Yinyin Bao

Elise Guégain

Julien Nicolas
email: julien.nicolas@u-psud.fr

Drug-Initiated Synthesis of Cladribine-Based Polymer Prodrug Nanoparticles: Biological Evaluation and Structure Activity Relationships

By using two reversible deactivation radical polymerization techniques, either nitroxide-mediated polymerization or reversible addition-fragmentation chain transfer polymerization, the "drug-initiated" approach was applied to cladribine (CdA) as an anticancer drug to synthesize small libraries of well-defined and self-stabilized CdA-based polymer prodrug nanoparticles, differing from the nature and the molar mass of the grown polymer, and the nature of the linker between CdA and the polymer, thus allowing structurecytotoxicity relationships to be determined. Their biological evaluation was investigated in vitro on L1210 cancer cells. The preparation of fluorescent CdA-based nanoparticles with excellent imaging ability was also reported by applying the "drug-initiated" approach to an aggregation-induced emissionactive dye.

Polymer prodrug nanocarriers have emerged as promising drug delivery systems that aim to circumvent the drawbacks of traditional drug-loaded nanocarriers (i.e., relying on a physical encapsulation of the drug into the polymer matrix) that usually exhibit significant limitations such as the "burst effect" and poor drug loadings. 1,2 Polymer prodrug nanocarriers are obtained by covalent conjugation of drugs to a polymer chain by different approaches: (i) the post-functionalization of preformed polymers ("grafting to"); [3][4][5] (ii) the polymerization of drug-conjugated monomers ("grafting through") [6][7][8][9][10] and (iii) the polymerization from drug-conjugated initiators ("drug initiated" or "grafting from"). 11 In all cases, the resulting polymer prodrugs can be formulated into narrowly dispersed and stable nanoassemblies in aqueous solution. Recently, our group further developed the "drug-initiated" approach and applied it to vinyl polymers by reporting on the design of a gemcitabine (Gem)-conjugated alkoxyamine 12 or chain transfer agent (CTA) 13,14 to prepare well-defined polymer prodrug nanoparticles by either nitroxide-mediated radical polymerization (NMP) of isoprene or reversible addition-fragmentation chain transfer (RAFT) polymerization of squalenyl methacrylate (SqMA), respectively. The resulting self-stabilized, Gem-polymer prodrug nanoparticles exhibited great colloidal stability and significant in vitro and in vivo anticancer activity. Nevertheless, to further confirm the robustness and the versatility of the "drug-initiated" method from reversible-deactivation radical polymerization (RDRP) techniques, it was then applied to cladribine (CdA, 2-chloro-2'deoxyadenosine) as another anticancer drug.

CdA, which can inhibit DNA synthesis, cause cell death and induce apoptosis, has been used as a first-line therapy in hairy cell leukemia and B-cell chronic lymphocytic leukemia. [15][16][17][18][19] However, because of its rapid clearance from the bloodstream, CdA is administered at high doses, inducing cytotoxicity to resting and proliferating cells. [20][21][22] Therefore, new strategies are needed to extend the circulation time and reduce the dose of CdA, as well as to induce higher toxicity to cancer cells compared to healthy cells.

In this context, we report on the synthesis of different series of CdA-based polymer prodrugs, either by NMP or RAFT polymerization (Figure 1). 23,24 The impact of the chemical structure of the polymer prodrugs on the cytotoxicity of the corresponding nanoparticles to cancer cells was systematically investigated by comparing: (i) two different linkages between the drug and the polymer; (ii) two different polymers and (iii) different polymer chain lengths. It enabled the establishment of preliminary structure-cytotoxicity relationships. In addition, we also prepared fluorescent polymer prodrug nanoparticles by growing a welldefined polymer from a naphthalimide-based fluorophore with aggregationinduced emission (AIE) activity, 25 which enabled the successful tracking of CdA-polymer prodrug nanoparticles in living cells by a co-nanoprecipitation process. 26

Experimental Part

Materials

Cladribine was purchased from Sequoia Research Products, Limited (U.K.). 4-Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl] pentanol (CDP) and 4cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl]pentanoic acid (CDPA, 97%) were obtained from Sigma-Aldrich. Diglycolic anhydride and tetra-nbutylammonium fluoride were purchased from Thermo Fisher Scientific (USA). SqMA was prepared according to published methods. 13 N-tert-Butyl-N-(1diethyl phosphono-2,2-dimethylpropyl)nitroxide (SG1, 86%) was kindly provided by Arkema. The AMA-SG1 (2-(N-tert-butyl-N-(1-diethoxyphosphoryl-2,2-dimethylpropyl) aminoxy)propionic acid)alkoxyamine was prepared according to a published method. 27 CdA-AMA-SG1, CdA-digly-AMA-SG1, digly-AMA-SG1, CdA-CDPA, CdA-digly-CDP, digly-CDP, and Napht-AMA-SG1 were prepared as reported elsewhere. [23][24][25][26] LysoTracker Red was purchased from Life Technologies. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and human serum were purchased from Sigma (France). Penicillin was purchased from Lonza (Verviers, Belgium). All other materials were purchased from Sigma-Aldrich at the highest available purity and used as received.

Synthesis

Polymerization of isoprene from CdA-AMA-SG1. CdA-AMA-SG1 (55 mg, 0.075 mmol) was placed in a 15-mL-capacity pressure tube (Ace Glass, Model 8648-164) fitted with plunger valves and thermowells. Isoprene (0.75 mL, 7.5 mmol) and dioxane (0.75 mL) were added and the tube was subjected to three freeze-pump-thaw cycles, then backfilled with argon. The tube was placed in an oil bath at 115 °C for 2 h (PI1), 8 h (PI2), or 16 h (PI3) and then cooled to room temperature by placing them in a cold-water bath. The residue was concentrated under reduced pressure to give CdA-polyisoprene (PI) as a colorless product. Another polymerization was performed for 16 h with [isoprene] 0 /[CdA-AMA-SG1] 0 = 200/1 (PI4).

Polymerization of isoprene from CdA-digly-AMA-SG1. CdA-digly-AMA-SG1 (54 mg, 0.06 mmol) was placed in a 15-mL-capacity pressure tube (Ace Glass, Model 8648-164) that was fitted with plunger valves and thermowells. Isoprene (0.6 mL, 6.0 mmol) and dioxane (0.6 mL) were added, and the tubes were subjected to three freeze-pump-thaw cycles, then backfilled with argon. The tubes were placed in an oil bath at 115 °C for 4 h (PI1d) or 16 h (PI2d) and then cooled to room temperature by placing them in a cold-water bath. The residue was concentrated under reduced pressure to give CdA-digly-PI as a colorless product. Two other polymerizations were performed for 16 h with [isoprene] 0 /[CdA-digly-AMA] 0 = 200/1 (PI3d) and 400/1 (PI4d).

Polymerization of SqMA from CdA-CDPA. CdA-CDPA (31.8 mg, 0.04 mmol), AIBN (2.3 mg, 0.014 mmol), SqMA (0.11 g, 0.24 mmol) and dioxane (0.75 mL) were mixed together and equally distributed among four 7 mL glass vials. The solutions were degassed with argon bubbling for 30 min. The tubes were placed in an oil bath at 70 °C for 0.5 h (PSq1), 1 h (PSq2), 2 h (PSq3) or 22 h (PSq4), and then cooled to room temperature by placing them in a coldwater bath. For each polymer, the residue was concentrated under reduced pressure and precipitated in cold MeOH to give protected CdA-PSqMA as a yellow viscous liquid.

