/CT images were acquire at early time points (4 h pi), early-intermediate (24 h pi) and intermediate (48 h pi). Three mice from the IT injection and three mice from IV injection group were randomly chosen and further subjected to later time points of 72 h and 120 h pi respectively. Biodistribution studies were performed at intermediate (48 h pi) and at late time points (72 or 120 h pi) (Fig 2). After the corresponding SPECT/CT scans, mice were sacrificed by cervical dislocation, and blood along with a selection of organs were collected. The radiation arising from the different organs was measured with the aid of a gamma counter (Wallac, 1480 Wizard 3", PerkinElmer, Groningen, The Netherlands). For each tissue, the polymer uptake was calculated as a percentage of the injected dose per gram of tissue (% ID/g).

Images processing and quantification

SPECT/CT images were reconstructed using dedicated software Posem [START_REF] Maeda | Macromolecular therapeutics in cancer treatment: The EPR effect and beyond[END_REF] algorithm [START_REF] Branderhorst | Pixel-based subsets for rapid multipinhole SPECT reconstruction[END_REF].

Based on the acquired images, the uptake and accumulation of P94 radiolabeled copolymers after IV and IT injection was monitored in selected organs (heart, bladder, tumor, liver, lungs, liver) by drawing a region of interest around the specific organ based on CT images. The quantification was performed at 4, 24, 48 and 120 h after IV injection and at 24, 48, and 72 h after IT injection. The intensity of signal per unit of tissue was measured and the uptake calculated as a percentage of the injected activity % ID/mm 3 . The quantification of images was performed using VivoQuant iPACS Software Version 1.23 (INVICRO, Boston, MA, USA).

Software and statistical analyses

All the data collected were processed with Microsoft Excel 2010 and Prism version 5 (GraphPad Software, La Jolla, California, USA). Data from the in vivo evaluation were analyzed using either exponential fitting or linear regression. Ex vivo based biodistribution results were analyzed with Mann-Whitney U test, setting p<0.05 as significantly different. Outliers were determined using the modified Thompson Tau Technique.

Results

Functionalization of P94 triblock copolymers with DTPA chelator and radiolabeling (P94-111 In-

DTPA)

Pluronic P94 triblock copolymers were purified from low molecular weight contaminants resulting in compounds with high purity (98%) [START_REF] Arranja | Lack of a unique kinetic pathway in the growth and decay of Pluronic micelles[END_REF]. These were functionalized with the chelator DTPA and successfully radiolabeled with the radionuclide 111 In using standard conditions, reaching a labeling efficiency of > 91%. Because of the good yield, no further purification of the compound was needed (Fig 1).

SPECT/CT imaging and in vivo quantification

After tumor inoculation and IV or IT injections of radiolabeled Pluronic P94 copolymers (Fig. 2), SPECT/CT scans of the total body distribution of radioactivity were obtained. in parallel, a negative control group injected intratumorally with 111 In-DTPA revealed a very fast clearance of the compound from the tumor tissue at the first time point (0.5 h) (Supplementary information). After IV injection of the radiolabeled copolymers, we observed at the first time point (4 h pi) a substantial accumulation of the copolymers in the bladder, which is associated to the fact that these nano-sized compounds (~4 nm) are below the renal clearance cut-off as observed previously [START_REF] Arranja | SPECT/CT Imaging of Pluronic Nanocarriers with Varying Poly(ethylene oxide) Block Length and Aggregation State[END_REF]. At the same time point, we also observed a sustained accumulation in the liver with minor involvement of the tumor (Fig 3; Table 1). The polymer uptake in the offtarget tissues was relatively high (> 0.1 kBq/mm 3 ) as observed by the elevated accumulation in the heart, lungs and kidney (Table 1). However, we observed an overall decrease of activity in all organs indicating that these compounds are not retained over time and follow a negative exponential trend, including the tumor (Fig 3B). 2). Also, for the other selected organs, the amount of radioactivity measured between 24 and 72 h was low and fairly constant (Fig 4C).

Biodistribution results

The uptake in the different organs following IV injection at 48 and 120 h pi was confirmed by performing ex vivo quantification. As expected for this type of compounds, we observed elevated uptake in the liver, spleen and kidney as these organs are involved in the clearance of polymers. The uptake in off-target tissues was relatively high being in general above 1% ID/g, with only the muscle as an exception (0.05% ID/g). Especially, the liver and spleen presented the highest uptake (Table 3). As observed in the in vivo studies, quantification based on ex vivo data confirmed that Pluronic P94 copolymers uptake in the tumor after IV injection at 48 h and 120 h pi was not higher than that in any other off-target tissue (Table 3

; Fig 5A).
In contrast, the biodistribution after IT injection showed a different pattern. The majority of the off-target organs presented low uptake, in general below 0.5% ID/g (except for skin and muscle), and a substantial tumor retention. Among the clearance organs, after IT injection only the liver presented considerable uptake (Table 3; The retention of radioactivity, which is directly related to the retention of the P94 copolymers, was compared between the two latest time points in all the investigated tissues. The results indicated that, independently from the injection route, in the majority of the off target tissue we observe a significant reduction (p<0.05) of measured radioactivity. That behavior suggests a physiological clearance effect in the different tissues. The only exceptions to that are represented by the clearance organs (liver, spleen and kidney) and the skin that shows no statistical difference (p> 0.05) between 48 and 72/120 h pi.

The retention of radioactivity in the tumor was different from that observed in the other off target tissues. In fact, independently from the injected routes, we observed no changes in the retention in the tumor mass between the last two pi time points.

In parallel, the ex vivo evaluation of the control group confirmed the fast clearance of the 111 In-DTPA, with no tumor retention or accumulation in any of the investigated organs (Supplementary Information).

Discussion

Below a certain critical temperature (CMT) and concentration (CMC), Pluronic copolymers consist of free polymer chains in solution that are highly compact with the terminal PEO blocks surrounding the non-hydrated PPO block [START_REF] Alexandridis | Poly(ethylene oxide) poly(propylene oxide) poly(ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces: thermodynamics, structure, dynamics, and modeling[END_REF]. In the case of the P94 triblock copolymers, in aqueous solution at 15 o C, these aggregated polymer chains present an average diameter of 4 nm ( 19) and a neutral zeta potential. In this work, we evaluated the potential of these nanosized polymer chains to be used for imaging and therapeutic application. The radiolabeled P94-111

In-DPTA triblock copolymers were administered to tumor bearing animals using two different injection routes, IV and IT, followed by SPECT/CT imaging and ex vivo quantification of the total body biodistribution over time. In clinical practice, both systemic and local injections are being used for tumor treatment [START_REF] Kwekkeboom | Peptide Receptor Radionuclide Therapy in the Treatment of Neuroendocrine Tumors[END_REF][START_REF] Cordier | Targeted Radiolabeled Compounds in Glioma Therapy[END_REF]. The choice between the two injection routes is mainly determined by specific needs. Systemic application, such as IV injection, is the route of choice for diagnosis and also for treatment of (potentially) metastasized lesions [START_REF] Kwekkeboom | Peptide Receptor Radionuclide Therapy in the Treatment of Neuroendocrine Tumors[END_REF][START_REF] Cordier | Targeted Radiolabeled Compounds in Glioma Therapy[END_REF]. Local (intratumor) injections on the other hand have shown beneficial outcomes for the treatment of tumors as well as they may result in higher concentrations of drugs in the tumor with limited the effects in off-target organs [START_REF] Cordier | Targeted Radiolabeled Compounds in Glioma Therapy[END_REF]. The biodistribution results obtained after IV versus IT in vivo administration of radiolabeled Pluronic P94 copolymers confirmed a profound difference in distribution profiles and in the potential therapeutic application effect.

After IV injection, radiolabeled P94 copolymers presented limited uptake in the tumor (~1% ID/g), which is probably resulting from the poor EPR-mediated extravasation of these nanosized triblock copolymers (~ 4 nm) and/or low EPR of the type of tumor used in this study. As expected, uptake in the liver, spleen and kidneys was considerable as they represent the main clearance organs for these nano compounds [START_REF] Arranja | SPECT/CT Imaging of Pluronic Nanocarriers with Varying Poly(ethylene oxide) Block Length and Aggregation State[END_REF][START_REF] Grindel | Distribution, metabolism, and excretion of a novel surface-active agent, purified poloxamer 188, in rats, dogs, and humans[END_REF][START_REF] Wang | Disposition in rats of a polyoxypropylene-polyoxyethylene copolymer used in plasma fractionation[END_REF][START_REF] Batrakova | Distribution kinetics of a micelle-forming block copolymer Pluronic P85[END_REF]. The relatively high radioactivity measured in the stomach (~1% ID/g) may result from unspecific uptake, although it can also be partially due to the phenomenon of self-and social grooming in mice [START_REF] Blanchard | Animal models of social stress: effects on behavior and brain neurochemical systems[END_REF]. The uptake in the digestive tract, particularly in caecum, colon and rectum, might be due to liver and bile clearance [START_REF] Bertrand | The journey of a drug-carrier in the body: an anatomophysiological perspective[END_REF][START_REF] Longmire | Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats[END_REF] as also observed previously in healthy mice after IV injection of Pluronic P94 and F127 [START_REF] Arranja | SPECT/CT Imaging of Pluronic Nanocarriers with Varying Poly(ethylene oxide) Block Length and Aggregation State[END_REF], and other Pluronic copolymers [START_REF] Grindel | Distribution, metabolism, and excretion of a novel surface-active agent, purified poloxamer 188, in rats, dogs, and humans[END_REF][START_REF] Wang | Disposition in rats of a polyoxypropylene-polyoxyethylene copolymer used in plasma fractionation[END_REF].

