Amphiphilic and Perfluorinated Poly(3-Hydroxyalkanoate) Nanocapsules for 19F Magnetic Resonance Imaging

Nanoparticles have recently emerged as valuable tools in biomedical imaging techniques. Here PEGylated and fluorinated nanocapsules based on poly(3-hydroxyalkanoate) containing a liquid core of perfluorooctyl bromide PFOB were formulated by an emulsion-evaporation process as potential 19F MRI imaging agents. Unsaturated poly(hydroxyalkanoate), PHAU, was produced by marine bacteria using coprah oil and undecenoic acid as substrates. PHA-g-(F; PEG) was prepared by two successive controlled thiol-ene reactions from PHAU with firstly three fluorinated thiols having from 3 up to 17 fluorine atoms and secondly with PEG-SH. The resulting PHA-g-(F; PEG)-based PFOB nanocapsules, with a diameter close to 250–300 nm, are shown to be visible in 19F MRI with an acquisition time of 15 min. The results showed that PFOB-nanocapsules based on PHA-g-(F; PEG) have the potential to be used as novel contrast agents for 19F MRI.


Introduction
F magnetic resonance imaging (MRI) has considerably developed in recent years in clinical practice [1][2][3][4]. The most frequently used contrast agents are liquid perfluorocarbons, such as perfluoro-15-crown-5-ether, perfluorooctyl bromide (PFOB) (17 fluorine atoms) and perfluoropolyethers (PFPE) [5][6][7]. These molecules have intrinsic physico-chemical and biological properties that are particularly well suited for in vivo applications, such as non-toxicity and biological and chemical inertness [8][9][10][11]. Due to their hydrophobic and lipophobic character, they are formulated with surfactants or encapsulated in polymer particles [12,13]. Thus, several surfactants have been studied to ensure capsule formation with a PLGA polymer shell and a PFOB liquid core such as polyvinyl alcohol and sodium cholate [14]. Systemic administration of these systems requires coatings to prevent uptake by macrophages. For this, different strategies have been developed including surface coating with polysaccharides or polyethylene glycol (PEG) chains [15]. PEGylated polyester nanocapsules have been shown to be of interest for MRI and their long circulating properties and capacity to diagnose tumors have been demonstrated in vivo [15,16]. Furthermore, theranostic capabilities to observe drug accumulation and release from a PFOB-loaded nanocarrier have been shown [17][18][19]. Fluorinated copolymers containing fluorinated units based on acrylic acid derivatives as 2,2,2-trifluorethylacrylate [20], 2,2,2trifluorethyl methacrylate [21,22], poly[N-(2,2-difluorethyl) acrylamide] [23] or octafluoropentyl methacrylate [24] were synthesized. Although a high density of fluorine atoms is required to be MRI visible [25][26][27], it may lead to an aggregation in aqueous medium because of the hydrophobic character of the fluorinated groups. Moreover, Houvenagel et al. showed that the presence of fluorinated groups into the polymer itself considerably improves the encapsulation rate of PFOB inside the nanoparticles compared to the native polymer [28]. In order to increase the stability in water, hydrophilic blocks were added.

PHA (72) U (28) Biosynthesis
Pseudomonas raguenesii sp. nov. (strain CNCM I-4063 in the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Paris, France) [46] was cultivated in a two-steps batch cultivation process. In the first step, the cells were inoculated at 10% (v/v) with a suspension of cells in exponential phase and grown in 5 L Erlenmeyer flasks containing 2 L of marine broth medium (for 1 L: 5 g peptone, 1 g yeast extract, 20 g Sea Salts, pH 7.6) in an incubator at 34 • C, 160 rpm. After the cultivation for 8 h, cells were harvested by centrifugation (7000 rpm, 15 min) and transferred into 30 L fermenter containing 18 L nitrogen free medium (15 g.L −1 sea salts) enriched with a mixture of coprah oil (5 g.L −1 ) and 10-undecenoic acid (0.6 g.L −1 ). Culture was incubated at 34 • C, pH is adjusted at 7.6 throughout the fermentation process by automatic addition of 1M NaOH or 1M H 2 SO 4 and the oxygenation is ensured by a light air flow. Following cultivation for 72 h, cells were harvested by centrifugation (7000 rpm for 15 min) and the pellets lyophilized prior to PHA extraction. Pellets of freeze-dried cells were ground with a mortar and pestle; the resulting powder was extracted with chloroform for 4 h at 50 • C. The PHA-containing chloroform phase was washed once with water and concentrated. The organic phase was evaporated to dryness, and purified PHA were obtained by repeated precipitations in 10 volumes of cold methanol.

