Dr Paul Cressey

Dr Wasim Abuillan

Dr Nada Ibrahim

Jana Alhoussein

Dr Oleg Konovalov

Prof. Dr Gang Zheng

Dr Ali Makky
email: ali.makky@universite-paris-saclay.fr

Self-Organization of Lipid-Porphyrin Conjugates at the Air/Water interface

Keywords: Lipid-porphyrin conjugates, air/water interface, XRR, GISAXS, domains

Lipid-porphyrin conjugates are versatile compounds which can self-assemble into liposome-like structures with multifunctional properties. Most of the conjugates that have been described so far, consisted in grafting pyropheophorbide-a (Pyro-a) or other porphyrin derivatives through the esterification of the hydroxyl group in the sn-2 position of a lysophosphatidylcholine. However, despite the versatility of these conjugates, less is known about the impact of the lipid backbone structure on their 2D phase behavior at the air/water interface and more precisely on their fine structures normal to the interface as well as on their in-plane organization. Herein, we synthesized a new lipid-porphyrin conjugate (PyroLSM) based on the amide coupling of Pyro-a to a lysosphingomyelin backbone (LSM) and we compared its interfacial behavior to that of Pyro-a and Pyro-a conjugated lysophosphatidylcholine (PyroLPC) using Langmuir balance 2 combined to a variety of other physical techniques. Our results provided evidence on the significant impact of the lipid backbone on the lateral packing of the conjugates as well as on the shape and size of the formed domains. Compared to Pyro-a and PyroLPC monolayers, PyroLSM exhibited the highest lateral packing which highlights the role of the lipid backbone in controlling their 2D organization which in turn may impact the photophysical properties of their assemblies.

Introduction

Lipid-porphyrin conjugates have gained recently tremendous attention for the development of multifunctional nanomaterials with light activatable properties. These unique building blocks can self-assemble in aqueous media into liposome-like structures either by their own or when mixed with helper phospholipids such as cholesterol. Compared to their disassembled counterparts, the lipid-porphyrin assemblies exhibit unique photophysical properties and biomedical outcomes. [1] In these assemblies, the porphyrin cores adopt a densely packed arrangement which quenches their fluorescence ability. Thus, upon their illumination at their absorbance wavelengths, the absorbed photonic energy is dissipated thermally through vibrational relaxation. [2] This leads to the generation of heat upon the illumination of the lipidporphyrin assemblies. [2][3][4] Therefore, the nanoassemblies may act as highly efficient cytotoxic photothermal agent and can be used in photothermal therapy (PTT). [4] Interestingly, once the liposomal structures are passively disrupted in the body, both the fluorescence and photosensitizing properties of porphyrin pigments are regenerated thus enabling another phototherapeutic modality called photodynamic therapy (PDT). [2,5] Most of the lipid-porphyrin conjugates that have been described so far for PTT/PDT and photoacoustic imaging applications were usually synthesized through esterification of the sn-2 position of lysophosphatidylcholine (LPC) with either pyropheophorbide-a (Pyro-a), [2] bacteriochlorophyll a [2] or benzoporphyrin derivative. [6,7] PyroLPC also named Pyrolipid [2] which consists in conjugating Pyro-a directly to LPC, is the most successful conjugate reported to date. Indeed, the PyroLPC conjugates were shown to form stable supramolecular assemblies with multifunctional properties. [2] Other conjugates consisting of either pheophorbide-a [8] (Ph x LPC) or pyropheophorbide-a [2] (Pyr x LPC) coupled to the hydroxyl group in the sn-2 position of LPC via different linkers exhibiting different lengths have also been described in our previous works. [9][10][11][12] Compared to pheophorbide-a conjugated phospholipids (Ph x LPC) which formed monolayers in the liquid expanded state, the Pyr x LPC compounds formed films in the liquid-condensed state with subsequent appearance of well structured domains at the air/water interface. [11] This highlighted the importance of - interactions between the Pyro-a chromophores in dictating the organization of the conjugates at the air/water interface [11] and thus their self-assembling properties when suspended in water. [12] Besides LPC backbone, lysosphingomyelin (LSM) has also been employed. LSM based conjugates exhibited promising properties in favoring the assemblies of conjugated porphyrins into liposome-like structures [9,10,13] with multifunctional properties.

Nevertheless, despite the versatility of these lipid-porphyrin conjugates, less is known about the impact of lipid backbone on their 2D phase behavior at the air/water interface and more precisely on their fine structures normal to the interface as well as on their in-plane organization. Herein, we synthesized a new lipid-porphyrin conjugate based on the amide coupling of Pyro-a to a LSM backbone, [9,10,13] referred in this work as the PyroLSM conjugate. The 2D phase behavior of Pyro-a, PyroLSM and PyroLPC was assessed at the air/water interface using a Langmuir balance.

The morphology of the monolayers was thoroughly studied using Brewster angle microscopy (BAM) and atomic force microscopy (AFM) on Langmuir-Blodgett transferred films. In addition, the fine structures of the monolayers were studied at the air/water interface by X-ray reflectometry (XRR) and Grazing-Incidence Small-Angle X-ray Scattering (GISAXS).

Experimental Section

Materials

Pyropheophorbide-a (Pyro-a, ≥ 95%, Mw = 534.66 g/mol) was purchased from Livchem Logistics GmbH (Frankfurt, Germany). Egg sphingomyelin (Egg SM, 99%, Mw=710.965 g.mol - 1 ) was purchased from Avanti Polar Lipids (Alabaster, AL). N-(3-Dimethylaminopropyl)-N′ethyl carbodiimide hydrochloride (EDC.HCl, 99%, Mw= 191.70 gmol -1 ) 1-hydroxybenzotriazole hydrate (HOBt, 97%, Mw= 135.12 gmol -1 ), triethylamine (TEA, 99%, Mw= 101.19 g.mol -1 ), HEPES (99.5%, Mw= 238.31 gmol -1 ), sodium chloride (NaCl, 99%, Mw= 58.44 g.mol -1 ), methanolic hydrogen chloride (0.5M) were provided by Sigma (St. Louis,MO, USA). The ultrapure water (γ = 72.2 mN/m at 22° C) used in all experiments was produced by a Millipore Milli-Q® Direct 8 water purification system, with a resistivity of 18.2 MΩ.cm. Solvents were purchased from CARLO ERBA Reagents (Val de Reuil, France). All compounds and solvents were used without further purification. The synthesis of lysosphingomyelin (LSM) was carried out according to the previously reported procedure. [9] PyroLPC (also named Pyrolipid [2] ) was supplied by Gang Zheng's lab.

Synthesis of PyroLSM

The lysosphingomylein (LSM) was prepared by acidic hydrolysis of egg sphingomyelin (Nhexadecanoyl-d-erythro-sphingosylphosphorylcholine) in anhydrous methanolic hydrogen chloride at 50 °C (Scheme S1) following the procedure described previously. [9] Pyro-a (48 mg, 0.09 mmol), EDC.HCl (24 mg, 0.12 mmol) and HOBt (17 mg, 0.12 mmol) were mixed in 2 mL of anhydrous choloform and stirred for 1 hour at room temperature under Argon, in the dark. LSM (40 mg, 0.08 mmol) was dissolved in 2 ml of anhydrous chloroform with 0.2 mL of TEA, and then added to the pyro-a/ EDC.HCl mixture, and stirred in the dark, at room temperature, under Argon for 24 hours. Chloroform was then removed under vacuum. The crude was resuspended in minimum amount of chloroform and purified by chromatography on silica gel (eluted with chloroform-methanol-ammonia 70:30:4, volume ratio; Rf=0.3). PyroLSM was obtained (21 mg, dark green powder, yield 26%). 1

Surface Pressure Measurements

Pressure-area (π-A) isotherms of the pure components were recorded using a thermostated KSV-Nima Langmuir film balance (Biolin Scientific, Finland), composed of a Teflon trough (775.75 cm 2 ) equipped with two 145 mm long Delrin barriers. Fresh solutions of Pyro a and its derivatives at a concentration of 1 mg/ml in chloroform/methanol (9:1) were prepared. Then, 50 µL of Pyro a, 100 µL of PyroLSM or 80 µL of PyroLPC solutions were spread (~510 16 to 610 16 molecules) onto aqueous HEPES buffer solution (10 mM HEPES, 150 mM NaCl, pH = 7.4). After deposition, the solvents were left to evaporate for 15 min before compression of the monolayer at a rate of 10 Å 2 /molecule/min. All experiments were performed at 22±1 °C and the results reported are mean values of at least three measurements. From the surface pressure-area data, the surface compressional moduli (K or Cs -1 ) of monolayers were calculated, using Eq. 1 with A the molecular area and π, the surface pressure:

 (Eq. 1)

Brewster Angle Microscopy (BAM)

The morphology of the lipid-porphyrin monolayers at the air/buffer interface was monitored using a Brewster angle microscope (Micro-BAM 3, NimaTechnology Ltd, Coventry, UK) mounted on a Langmuir trough. The microscope was equipped with a single frequency laser diode ( = 659 nm, 30 mW optical power) generating a collimated beam of approximately 6 mm diameter, with a p-polarizer, analyzer, and a USB camera. The refracted beam was absorbed by a black glass plate placed in the subphase. The field of view of BAM was 3.6 × 4.1 mm 2 . The image size was rescaled and the zone of interest was cropped using imageJ software to get a final image size of 2.0 × 2.0 mm 2 with a resolution of 5.6 µm/pixel.

Langmuir-Blodgett Transfer and Atomic Force Microscopy (AFM) Imaging

The Langmuir-Blodgett (LB) film transfer of the lipid-porphyrin monolayers was performed on freshly cleaved mica surfaces (1.5 × 1.5 cm 2 ) using the Langmuir trough equipped with a Teflon well. The freshly cleaved mica substrate was immersed into the buffer subphase, and then the corresponding lipid-porphyrin conjugate solution was spread (total number of spread molecules ~ 5×10 16 ) onto the subphase. After solvent evaporation, the monolayer was compressed at a constant rate (10 Å 2 /molecule/min) and the pressure-area (π-A) isotherm was simultaneously recorded. When the surface pressure of 30 mN/m was reached, it was maintained constant and the mica substrate (1.5×1.5 cm 2 ) was lifted from the subphase at a speed of 1 mm/min. The transfer ratio [14] which is defined as the ratio between the decrease in the monolayer area during the deposition stroke and the surface area of the substrate was close to 1 (0.93 for Pyro-a; 0.92 for PyroLPC and 0.94 for PyroLSM). This indicates that the films are almost perfectly transferred onto the mica substrates. Afterwards, atomic force microscopy (AFM) imaging of the transferred lipid-porphyrin films was performed in ambient conditions at room temperature using a JPK Nanowizard Ultraspeed atomic force microscope from JPK Instruments (Berlin, Germany) in amplitude modulation AFM (AM-AFM) with force settings comprised between 60-65% of the free amplitude A ~12 nm. Gold-coated silicon ACTG cantilevers (nominal spring constant ~ 37 nN/nm, tip radius < 10 nm) from AppNano (Mountain View, CA) were used. For each transferred film, different regions of the mica substrate were systematically imaged at high resolution (5× 5 µm 2 , 10 × 10 µm 2 and 15 × 15 µm 2 at 1024 × 1024 pixels) with the AFM. AFM images were then processed using the JPK-SPM Data Processing software (version 6.1.131). All AFM images were flattened by subtracting a polynomial fit from each scan line at a 2 nd regression order.

