Hydration effects under near-infrared radiation

Colloids and Surfaces B: Biointerfaces 26 (2002) 223–233

Hydration effects under near-infrared radiation

Małgorzata Komorowska *, Magdalena Gałwa, Blanka Herter,Urszula Wesołowska

Institute of Physics, Wroclaw Uni�ersity of Technology, Wyb. Wyspianskiego 27, 50-370 Wrocław, Poland

Received 14 May 2001; received in revised form 7 November 2001; accepted 10 December 2001

Abstract

Changes in liposome membrane surface properties induced by near-infrared radiation (NIR) have been observedusing two methods: spin labels and microscope observations. Multilamellar liposomes were prepared from egg yolkphosphatydylcholine (PC). Isotropic tumbling correlation time (�c) for PC liposome membranes, calculated from EPRspectra, increased after irradiation (700–2000 nm). Both the Arrhenius plot temperature of discontinuity for theTEMPO-palmitate (TP) spin probe correlation time incorporated into the PC bilayer, and calculated activationenergy of its mobility are shifted towards higher values after irradiation, but only at neutral pH. The ability ofliposomes to agglomerate was modified considerably by irradiation when observed under optical microscope. Allobserved phenomena have been discussed as a result of NIR hydration effects. © 2002 Elsevier Science B.V. All rightsreserved.

Keywords: Hydration; Liposomes; Near-infrared; Spin labels; Phase transition; Egg yolk phosphatydylcholine; Agglomeration

www.elsevier.com/locate/colsurfb

1. Introduction

The effect of light, ranging from visible tonear-infrared (NIR), on tissue has been studiedintensively for medical and biological reasons.

(i) The regulation of circadian-rhythm by light[1].

(ii) Light therapy [2–5].(iii) The stimulating activity of light [6–8].(iv) Diagnostics and medical analysis [10,11].

The absorption and scattering spectra of vari-ous human tissues (in vivo and in vitro) show thatthey absorb radiation from the region 750 to 2000nm. For example, hemoglobin is characterized byabsorption bands of 760, 805, 820, 910, 1020 nm.Lipids absorb 770, 920, 1040 nm wavelengths,proteins: 910, 1020 nm and water: 749, 880, 980,1211, 1450, 1787 nm. Only hemoglobin absorp-tion bands include electronic transitions (except910 nm), the others being vibration overtones orcombination bands [9,12–14].

Our earlier studies on spin-labeled bovine ery-throcytes clearly showed that exposure to infraredradiation (700–2000 nm) leads to structuralchanges inside the hydrophobic region of the ery-throcyte membrane and to decrease in polarity in

* Corresponding author. Tel.: +48-71-320-3186; fax: +48-71-328-3696.

E-mail address: [email protected] (M. Ko-morowska).

0927-7765/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.

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M. Komorowska et al. / Colloids and Surfaces B: Biointerfaces 26 (2002) 223–233224

the vicinity of the polar heads [15]. The structuralchanges observed in the erythrocyte membraneare probably due to NIR absorption byhemoglobin or �NH, �OH, �SH or �POHprotein/lipid groups.

The primary points of discussion are mecha-nisms that can occur after excitation of the groupsmentioned by radiation. All of them are involvedwith hydrogen bonds. Zundel et al. has studiedeasily polarizable hydrogen bonds [16,17], whichare characterized by the presence of continua intheir infrared spectra. For this type of hydrogenbonds, proton transfer can be easily controlledwith local electric fields generated by fixedcharged groups, cations, polar molecules or evenby coupling proton motion with hydrogen bondstretching vibration. The hydration or dehydra-tion of such systems causes various changes. As aresult of interaction with water, the proton polar-izability of hydrogen bonds usually decreases. Forexample, in tyrozine–phosphate systems, watermolecules break the H-bonds with large protonpolarizability [16]. For this reason, even minimalchange in the hydration of such systems stronglyinfluences the presence of polar structure, the finalproduct of proton transfer.

