Sebastien Blanquer

Julien Tailhades

Vincent Darcos

Muriel Amblard

Jean Martinez

Benjamin Nottelet
email: benjamin.nottelet@univ-montp1.fr

Jean Coudane

Easy synthesis and ROP of 5-Z-amino-δ-valerolactone: new degradable aminofunctionalized (co)polyesters

Keywords: lactones, polyelectrolytes, aliphatic polyesters, amino functional polymer, degradable polymers

Novel 5-Z-amino-δ-valerolactone (5-NHZ-VL) was synthesized with aim to prepare degradable polyesters and copolyesters having amino pendant groups. Following a straightforward and efficient synthetic pathway, 5-NHZ-VL was obtained in only two steps and up to 50 % yield. The monomer was fully characterized by 1 H NMR, 13 C NMR, ESI mass spectrometry and HPLC. Various conventional conditions were tested for this lactone ring opening polymerization and led to the novel corresponding poly(5-NHZ-VL) (Mn = 7000 g/mol ; PD = 1.2). Following this homopolymerization, 5-NHZ-VL was copolymerized with ε-caprolactone to generate a family of copolyesters with an amino-group content ranging from 10 to 80 %. Finally, the polyelectrolyte poly(5-NH3 + -VL) was recovered by removal of the protecting group under acidic conditions and integrity of the polyester backbone was confirmed by 1 H NMR.

INTRODUCTION

Biodegradable aliphatic polyesters have been the subject of a great deal of development work over the past 30 years due to their use in environmental and biomedical applications. However, these polyesters suffer from a lack of functional groups that could broaden the range of their properties, for instance conferring hydrophilicity or chemical reactivity. Considerable effort has therefore been made in recent years to prepare new functionalized polyesters. For example, few years ago, and as first strategy, we described a versatile and rapid method to chemically modify poly(ε-caprolactone) (PCL) chains via a polycarbanionic intermediate with up to 20 % functionalized units. 1,2 To obtain higher contents of functional groups, a second strategy is preferred, based on the synthesis and polymerization of previously functionalized lactones. 3 However, the chemistry involved in their synthesis often results in poor yields, both due to the number of steps required and the necessary careful purification of the monomers. Also, whether or not polymerization is possible, and the ease with which it can be performed, depends on the size of the lactone ring and the nature of the lactone functionalization.

Despite these drawbacks, a large variety of functional lactones has been described ranging from 4membered β-malolactonate 4 to 7-membered γor α-functionalized ε-caprolactone. [5][6][7][8] Kimura's early work 9 has most recently been followed by efforts concentrated on lactide-type structures giving access to functional poly(lactide)s with carboxyl, 10 hydroxyl, 11,12 or even alkyl substituted units. 13 However, and to the best of our knowledge, although amino-polyesters have great potential in biomedical applications, only two examples of polymerizable amino-functionalized lactones have been reported in the literature.

These monomers take the form of a butyrolactone derived from serine 1, [14][15][16][17] and a functional lactide derived from lysine 2 (Scheme 1). 10 Various strategies employed to polymerize these lactones have been reported, but none is fully satisfactory with either i) low yields after the final purification step used to obtain polymerizable monomers, ii) low polymerizability, or iii) marked degradation during the amino group deprotection step. 10,[14][15][16][17] In order to overcome some of these drawbacks and increase the diversity of aminofunctionalized lactones, we report here a rapid and simple two-step synthesis yielding a novel amino-δvalerolactone. In a second part, polymerization conditions towards the resulting aliphatic aminofunctionalized polyester are discussed. To finish, examples of copolyesters of amino-δ-valerolactone and ε-caprolactone are presented with molar ratios of aminated units ranging from 10 % to 80 %.

EXPERIMENTAL Materials

N-benzyloxycarbonyl-glutamic acid γ-tert-butyl ester (ZGlu(OtBu)OH, 99 %), N-N'diisopropylethylamine (DIPEA), sodium borohydride powder (NaBH4, 99 %), sodium bicarbonate (NaHCO3), magnesium sulfate (MgSO4), ε-caprolactone (ε-CL), tin(II) 2-ethylhexanoate (Sn(Oct)2, ≈ 95 %), tin(II) trifluoromethanesulfonate (Sn(OTf)2, ≥97.0 %), trifluoroacetic acid (TFA), solution of hydrobromic acid in acetic acid (HBr/AcOH, 33 wt.%), propan-2-ol (i-PrOH), ethyl acetate, THF, toluene, DMF, diethyl ether and dichloromethane (DCM) were purchased from Sigma-Aldrich (St-Quentin Fallavier, France). Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) was purchased from Merck (Strasbourg, France). All chemicals and solvents were used without purification with exception of the polymerization solvents, i-PrOH and ε-CL which were dried over calcium hydride for 24 h at room temperature and distilled under reduced pressure.

Characterizations

Molecular weights of the polymers were determined by size exclusion chromatography (SEC) using Waters Inc. equipment fitted with a 5 µm mixed C PLgel 5 µm MIXED-D (300×7.5 mm) and SHODEX RI 101 refractometric detector. The mobile phase was DMF at 1 mL/min flow. Typically, the polymer (10 mg) was dissolved in DMF (2 mL) and the resulting solution was filtered through a 0.45 µm Millipore filter before injection of 20 µL of filtered solution. Mn and Mw were expressed according to calibration using poly(methyl methacrylate) standards. For water soluble polycations, molecular weights were determined by SEC using a LCC-501 System (Pharmacia Biotech) equipped with a UV-visible detector operated at 214 nm and a column packed with cationic DEAE Sepharose gel. The mobile phase was a 1.6 M NaCl aqueous solution at 0.25 mL/min. Mn and Mw were expressed according to calibration using poly(L-Lysine) standards. Differential scanning calorimetry (DSC) measurements were carried out under nitrogen at a 10 °C/min heating rate on a Perkin Elmer Instrument DSC6 Thermal Analyser. 1 H NMR and 13 C spectra were recorded using an AMX300 Brücker spectrometer operating at 300 MHz and 75 MHz, respectively. Deuterated dimethyl sulfoxide or chloroform were used as solvent. LC/MS analyses were performed on a Quatromicro (Micromass, Manchester, U.K.) triple-quadrupole mass spectrometer fitted with an electrospray interface and thanks to a Waters Alliance 2690 system (Electrospray : 30 V, 80 °C, 100-200 µL/min; HPLC : Phenomenex Onix Monolith C18 (25 x 4.6 mm, macropore : 2 µm), 3 mL/min, gradient : 100 % (H2O, 0.1 % HCOOH) to 100 % (ACN, 0.1 % HCOOH), UV detection at λ = 214 nm ).

Synthesis of tertiobutyl-4-(benzyloxycarbonlylamino)-5-hydroxypentanoate (4)

The selective reduction of the main chain carboxylic acid group is based on the formation of an activated ester thanks to the use of BOP as a reagent as previously described in literature. 18 Typically, DIPEA (1.18 ml, 7.1 mmol) and a BOP solution (2.87 g, 6.5 mmol) in 10 ml of THF were slowly added on a stirred suspension of Z-Glu(OtBu)OH 3 (2 g, 6 mmol) in 20 ml of THF at room temperature. After 10 minutes of stirring, NaBH4 (1.1 g, 30 mmol) was slowly added to the reaction medium which was then stirred for 2 hours at room temperature. The resulting mixture was then diluted in CH2Cl2 (150 mL) and carefully washed with a 5 % HCl solution (5×100 mL), followed by NaHCO3 saturated solution (3×100 mL) and brine (3×100 mL). The organic phase was then dried over MgSO4 and concentrated under reduced pressure to yield a clear oil. Final purification of compound 4 was obtained by flash column chromatography using in a first step CH2Cl2 to elude by-products and in a second step ethyl acetate to recover pure 4 with a 90 % overall yield. 1

Synthesis of benzyl 6-oxotetrahydro-2H-pyran-3-ylcarbamate (5-Z-amino-δ-valerolactone) (5)

Lactonization of 4 was done via the simultaneous removal of the tertiobutyl group and cyclization of the resulting intermediate to yield 5. In a typical experiment, 4 (2 g) was dissolved in a mixture of trifluoroacetic acid (10 mL) and CH2Cl2 (10 mL) and stirred for 3 hours at room temperature. After reaction completion, cold water (80 mL) and CH2Cl2 (80 mL) were added. The organic layer was washed with water (5×50 mL), dried over MgSO4, filtered, and concentrated under reduced pressure.

