Alexandre Barras
email: alexandre.barras@univ-lille1.fr

Nadia Skandrani

Mariano Gonzalez Pisfil

Solomiya Paryzhak

Tetiana Dumych

Aurélien Haustrate

Laurent Héliot

Tijani Gharbi

Hatem Boulahdour

V'yacheslav Lehen'kyi

Rostyslav Bilyy

Sabine Szunerits

Gabriel Bidaux

Rabah Boukherroub
email: rabah.boukherroub@univ-lille1.fr

Improved Photodynamic Effect through Encapsulation of Two Photosensitizers in Lipid Nanocapsules

Keywords: Lipid nanocapsules, hypericin, protoporphyrin IX, singlet oxygen, photodynamic therapy

Photodynamic therapy (PDT) has developed into a new clinical and non-invasive treatment for cancer over the past 30 years. By the combination of three non toxic partners i.e. a photosensitizer (PS), molecular oxygen (O 2 ) and light, cytotoxic reactive oxygen species (ROS) are locally produced leading to irreversible vascular and cellular damages. In the present study, we report for the first time that the combination of two photosensitizers (2 PSs) loaded in the same lipid nanocapsules (LNCs) leads to enhanced photodynamic therapy efficiency when compared with previously reported systems. The 2 PSs-loaded LNCs are shown to increase the in vitro phototoxicity at the nanomolar range (IC 50 = 274 and 278 nM on HeLa and MDA-MB-231 cell lines, respectively), whereas the corresponding single PS-loaded LNCs at the same concentration exhibit a phototoxicity two times lower. Intracellular localization in HeLa cells indicates a subcellular asymmetry of PpIX and Hy, in the plasma, ER membranes and in round internal structures. Biodistribution of LNCs was studied upon different ways of injection into swiss nude mice; based on obtained data LNCs were injected intratumorally and used to slow the grow of xenograft tumors in mice. The results obtained in this study suggest that the combination of two or more PSs may be a promising strategy to improve the efficacy of the conventional photodynamic therapy as well as to reduce dark toxicity.

INTRODUCTION

Photodynamic therapy (PDT), involving the excitation of a non-toxic photosensitizer (PS) at an appropriate wavelength in presence of molecular oxygen, has become a promising method for the treatment of tumor cells. The photochemical reactions in PDT are responsible for the formation of singlet oxygen as well as the highly reactive oxygen species (ROS) that can cause lethal damage to the targeted cells. 1 Photofrin was the first PS to receive regulatory approval for PDT treatment of various cancers throughout the world, including the United States (approved by Food and Drug Administration [FDA] in 1995. 2 Even though this first-generation of PS has proven to be efficient in the treatment of various cancer types, Photofrin has some drawbacks such as low absorption of light and high photosensitivity due to the poor clearance of the compound. Therefore, to overcome these limitations, the development of improved (second-generation) PSs has been the focus of research as this would allow optimizing the amount of singlet oxygen formed and will consequently positively impact PDT. 3 Ideally, the PS should absorb in the visible to the nearinfrared region with high molar extinction coefficient and high singlet oxygen quantum yields (Φ Δ ). Porphyrins, a group of organic macromolecules composed of pyrrole conjugated molecules, have been chemically modified in order to increase and shift the light absorption to the nearinfrared therapeutic region and/or their solubility. Other formulations have been performed by addition of metal ions to form metallo-porphyrin complexes. In general, the second generation of PSs exhibited improved photophysical properties and allowed to address some of the shortcomings associated with the first generation PSs. Conjugation of the second generation PSs to biomolecules such as monoclonal antibodies (MAb) allowed to increase the affinity and selective delivery of the third-generation PS for malignant tissue. 4 Nonetheless, to date, only three PS (Aminolevulinic acid (ALA) and derivatives of ALA, a prodrug of porphyrin; photofrin®, a mixture of porphyrins and verteporfin, a porphyrin derivative) are FDA-approved.

Even though, the combination of PS with other active drugs (chemotherapy, immunotherapy) or techniques (radiotherapy) are good alternatives for diagnostics and enhancement of therapeutic effects, 5 we believe that a simple combination of two or more PS molecules with safe lipid nanocarriers such as liposomes or lipid nanoparticles might represent an interesting approach worth of investigation.

The literature survey is limited to a few reports using the combination of free PS molecules. In 2009, Schneider-Yin et al. reported the enhancement of phototoxicity on HEC-1A cell colony formation by addition of hypericin (60 nM) to 5-aminolevulinic acid (5-ALA, 0.5 mM) after 10 days of post-illumination with a white light. In that case, the nanomolar concentration of hypericin may just increase the amount of protoporphyrin IX (PpIX) produced by cells during PDT treatment, as demonstrated by PpIX quantification. 6 The dark toxicity of a mixture of hypericin and Foslipos® (1:1) was studied on two different head and neck squamous cell carcinoma cell lines. 7 The obtained data suggested that this combination decreases the dark toxicity effect. Nevertheless, cell viability differences between each PS alone and the mixture were not biologically significant on UMB-SCC 745 and 969 under these conditions. 8 By combining two highly efficient PSs (zinc(II)-phthalocyanine and meso-tetrakis(4-Nmethylpyridyl)porphine), Acedo et al. demonstrated a synergistic tumor cell phototoxicity on three cell lines (HeLa, HaCat and MCF-7). 9 We have recently demonstrated that lipid nanocapsules (LNCs) loaded with hypericin can be successfully used in PDT experiment in vitro. 10 Indeed, nanoparticles-based PDT has several advantages over traditional PDT. 11,12 For example, solubility and aggregation problems of PSs in aqueous media can be overcome by encapsulating the PSs.

Due to the high surface/area ratio of nanocarriers and the possibility to add targets onto their surface, selective targeting toward specific cells can be achieved, limiting the damage of healthy tissues. In this study, we investigate the possibility to encapsulate two PSs (hypericin and protoporphyrin IX) in lipid nanocapsules (LNCs) with the aim to achieve enhanced PDT in vitro and in vivo. The physical and photophysical properties of the resulting nanocapsules were characterized in terms of drug loading, size, charge and optical properties. Singlet oxygen yield as well as photobleaching were also evaluated. The in vitro phototoxicity was then measured on two cell lines (HeLa and MDA-MB-231). In order to understand these results, intracellular localization, kinetic measurements on endoplasmic reticulum and mitochondrial networks, and reactive oxygen species (ROS) production were performed by confocal and video-microscopies.

The in vivo biodistribution and tumor evolution were finally investigated in swiss nude mice bearing xenograft tumors.

EXPERIMENTAL SECTION

Materials. Hypericin (Hy, purity >99%) and Protoporphyrin IX (PpIX, purity >95%) were provided by Planta Natural Products (Wien, Austria) and Sigma-Aldrich (Saint-Quentin Fallavier, France), respectively. A stock solution of Hy and PpIX were prepared in dimethylsulfoxide (DMSO) at 10 mM and stored at -20 °C in the dark. Labrafac TM Lipophile WL 1349 (caprylic/capric triglyceride), Phospholipon® 90G (soybean lecithin at 97.1% of phosphatidylcholine), and Solutol® HS15 (a mixture of free polyethylene glycol 660 and polyethylene glycol 660 hydroxystearate) were generously provided by Gattefosse S.A.S. (Saint-Priest, France), Phospholipid GmbH (Köln, Germany), and Laserson (Etampes, France), respectively. Deionized water was obtained from a Milli-Q plus system (Millipore, Paris, France).

Rose bengal (RB, purity 95%) and all other chemical reagents and solvents were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France) and used as received.

Lipid nanocapsules (LNCs) preparation. Hy and PpIX were mixed in Labrafac prior to all preparation steps. Thereafter, LNCs were formulated at a nominal size of 25 nm using a phase inversion method of an oil/water system, as described by Heurtault. 13 Briefly, the oil phase containing Labrafac (126 mg) and Phospholipon 90G (18.75 mg) was mixed with the appropriate amounts of Solutol (204 mg), H 2 O (270 µL) and NaCl (22 mg), and heated under magnetic stirring up to 85 °C. The mixture was subjected to 3 temperature cycles from 70 to 90 °C under magnetic stirring. Then it was cooled to 78 °C and 1.65 mL of distilled cold water (0 °C) were added. The resulting suspension was stirred at room temperature for another 10 min before further use. The percentage of Hy and PpIX corresponds to the total initial mass percentage of the components of the lipid nanocapsules (i.e. 0.63 g). LNCs were purified from supernatant using a disposable PD-10 desalting column (Sephadex® G-25 for gel filtration as stationary phase, Amersham Biosciences). LNCs were sterilized in 0.2 µm filter syringe before cell experiments.