Synchrotron X-ray Experiments

X-ray experiments were carried out at the beamline ID10 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). The samples were irradiated with a monochromatic synchrotron beam with an energy of 8 keV (λ = 1.55 Å). The experiments were performed on monolayers of lipid-porphyrin conjugates spread on the surface of HEPES buffer (HEPES 10 mM, NaCl 150 mM, pH 7.4) and compressed to a surface pressure of 30 mN/m at a temperature of 293 K. The film balance was kept in a helium atmosphere to minimize air scattering and maintain consistent environment for all measurements. Specular X-ray Reflectivity (XRR): XRR was measured with a MAXIPIX 2D detector (Multichip Area X-ray detector based on a photon-counting Pixel array). [15] The error bars in the XRR curves correspond to the error resulting from the counting statistics and were calculated as described previously. [16] After background subtraction, the specular reflectivity was analyzed using a genetic minimization algorithm implemented in the MOTOFIT software package [17] by choosing a two-slab model. [11] Grazing Incidence Small Angle Scattering (GISAXS): The out-off specular plane scattered intensity were collected with a MAXIPIX 2D detector. The intensity from direct beam (0.1×2 mm 2 ) was blocked using a beam stop. The beam impinges on the sample at angle of incidence of α c = 0.125° which close to the critical angle of total external reflection (α c = 0.11°). Under these conditions, the beam footprint is estimated to be 92 mm 2 . From the experimental geometry considerations, the GISAXS signal is translated into reciprocal space map [18] (supporting information, Figure S1).

Results and Discussion

Interfacial Behavior of Pyropheophorbide a and its Conjugates

The two-dimensional phase behavior of the Pyro-a, PyroLPC and PyroLSM compounds at the air/buffer interface was studied by surface pressure measurements using Langmuir balance. The π-A isotherms obtained for Pyro-a and the two lipid-porphyrin conjugates (PyroLPC and PyroLSM) are shown in Figure 2. From these isotherms we estimated the molecular area at the surface pressure onset (A 0 ), the molecular area at surface pressure of 30 mN/m (A 30 ), the limit area (A limit ) which is obtained from the zero intersection by extrapolating the linear steep increase of the surface pressure [19,20] and the compressional modulus (K or Cs -1 ). Their characteristic values are summarized in Table 1. Pyro-a monolayer displays similar isotherm characteristics as previously reported [11] with A limit = 56 Å 2 and A 30 = 47 Å 2 . These small molecular areas correspond to the formation of closely packed films of Pyro-a molecules that exhibit side-on orientation while exposing the carboxyl group to the buffer subphase.

Table 1:

The mean values (n=3) of the limit molecular area (A limit ), molecular area at surface pressure of 30 mN/m (A 30 ) and the maximal compressional modulus (K max ) for Pyro-a, PyroLPC, and PyroLSM monolayers. The standard errors of the molecular areas are for three trials.

Monolayer composition

A 0 (Å 2 ) A 30 ( Compared to the Pyro-a, PyroLPC and PyroLSM conjugates showed significantly more expanded monolayers with A limit values of 84 Å 2 and 70 Å 2 , respectively. In addition, while the PyroLPC displayed -A isotherm features similar to that of Pyro-a, PyroLSM exhibited a sharper phase transition between the gaseous and the liquid-condensed phase. These observations were further supported by the calculation of the compressional modulus (Eq. 1), where PyroLSM exhibited the highest maximal value (K max = 425 mN/m) followed by that of PyroLPC (K max = 247 mN/m) and Pyro-a (K max = 166 mN/m), respectively. Such compressional modulus values indicate that the three compounds form monolayers in the liquid condensed state. [START_REF] Davies | Interfacial Phenomena[END_REF] Whereas the molecular area expansion of the conjugates compared to Pyro-a can be explained by the presence of the phosphatidylcholine headgroup in the vicinity of the porphyrin core, the difference in their compressional modulus implies different lateral packing between the molecules which is favored by the presence of LysoSM backbone for PyroLSM. Indeed, it has been demonstrated that sphingomyelins exhibit tighter interfacial packing compared to the chain-matched phosphatidylcholines. [START_REF] Steinbauer | [END_REF]23] This was related to the presence of the interfacial hydroxyl and amide residues that promote the formation of hydrogen bonds with the surrounding molecules as previously reported by Steinbauer et al. [START_REF] Steinbauer | [END_REF] and Li et al. [23] . The stability of the monolayers as well as the reversibility of their two-dimensional surface phase transitions were assessed by subjecting the monolayers to three repetitive compression-expansion cycles (Figures 2C-E). The monolayers were first compressed to a surface pressure of 30 mN/m where a liquid-condensed phase is obtained and then expansed to zero surface pressure. Following the first expansion step, Pyro-a exhibited more significant hysteresis compared to PyroLSM and PyroLPC which indicates the higher lateral interaction between the Pyro-a molecules. Interestingly, the isotherms in the subsequent compression-expansion cycles were almost superimposed with a slight reduction in the limit molecular area (A limit < 5Å 2 ), thus evidencing the reversibility and the stability of the three monolayers. The morphology of the monolayers of Pyro-a, PyroLPC and PyroLSM was assessed using Brewster angle microscopy and the images were recorded at increasing surface pressures (Figure 3). At the beginning of the monolayer compression ( = 0.1 mN/m), Pyro-a film appeared smooth with the presence of bright and dark regions which may be explained by the different molecular orientation relative to the incident p-polarized laser. As the surface pressure increases, the formation of bright island-like structures was observed and they grow gradually with the surface pressure up to 30 mN/m. The PyroLSM monolayer showed similar island-like structures but with bigger size that were present even at very low surface pressures (< 1 mN/m). Conversely, PyroLPC monolayer exhibited smooth film during the whole compression which becomes brighter with the increase in surface pressure.

Å 2 A limit (Å 2 ) K max (mN/m) Pyropheophorbide-a 69±4 48±1 57±2 190±26 PyroLPC 103±3 75±2 87±2 225±21 PyroLSM 80±3 63±2 70±2 394±31

Brewster Angle Microscopy Analysis

AFM Imaging of Langmuir-Blodgett Transferred Films

In order to get higher resolution of the monolayers morphology, the films made of Pyro-a and the conjugates were transferred at a surface pressure of 30 mN/m (liquid-condensed phase) onto mica substrates by the Langmuir-Blodgett technique and they were imaged afterwards by AFM at room temperature in air conditions (Figure 4). The topographic AFM images of Pyro-a film

Monolayers

The fine structures perpendicular to the plane of Pyro-A, PyroLSM and PyroLPC monolayers compressed to surface pressure of 30 mN/m were studied using specular X-ray reflectivity (XRR). Figures 4A shows the XRR curves of Pyro-a, PyroLSM and PyroLPC monolayers, fitted using a two-slabs model. [9,11] The corresponding electron density profiles ( ) reconstructed from 2. For the Pyro-a monolayer, the obtained total thickness is 16.4 Å, which is in agreement with the thickness of pheophorbide-a monolayer (thickness ~15.7 Å), [9] a porphyrin derivative with a similar structure to that of Pyro-a.

In addition, this thickness value is consistent with the AFM height values and corresponds to the formation of Pyro-a film where the porphyrin molecules are stacked to each other with an upright orientation with the carboxyl group directed toward the water subphase. PyroLSM monolayer exhibits a total thickness of 19.3 Å. Such thickness is in agreement with that of similar compound named PhLSM [9,10] which consists in grafting Pheo-a chromophore to LSM. The reconstructed electron density profiles along the Z-axis.

Compared to PyroLSM, the PyroLPC monolayer has a total thickness of 18.5 Å. This ~ 1 Å difference in the total thickness between the two Lipid-Por conjugates is mainly related to the structural difference between the lipid backbones. PyroLPC consists of a C16 Lyso-PC which is esterified in the sn2 position with the carboxylic group of the Pyro-a. However, PyroLSM has a LSM backbone which is consisting of a sphingosine chain of 18 carbons long. In addition, the interfacial hydroxyl group and amide residue of the sphingosine backbone could promote the formation of hydrogen bonds between the PyroLSM molecules thus leading to tighter packing with subsequent increase in the thickness. Similar behavior has been previously observed by Niemelä et al. [24] when comparing DPPC bilayer to that made of palmitoylsphingomyelin (PSM).

By the mean of molecular dynamics simulations, the authors demonstrated that PSM molecules exhibit strong hydrogen bonding properties that led to specific ordering effects in the vicinity of the polar headgroup with subsequent increase in the total thickness. [24] Besides, the electron density of the hydrophobic regions of the PyroLSM monolayer ( HC = 0.194 e-× Å -3 ) is lower than that of the PyroLPC ( HC = 0.224 e-× Å -3 ). Both lipid conjugates showed high electron densities in the polar region which could be related to the presence of the porphyrin core taking an upright orientation in the vicinity of the polar headgroup similar to other lipid-porphyrin conjugates. [11] However, it should be noticed that the electron density of the hydrophilic region of PyroLSM ( polar = 0.481 e-× Å -3 ) is higher than that of PyroLPC ( polar = 0.467 e-× Å -3 ).

Such behavior could be explained by the structural differences in the polar headgroup causing a difference in the relative position of the porphyrin core at the air/buffer interface. To get further insight on the in plane lateral correlation between the domains imaged by AFM, grazing incidence small angle X-ray scattering (GISAXS) measurements (Figure S1) on the three monolayers were performed at a surface pressure of 30 mN/m. Whereas for PyroLPC and PyroLSM monolayers GISAXS signals could not be detected, a significant signal could be observed for Pyro-a film. This is due to the electron density contrast between the molecules and their surrounding. For instance, the electron density of Pyro-a obtained from XRR for the hydrophobic core is approximately two times higher the correspondings values of PyroLPC and PyroLSM monolayers (Table 2). Taking the average electron density for the whole monolayer then only Pyro-a (ρ avg = 0.428 e -/Å 3 ) has a good contrast with respect to water while it is not the case for PyroLPC (ρ avg = 0.349 e -/Å 3 ) and PyroLSM (ρ avg = 0.328 e -/Å 3 ).Though the PyroLSM showed a weak GISAXS signal (Fig S2). Indeed, in the case of Pyro-a, molecules can interact with each other via face to face stacking but also via edge to edge interactions on both sides of each molecule. However in the case of the conjugates, due to the presence of a lipid backbone the interactions between the porphyrin cores will be limited which leads to the formation of smaller size domains. The GISAXS reciprocal space map is shown in Figure 6A. The intensity is integrated between the two dashed lines along q xy and presented in Figure 6B. The intensity profile exhibits a peak as indicated by the arrow. In order to accurately determine the peak position, a linear offset is subtracted from the intensity profile and then it is fitted with Gaussian (inset, Figure 6B). The peak position is found to be located at q xy = 0.0091 Å -1 corresponding to inter-domain spacing of 69.1 nm. The correlation length can be estimated from the peak width using Scherrer formula as (Eq. 2) where Δq xy is the full-width at half maximum. The correlation length is estimated to be L = 111 nm which means that the interaction between the domains reach only up to the second neighbors. On the other hand, PyroLSM monolayer exhibited a weak GISAXS peak at q xy = 0.0213 Å -1 corresponding to inter-domain spacing of 29.5 nm which agrees very well with AFM results where the domains of PyroLSM is smaller to that of Pyro-a.