Column chromatography with AcOEt/Et2O (5:5), yielded compound 5 (0.85 g, 55 %) as white crystals with a minimum 98 % purity. mp 65°C (from DSC). 1

Synthesis of poly(5-Z-amino-δ-valerolactone) (6)

All polymerizations in solution have been carried out using 2 % of either Sn(Oct)2 (35 % (w/v) THF stock solution) or Sn(OTf)2 (36 % (w/v) THF stock solution) as a catalyst and dry propan-2-ol (2 % (v/v) THF stock solution) as an initiator. Details of polymerization conditions as well as corresponding results are summarized in Table S1. † Typically, a schlenk flask containing a stir bar was fitted with a septum, flame-dried under vacuum, and placed under nitrogen. Monomer and all solutions were added under nitrogen. In a typical procedure (Table S1, 10), 5 (0.3 g, 1.2 mmol) was dissolved in dry NMP (1.5 mL) and heated to 140°C. 28 µL of Sn(OTf)2 stock solution and 57 µL of propan-2-ol stock solution were added under argon atmosphere. At the desired reaction time the reaction was stopped by adding 3 mL of THF, followed by precipitation of the polymer in cold methanol. Polymer was dried at room temperature under vacuum after solvent decantation. For bulk polymerizations the same procedure was followed without solvent. In addition, no initiator was used and reaction was carried out in sealed flasks when reduced pressure was used. Monomer conversion was determined by 1 H NMR analysis via the signal shift of the methylene protons of the benzyl group from 5.05 ppm to 4.95 ppm in 5 and in poly(5-Z-amino-δ-valerolactone), respectively. Molecular weights and polydispersities were determined by SEC: Mn = 7000 g/mol, PDI = 1.2 (Table S1,4

Synthesis of poly(5-NH3 + -δ-valerolactone) (7)

Z-amino protected polymer was deprotected following a classical procedure under acidic conditions to yield the corresponding polyester with free ammonium pendant groups. Typically, a solution of hydrobromic acid (0.23 mL, 4.0 mmol) in acetic acid (33 wt.%) was slowly added on a stirred suspension of 6 (0.1g, 0.4 mmol of Z group) in dichloromethane (3 mL). Deprotection was carried out at room temperature and was stopped when no more CO2 bubbling was detected. The reaction medium was precipitated and washed with cold diethyl ether. The obtained polymer was then dissolved in distilled water and dialyzed against water before freeze-drying to yield the final polymer 7 as white powder (80 % yield). 1 H NMR : (300 MHz; DMSO-d6): δ (ppm)= 4.2 (m, 1Ha, CH2-O), 4.1 (m, 1Hb, CH2-O ), 3.5 (m, 1H, CH-NH3 + ), 2.6 (m, 2H, CH2-CO), 1.9 (m, 2H, CH2-CH2-CH). 13

Synthesis of poly(5-Z-amino-δ-valerolactone-co-ε-caprolactone)

In a typical procedure, 5 (99 mg, 0.39 mmol) and ε-CL (407 mg, 3.5 mmol) were added in a schlenk flask under nitrogen atmosphere (Table 2, entry 1). Catalyst (Sn(Oct)2, 0.64 M in THF, 9µL, 7.8.10 -6 mol) and initiator (i-PrOH, 2,6 M in THF, 15µL, 3.9.10 -5 mol) were added through the septum. Reaction was carried out for 24 hours at 110°C under nitrogen and atmospheric pressure. At the desired reaction time reaction was stopped by adding 3 mL of THF, followed by precipitation into cold methanol. The obtained polymer was filtrated and dried at room temperature under vacuum. Monomer conversions were determined by 1 H NMR analysis via the signal shift of the methylene protons of the benzyl group from 5.05 ppm to 4.95 ppm in 5 and in the copolyesters, respectively, and the shift of the methylene protons from 4.20 ppm to 4.00 ppm in ε-CL and in the copolyesters, respectively. Comonomers ratios in the copolyesters were determined by 1 H NMR analysis using the intensities of the signals at 4.95 ppm (s, 2H, OCH2Ph, 5) and at 4.00 ppm (t, 2H, OCH2-CH2 , ε-CL and m, 1Ha, CH2-O, 5).

RESULTS AND DISCUSSION

5-Z-amino-δ-valerolactone synthesis

Synthesis of the novel 5-Z-amino-δ-valerolactone was performed according to a well-known strategy based on the use of α-amino-acids. Functional lactones with hydroxyl, carboxyl or even amino groups were successfully prepared in the past, thanks to this technique. However, in most cases after lactonization, a very careful final purification with several successive column chromatographies is necessary to get a polymerizable monomer. For the few existing examples of amino-protected lactones, typical yields of 60 to 80 % are generally claimed, but these yields correspond to monomers purified by a single column chromatography. 10,14,16,17 Authors further purify their monomers to get a polymerizable grade but without any mention of the new yields, or when it is the case with yields typically inferior to 10 %. 15 Thus, despite good initial yields, the required high purity of the lactone for an efficient ring opening polymerization generally leads to low final yields. Thanks to a careful choice of the starting αamino-acid and its protecting groups, we provide here a new amino-functionalized lactone obtained in high purity with up to 50 % yield. This easy synthesis of 5 was based on the use of a commercially-available glutamic acid derivative, namely N-benzyloxycarbonyl-glutamic acid γ-t-butyl ester 3 (ZGlu(OtBu)OH) (Scheme 2). In the first step, the free carboxylic group of 3 was reduced after formation of an activated ester intermediate by following a method described in the literature. 18,19 In the present work, N,N'diisopropylethylamine/BOP and NaBH4 were used for activation and reduction, respectively. Flash column chromatography of the step 1 products gave t-butyl 4-(benzyloxycarbonylamino)-5hydroxypentanoate 4 with a 90 % yield. † The second step consisted in simultaneous in situ deprotection of the carboxylic acid and intramolecular cyclization of 4 in a CH2Cl2/TFA (1:1) mixture at room temperature. The final benzyl 6-oxotetrahydro-2H-pyran-3-yl carbamate 5 (5-NHZ-δ-VL) was purified by column chromatography and recovered as white crystals with a 55 % yield and a high purity (> 98 %). The 1 H NMR spectrum and corresponding signals assignments were consistent with the expected structure of 5 (Figure 1). The structure and the purity were also confirmed by 13 C NMR, HPLC and mass spectrometry † . The high purity of 5 allowed its spontaneous crystallization together with its polymerization under standard ring-opening polymerization (ROP) conditions without further purification. It is noteworthy that such relative ease in obtaining a pure functionalized lactone is rare and that the overall 50 % yield for the polymerizable lactone confirms the efficiency of the short synthetic route proposed here.

Ring opening polymerization of 5-Z-amino-δ-valerolactone

Homopolymerization of 5 was carried out by ring-opening polymerization. Given previous reports concerning the inherent difficulties of polymerizing amino-functionalized lactones without deprotection/degradation and/or limitation of molecular weights, 10,14-17 we were interested in testing both polymerization conditions of 5 and subsequent amino group deprotection (Scheme 3). ROP studies were therefore conducted in bulk under conditions conventionally used to obtain high molecular weight PLA, or in solution with toluene, NMP or DMF as solvent. Tin-based catalysts, namely Sn(Oct)2 and Sn(OTf)2, were used with i-PrOH as initiator (Table 1). For clarity reasons, only the best conditions are listed in Table 1 and other ROP conditions are supplied as supplementary data. † Polymerization was followed by 1 H NMR in DMSO-d6 using the chemical shifts of the peak corresponding to the methylene protons of the Z protecting group, which were found at 5.05 ppm and 4.95 ppm in 5 and poly(5-NHZ-δ-VL), respectively. Molecular weights were determined by SEC and calculated using 1 H NMR spectra (Figure 2). It is to note that initiator peaks could not be used for this purpose as they are masked by polymer signals. Instead, the peaks corresponding to the alcohol chainend were used. These signals were found at 4.65 ppm and 3.40 ppm for (-CH(NHZ)-CH2-OH) and (CH2-OH), respectively. These assignments were confirmed by COSY-NMR experiments † .

First of all it is to note that molecular weight distributions (Mw/Mn) of polyesters were narrow, indicating a fast initiation compared to propagation, and a fast propagation compared to transfer or termination reactions. When conventional Sn(Oct)2 was used as the catalyst, polymerizations were slow under all the conditions employed with even no polymerization for the lowest temperature of 110°C in toluene or bulk under atmospheric pressure. † In an attempt to increase the reaction rate, the temperature was raised to 140°C. Despite this higher temperature, polymerizations were still slow with low conversions after 24h of polymerization. † For example, conversions of 20 % in bulk and 12 % in DMF were found with only oligomers (MnSEC = 1 200-1 700 g/mol) being produced in DMF, possibly due to transfer and/or depolymerization reactions (Figure 3). Longer reaction times and bulk polymerization were the only ROP conditions leading to elevated final conversions and higher molecular weights, up to 7 000 g/mol (PDI = 1.2) (Table 1, Figure 3). It is to note that slightly higher molecular weights were obtained under reduced pressure (7 000 g/mol) compared to atmospheric pressure (5 000 g/mol) as a result of the better removal of potential contaminants.

