Antoine Rached

Virginie Lattard
email: virginie.lattard@vetagro-sup.fr

Ambre Fafournoux

Herv É Caruel

Isabelle Fourel

Etienne Benoit

S Ébastien Lefebvre

Comparative pharmacokinetics of difethialone stereoisomers in male and female rats and mice: development of an intra-and inter-species model to predict the suitable formulation mix

Keywords:

The ecotoxicity of anticoagulants used for rodent pests' management is a major concern, particularly with second generation anticoagulants, which are more persistent in the body of rodents and therefore more likely to cause secondary exposure in their predators. One of the solutions envisaged to mitigate this risk is to use stereoisomers of these anticoagulants, each of which has particular pharmacokinetics. However, the few studies published to date have considered only one species and one sex. Here, we study the pharmacokinetics of the 4 stereoisomers of 3.4 mg/kg of difethialone in rats (Rattus norvegicus) and 3 mg/kg in mice (Mus musculus) in both sexes and propose a model to choose the optimal stereoisomer efficacy/ecotoxicity mixture for the management of all these animals. Our results show that while the most persistent stereoisomer (E3-cis) is common to both species and sexes, the pharmacokinetics of the other stereoisomers show marked differences between sexes and species. Thus, the area under curve (AUC) of E4-trans in male rats is four times lower than in females or mice, making it a priori unusable in male rats. Conversely, our modeling seems to show that the E1-trans stereoisomer seems to offer the best compromise AUC persistence. In conclusion, we highlight that studies on anticoagulants must necessarily integrate research on the effect of gender and species both on efficacy and with regard to the ecotoxicity of these molecules.

Introduction

Anticoagulant rodenticides are highly effective for rodent control in agriculture and urban environment because of their delayed action that prevents rodents from developing food aversion. The first generation of anticoagulant rodenticides (FGARs) was developed and used from the 1950s. Unfortunately, their efficacy was rapidly limited by resistance phenomena associated with mutations in the Vkorc1 gene, present in some rodent populations.

To overcome this resistance, a "second generation" of anticoagulant rodenticides (SGARs) -including difenacoum, bromadiolone, flocoumafen, brodifacoum and difethialonewas developed. This generation is more toxic (lower concentration to induce mortality) and presents greater duration of action (longer persistence in target organ) [START_REF] Hadler | Forty-Five Years of Anticoagulant Rodenticides-Past, Present and Future Trends[END_REF][START_REF] Watt | Anticoagulant Rodenticides[END_REF][START_REF] Rattner | stereoisomers in male and female rats and mice: development of an intra-and inter-species model to predict the suitable formulation mix -9/10 Times Cited: 0 Cited Reference Count[END_REF][START_REF] Buckle | Control Methods: Chemical[END_REF][START_REF] Douglas | The Emerging Threat of Superwarfarins: History, Detection, Mechanisms, and Countermeasures[END_REF][START_REF] Berny | Development of Resistance to Anticoagulant Rodenticides in Rodents[END_REF] . This group of molecules was also referred to as superwarfarins and longacting anticoagulant rodenticides. Despite these properties, resistance to some SGARs has been reported in different regions due to their widespread use [START_REF] Roger | Control of a Population of Norway Rats Resistant to Anticoagulant Rodenticides[END_REF][START_REF] Mooney | VKORC1 Sequence Variants Associated with Resistance to Anticoagulant Rodenticides in Irish Populations of Rattus Norvegicus and Mus Musculus Domesticus[END_REF] . In addition, intoxication of non-target animals is the most serious drawback of SGARs. Indeed, direct access of domestic or wild animals to the poisoned bait or repeated consumption of poisoned rodents by some predators and scavengers lead to fatal poisonings [START_REF] Jhon | Secondary Exposure to Anticoagulant Rodenticides and Effects on Predators[END_REF][START_REF] Rattner | Anticoagulant Rodenticide Toxicity to Non-target Wildlife Under Controlled Exposure Conditions[END_REF][START_REF] Rached | Biomarkers Potency to Monitor Non-target Fauna Poisoning by Anticoagulant Rodenticides[END_REF] . Although ingestion of intoxicated rodents does not lead systematically to the death of the animal, many predators and scavengers have been shown to be chronically exposed to SGARs due to the long tissue-persistence of these molecules and the consequences of this exposure on populations are not fully understood [START_REF] Rattner | Toxicokinetics and Coagulopathy Threshold of the Rodenticide Diphacinone in Eastern Screech-Owls (Megascops Asio)[END_REF][START_REF] Barnett | Challenges in the Interpretation of Anticoagulant Rodenticide Residues and Toxicity in Predatory and Scavenging Birds[END_REF] .

To overcome this drawback, several studies have suggested the development of ecological anticoagulant rodenticides based on the stereochemical principle. Thus, since the SGARs are all composed of 4 stereoisomers, it has been shown that these stereoisomers associated by couple of transor cis-isomers presented different biological properties within the same SGAR. Indeed, they presented a different hepatic half-life with systematically a couple of stereoisomers eliminated more rapidly than the other, while their inhibitory efficacy remains comparable [START_REF] Damin-Pernik | Development of an Ecofriendly Anticoagulant Rodenticide Based on the Stereochemistry of Difenacoum[END_REF][START_REF] Damin-Pernik | Management of Rodent Populations by Anticoagulant Rodenticides: Toward Third-Generation Anticoagulant Rodenticides[END_REF] . This difference is clear for bromadiolone, difenacoum and brodifacoum: the systematic use of the least persistent diastereoisomeric pair would reduce the ecotoxicity associated with their use.

Surprisingly, this difference is less clear for difethialone, which is the most recent SGAR. This limited difference prompted Lefebvre et al. to investigate the pharmacokinetic properties of the two enantiomers of each diastereomer of difethialone. In contrast to the diastereomers, the four stereoisomers individually have different liver half-lives in male rats and reformulation of the active ingredient in favor of certain stereoisomers would improve its ecotoxicological profile. A mathematical modeling approach based on liver half-lives has been proposed to optimize the formulation of rodenticide mixtures [START_REF] Lefebvre | Comparative Biological Properties of the Four Stereoisomers of Difethialone, a Second-Generation Anticoagulant Rodenticide, in Rats: Development of a Model Allowing to Choose the Appropriate Stereoisomeric Ratio[END_REF] .

Could this bait improvement be generalized to female rats, or even mice whose populations are also managed primarily with anticoagulant rodenticides? This seems crucial as both the Norway rat (Rattus norvegicus) and the house mouse (Mus musculus) are commensal rodents. They both live near humans, even though their habitat is often separated because of the aggressiveness of one towards the other. The half-lives of current anticoagulants have been described as very different between species [START_REF] Horak | Pharmacokinetics of Anticoagulant Rodenticides in Target and Non-target Organisms[END_REF] . Optimizing the composition of an active ingredient must therefore take this into account. In addition, as sex differences in response to anticoagulant rodenticides have been demonstrated in rats [START_REF] Hara | Sex Differences in the Anticoagulant Effects of Warfarin[END_REF][START_REF] Gill | Blood-Clotting Response Test for Bromadiolone Resistance in Norway Rats[END_REF][START_REF] Gerard | Comparison of the Half-Lives and Regeneration Rates of Blood Clotting Factors II, VII, and X in Anticoagulant-Resistant and Susceptible Norway Rats (Rattus Norvegicus Berk.)[END_REF][START_REF] Zhu | Gender Differences in Pharmacokinetics of Oral Warfarin in Rats[END_REF] , this factor must also be considered. Would the composition of a remixed difethialone bait be the same if all of these criteria were considered, or should a compromise be sought?

In this work, we compared first the pharmacokinetics of the four individual stereoisomers of difethialone in male and female rats and mice, and thus we tried to identify the best theoretical mix composition of difethialone stereoisomers to satisfy the need for efficiency of rodenticides while controlling the ecotoxic risk as best as possible.

Material and methods

Chemicals

Difethialone (3-[3-[4-(4-bromophenyl) phenyl]-1,2,3,4tetrahydronaphthalen-1-yl]-2-hydroxythiochromen-4-one) with a purity above 98 % composed of the four stereoisomers was provided by Liphatech (Pont-du-Casse, France). It was a mixture containing 45 % of the cis-isomers in racemic proportion (1S, 3R and 1R, 3S) and 55 % of the trans-isomers in racemic proportion (1S, 3S and 1R, 3R). Isoflurane ®and vitamin K1 were purchased from Alcyon, (Miribel, France).

Animals

Forty male and female, seven-week-old, CD 1-mice and 21 eight-week-old female OFA-Sprague Dawley rats were obtained from a commercial breeder Charles Rivers, (l'Arbresle, France). Animals were housed three per standard cage (Eurostandard, Type IV, Tecniplast, Limonest, France) for rats and five per cage for mice in the institutional animal facility in a controlled environment (ambient temperature 20 ± 1 °C and 12-h light/dark cycle) with access to standard food (Scientific Animal Food and Engineering, reference A04) and water ad libitum. Animals were acclimatized for at least one week prior to use and were maintained in accordance with the European and French legislation guidelines on animal studies.

Pharmacokinetic study

Experimental research on the rodents was carried out according to an experimental protocol following international guidelines and approved by the ethics committee of the Veterinary School of Lyon (authorization n°201704190941578).

Female OFA-Sprague Dawley rats received through per os administration 3.4 mg/kg of difethialone dissolved in 10 % DMSO and 90 % vegetable oil. Rats were maintained in life by daily subcutaneous administration of vitamin K1 (10 mg.kg -1 ). 4, 9, 24, 48, 120, 168, and 216 hours after difethialone administration, three rats were anesthetized with isoflurane, and blood was taken by cardiac puncture into citrated tubes. After blood sampling, rats were euthanized with CO2 and the liver of each rat was immediately collected and stored at -80 °C until analysis.

Data concerning the pharmacokinetic study of difethialone in male rats are taken from our previously published article Lefebvre et al. 2020 [START_REF] Lefebvre | Comparative Biological Properties of the Four Stereoisomers of Difethialone, a Second-Generation Anticoagulant Rodenticide, in Rats: Development of a Model Allowing to Choose the Appropriate Stereoisomeric Ratio[END_REF] .

Male and female CD1-mice received through per os administration 3 mg/kg of difethialone dissolved in 10 % DMSO and 90 % vegetable oil. Mice were maintained in life by daily subcutaneous administration of vitamin K1 (10 mg.kg -1 ). 24, 72, 168 and 336 hours after difethialone administration, five mice of each sex were anesthetized with isoflurane and blood was taken by cardiac puncture into citrated tubes. After blood sampling, mice were euthanized with CO2 and the liver of each mouse was sampled and stored at -80 °C until analysis. Concentration of each stereoisomer of difethialone over the time after a per os administration of 3.4mg/kg in rats or 3mg/kg in mice of difethialone. Values are presented as mean and standard deviation. The grey area is the one that is analyzed for interspecies comparison and modeling in the rest of the article. Data for male rats are from Lefebvre 2020 [START_REF] Lefebvre | Comparative Biological Properties of the Four Stereoisomers of Difethialone, a Second-Generation Anticoagulant Rodenticide, in Rats: Development of a Model Allowing to Choose the Appropriate Stereoisomeric Ratio[END_REF] 1.4 Dosages Difethialone stereoisomers were dosed as described previously in Fourel et al. [START_REF] Fourel | Enantiomeric Fraction Evaluation of the Four Stereoisomers of Difethialone in Biological Matrices of Rat by Two Enantioselective Liquid Chromatography Tandem Mass Spectrometry Methods: Chiral Stationary Phase or Derivatization[END_REF] .