After IT injection, the P94 copolymers remained mostly confined to the tumor and reach the liver only at late time points, while the uptake in the other organs remained low. The radiolabeled P94 copolymers for this approach were administered in one single injection in the center of the tumor mass. At later time points, the re-distribution of the radioactivity to the tumoral rim indicated that there is probably a significant effect of the pressure driving fluids to the periphery of the tumor mass, thus pushing the copolymers to the tumor margins [START_REF] Boucher | Microvascular pressure is the principal driving force for interstitial hypertension in solid tumors: implications for vascular collapse[END_REF]. This mechanism implies movement of liquid within the tumor [START_REF] Munson | Interstitial fluid flow in cancer: implications for disease progression and treatment[END_REF], from the inside to the outside, as a consequence of the differences in hydrostatic pressure. The same mechanism might also be responsible for release of the copolymers in the systemic circulation, and ultimately for the whole body distribution as observed. However, the relatively high and long retention of the radioactivity in the tumor mass suggests that this compound is internalized by the tumor cells,

as it has been observed previously in vitro as well [START_REF] Arranja | Interactions of Pluronic nanocarriers with 2D and 3D cell cultures: Effects of PEO block length and aggregation state[END_REF].

The total body retention of P94 copolymers over time after IV injection declined gradually, indicating constant clearance of the compound. This effect was less pronounced after IT injection. Interestingly, independently from the injection route, the tumor retention between 48 h and the latest time point (being 120 and 72 h for IV and IT injection, respectively) was not statistically different meaning that the compounds were not cleared from the tumor tissue.

This finding might have an important implication in view of tumor treatment potentially leading to higher therapeutic efficacy.

Regarding potential toxicity in the off-target organs, our results indicate that using either IV or IT injections, healthy tissues are inevitably involved in the uptake of P94 copolymers. Resulting potential off-target toxicity might be more relevant in those organs and tissues that have shown reduced clearance, such as the liver, spleen and skin. In the case of the liver, it is known that this organ has a wide-ranging resistance to drugs, as observed with other nanocarriers [START_REF] Yu | Clearance Pathways and Tumor Targeting of Imaging Nanoparticles[END_REF], and thus low liver toxicity is to be expected. In the case of skin and spleen, however, offtarget toxicity might be a dose-limiting factor for long term therapy as observed or suggested in previous investigation with other nanoparticles [START_REF] Bertrand | The journey of a drug-carrier in the body: an anatomophysiological perspective[END_REF][START_REF] Lotem | Skin toxic effects of polyethylene glycol-coated liposomal doxorubicin[END_REF].

The copolymer P94, in contrast with many other carries, can be used as a therapeutic compound or an adjuvant in chemotherapeutic treatments due to its ability to be internalized by the cells, to reach the cytoplasm and nucleus [START_REF] Arranja | Interactions of Pluronic nanocarriers with 2D and 3D cell cultures: Effects of PEO block length and aggregation state[END_REF], and to possess cytotoxic effects at high concentrations [START_REF] Arranja | Cytotoxicity and internalization of Pluronic micelles stabilized by core cross-linking[END_REF]. In this study, we have radiolabeled the P94 copolymers with 111 In and showed its potential for imaging purposes, such as follow-up imaging of treatment progression. Because of the specific behavior of P94 copolymers once internalized by the cells [START_REF] Arranja | Interactions of Pluronic nanocarriers with 2D and 3D cell cultures: Effects of PEO block length and aggregation state[END_REF], this carrier could also be used, for instance, to provide Auger electron therapy by selecting appropriate radionuclides [START_REF] Hoang | Block Copolymer Micelles Target Auger Electron Radiotherapy to the Nucleus of HER2-Positive Breast Cancer Cells[END_REF].

We conclude that the novel Pluronic P94 nanocarrier is very promising as a drug providing toxicity to tumor cells, as an imaging agent to monitor the safety and efficacy of the therapy, and as a carrier for many different radiotherapeutics.

Conclusion

Our results indicate radiolabeled P94 copolymers can be ligand for drugs and can be employed as carrier for drug delivery after IV and IT injection. After IV injection, the relatively long circulation time and significant uptake in healthy organs might hamper their further application and thus the right radionuclide should be carefully selected. In contrast, when administered via IT injection, P94 copolymers present extremely high and long lasting radioactivity in the tumor and minimal uptake in off-target tissues, providing excellent prospective for application as a carrier for radionuclide imaging and/or treatment.

The possibility to easily modify and radiolabel P94 with specific radionuclides suitable for imaging (i.e. 111 In) and therapy suggests the potential use of radiolabeled P94 not only as (chemo)therapeutics agent itself, but also as a molecular/nuclear imaging and theranostic agent. Further studies will be focused on the therapeutic efficacy to determine the effective anticancer potency of this promising compound, and its future applicability in the clinics.

Acknowledgments

This work was supported using imaging equipment provided by the Applied Molecular Imaging

Fig 1 .

 1 Fig 1. Schematic representation of the successive modifications of Pluronic P94 triblock copolymer

Fig 2 .

 2 Fig 2. Temporal diagram of injection and scans.

Fig 3 .

 3 Fig 3. P94-111 In-DPTA biodistribution after intravenous (IV) injection. A) Whole body SPECT/CT images of IV injected animals at different time points post injection. B) Quantitative distribution of P94-111 In-DPTA based on images in selected organs. MIP: maximum intensity projection.

Fig 4 .

 4 Fig 4. P94-111 In-DPTA biodistribution after intratumoral (IT) injection. A) Whole body SPECT/CT images of IT injected animals at different time points post injection. B) SPECT/CT images of tumor region, at different time points post injection. C) Quantitative distribution of P94-111 In-DPTA based on images in selected organs. MIP: maximum intensity projection. Cr: coronal view; Tr: transversal view.

  Fig 5B), while the uptake in the spleen and the kidney was low (< 1 % ID/g) and in line with that of the other off-target tissues. As already indicated by the in vivo studies, we could confirm the elevated retention of Pluronic P94 in the tumor (Fig 5B).

Fig 5 .

 5 Fig 5. Biodistribution results in selected organs at 48 and 72/120 h after (A) intravenous (IV) or (B) intratumoral (IT) injection of P94-111 In-DPTA.

  Erasmus MC facility. The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no. PITN-GA-2012-317019'TRACE 'n TREAT. The authors would like to thank all the members of the consortium under the Marie Curie Actions, ("Trace'n'Treat") for their support, collaboration and fruitful discussions.

Fig 1 .

 1 Fig 1. Result of negative control group. A-D) SPECT/CT images: A) Focus scan in the tumor region, transversal view ; B) Focus scan in the tumor region, maximum intensity projection; C-D) Whole body images, maximum intensity projection; E) Biodistribution data at 48 h pi.

Table 1 .

 1 Accumulation of P94-111 In-DTPA in selected organs at different time points, after intravenous injection (IV). sd: standard deviation.

		4 h		24 h		48 h		120 h
	KBq/mm 3	mean	sd	mean	sd	mean	sd	mean	sd
	Bladder	0,618	0,102	0,060	0,014	0,032	0,003	0,041	0,025
	Heart	0,183	0,015	0,080	0,023	0,035	0,004	0,02	0,005
	Liver	0,360	0,011	0,308	0,031	0,270	0,044	0,190	0,023
	Lungs	0,141	0,008	0,062	0,012	0,041	0,015	0,022	0,004
	Kideny	0,122	0,022	0,069	0,015	0,050	0,012	0,033	0,006
	Tumor	0,053	0,003	0,039	0,010	0,032	0,008	0,018	0,001

Table 2 .

 2 Accumulation of P94-111 In-DTPA in selected organs at different time points after intratumoral (IT) injection. sd: standard deviation; *: not available.