Particle size, zeta potential, UV-Vis and fluorescence measurements. LNCs were characterized in terms of size, surface charge and spectroscopic properties. The average diameter and polydispersity index (PI) were determined by dynamic light scattering using a Zetasizer® Nano ZS (Malvern Instruments S.A., Worcestershire, UK). The zeta potential was measured using the electrophoretic mode with the Zetasizer®. All the batches were diluted at 1/100 (v/v) in distilled water (filtered over 0.22 µm) prior to the analysis and performed in triplicate. UV-Vis measurements were carried out on a Perkin Elmer Lambda 950 dual beam spectrophotometer operating at a resolution of 1 nm in a 1 cm spectrometric cuvette. Fluorescence spectra were recorded using a Cary Eclipse spectrometer (Agilent, France) at room temperature (emission and excitation slit 5 nm, scan rate 600 nm/min).

Drug loading and encapsulation efficiency. LNCs were also characterized in terms of drug loading and encapsulation efficiency. The drug loading was directly determined by reversed phase high performance liquid chromatography (RP-HPLC). RP-HPLC analyses were realized on a Shimadzu LC2010-HT (Shimadzu, Tokyo, Japan). A 5 µm C 4 QS Uptisphere® 300 Å, 250×4.6 mm column (Interchim, Montluçon, France) was used as the analytical column. The column was heated to 40 °C. The mobile phase was a mixture of eluent A (trifluoroacetic acid 0.05% in H 2 O) and eluent B (trifluoroacetic acid 0.05% in CH 3 CN) at a flow rate of 1 mL/min. The linear gradient was 0 to 80% of eluent B in 30 min and detection was performed at 590 nm for Hy and at 402 nm for PpIX. A 10 mM stock solution of Hy and PpIX were prepared in dimethylsulfoxide for the calibration curve. Concentrations of 1 -100 µM of Hy and PpIX in dimethylsulfoxide were prepared from this stock. Each sample was injected (40 µL) into the RP-HPLC column.

Calibration curves were obtained by linear regression of drug concentration (µM) versus the peak area and are shown in Table 1 For the determination of Hy and PpIX encapsulation rates, LNCs were separated from supernatant using a disposable PD-10 desalting column (Sephadex® G-25 for gel filtration as stationary phase, Amersham Biosciences). The column was stabilized with 25 mL of distilled water. Then 2 mL of the LNCs suspension was deposited on the column and 0.5 mL of water were added to fill in the dead volume of the column. Finally, the LNCs were collected with 4 mL of distilled water as eluent. We measured Hy and PpIX concentrations by RP-HPLC before and after filtration to determine the encapsulation efficiency (EE) using the equation (1). Detection of singlet oxygen. The p-nitroso-dimethylaniline (RNO)-bleaching method was used to measure the relative 1 O 2 generation efficiency of PS-loaded LNCs (Hy or PpIX or both). 14,15 The 1 O 2 generation efficiency was followed by the bleaching of RNO at 440 nm with the transannular peroxide intermediate formed as a result of the reaction between singlet oxygen and imidazole. The solution containing Hy-loaded LNC25, PpIX-loaded LNC25 and PpIX-Hy-loaded LNC25 was prepared in the presence of imidazole (8 mM) and RNO (12.5 µM) in a 50 mM phosphate buffer (pH = 7.4). Samples in a 1 cm spectrometric cuvette, placed at a distance of 1.8 cm, were irradiated at room temperature in air for 60 min using a visible lamp (Spot Light Source 400-700 nm, L9566-03, Hamamatsu, Japan). The intensity of the light was measured using a PM600 TM Laser Fiber Power Meter (Coherent Inc, USA) and was determined as being 0.6 W.

Blue and red photo-irradiations were done using a visible lamp (Spot Light Source 400-700 nm, L9566-03, Hamamatsu, Japan). A band pass optical filter (BG7 Colored Glass Bandpass Filter, 435 -500 nm) and a long pass optical filter (FGL515 Colored Glass Longpass Filter, 515 nm) were used to filter off ultraviolet/red radiations and ultraviolet/blue radiations, respectively. Blue and red light intensities were measured using a PM600TM Laser Fiber Power Meter (Coherent Inc, USA) and was determined as being 0.15 W and 0.45 W, respectively. The decrease of the absorbance caused by RNO bleaching was monitored spectrophotometrically on a Perkin Elmer Lambda 950 dual beam spectrophotometer operating at a resolution of 1 nm in the region of 350-550 nm. All measurements were performed in triplicate. In order to estimate the 1 O 2 quantum yield of PS-loaded LNC25 aqueous formulations, the natural logarithm values (ln A/A 0 ) of RNO at 440 nm were plotted vs the photoirradiation time and fitted with a pseudo-first-order kinetic model. The 1 O 2 quantum yield of PS-loaded LNC25 in aqueous solution can be estimated using Rose Bengal (RB) as a standard (Φ RB = 0.76 in aqueous solution) 16 using the equation (2). 17 In a control experiment, the temperature increase of serum-free DMEM irradiated with visible light (λ>400 nm, 10 mW) for 12 min was captured by an Infrared Camera (Thermovision A40) and treated using ThermaCam Researcher Pro 2.9 software.

DNA cell cycle analysis. The cell cycle was analysed according to Sackova et al. 18 Briefly, the HeLa and MDA cells were treated in the same manner as before with Hy-loaded LNC25, PpIXloaded LNC25 and Hy-PpIX-loaded LNC25 at 0.5 µM for 2 h, washed two times with PBS and irradiated with visible light (λ>400 nm, 10 mW) for 12 min. Then, the cells were harvested at 2 h post irradiation, washed twice with PBS, trypsined and fixed in ice-cold 70% ethanol at -20 °C.

The cell suspension was then centrifuged, washed twice with PBS and resuspended in 500 µL of a PBS solution containing ribonuclease A (200 µg/mL) and propidium iodide (100 µg/mL). The This method offers a good control over the nanocapsules' size (between 27 to 31 nm) with monodisperse size characteristics for all batches (PI<0.17) (Table 2). Furthermore, the study of the behaviour of lipid nanocapsules in biological media was performed by measuring the size of the LNCs in different media (ultrapure water, PBS pH 7.4, serum-free DMEM and DMEM with serum) at room temperature (25 °C) and at 37 °C after 1 h incubation (Figure S1). The results

showed that the particle size decreases slightly in PBS, while it increases slightly in medium (with or without serum) with no visible aggregation at 25 °C, indicating the good stability of the LNCs. The two types of PS were entrapped in the LNC25 with high encapsulation efficiency (>97%) due to their high hydrophobicity. The physicochemical properties of PS-loaded LNC25 are quasi identical to the blank LNC25; the presence of Hy or PpIX had no effect on the mean diameter and the polydispersity index. The presence of Solutol HS-15, which exposes PEG chains, confers a negative zeta potential for blank LNC25 (-6.1±1.5 mV). This value is similar to the zeta potential of other pegylated vectors such as liposomes 19 and identical to the previously reported values in the literature. 20 However, the zeta potential values of PS-loaded LNC25 are more negative as compared to blank LNC25. In spite of the high hydrophobicity of Hy, the amphiphilic molecules are preferentially located in the layers near the membrane/water interface. 21,22 In view of the results, it seems probable that Hy, an amphiphilic compound, and PpIX, containing charged hydrophilic propionate chains, are arranged at the interface which exposes hydroxyl-and carboxyl-moieties, respectively, as shown previously with amiodarone-, quercetin-and Hy-loaded LNCs. 10,23,24 The UV/Vis absorption spectra of Hy-loaded LNC25, PpIX-loaded LNC25 and PpIX-Hyloaded LNC25 are examined in the 300-700 nm range and are compared to those of the corresponding free molecules when dissolved in DMSO (Figure 1 andS2). Major absorption bands for Hy (Figure S2A) are found in the range of 300-400, 450-500 and 550-600 nm for both free (dissolved in DMSO) and encapsulated Hy. The shape of absorption spectra of Hy-loaded

LNCs is identical to that of free Hy in DMSO. A major influence of the absorption measurements between 300 and 500 nm is due to the turbidity of the suspended LNCs that causes light scattering. The characteristic absorptions of Hy in DMSO correspond to an intense band at 599 nm and a less intense band at 555 nm. A hypsochromic shift of 3 nm in absorption maxima can be observed due to different molecular environment surrounding the Hy molecule and solvent. 25- 27 For PpIX in DMSO, the UV/Vis absorption spectrum is dominated by the characteristic porphyrin Q-bands in the range of 450-650 and B-band (Soret band). In DMSO, where PpIX is a monomer, 28 the intense B-band is located at 407 nm, together with four weaker Q-bands at 504, 539, 573, and 628 nm (Figure S2B). The fluorescence emission spectra of Hy-loaded LNC25, PpIX-loaded LNC25 and PpIX-Hyloaded LNC25 between 550 and 750 nm are seen in Figure 2 and S3 and were compared to the corresponding free molecules in DMSO. In the case of free Hy and Hy-loaded LNC25, a fluorescence emission is observed between 550 and 750 nm using an excitation wavelength at 330 nm (Figure S3A). In pure DMSO, Hy displays two emission bands located at 602 and 652 nm, in accordance with previously reported data. 29 Hy dissolved in LNCs exhibits also two emission bands located at 599 and 649 nm. The hypsochromic shift (3 nm) in emission bands is due to the different molecular environment surrounding the Hy molecule and the solvent used. 27 It is known that a blue shift corresponds to the decrease of the solvent polarity from DMSO to Labrafac. Interestingly, Hy-loaded LNC exhibit approximatively the same fluorescence intensity at the same concentration compared to free Hy in DMSO. All together, the results suggest that Hy encapsulation in LNCs enhances its solubilisation and the fluorescence emission is predominated by the monomeric form of Hy molecules inside the nanocapsules.