Data Analysis

Data handling and statistical analyses were performed with R 4.0.5 [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF] and the following package PK (1.3.5) [START_REF] Jaki | Basic Non-Compartmental Pharmacokinetics[END_REF] . The non-compartmental analysis was performed by applying the Bailer's method for sparse sampling as described in Nedelman [START_REF] Nedelman | Applying Bailer's Method for AUC Confidence Intervals to Sparse Sampling[END_REF] . Results were expressed as mean values ± Standard Error or fitted value and Confidence Interval. Wilcoxon test or if relevant Dunn's multiple comparison test was used with α < 0.05 in order to statistically compare the results among groups.

Results

General pharmacokinetics of each stereoisomer

and effect of sex on pharmacokinetic parameters Hepatic concentrations of each stereoisomer of difethialone (E1-trans, E4-trans, E2-cis and E3-cis, called according to their order of elution on the chromatographic chiral column and according to their belonging to the diastereoisomeric couple) versus time are shown in figure 1 A, B, C andD, respectively. Remarkably, the stereoisomer with the highest concentration and the most persistent by far was E3-cis, regardless of species or sex. Considering that the sample times and doses were different among species, the results were first analyzed according to the sex within a species. Tables 1 and2 present the non-compartmental analysis of the pharmacokinetics of each stereoisomer for rats and mice, respectively. In rats, the AUC 4-216h were significantly higher in females than in males for all four stereoisomers as the AU C4h-in f inite except for the E1-trans stereoisomer where no significant difference was found. Cmax was statistically different between males and females for only the E4-trans stereoisomer. T max was also different for the latter stereoisomer with a Tmax of 4h and 9h in males and females, respectively. The mean residence time (MRT) was significantly lower in females for both trans-stereoisomers and higher for E2-cis stereoisomer.

In mice, significant differences in AUCs were highlighted only for the E1-trans and E2-cis stereoisomers with higher AUCs in males compared to females. These higher AUCs were associated with significantly higher Cmax in males for both stereoisomers but no statistical differences for MRT. MRT and half-life of each difethialone stereoisomer according to species and sex between 24 and 168 h after a per os administration of difethialone.

λ (h -1 ) R 2 MRT 24-168h (h) Half-life (h) λ (h -1 ) R 2 MRT 24-168h (h) Half-life (h) λ (h -1 ) R 2 MRT 24-168h (h) Half-life (h) λ (h -1 ) R 2
Comparative pharmacokinetics of difethialone stereoisomers in male and female rats and mice: development of an intra-and inter-species model to predict the suitable formulation mix -5/10

Non-compartmental analysis of the four individual stereoisomers of difethialone according to species

Since the 24-hour to 168-hour period of the pharmacokinetics (i.e., corresponding to 24, 72, 168-hours sample times for mice and 24, 48, 120, and 168-hours sample times for rats) was common among the four groups, the analysis of the results was continued by considering only this relevant part in order to compare the efficacy and the ecotoxicity of difethialone between species and gender. Because the amount of difethialone administered per weight was higher in rats than in mice, only the non-inferiority of the concentrations or AUC results obtained in rats versus mice (i.e., intra-or inter-gender comparison) was statistically analyzed. Results are presented in figure 2. Parameters of the non-parametric pharmacokinetic between 24 and 168h for each stereoisomer according to the sex and the species. For each stereoisomer, for all pairs (rats-mice) of histograms designated < symbol, the values of the mice are significantly higher than those of the rat Considering the E1-trans stereoisomer, both male and female mice (9.3 and 6.6 µg/g, respectively) had higher liver concentration than male rats (3.6 µg/g) 24 hours after the difethialone administration. The significance of difference was not tested at 168h as the E1-trans concentration was not tested. Indeed, at this time the rats had hepatic concentrations of the order of the microgram (0.7 µg/g for males and 1.1 µg/g for females) when the mice had a hepatic concentration of the order of a hundred of nanograms (170 ng/g for males and 60 ng/g for females). Moreover, only the AUC 24-168h of male mice was higher than that in male rats (453 µg.h/g versus 314 µg.h/g respectively).

For the E3-cis stereoisomer, the liver concentrations 24 hours after the administration were significantly greater in mice (both sexes, 12.3 µg/g in males and 12.1 µg/g in females) than in rats (both sexes, 7.1 µg/g in males and 9.1 µg/g in females). However, at 168 hours if male and female mice liver concentrations (5.4 µg/g and 6.6 µg/g respectively) were significantly higher than that of male rats (1.7 µg/g), only female mice concentration was greater than that of female rats (4.3 µg/g). AUC 24-168h of male and female mice (1170 µg.h/g and 1346 µg.h/g respectively) were twice as high as that of male rats (662 µg.h/g), however no significant difference was highlighted with female rats (1093 µg.h/g).

Considering E2-cis stereoisomer, concentration at 24 hours and 168 hours and AUC 24-168h were lower in mice than in rats so they have not been tested for a significant difference. However, it is noteworthy that the concentration at 168h was in the order of a few hundred nanograms for both male and female rats (0.17 µg/g and 0.61 µg/g respectively), versus a few nanograms for both male and female mice (0.015 µg/g and 0.003 µg/g respectively).

Finally, considering E4-trans stereoisomer, the liver concentrations at 24 hours of both male and female mice (2.7 µg/g and 2.2 µg/g respectively) were significantly higher than that of male rats (0.29 µg/g). The same kind of differences were observed for the concentration at 168 hours with values of 0.18 µg/g for male mice and 0.32 µg/g for female mice that were both significantly higher than the value of 0.046 µg/g obtained for male rats. The AUC 24-168h with values of 127 µg.h/g for male mice and 117 µg.h/g for female mice were also both significantly higher than the 23 µg.h/g for male rats.

In addition to the MRTs, the half-lives of each stereoisomer was determined between 24h and 168h for each speciessex pair (Table 3 and figure 3). Statistical differences were observed between rats and mice considering the MRTs and half-lives of E1-trans and E2-cis. For these two stereoisomers, MRTs and half-lives were shorter in mice than in rats for both sexes (two times more regarding the half-lives), while no difference was pinpointed for others (table 3). Even if the half-life of E4-trans stereoisomer (63.7h) in male rats seemed to be higher than that for female rats or mice, the difference was not statistically significant.

Discussion

In our previous article, that included only the data of male rats, we have proposed a model to predict the efficacy and the ecotoxicity of different mixes of difethialone to optimize the mix of difethialone by increasing its AUC and thus their efficacy while decreasing their persistence and thus their ecotoxicity.

The results of this previous study showed that a mixture of E1 and E2 could achieve a good compromise between these two opposing criteria [START_REF] Lefebvre | Comparative Biological Properties of the Four Stereoisomers of Difethialone, a Second-Generation Anticoagulant Rodenticide, in Rats: Development of a Model Allowing to Choose the Appropriate Stereoisomeric Ratio[END_REF] . Considering the results of the current study, is this mix still a good opportunity if we extend the use of such a bait to female rats or mice of both sexes? Should we evolve the model to adapt it to the constraints of sex and species? Is there an optimal mix for managing rat populations on the one hand, and mouse populations on the other hand, or even the infestation of both populations? This study was conducted to answer these questions and to extend the first proposed model to the different rodent populations that can be managed with anticoagulant rodenticides (ARs). Nevertheless, two major limitations of this study must be mentioned, the dose of difethialone administered orally to rats and mice at T0 slightly differs (3,4 mg/kg for rats and 3 mg/kg for mice) and that the sampling times are different for each species. These two aspects of the experiment have been considered in the analysis of pharmacokinetics and particularly in comparisons between the two species. One of the purposes of our modeling is to limit the use of laboratory animals in rodenticide research by predicting the best mixture before testing. In accordance with this objective and the principle of the 3R that rules the experiments on animals, we chose not to retest but to use unpublished results to build our model. However, in order to reduce the importance of these biases, we performed the analysis in two steps. A first analysis was performed by comparing the sexes within each species (same dose and same sampling time). Then a second one by comparing the species between them in period where the sampling times overlap for both species with a common start and finish.

In male rats, a mixture of E1 and E2 allows by modulating the ratio between both stereoisomers to reach a wide range of AUC, from simple to double, with a simple to triple increase in persistence at 168h. Thus, this simple pair of stereoisomers would allow creating a variety of baits that adapt to the various conditions by an optimization of the effectiveness/ecotoxicity compromise. If such baits were used in female rats they would, a priori, retain their efficacy, since the AUC 24-168h of each of the two stereoisomers are higher than those of the males. However, in female rats, the difference between E1trans and E2-cis, although significant (p=0.035), is greatly reduced, the AUC 24-168h of E1-trans being only 20 % higher than that of E2-cis. Moreover, the persistence of the E2-cis stereoisomer is only 30 % lower than that of the E1-trans (p=0.040). Thus, in females, if a mixture of difethialone composed only of E1-trans and E2-cis (i.e., a stereoisomer that is a priori more effective but more persistent and another that is less effective and less persistent) was recommended as in male rats, part of the modularity and flexibility of use that such a mixture allows in male rats would be lost. Indeed, the difference in properties between the two stereoisomers in female rats is much smaller than in male rats. In mice, the persistence of both stereoisomers is lower in both sexes than in rats, especially for E2-cis. However, for the latter this low persistence is accompanied by a strongly decreased AUC 24-168h . Thus, the effectiveness and value of adding the E2-cis stereoisomer in a bait for mice is questionable. Again, the benefit of a mixture composed of E1-trans and E2-cis, as presented in our previous article, cannot be extended from the male rats. The mechanisms underlying these sex and species differences have not been explored here. However, the literature provides different hypotheses to explain those differences. Considering the effect of sex on pharmacokinetics, in agreement with previous studies on the pharmacokinetics of difethialone, each of the stereoisomers has a higher AUC in female rats compared to male rats. The difference between males and females is particularly large in rats, with a 1.4-(E3-cis) to 4-fold (E4-trans) higher AUC 4-216h in females. Nevertheless, the origin of this difference is not clearly identified here. Indeed, the MRT 4-216h is only 21 % higher in females than in males for the E2-cis stereoisomer and the MRT 4-216h of the other stereoisomer is lower in females or not significantly different. The origin might be intestinal absorption or hepatic capture efficiency of the female, although the statistical power of our data does not allow us to conclude. Indeed, although the maximum concentration at peak is 1.3, 1.5, 1.7, and 2.3 times higher in females for E3-cis, E1-trans, E2-cis, and E4-trans respectively, there is only a statistically significant difference for E4-trans. However, the pharmacokinetics of the latter stereoisomer seems to be strongly sex-dependent in rats. Indeed, the hepatic elimination rate of this stereoisomer appears to be much higher in males, resulting in an earlier Cmax and a low liver concentration at 24h. Although the stealthiness of the passage of this stereoisomer in the liver seems to argue against its use as a rodenticide in male rats, it raises many questions about its hepatic metabolism. This difference could suggest that difethialone E4-trans metabolic enzymes are part of the sex-specific cytochromes P450 in rats (CYP2C11, CYP2C13, CYP3A2, and CYP2C12) [START_REF] Czerniak | Gender-Based Differences in Pharmacokinetics in Laboratory Animal Models[END_REF] or that the tissue distribution is different between sexes. Considering the mice, significant differences are only pinpointed for AUC 24-336h of the E1-trans and E2-cis stereoisomers, with a lower value in females. The AUC results are consistent with the maximal concentrations, which are significantly lower in females for both stereoisomers. The lack of significant difference for the corresponding MRT may suggest, as for rats but conversely for the genders, that a higher liver bioavailability of these two stereoisomers in male mice might explain the observed difference on AUC. Further studies focusing on the absorption of the different stereoisomers and their metabolism are needed to confirm, or not, this hypothesis. These observations, in addition to ruling out the stereoiso-Comparative pharmacokinetics of difethialone stereoisomers in male and female rats and mice: development of an intra-and inter-species model to predict the suitable formulation mix -7/10

