Investigating the possible use of a tetra (hydroxyphenyl) porphyrin as a fluorescence probe for the supramolecular detection of phospholipids

Journal of Luminescence 131 (2011) 2528–2537

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Investigating the possible use of a tetra (hydroxyphenyl) porphyrin as a fluorescence probe for the supramolecular detection of phospholipids Hanadi Ibrahim a, Athena Kasselouri a,n, Bertrand Raynal b, Robert Pansu c, Patrice Prognon a a

Laboratoire de Chimie Analytique, EA 4041, IFR 141, Univ Paris-Sud, 5 rue J-B. Clement, 92290 Chatenay-Malabry, France Plateforme de Biophysique des Mole´cules et de leurs Interactions, Institut Pasteur, 28 rue Docteur Roux, 75724 Paris Cedex 15, France c PPSM, UMR 8531 CNRS, Ecole Normale Supe´rieure de Cachan, F-94235 Cachan cedex, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 December 2010 Received in revised form 20 May 2011 Accepted 9 June 2011 Available online 15 June 2011

The present work explores the interest of using the 5,10,15,20-tetrakis(3-hydroxyphenyl)-21H,23Hporphine (m-THPP), a commercially available porphyrin, as a polarity probe for the detection of phospholipids. Feasibility of the process was first tested by direct fluorimetry and it was established that emission enhancement in the presence of lipids was maximum for a methanol content of 15% (v/v), a neutral pH (6–8) and a probe concentration of 2  10  7 M in the working solution. Detection with m-THPP in these conditions presents higher response factor but shorter linear range than diphenylhexatriene (DPH), the reference dye for the supramolecular detection of lipids. Insights on the mechanism of m-THPP–lipid association demonstrated that, at high lipid concentrations, porphyrin is associated to lipid assemblies in monomer form and located in the interior of bilayer; in low lipid content, porphyrin aggregates coexist with the monomer. Finally it was assessed that the method can be successfully coupled with micro-bore liquid chromatography. & 2011 Elsevier B.V. All rights reserved.

Keywords: Fluorescence probe Porphyrin Phosphatidylcholines Supramolecular assemblies m-THPP DPH

1. Introduction Phospholipids (PLs) are the main components of cell membranes and exhibit important biological functions. Lipid bio-analysis (lipidomics) is now a rapidly growing field providing some insights as to how specific phospholipids play roles in normal physiological and disease states [1]. As a consequence, an increased focus has been observed on the development of analytical methods for characterisation and quantitation of the individual molecular species [2,3]. Phospholipids are molecules that lack chromophores making a sensitive UV detection impossible. Usual detection techniques after chromatographic separation are mass spectrometry, evaporating light scattering detector (ELSD) and fluorescence detection by means of a covalent or a non-covalent association with a fluorescent reagent [4]. In this study, we are interested in the use of polarity probes in order to indirectly detect lipids. Lipid detection can be performed from the emission enhancement of the probe in presence of hydrophobic phospholipids compounds in polar media, due to supramolecular assemblies between probe and lipids. To date, 1,6-diphenyl-1,3,5hexatriene (DPH, Fig. 1a) is the most popular hydrophobic probe used in both direct fluorimetric detection of lipids [5] and after chromatographic separation by mean of a post-column device [6]. In this work, we explore the interest of using a tetraphenyl porphyrin (TPP) derivative, the 5,10,15,20-tetrakis(3-hydroxyphenyl)-21H,23H-

n

Corresponding author. Tel.: þ33146835849. E-mail address: [email protected] (A. Kasselouri).

0022-2313/$ - see front matter & 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2011.06.016

porphine (m-THPP, Fig. 1b), for probing phospholipids as an alternative to DPH. The advantage of this tetrapyrolic derivative is that it shows an emission at 600–800 nm, which is of particular interest for bio-analysis. Porphyrins are a class of compounds that have already found numerous applications in biology and analysis; for example they are used as probes in optical biosensors [7]. Porphyrin macrocycle has a 22p-electron conjugated system (Fig. 1b) that serves as a probe for the detection of interactions with host molecules. It is well known that porphyrin aggregates in polar media and aggregation– disaggregation processes can take place in interaction with ligands; the phenomenon can be studied from fluorescence and absorbance intensities variations. In addition, supramolecular assemblies on the basis of amphiphilic porphyrin derivatives and lipids are prospective subjects for numerous fields. The introduction of tetrapyrrol compounds in monolayer [8], bilayer [9] and micellar [10] lipid aggregates yield to supramolecular assemblies with unique physicochemical properties. These complexes have been widely applied to the modelling of biological processes. In a recent work [11], we demonstrated that some tetraphenyl porphyrin (TPP) derivatives exhibit an important enhancement of their fluorescence emission in the presence of dimyristoylphosphatidylcholine (DMPC) liposomes. The emission enhancement is due to the fact that the porphyrin is almost not fluorescent in aqueous media due to the self-aggregation process, but associated to lipids a progressive regeneration of the fluorescent monomer occurs inducing a very important increase in fluorescence emission. Among the porphyrin derivatives studied, m-THPP is chosen as a probe for our

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HO

HO

N

N

OH

H H N

N

OH Fig. 1. A (a) 1,6-diphenyl-1,3,5-hexatriene (DPH), (b) 5,10,15,20-tetrakis(3-hydroxyphenyl)-21H,23H-porphine (m-THPP).