		0,5 h		24 h		48 h		72 h
	KBq/mm 3	mean	sd	mean	sd	mean	sd	mean	sd
	Bladder	*	*	0,035	0,021	0,019	0,007	0,019	0,004
	Heart	*	*	0,045	0,011	0,041	0,017	0,023	0,005
	Liver	*	*	0,126	0,021	0,126	0,027	0,120	0,010
	Lungs	*	*	0,046	0,015	0,049	0,015	0,028	0,004
	Kideny	*	*	0,050	0,016	0,043	0,015	0,049	0,004
	Tumor	*	*	0,662	0,105	0,541	0,049	0,493	0,059

Supplementary

Injection route determines biodistribution and tumor accumulation of Pluronic copolymers

C. Santini 1 , A. Arranja 2,3,4 , A. Denkova 2 , F. Schosseler 3 , K. Morawska 5 , P. Dubruel 5 , E. Mendes 4 , M. de Jong 1 , M. Bernsen 1


Laurence Jennings

Gilles Waton

François Schosseler

Eduardo Mendes

Towards a Rational Morphology Control of Frozen Copolymer Aggregates †

Kinetically frozen copolymer micelles are commonly prepared by confining amphiphilic block copolymers in the evaporating dispersed phase of oil-in-water emulsions. We revisit the mechanisms of this process by examining its successive steps separately: the formation of the solvent/water interface, the emulsification, the solvent evaporation and the formation of the aggregates. We bring into evidence that: i) Spontaneous water in solvent emulsification, i.e., the formation of a double emulsion, is a necessary step for the subsequent assembly of the copolymers into kinetically frozen aggregates with certain morphologies far from equilibrium. ii) Equilibration of the copolymer conformation at the solvent-water interfaces is a relatively slow process that can be outpaced, or even quenched before completion, by fast solvent evaporation rates. iii) Rather than dictated by the packing parameter at equilibrium, the morphology of the aggregates is determined by the effective copolymer conformation at the solvent-water interface when they form. iv) Ultra-long worm-like micelles do not form by a direct digitation of the dispersed oil phase into the water continuous phase but through the inversion of the double emulsion. From these findings, we design a simple setup that allows to control the morphology of the frozen aggregates obtained from a given copolymer composition by simply tuning the solvent evaporation rate.

Introduction

Surfactant micelles are generally defined by their dynamic nature involving a constant exchange of surfactant molecules between each other. The exchange kinetics are largely dominated by the strength of the incompatibility between the solvent and the insoluble surfactant molecule moiety. For surfactant molecules or amphiphilic copolymers with a large enough molecular weight, kinetics can become so slow that micelles cannot form by direct dissolution in a block selective solvent.

Eisenberg et al. demonstrated the concept of kinetically frozen polymer micelles and showed the wide range of kinetically stable morphologies obtained by progressively adding water to solutions of strongly hydrophobic block copolymers in an organic solvent of both blocks. [1][2][3] For high enough water fraction, unimer exchange is prevented and micelles with frozen morphology and fixed aggregation number are obtained. † Electronic Supplementary Information (ESI) available: Microscopy images of the spontaneous emulsification process, SEM imags of the aggregates, LSCM of adsorbed droplets, and videos of aggregates formation. See DOI: 10.1039/b000000x/ A decade later, Zhu et al. introduced the emulsion evaporation method where the common solvent of both blocks is immiscible with water and constitutes the dispersed phase of an oil in water emulsion. By further evaporating the organic solvent, the confined copolymers can be assembled into kinetically frozen micelles of different morphologies and efficiently loaded with hydrophobic compounds or nanoparticles. 4,5 Currently however, there is no clear understanding of the parameters governing the aggregation of the unimers into one or another morphology. Zhu et al. suggested that the local increase in copolymer concentration, due to the evaporation of the dispersed phase, decreases the interfacial tension between the two solvents, which could reach null or even transiently negative values, thus generating the driving force for the formation of out of equilibrium morphologies as a means to increase the total surface of the system. 6 This interpretation is based on a theoretical model by Granek et al. which describes the spontaneous emulsification of oil droplets in a water solution of surfactant molecules. 7 There are two main differences between the model and the experimental conditions. In the model the continuous phase is the reservoir of surfactant molecules; conversely, in the experimental case the dispersed phase contains the copolymers. Moreover, the model describes low molecular weight surfactants and links the characteristic size of the formed micelles to the characteristic length of the grow-ing fluctuations of the interface. The calculation of the latter does not include any specific effects associated with the constraints of chain conformation for high molecular weight block copolymers under confinement. 7 Indeed, the confinement of amphiphilic block copolymers at a solvent-solvent interface affects its local curvature. 8 Parallel to the explanation based on interfacial tension, Su et al. have shown that using solvents with different boiling points has a clear effect on the morphology of the assembled objects. 9 Their results, together with previous reports on the budding of oblate vesicles, 8 suggest that increasing surface coverage results in the formation of micelles by budding, tubing or fingering of the solvent/water interface due to changes in its local curvature rather than changes in its interfacial tension.

One puzzling and common feature of both solvent replacement and emulsion evaporation methods is the often observed coexistence of different frozen aggregates morphologies. 4,10 This diversity decreases considerably the usefulness of these methods since potential applications, e.g., in pharmaceutical formulations of nano-carriers, require often a strict control on size and morphology of the aggregates.

Clearly the formation of kinetically frozen micelles by evaporating emulsion droplets involves both thermodynamic equilibrium parameters and kinetic aspects in obtaining specific morphologies. 11,12 However our understanding of the relative importance of thermodynamics and kinetics is still empirical and will remain so until an attempt to vary them independently. This is the purpose of our present study. We used a system with the minimal number of components, consisting in poly(styrene-b-ethylene oxide) (PS-PEO) copolymers dissolved in a chloroform phase in contact with a pure water phase. After evaporation of the organic phase, the resulting aggregates are dispersed in water and are kinetically stable due to the glassy hydrophobic PS core. We used two PS-PEO copolymers with respective PEO weight fraction of 65% and 30%. These two copolymers, consistently with the packing parameter concept developed for small surfactants, 13 form mainly spherical and elongated micelles, respectively, through the emulsion evaporation method. 4,14 We studied the problem step-by-step by directly observing each key stage: from the initial formation of chloroform/water interfaces to the evaporation of the chloroform emulsion droplets and the formation of copolymer assemblies in water. Each stage was investigated for the two copolymer compositions. We show that we can control the morphology of the aggregates independently of the copolymer's block length ratio by simply tuning the evaporation rate of the organic solvent.

Materials and methods

Materials

Poly(styrene-b-ethylene oxide) block copolymers PS 16k -PEO 7.5k (PDI = 1.09) and PS 9.5k -PEO 18k (PDI = 1.04) were purchased from Polymer Source Inc, Canada. The subscripts indicate the molecular weight of the blocks in Dalton. Anhydrous chloroform ≥ 99%, 0.5-1% ethanol as stabiliser, and the fluores-cent dye 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were purchased from Sigma Aldrich. Quartz cuvettes were purchased from Hellma and made of Suprasil R quartz, with spectral range 200-2500 nm. The imaging chambers, CoverWell R Perfusion Chamber height x diameter: 2.5 mm x 9 mm and a volume of 150 µL, were purchased from Sigma Aldrich. Silicon chips (5x5 mm) for the SEM experiments were obtained from SIL'TRONIX ST.

Planar interface microscopy experiments

We performed these experiments in quartz cuvettes placed in a thermostated holder (25 • C) and used a horizontal backilluminated microscope setup to observe the evolution of the chloroform/water interface with a 5x objective. An Infinity2 camera (Lumenera) captured the images.

In the absence of evaporation

We prepared 100 mg/mL stock solutions of each copolymer in chloroform. The initial dissolution was facilitated using a vortex mixer. The solution was subsequently left overnight to fully dissolve the block copolymer. Using a glass syringe 100 µL of stock solution were placed on the bottom of a quartz cuvette (path length 1 mm, volume 350 µL) and 100 µL of water were immediately but gently added to the chloroform/air interface. The cuvette was immediately sealed with a PTFE cap and put into the thermostated holder.

With controlled evaporation rate

For these experiments the copolymer concentration in the stock chloroform solutions was 10 mg/mL. We filled 1 mL of milliQ water into a 3.5 mL 1x1 cm quartz cuvette and added then 10 µL of copolymer stock solution at the water-air interface using a glass syringe. We sealed the cuvette with a PTFE cap fitted with a syringe needle connected to a peristaltic pump REGLO Digital MS-2/8 (ISMATEC). The tubing used with the peristaltic pump had an inner diameter of 1.52 mm. The cuvette was placed in the thermostated holder.

Observation of emulsion destabilisation

For these experiments we prepared a stock solution of 10 mg/mL PS-PEO in chloroform containing also 0.02 wt% of the fluorescent dye DiI. From this solution, 10 µL were placed in a vial containing 1 mL of MilliQ water. An emulsion was formed using a vortex for 30 seconds. The emulsion was then transferred, using a glass syringe, into a perfusion chamber stuck onto a glass coverslip. The chambers were not sealed allowing solvent evaporation. Instability of the droplets occurred within a few minutes from the preparation of the perfusion chamber and was observed in real time with a Nikon Eclipse TE2000E inverted fluorescence microscope.

Scanning Electron Microscopy

Plasma treated silicon chips were coated with 5 µL of the aqueous solutions containing the aggregates. These were imaged using a SU8000 UHR Cold-Emission FE-SEM Scanning Electron Mi- croscope (Hitachi, Japan). The micellar samples were imaged at 1 kV acceleration voltage without the addition of a conductive coating. The larger aggregates were sputtered with gold using a Bal-Tec SCF 050 Sputter Coater before imaging. A coating of approximately 10 nm was applied using a current of 40 mA and a sputtering time of 30 seconds at a working distance of 50 mm.