C d, f 1...4 (t) = d D 4 ∑ i=1 f i F i * C D,i,24 * e -λ i (t-24)
1. Equation used to model the liver concentration evolution in time of different mixes of difethialone stereoisomers. i is the index of each stereoisomer; f the fraction of each stereoisomer in the modelized mix; F the fraction of each stereoisomer in the experimental mix (F1 = 0.275, F2 = 0.225, F3 = 0.225, F4 = 0.275); d is the dose of difethialone to model; D is the dose of difethialone used in our experimental plan (in mice D = 3.0 mg/kg, in rats D = 3.4 mg/kg); C (D,i,24 ) is the average concentration obtained experimentally for the respective stereoisomer at 24h and with the D dose of difethialone; λ i is the decrease constant of the respective stereoisomer obtained experimentally with data between 24 and 168 hours; t is the time variable that can take values between 24 and 168 hours.).

meric pair selected with male rats for use in female rats or mice, also eliminate the use of the model in a multi-gender, multi-species approach. Indeed, one of the assumptions used in its construction is that absorption is not a determining factor in the observed differences in AUC and residues. However, our analysis shows that the differences in pharmacokinetics observed between gender and species cannot be explained by elimination alone. Thus, it is necessary to reconstruct a multigender and multi-species model considering an absorption criterion while keeping the spirit of the previous model to plot a graph with an efficiency indicator (the AUC) expressing as a function of an ecotoxicity indicator (the amount of molecule remaining in the liver five days after taking the bait, i.e., when the animal dies). To take into account the differences in hepatic capture between species and sexes and as there are not enough early samples (between 0 and 12 hours after per os administration) to determine an absorption coefficient, we have added the consideration of the observed C 24 concentration of each stereoisomer and included the notion of the administered dose in the model. For the administered dose and its relationship with C 24 , we chose a linear relationship, which seems relevant for an oral administration of difethialone in the order of milligrams. Indeed, in one of our previous studies in rats, a dose of 10 mg/kg was administered orally and the liver concentrations of difethialone at 24h were about 3 times higher than those observed in the present study [START_REF] Lefebvre | Origin of the Gender Differences of the Natural Resistance to Antivitamin K Anticoagulants in Rats[END_REF] . Then, equation 1 is used to model the pharmacokinetic of difethialone stereoisomeric mixture. For each stereoisomer i, C D,i,24 is defined with C 24 concentration from figure 2 and λ i with elimination constants of table 3.

The implementation of the model results in the creation of the graphs presented in figure 4. First, if a mixture with an AUC similar to the commercial product (represented with a star in figure 4) but with a maximum residue reduction at 5 days was created, the formulation would be identical for rats. However, for mice, it would be preferable to use an E1-trans/E3-cis mixture instead. For example, a mixture of 65 % E1-trans and 35 % E3-cis would decrease the hepatic residues of difethialone in male and female mice by 20 % without decreasing the AUC compared to the current product. However, if such a product was used in rats, the concentration at 120h in male rats would be increased by 15 %. But, should the AUC of difethialone baits be maintained at all costs, or should a more balanced approach towards low ecotoxicity be preferred when, in some urban territories, difethialone is the main anticoagulant found in birds of prey [START_REF] Okoniewski | Anticoagulant Rodenticides in Red-Tailed Hawks (Buteo Jamaicensis) from New York City[END_REF] ? Thus, to continue using difethialone, which is an effective method of controlling resistant rodent populations, it is essential to reduce its ecotoxicity. This ecotoxicity is a priori largely due to the E3-cis stereoisomer, which is the most persistent stereoisomer among sexes and species. Therefore, as much as technically possible, baits should be formulated without E3cis. Based on this assumption, E1-trans seems to be the best compromise for mice and rats with an AUC corresponding to 70 % or 53 % of that of the current product for rats and mice respectively at a dose of 3 mg/kg. Nevertheless, this decrease in AUC is accompanied by a reduction in residues at 120h of 48 % for female rats, 82 % for male mice, and 90 % for female mice. Only the residues at 120h of male rats decreased in the same proportion as the AUC, i.e., by about 20 %. Since the reported LD50 of the current product are 0.5 mg/kg in rats and 1.3 mg/kg in mice [START_REF] Pelfrène | Chapter 100 -Rodenticides[END_REF] , corresponding, according to our model, to liver AUC values, of 226 h.µg/g in male rats, 332 h.µg/g in female rats, 875 h.µg/g in male mice and 956 h.µg/g in female mice, a male rat (≈300 g) or a female rat (≈250 g) would have to eat 10 g of bait; a male mouse (≈30 g), 3.6 g of bait and a female mouse (≈25 g), 2.9 g of bait to achieve such concentrations with a 25 ppm E1-trans bait. These amounts are about twice as much as with the current bait, but the residual hepatic dose at 5 days would be equivalent for the male rat, decreased by 20 % for the female rat, divided by 3 for the male mouse and by 5 for the female mouse. Of course, it will be necessary to verify experimentally the effectiveness of this product and technically assess its production challenges in order to provide a cost-effective commercial AR.

Finally, the E2-cis stereoisomer may also be of interest. Indeed, compared to the current product, this stereoisomer allows in rats, for a reduction of about half of the AUC, to decrease the residues at 120h of about 75 % in males and 60 % in females compared to the same dose of the current product. However, the decrease in AUC is even more drastic in mice, over 90 %. This might seem to be a disadvantage, but it could also be a sign of the selectivity that this stereoisomer would allow. Indeed, if this mouse specificity is shared with other small mammals that are often victims of primary intoxication to ARs [START_REF] Rached | Biomarkers Potency to Monitor Non-target Fauna Poisoning by Anticoagulant Rodenticides[END_REF][START_REF] Geduhn | Spatial and Temporal Exposure Patterns in Non-Target Small Mammals during Brodifacoum Rat Control[END_REF] , this would provide the way to the creation of ARs effective on rats but with limited primary and secondary ecotoxicity on non-target fauna. Further investigations on non-target species should be implemented to explore this In conclusion, and despite the biases of our study, we highlight that studies on anticoagulants must necessarily integrate research on the effect of gender and species both on efficacy (which is already done in regulatory tests), but also with regard to the ecotoxicity of these molecules. To date, many studies have simply extended the pharmacokinetic results for all rodents from rat or mouse data, often obtained only in males. However, as if significant differences are observed in our study performed on two laboratory strains belonging to the same phylogenetic super-family, one must question the need to avoid extrapolation from one species to the other, at least until the underlying mechanisms of SGAR stereoisomers metabolism and distribution, which are still in question, have been elucidated. Furthermore, as it is not possible to perform pharmacokinetic studies on all non-target species that may be exposed to anticoagulants, it is essential to characterize which stereoisomers are found in wildlife and which are not in order to assess the ecotoxic potential of each stereoisomer, as it is performed today with diastereoisomers [START_REF] Fourel | Monitoring the Hepatic Residues of Cis-and Trans-Diastereoisomers of Second Generation Anticoagulant Rodenticides Reveals a Different Bioaccumulation of Diastereoisomers in the Food Chain of the Réunion Harrier (Circus Maillardi)[END_REF][START_REF] Fourel | Cis-Bromadiolone Diastereoisomer Is Not Involved in Bromadiolone Red Kite (Milvus Milvus) Poisoning[END_REF][START_REF] Fourel | Liver and Fecal Samples Suggest Differential Exposure of Red Fox (Vulpes Vulpes) to Trans-and Cis-Bromadiolone in Areas from France Treated with Plant Protection Products[END_REF][START_REF] Alabau | Accumulation of Diastereomers of Anticoagulant Rodenticides in Wild Boar from Suburban Areas: Implications for Human Consumers[END_REF] and ultimately improve pest management by limiting effects on non-target fauna.

Author contributions

Figure 1 .

 1 Figure 1.Concentration of each stereoisomer of difethialone over the time after a per os administration of 3.4mg/kg in rats or 3mg/kg in mice of difethialone. Values are presented as mean and standard deviation. The grey area is the one that is analyzed for interspecies comparison and modeling in the rest of the article. Data for male rats are from Lefebvre 2020[START_REF] Lefebvre | Comparative Biological Properties of the Four Stereoisomers of Difethialone, a Second-Generation Anticoagulant Rodenticide, in Rats: Development of a Model Allowing to Choose the Appropriate Stereoisomeric Ratio[END_REF]

Figure 2 .

 2 Figure 2.Parameters of the non-parametric pharmacokinetic between 24 and 168h for each stereoisomer according to the sex and the species. For each stereoisomer, for all pairs (rats-mice) of histograms designated < symbol, the values of the mice are significantly higher than those of the rat

Figure 3 .

 3 Figure 3. Linearization of the concentration of each stereoisomer as a function of time by using a logarithmic scale base ten. Values are presented in percent of the concentration at 24h post-administration as mean and standard deviation

Figure 4 .

 4 Figure 4. AUC 24-168 and concentration of difethialone at 168h, obtained by calculation after a theoretical administration of 3 mg/kg of a couple of difethialone stereoisomers. Points represent the values for one stereoisomer alone. Lines represent values obtained with mixed of connected stereoisomer. Star point represents the results obtained with actual commercial difethialone

  Conceptualization: VL, EB, SL; Methodology: AR, VL, AF, IF, SL; Data curation: AR, SL; Formal analysis and investigation: AR, VL, SL; Writing -original draft preparation: AR, VL, SL; Writing -review and editing: AR, VL, AF, HC, IF, EB, SL; Funding acquisition: VL, HC; Resources: VL; Supervision: VL, SL.

Table 1 .