PEG-SH Synthesis
PEG-SH was synthesized according to a method described elsewhere [47] carried out in three steps. Tosylation of PEG 550 was carried out by mixing 30 g of MeO-PEG 550 (54.5 mmol) and 33 mg of 4-dimethylamino pyridine (DMAP) (2.7 mmol) dissolved in 160 mL of CH 2 Cl 2 /pyridine (1:1) solution at 0 • C under argon atmosphere. Furthermore, 18.7 g of 4-toluenesulfonyl chloride (TsCl) (98.1 mmol) was dissolved in 50 mL of CH 2 Cl 2 and added dropwise and the solution was stirred overnight until brought back at RT. Then, 200 mL of cold water were added and the aqueous phase was extracted 3 times with 150 mL of CH 2 Cl 2 . The accumulated organic phases were washed successively with 3 × 250 mL of a 1M HCl solution and 3 × 250 mL of a saturated NaCl solution. The solution obtained was dried over MgSO 4 and then concentrated under reduced pressure to obtain 37.2 g of product (PEG 550 -OTs). To carry out the synthesis of PEG 550 -thioacetate, 37.2 g of PEG 550 -OTs (53 mmol) was dissolved in 200 mL of THF under argon. Then, 11.6 g of potassium thioacetate (102 mmol) was added and the solution was refluxed for 20 h, inducing the formation of a white precipitate. After returning to RT, 400 mL of water were added. Contents of the flask were extracted with 3 × 150 mL of CH 2 Cl 2 and the organic phase was washed with 3 × 200 mL of water, treated with activated carbon, filtered and evaporated until an oil was obtained (15.3 g, yield 36%).
To obtain the final PEG 550 -SH, 15.3 g of PEG 550 -thioacetate (25 mmol) was dissolved in 200 mL of anhydrous MeOH under argon. Then 15 mL of 37% HCl were added and the resulting solution was refluxed for 2 h. Furthermore, 200 mL of water were added and the solution was extracted with 3 × 150 mL of CH 2 Cl 2 . The combined organic phases were washed with 3 × 200 of water and 3 × 250 mL of NaCl solution, dried over MgSO 4 and filtered. The solution was evaporated until a PEG 550 -SH oil was obtained (11 g, yield 77%).

Synthesis of PHA-g-(F; PEG)
Next, 200 mg of PHA previously grafted with fluorinated molecule, AIBN and PEG-SH were dissolved in 6 mL of anhydrous toluene under argon in the following proportions: for PHA (72) C 8 F 17 (12) PEG (16) 170 mg of PEG-SH, for PHA (72) U (20) C 6 F 13 (8) 230 mg of PEG-SH and for PHA (72) U (20) CF 3(8) 242 mg of PEG-SH were added. Solutions were heated at 80 • C for 20 h. Toluene was rotary evaporated and grafted polymers were dissolved in 30 mL of acetone. Solutions were poured in 30 mL of water and resulting colloidal suspensions were transferred into dialysis tube (molecular weight cut off, 6-8000 Da) and dialyzed against water for 3 days prior to lyophilization. GPC K-805 L columns (5 µm Mixte-C) using two PL aquagel-OH 40-30 columns (8 µm Mixte-C), at a concentration of 10 mg/mL. A Wyatt Technology (Santa Barbara, CA, USA) Optilab Rex interferometric refractometer was used as detector, and low polydispersity index polystyrene standards (3 × 10 4 -2 × 10 6 g/mol) were used to calibrate the system. Grafting of fluorinated compounds was assessed by Raman spectroscopy using a Raman microscope LabRAMHR from Horiba Jobin Yvon (Longjumeau, France) equipped with a laser emitting at 633 nm. Water contact angle measurements were performed using the Drop Shape Analysis system on a Krüss goniometer (Villebon-sur-Yvette, France).