Conclusion

In this study, we demonstrated that Pyro-a and its lipids conjugates can form stable monolayers at the air/buffer interface. PyroLSM compounds formed a more condensed monolayer with lateral packing compared to the PyroLPC. This shed light on the impact of the lipid backbone on their organization. Indeed, from the X-Ray reflectivity data, we could determine that the Pyro-a molecule take an upright orientation in the vicinity of the polar headgroup for both PyroLPC and PyroLSM. The compression of Pyro-a and its conjugates led to the formation of 2D domains at the air/water interface which size and shapes depended significantly on the chemical structure of the conjugate. The AFM topographic images revealed that Pyro-a molecules do not form homogenous film as the conjugates do. While Pyro-a monolayer compressed at 30 mN/m exhibited separated leaf-like structures, PyroLSM and PyroLPC ones displayed scales-like and flakes-like structures respectively. In addition, PyroLSM domains were larger than those of PyroLPC which could be attributed to an increase in the line tension for PyroLSM due to the higher van der Waals interactions offered by the LSM compared to LPC backbone. GISAXS signals were only detected for Pyro-a monolayer which could be due to the big size of domains.

The GISAXS signals collected for Pyro-a at the air/buffer interface enabled the calculation of the inter-domain correlation length which was ~ 111 nm consistent with the AFM data. Taken together, our results demonstrated the significant impact of the lipid backbone on the lateral packing of the conjugates as well as on the shape and size of the formed domains. This could have a great impact on the photophysical properties of the conjugates when assembled into supramolecular structures. Indeed, both PyroLPC and PyroLSM conjugates can form liposomelike structures however due the significant difference in the lateral packing of the two conjugates, they may interact and orient differently in the lipid bilayer matrixes which in turn impacts their photophysical properties but also their stability in biological media.

Twitter handles:

@Alimakky_LPCS, @umr8612

Table of contents (ToC) entry:

The impact of the lipid backbone structure on the interfacial behavior of lipid-porphyrin conjugates (PyroLSM and PyroLPC) monolayers has been investigated. By combining Langmuir balance to a variety of other physical techniques, we could demonstrate that the lipid backbone play an important role in controlling their lateral packing but also the shape and the size of formed domains.

Figure 1 :

 1 Figure 1: Chemical structures of pyropheophorbide-a and its lipid conjugates.

Figure 2 :

 2 Figure 2: -A isotherms of (A) and the corresponding compressibility modulus (B) for Pyro-a,

Figure 3 :

 3 Figure 3: Representative BAM images for Pyro-a, PyroLSM and PyroLPC monolayers spread on

(

  Figure 4A) showed separate crystalline structures exhibiting leaf-like structures with an average height of ~ 1.4 nm (Figure 4 A, lower panel). Conversely, both PyroLSM and PyroLPC conjugates showed continuous monolayer with the formation of structured domains exhibiting scales-like and flakes-like structures respectively with an average height of 0.15 -0.25 nm (Figure 4B, 4C).

Figure 4 :

 4 Figure 4:AM-AFM topography images in air of (A) Pyro-a, (B) PyroLSM and (C) PyroLPC

  the best fit results (solid black lines in Figures 4A) along the surface normal are shown in Figures 4B. The thickness (d), electron density ( ) and root mean square roughness ( ) of each interface deduced from the best matching fits are summarized in Table

Figure 5 :

 5 Figure 5: (A) XRR curves of Pyro-a, PyroLSM and PyroLPC monolayers on HEPES buffer at a

Figure 6 .

 6 Figure 6. (a) GISAXS reciprocal space map of Pyro-a monolayer recorded at a surface pressure

Table 2 :

 2 Best fit parameters for the XRR Results for Pyro-a, PyroLSM and PyroLPC monolayers at 30 mN/m as presented in Figure4.

		d (Å)	ρ (e -× Å -3 )	σ (Å)
		Pyro-a
	Hydrophobic core	9.8±0.3	0.455±0.005	4.1±0.1
	Choline group	6.6±0.4	0.389±0.007	3.4±0.2
	Buffer		0.335	5.6±0.2
		PyroLSM
	Hydrophobic core	10.3±0.2	0.194±0.009	4.5±0.2
	Hydrophilic groups	9.0±0.3	0.481±0.008	4.2±0.2
	Buffer		0.335	3.6±0.1
		PyroLPC
	Hydrophobic core	9.0±0.2	0.224±0.012	4.9±0.2
	Hydrophilic groups	9.5±0.3	0.467±0.010	4.2±0.3
	Buffer		0.335	3.5±0.3

Grazing Incidence Small Angle X-ray Scattering (GISAXS) Measurements

Acknowledgements

LGB is thankful to the French Ministry of Research for the financial support of his PhD thesis.

The financial supports for Lipid-Porphyrin conjugates research from the ANR JCJC Grant (ANR-19-CE09-0015 LISiNA) and from the Laboratory of Excellence LERMIT via an ANR grant (ANR-10-LABX-33) under the program of ''Investissements d'avenir'' are gratefully acknowledged. This project has also received financial support from the CNRS through the MITI interdisciplinary programs. We thank the ESRF for the synchrotron beam time. We are also thankful to the CNRS to fund the exchange program between the CNRS and the university of Toronto under the 80 PRIME scheme.


Louis-Gabriel Bronstein

Paul Cressey

Wasim Abuillan

Oleg Konovalov

Maciej Jankowski

Véronique Rosilio

Ali Makky
email: ali.makky@universite-paris-saclay.fr

Influence of the porphyrin structure and linker length on the interfacial behavior of phospholipid-porphyrin conjugates

Keywords: Phospholipid-porphyrin conjugate, monolayer, X-ray reflectometry, Atomic force microscopy, Langmuir-Blodgett film

Hypothesis: Phospholipid-porphyrin (Pl-Por) conjugates consist of porphyrin derivatives grafted to a lysophosphatidylcholine backbone. Owing to their structural similarities with phospholipids, Pl-Por conjugates can self-assemble into liposome-like assemblies. However, there is a significant lack of information concerning the impact of the porphyrin type and the length of the alkyl chain bearing the porphyrin on the interfacial behavior of the Pl-Por conjugates. We hypothesized that changing the chain length and the porphyrin type could impact their twodimensional phase behavior and modulate the alignment between the two chains.

Experiments: 6 Pl-Por conjugates with different alkyl chain lengths in the sn2 position of C16 lysophosphatidylcholine and coupled to either pheophorbide-a or pyropheophorbide-a were synthesized. Their interfacial behavior at the air/water interface was assessed using Langmuir balance combined to a variety of other physical techniques including Brewster angle microscopy, atomic force microscopy and X-ray reflectometry.

Findings: Our results showed that all 6 Pl-Por form stable monolayers with the porphyrin moiety at the air/water interface. We also showed that changing the porphyrin moiety controlled the packing of the monolayer and thus the formation of organized domains. The chain length dictated the structure of the formed domains with no evidence of the alignment between the two chains.

Introduction

Porphyrins and their derivatives are the most abundant pigments in nature [START_REF] Milgrom | The Colours of Life: An Introduction to the Chemistry of Porphyrins and Related Compounds[END_REF][START_REF] Cai | Chlorophylls derivatives: Photophysical properties, assemblies, nanostructures and biomedical applications[END_REF]. These ubiquitous macrocycles play a vital role in many biological functions such as photosynthesis, oxygen transport, oxygen storage [START_REF] Krishnamurthy | The role of transporters in cellular heme and porphyrin homeostasis[END_REF] and cell respiration [START_REF] Milgrom | The Colours of Life: An Introduction to the Chemistry of Porphyrins and Related Compounds[END_REF]. In addition, many porphyrin derivatives have been used as photosensitizers in photodynamic therapy which is considered an efficient and minimally invasive treatment modality for both cancer and local bacterial infections [START_REF] Cai | Chlorophylls derivatives: Photophysical properties, assemblies, nanostructures and biomedical applications[END_REF][START_REF] Jiang | A Perspective on the Trends and Challenges Facing Porphyrin-Based Anti-Microbial Materials[END_REF]. Moreover, grafting porphyrin derivatives to a lipid backbone such as lysophosphatidylcholine (LPC) led to phospholipid-porphyrin (Pl-Por) conjugates [START_REF] Massiot | Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro[END_REF][START_REF] Massiot | Photo-triggerable liposomal drug delivery systems: from simple porphyrin insertion in the lipid bilayer towards supramolecular assemblies of lipid-porphyrin conjugates[END_REF] that were shown to self-assemble into liposome-like nanostructures named "porphysomes" [START_REF] Lovell | Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents[END_REF] thanks to their phospholipid structure and the intermolecular interactions between the grafted porphyrins.

These assemblies possess unique optical and photophysical properties [START_REF] Lovell | Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents[END_REF][START_REF] Rajora | Advancing porphyrin's biomedical utility via supramolecular chemistry[END_REF] compared to their monomeric counterparts or to the unconjugated porphyrins. Indeed, depending on the porphyrin derivative, porphysomes show an intensive red-shift of the Q y -absorption band due to the formation of well-organized porphyrins aggregates called J-aggregates [START_REF] Würthner | J-Aggregates: From Serendipitous Discovery to Supramolecular Engineering of Functional Dye Materials[END_REF][START_REF] Charron | Photophysics of J-Aggregating Porphyrin-Lipid Photosensitizers in Liposomes: Impact of Lipid Saturation[END_REF][START_REF] Shakiba | Stable J-aggregation enabled dual photoacoustic and fluorescence nanoparticles for intraoperative cancer imaging[END_REF]. The dense packing of porphyrin molecules within these nanostructures provokes an intense fluorescence quenching of the porphyrin chromophores. Upon their illumination, the absorbed photonic energy usually released as fluorescence and singlet oxygen is dissipated thermally through vibrational relaxation [START_REF] Lovell | Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents[END_REF]. Therefore, the nanoassemblies may act as highly efficient cytotoxic photothermal agents and can be used in photothermal therapy. Interestingly, once the porphysomes have been passively disrupted in the body, both the fluorescence and photosensitizing properties of the porphyrin pigments are regenerated thus enabling their photodynamic properties. Consequently, Pl-Por conjugates are nowadays considered as versatile building blocks for the conception of multifunctional nanomaterials for several biomedical applications [START_REF] Lovell | Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents[END_REF][START_REF] Cui | A PEGylation-Free Biomimetic Porphyrin Nanoplatform for Personalized Cancer Theranostics[END_REF][START_REF] Rajora | Tailored theranostic apolipoprotein E3 porphyrin-lipid nanoparticles target glioblastoma[END_REF][START_REF] Paproski | Porphyrin Nanodroplets: Sub-micrometer Ultrasound and Photoacoustic Contrast Imaging Agents[END_REF][START_REF] Rizvi | Photodynamic therapy: Promoting in vitro efficacy of photodynamic therapy by liposomal formulations of a photosensitizing agent[END_REF][START_REF] Obaid | Nanolipid Formulations of Benzoporphyrin Derivative: Exploring the Dependence of Nanoconstruct Photophysics and Photochemistry on Their Therapeutic Index in Ovarian Cancer Cells[END_REF] including photothermal therapy, photodynamic therapy, photo-triggered drug release, fluorescence and photoacoustic imaging [START_REF] Lovell | Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents[END_REF]. The Pl-Por conjugates that have been described so far for biomedical applications were usually synthesized through esterification of the sn-2 position of lysophosphatidylcholine with either pyropheophorbide-a (Pyro-a) [START_REF] Lovell | Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents[END_REF] or bacteriochlorophyll a [START_REF] Lovell | Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents[END_REF]. Other conjugates with benzoporphyrin derivative [START_REF] Rizvi | Photodynamic therapy: Promoting in vitro efficacy of photodynamic therapy by liposomal formulations of a photosensitizing agent[END_REF][START_REF] Obaid | Nanolipid Formulations of Benzoporphyrin Derivative: Exploring the Dependence of Nanoconstruct Photophysics and Photochemistry on Their Therapeutic Index in Ovarian Cancer Cells[END_REF] have also been reported for photodynamic therapy applications. However, despite the versatility of these conjugates, there is a significant lack of information concerning their interfacial behavior. Studying the interfacial behavior of these compounds would offer a better understanding on how these compounds interact with each other and what conformation they adopt at the interface which in turn may give answers about the impact of the chemical structure on their assembling properties. In previous work we synthesized two lipid-porphyrin conjugates that were obtained by coupling pheophorbide-a (Pheo-a) [START_REF] Massiot | Impact of lipid composition and photosensitizer hydrophobicity on the efficiency of light-triggered liposomal release[END_REF] to either lyso-sphingomyelin (PhLSM) or a modified lysophosphatidylcholine with a C6 alkyl chain length linker in sn2 position (Ph 6 LPC, n=5) [START_REF] Massiot | Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro[END_REF][START_REF] Massiot | Photo-triggerable liposomal drug delivery systems: from simple porphyrin insertion in the lipid bilayer towards supramolecular assemblies of lipid-porphyrin conjugates[END_REF][START_REF] Massiot | Photo-triggerable liposomes based on lipidporphyrin conjugate and cholesterol combination: Formulation and mechanistic study on monolayers and bilayers[END_REF].