Although reasonably high and suitable for a variety of applications, this molecular weight obtained with Sn(Oct)2 was below 20 000 g/mol. Consequently, and to overcome the presumed unfavourable polymerization-depolymerization equilibrium for our lactone, Sn(OTf)2 was used as it is known to greatly accelerate the lactone polymerization rate even at lower temperature. As expected, Sn(OTf)2 gave far faster polymerization rate that Sn(Oct)2 at high temperatures, with 32 % conversion in DMF after 4 h, and up to 75 % conversion in NMP after only 1 hour. † However, rapid degradation occurred within the first hours of reaction as confirmed by low Mn and unexpected signals in the NMR spectra, indicating deprotection of the amino group concomitant with the polymerization. Considering these phenomena, and taking into account the lower temperature required with Sn(OTf)2, experiments were carried out in NMP at 30°C, and in bulk at 100°C under reduced pressure. † These milder conditions resulted in moderate polymerization rates, with for example 58 % monomer conversion obtained after 21 hours at 30°C in NMP, without any degradation of the polymer backbone and no amino group deprotection as confirmed by 1 H NMR. However, despite these more favorable parameters, no improvement in terms of molecular weights was observed for poly(5-NHZ-δ-VL). Limited molecular weights of ca. 2 000 g/mol in solution and up to 5 000 g/mol in bulk were obtained (Figure 3). Again, transfer reactions to the solvents might explain the low molecular weights obtained in DMF and NMP.

To complete our study, poly(5-NHZ-δ-VL) with Mn in the range 3 000 to 7 000 g/mol were analysed by DSC. † A strong influence of the molecular weight on the melting temperature (Tm) and glass transition temperature (Tg) was observed. Tm increase from 146 to 180°C and Tg from 27 to 45°C when molecular weights increase from 3 000 to 7 000 g/mol, respectively. In the same time enthalpy of fusion almost doubles passing from 34 to 65 J/g. These very strong variations with molecular weight can be explained by the possibility to form hydrogen bonds between the amido and ester group, which strongly increase the cristallinity of longer polymer chains.

In summary, homopolymerization of 5 led to polymers with molecular weight limited to Mn = 7000 g/mol under the investigated conditions. Other conditions, especially other catalytic systems might be of interest to obtain higher molecular weights and this will be studied further in our lab. The main aim of this work is to provide a new class of degradable aminated-polyesters based on the easily obtained 5-NHZ-δ-VL. Indeed, even if molecular weights below 20 000 g/mol are too low for some applications, for most of the herein targeted biomedical applications, low molecular weights polycations, and above all degradable ones like poly(5-amino-δ-valerolactone), are very attractive. For example, short degradable amino-functionalized polyesters are of interest for transfection of cells and gene delivery due to their ability to complexe DNA while being less toxic than high molecular weights and non degradable polycations [20][21][22] , or to prepare bioconjugates. 23,24 They also find one of their main applications in the field of amphoteric and/or amphiphilic block copolymers with applications in inter-polyelectrolytes complexes and micelles for drug delivery. [25][26][27] All these potential applications could be considered with poly(5-amino-δ-valerolactone) having a molecular weight below 10 000 g/mol. Besides, since copolyesters containing 5-amino-δ-valerolactone units could also be of great interest, we decided to evaluate the feasibility of the copolymerization of 5 with ε-CL.

Copolymerization of 5-Z-amino-δ-valerolactone and ε-caprolactone

Polymerizations of 5 with ε-CL were performed in bulk at 110°C by ROP initiated by i-PrOH and catalyzed by Sn(Oct)2. Copolyesters with molar ratios of aminated units ranging from 10 % to 50 % were prepared by adjusting the initial amounts of 5 and ε-CL. This family of copolyesters, with various proportions of amino-group content, is expected to cover a broad range of physico-chemical properties in adequation with some of the above mentioned applications. A degree of polymerization DP = 100 was targeted. Results are shown in Table 2. Experimental molecular weights were determined by SEC.

More accurate values would have been provided by 1 H NMR, unfortunately due to the high number of proton signals in the copolyesters, it was not possible to calculate the molecular weight by NMR. The theoretical molecular weight was calculated using Equation (1).

( )

0 0 5 0 ] Pr [ 114 ] [ 249 ] 5 [ OH i conv CL conv VL NHZ Mn CL VL NHZ th - × × - + × × - - = - - - ε ε (1)
After 24h, conversions were high for all copolymers with surprisingly higher conversions for 5 than for ε-CL. Experimental molar compositions (F5-NHZ-δ-VL) measured by NMR showed a deviation when compared with the theoretical composition (f5-NHZ-δ-VL) calculated by Equation (2), with 5 being preferably incorporated in the copolymer (Table 2, Figure 4). Although no reactivity study has been carried out at that point, this higher incorporation of 5 might allow the presence of small blocks of 5 in the copolymeric structure.

0 0 0 5 ] [ ] 5 ([ ] 5 [ CL VL NHZ VL NHZ f VL NHZ - + - - - - = - - - ε δ (2)
The composition deviation was accompanied by a molecular weights deviation (Figure 5). It is to note that lower molecular weights, below 5000 g/mol, were obtained for the highest initial 5/ε-CL ratio, in good agreement with the results found for the homopolymerization of 5. Hydrodynamic volumes of the copolymers changing with their composition, given molecular weights should be considered with care. However, copolymerization of 5 and ε-CL gave access to functional copolyesters with functionalities ranging from 12 % to 83 % and molecular weights up to 8600 g/mol, which might be of interest for further development of biodegradable bioconjugates after recovering of the amino-group.

Amino-group deprotection and poly(5-NH3 + -δ-valerolactone) recovery

Poly(5-NHZ-δ-VL) (Table 1, 1) was shortly treated under acidic conditions using a HBr/AcOH mixture on a suspension of the polymer in dichloromethane. The resulting water-soluble poly(5-NH3 + -δ-VL) was obtained as a white powder after dialysis and freeze-drying. Quantitative deprotection was confirmed by 1 H NMR with no protecting group being detected in the final poly(5-NH3 + -δ-VL) as shown by the absence of signal at 7.4-7.3 ppm and 4.95 ppm (Figure 6). This was further confirmed by 13 C NMR where some peaks are split in doubled peaks. † This is attributed to the existence of two configurations of carbon atoms involved in the hydrogen bonds between the ammonium group and carbonyl group of poly(5-NH3 + -δ-VL) formed after deprotection. Furthermore, as deprotection was carried out in a strong acidic medium, a major concern was the integrity of the polyester backbone.

Reaction time was kept short, typically below 30 minutes, to avoid degradation while insuring the quantitative deprotection of the amino-groups. Degradation was also avoided by using a suspension instead of a solution for the deprotection step. The absence of degradation was checked by 1 H NMR analysis in DMSO, taking into account the methylene protons signals of the alcohol chain end at 3.2 ppm (Figure 6). Polymerization degree was ca. 19 in the starting poly(5-NHZ-δ-VL) (Table 1, 1), which corresponded to MnNMR = 4 900 g/mol, and was found equal to 17 in the corresponding poly(5-NH3 + -δ-VL), which corresponded to MnNMR = 3 300 g/mol. This result confirms the efficiency of the deprotection strategy to remove the protecting groups while conserving the polyester backbone. Finally, SEC analysis of a water-soluble poly(5-NH3 + -δ-VL) was carried out using a dedicated low pressure SEC system with NaCL 1.6 M as eluent and cationic DEAE Sepharose gel as stationary phase. † Chromatogram showed two characteristic peaks corresponding to the polymer and the bromide counterion as previously described by our group. 28 A molecular weight of MnSEC = 1 100 g/mol (PDI = 2.3) was calculated using poly(L-Lysine) standards. This value is lower than the one calculated by NMR, however it has to be critically considered taking into account the structural differences of poly(L-Lysine) and poly(5-NH3 + -δ-VL), and the possible strong influence of the position of the amino groups towards the backbone on the hydrodynamic radius.

