Colloids and Surfaces B: Biointerfaces 20 (2001) 309–314

Near-infrared induced membrane surface electrostaticpotential, fluorescent measurements

Mal*gorzata Komorowska *, Adam Czarno l*eskiInstitute of Physics, Wroc l*aw Uni6ersity of Technology, Wyb. Wyspianskiego 27, 50-370 Wroc l*aw, Poland

Received 10 April 2000; received in revised form 18 May 2000; accepted 31 July 2000

Abstract

The change of the electrostatic surface potential induced by near-infrared radiation was monitored by thefluorescence probe technique. Fluorescence intensity of 1-anilinonaphtalene-8-sulfate (ANS) was studied in the pHrange 4.8–9.5 before and after exposition to NIR (700–2000 nm). The intensity of fluorescence changed (decreasedafter exposition on radiation) only at pH 7.4. The effect is due to decreasing concentration of ANS in liposomemembrane after irradiation. The modified distribution of ANS in liposome membrane upon irradiation is attributedto the dehydration of membrane surface. Dehydratation diminishes the electrostatic surface potential about 36915mV. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Fluorescence; ANS; Electrostatic potential; Hydration effects; Liposomes; Near-infrared

www.elsevier.nl/locate/colsurfb

1. Introduction

The near-infrared region (NIR) is known as aradiation very easily penetrating into tissues [1],however, significant absorption of this spectralregion by water, proteins and lipids [2–4] is usedfor chemical [5–7] as well as medical analysis [8]and diagnostics [9–11]. In fact, this radiation isabsorbed mainly by overtones of stretching orcombination vibrations �CH, �OH, �POH, �NHand SH groups; all of these molecules are in-volved in hydrogen bonds [3]. Zundel et al. stud-

ied the easily polarizable hydrogen bonds betweenvarious proton donors and acceptors which areresponsible for the presence of continua in IRspectra [12–16]. Studies on these phenomena havedemonstrated that many types of H bonds, whichform in proteins and H-bonded systems betweenside chains and phosphates, show large protonpolarizability. When H bonds with large protonpolarizability are present, what is common inbiological systems like membrane surface, theproton within the system of H-bonds shifts andsubsequently conformational changes are stronglyinterdependent. The proton transfer process caneasily be controlled by local electric fields, causedby fixed charged groups, cations, polar molecules,even coupling proton motion with hydrogen bond

* Corresponding author. Tel.: +48-71-3203186; fax: +48-71-3283696.

E-mail address: kom@rainbow.if.pwr.wroc.pl (M. Ko-morowska).

0927-7765/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S0927 -7765 (00 )00190 -9

M. Komorowska, A. Czarno l*eski / Colloids and Surfaces B: Biointerfaces 20 (2001) 309–314310

stretching vibration [12,15]. For example, absorp-tion of NIR, (what is equivalent with excitation ofNH stretching vibration overtones) leads to thedissociation of hydrogen bonded complexes ofaminoacids and TEMPO-spin label due to protontransfer induced by light from protonated aminein water solution to nitroxide group [17]. En-hanced protonation induced by NIR leads todehydration of the membrane surface [18]. Oursuggestion corroborates fact that the energy ofNIR (6–17 kJ per Avogadro’s number of pho-tons) is comparable to the energy of hydrogenbonds (1.3×104–2.1×104 J/Mol) [19] thus, ab-sorption of this radiation could lead to modifica-tion of the hydrated surface of membranes.Dehydration of the surface influences the value ofthe electrostatic membrane potential [20] there-fore, we have studied the changes of the electro-static potential on liposome surface afterirradiation, using fluorescent probe ANS (1-anili-nonaphtalene-8-sulfate) according to the methodproposed in [21].

When liposomes are suspended in aqueoussoluation of ANS, negativedly charged ANS ionspartition between liposome membrane and sol-vent. The benzene and naphtalene rings are lo-cated into hydrophobic region of membrane andcharged sulfonate groups into the polar region ofthe membrane [22]. The fluorescence efficiency ofANS strongly depends on the solvent polaritywhere ANS molecule is excited. The intensity offluorescence of ANS incorporated into liposomemembrane increases while the intensity of ANS inwater solution is negligible. The amount of ANSdissolved in hydrophobic part of membrane de-pends on the ionic strenght of electrolyte dis-solved in surrounding solvent and on the surfacecharge, it increases for positive charged mem-branes and decreases when negative charge ispresent [21]. Binding equilibrium of ANS to themembrane is described by reaction