Their interfacial behavior at the air/water interface and self-assembling efficiency in aqueous medium were characterized [START_REF] Massiot | Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro[END_REF][START_REF] Massiot | Photo-triggerable liposomal drug delivery systems: from simple porphyrin insertion in the lipid bilayer towards supramolecular assemblies of lipid-porphyrin conjugates[END_REF][START_REF] Massiot | Photo-triggerable liposomes based on lipidporphyrin conjugate and cholesterol combination: Formulation and mechanistic study on monolayers and bilayers[END_REF]. Our results demonstrated that both conjugates were able to self-assemble into liposome-like structures in buffer, but these assemblies were unstable with time. By combining Langmuir balance with X-ray reflectometry, we demonstrated that Ph 6 LPC formed a thicker monolayer with a higher electron density in the alkyl chains region than that of PhLSM. This was explained by a conformational difference between the two compounds. For Ph 6 LPC, the Pheo-a moiety aligns with the sn2 C16 alkyl chain whereas for PhLSM it remains at the interface near the polar head group. The instability of these nanoassemblies was interpreted as the result of a mismatch between the length of the alkyl chain in sn-1 position and the adjacent porphyrin, leading to an inadequate packing parameter for bilayer stability [START_REF] Massiot | Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro[END_REF][START_REF] Massiot | Photo-triggerable liposomal drug delivery systems: from simple porphyrin insertion in the lipid bilayer towards supramolecular assemblies of lipid-porphyrin conjugates[END_REF]. However, since the lipid backbones in PhLSM and Ph 6 LPC conjugates were structurally very different, a clear conclusion about the effect of the chemical structure on their assembling and interfacial properties could not be drawn. Here, we specifically focus on addressing the impact of i) the length of the chain separating the porphyrin from the polar headgroup and ii) the type of porphyrin on the interfacial behavior of Pl-Por conjugates. In this aim, we synthesized two new series of Pl-Por conjugates exhibiting different alkyl chain lengths in sn2 position and linked to either pheophorbide-a [START_REF] Massiot | Impact of lipid composition and photosensitizer hydrophobicity on the efficiency of light-triggered liposomal release[END_REF] (Ph x LPC) or pyropheophorbide-a [START_REF] Lovell | Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents[END_REF] (Pyr x LPC) via amide coupling (Figure 1). Our motivation for changing the length of the linker in sn2 position is to reduce the chain length mismatch between the sn1 and sn2 alkyl chains. Moreover, changing the porphyrin structure could play an important role in the interaction between the conjugates at the interface and influence molecules packing. Herein, the synthesized conjugates were characterized as monolayers at the air/water interface using a Langmuir balance. The morphology of the monolayers was thoroughly studied using Brewster angle microscopy (BAM) and atomic force microscopy (AFM) on Langmuir-Blodgett transferred films. In addition, the fine structure normal to the interface was studied by X-ray reflectometry (XRR).

Material and methods

Chemicals

Pheophorbide-a (Pheo-a, ≥ 95%, mixture of diastereomers, Mw = 592.69 g/mol) and pyropheophorbide-a (Pyro-a, ≥ 95%, Mw = 534.66 g/mol) were purchased from Livchem Logistics GmbH (Frankfurt, Germany).

Synthesis of lipid-porphyrin conjugates

Ph x LPC and Pyr x LPC were synthesized as described previously [START_REF] Massiot | Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro[END_REF] with some modifications as shown in the Figure S1. The modified phospholipids were synthesized by conjugating the corresponding tert-butyloxycarbonyl (BOC)-protected linker to the secondary alcohol in sn2 position of the 1-lysophosphatidylcholine backbone via direct acylation of the carboxylate group.

This acylation was carried out following a modified procedure outlined by Rosetto et al. [START_REF] Rosseto | Synthesis of oligo(ethylene glycol) substituted phosphatidylcholines: secretory PLA2-targeted precursors of NSAID prodrugs[END_REF][START_REF] Rosseto | Synthesis of phospholipids on a glyceric acid scaffold: design and preparation of phospholipase A2 specific substrates[END_REF][START_REF] Rosseto | Peptidophospholipids: synthesis, phospholipase A2 catalyzed hydrolysis, and application to development of phospholipid prodrugs[END_REF].

In brief, the reaction mixture was sonicated at 25°C in the presence of glass beads, under basic conditions using 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide . HCl (EDC.HCl) to activate the carboxylic acid. The BOC protected amine was deprotected under acidic conditions using HCl:dioxane. Following the deprotection, the attachment of pheophorbide-a or pyropheophorbide-a was carried out following regular amide coupling reaction conditions using EDC.HCl/ 1-hydroxybenzotriazole (HOBt) as the coupling agents.

Surface pressure measurements

Surface pressure-surface area isotherms of the pure components were recorded using a thermostated KSV-Nima Langmuir film balance (Biolin Scientific, Finland), composed of a Teflon trough (775.75 cm 2 ) equipped with two 145 mm long Delrin barriers. The free porphyrins and their conjugates in chloroform/methanol (9:1) solutions were spread (~510 16 to 610 16 molecules) onto aqueous HEPES buffer solution (10 mM HEPES, 150 mM NaCl, pH = 7.4).

After deposition, the solvents were left to evaporate for 15 min before compression of the monolayer at a rate of 10 Å 2 /molecule/min. All experiments were performed at 22±1 °C and the results reported are mean values of at least three measurements. From the surface pressure-area data, the surface compressional moduli (K) of monolayers were calculated, using Eq. 1 with A the molecular area and π, the surface pressure:

(Eq. 1)

Brewster angle microscopy (BAM)

The morphology of the lipid-porphyrin monolayers at the air/water interface was monitored using a Brewster angle microscope (Micro-BAM 3, NimaTechnology Ltd, Coventry, UK) mounted on a Langmuir trough. The microscope was equipped with a frequency laser diode ( = 659 nm, 30 mW optical power) generating a collimated beam of approximately 6 mm diameter, with a ppolarizer, analyzer, and a USB camera. The refracted beam was absorbed by a black glass plate placed in the subphase. The field of view of BAM was 3.6 × 4.1 mm 2 . The image size was rescaled and the zone of interest was cropped using imageJ software to get a final image size of 2.0 × 2.0 mm 2 with a resolution of 5.6 µm/pixel.

Langmuir-Blodgett transfer and atomic force microscopy (AFM) imaging

The Langmuir-Blodgett (LB) film transfer of the lipid-porphyrin monolayers was performed on freshly cleaved mica surfaces (1.5 × 1.5 cm 2 ) using the Langmuir trough equipped with a Teflon well. The freshly cleaved mica substrate was immersed into the buffer subphase, and then the corresponding lipid-porphyrin conjugate solution was spread (total number of spread molecules ~ 5×10 16 ) onto the subphase. After solvent evaporation, the monolayer was compressed at a constant rate (10 Å 2 /molecule/min) and the pressure-area (π-A) isotherm was simultaneously recorded. When the desired surface pressure was reached, it was maintained constant and the mica substrate (1.5×1.5 cm 2 ) was lifted from the subphase at a speed of 1 mm/min. Afterwards, atomic force microscopy (AFM) imaging of the transferred lipid-porphyrin films was performed in ambient conditions at room temperature using a JPK Nanowizard Ultraspeed atomic force microscope from JPK Instruments (Berlin, Germany) in amplitude modulation AFM (AM-AFM)

with force settings comprised between 55-65% of the free amplitude A ~12 nm). Gold-coated silicon SCOUT-350 cantilevers (nominal spring constant ~ 42 nN/nm, resonant frequency ~ 350 KHz, conical shape tip radius <10 nm) from NuNano (Bristol, UK) were used. For each transferred film, different regions of the mica substrate were systematically imaged at high resolution (5× 5 µm 2 , 10 x 10 µm 2 and 15 × 15 µm 2 at 1024 x 1024 pixels) with the AFM. AFM images were processed using the JPK-SPM Data Processing software (version 6.1.131). All AFM images were flattened by subtracting a polynomial fit from each scan line at a 2 nd regression order. For the orientation determination of the imaged objects, the processed AFM images were saved as grayscale images. The grayscale AFM images were then analyzed using OrientationJ [START_REF] Rezakhaniha | Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy[END_REF] which is an imageJ plug-in. We choose the "OrientationJ Analysis" option and the "Cubic Spline" function [START_REF] Rezakhaniha | Experimental investigation of collagen waviness and orientation in the arterial adventitia using confocal laser scanning microscopy[END_REF] to compute the gradient. The structure tensor was set to 3 pixels which corresponds to the width of the smallest fibril.