CONCLUSION

A novel amino-functionalized polyester, poly(5-NH3 + -δ-VL), was obtained by ROP of the corresponding protected valerolactone. Compared to all functional lactones previously described, the synthesis proposed here, with only two steps and high yields, is remarkably simple and renders the novel 5-NHZ-δ-VL very attractive for future polymerization and copolymerization studies intended to produce fully degradable and functional polyesters. In this purpose, various polymerization conditions were tested. Poly(5-NHZ-δ-VL) with narrow polydispersities and molecular weights in the range 5 000-7 000 g/mol were obtained in bulk. Copolymers of 5-NHZ-δ-VL and ε-CL were also prepared to generate a novel family of copolyesters having from 10 % to 80 % of aminated units. Finally, removal of the protecting group under chosen acidic conditions was found to be quantitative and respectful of the polyester backbone. Future work will focus on further improvements in molecular weights and potential applications related to degradable polyelectrolytes including degradation studies. Table S1. Thermal properties of poly(5-Z-amino-δ-valerolactone) as a function of Mn. Table S2. Conditions used for the ring-opening polymerization of 5. Table S1. Thermal properties of poly(5-Z-amino-δ-valerolactone) as a function of Mn.

  H NMR for 4: (300 MHz; CDCl3): δ (ppm)= 7.3 (m, 5H, Ph), 5.1 (m, 1H, NH), 5.0 (s, 2H, OCH2Ph), 3.8-3.7 (m, 1H, CH-NHZ), 3.6-3.5 (m, 2H, CH2-OH), 2.3-2.2 (m, 2H, CH2-CH2-CH), 1.9-1.7 (m, 2H, CH2-COOtBu), 1.4 (s, 9H, tBu). HPLC for 4: 1.52 min. Calculated monoisotopic mass for 4 (C17H25NO5) 323.17 g/mol, ES-MS (70 eV, m/z) found for 4: 324.3 [M+H] + , 346.3 [M+Na] +

  ). Molecular weights were also calculated using the relative intensities of the signals at 3.40 ppm (m, 1 Ha, CH2-OH) and 4.65 ppm (m, 1H, CH(NHZ)-CH2-OH) of the chain end and the intensity of the signal at 1.5-1.6 ppm (m, 2H CH2-CH2-CH). Final polymerization yield was 81 %. Melting point and glass transition temperature were measured by DSC at 180°C and 45°C, respectively. 1 H NMR : (300 MHz; DMSO-d6): δ (ppm)= 7.4-7.3 (m, 5H, Ph), 7.2 (m, 1H, NH), 4.95 (s, 2H, OCH2Ph), 4.0 (m, 1Ha, CH2-O), 3.8 (m, 1Hb, CH2-O), 3.6 (m, 1H, CH-NHZ), 2.2 (m, 2H, CH2-CO), 1.6 (m, 1Ha, CH2-CH2-CH), 1.5 (m, 1Hb, CH2-CH2-CH). 13 C NMR : (75 MHz; DMSO-d6): δ (ppm)= 26.4 (CH2-CH2-CH), 30.4 (CO-CH2-CH2), 49.6 (CH-NHZ), 65.8 (CH2-O and OCH2Ph), 128.1-128.8 (CH of Ph), 137.6 (C of Ph), 156.5 (COCH2Ph), 172.8 (CH2-CO-CH2).

  C NMR : (75 MHz; DMSO-d6): δ (ppm)= 24.8 (CH2-CH2-CH), 29.8 (CO-CH2-CH2), 49.5 and 52.0 (CH-NH3 + ), 60.8 and 63.5 (CH2-O), 172.5 and 174.2 (CH2-CO-CH2).

Scheme 1 .Scheme 2 .Scheme 3 .

 123 Scheme 1. Amino-functionalized lactones described in the literature and used for polyester synthesis.

Figure 1 .

 1 Figure 1. 1 H NMR (300 MHz; DMSO-d6) spectrum of 5-Z-amino-δ-valerolactone 5.

Figure 2 .

 2 Figure 2. 1 H NMR (300 MHz; DMSO-d6) spectrum of poly(5-Z-amino-δ-valerolactone).

Figure 3 .

 3 Figure 3. Molecular weights of final polymers obtained from 5-NHZ-δ-VL with Sn(Oct)2 or Sn(OTf)2 as a catalyst and different solvent/temperature/pressure conditions (B for bulk, A for atmospheric pressure and R for reduced pressure 1.10 -3 bar).

Figure 4 .Figure 5 .Figure 6 . 1 H

 4561 Figure 4. 1 H NMR (300 MHz; DMSO-d6) spectra of the poly(5-NHZ-δ-valerolactone-co-εcaprolactone) (f5-NHZ-δ-VL = 10 to 50) and of poly(5-NHZ-δ-valerolactone) (f5-NHZ-δ-VL = 100). (Signals at 2.5 and 3.3 ppm correspond to DMSO-d6 and residual water of DMSO-d6, respectively)

Figure 6

 6 Figure 6

Figure S10 .

 S10 Figure S10. DSC thermogram of poly(5-Z-amino-δ-valerolactone).

Figure S11 .

 S11 Figure S11. Chromatogram of poly(5-Z-amino-δ-valerolactone) (TableS1, entry 4, SEC

Figure S12. 1 H

 1 Figure S12. 1 H NMR (300 MHz; DMSO-d6) spectrum of poly(5-NH3 + -δ-valerolactone)

Figure S15 .

 S15 Figure S15. Conversion and molecular weight vs time for the ROP of 5 using Sn(Oct)2 as a

Figure S16 .

 S16 Figure S16. Conversion and molecular weight vs time for the ROP of 5 using Sn(OTf)2 as a

Figure S3. 13 C

 13 Figure S3. 13 C NMR (300 MHz; DMSO-d6) spectrum of 5-Z-amino-δ-valerolactone 5. (δ : 40 ppm = DMSO-d6).

Figure S4 .

 S4 Figure S4. HPLC chromatogram of 5-Z-amino-δ-valerolactone 5.

Figure S5 .

 S5 Figure S5. ESI mass spectrometry spectrum of 5-Z-amino-δ-valerolactone 5.

Figure S6 .

 S6 Figure S6. DSC thermogram of 5-Z-amino-δ-valerolactone 5.

Figure S11 .

 S11 Figure S11. Chromatogram of poly(5-Z-amino-δ-valerolactone) (TableS1, entry 4, SEC carried out in DMF, PMMA standards).

Figure S13. 13 C

 13 Figure S13. 13 C NMR (75 MHz; DMSO-d6) spectrum of poly(5-NH3 + -δ-valerolactone). (δ : 40 ppm = DMSO-d6)

Figure S14 .

 S14 Figure S14. Chromatogram of poly(5-NH3 + -δ-valerolactone) (TableS1, entry 4, SEC carried out in NaCL 1.6 M, PLL standards). Due to ion exchanges with the column, part of the initial counter-ions are no longer paired with the ammonium groups, which explains that they are visible in the chromatogram.28

Figure S15 .

 S15 Figure S15. Conversion and molecular weight vs time for the ROP of 5 using Sn(Oct)2 as a catalyst.

Figure S16 .

 S16 Figure S16. Conversion and molecular weight vs time for the ROP of 5 using Sn(OTf)2 as a catalyst.

Table 1 .

 1 Synthesis of poly(5-NHZ-δ-VL) in bulk.

	Entry Catalyst Temperature	Pressure	Reaction	Conversion	MnNMR a	MnSEC b	Mw/Mn b
			(°C)	(bar)	time (h)	(%)	(g/mol)	(g/mol)
	1	Sn(Oct)2	140	1	96	93	4900	5 000	1.2
	2	Sn(Oct)2	140	1.10 -3	120	91	5600	7 000	1.2
	3 Sn(OTf)2	100	1.10 -3	120	88	4750	5 000	1.3
	a. calculated using 1 H NMR signals at 4.65 ppm and 3.40 ppm
	b. SEC in DMF, calibration with PMMA standards

Table 2 .

 2 Synthesis of poly(5-NHZ-δ-VL-co-ε-CL) in bulk (catalyst Sn(Oct)2, initiator iPrOH, 1 bar, 110°C, 24 h).

	Entry f5-NHZ-δ-VL a F5-NHZ-δ-VL b Conversion b	Conversion b	Mnth c	MnSEC d	Mw/Mn d
				5-NHZ-δ-VL	ε-CL	(g/mol)	(g/mol)
				(%)	(%)
	1	10	12	95	89	11500	8600	1.2
	2	25	40	90	60	10700	5800	1.1
	3	50	83	80	70	13950	3300	1.6
	a. calculated using Equation (2)
	b. calculated using 1 H NMR
	c. calculated using Equation (1)
	d. SEC in DMF, calibration with PMMA standards

† Supporting Information Available: Additional synthetic data and spectral data. This material is available at http://www3.interscience.wiley.com

Acknowledgements

The authors wish to thank the French Ministry of Education and Research for Sebastien Blanquer's fellowship, Sylvie Hunger, Laurent Leclercq and Cédric Paniagua for their help on NMR, low pressure SEC analyses and lactone synthesis, respectively.