Results

Macroscopic planar interface without evaporation

We studied first the emulsification process at a planar macroscopic interface without evaporation. According to the current model of spontaneous emulsification, 4,5,7 this phenomenon should occur at high enough interface coverage by the copolymer. Therefore we chose a rather high initial copolymer concentration (100 mg/mL) to observe it in the absence of evaporation. We built carefully the planar interface in a sealed cuvette as described in section Materials and Methods. The upper row of Fig. 1 shows views of the biphasic systems obtained with the two copolymers 24 hours after the formation of the interface. The second row shows microscopy images of the bottom PS-PEO/chloroform phase.

With this system the emulsion evaporation method yields aqueous suspensions of copolymer aggregates with a glassy PS core and a PEO corona. Based on the current understanding of this method, [4][5][6][7] we would therefore expect to observe spontaneous formation of a chloroform in water emulsion in Fig. 1.

Instead, both copolymers initially formed spontaneously water in chloroform emulsions, with water droplets still clearly visible after 24 hours (Fig. 1, 2nd row). This formation of water droplets in the chloroform phase occurs almost immediately after the two solvents are placed in contact (Fig. S1 and Movie 1 † ).

The spontaneous formation of water in oil emulsions in similar systems is a known phenomenon and various mechanisms for it were reviewed in the literature. 15,16 These include the above mentioned transiently null or negative interfacial tension, or the presence of impurities driving osmotic transport of water to oil containing salt impurities. 17 In the present case, however, the occurrence of interfacial turbulence driven by, e.g., concentration gradients 15 seems a more likely mechanism in view of the strong flow of water droplets through the water/chloroform interface (Movie 1 † ).

Only after several hours, a bluish colloidal suspension appeared in the water phase of the sample containing PS 9.5k -PEO 18k (Fig. 1, top left) and optical microscopy confirmed the presence of chloroform droplets in this phase. After evaporation of a small quantity of the upper transparent water region, scanning electron microscopy confirmed the formation of spherical micelles from the water phase for this sample (Fig. S2 † ). Thus upon formation of the macroscopic interface with PS 9.5k -PEO 18k dissolved in chloroform, two phenomena occur at the interface on very different time scales: a violent mixing creating water droplets in the chloroform phase and a much slower emulsification of chloroform droplets in the water phase. Interfacial tension measurements on evaporating droplets showed that micelles form once the copolymer concentration reaches a certain value, independently of the copolymer's molecular weight or block length ratio. 6 Thus our results suggest that this critical copolymer concentration at the macroscopic interface is reached only after a few hours in the absence of evaporation.

In the case of PS 16k -PEO 7.5k , no chloroform droplets formed in the upper water phase contrary to our expectations based on the current understanding of micelle formation by the emulsion evaporation method. [4][5][6][7] These observations indicate a fundamental difference between the formation pathway of spherical or elongated micelles. The latter cannot form through interfacial destabilisation induced by null or transiently negative interfacial tension due to copolymer accumulation at the interface, as proposed in earlier reports. [4][5][6][7]

Micelle formation by emulsion evaporation

In light of these findings, we examine now the more complex conditions of evaporating emulsion droplets. Using a vortex, we prepared chloroform in water emulsions with droplets having diameters between 5 and 20 µm. A small amount of hydrophobic DiI dye added to the chloroform allowed the observation of droplet destabilisation by fluorescence microscopy. An overview of the results is shown in Fig. 2 and videos for each situation are available (Movies 2-5 † ). In the case of spherical micelles, fluorescence microscopy showed only shrinking droplets and the presence of micelles with spherical morphology was confirmed by SEM on the dried sample (Fig. 2, top left). For all the other cases, Fig. 2 shows snapshots extracted from movies shot during the formation of the aggregates. The floating emulsion droplets formed micelle morphologies in agreement with those reported in the literature. 4,14 However, a fraction of the droplets, which were adsorbed on the glass slide of the imaging chamber, formed objects of lower curvature: elongated micelles for PS 9.5k -PEO 18k and vesicles, implying bilayer formation, for PS 16k -PEO 7.5k .

Scanning electron microscopy of the aggregates formed from adsorbed droplets containing PS 16k -PEO 7.5k , confirmed the presence of budding vesicles and strings of vesicles (Figs. S3 and S4 † ). Zhu et al reported the formation of similar morphologies from chloroform droplets containing PS-PEO and SDS. 18 However, from purely topological considerations, the formation of these structures is not fully compatible with the spontaneous emulsification theory. 7 While a fingering instability of the polymer monolayer at the emulsion interfaces can yield spherical or elongated micelles, interconnected bilayers (multi-vesicles and strings of vesicles) cannot form without a series of folding deformations (Fig. 3). These are especially unlikely to occur if their driving forces are low interfacial energies or spontaneous curvatures.

On the other hand, the presence of smaller water droplets in the chloroform droplets, i.e., a double emulsion, would naturally yield these topologies (Fig. 3, bottom). The formation of such a double emulsion after the initial chloroform in water emulsification could happen either through violent interfacial mixing as described above for the planar interface or through osmotic transport. 17 Investigating the fine structure of emulsion droplets was not possible in the case of copolymer PS 9.5k -PEO 18k . However for PS 16k -PEO 7.5k , we could remove abruptly the more volatile organic solvent by vacuum drying. This procedure allowed us to recover intermediate structures of the chloroform droplets frozen before their destabilisation. The frozen droplet structure looks like a "ball of yarn" of interconnected domains prior to the formation of elongated micelles (Fig. 4). Therefore, elongated structures form already within the chloroform droplets before these become visibly unstable. These frozen structures are reminiscent of a complex arrangement of entangled chloroform and water domains in the evaporating chloroform droplets.

The scheme in Fig. 5 summarises the effects of our experimental conditions on the aggregates morphology. The left column shows the case of the planar interface without any solvent evaporation. The two right columns present the results for floating or adsorbed droplets with evaporation. In each row, for a given copolymer composition, the aggregates morphology depends on the evaporation conditions and on the adsorption state of the droplets, i.e., physical parameters override copolymer composition in determining the aggregates morphology. To our knowledge, this is the first report of such effects. This finding could explain the polymorphism commonly observed in samples formed through the emulsion evaporation method. Fig. S5 † and Movies 6-7 † show typical 3D reconstructions of confocal slices imaged close to the bottom of the imaging chamber, before and after the micelle formation. The polymorphism observed in a given sample will likely depend on the effective surface/volume ratio in the preparation vessel.

Controlling aggregate morphology through the evaporation rate

To distinguish the effects of the adsorption state of the droplets from those of evaporation, we designed a new experiment: depositing a single droplet of chloroform at the air/water interface we controlled the solvent evaporation using a peristaltic pump (Fig. 6, top). With this setup we eliminate any adhesion and interaction of the solvent with the glass vial.

Chloroform has a higher density than water (1.489 g/mL at 25 • C), but, at 25 • C and without the addition of any surfactants, the surface tension of chloroform with air is 26.7 mN/m which is smaller than that of water with air: 72 mN/m. 19,[START_REF] Adamson | Physical Chemistry of Surfaces[END_REF] This makes it possible to deposit and balance chloroform droplets onto a water surface, provided that their mass is small enough to be balanced by the energy gained from the formation of the chloroform/air interface. [START_REF] Phan | [END_REF] All the above continues to be possible when using a chloroform solution containing PS-PEO copolymers.

We used three increasing flow rates from the pump. Since the nominal flow values for the pump are only applicable to liquids, we denote the flow rates used herein as x1, x2 and x6 (Fig. 7) indicating their relative increase. Indeed, under these conditions the 10 µL chloroform droplet was evaporated in approximately 40, 20 or 7 minutes, respectively.

As soon as the chloroform is deposited onto the water, a water in chloroform emulsion starts forming. During the evaporation of the organic solvent we observe a continuous change in the surface area of the chloroform droplet. Both the reduction of the solvent's mass and the decrease of its surface tension with water provide a driving force for a spreading of the chloroform droplet.

When using the x1 flow rate, the chloroform droplet shrinks and spreads on the water surface for the first 40 minutes. After this stage spherical micelles are present in the water phase when using the PS 9.5k -PEO 18k . With the more hydrophobic PS 16k -PEO 7.5k that forms elongated micelles, we observe an abrupt transition from a thin chloroform layer at the water surface into a series of filaments growing towards the bottom of the vial. Fig. 6 shows two snapshots taken before (bottom left) and after (bottom right) the formation of the elongated micelles.

After the total evaporation of the organic solvent we performed SEM to image the objects formed in the water phase (Fig. 7). At the lowest solvent evaporation rate, each copolymer formed the same morphologies as reported in the literature 4,14 and as expected from their packing parameter: 13 spherical for the more hydrophilic copolymer and elongated for the more hydrophobic one (top row). This suggests that the lowest evaporation rate provides enough time for the copolymers to rearrange into a conformation consistent with their packing parameter. Increasing the evaporation rate we observe a transition from spherical micelles to elongated ones for the PS 9.5k -PEO 18k and a transition from elongated micelles to vesicles for the PS 16k -PEO 7.5k (middle row). Finally, at the highest evaporation rates we obtain vesicles for both the copolymers used (bottom row). The characteristic diameters of the aggregates were estimated from SEM images and are listed in Table 1. The trend of a decreasing object curvature with increasing evaporation rate is consistent with the results obtained by Su et al. 9 In the latter study however, the evaporation rate was varied by choosing solvents with different boiling temperature. Here our conclusion is stronger since the solvent quality remains the same. The observed effects hint to the importance of the relative rates of copolymer diffusion to the interface, conformational rearrangement at the interface, and solvent evaporation.