Our earlier studies evidently showed that NIRirradiation reduces the electrostatic surface poten-tial of liposome membranes. Change in electro-static potential was measured using thefluorescence probe technique [18]. The fluores-cence intensity of 1-anilinonaphthalene-8-sulfate(ANS) was studied from 4.8 to 9.5 pH before andafter exposure to NIR (700–2000 nm). Fluores-cence intensity decreased after exposure only atpH 7.4. Such an effect is due to the decreasingconcentration of ANS in the liposome membraneafter irradiation. Modified ANS distribution inthe liposome membrane upon irradiation is at-tributed to membrane surface dehydration, whichdiminishes electrostatic surface potential by about36�15 mV [18].

The experiments presented are a continuationof our studies on the action of NIR on theliposome membrane. The aim is to investigate theinfluence of this radiation on the structural,colloidal and thermotropic properties of multil-amellar liposomes formed from egg yolk phos-

phatydylcholines. All these properties are stronglydependent on the interaction of water with lipidpolar heads. The spin probe method was em-ployed, as it is sensitive to local structural changesin the probe’s vicinity. Lipid mobility and thus themobility of the spin probes incorporated into theliposome bilayer are functions of environmentfluidity. Such factors and environment polarityinfluence the shape of the spin probe EPR spectra[19]. The spectra of the TEMPO-palmitate spinprobe were studied for changes within the lipo-some membrane induced by NIR. This spin probeis located in the interface area where interactionbetween lipid polar heads with water takes place.

2. Materials and methods

2.1. Materials

Egg yolk phosphatydylcholine (PC) andpalmitic acid (PA) was obtained from Sigma,TEMPO-palmitate (TP) spin label was producedat the University of Todz (Poland), the bufferchemicals employed were of reagent grade purity,deionized and redistilled water was used.

2.2. Liposome preparation

Multilamellar liposomes were used in all experi-ments. PC in chloroform solution was dried undervacuum in a glass tube and resuspended in a 310mosm phosphate buffer at appropriate pH underan argon atmosphere. The mixture was mechani-cally shaken at room temperature until a milkysuspension was obtained. Final lipid concentra-tion was 50 mg/ml. Liposomes containingpalmitic acid (PA), molecular ratio of lipid to acidbeing 200:1, acid was added to the lipid solution,which was then mixed and dried under vacuum.Then the same procedure as described above wasapplied.

Liposomes were labeled by a 1×10−3 Methanolic solution of the TP spin probe (molecu-lar ratio of lipid to spin probe being 200:1). Thespin probe was added to the lipid solution beforeliposome formation. In all experiments, 310 mosm

M. Komorowska et al. / Colloids and Surfaces B: Biointerfaces 26 (2002) 223–233 225

phosphate buffers were used at pH 4.8, 6.3, 7.3and 8.3. The suspensions thus prepared were ex-posed to radiation.

2.3. Irradiation procedure

In order to investigate the effect of NIR, lipo-somes under argon atmosphere were exposed tothe radiation of a halogen lamp with a 700–2000nm filter. Samples were kept in a dark cell andlight was focused on the flat glass tube containingthe suspension. The power density of the incidentlight on the glass tube was 6.9 mW/m2. Thesuspension was gently stirred and cooled duringexposure to light. Irradiation temperature waskept constant 298�2 K by means of a water-cooling system. Except for irradiation, the controlsamples were treated identically. Samples wereirradiated 60 min for microscope observationsand 5, 15, 30, 45 and 60 min. for EPR measure-ments. After each exposure, six samples weretaken for both microscope observations and EPRmeasurements.

2.4. Microscope obser�ations

Vesicles in buffer solution were studied byphase contrast microscopy using an optical micro-scope Olympus BH-2 with camera. Pictures weretaken under an augmentation of 250. To avoidthe sedimentation of multilamellar liposomes, thesuspension was gently stirred and then placed inthe objective of the microscope.

2.5. EPR measurements

All spectra were recorded at 300 K using astandard SE/X-28 ESR spectrometer operating inthe X-band (manufactured by the Wroclaw Uni-versity of Technology). In order to estimate spinprobe mobility during its isotropic rotational mo-tion, tumbling correlation time was calculated[19,20] as

tc=6.5×10−10�H0[(H0/H−1)0.5−1]

where H0 and H−1 are parameters taken from theEPR spectrum, relatively mid-field and high-fieldline amplitudes. �H0 is the line width of the

mid-field signal. The thermal dependence of �c

was taken at the temperature range 283–333 K.Thermotropic properties were studied using aBruker ESP-300E X-band spectrometer with avariable temperature unit, model B-T 2000(Bruker, Karlsruhe, Germany). Isotropic hy-perfine nitrogen splitting was monitored as a mea-sure of environment probe polarity [19],

Aiso=1/3(A�+2A�)

where A� and A� are outer and inner hyperfinesplitting taken from the EPR spectra of the TPspin probe.