Spectral data, chromatograms and thermograms a) Benzyl 6-oxotetrahydro-2H-pyran-3-ylcarbamate (5-Z-amino-δ-valerolactone) (5):

Figure S2. 1 H NMR (300 MHz; DMSO-d6) spectrum of 5-Z-amino-δ-valerolactone 5.

c) Poly(5-NH3 + -δ-valerolactone) (7):


Barbara Porsio

Laurent Lemaire

Sarah El Habnouni

Vincent Darcos

Florence Franconi

Xavier Garric

Jean Coudane

Benjamin Nottelet
email: benjamin.nottelet@univ-montp1.fr

MRI-visible nanoparticles from hydrophobic gadolinium poly(-caprolactone) conjugates

Keywords: nanoparticles, MRI, polyester

In this work we report on the synthesis of two hydrophobic and degradable gadolinium poly(-caprolactone) conjugates and their use for the preparation of MRI-visible nanoparticles intended for diagnosis applications. Advantage has been taken from functional poly(caprolactone)s (PCL) bearing propargyl (PCL-yne) or amine groups (P(CL-co-NH2VL)) to yield conjugates by following two strategies. In a first approach, an azido-chelate of gadolinium (Gd(III)) has been conjugated by CuAAC to PCL-yne to yield a polymeric chelate containing 2.6 wt% of Gd(III). In a second approach, a dianhydride Gd(III)-ligand was reacted with P(CL-co-NH2VL) to yield, after complexation with Gd(III) salts, a polymeric chelate containing 15.4 wt% of Gd(III). The polymers biocompatibility was assessed against L929 fibroblasts. In a second part, advantage was taken from the PCLs conjugates hydrophobicity to easily prepare by nanoprecipitation nanoparticles with diameters ranging from 120 to 170 nm. The nanoparticles MRI-visibility was then evaluated and confirmed under the spin-echo and the clinically relevant gradient-echo MRI sequences.

Introduction

Magnetic resonance imaging (MRI) is the leading imaging technique to provide high spatial and temporal resolutions in clinical diagnosis and enhance the detection and characterization of lesions within the body. 1 In this frame, MRI-visible nanoparticles are proposed for early tumor diagnosis (especially in liver), thrombus diagnosis, as well as for their potential as platform for multifunctional biomedical applications with simultaneous drug-delivery and imaging capabilities. 2,3 Polymeric nanoparticles are generally associated with negative contrast agents (CAs), that contain ferrous superparamagnetic compounds (T2/T2* agents), or positive CAs, that contain paramagnetic elements (e.g., gadolinium Gd(III)) (T1 agents), embedded or grafted to the materials. T1 agents are however generally preferred over T2/T2* agents as they induce a positive enhancement of the signal by modulating the longitudinal relaxation time of the water protons in the tissues. 4 This explains why the blood pool agents currently approved for clinical uses are mainly based on hydrophilic and low molecular weight Gd(III) chelates (Magnevist®, Dotarem®, Omniscan TM ) that are injected prior to MRI scanning in order to increase the signal. However, large amounts of these potentially toxic contrast agents are needed per injection. 5 Various strategies have therefore been proposed to address this problem in particular with water soluble macromolecular contrast agents (MMCAs) that are known to remain in the vascular system for a longer period, and may thus provide a longer imaging window and a more favorable signal-to-noise ratio. In addition, polymeric systems are believed to allow higher sensitivity by increasing the relaxivity of Gd(III)-CAs and allow modulating the pharmacokinetics of Gd(III)-CAs. 6 More in details, a common strategy to get higher relaxivity relies on the reduction of Gd(III)-CAs tumbling rate in solution. This can be obtained by the design of polymeric systems whose higher conformational rigidity over low molecular weight chelates is expected to restrict the internal rotation of the Gd(III)-CA. 7 MMCAs with multiple Gd(III) centres have thus extensively been studied, 4,8,9 and include water-soluble polycondensates of chelates, [10][11][12][13] water-soluble linear, brush, star or dendrimer conjugates, [14][15][16][17][18][19][20] or amphiphilic copolymeric conjugates able to self-assemble into the form of core-shell micelles. [21][22][23][24][25][26] However, one drawback resides in the limited relaxivity enhancements because of the flexibility of the copolymers used, especially poly(ethylene oxide), and the fact that Gd(III) centers are in most cases linked as chain-ends moieties, which limits their conformational restriction. In that sense, crosslinked structures can be an interesting alternative to slow down Gd(III) centers rotational motions. 27 The use of hydrophobic polymer backbone bearing Gd(III) centers to generate MRI nanoparticles may be another alternative as recently illustrated by our group. 28- 30 Indeed, although hydrophobic environment do not favor the formation of hydration sphere and water molecule exchanges between the inner and second hydration sphere, it could be of benefit to provide a more constraint environment. 31 This approach is to date limited to the use nanoparticles made of FDA approved hydrophobic, non-cytotoxic and biodegradable polyesters like PLA and PLGA to encapsulate Gd(III) chelates. However, hydrophilic lowmolecular weight Gd(III) chelates incorporated into PLGA micro-or nanospheres have been shown to rapidly diffuse out leading to a loss of MRI-visibility. 2,[31][32][33][34] To avoid this, surface modification of PLGA nanospheres was recently proposed by Ratzinger et al. who immobilized diethylenetriaminepentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ligands on spacers prior to Gd(III) chelation. 35 Although very promising, two main drawbacks were reported. First, Gd(III) final dose was highly dependent on the type of ligation, with efficient loading obtained only with poly(ethylenimine) spacers leading to core-shell amphiphilic structures. Second, extensive washes had to be performed by successive dialyses to eliminate the non complexed Gd(III) which resulted in partial degradation of the PLGA particles.

The present work aims at an alternative strategy for the preparation of MRI-visible nanoparticles directly generated from original multivalent PCL-based MMCAs. By contrast with others MMCAs, we take advantage of two multifunctional PCLs, namely a propargylated PCL and an aminated PCL, to yield hydrophobic PCL-based multicenters MMCAs by Cu I -catalyzed [3+2] cycloaddition (CuAAC) and amidification reaction, respectively. MMCAs are characterized in particular with respect to their Gd(III) content and their cytocompatibility. Thanks to their hydrophobicity, the PCL MMCAs are then used to easily prepare MRI-visible nanoparticles that are characterized in terms of size and surface charges. Finally, the nanoparticles MRI-visibility is evaluated in vitro under the spin-echo and the clinically relevant gradient-echo MRI sequences.

Materials and methods

Materials

Benzyl alcohol, ε-caprolactone and toluene were dried over calcium hydride for 24 hours at room temperature and distilled under reduced pressure. Tetrahydrofuran (THF) was dried by refluxing over a benzophenone-sodium mixture and distilled. All other chemicals were obtained from Aldrich and were used without any further purification. Deuterated chloroform (CDCl3) and dimethyl sulfoxide (DMSO6D) were purchased from Eurisotop (Saint-Aubin, France). Spectra/Por dialysis tubes (cut-off 1000, 3500, 5000 g/mol) were purchased from Spectrum Labs (Breda, The Netherlands). PrestoBlueTM, Dulbecco's Modified Eagle Medium (DMEM ⁄ F-12), Phosphate Buffered Saline (PBS), sterile Dulbecco's Phosphate Buffered Saline (DBPS), Foetal Bovin Serum (FBS), penicillin, streptomycin, and glutamine were purchased from Invitrogen (Cergy Pontoise, France). BD Falcon™ Tissue Culture Polystyrene (TCPS) 24-well plates were purchased from Becton Dickinson (Le Pont de Claix, France).

Characterization

1 H NMR and 13 C NMR spectroscopy was performed on a Bruker spectrometer (AMX300) operating at 300 MHz and 75 MHz, respectively. Deuterated chloroform or deuterated dimethyl sulfoxide were used as solvents. Chemical shifts were expressed in ppm with respect to tetramethylsilane (TMS).

Infrared spectroscopy was performed on a Perkin Elmer Spectrum 100 FT-IR spectrometer using the attenuated total reflectance (ATR) technique.

Size exclusion chromatography (SEC) was performed at room temperature on a Waters system equipped with a guard column, a 600 mm PLgel 5 mm Mixed C column (Polymer Laboratories), and a Waters 410 refractometric detector. Calibration was established with poly(styrene) standards from Polymer Laboratories. THF was used as eluent at a flow rate of 1 mL.min -1 .

LC/MS analyses were performed on a Q-TOF (Waters) spectrometer fitted with an electrospray interface. Solvents used for HPLC and LC/MS were HPLC grade. MALDI analyses were performed on a Ultra-Flex III (Bruker) spectrometer using a dithranol matrix. Particle size and zeta potential determination were performed on a Zetasizer Nano ZS (Malvern Instrument, UK). For mean particle size and polydispersity index (PDI) measurements, the samples were diluted in milliQ water. For zeta potential analyses, the samples were diluted in milliQ water or PBS (pH = 7.4). Solutions were filtered through a 0.45µm filter. All measurements were performed in triplicate.