Preparation of Nanoparticles
Nanoparticles of perfluorobromide (PFOB) were prepared by an emulsion evaporation process adapted from [14]. Then 30 mg of PHA-F-PEG were dissolved into 2 mL of methylene chloride with 30 µL of PFOB. Organic phases were vortexed in 10 mL of 1.5% sodium cholate (w/v) aqueous solution for 1 min and then emulsified 1 min by sonication (Ultrasonic processor) at 20% of maximal power, over ice. Solvent was evaporated by magnetic stirring at room temperature for 3 h, at 300 rpm. Suspensions were purified to remove sodium cholate and putative non-encapsulated PFOB by ultracentrifugation for 45 min at 18,000× g at 4 • C. Pellets were resuspended in distilled water at the desired concentration and supernatants were analyzed by 19 F NMR to ensure absence of residual PFOB.

Nanoparticles Characterization
Nanoparticles size and surface charge were measured by dynamic light scattering (DLS) and zeta potential (ZP) on a Malvern Zetasizer Nano ZS (Malvern, UK). Measurements were performed at 20 • C, at an angle of 173 • . PHA-F-PEG nanoparticles morphology was investigated by transmission electron microscopy (Tecnai F20 microscope, FEI Company, Hillsboro, OR, USA). Samples were deposited on copper grids, dried at room temperature and then were colored with uracyl acetate (1 wt.%).

1 H/ 19 F MRI Imaging
Images of phantoms containing nanoparticles solutions were acquired on a 7-T small animal MRI scanner (Bruker, Ettlingen, Germany) using a double tuned, 1 H/ 19 F coil at Neurospin (CEA, Saclay, France). Sample solutions (at 30 mg • mL −1 ) were placed in 1 mL syringes, water and commercial PFOB were used as references. Images were acquired by cross sections of the syringes containing solutions to be analyzed. 1 H MRI images were acquired for localization of the samples and 19 F MRI images were acquired using a turbo spinecho sequence, (RARE factor = 64, TE = 23.3 ms, TR = 4550 ms, number of average = 100, FOV = 0.2 × 0.2 mm, slice thickness = 8 mm, measurement time = 15 min 10 s).

PHAU Production
The ability of Pseudomonas raguenesii to produce unsaturated PHA (PHAU) was tested using coprah oil (5 g·L −1 ) that is a vegetable oil from coconut composed of more than 90% of saturated fatty acids, in mixture with 10-undecenoic acid (0.5 g·L −1 ) to induce PHAU production. After 72 h of culture, cells are harvested by centrifugation and PHAU is extracted from these freeze-dried pellets. After extraction and purification, PHAU production has a yield of 17% (w/w). The molar mass is 123,000 g.mol −1 with polydispersity index of 1.8. The 13 C NMR spectrum of the polymer is shown in Figure 1 (HSQC spectrum is provided in the Supplementary Material, Figure S1). Chemical shifts of the C1 carbon (C=O) were detected at 169 ppm, that of C2 at 39 ppm and C3 at 71 ppm. C4 carbon of the side chain methylene groups is detected at 33 ppm. The chemical shifts at 14 ppm correspond to the terminal methyl groups (CH 3 ) of the side chains and the chemical shifts observed between 22 ppm and 31 ppm correspond to the different methylene groups (CH 2 ) of the side chains. The presence of a double bond is confirmed by the presence of peaks at 114 ppm and 139 ppm corresponding respectively to the methylene (=CH 2 ) and methine (=CH-) groups of the double bond. The chemical shift at 34 ppm corresponds to the α-methylene groups of the double bond.
(C=O) were detected at 169 ppm, that of C2 at 39 ppm and C3 at 71 ppm. C4 carbon of the side chain methylene groups is detected at 33 ppm. The chemical shifts at 14 ppm correspond to the terminal methyl groups (CH3) of the side chains and the chemical shifts observed between 22 ppm and 31 ppm correspond to the different methylene groups (CH2) of the side chains. The presence of a double bond is confirmed by the presence of peaks at 114 ppm and 139 ppm corresponding respectively to the methylene (=CH2) and methine (=CH-) groups of the double bond. The chemical shift at 34 ppm corresponds to the αmethylene groups of the double bond.