Polymerization of SqMA from CdA-digly-CDP. CdA-digly-CDP (52.5 mg, 0.06 mmol), AIBN (1.2 mg, 0.007 mmol), SqMA (0.16 g, 0.36 mmol) and dioxane (0.8 mL) were mixed together and equally distributed among three 7 mL glass vials. The solutions were degassed with argon bubbling for 30 min. The tubes were then placed in an oil bath at 70 °C for 1 h (PSq2d), 4 h (PSq3d) and 30 h (PSq4d) and then cooled to room temperature by sinking them in a cold-water bath. For each polymer, the residue was concentrated under reduced pressure and precipitated in cold MeOH to give protected CdA-digly-PSqMA as a yellow viscous liquid. Another polymerization was performed for 4 h with [SqMA] 0 /[CdA-digly-CDP] 0 = 3/1 (PSq1d).

Polymerization of isoprene from Napht-AMA-SG1. Napht-AMA-SG1 (38.7 mg, 0.056 mmol) was placed in a 15 mL capacity pressure tube (Ace Glass 8648-164) fitted with a plunger valve and a thermowell. Isoprene (PN1: 2.4 mL, 23.1 mmol; PN2: 1.2 mL, 11.5 mmol) and dioxane (PN1: 2.4 mL; PN2: 1.2 mL) were added and the tube was subjected to three freeze-pump-thaw cycles, then backfilled with argon. The tube was placed in an oil bath at 115°C for 18 h and then cooled down to room temperature by placing the tube in a cold-water bath. The residue was concentrated under reduced pressure and precipitated in MeOH to give Napht-PI as a yellow viscous liquid.

Deprotection of TBDMS protected polymer prodrugs. TBDMS-protected CdA-PI, CdA-digly-PI, CdA-PSqMA, or CdA-digly-PSqMA (100 mg) was dissolved in 0.5 mL of THF, then tetra-n-butylammonium fluoride (1 M in THF, 50 μL) was added and the solution continued to be stirred for 15 min before precipitation into 10 mL of MeOH and storage at 4°C for 36 h. The purified polymer was then collected by centrifugation, dried under high vacuum and analyzed by 1 H NMR and SEC. 1 H NMR analysis showed a complete disappearance of TBDMS protecting groups.

Analytical Methods

Nuclear magnetic resonance spectroscopy (NMR). NMR spectroscopy was performed in 5 mm-diameter tubes in CDCl 3 or DMSO-d 6 at 25 °C. 1 H and 13 C NMR spectroscopy was performed on a Bruker Avance 300 spectrometer at 300 MHz ( 1 H) or 75 MHz ( 13 C). The chemical shift scale was calibrated on the basis of the internal solvent signals.

Mass spectrometry (MS). Mass spectra were recorded with a Bruker Esquire-LC instrument.

Size exclusion chromatography (SEC). SEC was performed at 30 °C with two columns from Polymer Laboratories (PL-gel MIXED-D; 300 mm × 7.5 mm; bead diameter = 5 mm; linear part, 400 to 4 × 10 5 g mol -1 ) and a differential refractive index detector (SpectraSystem RI-150 from Thermo Electron Corp). The eluent was chloroform at a flow rate of 1 mL min -1 (Waters 515 pump) and toluene was used as a flow-rate marker. In the case of PI, the calibration curve was based on polystyrene (PS) standards (peak molar masses, M p = 162-523 000 g mol -1 ) from Polymer Laboratories. A PI calibration curve was constructed by converting the PS standard peak molecular weights (M PS ) to PI molecular weights (M PI ) using Mark-Houwink-Sakurada (MHS) constants determined for both polymers in CCl 4 at 25 °C. For PI, the MHS constants used were K PI = 2.44 × 10 4 and α PI = 0.712. For PS, K PS = 7.1 × 10 4 and α PS = 0.54 (MW < 16 700 g mol -1 ) or K PS = 1.44 × 10 4 and α PS = 0.713 (MW > 16 700 g mol -1 ). 28 In the case of PSqMA, the calibration curve was based on polymethyl methacrylate (PMMA) standards (peak molar masses, M p = 625-625 500 g mol -1 ) from Polymer Laboratories. This technique allowed the number-average molar mass (M n ), the weight-average molar mass (M w ) and the dispersity (Đ = M w /M n ) to be determined.

Dynamic light scattering (DLS) and zeta potential. Nanoparticle diameters (D z ) and zeta potentials (ζ) were measured by DLS with a Nano ZS from Malvern (scattering angle = 173°) at a temperature of 25 °C. The surface charge of the nanoparticles was investigated by ζ-potential (mV) measurement at 25 °C after dilution with 1 mM NaCl, using the Smoluchowski equation. For long-term colloidal stability studies, nanoparticles were stored at 4 °C between each measurement.

Cryogenic transmission electron microscopy (cryo-TEM).

The morphology of the different nanoassemblies was examined by cryo-TEM. Briefly, 5 μL of the nanoparticle suspension (5 mg mL -1 ) was deposited on a lacey formvar/carbon 300 mesh copper microscopy grid (Ted Pella). Most of the drop was removed with blotting filter paper and the residual thin film remaining within the holes was vitrified by plunging into liquid ethane. Samples were then observed using a JEOL Model 2100HC microscope.

Fluorescence spectroscopy. Emission spectra were obtained using a LS 50B fluorescence spectrometer from PerkinElmer. All measurements were conducted at room temperature (∼25 °C).

Nanoparticle Preparation

Nanoparticles were prepared by the nanoprecipitation technique. Unless otherwise mentioned, a final nanoparticle concentration of 2.5 mg mL -1 was targeted. Briefly, 2.5 mg of polymer was dissolved in 0.5 mL of THF and added dropwise to 1 mL of MilliQ water under stirring. THF was then evaporated at room temperature using a Rotavapor (Buchi). Average diameter (D z ) and zeta potential measurements were carried out in triplicate. For the conanoprecipitation of Napht-PI/CdA-digly-PI, 2.23 mg of PI4d and 0.27 mg of PN2 were dissolved in 0.5 mL of THF. A similar procedure was then applied. The resulting PN2/PI4d nanoparticles had a composition of 10.8 wt.% PN2 and 89.2 wt.% PI4d, thus giving an overall drug loading of 5.3 wt.%.

Biological Evaluations

Cell lines and cell culture. The murine leukemia L1210 cells were kindly provided by Dr Lars Petter Jordheim (INSERM U590, Université Claude Bernard Lyon I, Lyon, France), and maintained as recommended. Briefly, L1210 cells were cultured in DMEM. All media were supplemented with 10% heat-inactivated FBS (56 °C, 30 min) and penicillin (100 U mL -1 ). Cells were maintained under a humid atmosphere at 37 °C with 5% CO 2 .

In vitro anticancer activity. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] was used to test cytotoxicity of polymer prodrug nanoparticles and cell viability. Briefly, L1210 cells (5 × 10 3 per well) were seeded in 96-well plates. After 1 h of incubation, cells were exposed to a series of different concentrations of polymer prodrug nanoparticles or free CdA for 72 h. After exposure, the medium was added with 20 μL of MTT solution (5 mg mL -1 in PBS) to each well. The plates were incubated for 1 h at 37 °C and the medium was removed after centrifugation (200g, 5 min, 25 °C). 200 μL of DMSO were then added to each well to dissolve the formazan crystals. Absorbance was measured at 570 nm using a plate reader (Metertech Σ 960, Fisher Bioblock, Illkirch, France). The percentage of surviving cells was calculated as the absorbance ratio of treated to untreated cells. The inhibitory concentration 50% (IC50) of the treatments was determined from the doseresponse curve. All experiments were set up in sextuplicate to determine means and SDs.