Gd(III) quantification was performed on an

Transmission electron microscope (TEM) micrographs were obtained with a JEOL 1200 EXII (working voltage of 120 kV). A drop of nanoparticles solution was placed onto a carbonsupported copper grid for 5 min. The excess liquid was removed by capillarity with a filter paper. Mean particle size was determined by measuring the particles diameter with Image J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2012) 2.3 Synthesis of MRI-visible P(CL[DTPA(Gd)]) 3 3 was prepared by reaction between poly(α-propargyl-ε-caprolactone-co-ε-caprolactone)s P(Pg-CL) 2 (FPrCL = 5%, MnSEC = 25000 g.mol -1 . Ð= 1.9) and a clickable Gd(III) chelate (diN3-DTPA(Gd(III))) 1 (full synthetic details for compounds 1 and 2 can be found in the Supporting Information). Briefly, copolymer 2, complex 1 (3 eq./αPgεCL units) and CuBr (2eq./αPgεCL units) were solubilized in a large amount of dimethylformamide (DMF). The solution was degassed by three freeze-pump-thaw cycles. N,N,N′,N′′,N′′pentamethyldiethylenetriamine (PMDETA) (2eq./ αPgεCL units) degased by argon bubbling was added to the reaction medium. The reaction was carried out for 48 hours at room temperature under stirring. The crude medium was dissolved in THF for dialysis (CO 3500 g.mol -1 ) against distilled water. 3 was recovered after removal of the solvents and dried in vacuo. The content of complexed Gd(III) in 3 was quantitatively determined by ICP-MS : 2.6 wt%.

Synthesis of MRI-visible P(CL-co-VL[DTPA(Gd)]) 5

5 was prepared by reaction between poly(-caprolactone-co-5-amino--valerolactone) P(CLco-NH2VL) 4 (F-VL = 33%, MnSEC = 6000 g.mol -1 . Ð= 1.2) and DTPA dianhydride (full synthetic details for compound 4 can be found in the Supporting Information). More in details, a solution of DTPA dianhydride (162.1 mg, 1.2 eq. with respect to NH2 groups) in anhydrous DMF was added dropwise to a solution of 4 (100 mg, 0.37 mmol of NH2) in a large amount of anhydrous DMF (4 mL) and in the presence of triethylamine (0.1 mL; 3eq.

with respect to NH2 groups). The reaction was left to stir for 24 hours at room temperature under an inert atmosphere of argon. The resulting polymer was purified by dialysis against an HCL solution (0.1M) and distilled water (CO 1000 g.mol -1 ). The polymer was finally recovered by freeze-drying in good yield (205 mg). The extent of amidification was determined by 1 H NMR analysis (60% yield). 1 Complexation of the macromolecular ligand with Gd(III) was then carried out. In a typical experiment, the copolymer (100 mg, 9.3×10 -5 mol of mono-amide DTPA) was solubilized in DMSO (5 mL) before addition of GdCl3-6H2O (63 mg, 1.9×10 -4 mol, 2eq. with respect to mono-amide DTPA). Complexation was let to run for four days under stirring at 40 °C. The polymer was purified by dialysis (CO 1000 g.mol -1 ) against methanol for 24 hours before final recovery of 5 by solvent removal under reduced pressure. The content of complexed Gd(III) in 5 was quantitatively determined by ICP-MS : 15.4 wt%.

Preparation of MRI-visible nanoparticles

Nanoparticles were prepared with a concentration of Gd(III) of 0.1 wt% with respect to PCL.

In a typical experiment, 477.5 mg of PCL (MnSEC = 35 000 g/mol, Ð = 1.86), 22.5 mg of 3 and 180 mg of SPAN ® 80 were dissolved in 90 mL of acetone. This organic solution was added dropwise to an aqueous phase containing Tween ® 80 (360 mg in 180 mL) under magnetic stirring and at room temperature. The mixture was left under stirring for 2 hours to form the nanoparticles by solvent diffusion. Acetone and part of the water were eliminated at 30 °C under reduced pressure to a final suspension volume of 100 ml. The suspension was dialyzed for 24h against distilled water (CO = 5 000 g/mol) before freeze-drying and recovery of the nanoparticles (NP3) in good yield (83%).

Similar conditions were used to prepare nanoparticles (NP5) from polymer 5 with adjusted ratios of PCL and 5.

MR imaging protocols

MR imaging experiments were performed on a Bruker Biospec 70/20 system operating at a magnetic field of 7T (Bruker, Wissembourg, France). The resonant circuit of the NMR probe was a 35-mm diameter birdcage resonator. MRI nanoparticles, under the form of small clusters, were embedded in a degassed 1 % (w/w) agarose gel prior to imaging. To test signal enhancement, samples were analyzed using either a three-dimensional ( Sample disks (ø 15 mm) were cut from copolymer films and disinfected in ethanol for 30 minutes before immersion in a solution of sterile PBS containing penicillin and streptomycin (1 mg/mL) and incubation for 48 hours at 37 °C. Films were then rinsed 2 times with sterile PBS before soaking for 12 hours in sterile PBS. After disinfection, disks were placed in TCPS 12-well plates and Viton ® O-rings were used to maintain the samples on the bottom of the wells and avoid cells growing on TCPS underneath the samples. Disks were finally seeded with 1.10 4 cells and viability was evaluated after 1, 2 and 3 days using PrestoBlue® assay, which reflects the number of living cells present on a surface at a given time point. At scheduled time points, culture medium was removed and replaced by 1 mL of fresh medium containing 10 % of PrestoBlue®. After 2 hours of incubation at 37 °C, 200 µL of supernatant were taken from each well and analyzed for fluorescence at 530 nm (ex.) and 615 nm (em.) with a Victor X3 (Perkin Elmer).

Results and discussions

Synthesis of the PCL MMCAs

The objective of the present work was to propose an alternative strategy to hydrophilic MMCAs or mixtures of low molecular weight CAs and polymer matrices for the preparation of MRI-visible nanoparticles. In addition, attention was paid to provide MRI-visible nanoparticles that would also be degradable and biocompatible. Therefore, two multivalent hydrophobic PCL MMCAs have been prepared (Scheme 1). In a first approach, the ligation chemistry between the PCL backbone and the Gd(III) chelate was based on azide-alkyne Huisgen cycloaddition. An azide-functionalized bis-amide DTPA(Gd(III)) chelate (1) and PCL-yne (2) have been prepared and used for the CuAAC according to a previously reported methodology. 29 Thanks to the high efficiency of the click chemistry ligation and of the use of a preformed clickable Gd(III) chelate, this strategy allows to finely tune the final Gd(III) content as well as the density of Gd(III) chelate along the polymer backbone. To ensure MRI-visibility of the targeted MMCA, a PCL containing 5 mol% of propargylated units (MnSEC = 25000 g.mol -1 . Ð= 1.9) was synthesized and conjugated to the azide-functionalized bis-amide DTPA(Gd(III)). This composition was chosen to yield a final Gd(III) content of ca. 2 wt%, as it was shown in previous studies that this ratio provides a good MRI signal enhancement while minimizing the overall loading of the toxic Gd(III) species. 29 The copolyesters were characterized before and after ligation by 1 H NMR analyses (Supporting Information Figure S1). The final content of Gd(III) in 3 was determined by ICP-MS and found to be 2.6 wt% in the copolyester to be compared with the targeted 2.1 wt%. This small discrepancy may be due to an initial underestimation of the propargyl group content, resulting from the low intensity of the signal corresponding to the methine proton of the propargyl in the 1 H NMR spectra.

In a second approach, the ligation chemistry between the PCL backbone and the Gd(III) chelate was based on the reaction between the primary amine groups of poly(-caprolactoneco-5-amino--valerolactone) 36,37 (P(CL-co-NH2VL)) and the activated carboxylic groups of DTPA dianhydride to yield an original Gd(III)/PCL amide conjugate. P(CL-co-NH2VL) (4) was synthesized by ring opening polymerization of CL and amino-protected 5-Z-amino-valerolactone (NHZ-VL), followed by the acidic recovery of the free amine groups. The molar ratio of NH2-VL was 33% while the final molecular weight was MnSEC = 6000 g.mol -1 with Ð= 1.2 (Supporting Information Figure S2a). In opposition to the first strategy, the PCL MMCA was obtained in two steps by reaction of 4 with the Gd(III) ligand, namely DTPA dianhydride, and the subsequent complexation of the macromolecular ligand with GdCl3-6H2O. 1 H NMR analysis was used to evaluate the extent of DTPA grafting on 4 (Supporting Information Figure S2b). By comparison between the intensities of the signals at  = 3.0 to 3.50 ppm corresponding to the methylene protons of mono-amide DTPA and of the methine proton of NH2-VL units and the signal at  = 4.00 ppm corresponding to the methylene protons of εCL and NH2-VL units, a 60% yield was calculated. 5 was finally obtained by complexation of GdCl3-6H2O with the PCL macroligand. The final content of Gd(III) in 5 was determined by ICPMS and found to be 15.4 wt% in good accordance with the expected value of 14.7 wt%.