X-ray reflectivity experiments at the air/buffer interface

XRR experiments were carried out at the beamline ID10 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France). The samples were irradiated with a monochromatic synchrotron beam with an energy of 8 keV (λ = 1.55 Å). The XRR experiments were performed on monolayers of lipid-porphyrin conjugates spread on the surface of HEPES buffer (HEPES 10 mM, NaCl 150 mM, pH 7.4) and compressed to a surface pressure of 30 mN/m. The film balance was kept in a helium atmosphere to minimize air scattering and maintain a consistent environment for each measurement. XRR was measured with a MAXIPIX 2D detector (Multichip Area X-ray detector based on a photon-counting Pixel array) [START_REF] Ponchut | MAXIPIX, a fast readout photon-counting X-ray area detector for synchrotron applications[END_REF]. The error bars in the XRR curves correspond to the error resulting from the counting statistics and were calculated as described previously [START_REF] Abuillan | Physical interactions of fish protamine and antisepsis peptide drugs with bacterial membranes revealed by combination of specular x-ray reflectivity and grazing-incidence x-ray fluorescence[END_REF]. After background subtraction, the specular reflectivity was analyzed using a genetic minimization algorithm implemented in the MOTOFIT software package [START_REF] Nelson | Co-refinement of multiple-contrast neutron/X-ray reflectivity data using MOTOFIT[END_REF] by choosing a two-slab model (see supporting information). To assess the impact of the porphyrin type and the spacer length on the interfacial behavior of the two series of lipid porphyrin-conjugates, we compared the molecular organization of the conjugates and unmodified porphyrins spread at the air/buffer interface, using the Langmuir film balance. The π-A isotherms obtained for the two porphyrins (Pheo-a and Pyro-a) and the two synthesized lipid-porphyrin conjugates series (Ph x LPC and Pyr x LPC) are shown in Figure 2. To compare the features of these isotherms, we estimated the molecular area at the surface pressure onset also named lift-off area (A 0 ) and the limit area (A limit ) which is obtained from the zero intersection by extrapolating the linear steep increase of the surface pressure [START_REF] Chifu | Xanthophyll films: I. Single-component monolayers at the air/water interface[END_REF][START_REF] Jurak | Analysis of Molecular Interactions between Components in Phospholipid-Immunosuppressant-Antioxidant Mixed Langmuir Films[END_REF]. The compressibility modulus (K) was also determined. These parameters are summarized in Table 1.

Results

Surface pressure measurements

Pheo-a exhibited similar isotherm characteristics as reported previously [START_REF] Massiot | Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro[END_REF] with A 0 ~ 80 Å 2 and A limit ~ 60 Å 2 . This corresponds to a side-on orientation of the Pheo-a molecules exposing their carboxylic and ester groups to the buffer subphase with subsequent formation of closely packed films of Pheo-a molecules [START_REF] Massiot | Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro[END_REF].

Comparing the Ph x LPC isotherms with that of the parent Pheo-a molecule, it can be readily seen that all Ph x LPC compounds show similar isotherm shapes but form more expanded monolayers (Table 1). Moreover, the isotherms exhibit a monotonic shift toward larger molecular areas as the length of the spacer separating the polar headgroup from Pheo-a increases. Ph (LC) states coexist during the first-order phase transition between the two monolayer states [START_REF] Albrecht | Polymorphism of phospholipid monolayers[END_REF][START_REF] Tsukanova | Monolayer Behavior of NBD-Labeled Phospholipids at the Air/Water Interface[END_REF]. Interestingly, the plateau-like pressure of Pyr 3 LPC and Pyr 4 LPC increases with subsequent reduction of the plateau region when the temperature of the buffer subphase is augmented from 17°C to 29°C (Figure S2). This confirms that the plateau-like region originates from the coexistence of the LE and LC states. Surprisingly, the isotherm of the Pyr 2 LPC with the shorter spacer length does not display any plateau-region. Moreover, while the grafting of Pyro-a to the sn2 chains of the modified lipids induces larger A onset , A limit does not seem to be significantly affected by the spacer length. Thus, Pyr x LPC may adopt similar molecular packing at high surface pressures independently of the linker length. Unlike monolayers of Ph x LPC compounds showing lower compression modulus than those of pure Pheo-a, Pyr x LPC ones display similar maximal compression modulus (K max ~200 mN/m) as Pyro-a. These monolayers are in the liquid condensed state [START_REF] Davies | Interfacial Phenomena[END_REF]. This indicates that the grafting of Pyro-a on the sn2 chains via different spacer length does not affect the lateral packing of the Pyro-a molecules at the air/water interface and thus the monolayer rigidity.

Table 1: Molecular area at surface pressure onset (A 0 ), molecular area at surface pressure of 30 mN/m (A 30 ), limit molecular area (A limit ), and maximal compressional modulus (K max ) for Pheoa, Ph x LPC, Pyro-a and Pyr x LPC monolayers. The reported values are the average ± standard deviation of n = 3 independent experiments.

Monolayer composition

A 0 (Å 2 ) A 30 (Å 2 ) at 30 mN/m

A limit (Å 2 ) K max (mN/m) Pheophorbide-a 79±4 51±3 60±4 220±34 Ph 2 LPC 95±2 57±2 74±2 145±13 Ph 3 LPC 104±2 63±2 77±2 148±7 Ph 4 LPC 120±1 66±1 81±2 158±12 Pyropheophorbide-a 76±2 50±1 58±1 190±13 Pyr 2 LPC 80±3 61±1 71±1 208±6 Pyr 3 LPC 93±1 61±1 70±2 222±39 Pyr 4 LPC 108±2 64±1 74±2 250±36

Brewster angle microscopy analysis

To further investigate the different states of lipid-conjugates monolayers, Brewster angle microscopy (BAM) images were recorded at increasing surface pressures (Figure 3). Monolayers It should be noticed that some of these rod-like structures appear dark. This implies that these structures are 3D in shape and possess an inner anisotropy. In other words, the observation of dark or bright structures arises from the relative orientation of the rod-like structures at the interface relative to the incident p-polarized laser. At a surface pressure of 10 mN/m, the rods get close to each other and collapse at higher surface pressures. The 3 LPC monolayer shows the coexistence of both smooth condensed monolayers and 3D rod-like structures even at a surface pressure of 30 mN/m. These observations suggest that Pyr 3 LPC do not form homogenous monolayer but rather 3D rod like structures mixed with a 2D condensed monolayer.

AFM assessment of the conjugates' domains morphology

BAM enables the detection of microdomains that grow upon compression at the air/water interface, however due to its resolution limit, it does not allow the visualization of the nanodomains or the structure of the nuclei that may form during monolayer compression. Thus, the monolayers of the various lipid-porphyrin conjugates studied were transferred at a surface pressure of 30 mN/m onto mica substrates by the Langmuir-Blodgett technique and then, they were imaged by AFM in air conditions (Figure 4). While the topographic AFM images of Ph x LPC films (Figure 4, upper panel) show smooth homogenous monolayers confirming the absence of structured domains, Pyr x LPC films exhibit domains with different shape depending on the spacer length (Figure 4, lower panel). Pyr 2 LPC compound forms a stratified monolayer with the presence of condensed domains that are consistent with BAM images (Figure 3). In order to get a better understanding on the formation of these elongated domains for Pyr 3 LPC and Pyr 4 LPC, Langmuir-Blodgett films were transferred onto mica substrates at surface pressures near the plateau-like region ( = 2 mN/m, Figure 5) and above the transition ( = 15 mN/m, Figure 6). The transferred Pyr 3 LPC film at 2 mN/m shows the appearance of nucleated molecules into well-defined structures (Figure 5A) with both straight fibrils-like and tape-like structures, with average heights of ~ 1.5 nm and ~ 2.0 nm, respectively. It is worth noting that these fibrils and tape-like structures have a specific orientation. While the fibrils are oriented toward the same direction, tape-like ones are aligned at an angle of 30 °C (Figures 5, B andC). In the case of Pyr 4 LPC film, twisted fibril-like structures of ~ 1.5 nm in height are revealed with the presence of accumulated branched structures (Figures 5D-F). (Figure 6A). Surprisingly, some regions also show 3D rod-like structures of ~ 25 nm high (Figure 6B) indicating the presence of piling up of these elongated structures. The compression of Pyr 4 LPC monolayer up to 15 mN/m results in the formation of similar elongated structures with ~1.5 nm height and several micrometers in length (Figure 6C). In addition, Pyr 4 LPC shows similar structure shape with an average height of ~10 nm in some regions (Figure 6D). However, they are narrower than those observed in the Pyr 3 LPC films and show a shallow groove throughout the rod. Thus, both Pyr 3 LPC and Pyr 4 LPC compounds can self-assemble into rod-like structures at the air/water interface with clear evidence that the linker length has an impact on the morphology of the rods.

Fine structures of PL-Por conjugate monolayers

The fine structures perpendicular to the plane of Ph x LPC and Pyr x LPC monolayers were studied using specular X-ray reflectivity (XRR). A monolayer of dipalmitoyl phosphatidylcholine (DPPC), a phospholipid with similar polar headgroup (i.e., choline headgroup) and alkyl chain length (16 carbons), was compressed to 30 mN/m and characterized by XRR for the sake of comparison. Figures 7A and7B, show the XRR curves of DPPC, Ph x LPC and Pyr x LPC monolayers, fitted using a two-slab model. The corresponding electron density profiles ( ) reconstructed from the best fit results (solid black lines in Figures 7A-B) along the z-axis are also shown in Figures 7C-D. The thickness (d), electron density ( ) and root mean square roughness ( ) of each interface deduced from the best matching fits are summarized in Table 2. For the DPPC monolayer, the calculated total thickness is 24.2 Å, which agrees with literature values for DPPC in the liquid condensed phase. Ph x LPC monolayers have a total thickness of 16 to 17 Å depending on the spacer length. This 30 % difference in the total thickness of the Ph x LPC monolayer compared to DPPC is most probably related to the difference in the packing density of the two compounds at 30 mN/m. Indeed, at this surface pressure, Ph x LPC monolayers display larger molecular areas (57-66 Å 2 ) than DPPC (A 30 = 48 Å 2 ). Thus, Ph x LPC molecules are less ordered than DPPC ones where the two alkyl chains are aligned in the liquid condensed state.

These thickness values are in good agreement with those estimated by AFM. The electron density of the hydrophobic regions of the Ph x LPC monolayers ( HC = 0.202-0.235 e -.Å -3 ) remains almost unchanged compared to DPPC ( HC = 0.216 e -.Å -3 ) even though Ph x LPC compounds possess only one alkyl chain per molecule and form more expanded monolayers than DPPC. Conversely, the electron densities of the hydrophilic region ( polar = 0.475-0.506 e -.Å -3 ) are significantly higher than that of the DPPC choline group ( choline = 0.418 e -.Å -3 ). Given that the total thickness of a Pheo-a monolayer is ~16 Å at 30 mN/m where the molecules take an upright orientation with respect to the interface, it is plausible that Pheo-a molecules remain at the air/water interface when conjugated to the modified phospholipids. This induces an increase in the electron density in both polar and hydrocarbon regions. Unlike the previously synthesized Ph 6 LPC compound (n = 5) [START_REF] Massiot | Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro[END_REF], the spacer lengths for Ph x LPC compounds (n=1, 2 or 3) are not long enough to allow the porphyrin core to align with the sn-1 C16 carbon chain. This result is in accordance with the isotherm's shape that remains expanded even at high surface pressures. A similar trend is observed for Pyr x LPC monolayers, indicating that the latter molecules adopt similar conformations as those of Ph x LPC. Again, the average thickness of Pyr x LPC monolayers is consistent with AFM data, without indication of the presence of regions with 10-25 nm thicknesses. This is because XRR measures an ensemble average [START_REF] Daillant | X-ray and Neutron Reflectivity: Principles and Applications[END_REF]