At the higher evaporation rates, yielding vesicles in the water phase, part of the initial chloroform droplet solidified into one single dome-like aggregate. This is easily understood: chloroform acts as a plasticiser for the PS blocks allowing the copolymers to rearrange their conformation at the solvent/water interface and to form vesicles. However, for low enough chloroform content, mobility is finally prevented and a frozen single aggre- gate remains in the later stage.

Further inspection of these aggregates by SEM, Fig. 8, showed that they have a rough surface reminiscent of the budding vesicles shown by Zhu et al. 22 Comparing with Fig. 4, there is a clear correlation between the structure of the dried droplets and the morphology of the formed aggregates in each case. These structures are only possible because of the initial presence of a water in chloroform emulsion which remains confined in the fast evaporating chloroform droplet.

Discussion

Copolymer conformation at water/chloroform interface

We can explain the surprising formation of water in chloroform emulsions, irrespectively of the copolymer composition, by considering the peculiar solubility of PEO in water. PEO is generally assumed to be a hydrophilic polymer. This is indeed true only for low molecular weights (<2000 g/mol) when the OH termination provides a strong contribution to water solubility. 23 For higher molecular weights however, methanol, DMSO, chloroform and THF are increasingly better solvents for PEO than water. 24 The better solubility of PEO in organic solvents is due to the presence of the CH 2 groups, which contribute significantly to the polymer's hydrophobicity. However the backbone oxygens can also bind through hydrogen bonds to water molecules with minimal disturbance of the tetrahedral arrangement of bulk water. 25 This combination causes PEO to be a weakly amphiphilic polymer on its own. [START_REF] Israelachvili | Proc. Nat. Acad. Sci[END_REF] An organic solvent capable of donating H-bond, like chloroform, can accommodate both the CH 2 and O moieties in the PEO backbone. Therefore, pure chloroform is a better solvent for PEO than pure water. 23 Nevertheless, the intrinsic strength of the H-bond with chloroform is much weaker than the one with water. [START_REF] March | Advanced organic chemistry: reactions, mechanisms, and structure[END_REF] Therefore the better location for the PEO block should be along any interface between chloroform and water where both the CH 2 groups and the O atoms have their preferred environ-

ment.

As far as we know, no report investigated the conformation of PS-PEO copolymers at the chloroform/water interface. Earlier studies of these copolymers at a liquid-liquid interface focused on the toluene/water, [START_REF] Prokop | [END_REF]29 the heptane/water, 30 or the cyclohexane/water 31 interfaces. None of these interfaces can mimic our experimental conditions where the organic phase is a good solvent for both blocks.

The best approximations we could find are studies of poly(styrene oxide)-PEO copolymers at water/chloroform interface, 32 and of PEO homopolymers at water/dichloromethane interface. 33,34 In both cases, results hint to a spreading of the PEO chains at the interface. Upon compression of the interfacial area, the interfacial films made of PEO show fast reorganisation and low energetic dissipation, consistent with the expulsion of a limited number of chain segments in the water phase, likely in the vicinity of the hydroxy end-groups. 33,34 For the copolymers the surface activity is decreasing (lower surface coverage) with increasing hydrophobicity: rather large bulk concentrations are necessary to obtain a dense surface coverage and lower significantly the interfacial tension. 32 Although the molecular weight and composition of our samples do not match those of these studies, the general trends should remain the same.

Therefore the conformation of the copolymers at the interface could be likened to a large floating PEO raft with a dangling PS tail extended in the chloroform phase, Fig. 9 left. In this conformation, the PEO favours a flat interface while the PS block promotes a convex curvature towards the chloroform phase. It becomes then clear that at low surface coverage the local curvature induced by the copolymers sitting at the interface promotes the formation of a water in oil emulsion as opposed to the higher surface coverage brush that favours the formation of a chloroform in water emulsion, Fig. 9 right.

The behaviour of copolymers at interfaces is often rationalised by introducing the spreading parameter concept. This is the difference between the work of adhesion and the work of cohesion of a substance at an interface and provides an idea of its wetting properties on a given surface. 35 This parameter depends on the block copolymer block length ratio and has been linked to the morphology of micelles formed at the water-air interface. 36 Different surface conformations of the copolymers will affect the spreading parameter by changing the contribution of the blocks to the adhesion and the cohesion at the interface. Therefore, when the interfacial coverage increases, e.g., by evaporation" and drives a conformational transition at the interface, the spreading parameter depends on the instantaneous adsorption state of the copolymers at the interface.

Clearly, the spreading parameter becomes a dynamic concept in the case of the non-equilibrium phenomena considered here. Its instantaneous value depends on the respective rates of surface coverage variation and interfacial conformation equilibration.

Formation of the aggregates

In this section, we discuss how the concept of a dynamical spreading parameter can explain the results in the different experimental conditions probed in this work.

Planar interface without evaporation.

Several mechanisms contribute to the dynamics of interfacial concentration in the absence of evaporation (Fig. 10), the most obvious one being the ordinary diffusion of bulk copolymers to the interface.

Water making contact with the chloroform surface results in a chemical potential shock. Since the initial copolymer surface concentration is at an equilibrium corresponding to the surface tension of chloroform (≈ 26 mN/m) and the interfacial tension for chloroform/water is much higher (≈ 32 mN/m), turbulent convection appears as an initial equilibration mechanism of the interface that is more efficient than diffusion. Clearly the experimental evidence (Movie 1 † ) shows that this convection brings water droplets into the chloroform phase, which we attribute to a spontaneous interfacial curvature convex towards the chloroform phase as discussed above.

The creaming of water droplets brings copolymer chains back to the macroscopic flat interface. On the other hand, spontaneous water in chloroform emulsification depletes the copolymer concentration at this interface and slows down the increase of interface coverage. Once the instantaneous spreading parameter has reached a critical value, spontaneous chloroform in water emulsification can start if the copolymer composition is consistent with a convex curvature towards the water phase. This explains the delay between the formation of the interface and the formation of chloroform droplets in the water phase. For the more hydrophobic copolymer however, this never happens: the local interfacial curvature never reaches values allowing the digitation of chloroform into the water phase.

Emulsion evaporation.

The scheme in Fig. 11 describes the evaporation driven evolution of a solvent droplet (pink) in water (blue). For clarity, the droplet states are drawn the same size and the amount of copolymer (dark red) increases to mimic the concentration increase with evaporation (from top left to bottom right). We depict a situation where the copolymer composition is consistent with the formation of elongated micelles and the evaporation rate is slow compared to the conformation equilibration rate.

Initially, a double w/o/w emulsion is formed (Fig. 11, 1st row) following the mechanisms described in Fig. 10. For the more hydrophobic copolymers considered here, the curvature at low surface density is higher and favours the emulsification of smaller water droplets in the chloroform droplets, hence their cream- ing should be slower. As water droplets form and chloroform evaporates, both the water fraction and the copolymer concentration increase in the chloroform droplets until the phase inversion composition is reached. 37 At that time, in slow evaporation conditions, the copolymers have adopted a dense brush conformation at the interfaces and the obtained morphology corresponds to their composition, i.e., to elongated micelles in the case depicted here. These newly formed elongated micelles are swollen with chloroform for the initial stage. However, the removal of this solvent will cause the PS blocks to rearrange within the micelle core. This has the tendency to stretch out and to stiffen the glassy PS-PEO micelles, which gives the impression that the micelles are being ejected from the evaporating chloroform droplet (Movies 2,5 † ).

If the solvent is removed abruptly, the full inversion is prevented and the final morphology of the droplet is a ball of yarn of entangled elongated structures as shown in Fig. 4.

In the case of more hydrophilic copolymers, the solvent evaporation results in the same spherical morphology as obtained in the absence of evaporation as long as it is slow enough. This is simply because the direct formation of spherical micelles in the water phase is still possible. However the final diameter of the micelles can depend on the evaporation rate in this regime of slow evaporation. 38 Planar interface with controlled evaporation rate.

Considering a suspended chloroform droplet deposited at the air/water interface, there is first the formation of a water emulsion inside, as discussed above for the other experimental configurations.

Subsequent solvent evaporation increases the copolymer concentration at every water-chloroform interface inside the chloroform droplet. We hypothesise that the morphology of the aggregates is determined by the adsorption state of the copolymers at the onset of surface instability, once sufficient organic solvent has evaporated.

When the solvent evaporation is slow enough, the adsorption state of the copolymers assumes a dense, close to equilibrium, brush configuration. Therefore the formed micelles represent more closely the usual packing parameter of the copolymers estimated from their composition. At higher evaporation rates, this is no longer the case, the copolymers have a different spreading parameter and aggregates form with morphologies far away from the ones expected from their composition.