experiments, because it presents the highest affinity for DMPC, the lower emission in water, an important fluorescence enhancement in the presence of lipids and is commercially available. Our present work mainly focuses on the development of a direct fluorimetric protocol for the detection of lipids using m-THPP as a probe and on the study of the particularities of m-THPP–phospholipid association. The interest in coupling with liquid micro-chromatography has also been explored for further applications. 2. Experimental 2.1. Chemicals 1,6-diphenyl-1,3,5-hexatriene (DPH; Fig. 1a), and all phospholipids used [1,2-Dioctanoyl-sn-glycero-3-phosphocholine (8:0–8:0 PC; abbreviated DOPC), 1,2-Dicaprinoyl-sn-glycero-3phosphocholine (10:0–10:0 PC; abbreviated DCPC), 1,2-Dilauroyl-sn-glycero-3-phosphocholine (12:0–12:0 PC; abbreviated DLPC), 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (14:0–14:0 PC; abbreviated DMPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (16:0–16:0 PC; abbreviated DPPC) and 1,2-Distearoylsn-glycero-3-phosphocholine (18:0–18:0 PC), (abbreviated PSPC)] were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). 5,10,15,20-tetrakis(3-hydroxyphenyl)-21H,23H-porphine (m-THPP; Fig. 1b) was purchased from Frontier-Scientific (United Kingdom). All reagents are of analytical grade and used as received. 2.2. Methods 1 mM methanolic stock solutions of lipids were prepared, stored at –18 1C and monthly renewed. 1 mM methanolic solution of m-THPP and 1 mM solution of DPH in tetrahydrofuran were prepared in the dark and stored at 18 1C. Further dilutions were performed in water for the dyes and in methanol for the phospholipids. Working solutions are prepared 10 min before measurement by mixing an aqueous solution of the dye and a methanolic solution of lipid of various concentrations. For kinetic experiments measurements were performed immediately after mixing. If no other information is given, the final content in methanol is 15% (v/v) and the concentration of the dye is 2  10–7 M. 2.3. Apparatus Steady state spectroscopy: absorption spectra were recorded using a Cary Bio 300 spectro-photometer (Varian, USA). Steady state

fluorescence measurements were performed with a Perkin-Elmer LS50B spectrofluorimeter equipped with a red sensitive photomultiplier. Excitation and emission wavelengths were set at 419 and 648 nm, respectively, for m-THPP and at 357 and 428 nm for DPH. Time resolved fluorescence: measurements were carried on a single photon counting set-up, based on a Titanium sapphire laser (Tsunami 3950B Spectra Physics, Les Ulis, France) and a multichannel plate photomultiplier (R3809U, Hamamatsu, Massy, France). Excitation of porphyrins was performed at 420 nm. For details of the experimental set-up see Schoutteten et al. [12]. Data treatment was performed using Igors software. Dynamic light scattering (DLS) experiments were performed in a 3 mm Suprasil quartz cell (Hellma, France) using a DynaPro-MS800s apparatus (Wyatt, Santa Barbara CA) equipped with a gallium aluminium arsenide 825 nm laser. Scattered light was measured at 901. Each autocorrelation curve is the mean of 20 acquisitions and for each sample, 3 autocorrelation curves was recorded. These curves were then analysed with the Sedfits 9.3 software from www.analyticalultracentrifugation.com using the continuous I(Rh)-Distribution model, resulting in hydrodynamic radius distribution plots. Polydispersity indexes were calculated as the ratio between the standard deviation (second moment) and the mean of each peak of the distribution using the method of cumulants implemented in the Dynamics v7 software (Wyatt, Santa Barbara CA). The radius values were calculated from Stokes–Einstein equation. Viscosity of a methanol/water: 15/85 (v/v) mixture was measured by a Rheostress RS100 5 Ncm apparatus (Haake Fisons, Karlsruhe, Germany) equipped with a DG41 R&S cylindrical coaxial geometry and it was found to be 2.403 mPa s. All experiments in solution (steady state at time resolved fluorescence, DLS and viscosity) were performed at 25171 1C. High performance liquid chromatography: the mobile phase was composed of methanol containing 40 mM of choline chloride and was pumped at a flow rate of 0.1 mL/min by a SP8800 Spectra Physics (California, USA) pump. Injection was performed through a Prostar 420 (Varian, USA) auto sampler and analysis took place on a Nucleosils 100–5 C18 (150  1 mm) column (Interchim). Post-column mobile phase was pumped at 0.5 mL/min by a Shimadzu (Kyoto, Japan) LC-10AS pump and mixed with the column effluent via a Tee device, followed by a tubing of 1400  0.5 mm. Detection was performed with a Shimadzu RF-10AXL fluorescence detector, equipped with a red sensitive photomultiplier. Data acquisition was performed with Kroma system 2000 software (Kontron Instruments, Italy).

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2.4. Association constants determination

3.2. Method development

Association constant (K) of porphyrin to phospholipids can be defined as

The aim of this study was to develop a rapid, easy to perform and sensitive analytical protocol for the detection of lipids. The method is based on the enhancement of the fluorescence emission of the probe induced by the dissociation of probe aggregates and the probe association to lipid vesicles. In practice, a methanolic solution of the lipids is mixed with an aqueous suspension of the dye. The operating conditions have been adapted in order to obtain high values of I–I0, where I and I0 are, respectively, the fluorescence emission intensities of the probe in the presence and in the absence of the lipids.