Cell imaging. L1210 cells were cultured on a coverslip in a culture dish for 24 h to achieve confluence. Cells were then incubated with the fluorescent nanoparticles at a concentration of 20 μg.mL -1 at 37 °C for 24 h, then incubated with LysoTracker Red for 30 min under same incubation conditions. After treatment, the cells were washed with Dulbecco's phosphate buffered saline (PBS) and imaged using a confocal (SP8 TCS Leica, Germany) with a 63X/1.4 oil-immersion objective. The excitation wavelength was 405 nm and 543 nm, and the fluorescence was collected in the range of 415-530 nm and 569-682 nm, respectively. The pinhole diameter was set to 1 Airy Unit.

Results and Discussion

Design and Synthesis of CdA-Based Polymer Prodrugs

Two different RDRP techniques (i.e., NMP or RAFT polymerization) were employed to prepare CdA-polymer prodrugs because of their well-known advantages in terms of simplicity and robustness to achieve well-defined functional polymers. Two CdA-bearing alkoxyamines and two CdA-bearing CTAs were synthesized, with either ester (linear or -methyl substituted) or diglycolate linkers in between the drug and the RDRP moiety (Figure 2). Because of the poor reactivity of the C-6 amino group of CdA, the secondary hydroxyl group was selectively functionalized with the alkoxyamines or the RAFT agents, after protection of the primary hydroxyl group by tertbutyldimethylsilyl chloride (TBDMSCl). The functionalization was achieved by EDC/DMAP-assisted esterification with two SG1-based alkoxyamines (i.e., AMA-SG1 and digly-AMA-SG1) or two trithiocarbonates (4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl]pentanoic acid (CDPA) and diglycolate 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol (digly-CDP)). To position a diglycolate linker in between the drug and the RDRP moiety to enhance the drug release kinetics, AMA-SG1 and CDP were previously reacted with diglycolic anhydride, giving digly-AMA-SG1 and digly-CDP. The coupling yields ranged from 47% to 91% (those with the diglycolate linker gave higher yields, likely because of a lower steric hindrance). Two different polymer promoieties, polyisoprene (PI) and poly(squalene methacrylate) (PSqMA), were selected. Given its chemical degradability, 29 as well as its biocompatibility [START_REF] Yang | Low temperature heat shrinkable polymer material[END_REF] and its structural similarity with natural terpenoids, PI was chosen for the design of CdA-based polymer prodrugs by NMP. Because of the wide application of squalene (Sq) as building block for the construction of molecular prodrugs, 31,32 SqMA, 13 a methacrylate monomer with a pendant squalene moiety was chosen for the design of CdA-based polymer prodrugs by RAFT polymerization. NMP of isoprene in presence of either CdA-AMA-SG1 or CdA-digly-AMA-SG1, was performed at 115 °C in dioxane, followed by deprotection of TBDMS groups by tetra-n-butylammonium fluoride (TBAF) to give the CdA-PI (PIx, x = 1-4) and CdA-digly-PI (PIxd, x = 1-4) conjugates, respectively (Figure 2). The RAFT polymerization of SqMA in presence of either CdA-CDPA or CdA-digly-CDP, was carried out in 1,4-dioxane at 70 °C with AIBN as the initiator, resulting in CdA-PSqMA (PSqx, x = 1-4) and CdAdigly-PSqMA (PSqxd, x = 1-4) prodrugs after similar deprotection, respectively (Figure 2).

By varying the polymerization time and/or the [monomer] 0 /[functionalized CdA] 0 ratio, a library of well-defined CdA-based polymer prodrugs with variable chain lengths was obtained. Excellent control over the molecular weight of the polymers by both NMP and RAFT polymerizations was observed. The resulting polymerizations exhibited a linear relationship between monomer conversion and M n , with values close to theory. All polymer prodrugs were characterized by 1 H NMR and SEC, and the M n ranged from 1420 g mol -1 to 4980 g mol -1 , whereas CdA drug loading varied from 5.7 wt.% to 20.1 wt.%. It should be noted that, the drug loading could easily be adjusted by changing the polymer chain length, one of the feature of the drug-initiated approach.

/ M n,SEC . f [isoprene] 0 /[alkoxyamine] 0 = 200/1. g [isoprene] 0 /[alkoxyamine] 0 = 400/1. h [SqMA] 0 /[CTA] 0 = 3/1, for the other PSqMA polymers [SqMA] 0 /[CTA] 0 = 6/1.

Formulation and Colloidal Characteristics of the Nanoparticles

CdA-based polymer prodrug nanoassemblies were prepared by nanoprecipitation of a THF solution of the different CdA-based polymer prodrugs into water, targeting a final concentration of 2.5 mg mL -1 . As shown in Table 1, the nanoparticles exhibited an average diameters in the 70-200 nm range and narrow particle size distributions (PSD = ca. 0.1) as measured by DLS. Representative Cryo-TEM images of each type of nanoparticles showed spherical morphologies and colloidal characteristics in good agreement with DLS data (Figure 3). The colloidal characteristics of the different polymer prodrug nanoparticles were then monitored at room temperature with time; from 3 weeks to 3 months. As shown in Figure 4, they all exhibited very high colloidal stability in water in terms of average diameter and particle size distribution. The high stability is likely the result of an efficient electrostatic stabilization provided by the strongly negative surface charges (from -70 mV to -50 mV) of the different nanoparticles, as shown by zeta potential measurements (Table 1). Interestingly, the average diameter can be accurately tuned by varying the polymer chain length: the longer the chain length, the smaller the diameter. With CdA-digly-PI for example, the average diameter decreased from 143 nm to 111 nm along with increasing the M n from 1470 g mol -1 to 4980 g mol -1 (PI1d-PI4d) (Table 1). This trend could be assigned to an increasing compaction of the polymer chains when the M n is increased because of increased hydrophobic interactions and lower solubility.

In Vitro Anticancer Activity

The in vitro cytotoxic activity of the different polymer prodrug nanoparticles was then investigated by cell viability assay (MTT) on a murine leukemia (L1210) cell line (see Figure 5). On one hand, CdA-PI nanoparticles did not show any cytotoxicity up to 10 μM, even when the incubation time was increased from 3 days to 5 days, whereas CdA-digly-PI nanoparticles (PI1d-PI4d) with M n ranging from 1470 g.mol -1 to 4980 g.mol -1 led to significant cytotoxicity (Figure 5a) with half maximal inhibitory concentrations (IC50) varying between 660 nM and 1430 nM. These results are in line with the drug release profiles of the different CdA-based prodrugs 23,24 and also with previous literature about small CdA prodrug molecules. 33,34 The nanoparticles from control polymer AMA-digly-PI (PI0d) (M n = 2650 g mol -1 ) didn't give any cytotoxicity up to at least ∼6 μM. On the other hand, all PSqMA-based prodrug nanoparticles exhibited from moderate to significant anticancer acitivity (Figure 5b). For instance, CdA-digly-PSqMA nanoparticles PSq1d, PSq3d and PSq4d (M n = 2920, 3310 and 4470 g.mol -1 , respectively) gave IC50 values regularly decreasing from 2.17 to 1.36 µM. However, the cytotoxicity of CdA-PSqMA nanoparticles was much lower than that of CdA-digly-PSqMA. PSq1, PSq3 and PSq4 (M n = 2710, 3420 and 4040 g.mol -1 , respectively) indeed showed IC50 values gradually ranging from 4.1 µM to 8 µM. CdA-based polymer prodrug nanoparticles showed higher IC50 values than free CdA (IC50 = 145 nM), because of their prodrug nature, but high cytotoxicity to cancer cells was still obtained. Interestingly, for both of PI-and PSqMA-based systems, IC50 values decreased with increasing the polymer chain length (Figure 5). Several parameters are known to strongly influence the cellular uptake of the nanoparticles by cancer cells (and eventually the cytotoxicity), such as their average size and the composition of their corona. Whereas smaller nanoparticles usually give a better internalization in cancer cells, increasing the polymer chain length is likely to result in more hydrophobic nanoparticle surfaces, leading to a different protein adsorption pattern, and eventually change the nanoparticle uptake by cancer cells. 35,36 From all these results, a structure-cytotoxicity relationship was then established (Table 2). A decrease of cytotoxicity to cancer cells was observed in the following order: CdA-digly-PI > CdA-digly-PSqMA > CdA-PSqMA >> CdA-PI (data for CdA-PI was not plotted because no cytotoxicity was obtained). Not only this can be assigned to the lability of the linker (diglycolate > linear ester > methylsubstituted ester), but also to the nature of the monomer used (CdA-digly-PI > CdA-digly-PSqMA). Indeed, PI chains are likely to provide less steric hindrance than bulky PSqMA chains, thus giving more accessibility for the cleavage to proceed.