 1 Pharmacokinetic parameters of the non-compartmental analysis of each difethialone stereoisomer in the liver of rats. Data are presented with standard error. Data for male rats are from Lefebvre et al. 2020[START_REF] Lefebvre | Comparative Biological Properties of the Four Stereoisomers of Difethialone, a Second-Generation Anticoagulant Rodenticide, in Rats: Development of a Model Allowing to Choose the Appropriate Stereoisomeric Ratio[END_REF] . *Statistical significant between male and female < 0,05

		Cmax (µg/g)	Tmax observed (h)	AUC 4-216h (µg.h/g)	AUC 4-In f inite (µg.h/g)	MRT 4-216h (h)
		Male	Female	Male	Female	Male	Female	Male	Female	Male	Female
	E1-trans	5,64±0,57	8,64±1,00	9	9	458±27,1	650±25,1*	660±103	767±25,9	77,9±3,94	68,5±2,06*
	E2-cis	4,41±0,46	7,33±0,91	9	9	226±24,17	536±17,2*	252±30,1	606±19,4*	53,2±4,50	64,9±1,39*
	E3-cis	10,6±0,91	13,7±1,22	9	9	952±50,5	1558±50,3*	1434±166	2821±177*	82,3±3,23	87,8±2,50
	E4-trans	3,23±0,25	7,56±0,56*	4	9	54,5±4,52	219±9,21*	59,5±4,9	226±9,09*	45,6±2,71	39,2±1,39*
		Male	Female	Male	Female	Male	Female	Male	Female	Male	Female
	E1-trans	9,3±0,86	6,6±0,44*	24	24	469±32,5	264±13,4*	469±32,5	264±13,4*	54,4±2,58	47,4±1,63
	E2-cis	1,9±0,18	1,4±0,06*	24	24	73,2±5,16	43,2±20,4*	73,3±5,2	43,6±2,03*	44,0±1,93	39,6±1,65
	E3-cis	12,3±1,1	12,1±0,68	24	24	1996±103	2176±117	3429±384	2911±258	148±5,19	137±4,94
	E4-trans	2,7±0,44	2,2±0,39	24	24	157±15,9	149±15,5	160±15,9	154±15,8	83,5±5,71	90,3±6,51

Cmax (µg/g) Tmax observed (h) AUC 24-336h (µg.h/g) AUC

24

-In f inite (µg.h/g) MRT 4-216h (h)

Table 2 .

 2 Pharmacokinetic parameters of the non-compartmental analysis of each difethialone stereoisomer in the liver of mice. Data are presented with standard error.*Statistical significant between male and female < 0,05

		Male Rat	Female Rat	Male Mice	Female Mice
	MRT 24-168h (h)	Half-life (h)

Table 3 .

 3


Geoffrey Prévot

Talia Bsaibess

Jonathan Daniel

Coralie Genevois

Guillaume Clermont

Isabelle Sasaki

Sebastien Marais

Franck Couillaud

Sylvie Crauste-Manciet

Mireille Blanchard-Desce

Multimodal optical contrast agents as new tools for monitoring and tuning nanoemulsion internalisation into cancer cells. From live cell imaging to in vivo imaging of tumours

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Non-invasive molecular imaging has a great potential for human clinical use both for diagnosis and treatment. Among the diverse imaging techniques available, near-infrared fluorescence (NIRF) imaging is emerging as a promising tool to revolutionize the medical field, providing insights into cellular and molecular events in both physiological and pathological conditions. NIRF is highly sensitive, quantitative, cost-effective and easy to operate. It also represents a powerful intraoperative tool, allowing tumour visualization 1 and subsequently highly precise tumours resection with minimal peritumoral damage. 2 However, to become a clinical reality and a routine technology, optimized NIR fluorescent contrast agents are still needed. Several types of fluorescent tracers have been developed including organic dyes, molecular-based nanoparticles, 3 dye-loaded organic or inorganic nanoparticles and fluorescent quantum dots (QDs). 4 Among them, fluorescent QDs have become extremely popular in biological and biophysical studies thanks to their high brightness and photostability. Yet, the brightest ones raise concern in terms of safety as both acute and chronic toxicity have been demonstrated. 5 At opposite, NIRF dyes may show improved biocompatibility as is the case for indocyanine green and methylene blue which are approved fluorescent tracers. Still, these dyes present several restrictions including limited brightness in water, concentration-dependent aggregation and non-specific binding to plasma proteins. 6 Nanoemulsions (NEs) are one of the best candidates among the large variety of nanocarriers currently investigated. Oil-in-water (o/w) NEs are dispersed systems of two non-miscible liquids, consisting of an oily system dispersed in an aqueous system, forming oil droplets of nanometer sizes (with diameter between 20 and 500 nm) stabilized by surfactants. NEs have been used in pharmaceutical therapy for more than 50 years and, according to the Nanotoxicological Classification System (NCS), NEs belong to the low risk class (i.e., class 1) thanks to their size (over 100 nm) and their biodegradability. 7,8 NEs as drug carriers are known to have numerous advantages over others nanosystems, including liposomes, such as long term physical a. Univ. Bordeaux, Institut des Sciences Moléculaires (CNRS UMR 5255) 33405 Talence, France. mireille.blanchard-desce@u-bordeaux.fr b. ARNA Laboratory, Team ChemBioPharm, U1212 INSERM Please do not adjust margins Please do not adjust margins stability and high loading capacity of lipophilic substances in the oily core and amphiphilic substances in the corona. 9 Despite all these advantages, NEs have not been yet fully exploited in fluorescence imaging. For dual imaging applications, NEs has been designed as multimodal contrast agents for combined ultrasound and [START_REF] Pavone | Second harmonic imaging[END_REF] F MRI (Perfluorocarbon NEs) 10 or for combined MRI and X-Ray imaging (iodine and magnetite NEs). 11 Recently, Gianella et al. 12 developed multimodal NEs carrying iron oxide nanoparticles for MRI imaging and whose outside corona was labelled with lipid-PEG conjugated with Cy7 commercial dye for NIRF imaging. Particularly interesting systems, based on solid lipid nanoparticles (Lipidots) [13][14][15] have also been developed. These nanoparticles could be loaded with various hydrophobic cyanine dyes (up to a few hundred in the lipidot core), yielding a library of tunable lipidots emitting from the visible to the NIR. These bright and biocompatible NEs show low cytotoxicity and could be successfully used for in vivo NIR fluorescence (NIRF) imaging. 14,15 However, to the best of our knowledge, the design of NEs as multimodal optical contrast agents by using a single contrast agent to generate the different modalities has not been reported yet. Yet these different contrasts might offer interesting insight into the mechanisms of NEs internalisation into cells.

In this context, our goal has been to achieve ultrabright fluorescent NEs as translational multimodal optical contrast agents that could be used both in in vitro multiphoton imaging and NIRF in vivo imaging.

To this end, we designed new dedicated dyes that show far red to NIR1 fluorescence as well as large optical nonlinearities allowing their use for multimodal linear and nonlinear optical (NLO) imaging.

Results and discussion

Design and synthesis of the dyes for multimodal optical imaging

Two new fluorescent dyes have been designed to this aim. Dyes D1 and D2 are push-pull compounds which bear strong donor and acceptor end-groups as well as thiophene moieties in the conjugated system (Figure 1). This type of hemicyanine dyes is anticipated to show strong absorption in the visible region and fluorescence emission in the far-red to NIR region, 3,16 in relation with an intramolecular charge transfer (ICT) transition from the donor to the acceptor upon excitation. For this same reason, such dyes were expected to display large two-photon absorption cross-sections (2) in the first biological transparency widow, i.e., 700-1000 nm, [16][17] as well as large quadratic hyperpolarizability . 18 Thanks to the presence of the two alkyl (nBu) chains on the terminal nitrogen and of the positively charged opposite acceptor end-group (pyridinium or quinolinium moieties), the hemicyanine dyes show amphiphilic features which were meant to bestow them high affinity for the lipid corona of NEs. Due to their non-centrosymmetric structure and amphiphilic features (contrarily to cyanine dyes), these dyes were aimed to orientate in the lipid corona of NEs or outer leaflet of lipidic membranes and generate second harmonic generation (SHG). In the last two decades, SHG microscopy has gained recognition in the bioimaging community. [START_REF] Pavone | Second harmonic imaging[END_REF] SHG microscopy has been used to image structural organization in proteins, especially collagen. [START_REF] Cicchi | [END_REF] Exogenous SHG membrane probes, including push-pull amphiphilic dyes, have also been developed as membrane dyes to probe membrane potentials. [21][22][23][24][25][26] Hence amphiphilic push-pull dyes D1 and D2 were meant to act as multimodal optical contrast agents allowing both (i) in vitro multiphoton imaging by simultaneous tow-photon excited fluorescence (TPEF) and SHG microscopy, [23][24][25][26][27][28][29][30] (ii) monitoring the fate of the encapsulated dyes in cancer cells at the sub-cellular level and (iii) in vivo imaging of tumours in small animal using NIRF. The D1 and D2 dyes were synthetized as described in Scheme 1, starting from commercially available N,N-dibutyl-aniline. After iodination of N,N-dibutyl-aniline leading to 1 (67%), 1 was coupled to 5-formyl-2-thiopheneboronic acid or 5-formyl-2-2bithiopheneboronic acid using a Suzuki-Miyaura-type reaction to yield aldehyde 2a or 2b (60% and 73% respectively). The Wittig-Horner condensation between the aldehydes and the phosphine oxide 3p or 3q led to pyridine or quinoline 4a or 4b (85% and 81% respectively) which further gave dyes D1 and D2 after methylation with dimethylsulfate. Reagents 3p and 3q were prepared in two steps from 4-picoline or 4-methylquinoline in a 33 or 57 % global yield respectively: the first step involved lithiation of 4-picoline or 4methylquinoline with LDA followed by reaction with chlorodiphenylphosphine; in the second step, oxidation of the obtained intermediate phosphine with oxygen in refluxing toluene was carried out leading to compounds 3p and 3q.

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Absorption and fluorescence properties of the D1 and D2 dyes

As illustrated in Figure 2, the D1 and D2 dyes show an intense lowenergy absorption band in the visible region (with maximum molar absorption coefficient of 3.7 10 4 M -1 cm -1 and 3.9 10 4 M -1 cm -1 in chloroform for D1 and D2 respectively) whose position strongly depends on the polarity of their environment. A marked red-shift of the low-energy absorption band is observed on going from low polarity solvents to medium polarity solvents (Table 1). This behaviour is consistent with a photo-induced intramolecular charge transfer (or charge shift) from the donor end-group to the acceptor end-group resulting in an increase of the dipole moment upon excitation (Fig. 1).