Thus, at intermediate evaporation rates, the hydrophilic PS 9.5k -PEO 18k has not enough time to equilibrate a dense brush conformation associated with a high spontaneous interfacial curvature. Therefore it forms elongated micelles similar to the equilibrium ones obtained with the hydrophobic PS 16k -PEO 7.5k at low evaporation rates. Finally, when the evaporation rate is sufficiently fast, surface instability is met even before the spreading parameter corresponding to elongated micelles is reached. This corresponds to a situation in which the PEO blocks are largely parallel to the chloroform/water interfaces and thin bilayer vesicles are formed by the outwards pressure of the water droplets being confined in a copolymer rich evaporating solvent. Fig. 12 summarises the formation mechanisms for the hydrophilic PS 9.5k -PEO 18k covering all the morphologies observed. It is worth emphasising that, except for slow enough evaporation rates, the aggregates form through a phase inversion induced by solvent evaporation. For the more hydrophobic PS 16k -PEO 7.5k , aggregates cannot form without the prior w/o emulsion formation and its phase inversion.

Further comments

Returning to the case of emulsion evaporation, in light of the proposed mechanisms, we can also explain the different aggregates morphologies obtained with floating and adsorbed droplets. The latter, having a higher surface to volume ratio than the floating ones (Fig. S5 † ), evaporate faster and do not produce morphologies consistent with the copolymer composition.

Micelles and vesicles obtained in our experiments display very similar characteristic sizes (Table 1). This is indeed possible only because the copolymer conformations in both types of objects are very different, strongly non-equilibrium ones in the case of vesicles. This makes it possible to obtain thin bilayers that seem at first sight inconsistent with the macromolecular dimensions deduced from the micellar sizes. However the size similarity between the micelles and the vesicles could still be simply coincidental.

Finally, we would like to point out that our last experimental geometry, the chloroform droplet deposited on water, creates the conditions for viscous fingering phenomena, which are observed when a less viscous liquid is pushed against a more viscous one. 39 This phenomenon can also occur when the pressure is driven by gravity, e.g., when a less viscous higher density liquid layer is spread on a more viscous lower density layer. 40 This is precisely the case when we place a chloroform droplet on top of water. In such a geometry, we can expect a wide variety of interesting phenomena, depending on the interfacial tension be-tween the two liquids. 41 Thus viscous fingering could help the formation of elongated micelles in some conditions but does not invalidate the mechanisms proposed above. The investigation of the links between our results and viscous fingering would be very interesting but lies beyond the scope of our paper.

Conclusions

We studied the assembly of amphiphilic copolymers into kinetically frozen aggregates through the emulsion evaporation method. In addition to the common emulsion conditions, we made also experiments with a planar macroscopic interface, either without solvent evaporation or with controlled solvent evaporation rate.

From the observations of the interfacial behaviour by optical microscopy, we obtained three original insights about the mechanism of aggregate formation by the solvent evaporation method: i) spontaneous water in chloroform emulsification is a critical step for the subsequent formation of aggregates through emulsion inversion induced by solvent evaporation, ii) equilibration of the copolymer conformation at the interface is a relatively slow process that can be outpaced or even quenched before completion by fast evaporation rates, iii) morphology of the aggregates is determined by the effective copolymer conformation when emulsion inversion occurs.

We showed that, by controlling the solvent evaporation rate, we can form aggregates with morphologies different from the ones expected on the basis of copolymer composition.

Until now, the formation of pure block copolymer elongated micelles was restricted only to a very specific block length ratio of the copolymers. Our technique opens up new possibilities in the formulation of elongated micelles of very specific core/corona block length ratio. It provides the first steps towards the rational development of new approaches to control the morphology of micelles independently of their formulation.

Potential applications of these results can be foreseen in the field of nanomedecine where decoupling the respective sizes of hydrophobic core and hydrophilic corona can yield useful freedom in designing nano-carriers optimised for systemic circulation and cell penetration.

Furthermore, the previously unsuspected spontaneous emulsification step unveiled by our experiments opens new interesting perspectives for drug loading strategies.

a

  Université de Strasbourg, CNRS UPR 22, Institut Charles Sadron, 23 rue du Loess, 67034 Strasbourg cedex 2, France. Fax: 33 (0)3 88 41 40 99; Tel: 33 (0)3 88 41 40 39; E-mail: laurence.jennings@ics-cnrs.unistra.fr, francois.schosseler@icscnrs.unistra.fr b Advanced Soft Matter Group, Department of Chemical Engineering, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

Fig. 1

 1 Fig. 1 Top row: close view of the interfacial region in the two cuvettes containing a PS-PEO in chloroform solution (bottom phase) in contact with water (upper phase). Bottom row: optical microcopy images of the PS-PEO/chloroform phase. All images were taken 24 hours after the formation of the interface.

Fig. 2

 2 Fig. 2 Micelle morphologies formed during the evaporation of floating (left) or adsorbed (right) droplets of chloroform containing PS 9.5k -PEO 18k (top) or PS 16k -PEO 7.5k (bottom): spherical micelles (top left, SEM), elongated micelles (top right and bottom left), vesicles (bottom right).

Fig. 3

 3 Fig. 3 Schematic formation of elongated micelles and strings of vesicles (right) from a single or multiple emulsion droplet (left). Stretching the interface of a single emulsion droplet (top) can yield a cylindrical structure without tearing or rupturing the interface. Similarly, stretching a multiple emulsion droplet (bottom) can yield a string of vesicles. Other transformations (red arrows) would require tearing the interface.

Fig. 4

 4 Fig.4SEM image of a dry emulsion droplet of chloroform containing PS 16k -PEO 7.5k : an entangled structure suggests a complex arrangement of chloroform and water domains in the evaporating chloroform droplets.

Fig. 5

 5 Fig. 5 Schematic overview of the morphologies obtained with planar interfaces, floating and adsorbed emulsion droplets.

Fig. 6 (

 6 Fig. 6 (Top) Scheme of the experimental setup: a sealed cuvette, filled with water and a droplet of chloroform/copolymer solution deposited at the water air interface, is connected to a peristaltic pump extracting vapour. (Bottom) Photographs of a cuvette before (left) and during (right) the formation of the micelles. The fluorescent dye DiI was added to the PS 16k -PEO 7.5k /chloroform solution to enhance its visibility.

Fig. 7

 7 Fig. 7 Different morphologies obtained by varying the evaporation rate and visualised using Scanning Electron Microscopy. On the left PS 9.5k -PEO 18k and on the right PS 16k -PEO 7.5k . Ultimately both copolymers lead to similar vesicular morphologies. a) spherical micelles; b) elongated and spherical micelles; d) elongated micelles; c), e) and f) vesicles. Scale bar 500 nm.

Fig. 8

 8 Fig. 8 SEM image of a budding vesicle aggregate formed at high evaporation rate. Top: Overview of the aggregate formed as a pending droplet at the air/water interface (insert); Bottom: Detail of the surface populated with spherical aggregates.

Fig. 9

 9 Fig. 9 Schematic representation of the copolymer conformation at the water/chloroform interface under low (left) and high (right) surface concentration.

  J o u r a l N a me , [ y e a r ] , [ v o l . ] , 1-10 | 7

Fig. 10

 10 Fig. 10 Competing equilibration mechanisms at the planar interface in the absence of evaporation. From left to right: Copolymer diffusion from bulk to the interface; Water droplet formation in the chloroform phase, by either convection or spontaneous emulsification; Creaming of water droplets to the interface; Formation of chloroform in water droplets at high surface coverage.

Fig. 11

 11 Fig. 11 Formation of elongated micelles from an evaporating solvent droplet (light red) in water (blue). Copolymers at interfaces pictured in dark red. From top left to bottom right the bulk copolymer concentration is increasing by evaporation. For clarity, the droplet states are drawn with the same size. See text for details.

Fig. 12

 12 Fig. 12 Overview of the different formation mechanisms of the PS 9.5k -PEO 18k micelles with increasing evaporation rate from left to right. Each column represents the evolution of copolymer in a chloroform droplet suspended on top of a water volume. The bottom row shows the final aggregates morphology: spherical (1) or elongated (2) micelles, vesicles (3), obtained with the increasing evaporation rates. See text for details.

Table 1

 1 Morphologies and average diameters (nm) obtained as a function of the pump flow rate. The letters indicate the morphology of the aggregates: S for spherical, E for elongated and V for vesicles. When two morphologies are present the more frequent is listed as first.

	Polymer	x1	x2	x6
	PS 9.5k -PEO 18k PS 16k -PEO 7.5k	S: 45 ± 5 E: 55 ± 5	E/S: 33 ± 3 / 42 ± 4 V: 56 ± 16	V: 58 ± 18 V: 72 ± 18

[1][2][3][4][5][6][7][8][9][10]

J o u r n a l N a me , [ y e a r ] , [ v o l . ] ,

1-10 J o u r n a l N a me , [ y e a r ] , [ v o l . ] ,

Acknowledgments

We thank Dr. Marc Schmutz, from the electronic microscopy facility in Institut Charles Sadron, for his help with the SEM imaging. We thank also the reviewers whose comments helped to improve the original manuscript. The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no. PITN-GA-2012-317019 "TRACE and TREAT".