ANS+S�S−ANS (1)

where ANS is fluorescent probe with concentra-tion [CANS], S binding sites on the surface ofmembrane (concentration [CS]) and S�ANSbonded ANS to the membrane (concentration[CS�ANS]). The equilibrium constant K of thisreaction:

K=[CC�ANS]

[CANS][CS](2)

is a function of the electrostatic surface potentialC [21]:

K=Kc exp�−hBFc

RT�

(3)

where K is equilibrium constant of binding reac-tion (1), Kc the part of the constant related to thechemical potential, C the electrostatic surface po-tential, hB — parameter −1 for negative and +1for positive charge on the membrane surface [21].Because the concentration CANS is far of fromsaturation, we can assume that [CS] equals to thenumber of the total ANS binding sites in mem-brane thus, [CS] is proportional to the total con-centration of membrane lipids [L0] and thenumber of moles of binding sites per mole lipid b :

[CS]=b [L0] (4)

Eq. (2) can be transformed into form:

[CS�ANS][CANS]

=Kb [L0] (5)

and after substitution the Eq. (3)

[CS�ANS][CANS]

=b [L0]Kc exp�

−hBFc

RT�

(6)

The ratio of concentrations [CS�ANS]/[CANS] canbe obtained from fluorescence measurements.Hence, the difference in the electrostatic surfacepotential DC before and after irradiation may befound using Eq. (6).

2. Materials and methods

2.1. Materials

Egg phosphatydylcholine (PC) was obtainedand purified by the method described in paper[23], 1-anilino-8 naphthalene sulfonate (ANS) waspurchased from Molecular Probes, chemicalsbuffer employed were reagent grade purity anddeionised and redestilled water was used.

M. Komorowska, A. Czarno l*eski / Colloids and Surfaces B: Biointerfaces 20 (2001) 309–314 311

2.2. Preparation of liposomes

Multilammelar liposomes were used in all ex-periments. Phosphatydylocholine from egg (PC)in chloroform solution was dried under vacuum ina glass tube and resuspended in 310 mOsM phos-phate buffer at pH 5.5, 6.6, 7.0, 7.4, 8.2, and pH4.8 and 9.1 (pure NaHPO4 and Na2HPO4). Themixture was mechanically shaken at room temper-ature and later sonificated in supersonic washerunder full power for 5 min until a milky suspen-sion was obtained. The final lipid concentrationwas 1 mg/ml. The volume of 10 ml of suspensionprepared as above was exposed on radiation.

2.3. Irradiation procedures

A halogen lamp with a filter (cooled by streamof cold air) of transparency range 700–2000 nmwith the power density of incident light 6.9 mWon the flat glass tube with a sample was used as alight source. During exposition to the light sus-pension was gently stirred and cooled by thewater coat. Temperature was controlled andequals 30392 K. The samples were irradiated for15, 30 min. Except for irradiation, the controlsamples were subjected to the same conditions asthe experimental ones.

2.4. Fluorescence measurements

The irradiated or control suspension (0.5 ml)were dissolved in 2.5 ml of buffered solution offluorescent probe ANS (concentration −2.1×10−6 M) and shaken for 5 min at room tempera-ture. Subsequently, the same volume of theirradiated and control suspension (0.5 ml) pre-pared as described above except sonification wascentrifuged at 60 000×g for 60 min. Then 0.4 mlof supernatant, was dissolved in 1.5 ml of ethanoland intensity of fluorescence of ANS in ethanolicsolution was measured. All fluorescence measure-ments were performed on the spectrofluorimeterHitachi F-4500 at 480 nm under excitation of 372nm. After each exposure six samples were takenfor fluorescence measurements. The intensity offluorescence was calculated as a mean value from3 to 10 (for pH 7.4) independent measurements.

Fig. 1. Relative difference of the intensity of fluorescence ANS(calculated in %), DI, between dark and NIR exposed samplewith respect to the dark sample as a function of pH and timeexposition on radiation for ANS partitioning in liposomemembrane.

S.D. was changed from 9.9 to 3.1% thus thevalues of the relative changes of fluorescence be-low 10% are within experimental error.