Discussion

The surface pressure measurements show that all the studied Pl-Por conjugates can form stable films, but they exhibit different -A isotherm shapes and compressibility moduli depending on the linker length and the porphyrin chemical structure. All conjugates form more expanded monolayers than the respective free porphyrins. In addition, the monolayer expansion increases monotonically with the linker length. The lower values of compressional modulus of Ph x LPC compared to Pyr x LPC and free porphyrins demonstrate that Ph x LPC molecules exhibit a relatively low degree of condensation. The Langmuir isotherms experiments complemented with Brewster angle microscopy reveal that only Pyr x LPC compounds can form domains even at low surface pressures. These results unravel the impact of the porphyrin structure on the packing and order of the conjugates in the monolayers. Furthermore, obvious morphological differences between Ph x LPC and Pyr x LPC monolayers but also between the individual Pyr x LPC compounds were observed in the transferred Langmuir-Blodgett films. Therefore, according to these results, we may assume that the type of attached porphyrin controls the compressibility modulus but also whether domains are formed or not. It should also be noticed that the A limit occupied by Ph x LPC or Pyr x LPC conjugates (70-80 Å 2 ) is larger than that of monounsaturated phospholipid such as POPC or SOPC (~65 Å 2 ) [START_REF] Massiot | Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro[END_REF][START_REF] Korytowski | Impact of Lipid Oxidization on Vertical Structures and Electrostatics of Phospholipid Monolayers Revealed by Combination of Specular X-ray Reflectivity and Grazing-Incidence X-ray Fluorescence[END_REF][START_REF] Wnętrzak | Interactions of Alkylphosphocholines with Model Membranes-The Langmuir Monolayer Study[END_REF]. This larger molecular area can be related either to the presence of the bulky porphyrin moieties aligned with C16 alkyl chain or lying normal to the interface in the vicinity of the phosphatidylcholine headgroup. Indeed, it was demonstrated by Tsukanova et al. [START_REF] Tsukanova | Monolayer Behavior of NBD-Labeled Phospholipids at the Air/Water Interface[END_REF] that the monolayer of a chromophorelabeled phospholipid (1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}sn-glycero-3-phosphocholine) named NBD(C 12 )-PC (Figure 8) exhibits LE state with an A limit ~ 80 Å 2 similar to A limit for Ph x LPC and Pyr x LPC. In addition, their surface potential measurements and spectroscopic data revealed that the NBD group of the NBD-labeled phospholipid embeds into the phospholipid headgroup region. This was attributed to the intermolecular interaction provided by -cation electrostatic forces between the -electron of the NBD and the positive charge of the choline group [START_REF] Tsukanova | Monolayer Behavior of NBD-Labeled Phospholipids at the Air/Water Interface[END_REF]. Porphyrins are -electron rich moieties; thus, they may interact with the choline group via -cation as NBD-labeled phospholipids do. To verify such hypothesis, the fine structures perpendicular to the plane of Pl-Por monolayers were analyzed using XRR.

The XRR results demonstrated that the porphyrin moieties lie at the air/water interface in both Ph x LPC and Pyr x LPC monolayers. Thus -cation interactions could take place for the Pl-Por conjugates. However, if it is the case then why do Pyr x LPC compounds form domains and Ph x LPC exhibit only less ordered monolayers? One plausible explanation can be drawn from the difference in structure between the Pheo-a and Pyro-a chromophores. In particular the presence of the methyl ester group on the Pheo-a moiety results in two isomers which hinder the lateral packing of molecules and thus, the formation of the condensed monolayers observed for Pyr x LPC. This is also supported by the slight increase in the A limit of Ph x LPC compared to Pyr x LPC (Table 1). Besides, while the porphyrin type allows predicting the formation of domains, the linker length controls their structure and shape. Indeed, the Pyr 2 LPC monolayer exhibits a stratified structure with condensed domains, whereas Pyr 3 LPC and Pyr 4 LPC display predominantly elongated 2D structures where some of them can be piled up into 3D rod-like structures. Although we could not figure out how these compounds are organized within the domains, it can be speculated that changing the number of carbon atoms in the linker gives more flexibility to the attached porphyrin core to adopt different orientations during compression, but it also increases the Van der Waals interactions between the tail groups, promoting the formation of different domain structures.

Conclusions

The aim of this work was to assess the impact of the porphyrin structure and chain length separating the polar headgroup from the porphyrin moiety on the interfacial behavior of newly synthesized Pl-Por conjugates (Ph x LPC and Pyr x LPC). It was expected that chromophores grafted to a longer chain should align with the sn1 C16 alkyl chain improving the lateral packing properties due to the reduction of the length mismatch between the two chains. Indeed, Carter et al. [START_REF] Carter | Porphyrinphospholipid liposomes permeabilized by near-infrared light[END_REF] used molecular dynamics simulations to demonstrate that conjugating a pyro-a derivative (devinyl hexyloxyethy-pyropheophorbide, HPPH) which possesses an hexyl ether moiety at its extremity, enabled the formation of stable bilayers with superior packing properties when compared to the pyro-a conjugate. Such high packing properties of HPPH-conjugates were related to the hexyl ether moiety which provided a better space filling between the two bilayer leaflets [START_REF] Carter | Porphyrinphospholipid liposomes permeabilized by near-infrared light[END_REF]. To verify if a similar behavior can be observed by modulating the chain length between the polar headgroup and the porphyrin moiety, six new Pl-Por conjugates exhibiting different chain lengths in the sn2 position and coupled to 2 different porphyrins, were synthesized and their two-dimensional phase behavior at the air/water interface was studied. Our results revealed that owing to the structural similarities between Pl-Por compounds and conventional phospholipids, Pl-Por conjugates were able to form stable monolayers that were more expanded than those of free porphyrins, due to the presence of the lipid backbone at the interface. Using a variety of physical techniques combined to the Langmuir balance, we demonstrated for the first time that the structure of the porphyrin significantly impacts the lateral packing of the conjugates and controls whether they can form domains or not. Such domains could significantly impact the optical properties and the structure of the supramolecular assemblies made of either pure Pl-Por conjugates or their mixture with conventional phospholipids. Indeed, the presence of such Pl-Por domains in lipid bilayer may favor the formation of J-aggregates that are driven by edge to edge hydrophobic interactions between porphyrin moieties [START_REF] Charron | Photophysics of J-Aggregating Porphyrin-Lipid Photosensitizers in Liposomes: Impact of Lipid Saturation[END_REF][START_REF] Shakiba | Stable J-aggregation enabled dual photoacoustic and fluorescence nanoparticles for intraoperative cancer imaging[END_REF]. Interestingly, our results revealed for the first time that changing the number of carbon atoms in the linker separating the polar headgroup from the porphyrin core, controls the shape and structure of the organized domains of the conjugates. Although the molecular ordering of these domains could not be determined in this work, other X-ray techniques such as Grazing-incidence small-angle scattering (GISAXS) and Grazing Incidence X-ray Diffraction (GIXD) could be used to get better insight into their fine structure. Whereas GISAXS experiments would enable us to determine the size, the shape parameters as well as the electron density of the crystal domains, the GIXD technique would provide the molecular in-plane organization and the lateral ordering within the domains. Contrary

Figure 1 :

 1 Figure 1: Structure of lipid-porphyrin conjugates Ph x LPC and Pyr x LPC bearing pheophorbide-a and pyropheophorbide-a chromophores, respectively.

Figure 2 :

 2 Figure 2: -A isotherms (A, B) and the corresponding compressibility modulus (C, D) for pheophorbide-a and pyropheophorbide-a derivatives spread on HEPES buffer at 22±1°C.

  [START_REF] Krishnamurthy | The role of transporters in cellular heme and porphyrin homeostasis[END_REF] LPC and Ph 4 LPC exhibit an increase in the molecular area at the surface pressure onset (A 0 ) of 9 % and 26 %, respectively compared to Ph 2 LPC which possesses the shortest spacer. Besides, the maximal compression modulus (K max ) values for PhxLPC (K max ~ 150 mN/m) are lower than those for Pheo-a monolayer (K max ~ 220 mN/m). Such behavior could be explained by the presence of the PC polar head group with the acyl chain bearing Pheo-a at the interface which enlarge the molecular area of the Ph x LPC compounds and hinder the close packing of Pheo-a molecules, resulting in a compression modulus reduction. It is worth noting that the differences between the molecular areas of Ph x LPC conjugates remain constant up to the collapse unlike the previously synthesized Ph 6 LPC compounds with longer spacer (n = 5)[START_REF] Massiot | Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro[END_REF]. This suggests that the Pheo-a molecule attached to the lipid backbone remains at the interface even at high surface pressure without re-alignment with the sn-1 C16 alkyl chain or looping of the Pheo-a conjugated acyl chain toward the buffer subphase[START_REF] Massiot | Newly Synthesized Lipid-Porphyrin Conjugates: Evaluation of Their Self-Assembling Properties, Their Miscibility with Phospholipids and Their Photodynamic Activity In Vitro[END_REF]. The shorter spacer length provides less flexibility for the attached porphyrin core to adopt distinct local orientations at different surface pressures.Like Ph x LPC compounds, the surface pressure onset for the Pyr x LPC series are larger than the unconjugated chromophore (Pyro-a). Pyro-a displays a similar isotherm to Pheo-a but with slightly smaller A onset and A limit values, ~ 76 Å 2 and 58 Å 2 , respectively. This slight reduction of 3 Å 2 in the molecular area of Pyro-a compared to Pheo-a is most likely due to the absence of the methyl ester group on the porphyrin ring thus reducing the volume of the porphyrin hydrophilic group. The π-A isotherms of Pyr x LPC compounds show A onset values of ~ 80 Å 2 , 93 Å 2 and 108 Å 2 for Pyr 2 LPC, Pyr 3 LPC and Pyr 4 LPC respectively, all larger than that of the Pyro-a alone. Here again, the enlargement of the A onset is consistent with the increasing length of the linker from Pyr 2 LPC to Pyr 4 LPC, suggesting the effect of presence of the Pyro-a at the interface. However, while the Ph x LPC compounds display a monotonic increase in the surface pressure during monolayer compression with expanded-like profile, a slight plateau-like region appears for Pyr 3 LPC and Pyr 4 LPC isotherms at surface pressures of ~ 2-3 mN/m. Such behavior is usually observed with phospholipids such as DPPC where liquid-expanded (LE) and liquid-condensed

of

  Ph x LPC compounds appear homogenous and smooth, which confirms the LE state deduced from their isotherms and compression modulus values. Conversely, monolayers of Pyr x LPC compounds exhibit bright domain-like structures even before the surface pressure starts to rise (i.e. 0 mN/m) and change morphology upon further monolayer compression. The bright domains observed for Pyr 2 LPC grow gradually with monolayer compression to form homogeneous stratified monolayer at 1 mN/m. The Pyr 4 LPC monolayer shows similar morphology, but with smoother aspect at surface pressures between 1 and 5 mN/m. Interestingly, the Pyr 3 LPC monolayer exhibits different features during molecular compression at the air/buffer interface.Added to the homogeneous bright domains observed for the other two Pyr x LPC compounds, the Pyr 3 LPC monolayer displays supramolecular rod-like structures at a surface pressure of 0 mN/m, and their number increases when the surface pressure increases up to 10 mN/m.

Figure 3 :

 3 Figure 3: Representative BAM images recorded during compression of Ph x LPC and Pyr x LPC monolayers spread on HEPES buffer at 22±1°C. The scale bar corresponds to 500 μm.

  In the case of Pyr 3 LPC and Pyr 4 LPC, elongated structures are observed with lengths of several micrometers and maximal width up to 1 µm. It should be noticed that Pyr 3 LPC forms wider and relatively shorter domains than Pyr 4 LPC.

Figure 4 :

 4 Figure 4: Schematic illustration of the Langmuir-Blodgett technique used to transfer the compounds onto mica substrates (left). AM-AFM topography images of Ph x LPC and Pyr x LPC compounds transferred at a surface pressure of 30 mN/m (right).