PHA-g-(F; PEG) Synthesis from PHAU
Our strategy is to incorporate perfluoroalkyl side chains in the PHAU to enhance its tendency to self-assemble through the formation of fluorophilic interactions between perfluorinated side chains. Moreover, PEG grafting onto PHAU was performed to generate stealth material against the immune systems. The interest in preparing these amphiphilic copolymers that are both covalently grafted by PEG and fluorinated groups is 2-fold. It allows one both to increase the PFOB encapsulation and to avoid aggregation of the nanoparticles. The synthetic route to PHA-g-(F; PEG) is given in Figure 2. Different fluorinated thiols were grafted on PHAU (Table 1) in order to evaluate the importance of the length of the grafted moiety on the signal detected by MRI. The molar ratio of thiol was adjusted so as to functionalize only a part of the available double bonds because the other part will be further used to graft PEG on the side chains of PHAU.

PHA-g-(F; PEG) Synthesis from PHAU
Our strategy is to incorporate perfluoroalkyl side chains in the PHAU to enhance its tendency to self-assemble through the formation of fluorophilic interactions between perfluorinated side chains. Moreover, PEG grafting onto PHAU was performed to generate stealth material against the immune systems. The interest in preparing these amphiphilic copolymers that are both covalently grafted by PEG and fluorinated groups is 2-fold. It allows one both to increase the PFOB encapsulation and to avoid aggregation of the nanoparticles. The synthetic route to PHA-g-(F; PEG) is given in Figure 2. Different fluorinated thiols were grafted on PHAU (Table 1) in order to evaluate the importance of the length of the grafted moiety on the signal detected by MRI. The molar ratio of thiol was adjusted so as to functionalize only a part of the available double bonds because the other part will be further used to graft PEG on the side chains of PHAU.

Fluorinated Thiol Chemical Structure Abbreviation
Trifluoropropane-1-thiol CF3 Perfluorooctane-1-thiol C6F13 Perfluorodecane-1-thiol C8F17 Raman spectrum (Figure 3) of PHAU-g-(C8F17) clearly showed the presence of fluorinated groups by the appearance of characteristic bands at 735 and 765 cm −1 assigned to symmetric CF2 stretching and symmetrical vibration involving the trifluoromethyl group CF3 respectively. The total disappearance of the band at 2580 cm −1 assigned to the SH

Fluorinated Thiol Chemical Structure Abbreviation
Trifluoropropane-1-thiol CF3 Perfluorooctane-1-thiol C6F13 Perfluorodecane-1-thiol C8F17 Raman spectrum (Figure 3) of PHAU-g-(C8F17) clearly showed the presence of fluorinated groups by the appearance of characteristic bands at 735 and 765 cm −1 assigned to symmetric CF2 stretching and symmetrical vibration involving the trifluoromethyl group CF3 respectively. The total disappearance of the band at 2580 cm −1 assigned to the SH group of the perfluorodecane-1-thiol showed that the thiol-ene reaction is quantitative.