Fluorescence Imaging in Living Cells

To study the subcellular localization of CdA-based polymer nanoparticles in living cells, a fluorescent polymer (Napht-PI PN2, M n = 3700 g.mol -1 , Ð = 1.15) 26 exhibiting aggregation-induced emission (AIE) activity 25 was synthesized following a similar strategy and co-nanoprecipitated with CdAbased polymer prodrugs. L1210 cells were then incubated with fluorescent nanoparticles obtained by the co-nanoprecipitation of PN2 and PI3d. As shown in Figure 6, a bright green fluorescence signal from the nanoparticles (green channel, 500-560 nm, λ ex = 408 nm) was observed in L1210 cells, which indicated the efficient internalization of the nanoparticles into the cells. The presence of the nanoparticles in the lysosomes was confirmed by a yellow colocalization observed after overlaying the green channel with the red channel (staining of the lysosomes, 569-682 nm, λ ex = 543 nm) (Figure 6d), which suggested an endocytosis mechanism. Importantly, cytotoxicity to cancer cells was maintained as shown by MTT assay (whereas pure PN2 nanoparticles were not toxic to cells). 26 This general strategy enabled the facile production of AIE dye-based polymers for nanoparticle labeling by simple co-nanoprecipitation with drug-polymer conjugates.

Conclusion

Different libraries of CdA-based polymer prodrugs were successfully synthesized by the "drug-initiated" method via either NMP of isoprene or RAFT polymerization of a methacrylic ester bearing a naturally occurring isoprenoid moiety. The formed nanoparticles obtained by nanoprecipitation exhibited great colloidal stability in aqueous solution and in vitro cytotoxicity to cancer cells governed by their structural parameters. With the help of fluorescent nanoparticles from AIE-dye-initiated polymerization, the subcellular localization of CdA-based polymer nanoparticles was also successfully imaged in living cells. This study opens new perspectives for the design of a broad range of novel drug-initiated polymer prodrug nanocarriers by changing the nature of the drug, the linker and the polymer.

Figure 1 .

 1 Figure 1. Synthesis of CdA-based polymer prodrug nanoparticles from "druginitiated" NMP of isoprene or RAFT polymerization of SqMA.

Figure 2 .

 2 Figure 2. Structures of cladribine-polyisoprene (CdA-PI, PIx), cladribinediglycolate-polyisoprene (CdA-digly-PI, PIxd), cladribine-poly(squalenoyl methacrylate) (CdA-PSqMA, PSqx) and cladribine-diglycolate-poly(squalenoyl methacrylate) (CdA-digly-PSqMA, PSqxd).

Figure 3 .

 3 Figure 3. Cryo-TEM images of: (a) CdA-PI PI3, (b) CdA-digly-PI PI4d, (c)CdA-PSqMA PSq1 and (d) CdA-digly-PSqMA PSq1d nanoparticles.

Figure 4 .

 4 Figure 4. Evolution with time of the average diameter (solid symbols) and the particle size distribution (open symbols) determined by DLS of: (a) CdA-PI (PI1 and PI3) and CdA-digly-PI (PI1d-PI4d) nanoparticles and (b) CdA-digly-PSqMA (PSq1d-PSq4d) nanoparticles.

Figure 5 .

 5 Figure 5. Viability assay (MTT test) on L1210 cells with increasing concentrations of: (a) CdA-digly-PI nanoparticles PI1d-PI4d and PI0 (AMAdigly-PI) and free CdA and (b) CdA-digly-PSqMA nanoparticles PSq1d, PSq3d and PSq4d, CdA-PSqMA nanoparticles PSq1, PSq3 and PSq4 and free CdAs.

Figure 6 .

 6 Figure 6. L1210 cells Normarski image (a), confocal microscopy images [green channel (Napht, b), red channel (LysoTracker Red, c)] and merge of red and green fluorescence images with Nomarski image (d) after a 24 h incubation of L1210 cells with AIE-active polymer prodrug nanoparticles (PI3d/PN2, 1 wt.% of Napht-OH). Scale bars = 20 µm. Arrows indicate colocalization.

Table 1 Macromolecular and Colloidal Characteristics of CdA-PI, CdA- digly-PI, CdA-PSqMA and CdA-digly-PSqMA Polymer Prodrug Nanoparticles

 1 Calculated according to DP n,SEC = (M n,SEC -MW alkoxyamine + MW TBDMS ) / MW isoprene for PI and DP n,SEC = [M n,SEC -(MW CTA -MW TBDMS )] / MW SqMA for PSqMA. c Determined by DLS. d Zeta potential. e %CdA = MW CdA

	Expt.	M n a (g.mol -1 )	DP n,SEC	b	Ð a	D z (nm) c	PSD c	 d (mV)	%CdA e (wt.%)
	PI1	1420	12		1.17	167	0.06	-69	20.1
	PI2	1560	14		1.09	201	0.12	-70	18.3
	PI3	2270	24		1.15	152	0.08	-64	12.6
	PI4 f	2520	28		1.14	137	0.10	-71	11.3
	PI1d	1470	10		1.10	143	0.07	-66	19.4
	PI2d	1710	16		1.13	128	0.10	-68	16.7
	PI3d f	2960	34		1.17	127	0.08	-70	9.6
	PI4d g	4980	64		1.20	111	0.11	-66	5.7
	PSq1	2710	4		1.18	69	0.14	-51	10.5
	PSq2	3080	5		1.16	82	0.11	-54	9.3
	PSq3	3420	6		1.17	94	0.11	-58	8.3
	PSq4	4040	7		1.12	94	0.09	-61	7.1
	PSq1d h	2920	4		1.25	112	0.12	-62	9.8
	PSq2d	3090	5		1.25	107	0.14	-60	9.2
	PSq3d	3310	6		1.27	96	0.12	-54	8.6
	PSq4d	4470	8		1.27	84	0.10	-53	6.4

a Determined by SEC. b

Table 2 Evolution of the IC 50 as Function of the Nature and the M n of the CdA-Based Polymer Prodrug. a

 2 Note that CdA-PI is not represented because the IC 50 was impossible to determine because of absence of CdA release.

	Expt.	M n (g.mol -1 )	IC 50
			(M)
	CdA-PI	n.d.	n.d.
		4470	1.36
	CdA-digly-PI	3310	2.20
		2920	2.17
		4040	4.10
	CdA-PSqMA	3420	7.0
		2710	8.0
		4980	0.66
	CdA-digly-PSqMA	2960	0.72
		1710	1.43

a

Acknowledgments

This work is supported by Marie Curie Intra-European Fellowship (YB) from European Commission (No. 623501). The authors thank Julie Mougin (Institut Galien Paris-Sud) for technical assistance in HPLC and cryo-TEM (Service de Microscopie Electronique, UFR de Biologie Intégrative, Université Paris-Sud), Valérie Nicolas (Institut Paris-Sud d'Innovation Therapeutique (IPSIT), Université Paris-Sud) for technical assistance in confocal imaging, St phanie Denis (Institut alien aris-Sud) for technical assistance in cell culture and St phanie icola for mass spectrometry analyses (Service d' nalyses des M dicaments et M tabolites, Institut aris-Sud d'Innovation Therapeutique (I SIT), I 1 1, niversit aris-Sud). CNRS and University Paris-Sud are also acknowledged for financial support.