Supporting Information

Towards a Rational Morphology Control of Frozen Copolymer Aggregates

Laurence Jennings, Gilles Waton, François Schosseler and Eduardo Mendes Table of Contents: 1. Microscopy images of the spontaneous emulsification process 2. Scanning Electron Microscopy images of the aggregates 3. Laser scanning confocal microscopy of adsorbed droplets

List of videos

Videos can be downloaded form the publisher's website.

Microscopy images of the spontaneous emulsification process

A video of the spontaneous emulsification in presence of PS 16k -PEO 7.5k can be downloaded from the publisher's website. Here is shown a montage containing four frames of the video taken 5 seconds apart from each other.

Figure S1. Montage from the video of spontaneous emulsification. From top left to bottom right each frame is taken 5 seconds after the previous one. In the initial frame we can see the chloroform phase containing PS 16k -PEO 7.5k and the water phase being deposited from the top. In the second frame, 5 seconds after the formation of the chloroform-water interface the spontaneous formation of droplets of water in chloroform is clearly visible.

Scanning Electron Microscopy Images of the Aggregates

Laser Scanning Confocal Microscopy


Chaima Bensaci
email: ch.bensaci@univ-skikda.dz

Youcef Zennir
email: y.zennir@univ-skikda.dz

Denis Pomorski
email: denis.pomorski@univ-lille1.fr

Complex Safety Study of intelligent multi-robot navigation in risk's environment

Keywords: FMEA, Multi-mobile robots system, Navigation control/command, Risks analysis, Safety level, Simulator

The issue investigated in this paper concerns navigation, survey / control and the complexity associated with a mobile multi-robot coordination and cooperation in a complex environment (robotic analysis laboratory), which is little or no known with significant industrial risks, in the presence of human and machines. This group of mobile robots is mainly used to move chemicals products, which can lead dangerous accidents (toxic, flammable, explosive ...) between the different rooms of the laboratory. The objective of our study is to ensure a good precision in the robots navigation in order to optimize human efforts, reduced error and establishment safety while keeping an eye on robots with good functioning and a desired production. In the literature there are several risk analysis techniques. Among the most used techniques in robotics, the FMEA method (failure modes, effects and criticality analysis). We applied the method FMEA on one robot. Then, the method FTA was chosen to generalize dependability study on all robots. Finally, To manage this level of complexity, a control architecture based on controllers decomposition into a set of elementary behaviors / controllers (obstacles avoidance and collision between robots, attraction to a target, planning ...) was proposed.

I. INTRODUCTION

The human being ambition to make his life easier, and his inability to carry out different missions because of their difficulties or their influence on his health, urge them to discover and develop solutions to achieve this objective, mobile robotics is one of these solutions. With the technological development, mobile robotics invaded a variety of fields. Among these areas of possible application especially that requires research include robotics dedicated to hazardous environments where safety is a major step.

The test platform of our work is a robotic chemical analysis laboratory equipped with analysis machines; various risk products where the human being is present. Navigating safely these multi-robots under these conditions presents a great challenge. For a robots group capable of cooperate and navigate accurately and safely without accidents, the focus at first was on risk scenarios that could be considered, then a possible risk analysis was made in order to achieve better multi-robots surveying / (controlling) commanding .

II. DESCRIPTION OF RISKS SCENARIOS

A robotic chemical analysis laboratory is the dynamic simulation platform used in this work. A miniature model of the laboratory consists of two rooms was developed under V-Rep simulator. The laboratory contains robots and human beings collaborating together to perform complex risk tasks, these robots transport chemical products that are harmful to human health and to the environment (toxic, flammable, explosive) within the room as shown in the follows figure. During robots collaboration to perform the task requested, accidents are likely to generate between robots also between a failing robot and a human being stemming from the problems of communication, interaction and organization. The following figures present possible accident scenarios. Potential accidents can be broken down as follows:

1. Collision of empty robots with high speed;

2. Collision between a robot loaded with chemical products and empty robots and between robot and human being;

3. Collision of robots loaded with chemical products;

4. A robot fails when it is moving dangerous products, its main task is the transport of these products in a precise time.

The table below classifies the various possible accidents from the severity of its consequences.

III. DESCRIPTION OF THE ROBOT USED TURTLEBOT 2

TurtleBot 2 is a robot compatible with ROS. It consists of a Kobuki robot, a Kinect, a Laptop and trays for the installation of these two components. The TurtleBot 2 comes from the Canadian company ClearPath but the Kobuki is manufactured by Yujin in Korea and the Kinect by Microsoft in the United States. The Kobuki base is inspired by robot vacuums; it is a robot with two differential wheels with proximity sensors, encoders on the wheels and a gyrometer for each axis [START_REF] Wehbi | Architecture logicielle et matérielle pour fusion tolérantes au fautes pour systèmes multi-robots sous ROS[END_REF].

A. The computer of the robot

The computer attached to the TurtleBot is an ASUS eeePC which is a lightweight netbook. The main task of the robot computer is to receive data and send it to a desktop computer or save it to a hard drive. The robot computer also interacts with the TurtleBot hardware. Because of the computing power in the robot computer, some calculations can also advantageously be performed on this computer, instead of letting the computer desktop do all the work [START_REF] Wehbi | Architecture logicielle et matérielle pour fusion tolérantes au fautes pour systèmes multi-robots sous ROS[END_REF].

B. The Kinect

Originally, The Kinect is a device for the Xbox 360 console to control video games without using a joystick. It was designed by Microsoft in September 2008 [START_REF] Bertrand | [END_REF].

1) The components of a Kinect: The following figure shows the various components of the kinect.

a) An infrared emitter (IR) and an IR depth sensor:

The transmitter emits beams of infrared light and the depth sensor reads the reflected infrared beams towards the sensor. The reflected beams are converted into depth information measuring the distance between an object and the sensor. This makes capturing a depth image possible.

b) An RGB camera that stores data from three channels has a resolution of 1280x960. This makes capturing a color image possible. c) Microphone Row,which contains four microphones to capture the sound. Because there are four microphones, it is possible to record audio as well as find the location of the sound source and the direction of the sound wave.

d) A 3-axis accelerometer configured for a 2g range, where g is acceleration due to gravity. It is possible to use the accelerometer to determine the current orientation of the Kinect [START_REF] Wehbi | Architecture logicielle et matérielle pour fusion tolérantes au fautes pour systèmes multi-robots sous ROS[END_REF].

C. The Kobuki base

The mobile robot Kobuki is the mobile base of the TurtleBot 2. The robot iClebo Kobuki of Yujin Robot is a mobile development platform for education, research and for all those who want to develop their high-level applications. It is equipped with: a) Encoders: The encoder is a sensor attached to a rotating object to measure rotation. By measuring the rotation we can determine the displacement, speed, acceleration of the robot.

b) Bumpers: There are 3 bumpers distributed between the left, the center and the right sides of the base. c) Vacuum sensors: Likewise, 3 sensors are distributed between the left, the center and the right part of the base.

d) Wheel descent sensors: There are 2 of these sensors one for each wheel (left, right).

e) Gyroscope: It is a sensor of angular speeds on 3 axes x, y and z.

f) Dashboard: This is an interface tool provided by ROS that allows us to check the states of the sensors and different equipment such as the state of charge of the batteries (laptop + TurtleBot) [START_REF] Wehbi | Architecture logicielle et matérielle pour fusion tolérantes au fautes pour systèmes multi-robots sous ROS[END_REF].

IV. METHODS USED

The aim of this study is to ensure maximum accuracy in the navigation of robots also the dependability in working environment in order to optimize the human safety [START_REF] Froquet | Contribution à l'analyse des risques : Proposition d'une méthode par scénarios et capitalisation de la connaissance[END_REF], establishments and to preserve the proper of these agents (robots). To achieve this goal, we first need to ensure microscopic safety, to solve the navigation and control problems of each mobile robot so as to ensure macroscopic safety at the laboratory level, to take into account the interactions between the various agents to preserve the entire environment from the occurrence of accidents.

The following figures present the safety theory followed. Where: Microscopic safety means the dependability of each robot while macroscopic safety include the dependability of the whole laboratory. Dependability part is based on two methods of different approach, one of an inductive approach and the other of a deductive approach [START_REF] Froquet | Contribution à l'analyse des risques : Proposition d'une méthode par scénarios et capitalisation de la connaissance[END_REF]. First, we applied the FMEA method (Failure Mode and Effects Analysis Method) on one robot.Then,we complet this part by using the FTA method (Fault Tree Analysis) to analyze the multi-robot communication risks . Concerning the control / command part a behavioral approach has been adopted. This approach makes it possible to distribute the control over the robotic entities by decomposing the global task into a set of elementary tasks. the control architecture then has a set of behaviors / controllers. Each one can accomplish a corresponding elementary task (avoidance of obstacles and collision between robots, attraction to a target, planning, etc.) [START_REF] Mouad | Architecture de Contrôle/Commande dédiée aux systèmes Distribués Autonomes : application à une plate-forme multivéhicules[END_REF]. The following diagram summarizes the methods used in this paper as well as the relationship between dependability and intelligent control. with those of multi-agent systems, artificial intelligence and organizational models in order to bring a high level of coordination between the mobile agents / robots. The group of mobile robots is coordinated according to the different standards and specifications of the organizational model. Thus, the activation of an elementary behavior in favor of another is done by respecting the structural constraints of the robots in order to ensure the maximum precision and security of the coordinated movements between the different mobile entities (robots). Different cooperation strategies can be considered as examples:

• Cooperation carried out through a centralized supervisor so that the desired destination can be reached more quickly, unexpected events are managed individually by the mobile agents / robots in a distributed way.