Figure 5 :

 5 Figure 5: AM-AFM topography images of (A, B) Pyr 3 LPC and (D, E) Pyr 4 LPC compounds transferred on mica substrate at a surface pressure of 2 mN/m. The results of the features orientation were determined using OrientationJ[22], ImageJ plug-in which allowed getting HSB (Hue-Saturation-Brightness) color-coded maps (C, F) for Pyr 3 LPC and Pyr 4 LPC, respectively. At the surface pressure of 15 mN/m, the transferred Pyr 3 LPC monolayers show very long tape-

Figure 6 :

 6 Figure 6: AM-AFM topography images of Pyr 3 LPC (A, B) and Pyr 4 LPC (C, D) compounds transferred on mica substrate at a surface pressure of 15 mN/m.

Figure 7 :

 7 Figure 7: XRR curves of (A) Ph x LPC and (B) Pyr x LPC monolayers at a surface pressure of 30 mN/m. The solid lines represent the best model fits to the experimental data. Error bars for the reflectivity data represent the error resulting from the counting statistics. (C) and (D) are the reconstructed electron density profiles (e -refers to the number of electrons) along the Z-axis for DPPC, Ph x LPC and Pyr x LPC compounds.

Figure 8 :

 8 Figure 8 : Schematic illustration of possible orientations of Ph x LPC and Pyr x LPC at the air/water

  (see supporting information) over few square millimeters indicating that thicker regions (> 2 nm) are less dominant (illumination area ~ 12-75 mm 2 ). It should be noted that the roughness of the hydrophobic region of Pyr x LPC monolayers is increased by approximately 1 Å when compared to their Ph x LPC counterparts. Such increase in the monolayer roughness is consistent with the formation of 2D and 3D domains in the monolayer as revealed by BAM and AFM experiments.

Table 2 :

 2 Best fit parameters for the XRR Results for DPPC, Ph x LPC and Pyr x LPC monolayers at 30 mN/m as presented in Figure7. The errors are the standard deviations resulting from the Gaussian error propagation during the refinement of the reflectivity curves. (e -refers to the

	number of electrons)
		d (Å)	ρ (e -.Å -3 )	σ (Å)
		DPPC
	Hydrophobic core	13.6±0.2	0.216±0.025	4.4±0.3
	Choline group	10.6±0.4	0.418±0.016	4.4±0.5
	Buffer		0.335	3.3±0.2
		Ph 2 LPC
	Hydrophobic core	8.5±0.2	0.202±0.014	4.8±0.2
	Hydrophilic groups	7.5±0.3	0.506±0.014	4.7±0.4
	Buffer		0.335	4.1±0.4
		Ph 3 LPC
	Hydrophobic core	8.6±0.2	0.207±0.011	4.9±0.1
	Hydrophilic groups	8.3±0.2	0.481±0.006	4.2±0.1
	Buffer		0.335	4.0±0.1
		Ph 4 LPC
	Hydrophobic core	8.6±0.3	0.235±0.018	5.4±0.2
	Hydrophilic groups	8.5±0.5	0.475±0.011	4.0±0.2
	Buffer		0.335	3.9±0.3
		Pyr 2 LPC
	Hydrophobic core	8.2±0.1	0.234±0.014	5.0±0.2
	Hydrophilic groups	9.5±0.3	0.449±0.005	3.9±0.1
	Buffer		0.335	3.4±0.1
		Pyr 3 LPC
	Hydrophobic core	8.3±0.4	0.207±0.019	6.1±0.3
	Hydrophilic groups	7.9±0.7	0.475±0.019	4.7±0.1
	Buffer		0.335	3.7±0.3
		Pyr 4 LPC
	Hydrophobic core	8.3±0.3	0.198±0.015	5.5±0.3
	Hydrophilic groups	8.6±0.3	0.473±0.011	4.5±0.3
	Buffer		0.335	4.0±0.2

Acknowledgements

LGB is thankful to the French Ministry of Research for the financial support of his PhD thesis.

The financial supports for Lipid-Porphyrin conjugates research from the ANR JCJC Grant (Project-ANR-19-CE09-0015), from MITI Auto-organisation 2021 and from the Laboratory of Excellence LERMIT via an ANR grant (ANR-10-LABX-33) under the program of ''Investissements d'avenir'' are gratefully acknowledged. We also acknowledge Dr Nada Ibrahim for the fruitful discussion about the porphyrin conjugation protocol.

Appendix A. Supplementary material

to our expectations, the XRR results did not show evidence of an alignment between the sn1 and sn2 chains independently of the chain length and porphyrin moiety. This shed light on the importance of -interactions between chromophores and -cations between the chromophore and the choline group in controlling the organization of the Pl-Por conjugates at the air/water interface. Our findings pave the way for future research on the impact of the porphyrin moiety and the structure of polar headgroup on the molecular organization of the conjugates. Finally, although the monolayer study did not reveal an alignment between the two chains, this does not exclude its occurrence in the bilayer assembly. Indeed, the bilayer configuration may promote the inter-leaflet -stacking between the porphyrin moieties and thus leading to the alignment between the two chains. Hence, this work will be further complemented by bilayer studies to assess the impact of the chemical changes in the Pl-Por conjugates on the structure of their supramolecular assemblies and their optical properties.

CRediT authorship contribution statement

LGB, PC, OK, MJ, WA and AM performed the experiments. LGB, PC, OK, MJ, WA, VR and AM analyzed the data. AM conceptualized, supervised, funded the project and wrote the original manuscript. All the authors reviewed and edited the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Iona Thomas

Stéphane Ducasse

Pablo Tesone

Guillermo Polito

Technical report: A classification of runtime reflective operations in Pharo

This is a companion technical report of the article: "Pharo: a reflective language -a first systematic analysis of reflective apis" published at IWST 2023.

Reflective operations are powerful APIs that allow one to build new tools and alter the architecture of the language. They are used extensively in the Pharo tool ecosystem and new ones have been added when the need arose for new features. However, as the Pharo reflective API evolved organically with the language, we lacked information on which are the reflective operations inside Pharo. Pharo evolved from Squeak since 2008, and Squeak itself builds on the Smalltalk original API. At this point, the documentation of the reflective API was partial and outdated.

In this report, we propose a categorization and an up-to-date catalog of the available runtime reflective operations in Pharo. For each category, we provide a short description and a list of the corresponding reflective methods.

Overview of the reflective APIs and their categories

This is a companion technical report of the article: "Pharo: a reflective language -a first systematic analysis of reflective apis" published at IWST 2023 [START_REF] Thomas | Pharo: a reflective language -A first systematic analysis of reflective APIs[END_REF]. We extracted the catalog and the categories so that other articles can refer to such a long and dry analysis.

We present a catalog of the reflective features in Pharo. Figure 1 shows the categories we propose to classify these features. The following sections will each present a category briefly and list the corresponding methods. For a more in-depth presentation and analysis of these categories see [START_REF] Thomas | Pharo: a reflective language -A first systematic analysis of reflective APIs[END_REF].

Object inspection

The first family of reflective operations is centered around object inspection. Rivard describes these operations in the Meta-Operations category, but he groups together inspection and modification. Our category is composed of three subcategories: State inspection. These operations allow one to access the number of indexable variables, and the values of instance variables, if they include a specific object • Context»objectSize: iona.thomas@inria.fr (I. Thomas); stephane.ducasse@inria.fr (S. Ducasse); pablo.tesones@inria.fr (P. Tesone); guillermo.polito@inria.fr (G. Polito) • Object»instVarAt:

• Object>instVarNamed:

• ProtoObject»instVarsInclude:

Accessing object identity. These operations allow one to identify one object

• ProtoObject»basicIdentityHash • ProtoObject»identityHash
Accessing object class. This operation allows one to access the class of an object :

• ProtoObject»class • Context»objectClass:

Object modification

The second family of reflective operations is centered around object modification. It is the counterpart of the first one and it is composed of State modification, Manipulating object identity, and Object's class change.

State modification. These operations allow one to set the value of an instance variable.

• Object»instVarAt:put: Write an instance variable using its index • Object»instVarNamed:put: Write an instance variable using its name.

Manipulating object identity.

As a side effect of reference swapping, the reflective operation ProtoObject»becomeForward:copyHash: manipulates the hash of an object.

Object's class change. These operations allow one to change the class of an object to another one:

• Behavior»adoptInstance:

• Metaclass»adoptInstance:from:

• Object»primitiveChangeClassTo:

Class structural inspection

This category groups reflective APIs on the query over the class structure and its constituents: methods, variables (instance/class/slots). It is composed of the following APIs:

• Class/metaclass shift to support the navigation from a class to its metaclass and the inverse,

• Iterating and querying hierarchy to support hierarchy analysis with, in particular, testing for belonging to a specific class or class hierarchy (e.g., isKindOf:),

• Instance variable inspection, Class variable inspection, Shared pool inspection, Slot inspection

all deal about variables. Slot inspection provides a higher level view compared to instance variables. Some slots can come from traits. Therefore there is a difference between localSlots (not coming from traits) and slots (taking into account that they may come from traits), • Selectors and method inspection supports if a class has (abstract) methods, an object responds to a specific selector, and all the classes implementing a set of selectors. • Variable lookup supports the access to the binding of a variable.

• Pragmas supports the query and iteration of class annotations (named pragmas in Pharo).

• Class kind testing supports the testing of the state of a class from a system perspective (obsolete, anonymous...).

Class/metaclass shift. Those operations deal with testing and navigating between the class and instance side of classes.

• ClassDescription»classSide / Class»classSide / Metaclass»classSide • ClassDescription»instanceSide / Class»instanceSide / Metaclass»instanceSide • ClassDescription»isClassSide / CompiledMethod»isClassSide • ClassDescription»isInstanceSide • Metaclass»isMetaclassOfClassOrNil • ClassDescription»hasClassSide / Class»hasClassSide / Metaclass»hasClassSide • Object»isClass / Class»isClass / Trait»isClass / Metaclass»isClass • Behavior»isMeta / ClassDescription»isMeta / Metaclass»isMeta
Iterating and querying hierarchy. These operations allow one to query the hierarchy of a class and iterate over those. They allow one to access superclasses and subclasses, testing for common ancestry in the class hierarchy, and testing that an object is an instance or subinstance of a class. • Class»definesClassVariableNamed:

• Class»readClassVariableNamed:

• ClassDescription»classThatDefinesClassVariable:

• Behavior»whichClassDefinesClassVar:

• Class»usesClassVarNamed:

The methods ClassDescription»classThatDefinesClassVariable: and Behavior»whichClassDefinesClassVar: looks the same and the mix between variable and var is prejudicial.

Shared pool inspection. These operations allow one to query and test on sharedPools and

where there are used. They are important to navigate with the IDE.

• ClassDescription»sharedPools / Class»sharedPools • Behavior»allSharedPools / ctClassDescription»allSharedPools / ctClass»allSharedPools • Class»sharedPoolNames / Metaclass»sharedPoolNames

• ClassDescription»hasSharedPools / Class»hasSharedPools • ClassDescription»sharedPoolOfVarNamed / Class»sharedPoolOfVarNamed • Class»sharedPoolsDo: • Class»classPool / Metaclass»classPool • ClassDescription»usesLocalPoolVarNamed: / Class»usesLocalPoolVarNamed: • ClassDescription»usesPoolVarNamed: / Class»usesPoolVarNamed: • ClassDescription»includesSharedPoolNamed:
Slot inspection. These operations allow one to query and test on slots in a class, or read those in an object. This is a higher-level view compared to instance variables. Some slots can come from traits. Therefore there is a difference between localSlots (without traits) and slots (with).