AFM = scanning probe technique (nano-tip close to a surface): tip is located at the end of a micro-lever = cantilever A1-Tip-cantilever system

• AFM = scanning probe technique (nano-tip close to a surface): tip is located at the end of a micro-lever = cantilever Lowering the shearing force between tip and surface 2.

Increasing force sensitivity w.r.t. the contact mode 3.

Non-contact (attractive force mapping only)

 Difficulties: 1.
Understanding the cantilever non-linear dynamics 2.

Detection of the oscillation (system detection bandwidth> MHz) 3.

Complexity of the electronics scheme 4.

Origin of the image contrast ?

• Measures with the tip-surface distance:

1. Oscillation frequency 2.

Oscillation ampltidue 3.

Excitation amplitude 4.

Phase shift between excitation and oscillation •

The mechanical system is strictly similar to a forced damped harmonic oscillator (to be checked experimentally however) VdW (sphere-plane)+ Morse, after 1 VdW (sphere-plane) only • At room temperature, the noise level of the instrument (z, amplitude detection...) exceeds that value. The resolution onset then is rather 10 pm.

• This sensitivity is enough to achieve atomic resolution at room temperature

•A1••

  Mechanical analogy: recessed beam  Further simplification: spring with stiffness k subject to an interaction force F int with the surface, resonance frequency of the fundamental bending mode f 0 Curvature radius: 5nm (at best) • AFM = scanning probe technique (nano-tip close to a surface): tip is located at the end of a micro-lever = cantilever • Mechanical analogy: recessed beam  Further simplification: spring with stiffness k subject to an interaction force F int with the surface, resonance frequency of the fundamental bending mode f 0 Measure of the lever deflection by means of an optical setup  indirect measure of forces (1 pN..1 nN):  Electrostatic (due to charges) : attractive, long-range (> 100 nm)  Van der Waals (London, Debye, Keesom) : attractive, long-range (> 1 nm)  Chemical (interatomic) : attractive or repulsive, short-range (few Å)  Magnetic (long-range) • For a detailed work on forces involved in AFM:  "Scanning probe microscopy: the lab on a tip" (E.Meyer, H.J. Hug, R. Bennewitz, Springer-Verlag Berlin Heidelberg 2004  "Noncontact Atomic Force Microscopy" (S.Morita, R. Wiesendanger, E. Meyer, Springer-Verlag Berlin Heidelberg 2002  "Intermolecular and surface forces, 3 rd edition" (J.N.Israelachvili, Elsevier 2011) CNRS, Universités d'Aix-Marseille (AMU) et de Toulon (UTLN)A1-Tip-cantilever system• Tip-surface interaction force 1 :1 PhD thesis S.Burke, Mac Gill Univ., 2008 probe technique (nano-tip close to a surface): tip is located at the end of a micro-lever = cantilever• Mechanical analogy: recessed beam  Further simplification: spring with stiffness k subject to an interaction force F int with the surface, resonance frequency of the fundamental bending mode f 0 Scaling illustration:G.Binnig et al., Phys. Rev. Lett. 56, p930 (1986) the vertical feedback can be performed on various signals, not only on the deflection F N (see hereafter)!

Force

  pm ... 10 nm Frequency: 32 kHz … 1 MHz UMR 7334, CNRS, Universités d'Aix-Marseille (AMU) et de Toulon (UTLN) B1-Dynamic AFM specificities Cantilever quality factor and intrinsic dissipation: A -A 1. Polesel et al., Nanotechnology 14,1036 (2003) z(t) G 0 Harmonic solution: after 1 (f 0 =270 kHz, Q=45000)  5 variables : Excitation amplitude, Excitation frequency, Oscillation amplitude, Phase (excitation / oscillation) and Tip-surface distance D Differential equation for D→ D→ A exc (t) A exc (t) f -f 0 (Hz) Exercice Starting from the differential equation for the forced damped harmonic oscillator (Quality factor Q, pulsation at resonance w 0 = 2p n 0 ): with a forcing term of the form: and assuming a harmonic solution: 1-Prove that the normalized amplitude a(u) = A(u)/A 0 , with u = w/w 0 being the normalized frequency, and the phase of the oscillator write : 2-Plot these functions (with Igor Pro for instance) with Q = 100, then 100000. A exc (t) A exc (t) UMR 7334, CNRS, Universités d'Aix-Marseille (AMU) et de Toulon (UTLN) mode AFM vs. non-contact AFM B -Dynamic AFM UMR 7334, CNRS, Universités d'Aix-Marseille (AMU) et de Toulon (UTLN) B2-Force sensitivity (for cantilevers) • Orders of magnitude: • k = 30 N/m • B ~ 250 Hz • w 0 = 2p f 0 = 2p 150 kHz • Q = 40 000 (sous vide) • k B T = 25 meV (298 K) • Increasing the sensitivity :  k : risk for "jump to contact"  k B T : not easy  w 0 : technically demanding (detection bandwidth must be increased accordingly)  Q : depend upon the intrinsic dissipation of the material • Equipartition theorem on a cantilever subject to thermal fluctuations yields a minimum detectable force : F min ~ 60 pN UMR 7334, CNRS, Universités d'Aix-Marseille (AMU) et de Toulon (UTLN) B2-Force sensitivity (for cantilevers) dF int ~ 100 pN dz ~ 10 pm • Tip-surface interaction force: s /m s UMR 7334, CNRS, Universités d'Aix-Marseille (AMU) et de Toulon (UTLN) • « Jump to contact »: B2-Force sensitivity (for cantilevers) Instabilities, bifurcations, hysteresis  Modelization mode AFM vs. non-contact AFM B -Dynamic AFM UMR 7334, CNRS, Universités d'Aix-Marseille (AMU) et de Toulon (UTLN)