The D1 and D2 dyes show fluorescence in organic solvents of low to medium polarity and emit in the far-red NIR1 region (Fig. 2). Similar to the absorption, the emission shows a marked positive solvatochromism in low to medium polarity organic solvents. A pronounced red-shift is observed on going from a low polarity solvent (max em = 707 and 740 nm in toluene for D1 and D2 respectively) to a medium polarity solvent (max em = 915 and 876 nm in dichloromethane). Again, this is consistent with a charge shift from the dialkylamino donor moiety to the pyridinium or quinolinium acceptor end-group upon excitation leading to larger dipole moment. We also note that the fluorescence quantum yield decreases dramatically with increased polarity. As a consequence, dyes D1 and D2 show vanishing fluorescence in medium polarity environments and are not fluorescent in polar solvents. Due to the strong dependency of their absorption and fluorescence properties on the environment, the D1 and D2 dyes thus represent interesting environment probes. In addition, we note that the dyes show large Stokes shift values (Table 1), which is of interest for use in fluorescence imaging as it allows easier detection. This provides an advantage for fluorescence imaging compared to commonly used NIR fluorescent dyes such as cyanines which show very small Stokes shift values (typically 320 cm-1 for Cy5, 530 cm -1 for Cy5.5 and 400 cm -1 for Cy7 ). 1). This is further confirmed by the less marked solvatochromism of dye D2. This indicates that hemicyanine dye D2 has an electronic structure which is closer to the cyanine limit than dye D1. Hence the electronic distribution in the ground state of D2 is slightly different with small positive charge on the electron-donating dialkyamino end-group in the ground state. This difference in electronic distribution is consistent with the shortest conjugated path and stronger acceptor end-group of D2 as compared to D1. 17 This difference in electronic distribution of the positive charge along the molecular axis is expected to lead to more tilted orientation (or more disordered organisation) into the lipid corona of the NE droplet and thus lower SHG activity. 26,30 Thanks to their amphiphilic character, both dyes could be dissolved in water using SDS as a surfactant. We note that both absorption and emission spectra are close to those measured in a low polarity solvent like toluene (Table 1 and Fig. S1), indicating that the dyes are protected from the water environment. Furthermore, the fluorescence quantum yields are higher in micellar environment thanks to the local viscosity which reduces non-radiative decay processes

Nonlinear optical properties of the D1 and D2 Dyes

The two-photon absorption (2PA) properties of the dyes were determined by conducting two-photon induced fluorescence (TPEF) experiments. The measurements were carried out in micellar water which ensures both good solubility and fluorescence of the dyes (vide supra). The TPEF measurements demonstrate that the both dyes show large two-photon absorption responses in the 1050-1150 nm spectral region (Fig. 3). Both dyes show maximum 2PA cross-sections (2) of about 800 GM (830 GM at 1060 for dye D1 and 785 GM at 1160 nm for dye D2). The 2PA bands of dye D2 are red-shifted compared to that of dye D1 as noticed for the corresponding lowenergy intense one-photon absorption band. Interestingly, an intense 2PA band is observed at higher energy which corresponds to a one-photon weakly allowed transition (Fig. S2). As a result, both dyes also show large 2PA close to 800 nm (565 GM at 820 nm for dye D1 and 672 GM at 830 nm for D2). Yet, we stress that when getting close to 800 nm, dye D2 two-photon absorption's overlaps with onephoton absorption as noted from the deviation to quadratic dependency of the fluorescence signal on the excitation intensity (Fig. S3). A quadratic dependency is observed only at wavelengths longer than 830 nm. At opposite, dye D1 shows pure two-photon excitation at 800 nm (Fig. S4).

These features indicate that dyes D1 and D2 can be used for twophoton excitation both (i) at long wavelengths (1050-1100 nm) allowing improved penetration and reduced background fluorescence in deep tissues and (ii) close to 800 nm which is the preferred wavelength to allow maximum output power when using popular lasers used in two-photon microscopy (i.e. Ti-sapphire laser). This makes dyes D1 and D2 of much interest for two-photon imaging by allowing both excitation and detection in the NIR1 region. We note that dye D2 shows larger 2PA responses close to 800 nm than dye D1 whereas dye D1 shows larger 2PA responses at 950 nm and in the 1050-1100 nm region.

Preparation and characterisation of nanoemulsions loaded with dyes D1 and D2

NEs loaded with the D1 and D2 dyes were then prepared according to previously reported procedure. [START_REF] Prevot | Clofent-This journal is[END_REF] Briefly, the dye is dissolved in an oily phase containing Lipoid® E80 (a mixture of egg yolk lecithin designed for parenteral use) in Miglyol® 840. An aqueous phase containing Tween® 80 (a polysorbate emulsifier used in parenteral formulation) is added and the NEs formulations are obtained after emulsification and homogenisation by sonication. Turbid orange NE[D1] and purple for NE[D2] emulsions were realized.

The dye loading did not impair the NEs diameter or polydispersity index (PdI) (Table 2 and Table S1). TEM image (Fig. 4) showed that the nanodroplets have a spherical shape with a size ranging from 60 to 600 nm in line with the Gaussian distribution obtained by DLS. A colloidal stability study of the NEs was performed over a period of one year by evaluating the evolution of the droplet diameter and PdI (Table S2). A slight increase of PdI and mean diameter were noticed over time, but formulation diameter remained submicronic and monodisperse (PdI <0.2). 32

Table 2. Properties of the dye-loaded NEs

Linear and nonlinear optical properties of the NEs

The investigation of the optical properties of the NE[D2] further confirms the positioning of the D1 and D2 dyes within NEs and provides additional information on their orientation (Fig. 5). We note that the excitation spectra of the dye when encapsulated in NE emulsions are significantly blue-shifted (by 1250 cm -1 for both dye D1 and D2) in comparison with the absorption spectra of the dye in a toluene (Tables 12). This blue-shift reveals a destabilisation of the Franck-Condon excited state. This is consistent with a photo-induced intramolecular charge transfer from the donor end-group to the acceptor moiety leading to a fractional positive charge on the donor end-group in the excited state (Figure 1) and a decrease of the positive charge on the acceptor end-group. Due to the positioning of the donor end-group in the oily core (where no negative counter-ion is present), and the lower positive charge at the water interface, the FC excited state is thus destabilized. In contrast, we note that the emission characteristics are similar to those measured in toluene (Table 12). This suggests that the dyes tend to adopt a more tilted orientation in the relaxed emitting state. As a result, both dyes loaded NEs show NIRF emission (Fig. 5). The fluorescence of NE[D2] remains red-shifted compared to that of NE[D1], as is the case for its absorption band.

In order to evaluate the brightness of the NE droplets, the number of dyes incorporated in each droplet was determined. The droplet concentration was measured using nanoparticle tracking analysis. The dye concentration was measured by absorption after lyophilisation of a NEs aliquot and subsequent dissolution in chloroform of the dyes which are well soluble as isolated units in that solvent. This protocol allows to get rid of scattering and to rule out any shadowing or aggregation effect which may occur in NEs. Thanks to their amphiphilic character, high loading of dyes could be achieved; with loading amounting to 2000 to 3500 dyes per nanodroplet (Table 2). Due to the encapsulation of a large number of strongly absorbing dyes per droplet, the NEs were thus found to show very large brightness as NIRF contrast agents (respectively 7 10 6 M -1 cm -1 and 5 10 6 M -1 cm -1 for dye D1 and D2), demonstrating their potential as contrast agents for NIRF in vivo imaging.

To further attest the potential of NE[D1] and NE[D2] for in vitro multiphotonic bioimaging, we investigated the two-photon absorption properties of these nano-objects by using two-photon excited fluorescence (TPEF) spectroscopy. As illustrated in Figure6, both NEs show very large two-photon absorption with a lowest energy 2PA maximum located at 1050 and 1080 nm respectively for NE[D1] and NE[D2] with very large 2PA response in the 1000-1100 nm range. A second local maximum is observed around 950 nm.

As a result, the nanodroplets show giant two-photon brightness (Table 2) S5). The ability of the bare NE[D2] nanocarriers was estimated in epidermoid carcinoma A431 cancer cells. Cellular uptake of the NIRF-NEs was confirmed by both confocal and two-photon fluorescence microscopy on living cells. As shown in Fig. 7, intense fluorescence signal is detected in the whole cytoplasm but none in the nucleus (Fig. 7A-B). The NIR fluorescence of the dye and its large brightness allowed reducing autofluorescence thus revealing a significant uptake of the D2 dye into the cells (Fig. 7A-B). Moreover, the optical properties of the dye allow both 2P excitation (950 nm) and detection (650-800 nm range) in the NIR range, resulting in total suppression of background fluorescence (Fig. 7C) and revealing the precise localisation of the dye in the cellular environment. In particular, we observe that numerous "hot spots" concentrate a large amount of the fluorescence signals suggesting accumulation of the dyes into specific subcellular compartments. Their tiny size suggests that they are endosomes which have trapped the NEs during the endocytosis process. Interestingly, Second Harmonic Generation (SHG) imaging provides additional information. As noted from Fig. 7D, these spots generate SHG signal, indicating that the dye D2 is oriented in an asymmetric way. This reveals that dyes D2 are located in the outer leaflet of lipid membrane as a result of fusion between the NEs and the membrane of the endocytosis vesicles. The push-pull nature of the dye combined with its confinement and preferential orientation into the vesicle's membrane makes it a powerful SHG emitter [27][28][29] explaining the strong SHG signal which exclusively arises from the vesicles. In order to use these dye-loaded NEs for in vivo imaging of tumours, stealth strategies were developed to reduce the opsonisation with plasma protein and to prolong circulation time of the engineered NIRF-NE. The gold standard strategy is to create a dense layer of polyethylene glycol (PEG) macromolecules on the nanodroplet surface. PEGylation reduces non-specific protein adsorption and cellular uptake. 33 PEG have the advantages of low-toxicity and lowimmunogenicity. Based on this, we prepared PEGylated NEs loaded with dye D1 and D2. Their optical properties were found to be similar to those of bare NEs (Table S3). We note that the dye-loaded PEGylated NEs show negative surface potentials due both to the negative charge of the DSPE PEGylated lipid and negatively charged phospholipids (i.e. phosphatidylethanolamine) contained in lecithin used for the preparation of standard nanoemulsions (see experimental section).