• Behavior»instanceVariables (returns a collection of slots)

Class structural modification

Figure 1 :

 1 Figure 1: Overview of the reflective APIs.

  This category is the counterpart of the previous one. It is composed of the following APIs whose objectives are clear: Hierarchy modification, Instance variable modification, Shared pool modification, Slot modification, Selector/Method modification, Old class creation, Fluid class creation, and Anonymous class creation. It focuses on the modification of the structural relation a class has with its constituents. Hierarchy modification. These operations allow one to edit the class hierarchy either by adding/removing/changing subclasses, or changing the superclass. • Class»subclasses: • Behavior»superclass: • Behavior»basicSuperclass: • Class»addSubclass: / Metaclass»addSubclass: / UndefinedObject»addSubclass: • Class»removeSubclass: / Metaclass»removeSubclass: / UndefinedObject»removeSubclass: • Behavior»removeAllObsoleteSubclasses • Behavior»addObsoleteSubclass: / Metaclass»addObsoleteSubclass:

  These operations allow one to query and get instance variable names, iterate over those, and get their index and the class defining a specific instance variable.

	• ClassDescription»allInstVarNamesEverywhere
	• ClassDescription»classThatDefinesInstVarNamed:
	• Behavior»whichClassDefinesInstVar:
	• Behavior»instSize
	• Object»basicSize / Context»basicSize • Class»subclasses / MetaClass»subclasses / UndefinedObject»subclasses • Behavior»allSubclasses • Behavior»instVarNames
	• Behavior»withAllSubclasses Class variable inspection. These operations allow one to query and test class variables, • Behavior»obsoleteSubclasses / Metaclass»obsoleteSubclasses get class variable names, get their values, iterate over those, class defining a specific instance • Class»hasSubclasses • Behavior»superclass variable and who is using them.
	• Behavior»allSuperclasses • Class»classVariables / Metaclass»classVariables
	• Behavior»withAllSuperclasses • Behavior»allClassVarNames
	• Behavior»allSuperclassesIncluding: • Behavior»classVarNames / Class»classVarNames / Metaclass»classVarNames
	• Behavior»allSubclassesWithLevelDo:startingLevel: • Class»hasClassVariable:
	• Class»subclassesDo: / Metaclass»subclassesDo: / UndefinedObject»subclassesDo: • Class»hasClassVarNamed: / Metaclass»hasClassVarNamed:
	• Behavior»allSubclassesDo: • Class»classVariableNamed:ifAbsent:
	• Behavior»withAllSubclassesDo: • Class»definesClassVariable:
	• Behavior»allSuperclassesDo: / UndefinedObject»allSuperclassesDo:
	• Behavior»withAllSuperclassesDo:
	• Behavior»withAllSuperAndSubclasses
	• Behavior»withAllSubAndSuperclassesDo:
	• Object»isKindOf:
	• Object»isMemberOf:
	• Behavior»kindOfSubclass
	• Class»commonSuperclassWith: / UndefinedObject»commonSuperclassWith:
	• Behavior»whichSuperclassSatisfies:
	• Behavior»inheritsFrom:
	• Behavior»includesBehavior:
	• Behavior»isRootInEnvironment
	• Behavior»selectSuperclasses:
	• Behavior»selectSubclasses:
	Instance variable inspection. • Behavior»instVarNames / ClassDescription»instVarNames
	• Behavior»allInstVarNames / ClassDescription»allInstVarNames
	• ClassDescription»instanceVariableNamesDo:
	• ClassDescription»hasInstVarNamed:
	• Behavior»definedVariables / Class»definedVariables

  Selectors and method inspection. These operations allow one to test if a class has (abstract) methods, an object responds to a specific selector, and all the classes that implement a set of selectors.Variable lookup. These operations allow one to lookup a variable in the scope of the receiver.• Behavior»classBindingOf: / SharedPool class»classBindingOf:• Object»bindingOf: / Behavior»bindingOf: / Class»bindingOf: / MetaClass»bindingOf:Pragmas. Pragmas are method annotations that got introduced both in VisualWorks and Pharo over the year. These operations (Class»pragmasClass»pragmasDo:)allow one to get all the pragmas of a class and iterate over them. Test various properties of classes: usage, anonymity, obsolescence.• Behvior»isAnonymous / Class»isAnonymous /MetaClass»isAnonymous • Object»isClassOrTrait / Class»isClassOrTrait • Behavior»isUsed / Class»isUsed / Trait»isUsed / Metaclass»isUsed • Behavior»isObsolete / Class»isObsolete / Metaclass»isObsolete

	• Behavior»lookupVar: / ctContext»lookupVar: / ctSystemDictionnary»lookupVar:
	• Behavior»slots / ClassDescription»slots / TraitedMetaclass»slots • Behavior»lookupVar:declare: / ctContext»lookupVar:declare: / ctSystemDiction-
	• ClassDescription»localSlots nary»lookupVar:declare:
	• Behavior»allSlots / ClassDescription»allSlots • Behvior»lookupVarForDeclaration:
	• ClassDescription»slotNames
	• ClassDescription»hasSlotNamed:
	• ClassDescription»slotNamed:
	• ClassDescription»slotNamed:ifFound:
	• ClassDescription»slotNamed:ifFound:ifNone:
	• Object»readSlot: • Object»readSlotNamed: Class kind testing.
	• ClassDescription»definesSlot:
	• ClassDescription»definesSlotNamed:
	• Behavior»hasMethods / Class»hasMethods
	• Behavior»hasAbstractMethods / Class»hasAbstractMethods
	• Object»respondsTo:
	• ClassDescription»classesThatImplementAllOf:
	Getting the selectors and local selectors, iterating over them, and querying them :
	• Behavior»includesSelector:
	• Behavior»includesLocalSelector: / TraitedClass»includesLocalSelector: / TraitedMeta-
	class»includesLocalSelector:
	• Behavior»isDisabledSelector:
	• Behavior»isLocalSelector:	/ TraitedClass»isLocalSelector:	/ TraitedMeta-
	class»isLocalSelector:
	• Behavior»selectors
	• Behavior»selectorsDo:
	• Behavior»selectorsWithArgs:
	• Behavior»whichClassIncludesSelector:
	Getting the methods, iterating over them, and querying them :
	• Behavior»methods
	• Behavior»methodNamed:
	• Behavior»includesMethod:
	• Behavior»methodsDo:
	• Behavior»selectorsAndMethodsDo:

© 2023 Copyright for this paper by its authors. Use permitted under Creative Commons License Attribution 4.0 International (CC BY 4.0).

Instance variable modification. It allows one to add or remove an instance variable (by name).

• ClassDescription»addInstVarNamed:

/ Class»addInstVarNamed: / Metaclass»addInstVarNamed:

• ClassDescription»removeInstVarNamed: Class variable modification. These operations allow one to add or remove a class variable.

• Class»addClassVariable:

• Class»addClassVarNamed:

• Class»removeClassVariable:

• Class»removeClassVarNamed: Shared pool modification. These operations allow one to add, change, or remove shared pools.

• Class»sharedPools: • Class»newAnonymousSubclass / Metaclass»newAnonymousSubclass

Method creation

The API Compiled method creation is a low-level API that supports the definition of compiled methods. Such an API is often ignored but it is central because it is responsible for the creation of new compiled methods that can then be further installed and executed.

Compiled method creation These operations support the creation of compile methods.

• CompiledMethod class»newMethod:header: • CompileMethod»literalAt:put: • CompiledMethod»classBinding:

• CompiledMethod»at:put:

Structural queries on methods

This category supports the cross-referencing between methods, instance variables, and classes. It is composed of two APIs: Method slot uses (which supports the access to a variable and modification of a variable) and Method element references (which is a low-level API querying the internal implementation of methods).

Method slot uses.

Query on which method is using some specific slot and how.

• Behavior»allMethodsAccessingSlot:

Method element references. Query which method/selector is using a given literal.

• Behavior»whichMethodsReferTo:

• Behavior»whichSelectorsReferTo:

Class references. Behavior»usingMethods returns methods is referencing a given class.

Message sending and code execution

This category of operations allows us to explicitly send messages, handle lookup failures or execute compiled methods. It is composed of different APIs: Reflective message send (which supports the lookup and execution of a method), Arbitrary method/primitive execution (which supports the execution of methods without lookup), Method lookup (which simulate the method lookup), Control message passing (which supports the possibility to control message sent), and Message send reification (which provides ways to access message information).

Reflective message send

• Object»perform:

• Object»perform:orSendTo: • Object»perform:with: • Object»perform:with:with: • Object»perform:with:with:with: • Object»perform:with:with:with:with: • Object»perform:withArguments: • Object»perform:withArguments:inSuperclass: • Object»perform:withEnoughArguments: Arbitrary method/primitive execution. While the message perform: supports message sending, the following messages bypass the method lookup and execute a given compiler method.

• ProtoObject»withArgs:executeMethod: • ProtoObject»tryPrimitive:withArgs: • CompiledMethod»valueWithReceiver:arguments: • ProtoObject»executeMethod:

• CompiledMethod»receiver:withArguments:executeMethod: Method lookup. These operations look up for selectors, and query about this lookup.

• Behavior»lookupSelector:

Control message passing.

• ProtoObject»cannotInterpret: Is sent to the receiver if the lookup finds a nil method dictionary. Use a special lookup starting above the class with the nil method dictionary. Has been implemented for proxy implementation. • ProtoObject»doesNotUnderstand: / Object»doesNotUnderstand: Is sent to the receiver of the message if the lookup reaches the root of the class hierarchy without finding the receiver. • ReflectiveMethod»run:with:in: Message send reification. These operations allow one to query a message send to know if it is its arguments, receiver and selectors, and to get a corresponding message. The following methods perform explicit message sending (do the lookup and then apply a compiled method if any is found):

• Object»perform:(with: with: with: with:) • Object»perform: orSendTo: • Object»perform:withArguments: • Object»perform:withArguments: inSuperclass: • Object»perform:withEnoughArguments:

Chasing and atomic pointer swapping

The APIs in this category are Find pointers to and Bulk pointer swapping (supports the atomic swapping to references). The first one is rarely mentioned but Pharo supports the possibility to identify pointers to a given object (e.g., ProtoObject»pointersTo and ProtoObject»pointsTo:).

Find pointers to.

• ProtoObject»pointersTo • ProtoObject»pointersToAmong:

Bulk pointer swapping.

• ProtoObject»become: swaps the references in both ways, • ProtoObject»becomeForward: swaps only towards a given object.

• ProtoObject»becomeForward:copyHash:

Memory scanning

This category contains two API Memory scanning that support the traversal of the complete heap and Instances of a class that gives access to all the instances of a class.

Bulk pointer swapping. The operations allow one to traverse the heap.

• Object»someObject • ProtoObject»nextObject Instances of a class. These operations allow one to get or iterate over the instances and subinstances of a class.

Stack Manipulation

This category groups together all APIs that support traversing and modifying the execution stack. These APIs are accessible from two main entry points: the Process class that provides access to the existing processes and their suspended execution stack, and the thisContext pseudo-variable that provides access to the current method execution. Both these entry points provide instances of Context that represent a method execution and that represent the execution stack as a linked list.

Context These operations allow one to get information on the execution context (stack, sender, receiver, ...). The thisContext pseudo-variable supports access to the current execution context. This supports the creation of continuations and co-routines.