  1. Pérez et al., Phys Rev. B 58, 10835 (1998) ) = D -z(t) = D -A cos(wt + j) d'après 1 1. Polesel et al., Nanotechnology 14:1036 (2003) Nony et al. J.Chem.Phys.111,1610Phys.111, (1999)); "Analyse de la microscopie de force dynamique: application à l'étude de l'ADN", L.Nony, Thèse de l'univ.Bordeaux I (2000). UMR 7334, CNRS, Universités d'Aix-Marseille (AMU) et de Toulon (UTLN) Long-range: Dispersive Van der Waals L.Nony et al. J.Chem.Phys.111,1610 (1999) Short-range: Morse L.Nony et al., Phys.Rev.B 74, 235409 (2006) B3-Non-linear dynamics and modelization • Coupling terms (Fourier components of the interaction force @ f): mode AFM vs. non-contact AFM B -Dynamic AFM  5 variables : Excitation amplitude, Excitation frequency, Oscillation amplitude, phase (excitation / oscillation) and tip-surface distance  The variables that are kept constant set the mode : Excitation amplitude and Excitation frequency are constant ↓ • Measures : Oscillation amplitude and phase vs. tip-surface distance • Probed interaction: most generally, attractive+repulsive interactions (intermittent contact) Tapping (Amplitude Modulation-AFM) B4-Tapping mode AFM vs. non-contact AFM: Phase (=-p/2 : resonance) and Oscillation amplitude are constant ↓ • Measures : Resonance frequency shift and excitation amplitude required to keep the oscillation amplitude constant vs. tip-surface distance • Probed interaction: most generally, attractive interaction (non-contact) non-contact AFM (nc-AFM, or Frequency Mod.-AFM 1 ) results and current trends C -Dynamic AFM in non-contact mode Phase (=-p/2 : resonance) and oscillation amplitude are constant Measures : resonance frequency shift and excitation amplitude required to keep the oscillation amplitude constant (DISSIPATION image) vs. tip-surface distance A 0 Df A 0 z feedback (TOPOGRAPHY image): performed to maintain Df = 𝑓 0 -𝑓 0 constant (equivalent to tunneling current in STM) C1-Introduction • Method developed 1 in 1991 • Concept: 1 T.Albrecht et al., J.Appl.Phys.69, 668 (1991) results and current trends C -Dynamic AFM in non-contact mode • Vertical sensitivity in the short-range regime (order of magnitude): • The experimental control on Df is better than 0,1 Hz (around f 0 ~ 150 kHz), i.e. 1 pm vertical resolution ! • This is what is achieved at low temperature (liquid Helium : 4K).

  results and current trends C -Dynamic AFM in non-contact mode UMR 7334, CNRS, Universités d'Aix-Marseille (AMU) et de Toulon (UTLN) results and current trends C -Dynamic AFM in non-contact mode

T.Albrecht et al., J.Appl.Phys.69, 668 (1991)

• Oscillation amplitude and phase (=-p/2) are fixed: Solving the system {cos(j), sin(j)} w.r.t. A and j. Normalized frequency shift :

• Conservative interaction force (Attractive = non-contact) :

• Hence the analytic expression for Df:

C2-Connection with the modelization (1):

F.Giessibl (1997,2000); U.Dürig (1999); L. Nony (1999Nony ( , 2006) ) Long-range Short-range

Exercice

• The calculation of the frequency shift can be simplified upon linearization of the interaction force by assuming small oscillation amplitudes (A~50 pm << D).

• In this case, reconsider the differential equation of motion of the cantilever and prove that the shifted resonance frequency then writes :

• Deduce out of this the linearized formula for the frequency shift :

C4-Instrumentation: PLL & amplitude feedback  A PLL ensures the detection of Df w.r.t. a reference frequency which is fixed equal to the resonance frequency of the free cantilever f 0 , therefore matching the phase condition j = -p/2

 Frequency demodulation around f 0 !  In this excitation scheme of the cantilever, there are no bifurcations structures as seen in AM-AFM. Qualitatively, the interpretation of the frequency shift can be made out of the linear approach, but it is to be reminded that the measured shift is the one that derives from the non-linear approach...

Cantilever signal nc-AFM: keypoints

• Demodulation technic of the frequency of the cantilever around a central frequency which is the resonance frequency of the free cantilever:

• The oscillation amplitude and the phase are kept constant : resonance values

• True non-contact method :

 Attractive interaction regime with the surface  Df < 0

• Very large z sensitivity upon accurate detection of Df, which allows for large vertical and lateral contrasts (atomic resolution)

C5-Current trends :

• Small amplitudes (50 pm, sensitivity to the SR-regime) : piezoelectric probes (tuning fork, "qPlus" Omicron, "Colibri" Specs). Usually used at low temperature:  imaging + spectroscopy: quantitative interpretation (forces, charges...)

• Time resolution: high-frequency cantilevers or higher harmonics…


Min-I Lee

Alice Defresne

Oliver Plantevin

Denis Ceolin

Jean-Pascal Rueff

Pere Roca I Cabarrocas

Antonio Tejeda

Defect state analysis in ion-irradiated amorphous-silicon heterojunctions by HAXPES

The efficiency in HIT (Heterojunction with Intrinsic Thin film) solar cells strongly depends on the passivation of dangling bonds at the a-Si:H/c-Si interface by hydrogen, introduced in the plasma enhanced CVD process. Here in, we study controlled defects that are introduced by Ar ion irradiation. We observe by hard X-ray photoemission spectroscopy (HAXPES) that during Ar ion implantation, Si-H bonds in the a-Si:H layer are broken and become dangling bonds. We quantify the number of dangling bonds in the a-Si:H layer, and we identify the electronic states associated to them, which explains previously observed photoluminescence transitions.

I. INTRODUCTION

Since 1992, a new technology based on silicon heterostructures has partly solved the problems of high energy consumption when fabricating crystalline silicon solar cells, that was called "Heterojunction with Intrinsic Thin film (HIT)" [START_REF] Tanaka | Development of new a-Si/c-Si heterojunction solar cells: ACJ-HIT (artificially constructed junction-heterojunction with intrinsic thinlayer)[END_REF]. Heterojunctions are made by combining a wide gap material such as hydrogenated amorphous silicon (a-Si:H) with an optical band gap E g ⇡ 1.7 eV with crystalline silicon (c-Si) having a smaller band gap E g = 1.1 eV. The efficiency of a-Si:H/c-Si heterojunction solar cells has reached over 26%, which is close to the theoretical conversion efficiency limit of Si solar cells ~29.1% [START_REF] Yoshikawa | Silicon heterojunction solar cell with interdigitated back contacts for a photoconversion efficiency over 26%[END_REF]. One of the factors that limit the solar cell efficiency is defects. A better understanding of defects, especially at the interface between the c-Si wafer and a-Si:H could help to increase the open-circuit voltage (V oc ), and the short-circuit current (J sc ) of the heterojunction solar cells, resulting in an increase of their efficiency. Defects limit the efficiency through the additional electronic states that they generate in the band gap of a-Si:H and c-Si. At this interface, there are two kinds of defect states: mid-gap and bandtail states [START_REF] Stuckelberger | Progress in solar cells from hydrogenated amorphous silicon[END_REF][START_REF] Dalal | Measurement of Urbach edge and midgap states in amorphous silicon pin devices[END_REF]. Mid-gap states act as highly efficient recombination centers. They arise from dangling bonds [START_REF] Santos | Atomistic study of the structural and electronic properties of a-Si: H/c-Si interfaces[END_REF], and are problematic for a-Si:H solar cells and other electronic devices. Bandtail states are less detrimental for the efficiency [START_REF] Stuckelberger | Progress in solar cells from hydrogenated amorphous silicon[END_REF], and their concentration is related to the mid-gap states density [START_REF] Stutzmann | Weak bond-dangling bond conversion in amorphous silicon[END_REF]. They are a consequence of disordered localized electronic states appearing close to the edges of the conduction and valence bands [START_REF] Hernandez-Como | Simulation of hetero-junction silicon solar cells with AMPS-1D[END_REF].