3. Results

Changes in the membrane structure of lipo-somes induced by NIR were monitored by aparameter directly related to tumbling correlationtime,

t={[(H0/H−1)irr.− (H0/H−1)dark]/(H0/H−1)irr}

×100%

where (H0/H−1)irr is the ratio of mid-field andhigh-field line amplitudes for irradiated samples,and (H0/H−1)dark that for samples kept underdarkness. The line width of the mid-field signalwas stable during the experiment.

Fig. 1 illustrates the dependence of the parame-ter t calculated for PC liposomes as a function ofNIR exposure time and at pH 7.3. Positive per-centage values indicate that �c increases and nega-tive that �c, decreases with reference to the darksamples. During the first 5 min of irradiation theparameter increases by about 4% and furtherirradiation does not change it. At pH 4.8 theparameter t decreases after irradiation to a mini-mal value of 6.6% (Fig. 2). The addition of asmall amount of palmitic acid to the liposomes(molecular ratio PC/PA being 200:1) reverses theeffect of NIR on t. Parameter t decreases withincreasing irradiation time by approximately 4%,see Fig. 1. Tumbling isotropic correlation time �c,for dark liposome samples prepared from pristinePC is smaller than that for liposomes doped byacid (see Table 1). The irradiation of a sampledoped by palmitic acid (pH 4.8) shifts t down as


shown in Fig. 2. During the first few minutes ofirradiation parameter t decreases by 6%, and thedifference rises to 10.6% after 60 min ofirradiation.

The calculated isotropic hyperfine splitting con-stant Aiso for both pH values changes duringirradiation (Table 2). This constant drops from1.593�0.004 to 1.587�0.003 mT for liposomesat pH 7.3 and increases after irradiation at pH

4.8. When polarity around the spin label de-creases, the isotropic hyperfine splitting constantalso decreases and vice versa.

The spectra of TP incorporated into PC lipo-somes are evidently isotropic thus its thermalproperties can be presented as Arrhenius depen-dencies of �c. Breaks in these Arrhenius plotssuggest effects such as phase transitions at thecharacteristic temperature Tp (Fig. 3, Table 2).

Fig. 1. The dependence of parameter t on exposure time to NIR at pH 7.3: for pristine PC liposomes (upper points) and PCliposomes doped by palmitic acid (lower points). Experimental errors are standard deviations.

Fig. 2. The dependence of parameter t on exposure time NIR at pH 4.8: for pristine PC liposomes (upper points) and PC liposomesdoped by palmitic acid (lower points). Experimental errors are standard deviations.


Table 1Tumbling correlation time, �c (T=300 K), for dark samples atpH 7.3 and 4.8

pH 4.87.3Sample �c (s)×10−9 �c (s)×10−9

1.240.99PCPA 1.291.07

PC— liposomes formed from pristine egg yolk phosphatydylo-choline; PC+PA— liposomes doped by palmitic acid. Stan-dard deviation was ��c=0.02×10−9 s.

Fig. 3. Arrhenius plot of �c for PC liposomes at pH 7.3: (�)for the dark sample and (�) for the irradiated sample (1 h).

Table 3Characteristic temperature Tp for PC liposomes at various pHbefore and after irradiation (1 h)

Tp (K)pH

Dark Irradiated

298 2978.3 phosphate buffer2997.3 phosphate buffer 308

7.3 NaCl 294 3106.3 phosphate buffer 298301

Experimental error was �Tp= �2.5 K; liposomes were sus-pended in an isoosmotic phosphate buffer or physiologicalsolution equilibrated by NaOH to pH 7.3

Subsequently, the linear dependence of the loga-rithm of �c plotted against 1/T allows us to calcu-late the activation energy of spin label mobility(Table 4) at different solvents and pH values.Differences in Tp between control liposomes (keptin darkness) at pH 6.3, 7.3 and 8.3 are notsignificant. Exposure of liposomes to NIR in-creases Tp values from 299 to 308 K in buffersolution and from 294 to 310 in physiologicalsolution but only at neutral pH (Table 3).