Investigation of dye loaded NE internalisation in cancer cells by multimodal optical imaging

As our final goal was to develop nano-tools as theranostics agents and adjuvant treatment during surgery of glioblastoma cancer, we first performed multimodal optical imaging on U87 cells which are model cancer cells for glioblastoma. These studies were aimed at gaining information on the kinetics of the cell internalisation of the dye-loaded NEs into the cancer cells. Our experiments do show that the uptake of the PEGylated NEs in U87 cells is indeed fast. As soon as the U87 cells are incubated with NEs, we could observe the uptake of the NEs in the first few minutes (Fig. 8 The fate of the NEs formulated with DSPE-PEG can be monitored by two-photon (Fig. S7) and SHG microscopy as well (Fig. 9). Strikingly, the SHG imaging suggests that the dye D1 is localized both in the outer membrane cell (indicating fusion of the NEs with the cell membrane) and endocytosis vesicles, while the SHG response of generated by dye D2 is lower and located only in the endosomes. The lower SHG signal generated by dye D2 might be related to a less favourable orientation (tilt angle as well as orientational disorder) 26,30 in the lipid membrane due to its different electronic distribution and shorter size as compared to dye D1. Indeed the stronger electron-withdrawing hydrophilic moiety and the shorter conjugated connector of the push-pull dye D2 induce a more pronounced partial intramolecular charge transfer in the ground state. 34 This results in an electronic distribution closer to the socalled cyanine limit for dye D2. Such difference is confirmed by the red-shifted absorption and smaller Stokes shift value of dye D2 compared to dye D1 (Table 1). This different electronic distribution results in a less marked positive charge on the hydrophilic end-group of D2 dye and concomitant slight positive charge on the donating hydrophobic end-group. Such variation is less favourable to the ideal orientation shown in Scheme 2. We note that a tilted orientation was reported for an amphiphilic push-pull membrane probe having the same hydrophilic moiety. 26 The resonance factor is also expected to significantly increase the SHG action cross-section (SHG) of dye D1 compared to that of dye D2. Yet, increasing the incubation time of NE[D2]-PEG to two hours leads to an enhancement of the SHG response (Fig. S6). This further confirms its presence in endocytosis vesicles and slower internalisation. 12C). However, fluorescence signal in tumours remained low when compared to the fluorescent signal in the liver on exited organs 24 hours after injection (Fig. S12). The in vivo NIRF fluorescence pattern is consistent with previously reported data using the RM1 sub-cutaneous mouse model after injecting NE 15 or other types of nanoparticles. 35 This is also consistent Please do not adjust margins Please do not adjust margins with many other types of nanoparticles and tumour models in mice. Accumulation in the liver results from fluorescent probe capture by Kupffer cells while accumulation in the tumour originates from the enhanced permeability and retention (EPR) effect. 36 The much lower fluorescence signal observed after injection with NE[D2]-PEG cannot be related to the excitation (535 nm) or imaging conditions (detection with the Cy5.5 filter in the 695-770 nm range) as they are more favourable to NE-[D2]-PEG than NE-[D1]-PEG (see Figure 5). This suggests that the NE metabolism and carrier behaviour is influenced by the presence of the dyes which modify their in vivo properties. This parallels the in vitro observation of slower kinetics of internalisation of the dyes of NE[D2]-PEG in U87 cells compared to those of NE[D1]-PEG.

Conclusions

Tailor-made amphiphilic elongated dyes having thiophene units in their conjugated path have been synthesized. They combine NIR emission in the 600-800 nm range, sizeable Stokes shift values, large two-photon absorption in the whole 800-1100 nm range (thus allowing NIR to NIR excitation/emission by two-photon excitation) and large second-order NLO response. These dyes were demonstrated to show high loading in NEs which are attractive nanocarriers for pharmaceutical use. Few thousands of these dyes could be loaded in the lipid corona of NEs. As a result NIR emitting NEs showing giant one-and two-photon brightness' were obtained. Strikingly the incorporation of these dyes also was shown to affect the properties of the NEs, conferring positive surface potential to NEs.

Thanks to the unique linear and nonlinear optical properties of the dyes, the carrier behaviour of the dye-loaded NEs into live cancer cells could be monitored by multimodal nonlinear optical imaging (i.e. combined TPEF and SHG microscopies) clearly evidencing the endocytosis process via SHG monitoring of endocytosis vesicles. Interestingly, the kinetics of the internalisation of the PEGylated dyeloaded NEs into live U87 cancer cells (model cells for glioblastoma) was found to depend on the electronic distribution of the dye. The hemicyanine dye having the positive charge more confined on the acceptor hydrophilic end-group shows the fastest internalisation, intermediate accumulation in the cell membrane and subsequent uptake in lysosomes. In contrast, the dye having more delocalised distribution of positive charge (i.e. closer to the cyanine type) shows slower internalisation into cancer cells as well as partial uptake in the mitochondria and no accumulation in cell membranes. Interestingly, the NEs showing the fastest internalisation into U87 cancer cells were also shown to allow in vivo NIRF imaging of tumours in mice. As such the multimodal NEs are of much interest and could be further elaborated for the design of theranostic multifunctional nanoparticles by loading lipophilic cytotoxic drugs in the oily core and grafting targeting agents (such as fragment of antibodies 37 ) on the surface to yield new theranostics agents and adjuvant treatment during surgical resection of tumours.

Experimental

Materials and methods: All air-or water-sensitive reactions were carried out under argon atmosphere. Solvents were distilled freshly using standard protocols. The starting materials were purchased from Aldrich, TCI and Alfa Aesar. Reactions were monitored by thin layer chromatography on Merck silica gel 60F254 precoated aluminium sheets. Column chromatography was performed on Fluka silica gel 60 (40-63 μm). 1 H, 31 P and 13 C NMR spectra were recorded on Bruker AVANCE I 300, AVANCE II 400, AVANCE III 600 spectrometer. Chemical shifts are given in parts per million with respect to residual solvent peak and coupling constant (J) are given in Hertz. Melting points were measured on Stuart SMP 10. IR spectra were recorded on Perkin Elmer Spectrum 100 FT-IR spectrometer on solid samples. High-resolution mass spectra were performed by the CESAMO (Bordeaux, France) using an FD emitter. LC/MS analyses were performed on a Shimadzu LCMS-2020. Elemental Analyses were carried out by the ''Institut de Chimie des Substances Naturelles'' (Gif-sur-Yvette, France). Pharmaceutical neutral oil phase Miglyol® 840 (oil based on propylene glycol dieter of C8 and C10 saturated plant fatty acid) was provided by CREMER OLEO GmbH & Co.KG (Hamburg, Germany). FDA approved surfactants were acquired from the following companies: egg lecithin containing 82.3% phosphatidylcholine (Lipoid® E80, Lipoid GmbH Ludwigshafen, Germany); Polysorbate 80 (Tween® 80, SEPPIC, France). PEGylated phospholipids: PE 18:0/18:0-PEG 2000 (N-(Carbonylmethoxypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero-3phosphoethanolamine) were kindly provided by Lipoid. Glycerol was purchased at Cooperation Pharmaceutique Française (Melun, France). Glucose 5 (G5) was acquired from Baxter (Guyancourt, France).

Synthetic procedures

Synthesis of the dyes D1 and D2.

5'-(4-(dibutylaminophenyl)bis-2,2'-thiophene)carboxaldehyde (2b):

To an oven dried round bottom flask cooled under argon, were added 4-iodo-N,N-dibutylaniline (1.21 g, 3.65 mmol), 5'-formyl-2,2'bithiophene-5-boronic acid (0.95 g, 4.01 mmol), K2CO3 (1.25 g, 9.12 mmol), Pd(dppf)Cl2 (148 mg, 0.18 mmol), toluene (9 mL) and methanol (9 mL). The mixture was stirred at 80 °C for 6 h. The mixture was filtered through a pad of celite, rinsed with dichloromethane then the filtrate was concentrated under vacuum. The residue was further purified by silica-gel column chromatography using a 1/1 dichloromethane/petroleum ether mixture to yield 1 g (73 %) of the desired compound 2b as an orange solid. M.p = 110-111°C. 1 H NMR (300 MHz, CDCl3)  9.83 (s, 1H) 7.65 (d, J = 4 Hz, 1H), 7.45 (d, J = 8.9 Hz, 2H), 7.29 (d, J = 3.8 Hz, 1H), 7.20 (d, J = 4 Hz, 1H), 7.07 (d, J = 3.9 Hz, 1H), 6.63 (d, J = 8.9 Hz, 2H), 3.30 (t, J = 7.6 Hz, 4H), 1.59 (quint, J = 7.5 Hz, 4H), 1.37 (sext, J = 7.4 Hz, 4H), 0.96 (t, J = 7.3 Hz, 6H); 13 Please do not adjust margins Please do not adjust margins 4-(Diphenylphosphinoylmethyl)pyridine (3p): 4-picoline (3.07 g, 33 mmol, 3.2 mL) was dissolved in 100 mL of anhydrous THF. The solution was cooled to -78°C and 1 equivalent of LDA (obtained in situ with 33 mmol, 14 mL of 2.36 M of BuLi in hexane added to 33 mmol, 3.31 g (4.5 mL) of anhydrous diisopropylamine in 23 mL of anhydrous THF) was added dropwise. After leaving the orange solution at -78°C for 2 h, chlorodiphenylphosphine, (7.2 g, 33 mmol, 5.9 mL) was added and the solution was stirred for 30 minutes at -78°C. Then, the reaction mixture was slowly allowed to warm to room temperature. The solvent was concentrated under vacuum, and the residue was extracted with dichloromethane and the organic phase was washed with water, dried over anhydrous Na2SO4. The solvent was concentrated under vacuum. The obtained oily product was dissolved in 150 mL of toluene and the solution was refluxed while bubbling air for 16 h. After cooling to room temperature, the mixture was filtered and the solid was collected and rinsed with toluene to afford 2.95 g of 3p as a white solid (yield 33 %). 1 aniline (4a): 3q (306 mg, 1.04 mmol), 2a (300 mg, 0.95 mmol), NaH (60% in mineral oil, 46 mg, 1.90 mmol) and 18-C-6 crown ether (catalytic were suspended in 20 mL de THF. The mixture was stirred at room temperature overnight. The excess of NaH was quenched by slow addition of a small amount of water to the reaction mixture then THF was evaporated under vacuum. The product was extracted with dichloromethane, and the organic phase was washed with water, dried over anhydrous filtered, and concentrated under reduced pressure. The residue was purified by silica-gel column chromatography using 8/2 dichloromethane/ethyl acetate mixture to yield 350 mg (85 %) of 4 as a red oil. 1 H NMR revealed the presence of a small proportion of the Z isomer (less than 10%). 1 Dibutyl-4-((5-(pyridin-4-yl)bis-2,2'-thiophen-5'-yl)ethynyl) aniline (4b): 3p (280 mg, 0.86 mmol), 2b (361 mg, 0.91 mmol), NaH (60% in mineral oil,44 mg, 1.80 mmol) and 18-C-6 crown ether (catalytic amount) were suspended in 20 mL de THF. The mixture was stirred at room temperature for overnight. The excess of NaH was quenched by slow addition of a small amount of water to the reaction mixture then the THF was concentrated under vacuum. The obtained precipitate was collected by filtration, washed with diethyl ether to afford the product as an orange solid (330 mg, 81%). M.p = 190°C. 1 H NMR (300 MHz, CDCl3)  8.54 (d, J = 6.1Hz, 2H), 7.44 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 15.9 Hz, 1H), 7.31 (d, J = 6.1 Hz, 2H), 7.14 (d, J = 3.8 Hz, 1H), 7.05 (m, 3H), 6.75 (d, J = 16.0 Hz, 1H), 6.63 (d, J = 8.8 Hz, 2H), 3.30 (t, J = 7.6 Hz, 4H), 1.59 (quint, J = 7.6 Hz, 4H), 1.37 (sext, J = 7.4 Hz, 4H), 0.96 (t, J = 7.3 Hz, 6H). 13 C { 1 H} NMR (75 MHz, CDCl3):  150. 1, 147.8, 145.4, 144.3, 139.5, 138.6, 133.5, 129.2, 126.8, 125.9, 125.1, 124.6, 123.2, 121.1, 120.8, 120.4, 111.6, 50.7, 29.4, 20.3, 13.9