• Cooperation with reinforcement learning [START_REF] Zennir | Multi-Actors distributed control systems: Reinforcement signal by Shannon's Entropy[END_REF].

• Cooperation with a selfish or collaborative aspect.

V. APPLICATION AND RESULTS

A. Dependability part 1) Application of FMEA method :

Table II below shows the application of the FMEA method on a Turtelbot 2 robot.

2) Application of FTA method :

Figure 9 presents the FTA method applied over the entire laboratory environment, we have chosen collision of robot as a top event, this collision leads to the occurrence of serious industrial risks: explosion, fire, chemical spills, Hazardous waste (toxic gas, dust, smoke, etc.)....For exemple the risk of explosion can appear if one of these three cases happened (figure 8): -Bad image quality.

-Incomplete Image.

-Low luminosity.

-Failure of the motor responsible for moving the sensor.

-Bad detection of static and dynamic obstacles.

-IR Sensor.

-Dashboard Kobuki.

-It is necessary to make periodic tests especially with each sensors initialization.

Infrared Depth Sensor ( IR Sensor)

Measures distance between obstacle or object and sensor (detects depth)

-No sensor indication (null value).

-Internal sensor failure or connection.

-IR Transmitter Failure.

-False command.

-There is No Motion or undesired movement.

-Dashboard Kobuki.

-Testing sensors and transmitters during initialization.

-False sensor indication (Random or constant values).

-Inadequate calibration.

-Error calculating the coordinates of objects on particular points.

-Risk of collision with human beings or with the other robots (Human and material damage).

-Check the sensor calibration.

-Aberrant values or a deviation in the point cloud resulting.

-Strong lighting.

-Properties of the object surface (shiny surfaces).

-Appearance of laser speckles with low contrast in the infrared image.

Consider setting the laboratory lighting.

Voice recognition

Sensor (microphone) -Capture sound -Record audio -Find the location of the sound source and the direction of the sound wave.

-No or wrong sensor indication.

-Sensor internal failure. -Noise.

-The sensor can not detect the source and the sound direction which may arise from navigation errors. -False command.

-Dashboard Kobuki.

-IR Sensor.

-RGB Camera.

Robot Controller

Control the robot by issuing commands.

-Blocked or frozen (constant command)

-Blocking the program or the operating system.

-Sends constant commands.

-Movement blocked at one point (frozen robot).

- After the application of FMEA method on a robot; the recommendations that were obtained to ensure its proper functioning and minimize its risks are as follows:

• It is necessary to make periodic tests especially at each initialization of the various components (sensors, actuators ...) ; • The setting up of prevention and protection means against risks such as prohibition signs and fire extinguishers within the laboratory; • The use of fault prevention techniques for developed software; • Emergency shutdown by the operator in case of serious malfunction; • An alert that will indicate the existence or not of an internal robot default ; • A sensor must be added indicating the battery status.

According to the FTA method the fault tree which can lead to a collision of robots depend to a three major problems:

• False sensor indication;

• Sending a false command to the robot;

• Communication problems.

B. Intelligent control part

In the literature there are different navigation control approaches of a mobile robot where multi-robots in an unknown environment [START_REF] Dutta | Obstacle Avoidance of mobile robot using PSO based Neuro Fuzzy Technique[END_REF][START_REF] Zennir | Control of the trajectory of a hexapod robot based on distributed Q-learning[END_REF][START_REF] Zennir | Distributed reinforcement learning of a six-legged robot to walk[END_REF]. The approach proposed for controlling robot navigation in an unknown or partially unknown environment with the presence of obstacles is multi-agent systems approach and reinforcement learning [START_REF] Md | Autonomous Robot Path Planning Using Particle Swarm Optimization in Dynamic Environment with Mobile Obstacles & Multiple Target[END_REF]. The architecture control of a single robot is illustrated in the following figure: We take note that:

• Agent: It is the linear model around an operating point. • State's': It is a continuous state space.

• Action 'a': It is the local command around an operating point. • The operation point is the points of equilibrium of the system which may be stable or not. • The critical function: the set of criteria to reward or not the chosen command.

Among the existing methods in the reinforcement learning approach, we have chosen to use the Q-learning and Qlearning-multi-agent algorithms [START_REF] Gavrilov | Mobile Robot Navigation using Reinforcement Learning based on Neural Network with Short Term Memory[END_REF][START_REF] Xia | Intelligent Mobile Robot Learning in Autonomous Navigation[END_REF][START_REF] Zennir | Safety study of the industrial system with FMEA(C): Applied on the storage tank TK102[END_REF][START_REF] Altuntas | Reinforcement learningbased mobile robot navigation[END_REF][START_REF] Khriji | Mobile robot Navigation Based on Q-Learning Technique[END_REF] in the case of multirobots.

We are faced with a learning of a dynamic system which implies a space of state and continuous action. Following the learning a command will be sent to the nonlinear process (TurtleBot 2 mobile robot). The architecture control in the multi-agents case is represented by the following figure: The different signal are :

• The state of the system is known in a global or partial way from the various sensors, GPS and others. • The optimization criterion of the error: there are several criteria, for example the quadratic error. • The reinforcement signal: there are several methods to calculate or determinate this reinforcement signal, they still have to be adapted to our problem.

The interaction between agents is done through the environment, in such a way that an agent takes into account the actions of other agents in order to make the right decision (action).

VI. CONCLUSION

In this paper we have tried to develop a risk analysis using the FMEA method on a TurtelBot 2 robot and the FTA method for the multi-robot. So we use two main concepts which are macroscopic and microscopic safety. Next, we proposed control architecture with a set of elementary behaviors / controllers in order to ensure the maximum accuracy and safety of the coordinated movements between the different mobile entities. Microscopic safety is the dependability of each robot. As for the macroscopic safety means the dependability of the overall of laboratory. To minimize the major risks in the laboratory we must increase the safety level integrated. In order to ensure the proper functioning of the robots and Increase the level of safety in the laboratory recommendations have been obtained. In the future work we applied the architecture control for one robot and multi-robots (11 robots) In order to improve the control and safety of the system and compared with nether approach of control (Q-fuzzy-muli-agent algorithm for example).

Fig. 1 .

 1 Fig. 1. Scenario of a robots in normal operation within a laboratory room Exposed to various risks.Thus, these robots can move between the different laboratory rooms. The figure[START_REF] Mouad | Architecture de Contrôle/Commande dédiée aux systèmes Distribués Autonomes : application à une plate-forme multivéhicules[END_REF] shows a robot moving to the other rooms.

Fig. 2 .

 2 Fig. 2. Scenario represents a robot moving between different laboratory rooms where hazards exist.

Fig. 3 .

 3 Fig. 3. Scenario represents a robot moving between different laboratory rooms where hazards exist.

Fig. 4 .

 4 Fig. 4. TurtleBot 2 components [1].

Fig. 5 .

 5 Fig. 5. The components of a Kinect XBOX360 [1].

Fig. 6 .

 6 Fig.6. Schema of the safety theory followed.

Fig. 7 .

 7 Fig. 7. Diagram summarizing the methods used and the relation between intelligent control and dependability.The specificity of the theoretical approach consists in combining the advantages of multi-controller architectures

Fig. 8 .

 8 Fig. 8. Schema represents causes that can lead to an explosion.

Fig. 10 .

 10 Fig.10. Synoptic of the control architecture to be applied (case one robot (agent)).

Fig. 11 .

 11 Fig. 11. Synoptic of the multi-agents control architecture.

TABLE I .

 I Classification of possible accidents and its consequences

		Possible accidents	Consequences	Criticality
			of accidents
		Case 1	Robots	C 0
			degradation
		Robot loaded with	Risk of
		explosive product	explosion	C 2
	Case	Robot loaded with	Risk of fire
	2	Extremely or easily
		flammable product
		Robot loaded with	Risk of toxicity
		very toxic or toxic gas	and asphyxia
	Case	Robots Loaded with	Risk of	C 3
	3	explosives ;	explosion; fire;
		flammables products ;	toxicity and
		toxic gas or	asphyxia; dust;
		incompatible products	Flammable
			vapors or gases
	Case	Robot loaded with	Risk of	C 4
	4	explosive product	explosion,
			fatality
		Robot loaded with	Risk of fire,
		Extremely or easily	human burn
		flammable product
		Robot loaded with	Risk of human
		very toxic or toxic gas	toxicity and
			asphyxia
		Robot loaded with	Destruction of
		Corrosive and	human skin or
		irritating products	eye tissue
		Case 5	Change of	C 1
			chemical
			product
			properties

TABLE I .

 I Table of FMEA performed on a Robot "TurtleBot 2" .

	Component	Function	Failure Mode	Cause	Effect	Detection	Action
	Visual sensor	Visual detection and
	(RGB Camera)	obstacle tracking
		(object scanning and
		color detection).