Defects can be present on the c-Si substrates of the c-Si/a-Si:H interface and in the a-Si:H layer. In order to understand their effect and confirm their location in the stacking, we used ion implantation to generate defects. During Ar ion implantation, Si-Si and Si-H bonds in a-Si:H are broken and become dangling bonds, which is similar to the case of a-C:H, where hydrogen can be released after ionimplantation [START_REF] Adel | Ion-beam-induced hydrogen release from a-C: H: A bulk molecular recombination model[END_REF]. The generated dangling bonds can lead to mid-gap defect states. By controlling the energy of Ar ions, the depth of the defective region, i.e. the defect location, can be controlled [START_REF] Defresne | Interface defects in a-Si: H/c-Si heterojunction solar cells[END_REF]. Ion irradiation is therefore a convenient way to control defect formation.

Information from the defects can be obtained from photoluminescence [START_REF] Dunstan | Photoluminescence in hydrogenated amorphous silicon[END_REF]. In particular, this technique has allowed to identify defect induced states on a-Si:H heterostructures [START_REF] Nguyen | Characterizing amorphous silicon, silicon nitride, and diffused layers in crystalline silicon solar cells using microphotoluminescence spectroscopy[END_REF][START_REF] Street | Luminescence and ESR studies of defects in hydrogenated amorphous silicon[END_REF]. Photoluminescence in different a-Si:H heterostructures shows two main peaks [START_REF] Defresne | Interface defects in a-Si: H/c-Si heterojunction solar cells[END_REF][START_REF] Plantevin | Suppression of the thermal quenching of photoluminescence in irradiated silicon heterojunction solar cells[END_REF]. The most intense peak at 1.1 eV corresponds to the band to band recombination in c-Si (1.1 eV). A broader and less intense peak appears at 1.2 eV, an energy smaller than the band gap of hydrogenated amorphous Si (1.7 eV) that must be associated to defect states within its gap. These states are detrimental for the solar cell efficiency due to the enhanced recombination at the interface [START_REF] Wang | Insights into the role of the interface defects density and the bandgap of the back surface field for efficient p-type silicon heterojunction solar cells[END_REF]. The nature and the exact location of the states within

Complete Manuscript

the gap of a-Si:H is to be identified.

In order to access the defects buried at the a-Si:H/c-Si interface, we have used hard x-ray photoemission spectroscopy (HAXPES). Different photon energies allowed us to study the defects in different regions of the a-Si:H heterojunction. We have studied the chemical environments of Si atoms, in order to quantify the number of dangling bonds and of Si-H bonds. We have also analyzed the valence band states, to observe directly the electronic states induced there by defects.

II. EXPERIMENTAL DETAILS

We have studied amorphous-silicon heterostructures consisting of undoped hydrogenated amorphous silicon (a-Si:H) and a thin layer of hydrogenated amorphous silicon carbon alloy (a-SiC:H) grown on n-type (100) single crystalline silicon wafers. The thin silicon carbon layer (3 nm) serves as a buffer layer to avoid the epitaxy of the amorphous silicon [START_REF] Martın | Surface passivation of p-type crystalline Si by plasma enhanced chemical vapor deposited amorphous SiC x: H films[END_REF]. A 7 nm layer of a-Si:H is deposited on top of a-SiC:H layer. More details about the growth procedure can be found in [START_REF] Defresne | Interface defects in a-Si: H/c-Si heterojunction solar cells[END_REF].

The a-Si:H and a-SiC:H were deposited by Plasma-Enhanced Chemical Vapor Deposition (PECVD) method at 200 °C in the ARCAM reactor [START_REF] Roca I Cabarrocas | A fully automated hot-wall multiplasma-monochamber reactor for thin film deposition[END_REF]. Once the heterostructure was produced by PECVD, defects were introduced by implanting 1keV of Ar + ions in a microwave source plasma reactor [START_REF] Plantevin | Suppression of the thermal quenching of photoluminescence in irradiated silicon heterojunction solar cells[END_REF]. The Ar + fluence was controlled to 10 15 Ar.cm 2 .

When dealing with buried systems, conventional photoemission does not allow to probe the core levels or the valence band. Conventional photoemission only probes the first monolayers of the material, due to the small electron escape depth upon operation with low energy photons. In order to probe the amorphous silicon heterostructure (a-Si:H/ c-Si), we used hard X-ray photoemission spectroscopy (HAX-PES), a photoemission performed at high photon energies (of the order of several keV).

We measured HAXPES at GALAXIES beamline of SOLEIL synchrotron. The beamline is equipped with an in-vacuum U20 undulator delivering photons between 2.3 to 12 keV energy. The HAXPES end-station is equipped with a high energy/ high resolution electron analyzer to overcome the rapid decrease in photoionization cross section when increasing the photon energy [START_REF] Scofield | Theoretical photoionization cross sections from 1 to 1500 keV[END_REF][START_REF] Yeh | Atomic subshell photoionization cross sections and asymmetry parameters: 1< Z< 103[END_REF]. We settled a resolution of 100 meV in our experiments.

Information on depths in photoemission are obtained through the inelastic mean free path (IMFP), related to the distance that electrons travel before suffering an inelastic collision and exciting plasmons or vibrations. The IMFP of electrons in the different core levels can be calculated by taking into account the thickness and the atomic density of each layer in the heterojunction sample.

Fig. 1 shows the IMFP calculation for electrons excited from the different layers using SESSA [START_REF] Werner | NIST Database for the Simulation of Electron Spectra for Surface Analysis (SESSA)[END_REF]. We have considered a thickness of 7 nm of a-Si:H (L1 = 7 nm), a bottom layer of 3 nm of a-SiC:H (L2 = 3 nm), and a 280 µm thick substrate of c-Si. The atomic densities were set to 4.90⇥10 22 atom/cm 3 , 4.82⇥10 22 atom/cm 3 and 4.99⇥10 22 atom/cm 3 for L1, L2 and c-Si substrate, respectively [START_REF] Custer | Density of amorphous Si[END_REF][START_REF] Harris | Properties of silicon carbide[END_REF][START_REF] Hull | Properties of crystalline silicon[END_REF]. Fig. 1(b) shows the IMFP of electrons from different layers as a function of photon energy. With a photon energy below 3.5 keV, most of the excited photoelectrons come from the a-Si:H layer (L1, red area). The measured electrons mainly arise from the a-Si:H layer. For studying the a-SiC:H region, a convenient energy is 5 keV, since the a-SiC:H region is reached for photons above 3.5 keV and the signal from c-Si is minimal below 5.3 keV. Finally, to reach the c-Si substrate, 8 keV photons are required. From these calculations, we selected three photon energies (3 keV, 5 keV, and 8 keV) for studying the different layers.

III. ELECTRONIC STATES INDUCED BY DEFECTS IN THE BAND STRUCTURE

In a first step, we tested our calculation of the IMFP as a function of the photon energy. We have therefore experimentally studied a 3nm thick a-Si:H heterojunction sample (1 nm a-Si:H + 2 nm SiC) on a c-Si substrate with a photon energy of 3 keV (Fig. 2). After normalizing the raw data to the density of states that contribute significantly in high photon energy photoemission [START_REF] Woicik | Hard X-ray Photoelectron Spectroscopy[END_REF], dispersing E(k) spectral features can be observed, indicating that we indeed reach the c-Si substrate. Our IMFP calculations are therefore correct and we can successfully reach the buried interface in our heterostructure with a correct tuning of photon energy. The experimental density of states (measured from a combination of a-Si:H and bulk c-Si) and the calculation from c-Si are plotted together in Fig. 2(b). The density of states allows to identify the valence band maximum (VBM), which will be our energy reference in the following.