3. Results

Experimental results are shown on Figs. 1–3.Fig. 1 illustrates the relative difference of fluores-cence ANS (calculated in %), DI, between darkand NIR exposed sample with respect to the darksample as a function of pH and time expositionon radiation. The negative values of DI meansthat the fluorescence intensity of ANS probe forirradiated sample decreases. At pH 8.2 and 9.07and lower pH 5.5 and 4.8 the changes are about5–6%. These changes of the fluorescence intensity

Fig. 2. Relative changes of the intensity of fluorescence withrespect to the dark sample as a function of pH and timeexposition on radiation for ANS partitioning in buffer solu-tion.

M. Komorowska, A. Czarno l*eski / Colloids and Surfaces B: Biointerfaces 20 (2001) 309–314312

Fig. 3. The dependence of the relative intensity of ANSfluorescence on concentration of ions in buffer solution andtime exposition on radiation.

molecules [24]. The ion pairs are formed betweenpositively and negatively charged groups as asecond interaction [25]. Charge pairs link 93% PCmolecules in membrane. Water bridges andcharge pairs together form an extended networkbonding almost 98% of membrane lipids. Basedon the four region model of the lipid bilayermodel described by Marrink and Berendsen[26,27] the region where described phenomenashould occur is an interphase. The total waterdensity decreases and the lipid density reachesmaximal value within this area which leads to thehighest density in the membrane. Many partialcharges are present there. This region plays themost important role in the lipid phase transitionphenomena. All present water molecules are lo-cated within structure of hydration shells of thephospholipid groups and interaction with waterstrongly influences on the surface tension and thediffusion coefficients of water and small moleculeswhich are lower as compared with the first region,perturbed water. Penetration of the negativelycharged ANS into interphase could be restrictedby,� the additional negative charge [21,22];� the decreasing width of the membrane-solution

interphase [20].The PC molecule, zwitterionic lipid but electri-

cally neutral, is in equilibrium with H+ and OH−

ions depends on the external pH. The non-esterphosphate in head group of PC is the protonbinding site when pH is very low. Increasing pHdissociates proton from phosphate and PCmolecule from acidic; positively charged form be-comes neutral; zwitterionic. Anions OH− adsorbon the surface to form the basic form of PCmolecule at high values of pH [28]. The dissocia-tion constants of acidic and basic form of PC onthe liposome surface were determined by titrationand equal pKa=2.581 and pKb=5.687, respec-tively [28]. At pH 7.4, phosphates are fully depro-tonated so that no meaningful increase ofnegative charge could be detected.

In our earlier studies, the mobility of TEMPO-palmitate spin label (it monitors the interphasearea) decreases after exposition to NIR what sug-gests the increasing microviscosity within the po-lar heads area [18]. It could occur during

are within experimental error. At pH 7.4 theintensity of fluorescence decreases 15% after 15min and reaches 23% after 30 min irradiation.This value stabilizes for exposition times longerthan 30 min Fig. 2 shows that the intensity offluorescence of ANS in solution grows about 25%after 30 min irradiation. It indicates that thedecreasing intensity of fluorescence is due to lowerconcentration of ANS incorporated into liposomemembrane after irradiation. Growing concentra-tion of electrolytes, 14 and 140 mMol of NaClwere added into 310 mOsm phosphate buffersolution at pH 7.4, cancels the effect of radiation,Fig. 3.

4. Discussion

A PC molecule contains negatively chargedgroups, the non-ester phosphate oxygen and car-bonyl oxygen in ester bonds, positive charge islocated on choline groups. Two classes of interac-tions between PC headgroups in the liquid crys-talline phase were established from simulationstudy and confrontation with experimental dates[24,25]. In average 4.5 water molecules are hydro-gen bonded to every PC molecule (excluded thewater structure around choline groups). The shortdistance interaction occurs between negativelycharged oxygens via only one water moleculesimultaneously hydrogen bounded to two PCmolecules. About 70% of PC molecules are hydro-gen bonded into clusters of two to seven

M. Komorowska, A. Czarno l*eski / Colloids and Surfaces B: Biointerfaces 20 (2001) 309–314 313

dehydratation of the liposome surface [20,29].When the interfacial area diminishes, penetrationANS ions (dissolved in the bulk solution) into thebilayer is restricted. Thus, we suppose that thepartly dehydrated liposome surface is responsiblefor lower concentration of ANS in the interphase.Interfacial hydration can affect the electrostaticmembrane potential to a distance 0.5–1 nm andtends to increasing the small electrostatic poten-tial, which the electroneutral membranes are char-acterized [20].