K ¼ ½bound mTHPP=ð½free mTHPP½PLÞ

ð1Þ

The fraction of bound m-THPP ¼[bound m-THPP]/([total m-THPP]) depends on lipids content [PL] as the following equation: Fraction of bound mTHPP ¼

K½PL 1 þK½PL

ð2Þ

Association constants (K) can be estimated for all lipids, by fitting the values of the fraction of bound m-THPP, obtained by absorption measurements as a function of lipid concentration, to the Eq. (2).

3. Results and discussion: 3.1. Photophysical properties of the probe. Absorption spectra of m-THPP were recorded in various organic solvents and the derived photophysical parameters are presented on Table 1. m-THPP is totally insoluble in hexane and only slightly soluble in toluene; as a consequence molar absorptivity cannot be calculated in the latter solvent (Table 1). The halfheight width (o1/2) is small and almost independent of the nature of the organic solvent, suggesting that the dye is present in its monomer form [13]. The maximum of the Soret band presents a pronounced red shift in DMSO with respect to the other solvents. In water, the dye presents a large Soret band located at 420 nm (so a o1/2 of 40 nm is observed) due to the well-known aggregation of porphyrins in aqueous media [14]. Fluorescence yields and lifetimes of the excited state are of the same order of magnitude in all organic solvents (even the more polar ones) where the dye is in monomer form (Table 1). Fluorescence decays in these solvents are mono-exponential confirming the presence of only one species. In aqueous media a significant decrease in the fluorescence efficiency has been observed and the decay becomes multi exponential due to the self-assembling. The long-time part of the decay has a lifetime of 10.2 ns, attributed to the monomer. Methanol has been chosen as organic solvent for the solubilisation of lipids, due to the relatively high fluorescence yield of the probe in this solvent and its common use in reversed phase liquid chromatography for the separation of lipids.

3.2.1. Methanol content As discussed above, m-THPP presents in aqueous media a large Soret band of low intensity. In our experiments the addition of methanol is necessary in order to avoid precipitation of the dye as well as of the lipids. The presence of increasing amounts of methanol results in the regeneration of the monomer form (Fig. 2). As we have mentioned, aggregated porphyrins present a very low fluorescence yield (FF) compared to that of the free monomer. The release of the monomer in the presence of methanol results in an important enhancement of the fluorescence intensity (Fig. 2 box). In our analytical method the percentage of methanol should not exceed 20% (v/v). Above this level, m-THPP becomes freely soluble as well as the lipids and consequently no supramolecular association occurs between phospholipids and the dye. In addition, methanol content has to be kept as low as possible in order to limit fluorescence emission of the blank (I0). We found that 15% of methanol (v/v) is a good compromise, avoiding precipitation of phospholipids species and resulting in the maximum difference in fluorescence emission in the presence and in the absence of lipids (I–I0; see also supplementary information). 3.2.2. Kinetic considerations In the chosen experimental conditions (water/methanol 85/15 (v/v)), phospholipids are not completely soluble and supramolecular assemblies are formed. The study of the kinetics of m-THPP incorporation into phospholipid assemblies is feasible due to the enhancement of fluorescence emission generated by the dye–lipid association. In Fig. 3 an example of the association of m-THPP with DMPC assemblies is depicted. High level of PL (curves (c) and (d)) induces reaction duration of about 10 min at room temperature (2571 1C). After this time, fluorescence intensity is constant (curve (d)) or is slightly decreasing with time (curve (c)). With lower lipid

Table 1 Characteristics of m-THPP electronic spectra. kmax is the wavelength of maximum absorption or emission, emax is the maximum molar absorptivity, x1/2 is the width of Soret band at half height, UF the fluorescence yield and s the life time of the excited state. Intensities of Q*X01 and Q*X00 bands were taken at their maximum. e0 is the dielectric constant of the solvent. Solvent

Toluene Ethyl-acetate THF Isopropanol Ethanol Methanol Acetonitrile DMSO Water a b

e0a

2.7 6.1 7.52 20.2 25.3 33 36.6 47.24 80.1

Absorption (Soret band)

Emission

kmax (nm)

emax (  104; cm  1 M  1)

x1/2 (nm)

kmax (Q*X00; nm)

Q*X00/Q*X01

UF

s (ns)

420 415 417 416 415 414 414 421  420

– 37.5 37.7 38.1 38.8 37.3 35.6 35.0  11.0

12.5 11.5 12.0 12.0 11.5 12.0 12.0 12.5  40

651 649 651 649 649 649 648 651  650

2.8 3.0 3.0 2.8 2.8 2.9 3.2 3.6 3

– 0.04 0.04 0.05 0.05 0.05b 0.05 0.04 o 0.005

9.5 9.2 9.8 10.0 9.7 9.4 9.1 10.8 10.2

at 293.2 1K, except for toluene (296.4 1K) and THF (295.2 1K) [15]. taken as reference [16].

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Fluorescence (a.u.)

0.14 0.12 50%

Absorbance

0.1 0.08

CH3OH %

1000 900 800 700 600 500 400 300 200 100 0 0

10

20

30

40

50

CH3OH %

0.06 0.04 0.02 0 380

390

400

410

420 430 440 wavelength (nm)

450

460

470

480

Fig. 2. Evolution of absorption spectra of m-THPP as a function of methanol content in % (v/v); [m-THPP]¼ 2.5  10  7 M. Box: variation of fluorescence emission at 649 nm.