To study the defect states, we focus now on a realistic heterojunction. Fig. 3 shows the integrated valence band, that emulates the DOS, from a 10 nm a-Si:H deposited on a c-Si substrate (7 nm of a-Si:H and 3 nm of SiC, see inset in Fig. 3(c)). As measured with a photon energy of 3 keV, by normalizing the spectra to the structureless high binding energy background, it can be appreciated that the spectral weight near the maximum of the valence band is higher in the irradiated heterostructures. By estimating the area difference between the irradiated and the pristine sample (integral of the pink region in Fig. 3(c)), we determine that the increase is 29 ± 5% at 3 keV , while a measurement with a photon energy of 5 keV gives an increase of 23 ± 5%, the error bars being associated to the normalization process.

These gap states are located at 0.3 eV above the valence band maximum (Fig. 3(b) and(c)), and are probably associated to dangling bonds produced by irradiation. In order to determine if this atomic origin is correct, we study in the following the dangling bond variation upon irradiation.

IV. QUANTIFICATION OF DEFECTS IN THE a-Si:H LAYER

Core level spectroscopy allows to determine the different inequivalent Si atoms in the system. Moreover, core level spectroscopy is one of the rare techniques that is sensitive to H atoms. This sensitivity is obtained through the chemical shift that the bonding to a H atom induces on the core level of another element. We have therefore analyzed the Si 2p core level to identify Si-H bonds. This analysis needs the accurate decomposition of the core level into its components. Fig. 4 compares the core levels measured on non-irradiated and irradiated samples with different photon energies, all of them being normalized to the total intensity of the Si 2p core level. The spectrum on the irradiated sample at 8 keV (Fig. 4(f)) has a major contribution of a single spin-orbit splitted component which should be the one of c-Si, due to the extreme bulk sensitivity at such photon energy. The bulk component can be satisfactorily fitted with spin-splitted doublets of Voigt functions with 0.36 eV Gaussian width, 0.15 eV Lorentzian width, a spin-orbit splitting of 0.6 eV, and a branching ratio of 0.5, in agreement with [START_REF] Lay | Surface core-level shifts of Si (111) 7x7[END_REF]. After identifying the main component, residuals show the presence of other components, more explicit at other photon energies.

We have thus added the components known to be present in the a-Si:H layers and at the surface, i.e. dangling bonds, Si-H bonds and Si-O bonds. Dangling bonds appear at lower binding energies [START_REF] Redondo | Oxidation of silicon surfaces[END_REF], so we introduced a component S1 with a chemical shift of -0.3 eV with respect to neutral silicon. We also introduced a component S2 associated to Si-H, whose binding energy is known to appear at higher binding energy than the elemental silicon [START_REF] Redondo | Oxidation of silicon surfaces[END_REF][START_REF] Cerofolini | Si 2p XPS spectrum of the hydrogen-terminated (100) surface of device-quality silicon[END_REF]. With these components plus an S4 component associated to Si 4+ , usually appearing when SiO 2 is present in the system, the core level at 8 keV is satisfactorily described. By observing the Si 4+ component (Fig. 4), it is evident that the irradiated sample is more reactive towards oxidation, because of the increase in the number of dangling bonds.

Once the spectra at 8 keV of photon energy was properly fitted, we applied the same decomposition to the other spectra by varying the intensities of the different components. For the spectra at 5 keV, with a strong contribution from the a-SiC:H layer, a new component S3 of 100.0 eV binding energy is needed for reproducing the experimental spectrum, which is precisely associated to the Si-C bonds [START_REF] Swain | Study of structural and electronic environments of hydrogenated amorphous silicon carbonitride (a-SiCN: H) films deposited by hot wire chemical vapor deposition[END_REF]. However, a small variation can be observed in the shallower layer after the irradiation, possibly related to the carbon diffusion during the irradiation process. With these four components, all the Si 2p core levels are satisfactorily fitted. The absolute binding energy for the four components is shown in Table I. The resulting decomposition is shown in Fig. 4. Component B, associated to Si 0 bulk atoms, increases at the highest probing photon energy we used (8 keV) as expected. As for the surface sensitive component S1 and S2, which are related to dangling bonds and Si-H bonds respectively, if we compare their intensities by visual inspection, we observe that the amount of dangling bonds in a-Si:H layer increases and that of Si-H bonds decreases after irradiation.

In fact, irradiation reduces the S2 intensity by 4 ± 1% at both 3 keV and 5 keV (Fig. 5). Most of the intensity lost of the total area is transferred to S1 or to S4, i.e. the hydrogenated bonds are broken and become either dangling bonds or dangling bonds that interact with the oxygen when present at the surface, as seen in an increase of the S4 component at 3 keV by 7 ± 1% (Fig. 5 (c)). We therefore observe that irradiation breaks some of the existing Si-H bonds, and increases the number of dangling bonds in the hydrogenated layers. The number of dangling bonds is correlated to the increasing intensity of the defect states at the valence band. There is indeed an increase of dangling bonds in the core level, which also relates to the intensity increase at the top of the valence band. We therefore identify the atomic origin of irradiation induced electronic states.

V. CONCLUSIONS

In conclusion, we have studied the dangling bonds formation in a-Si:H heterostructures after ion irradiation using both DOS and core level measurements with HAXPES. After the irradiation, the decrease of Si-H bonds gives raise to the increase of dangling bonds and forms the defect states. Moreover, part of those dangling bonds close to the surface has reached and forms the oxidized states of silicon. We also observe that those defect states are located at 0.3 eV above the maximum of the valence band. Since we have evidenced that those states above the maximum of the valence band cannot explain alone the 1.2 eV optical transition observed in photoluminescence, we infer the presence of defect states below the conduction band minimum, which could be probed by inverse photoemission spectroscopy in future experiments.

Figure 1 .

 1 Figure 1. (a) The photoexcited electrons can be extracted from different depths of the heterojunction with different photon energies. (b) Simulation of the Inelastic Mean Free Path of electrons in Si2p for different layers (L1: a-Si:H (red), L2: a-SiC:H (green), and Substrate: c-Si (blue)) as a function of photon energy. Background colors represent the depth of each layer in the heterostructure (right axis).

Figure 2 .

 2 Figure 2. Electronic structure of 3 nm a-Si:H/ c-Si heterojunction sample (1 nm of a-Si:H and 2 nm of a-SiC:H) measured at 3 keV. (a) k-resolved valence band normalized to the density of states. The dispersing states from c-Si shows that we have successfully reached the buried layer of our sample. (b) Density of states along with the calculated DOS (from [24]).

Figure 3 .

 3 Figure 3. (a) Comparison of the experimental DOS between irradiated (red curve) and non-irradiated (blue curve) heterostructures on (d) the 10nm a-Si:H heterojunction sample. (b) Zoom at the VBM. (c) DOS in logarithmic scale. The defect states, located at 0.3 eV above valence band maximum are highlighted in pink.

Figure 4 .

 4 Figure 4. Si 2p core level decomposition for the nonirradiated and the irradiated heterostructures measured at photon energies of 3 keV, 5 keV and 8 keV. Five components are necessary: a bulk component which is meanly from c-Si (B), a dangling bond component (S1), a Si-H bond component (S2), a Si-C component (S3), and a Si 4+ component that is associated to SiO2 (S4).

Figure 5 .

 5 Figure 5. Intensity evolution as a function of the photon energy (top axis) and the escape depth (bottom axis) of (a) S1 component associated to dangling bonds, (b) S2 component associated to Si-H bonds, and (c) S4 component associated to silicon dioxide (Si 4+ ).