The emission spectra of PpIX using an excitation wavelength at 410 nm are depicted in Figure S3B. The emission spectrum of free PpIX in DMSO shows two fluorescence bands at 631 and 699 nm, in accordance with previously reported data. 30 The emission spectrum PpIX-loaded LNC25 displays strong fluorescence emission bands at 634 and 701 nm, confirming PpIX monomer form in these LNCs. 31 The fluorescence emission spectra of PpIX before and after encapsulation are almost the same in term of intensity; however after encapsulation slight changes can be observed. The emission peaks, which had red-shift 3 nm compared with the emission peaks of free PpIX in DMSO, can also be attributed to the the difference in molecular environment surrounding the PpIX molecule. The fluorescence measurements of the PS mixture in DMSO or encapsulated in LNC25 using an excitation wavelength of 330 or 410 nm (Figure Generation of singlet oxygen and quantum yield of PS-loaded LNC25. The amount of singlet oxygen generated can be determined quantitatively by using the p-nitroso-dimethylaniline (RNO) method as described previously. 10 The method is based on the "bleaching" of RNO induced by the reaction of singlet oxygen with imidazole as selective acceptor. The formation of singlet oxygen is determined by following the absorbance decrease at 440 nm. Control experiments using blank LNC25 indicate no singlet oxygen generation for concentrations up to 0.72 mg mL -1 after 60 min irradiation at λ>400 nm (data not shown). In the case of PS-loaded LNC25 with a PS concentration of 0.5 µM, irradiation at λ>400 nm at different time intervals (Figure 3A) resulted in a significant amount of singlet oxygen for all investigated formulations. This is contrast to free PS (1% DMSO) in phosphate buffer condition, where almost no singlet oxygen generation is observed. PpIX-loaded LNC25 exhibit the highest production yield of singlet oxygen compared to PpIX-Hy-loaded LNC25 and Hy-loaded LNC25 at the same PS concentration (Table 2). The hydrophobic molecular environment inside the LNCs suppresses PS aggregation and water-induced quenching of singlet oxygen. Singlet oxygen is generated without release of PS directly from the nanocapsules and the level of active singlet oxygen is higher than that of free molecules. The slope of the curve in Figure 3B is a good indication of the efficiency of generated singlet oxygen. The production rate of PpIX-loaded LNC25 is higher than Hy-loaded LNC25 within 10 min, whereas the Hy-loaded LNC25 reach a saturation after 60 min. The quantum yield of singlet oxygen generation of PS-loaded LNC25 is estimated using the comparative method where Rose Bengal (RB) is used as a standard photosensitizer reference (Φ RB = 0.76). 16 As described above, the depletion of RNO by 1 O 2 is monitored at 440 nm and plots of ln(A/A 0 ) as a function of irradiation time can be fitted with a pseudo-first order kinetics (Figure 3B). The rate of 1 O 2 generation is obtained from the slope of the pseudo-first-order plots.

A similar plot is generated for RB as a standard photosensitizer reference and the 1 O 2 quantum yields of PS-loaded LNC25 in aqueous solution are estimated using equation (2). The results are summarized in Table 3. The quantitative estimation of 1 O 2 generation varies in the range of 0.31-0.38 by comparing the first-order reaction rate constants of RNO bleaching with PS-loaded LNC25 and RB. For encapsulated Hy, the quantum yield compares well with the quantum yields of 0.35-0.43 reported for 1 O 2 generation by Hy entrapped in liposomes. [33][34][35] The values are much higher than Φ = 0.02 reported for free Hy in water. 36 For encapsuled PpIX, the quantum yield agrees well with the quantum yields of 0.36 reported for 1 O 2 generation by PpIX perfectly dissolved with Triton X-100 in Tris-HCl buffer in order to induce monomerization. 37 Thus, the LNCs seem to be a good alternative not only to solubilize Hy, but also PpIX. Finally, the values obtained for PpXI-loaded LNC25 are quite similar. We also checked the wavelength dependence of the quantum yield upon two different irradiation wavelengths. Table 3 shows the values of quantum yield determined from the photodegradation of RNO bleaching with PS-loaded LNC25 and RB. Upon blue and red irradiation, the Φ values obtained for PpXI-Hy-loaded LNC25 provide a better quantum yields of 1 O 2 in comparison with single PS encapsulated in LNCs.

Photobleaching of PS-loaded LNC25. Upon irradiation, the PS are chemically modified or degraded. This phenomenon, called photobleaching, occurs by a direct attack of singlet oxygen or reactive oxygen species (ROS) on the PS. 38 Experimentally, photomodification (also referred to as photoproduct formation) and photodegradation (destruction) can be followed by a decrease in fluorescence emission and/or absorbance of PS in solution, cells or tissues during light exposure.

The kinetics of photobleaching of PS-loaded LNC25 in phosphate buffer is determined by spectrophotometric analysis (Figure 4A-C). The results of photobleaching of PpIX and Hy by irradiation of Hy-loaded LNC25, PPIX-loaded LNC25 and PPIX-Hy-loaded LNC25 at 5 µM in 50 mM phosphate buffer as a function of time are displayed in comparison with the free molecules dissolved in DMSO (Figure 4D-E). The results indicate a continuous decrease in absorbance of the bands without the appearance of new bands and suggests a phototransformation and a photodestruction of both PS under irradiation over time. For PpIX photobleaching study (Figure 4D), there is no significant difference in the rate of photodegradation for PpIX when it is encapsulated alone or with Hy inside the lipid nanocapsules. However, the photobleaching is faster compared to free PpIX dissolved in DMSO. As shown in Figure 4E, the rate of Hy photobleaching is slightly faster when it is encapsulated in LNCs compared to the free molecule in DMSO. Moreover, the photodegradation of Hy increases with time when co-encapsulated with PpIX in the lipid nanocapsules. The higher photodegradation of the remaining Hy by PpIX inside LNC may be beneficial during PDT treatment since photobleaching may induce photosensibility of the patient for an extended period of time. 38,39 On the other hand, upon blue and red light irradiation, the photodegradation of Hy slightly decreases with time when co-encapsulated with PpIX in the lipid nanocapsules (data not shown). In vitro phototoxicity. The cytotoxicity of PS-loaded LNC25 is assessed against HeLa cervical cancer cells and MDA-MB-231 breast cancer cells (Figure 5). The viability of cells upon photoirradiation (λ>400 nm, 10 mW, 12 min) is evaluated by MTT colorimetric assay. Cell cytotoxicity is measured as the percentage of the viable cells over the untreated cell control (without LNC25). During PDT treatment with visible light (λ>400 nm, 10 mW), the temperature increase of serum-free medium was less than 1.6 ± 0.1 °C after 12 min irradiation (Figure S5). This result confirmed that heat generated during PDT is negligible and has no impact on cell viability. For blanck LNC25, both cell lines did not show any observable phototoxicity or dark toxicity up to 400 µg/mL for incubation time of 8 h (see supplementary information: Figure S6).

The cells are incubated with Hy-loaded LNC25, PpIX-loaded LNC25 and PpIX-Hy-loaded LNC25 for 8 h at concentrations ranging from 0.01 µM to 1 µM of PS and then washed to remove non-associated PS prior to irradiation. Figure 5 shows changes in cell viability caused by different treatments. Under dark conditions, cytotoxicities of PS-loaded LNCs are negligible.

Cells survival by MTT colorimetric assay is >95 % for all formulations except a small cytotoxicity for PpIX at 0.5 and 1 µM (> 85 %) for both cell lines. Also, a small cytotoxicity is found for Hy at 0.5 and 1 µM (> 85 %) on MDA cell line. But used in combination, no cytotoxicity is observed for the PpIX-Hy-LNC25 up to 1 µM for both cell lines.