Fluorescence intensity (a.u)

450 (d)

400 350 300 250

(c)

200 150 100 (b) (a)

50 0

0

500

1000 t (sec)

1500

2000

Fig. 3. Variation of fluorescence intensity of a 10  7 M m-THPP solution in CH3OH/ H2O: 15/85 (v/v) as a function of time: (a) without DMPC, (b) [DMPC]¼5  10  8 M, (c) [DMPC]¼ 5  10  7 M and (d) [DMPC]¼ 5  10  6 M.

concentrations (curve (b)), the reaction appears more rapid, as the fluorescence enhancement seems to occur in less than one minute (the shortest duration measurable with our instrumentation). Experiments were also performed in the presence of different phospholipids, tested for molar ratio PL/dye¼100, that is in large lipid excess (see supplementary information). It was found that m-THPP incorporation kinetics depends on the nature of the phospholipid but there is no direct relationship with the phospholipid chain length. Incorporation is very rapid for DCPC (10:0– 10:0) and DPPC (16:0–16:0) but quite slower for DOPC (8:0–8:0), DLPC (12:0–12:0), DMPC (14:0–14:0) and DSPC (18:0–18:0). Ten minutes after mixing at room temperature, almost constant values of fluorescence intensity were reached for all lipids.

3.2.3. pH effect In acidic media, m-THPP exists in its diprotonated form: H2THPP2 þ (pKa¼3.8 [17]). In our experiments, in the absence of lipids, the Soret band of this form is located at 442 nm and only one emission band occurs at 678 nm according to the literature [18]. Upon addition of DMPC in acidic media, a change in the spectral characteristics has been noted: the absorbance at 442 nm decreases

and Soret band is blue shifted to 419 nm, characteristic of the neutral form of tetraphenyl porphyrin derivatives [18,19]. This is confirmed by fluorescence measurements, which show that the 678 nm emission band recorded at pH¼ 2 and 3 in the absence of lipids disappears, with the apparition of 648 and 712 nm emission upon DMPC addition typical of the neutral form (see also supplementary information). All these features suggest that it is the neutral form of m-THPP that bounds to DMPC aggregates. Similar results have been obtained by Gandini et al. [20] as the result of the association of a tetra(4-sulfonatophenyl) porphyrin to various micelles. The addition of lipids increase the fluorescence emission almost in the entire pH range studied. The trend of the m-THPP fluorescence intensity as a function of pH in hydro-organic environment at 649 nm is presented in Fig. 4, in the absence (I0) and in the presence (I) of DMPC. The decrease in the emission at high pH values (9  1 1) is related to the ionisation of the phenol groups. As seen, the maximum difference in fluorescence emission in the presence and in the absence of lipids (I–I0) occurs at pH between 6–8, corresponding to the neutral form. 3.2.4. Probe concentration Different concentrations of m-THPP were tested, from 1  10  7 to 8  10  7 M. Increasing probe concentration increases the fluorescence emission in presence of lipids (I), but also increases the fluorescence intensity of the blank (I0). In addition, at high probe concentrations, the inner-filter effect can become important. The limits of detection, taken as 3sb, (sb taken as the standard deviation of the blank) were estimated for DOPC, DLPC, DMPC, DPPC and DSPC and the lower values were obtained when the dye concentration was equal to 2  10  7 M. For this concentration, absorbance is low (o0.1) and the inner-filter effect is considered as negligible. In summary, in the selected protocol, aqueous solution of the dye and methanolic solution of PL are mixed at a ratio of 85/15 (v/v) and fluorescence intensity is read 10 min after mixing. Concentration of the probe in the working solution is 2  10  7 M. As the maximum of emission was obtained at the pH region of 6–8, no buffer was used, in order to simplify the protocol.

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3.3. Insight on the supramolecular PL assemblies 3.3.1. Dynamic light scattering study As we have already mentioned, phospholipids are not completely soluble in methanol/water 15/85 (v/v) mixture and form supramolecular assemblies. In order to definitively demonstrate this feature, photon correlation measurements were performed and the hydrodynamic radius (Rh) of the phospholipidic assemblies were evaluated. As expected, it was found that the size depends on the phospholipid tested (Table 2), but no direct 500 Fluorescence emission (a.u.)

450

Blank DMPC

400 350 300 250 200

correlation between the hydrodynamic radius and the length of the aliphatic chain was found. Among the lipids tested, DOPC, DCPC, DLPC and DMPC are present in the liquid phase at room temperature (Tc o25 1C) and form small assemblies. Knowing that the thickness of a DMPC bilayer is estimated to be about 40 A˚ [21] we can suggest that these small assemblies are probably micelles and small liposomes. It should be noticed that DPPC and DSPC that are in the gel phase at room temperature (Tc 425 1C) form larger vesicles (see Table 2). The molecular volumes of the studied phospholipids were calculated by the addition of volume of the headgroup (VH ¼331 A˚ 3), the volumes of each methylene group (VCH2 ¼27.7 A˚ 3) and the volumes of terminal methyl groups (VCH3 ¼52.6 A˚ 3). VH, VCH2 and VCH3 values are taken from Kucerka et al. [22]. From these results the maximum number of phospholipids (Nmolecules) that can be contained in these assemblies has been calculated. Phospholipid assemblies were also studied in the presence of the dye for a PL/m-THPP molar ratio of 100. The size profiles of the phospholipid assemblies are similar to the values presented in Table 2, demonstrating the absence of influence of the dye.