The difference in a concentration of anionicprobe ANS illustrates the changes of the electro-static surface potential due to dehydration andthe changes of the potential can be evaluatedfrom Eq. (6). Total fluorescence intensity I fromANS probe with concentration [CANS] is a sum ofintensities of bonded form and free in solution.

I=IB+IF (7)

IB=a8B[CS�ANS] (8)

IF=a8F[CANS] (8a)

where 8B and 8F are fluorescence efficiencies ofbound and free ANS probe, respectively, a ; is theextinction coefficient which is the same for bondand free probe [21].

The maximal fluorescence Imax is obtained whentotal probe introduced to the solution is boundedinto membrane,

[C0]= [CS�ANS]+ [CANS] (9)

then

Imax=a8B[C0] (10)

Imax is measured by the liposome addition intothe constant amount of ANS (using during exper-iments) until total probe in solution is bonded tothe membrane (stable values of the fluorescenceintensity).

From Eqs. (7) and (10) is obtained,� I−IF

Imax−I�

=[CS�ANS]

{[CANS]1−8F/8B}(11)

Equation can be simplified because 8F�8B,for ANS 8F:10−2 8B thus, the ratio 8F/8B isignored and IF�I (IF=0.08 a.u., I=61 a.u.).The Eq. (6) is rewritten is the form,

I(Imax−I)

=b [L0]Kc exp!−hBFc

RT"

(12)

The surface charge is almost stable after irradi-ation and diminishing intensity of fluorescence isdue to the dehydration what decreases the concen-tration of ANS probe incorporated to the mem-brane. The difference of potential induced by lightcan be calculated according to following formulaobtained from Eq. (12) for dark sample and irra-diated one, respectively.

Iirr.

(Imax−Iirr.)=b [L0]Kc exp

�−hBFcirr.

RT�

(13)

For negative charge on the membrane surfacehB= −1 thus,

cdark−cirr.

=RTF�

ln! Idark

(Imax−Idark)"

− ln! Iirr.

(Imax−Iirr.)"n

(14)

After substitution experimentally obtained val-ues, Imax=16096 a.u., Idark=6196 a.u., Iirr.=4595 a.u.

cdark−cirr.=36915 mV

From described above calculations the surfaceelectrostatic potential decreases about 36 mV afterexposition on light what corroborates the sugges-tion that dehydration is manly responsible forchanges of electrostatic potential.

The action of NIR on the liposome membranesis most effective at neutral pH. The explanation ofthe strong dependence on pH of the observedphenomena is complicated by a number of factors[24]. The pH at the interfacial area is shifted tohigher values by as much as 3 units in comparisonto the bulk solution. The head group hydrophilic-ity strongly depends on pH due to direct electro-static effects as well as hydration effects. Thestrength of hydrogen bonds between water andlipid molecules or interlipid hydrogen-bondingalso depends on pH. At lower pH partly protona-tion of head groups may cause to form dehy-drated bilayers with tilted hydrocarbon chains dueto broken hydrogen bound [15]. When pH in-creases complete deprotonation of the non-esteroxygen from head groups of PC occurs whatincreases the membrane hydration and out of

M. Komorowska, A. Czarno l*eski / Colloids and Surfaces B: Biointerfaces 20 (2001) 309–314314

plane fluctuations [25]. Subsequently, increasingadsorption of OH−1 occurs what additionallyweakens the hydrogen bonds. Only for neutrallipid surface and neutral external pH the networkof hydrogen bonds on the surface is enhanced. Ifthe medium sensitive to NIR radiation is thesystem of hydrogen bonds on the membrane sur-face thus, the action of NIR should be restrictedto the neutral pH. The same dependence of pHwas observed for kinetics for dissociation hydro-gen bonded complexes of TEMPO-spin label andaminoacids and amines induced by NIR.Photodissociation of these complexes was alsorestricted to the neutral values of pH [17]. Increas-ing the bulk concentration of monovalent sodiumcations shifts the chain-melting transition temper-ature to the higher values even up to 50 K for theneutral phospholipids due to displacement of thebound water from the interface [29]. Thus, theaction of the radiation is cancelled by Na+ ionsfor the same reasons like the low values of pH. Inconclusion all effects of NIR radiation could beexplained qualitatively as changes of the dehydra-tion of the liposome surface.

Acknowledgements

The work has been sponsored by the PolishNational committee for Scientific Research(KBN) under the TU research projects.

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