Photodynamic treatments with Hy-loaded LNC25 and PpIX-loaded LNC25 exhibit a dosedependent effect on HeLa and MDA-MB-231 cells. Indeed, the IC 50 of Hy-loaded LNC25 for displays an IC 50 of 0.55 µM (HeLa) and 0.50 µM (MDA-MD-231). Interestingly, the in vitro photodynamic therapy effect of both PS encapsulated in LNCs (PpIX-Hy-loaded LNC25) displays a substantially higher phototoxicity for both cell lines in the nanomolar range (IC 50 = 274 and 278 nM for HeLa and MDA-MB-231 cell lines, respectively). Thus, these 2PS-loaded LNCs are shown to increase the phototoxicity and provide a synergistic effect in the combined nanoparticulate system, whereas the corresponding single PS-loaded LNCs at the same concentration exhibit a phototoxicity two times lower. Furthermore, this system allows to reduce the dark toxicity on both cell lines in comparison with 50/50 molar ratio of free Hy/PpIX (see supplementary information, Figure S7). DNA cell cycle analysis. After 2 h incubation of MDA-MB-231 and HeLa cells with PpIXloaded LNC25, Hy-loaded LNC25 and PpIX-Hy-loaded LNC25 at 0.5 µM, both cells were irradiated and treated. The cellular DNA flow cytometric analysis displays the percentage of cells in G 0 -G 1 , S and G 2 -M phases (Figure 6 andS8). An increase in the proportion of cell into cell cycle (S or G2-M phases) is observed in both cell lines treated with the PS-loaded LNC25. This suggests that cell cycle is either slowed down or arrested during DNA replication (both cell lines) in addition of G2-M checkpoint (HeLa). Several studies reported that ROS-mediated DNA damage induces such an increase of cells engaged in cell cycle. 40,41 By analogy, it is likely that PpIX or Hy photoactivation induces DNA damage through the production of singlet oxygen. These results demonstrate a difference in PpIX and Hy subcellular localization (Figure 7). Indeed, while PpIX is mainly retained in the plasma and ER membranes, Hy mostly accumulates around the internal structures. Although, we do not exclude that a fraction of Hy also reached ER membranes, the colocalization studies between Hy and ER or mitochondria-target fluorescent biosensors revealed that Hy-overloaded structures are neither in ER nor in mitochondria (Figure S9). The size and number of these Hy-loaded structures, their exclusion from mitochondria and erythrocytes, 42 suggest that Hy likely accumulates in lysosomes. PpIX lifetime shifted upon its localization in plasma membrane or in internal membranes, suggesting that its immediate environnement modifies its properties. A previous study suggested that under photoconversion, PpIX induced strong modifications in ER and mitochondria shape and network organization. 43 We next assessed this hypothesis in our model. We measured the kinetic of these network modifications induced by PpIX and Hy treated cells. Under a constant laser illumination at 405/488/560 nm, live recordings of ER and mitochondrial networks are achieved on PpIX-loaded LNC25 (PpIX), Hy-loaded LNC25 (Hy) and PpIX-Hy-loaded LNC25 (PpIX+Hy) treated Hela cells for 2 h. As reported in Figure 8A, ER network undergone fragmentation leading to ER vesicularization within a minute timescale. By comparison, the fragmentation of the mitochondrial network occurred within a period of 20 min (Figure S10). 590 nm while recording fluorescence images with the 488 laser line. Objective: ×60. Scale bar: 5 µm.

The high reactivity of 1 O 2 with its local environnement highly restrictes its diffusion. The faster kinetic of ER fragmentation than the kinetic of mitochondria fragmentation, upon PS entrapment, suggests that ER membranes are one of primary sites of photostimulated singlet oxygen production by PpIX and Hy in Hela cells. Moreover, a recent study suggested that ROS are implicated as an inducer of ER fragmentation. 44 We measured ROS production with the DFC probe in cells loaded concomitantly with PpIX and Hy. As shown in Figure 8B, the fluorescence increased in fragmented organnels that looked similar to the fragmented ER (Figure 8A). This suggests that 1 O 2 production via PpIX and Hy could in turn induce ROS generation in the ER compartment, leading to its fragmentation. The slight difference of kinetics of ER fragmentation between encapsulated PpIX-(2±0.5 min) and Hy-loaded cells (6±1 min) could originate from their rates of 1 O 2 production and/or their concentrations in ER membranes. It should also be noted that ER fragmentation is accompagned with a massive blebbing of the plasma membrane (Figure S11) within a similar period of time. Membrane blebbing is known to involve the rupture of tension forces between cytosqueletton and juxta-membranar protein coating, 43 and can be triggered by an oxidative stress. 45 In vivo applications of photosynthesizer nanocompositions. With the aim to utilize the observed toxic effect caued by combination of two photosynthesizers in nanocapsules we performed analysis on how the studied compounds are distributed in the body during different type of injection. For this reason swiss nude mice were injected with 100 µL of Hy-loaded LNC25 (1.25 mg/kg of Hy) subcutaneously (s.c.), intravenously (into the tail vein, i.v.) or intraperitoneally (i.p.). Since studied compounds possess fluorescent properties they were easily monitored in the body of hairless mice using InVivo Extreme fluorescent scanner (Bruker). We observe a strong fluorescent signal, attributable to hypericine (ex. 540 nm, em 600 nm) at the place of subcutaneous injection and around the place of injection during intraperitoneal injection.

(Figure 9A). In the next step we injected Hy-loaded LNC25 i.v. and i.p. and sacrificed the animal 2 h post injection, main organs were removed and accumulation of hypericine was analysed.

During intravenous injection, we have observed fluorescent signal in intestine, gallbladder, liver and urinary bladder. This indicates that nanocapsules in the blood are removed by both kidney filtration (less extent) and conjugation in liver with subsequent excretion into bile and then into intestine (main part of compounds) (Figure 9B). In contrast, at the place of subcutaneous injection, we observed strong retention of LNC-Hy signal, while during subcutaneous injection we observed retention of Hy nanocapsules in the place of injection and in the close vicinity (like adipose tissue, subcutaneous tissue, skin). Thus we decided to choose intratumor injection as the best administration route for LNC-Hy.

  where Φ RB is the 1 O 2 quantum yield of RB in an aqueous solution as standard (Φ RB = 0.76); k LNC and k RB re the rate constants for photoreaction of RNO with PS-loaded LNC25 and RB, respectively; I LNC and I RB are the rates of light absorption by PS-loaded LNC25 and RB in aqueous solution, respectively, which are estimated by integration of the absorption bands in the region of 400-700 nm, 400-550 nm or 515-700 nm.Cell culture. The HeLa (cervix carcinoma, human) and MDA-MB-231 (human breast adenocarcinoma) cells were maintained in DMEM supplemented with 10% (v/v) fetal-bovine serum (FBS) and 1% (v/v) penicillin/streptomycin in a 5% CO 2 atmosphere at 37 °C.In vitro phototoxicity. The HeLa and MDA cells were seeded into 96-well plates (1×10 4 cells/well) 48 h before experiments. Prior to incubation, cells were washed with PBS. The medium was replaced with 100 µL of serum-free medium containing Hy-loaded LNC25, PpIXloaded LNC25, Hy-PpIX-loaded LNC25, and free PS (Hy and PpIX) (1% DMSO) at concentrations ranging from 0.01 to 1 µM. After 8 h incubation, cells were washed three times with PBS and then maintained in DMEM supplemented with 10% FBS. Cells were irradiated for 12 min using a visible lamp (Spot Light Source 400-700 nm, L9566-03, Hamamatsu, Japan). The intensity of the light was measured using a PM600 TM Laser Fiber Power Meter (Coherent Inc, USA) and was determined as being 10 mW. Light positive controls (untreated cells or with blank LNC25) and dark controls were included. After 16 h post irradiation at 37 °C, cell viability was colorimetrically measured by performing MTT assays. After removing the medium and washing by PBS, 100 µL of DMEM containing MTT solution (1:10 dilution of the 5 mg.mL -1 fresh MTT stock solution in PBS) were added. After incubation for another 3 h, the resulting formazan dye was dissolved in dimethylsulfoxide (50 µL) and the absorbance intensity was measured by a microplate reader (PHERAstar FS, BMG LABTECH GmbH, Germany) at 570 nm with background subtraction at 650 nm. Cell viability was determined as the percentage of live cells per total non-treated cells.

  cellular fluorescence of PI was detected in a linear scale using a flow cytometer (FACS) equipped with an excitation laser line at 488-633 nm. At least 10 000 events were collected for each sample. The percentage of cells in G 0 -G 1 , S and G 2 -M were calculated using MODFIT computer software and are represented within the histograms. Statistical difference from the untreated controls: * p < 0.05; ** p < 0.01.Cell transfection.The HeLa cells were seeded on 35 mm glass bottom dishes (MatTek Corporation, USA) at 1.2×10 5 cells/well for 48 h before transfection. Cells were washed with PBS, maintained in DMEM supplemented with 10% FBS and incubated for 20 h in a CO 2 atmosphere at 37 °C in the presence of D1ER and 4mtD3cpv encoding plasmids DNA (1.2 µg per well). Plasmids DNA were added to 100 µL Opti-MEM, then 3.6 µL of transfection reagents (ratio 1:3 v/v DNA/FuGENE®) were added and incubated for 15 min. The mixtures were transferred in each well and gently mixed. The day after, cells were washed with PBS, maintained in serum-free medium and incubated for 2 h in a CO 2 atmosphere at 37 °C in the presence of PpIX-loaded LNC25, Hy-loaded LNC25 and PpIX-Hy-loaded LNC25 at 0.5 µM of PS. After incubation, the cells were washed two times with PBS and maintained in dark in L-15 medium without phenol red (Life technologies) at 37 °C using a stage incubator (Life Imaging Services, Switzerland) for futher video-microcopy and live-irradiation.Fluorescence-lifetime imaging microscopy and confocal imaging. Fluorescence-lifetime imaging microscopy (FLIM) and time-lapse imaging were performed with a Leica TCS SP5 X (Leica Microsystems, Germany) confocal head with the SMD upgrade, mounted on an inverted microscope (DMI6000, Leica Microsystems, Germany