In vitro cytocompatibility of PCL MMCAs

Tests were conducted on the L-929 fibroblasts cell line, as recommended by International and European standards. 38 Cytocompatibility of the MRI-visible polymers was assessed on films containing the same gadolinium concentration (0.1 wt%) as the one used in the MRI-visible nanoparticles (see 3.3 and 3.4). In addition, higher concentrations (0.4 wt% and 1 wt%) were also evaluated for films prepared with 5 as this MMCA was evaluated for the first time. Cell proliferation and its extent were compared with TCPS culture plates and PCL films controls.

As shown in Figure 1 the presence of PCL MMCAs 3 and 5 did not impede fibroblast proliferation. At a 0.1wt% Gd(III) concentration, similar results were observed for 3 and 5 although their chemical nature and ligation strategies were different. Although lower than on TCPS control, proliferations occurred on films prepared with 3 or 5. They were similar, if not higher, to the ones observed on pristine PCL, which is widely recognized as a biocompatible material. The same trend was observed for films prepared from 5 and having a higher (0.4 wt%) Gd(III) concentration. Only at the high 1.0 wt% Gd(III) concentration a lower proliferation was observed. Two conclusions may be pointed out from these results. First, concentrations of Gd(III) should be kept low to guaranty a maximal cytocompatibility. This should not be a problem considering the good MRI-visibility obtained with the lowest 0.1 wt% Gd(III) concentration (see 3.4). Secondly, taking into account the similar proliferation obtained with MRI-PCLs compared to PCL, the former would appear to be suitable for the growth of fibroblasts and cell-contacting applications.

MRI-visible nanoparticles

Nanoparticles were prepared by nanoprecipitation with acetone and water as a miscible solvents mixture. An overall concentration of Gd(III) equal to 0.1 wt% was targeted and obtained by mixing defined amounts of pristine PCL and MRI-visible PCLs 3 and 5. In addition to MRI-visible nanoparticles NP3 and NP5, control PCL nanoparticles were also prepared. Dynamic light scattering (DLS) measurements showed diameter of nanoparticles ranging from 120 to 170 nm with polydispersities around 0.250 (Table 1, Figure S3). The distribution of nanoparticles obtained by DLS was in agreement with the one reported in the literature under the same conditions. 39 Images obtained by TEM show spherical nanoparticles with a moderate size-distribution (Figure 2). Image analyses gave relatively smaller diameters compared to DLS measurements, typically in the range 55 to 80 nm. This difference is attributed to the presence of few larger nanoparticles aggregates (see Figure S4) that may form as a result of the hydrophobicity of the PCL nanoparticles and that are known to influence DLS measurements in the intensity mode. MR imaging was finally carried out on the same samples after storage for a prolonged period of 18 months at 4°C. Lower rows in Figure 3a and 3b show the signal observed for NP3 and NP5 after storage. It is to note that under the same MRI experiment conditions, the T1 signal enhancement was similar to the one initially observed. Moreover, as the signal of the gel did not significantly change with time and that no diffusion in the gel was observed, it may be assumed that no release of Gd(III) occurred, which qualitatively confirms the stability of the macromolecular contrast agents. Of course under physiological conditions different results may be expected, but this result is in agreement with a previous study carried out on films prepared with compound 3 that demonstrated a good stability at 37°C (PBS, pH 7.4) with only 0.1% of the total amount of Gd(III) released after 6 months from films. 29

Conclusion

In this work we reported on two hydrophobic and degradable PCL MMCAs that were

  Element XR sector field ICP-MS (inductively coupled plasma-mass spectrometry) at Géosciences in Montpellier (University Montpellier II). Internal standardization used an ultra-pure solution enriched with indium.

  H NMR (300 MHz; DMSO-d6 ), δ (ppm): 4.00 (m, -CH2-O)NH-VL & εCL, 3.75 (CH2-CH(NH2)-CH2)NH-VL, 3.60-3.00 (N-CH2, CH2-CH(N)-CH2 )NH-VL & DTPA, 2.20-2.40 (CO-CH2-CH2)NH-VL & εCL, 1.70 (CH2-CH2-CH(N))NH-VL, 1.30-1.60 (CH2-CH2-CH2-CH2-CH2)εCL.

  3-D) acquisition with relaxation enhancement (RARE) sequence (TR = 3000 ms; mean echo time (TEm) = 8 ms; RARE factor = 8; FOV = 3 × 3 × 1.5 cm; matrix 128 × 128 × 64) in which T1 weighting was introduced into the MR images using an inversion pulse (inversion time was set at 1100 ms, sufficient to allow canceling of the embedding gel) or a 3D-gradient echo sequence (TR = 110 ms; TE = 3 ms;  = 60°; FOV = 3 × 3 × 1.5 cm; matrix 128 × 128 × 64).

2. 7

 7 In vitro cytocompatibilityMurine fibroblasts cells (designated L929) were used to assess the in vitro cytocompatibility of the materials as recommended by the International and European Standards (ISO 10993-5:2009). L929 cells were cultured in DMEM alpha supplemented with 10 % Fetal Bovine Serum (FBS), penicillin (100 U/mL), streptomycin (100 µg/mL) and glutamine (2 mM).

Scheme 1 . 5 ,

 15 Scheme 1. Preparation of MRI-visible PCL nanoparticles : i) CuBr, PMDETA, DMF, RT, 48 hours ; ii) (PCL, 2, SPAN ® 80, acetone) / (Tween ® 80, water), RT, 2 hours ; iii) DTPA

Figure 1 .

 1 Figure 1. L929 proliferation on PCL MMCAs films compared to PCL films and TCPS

-

  25 mV in water and from -9 mV to -5 mV in PBS. These negative values are classically observed for PCL nanoparticles, likely due to the surface exposure of carboxylate groups of the chain-ends at the surface.40 In addition, in NP3 and NP5 the mono-amide DTPA carboxylic groups are also exposed. The presence of bis-amide DTPA in copolymer 3 decreased the zeta potential of NP3 compared to PCL nanoparticles as shown by a ca. 10 mV decrease. In opposition, the presence of partially protonated amine groups in PCL-co-VLDTPA(Gd) resulted in a shift of the zeta potential of NP5 towards less negative values, with a 10 mV increase compared to PCL. Upon addition of salts in PBS medium and as a result of charge screening, zeta potential values drifted towards more neutral values while remaining negative.

Figure 2 .

 2 Figure 2. TEM micrographs of (a) PCL nanoparticles (b) P(CL[DTPA(Gd)]) nanoparticles

Figure 3 .

 3 Figure 3. Spin echo (a) and gradient echo (b) MR-imaging at t0 and after 18 months storage.

  successfully synthesized for the straightforward preparation of MRI-visible nanoparticles. The two strategies, convergent click chemistry and divergent amidification, led to MMCAs suitable for the preparation of MRI-visible nanoparticles containing 0.1wt% Gd(III) and exhibiting hydrodynamic diameters in the range 120 to 170 nm. Although different in nature, all nanoparticles turned to be MRI-visible independently of the MRI sequence used, in particular when using the clinically relevant gradient-echo sequence. Although this first study should be completed in the near future by additional experiments (degradation assays, toxicity evaluation of NP, hydrophilization of NP surfaces, in vitro/in vivo evaluation), we believe that the proposed PCL MMCAs represent an attractive platform for the preparation of degradable theranostic agents and could open the way to new diagnostic tools.

Table 1 .

 1 Mean particle size and zeta potential of PCL and MRI-visible PCL nanoparticles. Determined by DLS ; b Determined by TEM image analysis Zeta potential of the nanoparticles was measured for all systems in milliQ water and in PBS (Table1). All nanoparticles had a negative zeta potential with values ranging from -46 mV to

	Particles	Mean particle size [nm]	PDI a	Zeta Potential [mV] mQ water PBS	Gd/PCL [wt%]
	PCL	123 ± 6 a (55 ± 15) b	0.236 ± 0.012	-38	-5	0
	P(CL[DTPA(Gd)]) NP3	153 ± 11 a (71 ± 18) b	0.276 ±0.016	-46	-8	0.1
	P(CL-co-VL[DTPA(Gd)]) NP5	170 ± 24 a (80 ± 20) b	0.246 ±0.055	-25	-9	0.1

a

Acknowledgements

The authors wish to thank the French Ministry of Education and Research for Sarah El Habnouni's fellowship, the Erasmus program for Barbara Porsio's fellowship. For analyses, authors thank Chantal Douchet and Olivier Bruguier for ICP-MS, Corine Tourné-Péteilh for Nano ZS, Sylvie Hunger and Cedric Paniagua for NMR and Frank Godiard for TEM.