150 100 50 0 0

2

4

6 pH

8

10

12

Fig. 4. Evolution of fluorescence emission at 649 nm of 10  7 M m-THPP in CH3OH/H2O: 15/85 (v/v) as a function of pH. [DMPC]¼ 10  5 M.

3.3.2. Photophysical properties study In order to get a better insight into phospholipid–m-THPP association, the visible absorption spectra of the dye were recorded at various lipids concentrations (Fig. 5), together with the fluorescence spectra. As we can see in Fig. 5, phospholipids dissociate the porphyrin aggregates and regenerate the Soret

Table 2 Characteristics of phospholipid vesicles in CH3OH/H2O: 15/85 v/v: Rh is the radius, Vvesicle the volume of the equivalent sphere and polydispersity obtained by DLS, VPL is the calculated volume of a single phospholopid molecule and Nmolecules the maximum number of molecules that can be contained per vesicle. Tc is the phase transition temperature taken from Ref. [23]. [PL] ¼10  5 M.

Rh (nm)a Polydispersity (%)a Vvesicle (nm3) VPL (nm3) Nmolecules Tc (1C)

DCPC

DLPC

DMPC

DPPC

DSPC

13.6 7 1.3 14 10 439 0.7686 13580 –

7.2 70.6 68 1 562 0.8794 1780  5.5

5.4 7 1 32 666 0.9902 673 1

5.3 70.4 13 626 1.101 569 23.7

40.5 7 11.1 35 278 121 1.212 229 510 41.5

63.9 7 15.7 60 1 094 426 1.323 827 480 55.5

average of three measurements.

0.08 2 x 10-5 M

0.07 0.06 0.05

Fluorescence (a.u.)

0.09

Absorbance

a

DOPC

[DMPC]

[DMPC] (M)

0.04 0.03

0

0.02 0.01 0.00 380

390

400

410

420 430 440 wavelength (nm)

450

460

470

480

Fig. 5. Absorption spectra of 2  10  7 m-THPP in the presence of increasing amounts of DMPC. Solvent: CH3OH/H2O: 15/85 (v/v).

H. Ibrahim et al. / Journal of Luminescence 131 (2011) 2528–2537

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Table 3 Characteristics of the absorption and fluorescence spectra of 2  10  7 M m-THPP (in CH3OH/H2O: 15/85 (v/v)) in presence of [PL] ¼ 2  10  5 M. Parameters are similar to that of Table 1. K is the binding constant to phospholipids calculated by absorption measurements. Absorption (Soret band)

Emission

Binding constant

Phospholipid (PL)

lmax (nm)

emax (  104) cm  1 M  1

o1/2 (nm)

lmax (QnX00; nm)

QnX00/QnX01

K (  10 þ 6)M  1

DOPC DLPC DMPC DPPC DSPC CH3OH/H2O: 15/85 (blank solution)

419 418 418 419 418  420

36.5 40.5 40 39.8 37.5  13

14 12.5 12.5 14.5 13  35

650 647 647 647 648  650

3.0 2.8 3.0 2.9 3.1 3

1.2 70.2 1.4 70.2 2.1 70.2 1.4 70.2 1.1 70.2 –

Fraction of bound m-THPP

1.2 1 0.8 0.6

fluorescence absorbance

0.4 0.2 0 0

1

2

3

4 5 6 [DMPC] (µM)

7

8

9

10

Fig. 6. Molar fraction of bound m-THPP as a function of the lipid concentration, calculated by absorption (Absorbance ¼f([DMPC])) and by fluorescence data (I¼ f([DMPC])).

band of the monomer form, together with a significant enhancement of fluorescence emission (Fig. 5, box). Photophysical properties of the dyes in a large excess of lipids are summarised on Table 3. In these conditions, absorption spectra present narrow Soret bands demonstrating that m-THPP binds to PL aggregates in monomer form. The maximum of Soret band is 2 nm red shifted with respect to the spectra in methanol or acetonitrile and close to the maximum obtained in THF (see Table 1). The variation of the absorbance as a function of phospholipid concentration [Absorbance¼f([PL])], as well as the corresponding evolution of fluorescence intensity [I¼f([PL])], have been used to calculate the fraction of the dye bound to PL bilayer. As an example, Fig. 6 illustrates the results obtained in the case of DMPC. The two estimations (from absorbance and from fluorescence) can be considered as equivalent. Surprisingly, a slight discrepancy between the two curves is observed for low PL concentrations. Time resolved measurements were performed using excitation at the Soret band, at 420 nm. In the absence of lipid vesicles, the fluorescence decay is bi-exponential demonstrating the presence of two species having fluorescence lifetimes of t1 ¼2.4 ns and t2 ¼5.6 ns (Fig. 7, decay (a) and residual plot (a0 )). These two species probably correspond to a mixture of aggregates. Measurements were also performed in the presence of three of the lipids studied: DOPC, DMPC and DSPC (having short, intermediate and long chain length) in three different concentration levels. Typical results are shown in Fig. 7 for DMPC. Fluorescence decay is monoexponential in a large excess of lipids (decay (d), residual plot (d0 )), indicating the presence of only one fluorescence species. The lifetime is 10.5 ns, similar to that obtained in organic solvents (Table 1) and one can deduce that only the fluorescence of the monomer associated to lipid vesicles is recorded.