  Xenogeneic tumor model. Five-week-old male swiss nude mice were injected subcutaneously (s.c.) with 1.2 × 10 6 PC3-M cells resuspended in 50 µL PBS / 50µL Matrigel (BD). The PC3-M cells were grown by subcutaneous injection of 1.2 × 10 6 cells/50 μL of sterile PBS with 50 μL of Matrigel (BD) on flank of the mice. Tumor growth was monitored with caliper every two days by measuring its dimensions and calculating the volume. Tumor volumes were calculated as follow:Tumor volume (mm 3 ) = π/6 × length × weight × height Nanocapsules (100 µL) at a low dose of 1.25 mg/kg of PS were administered after 30 days intratumorally to mice bearing tumors with an average tumor volume of 250±50 mm 3 . Then, the tumors were immediately irradiated with visible light (λ>400 nm, 0.35 W) for 6 min, which correspond to the light dose of 40 J/cm². Three test groups and one control group were subjected to injection as follows LNC-Hy (group 1, n=3); LNC-PpIX (group 2, n=3); targeted, LNC-PpIX-Hy (group 3, n=3) and finally sterile saline (control, n=3). Tumor volume was measured with calipers every 5-7 days for a period of 53 days, and then the animals were euthanized. The efficiency of the lipid nanocapsules was evaluated by measuring the tumor volume of xenograft tumors in five/six-week-old male swiss nude mice weighing 20-25 g.RESULTS AND DISCUSSIONPreparation and physical characterization of PS-loaded LNC25. Three different formulations of PSs-loaded LNC25 were made: one with hypericin (Hy) at 0.04 wt % (weight percentage of Hy over the total initial mass of components i.e. 0.63 g), one with protoporphyrin IX (PpIX) at 0.045 wt % and the third having both PS present in the same LNC25 at 0.042 wt % (0.02 wt % of Hy + 0.022 wt % of PpIX). The PSs-loaded LNC25 formulation was achieved by mixing the PSs with excipients at optimized concentrations using the phase inversion method as described in the Experimental section.

  The absorption spectrum of PpIX-loaded LNC25 displays a strong B-band at 407 nm and four weaker Q-bands at 505, 539, 576, and 631 nm, confirming the presence of PpIX in monomer form. For the mixture of Hy and PpIX encapsulated in LNC25, the absorption bands are the sum of Hy-loaded LNC25 and PpIX-loaded LNC25 bands with no significant shift (Figure 1A). This spectroscopic study of PS-loaded LNC25 dispersed in water suggests a good solubilization of both molecules in the lipid core of LNCs. The comparison of Hy, PpIX and Hy-PpIX-loaded LNC25 are summarized in Figure 1B.

Figure 1 .

 1 Figure 1. Absorption spectra (300 -700 nm) of PpIX (5 µM) + Hy (5 µM) dissolved in DMSO (dotted line) and PpIX-Hy-loaded LNC25 (continuous line) in water at 10 µM (A); Comparison of Hy-loaded LNC25 (red line), PpIX-loaded LNC25 (blue line) and PpIX-Hy-loaded LNC25 (green line) (B).

2A and 2B )

 2B reveal that the spectra correspond to the sum of Hy and PpIX emission peaks (see supplementary information: FigureS4). The fluorescence peak at 670 nm, resulting from the formation of PpIX photoproduct, is completely missing after encapsulation.32

Figure 2 .

 2 Figure 2. Fluorescence emission spectra (550-750 nm) of PpIX (1.25 µM) + Hy (1.25 µM) dissolved in DMSO (dotted line) and PpIX-Hy-loaded LNC25 (continuous line) in water at 2.5 µM. The emission spectra are recorded using an excitation wavelength  ex =330 nm (A) and 410 nm (B).

Figure 3 .

 3 Figure 3. Generation of singlet oxygen (ΔA at 440 nm) (A) and pseudo first order plots of ln(A/A 0 ) (B) from photo-irradiated Hy-loaded LNC25 (open triangles), PpIX-loaded LNC25 (open circles), PpIX-Hy-loaded LNC25 (black squares) and free PpIX-Hy (1% DMSO) (black cross) at 0.5 µM in a 50 mM phosphate buffer (pH = 7.4) as a function of irradiation time. RB (red triangles) as reference.

Figure 4 .

 4 Figure 4. Effect of photoillumination on the absorption spectra of PpIX-loaded LNC25 (A), Hyloaded LNC25 (B) and PpIX-Hy-loaded LNC25 (C) at 5 µM of each PS in 50 mM phosphate buffer as a function of irradiation time (400-700nm). Rate of change of maximum absorbance of

Figure 5 .

 5 Figure 5. In vitro phototoxicity of PS-loaded LNC25. MTT assay data for cells, Hy-loaded LNC25, PpIX-loaded LNC25 and PpIX-Hy-loaded LNC25: 50/50 molar at different

Figure 6 .

 6 Figure 6. DNA flow cytometric analysis. The cells (A) MDA-MB-231 and (B) HeLa are treated with PS-loaded LNC25 at 0.5 µM and irradiated with visible light (10 mW) for 12 min. After fixation and staining with PI, the cells are analysed by flow cytometry. The percentage of cells in

Figure 7 .

 7 Figure 7. Intracellular localization of PS-loaded LNC25 (0.5 µM) after 2 h of incubation in HeLa cells. Images show the average fluorescence lifetime (color bar) and fluorescence intensity (brightness as photon count intensity) of cells treated with PpIX-loaded LNC25 (PpIX), Hyloaded LNC25 (Hy) and PpIX-Hy-loaded LNC25 (PpIX + Hy). Objective: ×60. Scale bar: 5 µm.

Figure 9 .

 9 Figure 9. Biodistribution of LNC-Hy solution in the body. Distribution in the body of nude swiss mice 2 h after different types of injections: subcutaneous (s.c.), intravenous (i.v.), intraperitoneal (i.p.) (A). Distribution in different organs 2 h after intravenous or intraperitoneal injection (B). Fluorescence images were obtained by Bruker In-Vivo Extreme imager using ex. 540nm and em. 600 nm.

5 tr e a tm e n tFigure 10 .

 510 Figure 10. Growth of PC3-M tumor xenografts in swiss nude mice. At day 30 after the development of tumors, they were injected with studied compounds, treated with visible light (λ>400 nm, 0.35 W) for 6 min and tumor growth was further monitored till day 53.

Table 1 .

 1 . Calibration curves and linear regression of Hy and PpIX

	Compounds Retention time	Regression equation a	k drug	Linear range	r 2,b
	Hy	26.6 min	Y = 54260 X	54260	1 -100 µM	0.9962
	PpIX	20.0 min	Y = 373350 X	373350	1 -100 µM	0.9999
	a Equation, where Y is the peak area and X is the concentration of compounds.
	b Correlation coefficient (n = 5).

  ). In all experiments, a 63×/1.2NA water immersion objective was used and the confocal pinhole was set to 1 Airy. PicoQuant GMBH, Germany) software. FLIM Images are 256×256. Since, for statistics issues, final images were the result of the accumulation of 120 frames acquired at 200 Hz. The final acquisition time for one TCSPC image was about 100 s long.In time-lapse experiments, ER was labeled in HeLa cells using the D1ER genetically encoded biosensor, as described below. As lasers sources, a 488 nm laser was used for imaging, and a 594 nm laser was used to generate redox stress in cells via the LNCs. Time-lapse movies are composed of 60 images for a total of 4 min. Images are 512×512 with a pixel size of 0.18 µm for a zoom factor of 3. /oxygen mixture and imaged before, immediately after injection, and then after 1, 2, 4, 8, 24, 48, 72 and 166 h. In vivo tracking of hypericin-containing nanocapsules was performed with a Bruker In-Vivo Xtreme machine supplied with interline front-illuminated (FI) 16 MP CCD detector, 400W Xenon Fluorescence illuminator (excitation wavelength: 540 nm, emission wavelength: 600 nm with constant exposure of 3 seconds) and module for animal warming air 37 °C. The data was analyzed by high-performance Bruker Molecular Imaging (MI) software. The data represents mirror images of the mice. To evaluate distribution of nanocapsules in organs, animals were sacrificed under anesthesia, and main organs were removed and analyzed for fluorescence attributable to hypericin.

	For FLIM experiments, a 405 nm pulsed diode laser PDL 800-B (PicoQuant GMBH, Germany)
	was used as a laser source with a repetition rate of 10 MHz. Fluorescence was detected through a
	483/32 single-bandpass filter (Semrock, USA) on Single Photon Avalanche Photodiodes (SPAD)

(MPD, Italy). Single photon events were recorded by a HydraHarp 400 (PicoQuant GMBH, Germany). Measurements were made with LAS AF (Leica Microsystems, Germany), and SymPhoTime (For live-cell imaging, cells were placed on 35 mm glass bottom dishes (MatTek Corporation, USA), filled with L-15 medium without phenol red (Life technologies), and kept at 37 °C using a stage incubator (Life Imaging Services, Switzerland). ROS production was also measured with the DFC probe in HeLa cells loaded concomitantly with PpIX-Hy-loaded LNC25 at 0.5 µM for 2 h by adding 2′,7′-dichlorofluorescin diacetate at 40 µM for 30 min.