N,N-

D1:

To a solution of 4b (60 mg, 0. 12 mmol) in 1.5 mL of chloroform were added 23 μL of dimethyl sulfate (32 mg, 0.25 mmol) under argon and the reaction mixture was stirred at room temperature overnight. Then, the solvent was partially evaporated and diethyl ether was added. The obtained precipitate was filtered, rinsed with diethyl ether and dried under vacuum to give 64 mg (90 %) of compound D1 as a black solid. M.p = 176°C. 1

D2:

To a solution of 4a (54 mg, 0. 12 mmol) in 3.5 mL of diethyl ether were added 25 μL of dimethyl sulfate (34 mg, 0.27 mmol) under argon and the reaction mixture was stirred at room temperature overnight. The obtained precipitate was filtered, rinsed with diethyl ether and dried under vacuum to give 60 mg (86 %) of the dye as a black solid (m.p. = 207°C). 1 H NMR (300 MHz, CDCl3)  9.31 (d, J = 6.6 Hz, 1H), 8.41 (d, J = 8.5 Hz, 1H), 8.07-8.12 (m, 2H), 8.02 (t, J = 7.5 Hz, 1H), 7.91(d, J = 15.3 Hz, 1H), 7.78-7.84 (t, J = 7.5 Hz, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 3.9 Hz, 1H), 7.28 (d, J = 15.3 Hz, 1H), 7.07 (d, J = 3.9 Hz, 1H), 6.62 (d, J = 8.5 Hz, 2H), 4.49 (s, 3H) 3.80 (s, 3H). 3.32 (t, J = 7.4 Hz, 4H), 1.60 (quint, J = 7.3 Hz, 4H), 1.38 (sext, J = 7.4 Hz, 4H), 0.97 (t, J = 7.3 Hz, 6H). 13

Preparation of the nanoemulsions

Standard nanoemulsions: A stock solution of Lipoid® E80 in oil was obtained by dispersion of Lipoid® E80 (120 mg) in Miglyol® 840 (2 g). In order to obtain a complete dispersion, mixture was heated to 70°C. The dye (1.2 mg) was added to the stock solution (423 µL) in order to obtain 5 mM concentration. After 10 min sonication in an ultrasonic bath heated to 70°C, a purple dispersion was obtained. The aqueous phase was prepared by dispersion of Tween® 80 (125 mg) in 4 ml Milli-Q. Emulsion was then obtained by phase inversion (800 mg of aqueous phase (Milli-Q/ Tween® 80) was added to 200 Please do not adjust margins Please do not adjust margins mg of oily phase) followed by homogenization during 7 min sonication in the ice bath using a Sonic Vibra-Cell VC 250 set at 70% and output 7. A milky droplet suspension was obtained.

Stealth nanoemulsions:

To prepare stealth NEs incorporating the dye, 15 mg of PEGylated phospholipids (PE 18:0/18:0-PEG 2000) were added to the oily phase before homogenisation. For in vitro and in vivo experiments, the formulations pH was adjusted close to physiological value (pH: 7.2) using sodium hydroxide 0.1 N. Glycerol at 2% was added to adjust osmolality at 300 mOsm/kg. NE was visually inspected for eventual creaming, phase separation and/or dye precipitation. After preparation and prior to use in imaging, formulations were stored at 4°C, away from light and a colloidal stability assay (over 12 months) was accomplished by visual observation, granulometric analysis and zeta potential measurements over time.

NE characterization:

The droplet physical properties (sizes and polydispersity index) were measured using a dynamic light scattering (DLS) device from Malvern Instruments (Zetasizer Nano ZS). The droplets were determined directly after formulation (dilution 1/1000). The mean size was determined with three independent measurements performed at 25°C. The Zeta potential was determined by using a Zetasizer device from Malvern Instruments after dilution (1/1000) using a Folded Capillary Cell (DTS1070, Malvern Instruments). Droplets concentration was investigated using a NanoSight NS300 from Malvern Instruments and was determined with five independent measurements. Nanoparticle tracking analysis was performed at 22°C on samples after dilution (1/250 000).

TEM was performed at Bordeaux Imaging Center on a Hitachi H7650 linked to an ORIUS SC1000 11MPX (GATAN) camera run by Digital Micrograph (GATAN). All formulations were diluted 1/50 and transferred to a carbon-coated copper grid. Negative staining was performed with uranyl acetate for 1 minute.

Determination of the dye loading in

Photophysical studies

UV-Vis absorption and emission spectroscopy: All photophysical properties were investigated with freshly prepared air-equilibrated solutions at room temperature (293 K). UV/Vis absorption spectra were recorded with a Jasco V-670 spectrophotometer. Steady-state fluorescence measurements were performed on dilute solutions (optical density < 0.1) contained in standard 1 cm quartz cuvettes by using a Horiba (FluoroLog or FluoroMax) spectrometer in photoncounting mode. Fully corrected emission spectra were obtained for each compound at  ex =  Abs max with an optical density at  ex of ≤0.1 to minimize internal absorption. Fluorescence quantum yields were measured according to literature procedures 38,39 by using 4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM) in EtOH (f= 0.437) and Indocyanine green in DMSO (f = 0.11) as standards.

Two-photon absorption: Two-photon absorption cross sections (σ2) were determined from the two-photon excited fluorescence action cross-section (σ2f) and the fluorescence quantum yield (f). TPEF cross sections were measured relative to Fluorescein in 0.01 M aqueous NaOH in the 690-1000 nm spectral range and relative to Nile Red in DMSO in the 1000-1160 nm spectral range, using the method described by Xu and Webb 40 and the appropriate solventrelated refractive index corrections. 41 Reference values between 700 and 715 nm for Fluorescein were taken from literature. The quadratic dependence of the fluorescence intensity on the excitation power was checked at all wavelengths. Measurements were conducted using an excitation source delivering fs pulses. This allows avoiding excited-state absorption during the pulse duration a phenomenon that has been shown to lead to overestimated two-photon absorption cross-section values. To scan the 680-1080 nm range, a Nd:YVO4-pumped Ti:Sapphire oscillator was used generating 140 fs pulses at a 80 MHz rate. To scan the 1000-1400 nm range, an OPO (PP-BBO) was added to the setup to collect and modulate the output signal of the Ti:Sapphire oscillator. The excitation was focused into the cuvette through a microscope objective (10X, NA 0.25). The fluorescence was detected in epifluorescence mode via a dichroic mirror (Chroma 675dcxru) and a barrier filter (Chroma e650sp-2p) by a compact CCD spectrometer module BWTek BTC112E. Total fluorescence intensities were obtained by integrating the corrected emission. The experimental uncertainty of the absorption crosssection values determined from this method has been estimated to be ±10%.

Live cells multimodal optical imaging

The preparations of the A431 and U87 Human cells for in vitro live cell imaging are described in the SI.

The concentrations of the NEs for the imaging on live A431 and U87 cells were set at 1/100 and 1/1000 of the nominal concentration respectively. For co-localization experiments, we followed the staining procedure recommended by the MitoTracker and LysoTracker supplier (Molecular Probes). Thus the concentrations were set at 200 nM and 100 nM for MitoTracker and LysoTracker respectively. Complete description of the conditions and imaging procedure are given in the SI.

The confocal microscope used a Leica TCS SP5 on an upright stand DM6000 (Leica Microsystems, Mannheim, Germany), equipped with the objective HCX APO L U-V-I 63.0X water NA 0.90. For confocal microscopy, the excitation source was a 488 nm laser and the photons were collected in the 650-800 nm (A431 cells) and 610-800 nm (U87 cells) wavelength range. The multiphoton microscopy was performed with a tuneable pulsed laser Mai Tai HP (Spectra-Physics, Irvine, USA), tuned at 896 nm (output average power of 1.76W). Photons were collected by an internal PMT in the 650-800 nm (A431 cells) and 610-800 nm (U87 cells) wavelength range. The Second Harmonic Generation (SHG) acquisitions were done with the same excitation wavelength (896 nm) in the transmitted light

Fig. 1 :

 1 Fig. 1: Design of the amphiphilic push-pull dyes D1 and D2. Upon excitation, a charge shift from the donor end-group (in blue) towards the acceptor end-group (in red) occurs leading to partial positive charge on the donating amino moiety and corresponding decrease of positive charge on the pyridinum (or quinolinium) acceptor.

Scheme 1 :

 1 Scheme 1: Synthesis of the D1 and D2 dyes Thanks to the presence of the two nBu alkyl chains, the D1 and D2 dyes are soluble in a range of organic solvents of interest for their encapsulation in NEs. In contrast, the dyes are non-soluble in water but are readily dispersed in water in the presence of surfactants. Their amphiphilic character was meant to favour high loading into NEs corona and preferential radial average orientation (Scheme 2), thus ensuring maximal absorption.

Scheme 2 :

 2 Scheme 2: Incorporation of amphiphilic dyes D1 and D2 into NEs made from lecithins

Fig. 2 .

 2 Fig. 2. Absorption and emission spectra of the D1 (top) and D2 (bottom) dyes in solution in toluene (blue), CHCl3 (green) and CH2Cl2 (red).

Fig. 3 .

 3 Fig. 3. Two-photon absorption spectra of dyes D1 (black) and D2 (red).

  average diameter determined by DLS, b) average number of D1 or D2 dyes per droplet, c) NE droplet two-photon brightness, d) standard: DCM in EtOH (f =0.437).



  TPA (nm)Please do not adjust margins Please do not adjust margins

Fig. 4 .

 4 Fig. 4. TEM image of the NEs loaded with the D2 dye.

Fig. 5 .

 5 Fig. 5. Excitation and emission spectra of NE[D1] (black) and NE[D2] (red).

  , making them of high interest as fluorescent probes for NIR (two-photon excitation) to NIR (NIRF detection) in vitro imaging. In particular, both NE[D1] and NE[D2] show two-photon brightness of about 10 5 GM in the 1020-1050 nm, with NE[D2] showing somewhat larger brightness (Fig.6). On the other hand, NE[D2] shows larger two-photon brightness than NE[D1] close to 800 nm. Finally, NE[D1] and NE[D2] show similar two-photon brightness (i.e. 5 10 4 GM) around 950 nm. Hence these three wavelengths will be preferred for monitoring cellular uptake of the NEs by two-photon fluorescence imaging.

Fig. 6 .

 6 Fig. 6. Two-photon brightness of NE[D1] (black) and NE[D2] (red).

  ) This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins Their large (one-and two-photon) brightness's make NE[D1]s and NE[D2]s suitable for monitoring the fate of dye-loaded NEs in cellular environments and the kinetics of their cell internalisation. Having in mind their potential use as theranostics tools, we first investigated their internalisation into cancer cells. We previously checked the safety of the NE[D1], NE[D2] as well as the NE[D2]-PEG and NE[D2]-PEG nanocarriers (Fig.