One can notice that the fluorescence decay depends on the phospholipid concentration (Fig. 7, decays (b), (c) and (d)). This could be due to the increasing contribution of the dissolved monomer mixed with the decreasing contribution of the aggregates. From that a partition coefficient could be deduced. In this objective the intermediate decays (b and c) have been fitted with fixed lifetimes of 5.6 and 10.5 ns. But the fits (black lines with white dots, Fig. 7) are not satisfying, and the long-time parts of decays (b) and (c) are too fast. This is probably due to the incomplete dissociation of the m-THPP aggregates in the bilayer and a decrease in the lifetime of the excited state of the monomer due to the proximity of the aggregates. Further increase in lipid concentration leads to progressive ‘‘dilution’’ of m-THPP aggregates and, in great excess of lipids, the decay becomes monoexponential (decay (d)), attributed to the monomer form. As the lifetimes of fluorescence species change with lipid concentration, calculation of the fraction of the bound m-THPP is better to be performed by absorption measurements (Fig. 6). From the fraction of bound m-THPP¼f ([PL]) curves obtained by absorption, association constants (K) of the porphyrin to the phospholipids have be determined, using Eq. (2). K values of m-THPP–lipid association (Table 3) are found to be high (from 1.1  106 to 1.4  106 M  1) for DOPC, DLPC, DPPC and DSPC and even higher for DMPC (K¼2.1  106 M  1).

3.3.3. Incorporation mechanism As already seen, lipid vesicles are formed by mixing an aqueous solution of the dye with a methanolic solution of the lipids. An example of incorporation kinetics into DMPC vesicles is described in Fig. 8 (curve (a)). In order to get a further insight on the mechanism of dye incorporation, supplementary experiments were performed where DMPC assemblies are formed first, in the absence of the dye (curves (b), (c) and (d)) by mixing a methanol solution of lipids with water. The dye was added thereafter (i) as a small aliquot (10 ml) of methanolic solution (curve (b)), (ii) as an aqueous/methanol solution (85/15 v/v; curve (c)) and (iii) as an aqueous solution (curve (d)). As we can see, for curves (b) and (c), incorporation is more rapid than in the curve (a), suggesting that the dye penetration into the PL assemblies is not the slowest step of the process. In contrast, when m-THPP was added in aqueous solution (curve (d)), the curve is rather similar to (a). As it is well known, when the porphyrin is dissolved only in water, extended auto aggregation occurs. The incorporation of the dye should be a two-steps process: first, dissociation of dye aggregates to generate the monomer form and then, incorporation of the monomer form into the lipid vesicles. Our results suggest that the slow incorporation kinetic observed is related to the disaggregation process that appears as the limiting factor and not to the penetration of the dye into the phospholipids assemblies.

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Fluorescence (counts)

4 2 0 -2 -4 4 2 0 -2 10-44

(a')

(d')

103

(d)

10

2

(c)

(b)

101 (a)

0

10

20 Delay in ns

30

40

Fig. 7. Fluorescence decay of DMPC/m-THPP aggregates as a function of DMPC content: [m-THPP]¼2  10  7 M, ratio DMPC/m-THPP: (a) 0, (b) 0.25, (c) 2.5 and (d) 25. (a0 ; d0 ) the residual plots of decays (a) and (d).

Fluorescence intensity (a.u.)

800 700 600 500

(a)

400

(b) (c)

300

(d)

200 100 0 0

500

1000

1500

2000

t (sec) Fig. 8. Incorporation of m-THPP into DMPC vesicles using four different protocols. [m-THPP]¼2  10  7 M, [DMPC] ¼2  10  5 M. For preparation details see text.

3.3.4. m-THPP localisation In order to explore the location of m-THPP into phospholipid assemblies (at the exterior of the bilayer or between the hydrophobic chains), competition experiments were planned using DPH as competitor; this probe is known to be located between the hydrophobic chains of the lipid layers [24]. In the absence of lipids, the two dyes (m-THPP, DPH) show very low emission in the solvent used (85/15 water /methanol). When the two dyes are simultaneously present in this solvent, m-THPP emission remains unchanged and a slight decrease in the emission of DPH was observed (results not shown). In the presence of DMPC three different cases were studied: m-THPP alone (solution a), DPH alone (solution b) and m-THPPþ DPH mixture (solution c). Solutions containing m-THPP were excited at two wavelengths: 419 nm (maximum of porphyrins absorption, subscript 1) and 357 nm (maximum of DPH absorption, subscript 2).