Animals. Five-week-old male swiss nude mice (specific and opportunistic pathogen free, SOPF) were purchased from Charles River Laboratories. The mice were housed in cages covered with air filters in a temperature-controlled room with a 12-h light/12-h dark schedule and kept on a standard diet with drinking water available ad libitum. Studies involving animals, including housing and care, method of euthanasia, and experimental protocols, were conducted in accordance with the local animal ethical committee in the animal house (D59-00913) of the University of Lille, under the supervision of Dr V'yacheslav Lehen'kyi (Protocol: 201703021400830, Authorization: 59-009270).

Biodistributions studies.

100 µL of nanocapsules diluted in sterile PBS (5 mg/kg of Hy) were injected intraperitoneally (i.p.) and intravenously (i.v.) into the tail vein, or subcutaneously (s.c.). Animals were anesthetized with isoflurane

Table 2 .

 2 Characterization of PS-loaded lipid nanocapsules (PS-LNCs)

		Hy (%)	PpIX (%)	Mean diameter (nm)	PI a	Zeta potential (mV)	EE of Hy (%)	EE of PpIX (%)
	LNC25	0	0	26.6±0.8 0.088±0.006 -6.1±1.5	-	-
	LNC25-Hy	0.04	0	27.1±2.4 0.154±0.013 -19.5±2.4 97±2.5	-
	LNC25-PpIX	0	0.045 31.6±1.9 0.172±0.024 -12.6±1.9	-	99±1.0
	LNC25-PpIX-Hy	0.02	0.022 28.0±3.3 0.137±0.018 -18.6±3.0 98±1.6 100±0.3
	a Polydispersity Index
	mean±SD, n=3

Table 3 .

 3 Pseudo-first-order kinetic parameters and quantum yields of1 O 2 determined at 0.5 µM for PS-LNC25 with different irradiations (mean ± SD, n = 2).

		Rate constants	Quantum	Quantum	Quantum
		k obs (min -1 ) b	yields Φ b	yields Φ c	yields Φ d
	Hy-loaded LNC25	-0.029 ± 0.001	0.38 ± 0.01	0.16 ± 0.01	0.28 ± 0.01
	PpIX-loaded LNC25	-0.074 ± 0.005	0.31 ± 0.02	0.11 ± 0.01	0.22 ± 0.02
	PpIX-Hy-loaded LNC25 -0.046 ± 0.006	0.32 ± 0.04	0.22 ± 0.01	0.52 ± 0.02
	PpIX-Hy	-0.004	0.03	n.d.	n.d.
	RB	-0.164 ± 0.007	0.76 a	0.76 a	0.76 a
	a Literature value 16
	b Visible irradiation (400-700 nm)
	c Blue irradiation (400-550 nm) d Red irradiation (515-700 nm)
	n.d., not determined.

ACKNOWLEDGMENT N.S., A.B., S.S. and Ra.B gratefully acknowledge financial support from the Centre National de la Recherche Scientifique (CNRS), the Université Lille 1 and the Hauts-de-France region. S.S., T.D., S.P., Ro.B. acknowledge financial support from EUH2020-MSCE-RISE-2015 project no. 690836 PANG. We thank Profs P. Woisel and J. Lyskawa (Laboratoire de Chimie Macromoléculaire, UMR CNRS 8009) for technical support with fluorescence measurements.

Competing interests

The authors declare no competing financial interests.

SUPPORTING INFORMATION

Supporting information

Supporting Figures 1-11.


Jean-Max Redonnet
email: jean-max.redonnet@univ-tlse3.fr

Alejandro Gamboa Vázquez

Alberto Traslosheros Michel

Stéphane Segonds

Keywords: free-form surface, CNC machine-tool, torus-end mill, effective tool radius, workpiece orientation, optimisation

Machining by parallel planes is a widely used strategy for end milling of free-form surfaces on 3-axis Numerically Controlled (NC) machine. In industry, this type of machining is generally performed with a hemispherical tool. However, numerous studies have shown the benefits of torus end mill on ball end or flat end mills. More than anything, the machining direction has much influence on productivity while using a torus end mill. In this context, the choice of the machining direction is of paramount importance when using a torus end mill in machining of free-form surfaces. This paper presents an optimisation of part machining direction allowing minimizing the machining time within the respect of the maximum imposed scallop height. This optimisation methodology is then applied to an industrial part and measurements are performed on this part. The study highlights the interest of optimizing the machining direction and the benefits that can be drawn with respect to machining using a no optimized direction.

Introduction

Machining by parallel planes 1 is a common strategy for the machining of freeform surfaces on 3-axis NC machine tool, especially for the production of molds and dies. It is widely used in industry because it is reliable and robust, well-proven through years of practice. Indeed, this strategy allows on one hand to leave no uncut areas of the part, and on the other hand to ensure that the generated tool paths do not intersect (which is significant in terms of productivity).

When finishing freeform surfaces, the main criterion for assessing the quality of the machining is the scallop height, denoted s h (see figure 1). During machining of a rough surface, a ridge, formed by the residual material between two successive paths of the tool, appears regardless of the type of tool used: flat-end, ball-end or torus-end mill. The height of this scallop is a critical parameter because it gives an indication on the amount of material which has to be removed by polishing operations that are done after the machining finishing operation. It mainly depends on the step over distance (denoted s od ) which is the distance, in a plane normal to the feed direction, between two successive paths of the tool.

In industry, the machining strategy by parallel planes is usually implemented using a ball end mill. However, numerous studies have demonstrated the opportunity of using a torus-end mill rather than a hemispherical or flatend mill [START_REF] Bedi | Toroidal versus ball nose and flat bottom end mills[END_REF][START_REF] Jensen | Accurate tool placement and orientation for finished surface machining[END_REF][START_REF] Redonnet | Optimising tool positioning for end-mill machining of free-form surfaces Institution of Mechanical Engineers Part[END_REF][5][6] . Indeed, as the flat-end tool, the torus-end mill allows under certain conditions, for a given step over distance, to obtain a lower scallop height than that allowed by the ball-end one. For a given scallop height (i.e. at constant quality), it saves then a lot of productivity. In addition, the torus-end mill avoids unsightly marks left on the part by flat-end tool and preserves the surface integrity 7 . By combining the benefits of both of flat-end and ball-end tools, torus-end mill appears to be the ideal compromise for finishing operation by end milling of freeform surfaces.

The relationship between machining direction, step over distance and tool geometry is well known [START_REF] Senatore | Correlation between machining direction, cutter geometry and step-over distance in 3-axis milling: Application to milling by zones[END_REF] . Figure 2 illustrates this relationship for both ball-end and torus-end mills. This clearly highlights the fact that the torus-end tool is far more efficient when machining in the direction of greatest slope while it is less efficient when machining in a direction perpendicular to the direction of highest slope. But while the max slope direction is quite obvious for a plane, like in Figure 2, in the case of a free-form surface, this direction is different for each point. Thus, the best direction for the whole surface cannot be determined easily.

In [START_REF] Quinsat | Optimal selection of machining direction for three-axis milling of sculptured parts[END_REF] , the authors show that even in the case of a ball end mill, the machining direction is one of the most influential parameters on the machining time. In this case the tool effective radius (see section Context page 3 for comprehensive definition of effective radius) is invariant and only the angle between the direction of maximum slope and machining direction influences. In the case of torus-end mill use, this parameter is even more influential since the effective radius of the toric profile which leaves traces in the material varies with the working direction. For a torus end mill of major radius R and torus radius r, effective radius varies between r and infinity.

These considerations show that efficiency of torus-end mill is highly affected by the machining direction. Thus, this direction have to be carefully choosen to be relevant for the whole surface machining.

For 3-axes machining the milling direction is defined by position and orientation of the workpiece. Indeed, the issue of position and orientation of the workpiece is often considered in terms of dynamic behavior of the machine [START_REF] Anotaipaiboon | Optimal setup for five-axis machining[END_REF][START_REF] Hu | Improving the dynamics of five-axis machining through optimization of workpiece setup and tool orientations[END_REF][START_REF] Pessoles | Optimisation of workpiece setup for continuous five-axis milling: application to a five-axis BC type machining centre[END_REF] , or from the viewpoint of the visibility of surface to be machined 13;14 . Few studies address the issue of the optimal machining direction. Among them may be mentioned [START_REF] Quinsat | Optimal selection of machining direction for three-axis milling of sculptured parts[END_REF] , which is confined to the use of a ball-end tool. But these approaches are all based on local parameters and do not consider the surface as a whole. Therefore they are limited to provide the optimum machining direction at a point of the surface or calculating a first optimal path 15;16 . If they are, for most of them, relevant and meaningful, their extension to the whole surface is not the result of a real optimisation process.