Fluorinated Thiol Chemical Structure Abbreviation
Trifluoropropane-1-thiol CF3 Perfluorooctane-1-thiol C6F13 Perfluorodecane-1-thiol C8F17 Raman spectrum (Figure 3) of PHAU-g-(C8F17) clearly showed the presence of fluorinated groups by the appearance of characteristic bands at 735 and 765 cm −1 assigned to symmetric CF2 stretching and symmetrical vibration involving the trifluoromethyl group CF3 respectively. The total disappearance of the band at 2580 cm −1 assigned to the SH group of the perfluorodecane-1-thiol showed that the thiol-ene reaction is quantitative. Moreover, the absence of the characteristic disulfide bond signal, usually observed

Perfluorooctane-1-thiol C6F13
Perfluorodecane-1-thiol C8F17 Raman spectrum (Figure 3) of PHAU-g-(C8F17) clearly showed the presence of fluorinated groups by the appearance of characteristic bands at 735 and 765 cm −1 assigned to symmetric CF2 stretching and symmetrical vibration involving the trifluoromethyl group CF3 respectively. The total disappearance of the band at 2580 cm −1 assigned to the SH group of the perfluorodecane-1-thiol showed that the thiol-ene reaction is quantitative. Moreover, the absence of the characteristic disulfide bond signal, usually observed around 500-550 cm −1 , confirms the correct grafting. The value equal to the ratio of integration of the ester absorption band at 1749 cm −1 , characteristic of the ester in the PHAU backbone and the double bond at 1644 cm −1 assigned to the unsaturated group, were determined before and after the thiol-ene reaction. The results showed that 41% of the initial double bonds were involved during the thiol grafting which was in agreement with the results obtained by 1 H NMR. Raman spectrum (Figure 3) of PHAU-g-(C 8 F 17 ) clearly showed the presence of fluorinated groups by the appearance of characteristic bands at 735 and 765 cm −1 assigned to symmetric CF 2 stretching and symmetrical vibration involving the trifluoromethyl group CF 3 respectively. The total disappearance of the band at 2580 cm −1 assigned to the SH group of the perfluorodecane-1-thiol showed that the thiol-ene reaction is quantitative. Moreover, the absence of the characteristic disulfide bond signal, usually observed around 500-550 cm −1 , confirms the correct grafting. The value equal to the ratio of integration of the ester absorption band at 1749 cm −1 , characteristic of the ester in the PHAU backbone and the double bond at 1644 cm −1 assigned to the unsaturated group, were determined before and after the thiol-ene reaction. The results showed that 41% of the initial double bonds were involved during the thiol grafting which was in agreement with the results obtained by 1 H NMR.  Initial PHAU was characterized by 1 H NMR (Figure 4) to determine the percentage of double bonds by integrating protons corresponding to the methine peak (3) at 5.2 ppm and the signal relating to the terminal unsaturation group of side chain (10) at 5.7 ppm. PHAU is a statistical copolymer containing saturated and unsaturated side chains in the proportion 0.72/0.28 respectively. Perfluorodecane-1-thiol was grafted and the PHAU-g-(C 8 F 17 ) was analyzed by 1 H NMR ( 1 H and 19 F spectra of copolymers are provided in the Supplementary Materials). Signals relating to the CH 2 methylene (15), characteristic of fluorinated groups, appeared at 2.8 ppm and indicated the success of the grafting. The conversion percentages of the double bonds after grafting were calculated by comparing the integrations of the protons corresponding to the CH peak (3) of the PHAU at 5.2 ppm, with respect to the signal of terminal unsaturation side chain (10) at 5.7 ppm. PEG550-SH was prepared in three steps according to a method described elsewhere [46] (Figure 5). Thiolated PEG was characterized by 1 H NMR (Figure 6a) and the presence of signals at 2.60 and 1.55 ppm attested that the reaction is quantitative. In a second step PEG methyl ether thiol was grafted using the thiol-ene reaction to prepare PHA-g-(C8F17; PEG550-SH was prepared in three steps according to a method described elsewhere [46] ( Figure 5). Thiolated PEG was characterized by 1 H NMR (Figure 6a) and the presence of signals at 2.60 and 1.55 ppm attested that the reaction is quantitative. In a second step PEG methyl ether thiol was grafted using the thiol-ene reaction to prepare PHA-g-(C 8 F 17 ; PEG). Two peaks are observed at 3.6 and 3.3 ppm that are assigned to the CH= (19,20) and CH 3 (21) methyl terminal groups of the PEG confirming the success of the grafting (Figure 4b). The total disappearance of the peaks at 5.7 ppm (10) and 4.9 ppm (11) showed that there are no more unsaturated groups in the PHA. The compositions of the different copolymers determined by NMR are listed in Table 2.
Bioengineering 2021, 8, x FOR PEER REVIEW 9 of 16 PEG). Two peaks are observed at 3.6 and 3.3 ppm that are assigned to the CH= (19,20) and CH3 (21) methyl terminal groups of the PEG confirming the success of the grafting (Figure 4b). The total disappearance of the peaks at 5.7 ppm (10) and 4.9 ppm (11) showed that there are no more unsaturated groups in the PHA. The compositions of the different copolymers determined by NMR are listed in Table 2.   The water contact angle of solvent cast PHAU film was found to be 96.2 • , which was in agreement with literature, whereas the contact angle was about 132.5 • for PHA 72 U 20 -g-(C 8 F 17 ) 8 . This difference can be explained by the presence of very hydrophobic fluorinated groups. The water contact angle decreased to 106.3 • after PEG grafting but it remained higher than the value of the initial PHA, attesting to the global hydrophobic character of the copolymer.