Spectra of DPH alone or m-THPP alone present their well-known characteristics in hydrophobic media (see Fig. 9, curves b2 and a1). m-THPP emission spectrum remains unchanged in the presence of DPH as curves c1 and a1 overlap. On the other hand, excitation of solution c at the DPH maximum absorption (357 nm) exhibits quenching of DPH emission (curve c2) with respect to the emission of DPH alone (curve b2) and generation of the emission band of the porphyrin at 650 nm (curve c2). Curve c2 remains unchanged if the dyes are not introduced simultaneously but successively. Excitation at the same conditions (357 nm) of the solution containing only m-THPP leads to a low emission of the porphyrin (curve a2) induced by the weak absorption of the porphyrin at this wavelength. This emission is lower than that of the m-THPPþDPH mixture excited at 357 nm (curve c2). There are two possible interpretations for the quenching of DPH fluorescence: i) porphyrin hinders the incorporation of DPH into the lipid assemblies yielding for this latter to remain in the bulk solvent, where it poorly undergoes fluorescence; ii) porphyrin is located, at least partly, between the hydrophobic chains, close to DPH and resonance energy transfer takes place that quenches the DPH emission. Even if the latter is the most probable explanation, both hypotheses confirmed a location of m-THPP close to that of DPH in the interior of phospholipid bilayer, in agreement with our recent study in DPMC liposomes [11]. It is worth noting that further addition of PL in the system leads to a progressive regeneration of the DPH fluorescence (results not shown), certainly due to the enlargement of distance between the two dyes entailed by the increasing content of PL in the assemblies. In this section some aspects of the lipid–dye interactions has been evidenced. We demonstrated that in the environment used (methanol/water 15/85 (v/v)), phospholipids form supramolecular assemblies of 5–65 nm (see Table 2), the size depending on the chain length of the PL. In high content of lipids the dye is associated to lipids assemblies in its monomer form and located at the interior of the PL bilayer. Our results also suggest that the

H. Ibrahim et al. / Journal of Luminescence 131 (2011) 2528–2537

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500

Fluorescence emission (a.u)

450

m - THPP emission

DPH emission

a1

400 350

b2

300 250 c1

200 150 100 50 0 380

c2 430

c2 480

530

580 630 wavelength (nm)

a2

680

730

780

Fig. 9. Fluorescence spectra of m-THPP (solution a), DPH (solution b) and m-THPP þDPH mixture (solution c); subscript 1: excitation at 419 nm, subscript 2: excitation at 357 nm. [m-THPP]¼ [DPH] ¼2  10  7 M, [DMPC] ¼2  10  5 M; solvent: CH3OH/H2O: 15/85 (v/v).

PL assemblies are formed first and the dye is incorporated later, as the disaggregation process is the limiting step. In low concentrations of lipids, the monomer form of the dye coexists with aggregates in the PL bilayer.

3.4.1. Comparison with the DPH In order to explore the potential use of m-THPP in the detection of lipids, calibration curves were established for various lipids and the results were compared with the curves obtained with DPH. Typical results are illustrated in Fig. 10 using DMPC as an example: the fluorescence intensity enhancement of m-THPP (I–I0), obtained in the presence of lipids, increases with increasing amount of lipids, and this enhancement is higher to that obtained with DPH. Results for all tested lipids are presented in Table 4. Results obtained for DCPC showed poor reproducibility and are not presented; the reason remained unclear. Response factor (expressed as the slope of the standard curve) is better for m-THPP for all lipids except for DOPC. Linearity range is larger with DPH in all cases. It is to be noted that standard deviations of the fluorescence of blank solutions are two to three times higher for m-THPP (Relative Standard Deviation (RSD)¼ 10–30%) than for DPH (RSDo10%). The variation of intensity of porphyrin solutions can be attributed to the physical instability of porphyrin aggregates.

3.4.2. Copper addition The analysis performed above points out the importance of reducing the fluorescence of the blank solutions. It is known that the emission of a dye can be reduced by the use of appropriate quenchers. Copper(II) has already been reported to act as fluorescence quencher [25] with some examples on porphyrin quenching [26]. In our experiments, it was shown that a few mM of CuCl2 quenches the fluorescence of m-THPP in a methanol/water 15/85 (v/v) solution. Other chloride salts do not produce a similar effect, demonstrating that the quenching is provided by copper. Quenching of m-THPP by copper showed to be a static process, as the calculated Stern–Volmer constant (KSV) in methanol/water media

800 I-I0 (a.u.)

3.4. Lipid detection by direct fluorimetry

1000

600 400 DPH

200

m-THPP

0 0

0.5

1

1.5 [DMPC] (µM)

2

2.5

3

Fig. 10. Fluorescence enhancement in the presence of DMPC. [m-THPP]¼ [DPH] ¼2  10  7 M; solvent: CH3OH/H2O: 15/85 (v/v). I, I0 are the emission intensities in the presence and in the absence of lipids, respectively; n¼ 3 for each concentration level. m-THPP: lex ¼ 419 nm, lem ¼ 648 nm; DPH: lex ¼ 357 nm, lem ¼ 428 nm.

Table 4 Comparison of m-THPP and DPH. [Dye]¼2  10  7 M. S(m-THPP)/S(DPH) in the common linearity range (n¼ 3). m-THPP Linearity (r 40.99) (M) DOPC (1–10)  10  7 DLPC (1–7)  10  7 DMPC (1–10)  10  7 DPPC (0.5–6)  10  7 DSPC (2–12)  10  7

DPH Slope (M  1)

Linearity (r 40.99) (M)

2.31  108 (0.5–25)  10  7 4.15  108 (1–25)  10  7 4.69  108 (1–15)  10  7 4.96  108 (0.5–25)  10  7 1.63  108 (1–15)  10  7

Slope (M  1)

Slope (m-THPP)/ Slope(DPH)

2.75  108 1.42  108 1.91  108 2.57  108 0.94  108

0.84 2.92 2.46 1.93 1.73

is of the order of 3.5  105 M  1 (see supplementary information), too high to be attributed to a dynamic quenching mechanism. Quenching of m–THPP fluorescence emission by copper takes also place in the presence of lipids, but in a lesser extent (see supplementary information). Copper content should, then, chosen in a way to diminish the blank fluorescence (I0), but to not affect the slope of the standard curve (I–I0 ¼f([PL])). Experiments with

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should be taken into account, as the composition of the mobile phase necessary to obtain separation, the surfactant concentration in the post-column phase and the delay of the process. Experimental conditions of the post-column process should be optimised by experimental design and validated in a separated study.