Other authors consider optimisation of freeform surface machining process from other points of view than productivity. For example in [START_REF] Balic | Intelligent tool path generation for milling of free surfaces using neural networks[END_REF] , the authors seek to optimize the quality of the surface using neural networks. Others seek to optimize the machining quality by adjusting the feed rate of the tool [START_REF] Erkorkmaz | Feedrate optimization for freeform milling considering constraints from the feed drive system and process mechanics[END_REF][START_REF] Chen | Feedrate optimization and tool profile modification for the highefficiency ball-end milling process[END_REF][START_REF] Tounsi | Optimized feed scheduling in three axes machining. Part 1: Fundamentals of the optimized feed scheduling strategy[END_REF] .

In the context of machining free-form surface with a torus-end mill, global process optimization using total machining time as objective function has not already been studied.

Our study is thus conducted in the context of 3-axis machining by parallel planes with a torus-end mill. The objective of this study is to minimize the machining time while maintaining a maximal scallop height constraint respected. At first, we neglect dynamic effects and it is considered that the machining time is directly proportional to the length of the tool path. Other work in the laboratory [START_REF] Pessoles | Kinematic modelling of a 3-axis NC machine tool in linear and circular interpolation[END_REF] showed that this approximation is quite valid for the machining of freeform surfaces in most configurations. the surface, a key parameter in the value of the tool effective radius. To date, however, there is no method for determining the machining direction that have to be used to machine a freeform surface with a torus end-mill in a minimum time.

The main contribution of this paper is thus to provide a procedure for determining the machining direction that have to be used to machine a freeform surface with a torus end-mill in a minimum time.

The machining of the freeform surface is time consuming, especially during finishing, so this method could provide increased productivity. It is noticable that as far as we know no optimisation process of the milling direction is proposed by any commercial CAM software, especially using a toroidal cutter.

From an optimization point of view this problem can be stated as follows :

Min toolpath_length(θ) s.t. s hi s hmax ∀i (1)
where the optimisation variable θ is the angle between the X axis of the machine and the vector F and i is the index of the points of the tool path (see figure 3). In this publication, it is shown how this optimisation can be implemented and the potential benefits it can bring. Therefore, the computation time of a path planning for the entire surface must be low enough to allow the integration of this process with the goal of optimisation. It then will therefore becomes possible to solve in a reasonable time the optimisation problem (1).

Furthermore this optimisation problem is subject to numerous inequality constraints. Indeed for each point of the planed toolpaths the maximum scallop height allowed must be respected. The seconds objective of this paper is to ensure that these constraints are respected. In other words, the procedure we propose must ensure quality respectfull.

To ensure both of these objectives are respected we first propose a new procedure to determine optimum machining direction while respecting tolerance. Then, on a test case, we apply this procedure and verify maximum scallop height is actually respected by measuring the machined part.

Context of this study

A key parameter in the generation of machined scallops is the effective radius of the tool denoted R ef f , which can be defined as the projection in a plane normal to feed direction, of the trace left by the tool into the material [START_REF] Redonnet | Study of the effective cutter radius for end milling of free-form surfaces using a torus milling cutter[END_REF] . Indeed, the effective radius of the tool is characteristic of scallop height s h . Denoting d the distance defining the next parallel plane position, the step over distance s od can be readily determined as it is directly related to d by angle γ characterising the local inclination of the surface in a plane normal to the machining direction (figure 4). Showing that the step over distance is directly related to the cutter effective radius is thus equivalent to showing that distance d depends directly on that effective radius. Well as proven in [START_REF] Redonnet | Study of the effective cutter radius for end milling of free-form surfaces using a torus milling cutter[END_REF] the distance d can be calculated by

d = 4 R 2 ef f + 4 R ef f -2 s h -s h 2 (2 + s h ) s h + s h
where is the local curvature of the surface For a given machining plane, the position of the adjacent plane is defined by the value of the transverse step over distance s od . This value is calculated using the maximum permissible value of the scallop height at the worst of the trajectory points. In other words, at this point, the scallop height between two adjacent planes is equal to the maximum allowable value, while at all other points of the trajectory the scallop height are less than the maximum permissible value.

In a previous study [START_REF] Redonnet | Study of the effective cutter radius for end milling of free-form surfaces using a torus milling cutter[END_REF] , a novel approach to calculate analytically the effective radius of a torus milling cutter moving in pure translation is presented. This approach is based on the two following lemmas : Lemma 1. Let P be the mathematical operation for projection along the feed direction F t in a plane normal to F t . Let T p (v), be the curve resulting from the projection along P of the cutter envelope. Let E(t) be the ellipse resulting from the projection along P of the cutter centretorus circle, and oE(t) an offset exterior to that ellipse with a value equal to the radius of the cutter torus. Then the two curves T p (v) and oE(t) are coincident.

Lemma 2. The radius of curvature of a plane offset curve is equal to the radius of curvature of the original curve augmented by the offset value.

Once demonstrated these lemmas, the effective radius of the toroidal cutter can be calculated considering the projection of the torus center circle in a plane normal to the machining direction and the r-offset of the resulting ellipse. The validity of this approach is fully demonstrated in [START_REF] Redonnet | Study of the effective cutter radius for end milling of free-form surfaces using a torus milling cutter[END_REF] . It is also stated that this demonstration is only valid for pure translation movement of the cutter.

Simple analytical formula (2) resulting of this study saves considerable time in the calculation of the effective radius of the tool and thus the scallop height resulting from two side by side tool paths. Indeed, the only known alternative to accurately assess the scallop height is through numerical calculation of the intersection between the two envelope volumes of tool paths. The calculation time is considerably shortened using a simple analytical formula allowing to consider applications banned until now due to their cost in terms of computation time.

The parallel planes machining strategy (figure 3) is used to define each tool path as the intersection of the tool-center surface and a vertical machining plane oriented along an overall machining direction that is called F. For torus end mill, the tool-center surface is defined by:

S o (u, v) = S(u, v) + r n(u, v) + (R -r) n p (u, v) with n p (u, v) = Z × (n(u, v) × Z) Z × (n(u, v) × Z)
where S(u, v) is the surface to be machined and n(u, v), the unit normal to the surface in (u, v).

For simplicity, we consider the vector F in the (X, Y ) plane of machine axes, i.e. the direction of F corresponds to the projection of F z , feed direction vector in the plane (X, Y ):

The analytical expression of the effective radius of a torus-end mill moving in translation is [START_REF] Redonnet | Study of the effective cutter radius for end milling of free-form surfaces using a torus milling cutter[END_REF] :

R ef f = (R -r) cos(α) 2 sin(S) 1 -sin(α) 2 sin(S) 2 + r ( 2
)
where S is the slope of the workpiece surface at the tool/workpiece contact point and α the angle between the direction of steepest slope projected in the plane (X, Y ) and the vector F. It can be noted that if R = r, equation (2) corresponds to the well-known expression of effective radius for ball-end mill cutter. Using this formula, the calculation of step over distance s od at a point can be done instantly compared to the numerical methods previously used that required an important computation time.

Optimization methodology and implementation

Optimisation algorithm

As posed in (1), the problem that is proposed to be solved is a nonlinear constrained optimisation problem. The main difficulty is the evaluation of the objective function as it requires the full simulation of a machining planning for the entire surface. Considering i the point index of a given path, and j the index of the path, are defined:

• P(i, j) the i th point of the path j • p(j) the position of the plane containing the path j in the direction perpendicular to the machining planes. • s od (i) the step over distance value calculated for a point i of a given path so that the scallop height criterion is respected.

• s odp (j) the value of the step over distance used to calculate the position of the next plan, i.e. p(j) = p(j -1) + s odp (j). For a given path, s odp (j) should be defined as s odp (j) = Min s od (i) to be sure to respect the scallop height criterion. • length(j) the length of the path j

The evaluation of the objective function then proceeds as described in algorithm (1). This algorithm is also presented as a flow chart in figure 5.

This evaluation process of the objective function effectively enforced all conditions sh i sh max ∀i whatever the value of the variable optimisation θ should be. It allows integrating the optimisation problem constraints (1) into the objective function.

To carry out the optimisation procedure itself, it is then possible to use an unconstrained optimisation algorithm. We chose the Nelder-Mead algorithm [START_REF] Nelder | A simplex method for function minimization[END_REF] because it is known for its robustness and efficiency, due at least in part, to the fact that it does not require calculation of derivatives. Its main drawback, commonly accepted, is that it progresses relatively slowly compared to algorithms including the calculation of derivatives. It may be noted that in the context of our problem, the evaluation of derivatives require the full calculation of a machining planning, which significantly lengthen the overall time resolution.

Furthermore, the optimisation procedure described in algorithm 1 also provides a complete and optimized planning for finishing of the surface, which is an advantage over a procedure that would only provide the optimal machining direction.

In terms of computation time, the whole process optimisation / planning requires a few seconds to tens of seconds depending on the chosen parameters. The most influential parameters are:

• The scallop height s h : the smaller it is and the more the machining planes will be close the one to others, resulting in the calculation of a larger number of machining paths. A value commonly used in the industry is 0.01 mm. • The required accuracy on the optimisation variable θ.