Fig. 7 .

 7 Fig. 7. Confocal imaging of live A431 cells incubated with NE[D2] (A) Bright-field. (B) Fluorescence imaging ( exc = 488 nm). (C). Two-photon imaging ( exc = 950 nm). (D) Second Harmonic Generation (SHG) imaging ( exc = 896 nm). Except for bright-field and SHG, photons were collected in the 650-800 nm range.

Fig. 8 .

 8 Fig. 8. Confocal imaging of the accumulation of NEs in live U87 cells. Fluorescence imaging ( exc = 561 nm) 2 min (A) and 42 min (B) after the addition of NE[D1]-PEG. Fluorescence imaging ( exc = 561 nm) 7 min (C) and 47 min (D) after the addition of NE[D2]-PEG. (E) Averaged quantification of fluorescence signal of NE[D1]-PEG (red line) in U87 cells (grey error bars, n = 5). (F) Averaged quantification of fluorescence signal of NE[D2]-PEG (red line) in U87 cells (grey error bars, n = 5). The photons were collected in the 610-800 nm range. Changes of the averaged fluorescence signal with time was calculated on 5 cells and for both NEs it shows a linear increase of accumulation of fluorescence signal into the cells.

Fig. 9 .

 9 Fig. 9. Multimodal optical imaging of NEs in live U87 cells. Bright-field, Confocal imaging ( exc = 561 nm), Two-photon imaging ( exc = 1020 nm for NE[D1]-PEG,  exc = 820 nm for NE[D2]-PEG) and Second Harmonic Generation imaging ( exc = 896 nm) of live U87 cells incubated with NEs. Except for bright-field and SHG, photons were collected in the 610-800 nm range. Co-localization experiments were then conducted to gain information on the fate of the dye after internalisation of the NEs by endocytosis. The co-localization experiments with LysoTracker (Fig. 10 and Figs. S8-S9) clearly evidence the presence of the dyes in the lysosomes logically resulting from the fusion of endocytosis vesicles with lysosomes.

Fig. 10 .

 10 Fig. 10. Co-localization imaging of NEs with lysosomes in live U87 cells. Bright-field, Confocal imaging of NEs ( exc = 561 nm), Confocal imaging of LysoTracker Blue DND-22 ( exc = 405 nm) and merged image of NEs and LysoTracker confocal imaging of live U87 cells incubated with NE[D1]-PEG or NE[D2]-PEG and LysoTracker Blue DND-22. Values of Pearson's correlation coefficient are indicated as inset in the merged images. The photons were collected in the 610-800 nm range for NEs and 415-480 nm range for LysoTracker. Interestingly, we also observed co-localization of the fluorescence in the mitochondria in the case of U87 cells incubated with NE[D2]-PEG. Such phenomenon was not observed upon incubation with NE[D1]-PEG (Fig. 11 and Figs. S10-S11). We also note that U87 cell membranes are stained only when incubated with NE[D1]-PEG (and not with NE[D2]-PEG) which is in good agreement with SHG imaging data. This reveals that the nature of the dye modifies the internalisation pathways. NE-loading with dye D2 allows NE[D2]-PEG to partly escape from the lysosomes to reach other organelles and particularly mitochondria whereas fusion of NE[D1]PEG with the U87 cell membrane occur.

Fig. 11 .

 11 Fig. 11. Co-localization imaging of NEs with mitochondria in live U87 cells. Bright-field, Confocal imaging of NEs ( exc = 561 nm), Confocal imaging of MitoTracker GreenFM ( exc = 488 nm) and merged image of NEs and MitoTracker confocal imaging of live U87 cells incubated with NE[D1]-PEG or NE[D2]-PEG and MitoTracker Green FM. Values of Pearson's correlation coefficient are indicated as inset in the merged images. The photons were collected in the 610-800 nm range for NEs and 500-550 nm range for MitoTracker.

Fig. 12 .

 12 Fig. 12. Time course of D1 and D2 fluorescence signal in mice bearing subcutaneous tumours (A) Fluorescence reflectance imaging of a representative mouse at different time after NE[D1]-PEG or NE[D2]-PEG injection. (B) Bioluminescence imaging of a representative mouse. (C) Quantification of fluorescence signal in the tumour after NE[D1]-PEG (black) or NE[D2]-PEG (red) injection (Mean +/-standard deviation for n=3).

  H NMR (CDCl3, 300 MHz):  8.40 (d, J = 5.9 Hz, 1H), 7.66-7.73 (m, 4H), 7.43-7.54 (m, 6H), 7.48 (d, J = 8.1 Hz, 2H), 7.03-7.06 (m, 2H), 3.62 (d, J = 13.9 Hz, 2H), 31 P (CDCl3, 121.4 MHz):  28.6. N,N-Dibutyl-4-((5-(quinolin-4-yl)thiophen-2-yl)ethynyl)

  H NMR (600 MHz, CDCl3) E isomer:  8.86 (d, J = 4.6 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.56 (d, J = 4.6 Hz, 1H), 7.54 (d, J = 15.8 Hz, 1H), 7.50 (d, J = 8.7 Hz, 2H), 7.46 (d, J = 15.7 Hz, 1H), 7.12 (d, J = 3.6 Hz, 1H), 7.08 (d, J = 3.6 Hz, 1H), 6.65 (d, J = 8.7 Hz, 2H), 3.31 (t, J = 7.5 Hz, 4H), 1.60 (quint, J = 7.6 Hz, 4H), 1.38 (sext, J = 7.4 Hz, 4H), 0.97 (t, J = 7.3 Hz, 6H).

  NEs: The dye concentration in the NE[D] suspensions was determined by lyophilisation of an aliquot of the oil droplet suspension and dissolving the residue in chloroform. Measurements of the absorbance give access to the initial concentration of the dye NE[D]. This protocol ensures that the use of the Beer Lambert law to derive the dye concentration is valid. It allows circumventing scattering or flattening phenomena that may cause unreliability when measurements are conducted on NEs suspensions.

Table 1 . Photophysical properties of the dyes

 1

	Dye	max abs	max	max em	FWHM a)
		nm	M -1 cm -1	nm

  3 cm -1

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	We also note that dye D2 shows red-shifted, more intense and
	narrower low-energy absorption band compared to D1 as well as
	smaller Stokes shift values (Table
		1.0
	Em.	0.8
	Norm. Abs. &	0.2 0.4 0.6
		0.0
			400	600	800	1000
				(nm)
		1.0
	Norm. Abs. & Em.	0.8 0.6 0.4
		0.2
		0.0
			400	600	800	1000
				(nm)
						f	Stokes shift
							10 3 cm -1 
	D1 in toluene	528		707	4.7	0.06 b)	4.8
	D1 in CHCl3	560	3.70 10 4	804	4.6	0.04 c)	5.4
	D1 in CH2Cl2	585		915	5.2	<0.01	6.2
	D1 in H2O d)	523		717	4.6	0.11 b)	5.2
	D2 in toluene	579		740	3.8	0.07 b)	3.8
	D2 in CHCl3	616	3.95 10 4	788	3.7	0.03 c)	3.55
	D2 in CH2Cl2	668		876	3.6	<0.01	3.55
	D2 in H2O d)	584		722	3.8	0.14 b)	3.3
	a) Full width at half maximum. b) Standard: standard: DCM in EtOH (f =0.437). c) Standard:
	Indocyanine green in DMSO (f = 0.11). d) micellar water (SDS/butanol/water, 6: 5: 89 wt %)
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  H NMR (300 MHz, CDCl3)  8.62 (d, J = 6.7 Hz, 2H), 7.66 (d, J = 6.7 Hz, 2H), 7.68 (d, J = 15.5 Hz, 1H), 7.37 (d, J = 8.7 Hz, 2H), 7.23 (d, J = 3.9 Hz, 1H), 7.07 (d, J = 3.8 Hz, 1H), 7.06 (br, 2H), 6.52-6.59 (m, 1H8, 2H), 4.28 (s, 3H) 3.78 (s, 3H). 3.27 (br, 4H), 1.53-1.63 (quint, J = 7.3 Hz, 4H), 1.35(sext, J = 7.4 Hz, 4H), 0.96 (t, J = 7.3 Hz, 6H). 13 C { 1 H} NMR (50 MHz, CDCl3):  152.6, 148.0 , 146.6, 144.4, 142.9, 137.8, 134.6, 134.4, 132.6, 126.8, 126.4, 123.7, 123.0, 121.3, 120.3, 119.4, 111.5, 54.5, 50.7, 47.2, 29.3, 20.3, 13.9. IR (KBr): 1641, 1604, 1518, 1453, 1252, 1216, 1187, 1052, 1012, 806, 745 cm -1 . m/z calcd for [C30H35N2S2] + MS (M) + : 486.95; found: 486.95

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Acknowledgements

This study was achieved within the context of the Laboratory of Excellence TRAIL ANR-10-LABX-57. MBD gratefully acknowledges support from the Conseil Régional d'Aquitaine ("Chaire d'excellence" grant). GP and TB are recipients of a Ph.D. fellowship from the French Ministry of Education Research and Technology. The microscopic measurements were done in the Bordeaux Imaging Centre, a service unit of the CNRS-INSERM and Bordeaux University, member of the national infrastructure France BioImaging. CREMER OLEO GmbH & Co.KG (Hamburg, Germany) is acknowledged for kindly providing Miglyol® 840.

Notes and references

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Please do not adjust margins Please do not adjust margins pathway, equipped with a short-pass filter (SP 680 nm) and a bandpass filter (448/20 nm). The system was used with a conventional scanner.

In vivo imaging

Animal studies were performed in agreement with French and European directives on the care and use of animals and have been approved by the local ethical committee (CEEA 50) under agreement A50120196.

Murine prostate cancer cell line RM1, initially obtained from Dr. T.C. Thompson (Baylor College of Medicine, Houston, TX, USA), was genetically engineered for constitutive expression of firefly luciferase (RM1-CMV/Fluc) as previously described 15 and was maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen), 1% antimycotic-antibiotic mix (PSA, and blasticidin (10 µg/mL, Euromedex, Souffelweyersheim, France). Cell line was maintained in a humidified 5% CO2 incubator at 37 °C.

Black-6 Albino mice were anesthetized with 2% isoflurane (Belamont, Nicholas Piramal Ltd, London, UK) in air. A subcutaneous tumour was generated on the right leg (2.10 6 cells/100 μL). Prior to the imaging session, the regions of the mice to be imaged were shaved with clippers and a depilatory cream. Four days after injection of RM1 cells, the formulations (20 µL diluted in 80 µL of glucose 5%) were administered intravenously in tail vein (N = 3 per condition).

Fluorescence reflectance imaging (FRI) was performed at Vivoptic (UMS 3767-University of Bordeaux) using a Lumina LT (Perkin Elmer Inc., Boston, MA, USA). Excitation was performed at 535 nm and emission was detected with the Cy5.5 filter (695-770 nm).

Conflicts of interest

"There are no conflicts to declare".