4. Conclusion

Fig. 11. Chromatographic analysis of a 6.5 mg/mL DLPC test solution; post-column detection with (a) 1 mM m-THPP (lex ¼419 nm, lem ¼650 nm), (b) 1 mM DPH (lex ¼357 nm, lem ¼430 nm). Injection volume: 20 mL. Post-colunm phase flow: 0.5 mL/min. Temperature: 35 1C. Surfactant in the post-column mobile phase [Brijs]¼ 20 mM. For chromatographic separation conditions see experimental section.

DMPC demonstrated that addition of 1 mM Cu2 þ in the m-THPP solution did not significantly modified the slope of the standard curve but highly decreased the signal (until  85%) and the standard deviation (  60%) of the blank. 3.5. Coupling with high performance liquid chromatography (HPLC) In order to explore the possible interest of coupling such an approach with separation techniques, some tests were performed using micro-liquid chromatography. Lipids can be detected after HPLC separation by means of a dye introduced via a post-column device [4]. In practice, an aqueous post-column reagent containing the dye (and a surfactant in order to assure dye solubility) is mixed with the chromatographic effluent before the detector. Detection is performed measuring the significantly increased fluorescence emission issued from the dye insertion into lipids supramolecular assemblies [27]. Using the same experimental set-up and conditions as in a previous work [6] we tried to demonstrate that m-THPP can be used for the detection of lipids in coupling with micro-liquid chromatography. The injected solution of DLPC is of 6.5 mg/mL, concentration near the limits of quantification obtained for PLs with DPH [6]. The obtained results are promising as the obtained peak is much higher than the noise and than that obtained with DPH (Fig. 11) in the same conditions. Analysis of successive dilutions of DMPC solutions (lipid presenting the lower limits of detection with DPH) allows to obtain a limit of detection of about 0.25 mg/mL for DMPC, half of that reported with the DPH [6]. It can be underlined that baseline variations are similar with m-THPP and with DPH, contrary to blank solutions in the direct method. This is attributed to the presence of surfactant (Brij 35s) in the post-column phase that stabilizes the m-THPP aggregates and diminishes emission variations. In the two approaches (direct spectrometry or after chromatographic separation) the mechanism of the detection is the same: enhancement of the fluorescence emission of the dye after interaction with the lipids assemblies, formed in polar media. It is to note that detection cannot be performed exactly in the same conditions in direct spectroscopy and in chromatography after post-column introduction of the dye. In the latter case, supplementary parameters

The objective of the present work was to explore the use of a hydroxylated porphyrin the m-THPP as a fluorescence probe for the detection of lipids. The interest of m-THPP in comparison to the other probes used for lipid detection is, from a spectroscopic point of view, to present an emission at 600–800 nm and so potentially a better selectivity. A protocol for the determination of lipids by direct fluorimetry has been proposed, based on the mixing a methanolic solution of the lipids with an aqueous solution of the dye. Dye concentration, time of incubation, pH and methanol content in the working solution have been carefully chosen in order to assure a high sensitivity. The mechanism of the process has been extensively studied. With the low content of methanol used, PLs are not soluble and form supramolecular assemblies exhibiting sizes depending on the structure of the PL. Further insight into the m-THPP–phospholipid association demonstrates that for high lipid content, the formation of lipid assemblies is not affected by the presence of the dye that can be inserted into PL assemblies after their formation. In high contents of lipids also, the dye is incorporated in its monomer form and located, as DPH, in the interior of the bilayer. In low lipid contents, aggregates of the dye coexist with free monomer in m-THPP–PL assemblies. Finally, detection of lipids with m-THPP is compared to that with DPH, the most widely used molecular probe for the detection of lipids. The major advantage of m-THPP is to induce a higher response factor than DPH for all lipids except DOPC. The main drawback of the m-THPP use is its limited linear range and important variations of the blank solutions signal due to the aggregates formation. Further experiments demonstrated that the intensity and the variations of emission of m-THPP blank solution can be significantly reduced by the addition of an efficient fluorescence quencher the Cu2 þ . In the last part of this work it has been demonstrated that the method can also be coupled with micro-chromatography, leading to low limits of detection for DMPC studied as example. The performance of the system should be further improved after optimisation of the operating conditions of the post-column device. Even if the chromatographic approach implies different set-up from the direct one, the knowledge of the mechanism and the factors that influence the lipid–m-THPP interactions previously established, should help to envisage analytical applications in this field.

Acknowledgements The authors are grateful to Dr P. England (Institut Pasteur, Plateforme de Biophysique des Mole´cules et de leurs Interactions) for his contribution to dynamic light scattering experiments.

Appendix A. supplementary materials Supplementary data associated with this article can be found in the online version at doi:10.1016/j.jbiomech.2009.11.012.

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