For our calculations we used an accuracy of 1 degré. degree. Run the optimisation procedure with superior accuracy generates greater computation time without making any significant gain in productivity.

Implementation

In order to determine the starting point of the optimisation procedure, a rough estimate of the first objective function is performed for a set of values covering the solution domain.

In practice, the objective function is evaluated every 5°, between -90°and +90°, simulating a machining operation with a 0.1 mm scallop height. If it is not exactly realistic for industrial application, this value (0.1 mm) provides very quickly (about ten seconds) an acceptable starting point for the optimisation procedure. From this starting point, a first simplex is automatically generated and optimisation itself begins (see algorithm 1).

The test environment has been developed in Java.

Application to a real case: Machining a boat propeller blade

Introduction

The determination of the optimal machining direction for end-milling of a free-form surface with a torus-end mill using parallel planes machining strategy allows to significantly increase the productivity. Of course, the time savings that are to be expected depend largely on the geometry of the surface. For example, the application of the previously detailed optimisation procedure to a complex surface from an industrial environment is presented. This surface, taken from a boat propeller measuring 293 mm in diameter (Fig. 6), is constituted by the upper surface of a blade (Fig. 7). When it is defined by a ruled surface, this type of part can be machined using side milling process [START_REF] Monies | Improved positioning of a conical mill for machining ruled surfaces: application to turbine blades[END_REF] , but this one is defined by a 8x8 Bézier surface. The best choice to manufacture it is then to mill it using a end mill strategy.

Simulations and real part machining were carried out using a torus-end mill of major radius R = 5 mm and torus radius r = 2 mm.

The manufacture of the workpiece from the trajectories calculated by the optimisation procedure has been implemented with a zigzag trajectory planning. Before the finishing operation, a roughing and a contouring phase have been completed (Fig. 8).

The finishing operation was conducted using a spindle rotation of 7950 rpm and a feed-rate of 1600 mm/min.

Results and discussion

Machining time

Using a 0.01 mm scallop height parameter, the total duration of the optimisation process is 116 seconds for the tool (R = 5 mm; r = 2 mm). The obtained optimal value of the angle is θ = -27.53°. The path length of the machining is 12315 mm.

The machining planning obtained by the optimisation procedure is shown in figure 9.

Considering the scallop height value (s h = 0.01 mm) for the finishing operation, the trajectories are so close to each other that representing all of them in this figure would make it unintelligible. To maintain readability of this representation, we have chosen to represent only one path over five. Data: A surface S(u, v), a torical tool (R, r), a feed direction θ Result: l, the total tool path length for a machining simulation calculation of the position of extreme planes pInf and pSup / * pInf < p(j) < pSup ∀j * / j = 0 calculation of the first path with p(0) = pInf + δ / * typically δ = 0.01 * / l = length(j) j = 1 while p(j) < pSup do s odp (j) = R / * initialization of s odp (j) of the j path to its max value * / foreach point i of the tool path j -1 do calculation of the maximum allowable step over distance s od (i) for the current point i if s od (i) < s odp (j) then s odp (j) = s od (i) end end calculation of all points of the tool path j using s (j) l = l + length(j) j = j + 1 p(j) = p(j -1) + s odp (j) end Algorithm 1: Evaluation of the objective function Using the parameters described above (section Application to a real case page 5), the total duration of the machining cycle was 8 min and 16 s. The such machined workpiece is shown in figure 10.

The results presented here can give an idea of the potential gains that allows the optimisation of the machining direction during milling of the free-form surfaces using parallel planes strategy with a torus end-mill. The diversity of complex surfaces coming from CAD is such that no analysis of the performance of a machining algorithm can be generalized. So the potential contribution To evaluate the potential gain, the results of the machining planning for the entire domain of definition of the parameter θ are compared. These results are shown in figure 11.

By various tests, it has been found that for this surface, the total duration of the procedure is about 2 minutes.

To evaluate the potential gain that prior calculation of the optimal machining direction can offer, the most favorable case and the worst case are compared (Table 1): This comparison highlights several interesting points:

• for this workpiece, the potential gain can rise up to 1 -12315 17301 = 0.288 = 28.8% • for this workpiece, the total duration of the procedure including optimisation (calculus plus machining) is less than the total length of the simple machining procedure in the worst case. In other words, the optimisation of the machining direction saves time since the machining of the very first part. Of course our procedure saves even more time than the series is important. Indeed, the procedure for determining tool paths is performed only once while the machining time saved occurs on every workpiece. For example, on the workpiece studied here for the realization of a series of 50 pieces, the total time savings could This example allows highlighting the interest to optimize the machining direction before the completion of part machining itself. In addition, the comparison is limited to measurements of time. In terms of manufacturing costs, the hourly computing rate is far less than the hourly machining rate; the interest of the presented procedure is then even more evident.

It should however be noted that the numerical values in the presented results include only one example. Since each piece of CAD is special case, no overall performance value can be advanced. The example that is discussed here, however, allows highlighting the potential value of the detailed procedure. With this in mind, this surface has several attractive features:

• it is an industrial workpiece • the optimal machining direction is not obvious a priori • it has wide variations in normal direction Prepared using sagej.cls For all these reasons, this area is representative of the forms commonly encountered in the modeling of molds and dies; so it is a good example to illustrate the performance of the presented procedure.

Surface quality

To check the validity of the machined part, we conducted a series of measurements at different points of the part. Special attention have been paid to focus the measurements in the most critical areas. Indeed, as mentioned in section Context page 3, during the machining of a curved surface with parallel planes strategy, all the different points of the path do not generate the same scallop height and it is the most critical point that determines the position of the adjacent plane.

To make these measurements, a 3D optical Alicona Infinite SL was used.

Figure 12 shows one of these measurements results. This figure highlights the scallops that are typical of a free-form surface machining. On this screnshot, the left scallop is fully measured between left and 19.059 mm and 19.069 mm. The maximum allowable scallop height is 0.01 mm, it can be said that tolerance is respected at this point. In the same figure the right scallop is fully measured between 19.056 mm and 19.066 mm. At this measurement point, geometric specification is also respected.

On the whole part, 10 measurements were made and similar results were found for all these measures, i.e. that tolerance is respected at each measured point. We can therefore say that the tool paths calculated by the presented algorithm (see algorithm 1) allows to respect the constraints of our optimization problem (see equation 1), i.e. maximum allowable scallop height. The validity of the presented optimization procedure is therefore confirmed by experimentation.

Conclusion and perspectives

In this article, a procedure to determine the optimal machining direction using parallel planes strategy and torus end-mill is presented. This optimal direction is the one that allows the greatest productivity while respecting the maximum scallop height constraint. This methodology has several advantages: first, it is simple to implement because it is based on a simple optimisation algorithm. Also it provides quick results and full toolpath planning in the context of machining freeform. This simplicity of implementation and speed make this an easy procedure to integrate into a workflow process.

The optimisation procedure we propose has been validated on a test case. This experiment shows the potential gain that can be obtained using it.

For moment our method is only applied for parallel planes strategy. We are currently working on improvments to be able to apply this method to other machining strategies. This will be the purpose of future publications. For this publication, we choose the parallel planes strategy because it is widely used in industry (most people choose it because it is reliable and efficient) and the optimisation problem is quite simple to state (it depends only on one optimisation variable: the feed direction θ).

To improve the procedure that is presented here, it is also planned to integrate the dynamic behavior of the CNC machine in order not to optimize the distance traveled by the tool but the total machining time. In addition, it is also proposed to test and / or develop other optimisation algorithms in order to improve the computation time.

Figure 1 .

 1 Figure 1. Scallop height

Figure 2 .

 2 Figure 2. Relationship between machining direction and step over distance using a ball-end mill (left) and a torus-end mill (right)

Figure 3 .

 3 Figure 3. Machining strategy by parallel planes

Figure 4 .

 4 Figure 4. Step over distance calculation

Figure 5 .

 5 Figure 5. Flowchart representation of the algorithm

Figure 6 .

 6 Figure 6. Boat propeller

Figure 7 .

 7 Figure 7. Upper surface of the blade

Figure 8 .

 8 Figure 8. Contouring and roughing preliminary operations

Figure 9 .

 9 Figure 9. Machining tool path planning along optimal direction (1 path over 5 is drawn)

Figure 10 .

 10 Figure 10. Machined part

Figure 11 .

 11 Figure 11. Evolution of the objective function value on the whole domain

Figure 12 .

 12 Figure 12. Machined part profile measure

  6. Monies F, Mousseigne M, Redonnet JM et al. Determining a collision-free domain for the tool in five-axis machining. International Journal of Production Research 2004; 42(21): 4513-4530. 7. Chen X, Zhao J, Dong Y et al. Research on the machined surface integrity under combination of various inclination angles in multi-axis ball end milling. Proceedings of the

Table 1 .

 1 Comparison between extreme cases for a single part

	Most favorable case Worst case

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