Ismaïl Ben

Hassan Saïdï

Christian Tenaud
email: christian.tenaud@limsi.fr

Guillaume Fournier

Hassan Saïdi

Role of the recirculation bubble dynamics on the unsteadiness of the Shock Wave Boundary Layer Interaction

come   L'archive ouverte pluridisciplinaire

INTRODUCTION

The Shock-Wave Boundary-Layer Interaction (SWBLI) is a recurrent physical phenomenon present in aeronautical and space systems. If the shock wave is strong enough, the subsequent adverse pressure gradient can lead to a separation of the boundary layer. A separation bubble is then created surrounded by a complex system of shock waves. The highly 3-D separation bubble leads to a steep increase of the drag force, heat fluxes and pressure fluctuations at the wall. Previous studies have additionally shown that the recirculation bubble as well as the reflected shock wave are subjected to a low frequency streamwise motion called the unsteadiness of the SWBLI. This unsteadiness subjects structures to oscillating loads that can damage their integrity, justifying the importance of a careful study of SWBLIs and particularly its low frequency unsteadiness.

Even if this phenomenon is well known, the mechanism leading to the SWBLI unsteadiness is still not well understood. Mainly two kind of mechanisms have been referred to in order to explain the existence of the SWBLI unsteadiness [START_REF] Délery | Some physical aspects of shock wave/boundary layer interactions[END_REF]. The first one consists in an excitation of the SWBLI by the large scales of the incoming turbulent boundary layer. The second one is provoke by the dynamics of the recirculation zone that drives the unsteadiness of the whole SWBLI system. Despite arguments in favor of each mechanism, no definitive explanation have been yet provided. The aim of the present work is then to perform well resolved DNS of SWBLIs and analyze results in order to contribute to a better understanding of the SWBLI unsteadiness and the physical mechanism causing these low frequency oscillations. To this end, the canonical configuration of an oblique shock wave impinging a boundary layer developing on a flat plate is considered.

NUMERICAL APPROACH

The Navier-Stokes equations are solved by using a high order finite volume approach on cartesian meshes. The Euler part is discretized by means of a high-order one-step Monotonicity Preserving scheme, namely the OSMP7 scheme [START_REF] Daru | High order one-step monotonicity-preserving schemes for unsteady compressible flow calculations[END_REF], based on a Lax-Wendroff approach, which ensures a 7th-order accuracy in both time and space in the regular regions. Monotonicity-Preserving (MP) constraints, first developed by Suresh and Huynh [START_REF] Suresh | Accurate monotonicity-preserving scheme with Runge-Kutta time stepping[END_REF] and further extended in [START_REF] Daru | High order one-step monotonicity-preserving schemes for unsteady compressible flow calculations[END_REF], are applied; they locally relax the TVD constraints near extrema. The resulting OSMP7 scheme recovers the 7th-order accuracy in both time and space everywhere, except near discontinuities where the scheme is Monotonicity-Preserving. Besides, the discretization of the diffusive fluxes is obtained by means of a classical centered 2nd-order scheme. These numerical schemes are implemented in the in-house parallel (MPI) Finite Volume DNS solver (code CHORUS) that has been developed at LIMSI-CNRS. This numerical tool has intensively been validated on the simulation of high Reynolds number turbulent and shocked flows [START_REF] Hassan Saidi | Solving three dimensional turbulent compressible flows using a high order One Step Monotony Preserving scheme[END_REF]. Results demonstrate the correct accuracy of the numerical approach to predict turbulent features and the great efficiency of the MP procedure to capture discontinuities without spoiling the solution at an almost negligible additional cost (at most 2.5 %).

RESULTS ON SWBLI CONFIGURATIONS

In order to better understand the mechanisms leading to the unsteadiness of the SWBLI, it has been chosen to first perform a SWBLI simulation suppressing one of the two suspected mechanisms leading to the unsteadiness. By simulating the interaction between a laminar boundary layer and an incident oblique shock wave, the suspected influence of the large scale turbulent structures within the incoming boundary layer on the SWBLI unsteadiness has been suppressed. The only remaining suspected cause of the unsteadiness would be the dynamics of the separation bubble.

Shock-Wave Laminar Boundary-Layer Interaction:

We first chose to simulate a 3-D test-case similar to the one designed by Degrez et al. [START_REF] Degrez | The interaction of an oblique shock wave with a laminar boundary layer revisited. An experimental and numerical study[END_REF] (M ∞ = 2.15; Re x sh = 10 5 ) with however a higher shock angle (σ = 33.8 • ) to recover an unsteady behavior of the recirculation bubble that exhibits 3-D flow patterns. Results (Fig. 1-left) have shown that the separation point of the recirculation bubble and the foot of the reflected shock wave have a fixed location along time. This result shows that, in this configuration, the SWBLI unsteadiness has not been observed. Analyses of the dynamics of the recirculation bubble has been performed. Two regions have been identified in the recirculation bubble: the first part of the separation, upstream of the incident shock wave impingement and the second part downstream of the impingement. Although SWBLI unsteadiness has not been observed (no motion of the reflected shock wave), the characteristic low frequency of the SWBLI unsteadiness has however been recorded that characterizes the dynamics of the whole recirculation bubble: low frequency breathing (St L 0.03 -0.04) of the recirculation bubble and medium frequency flapping (St L 0.12 -0.15) of the shear layer in whole of the bubble, in addition to higher frequencies (St L 0.5 -0.6) characterizing the vortex shedding from the shear layer in the second part of the recirculation bubble. In particular, the reattachment location is sensitive to the instabilities of the recirculation bubble, namely the breathing, the flapping and the Kelvin-Helmholtz frequencies. Even though present in the first part, the low frequency activity is not intense enough to provoke the oscillations of the separation point and the subsequent oscillations of the reflected shock foot.

Shock-Wave Turbulent Boundary-Layer Interaction:

In order to reproduce and analyze the SWBLI unsteadiness, the simulation of the interaction between a incident shock wave and a turbulent boundary layer has secondly been undertaken (Fig. 1-right). In this context, the accurate simulation of a turbulent compressible incoming boundary layer is of great importance. A Synthetic Eddy Method (SEM) (Jarrin et al. [2006]) that we adapted to compressible flows, has efficiently been employed at the inlet boundary to generate a fully developed flat plate turbulent boundary layer (M ∞ = 2.33; Re θ = 2539) upstream of the impingement of the oblique shock-wave without a prohibitive additional computational cost. In the turbulent case, the SWBLI unsteadiness has clearly been recorded. The frequencies characterizing motion of the reattachment point are characteristic of the low (St L 0.03 -0.04) and medium frequency flapping (St L 0.12 -0.15) of the separation zone, while the frequencies of the separation point are characteristic of the low frequency flapping of the separation zone. Besides, the foot of the reflected shock wave oscillates in phase with the separation point. Motions of separation and reattachment points are mainly in opposite phase indicating successive enlargements and shrinkages of the recirculation bubble; the shrinkage phase being associated with a shedding of a large amount of fluid from the separated zone to downstream. This medium frequency flapping have been shown to be irregular in time; the maximum size of the recirculation bubble being submitted to discrepancies between successive cycles. This behavior of the separation bubble is responsible for the low frequency temporal modulation of the amplitude of the separation and reattachment points and thus to the low frequency breathing of the separation bubble.

CONCLUSION

We here show that the reflected shock-wave is passive and the SWBLI unsteadiness is related to the low frequency breathing of the recirculation bubble (and not the large scales of the incoming turbulent boundary layer); the oscillations of the reflected shocks foot being in phase with the motion of the separation point. This low frequency activity of the recirculation bubble also seems to be at play in the laminar case. Although in the laminar case the behavior in the second part of the recirculation bubble is the same as in the turbulent case, the low frequency activity in the laminar regime is counter-currently damped towards the first part of the bubble as the length of the bubble is much greater than in the turbulent case. It is then not intense enough to excite, in the first part of the bubble, the separation point and the subsequent oscillations of the reflected shock foot.

Figure 1 :

 1 Figure 1: Q criterion colored by the magnitude of the longitudinal velocity. Shock waves are highlighted by isosurfaces of ∇ • u: laminar case on the left, turbulent case on the right.

Role of the recirculation bubble dynamics on the unsteadiness of the Shock Wave Boundary Layer Interaction.