Synthesis and Characterization of the Nanoparticles
PHAU-g-(F; PEG) PFOB nanoparticles were prepared by an emulsion evaporation method. In these conditions, the graft copolymers will adopt a specific conformation in the aqueous medium. The perfluorinated side chains may orientate towards the PFOB core whereas the hydrophilic PEG grafts may orientate towards the aqueous medium. The 19 F NMR spectrum of the nanocapsules reveals peaks consistent with the presence of three different containing sites in the fluorinated moieties. The peak at −65 ppm originates from CF 2 near the bromide atom, the one at −84 ppm from CF 3 fluorine and the peaks between −118 and −127 ppm from CF 2 fluorine of the PFOB. Although the peaks characteristic of the PHAU-g-(F; PEG) were clearly distinguished on the spectrum of the copolymer alone, they were hidden by the PFOB ones (Figure 7).  Negative staining improved the TEM image contrast and allowed the visualization of the inner core in PFOB in light gray whereas the polymeric shell seemed darker, which confirmed the core-shell structure (Figure 8). Images show spherical objects with size in good agreement with the diameter measured by dynamic light scattering DLS. Smooth surfaces are observed for all particles. DLS measurements showed that the average size of PHA-g-(F; PEG) PFOB nanoparticles increased from 246 nm for the perfluorodecane graft to 294 nm for the trifluoropropane graft. The increase of the length of the fluorinated side chains certainly improves the interactions between these hydrophobic moieties. A wider distribution of sizes was also observed. All samples present very negative zeta potential regardless of the length of the perfluorinated groups, resulting in a very good stability in aqueous medium with a low tendency to aggregation [48].
Negative staining improved the TEM image contrast and allowed the visualizat of the inner core in PFOB in light gray whereas the polymeric shell seemed darker, wh confirmed the core-shell structure (Figure 8). Images show spherical objects with size good agreement with the diameter measured by dynamic light scattering DLS. Smo surfaces are observed for all particles. DLS measurements showed that the average size PHA-g-(F; PEG) PFOB nanoparticles increased from 246 nm for the perfluorodecane gr to 294 nm for the trifluoropropane graft. The increase of the length of the fluorinated s chains certainly improves the interactions between these hydrophobic moieties. A wid distribution of sizes was also observed. All samples present very negative zeta poten regardless of the length of the perfluorinated groups, resulting in a very good stability aqueous medium with a low tendency to aggregation [48].

In Vitro 19 F NMR
Phantom MRI studies were performed to evaluate in vitro the contrast properties PFOB nanocapsules from different PHA-g-(F; PEG). These nanocapsules were dissolv in distilled water and different acquisition times were evaluated: 1 min 31 s, 15 min 1 1 h 16 min and 10 h. It turns out that from 15 min 10 s, nanocapsules solutions are distinc visible and as detectable as after 10 h of acquisition. This relatively short analysis time very encouraging to consider a further clinical application even though fluorine conc tration would probably by lower after an injection in vivo. The 1 H MRI and 19 F MRI imag thus acquired in 15 min were presented in Figure 9. A more intense signal is observed the bottom of the images due to the nanocapsules sedimentation during the different quisitions. One of the simple solutions to increase the visibility in MRI could be to incre the number of fluorine atoms per nanocapsule. Here, the number of F atoms varied fr 3 to 17 per grafted fluorinated segment for the three imaged nanocapsules. The resu obtained show that this is not a determining parameter because the particles are visi whatever the length of the grafted fluorinated segment. The CF3 is volatile, which c

In Vitro 19 F NMR
Phantom MRI studies were performed to evaluate in vitro the contrast properties of PFOB nanocapsules from different PHA-g-(F; PEG). These nanocapsules were dissolved in distilled water and different acquisition times were evaluated: 1 min 31 s, 15 min 10 s, 1 h 16 min and 10 h. It turns out that from 15 min 10 s, nanocapsules solutions are distinctly visible and as detectable as after 10 h of acquisition. This relatively short analysis time is very encouraging to consider a further clinical application even though fluorine concentration would probably by lower after an injection in vivo. The 1 H MRI and 19 F MRI images thus acquired in 15 min were presented in Figure 9. A more intense signal is observed at the bottom of the images due to the nanocapsules sedimentation during the different acquisitions. One of the simple solutions to increase the visibility in MRI could be to increase the number of fluorine atoms per nanocapsule. Here, the number of F atoms varied from 3 to 17 per grafted fluorinated segment for the three imaged nanocapsules. The results obtained show that this is not a determining parameter because the particles are visible whatever the length of the grafted fluorinated segment. The CF 3 is volatile, which can appear as a disadvantage during the grafting reactions and therefore it would be preferable to use C 6 F 13 or C 8 F 17 . The preliminary results showed that these PFOB nanocapsules could be used as contrast agents for MRI. appear as a disadvantage during the grafting reactions and therefore it would be preferable to use C6F13 or C8F17. The preliminary results showed that these PFOB nanocapsules could be used as contrast agents for MRI.

Conclusions
An efficient and reproducible method for the synthesis of copolymers of amphiphilic PHA-g-(F;PEG) has been developed by two successive thiol-ene reactions. These copolymers will be used for the encapsulation of PFOB known to be a contrast agent for MRI. The graft copolymers based on unsaturated PHAs produced by marine bacteria have been functionalized with on one hand perfluorinated groups to promote interactions with PFOB and on the other hand with PEG to improve hydrophilicity and escape the phagocytic cells of the immune system. The molar composition of the copolymer is strictly controlled due to the efficiency of the thiol-ene addition. These nanocapsules based on PHAg-(F;PEG) would constitute a new generation of biocompatible therapeutic nanovectors that can be tracked by MRI thanks to their contrast agents capabilities.

Conclusions
An efficient and reproducible method for the synthesis of copolymers of amphiphilic PHA-g-(F;PEG) has been developed by two successive thiol-ene reactions. These copolymers will be used for the encapsulation of PFOB known to be a contrast agent for MRI. The graft copolymers based on unsaturated PHAs produced by marine bacteria have been functionalized with on one hand perfluorinated groups to promote interactions with PFOB and on the other hand with PEG to improve hydrophilicity and escape the phagocytic cells of the immune system. The molar composition of the copolymer is strictly controlled due to the efficiency of the thiol-ene addition. These nanocapsules based on PHA-g-(F;PEG) would constitute a new generation of biocompatible therapeutic nanovectors that can be tracked by MRI thanks to their contrast agents capabilities.