A closer look at variations in thermal proper-ties caused by the exposure of liposomes to NIR,is shown in Table 4. The mobility activationenergy of TP spin label, incorporated into controlliposomes, depends strongly on the surroundingmedium. A significant difference in activation en-ergy �E1 below Tp is visible when liposomes aresuspended in physiological solution at neutral pH:then �E1 equals 32.8�3.6 kJ/M while for lipo-somes in buffer solution �E1=21.0�3.2 kJ/M.No significant differences (within experimental er-ror) in �E1 between liposomes in buffer solutionat pH: 6.3, 7.3 and 8.3 are noticeable. Activationenergy above Tp (�E2) decreases and this effectstrongly depends on pH. For elevated pH (8.3),

the slope of the linear dependence of ln �c versus1/T are constant, in fact, the temperature breakdisappears. When pH falls, �E2 also decreasesfrom the value 27.5�4.8 kJ/M at pH 8.3 to15.4�2.7 kJ/M at pH 7.3 and 19.5�2.5 kJ/M atpH 6.3.

The exposure of liposomes suspended in buffersolution to NIR does not have any effect on thevalue of spin label mobility activation energybelow the temperature break at pH 6.3 and 8.3.

Table 2The isotropic hyperfine splitting constant Aiso before and after exposure to NIR for TP spin label incorporated into PC liposomesat pH 7.3 and 4.8

pH 7.3 4.8

Aiso (mT)

Dark Irradiated Dark IrradiatedSample

1.593�0.004PC 1.622�0.0021.587�0.003 1.617�0.004


This value increases significantly from 21.0�3.2kJ/M for dark samples to (34.3�3.6) kJ/M afterexposure to NIR, at neutral pH and in buffersolution. The same result is visible for liposomesin physiological solution. The activation energy ofspin probe mobility increases from 32.8�3.6 to42.8�4.3 kJ/M. Above the temperature break,activation energies for liposomes irradiated atneutral pH in both solutions markedly decreaseand reach values comparable with �E2 at pH 6.3and 8.3.

Optical microscope observations provided in-teresting results. In the field of vision, PC lipo-somes easily associated, forming a uniformlyspread surface (Fig. 4a). After 1-h exposure to

NIR at 298 K the observed associates are smaller(Fig. 4b). When PC liposomes are doped with PA,aggregates are small (Fig. 5a). The irradiation ofdoped liposomes induces agglomeration, makingaggregate size comparable to that of pure PC(Fig. 5b). Exposure to NIR at pH 7.3 evidentlymodifies the ability of the membrane to disperse.Both processes were reversible. The identical pic-tures as before modification were visible undermicroscope, however we noticed recovery the nextday after irradiation.

At pH 4.8 aggregates are smaller than at pH 7.3and no meaningful change in aggregation inducedby NIR between pure and doped liposomes wasobserved (see Figs. 6a, b and 7a, b).

Table 4Activation energies as determined from Arrhenius plot slopes for the isotropic correlation time of TEMPO-palmitate spin labelincorporated into the liposome membrane

Activation energy (kJ mol−1)pH

Control liposomes Liposomes exposed to NIR

�E1 �E2 �E1 �E2

24.3�5.0 27.5�3.68.3 phosphate buffer 24.8�3.7 27.5�4.825.9�4.234.3�3.615.4�2.77.3 phosphate buffer 21.0�3.2

42.8�4.3 29.7�3.37.3 NaCl 32.8�3.6 22.1�3.219.5�2.526.7�2.7 21.7�2.36.3 phosphate buffer 26.8�2.9

Activation energy �E1 (below Tp) and �E2 (above Tp) are shown; liposomes were suspended in an isoosmotic phosphate buffer orphysiological solution equilibrated by NaOH to pH 7.3.

Fig. 4. Aggregate distribution visible under an optical microscope at pH 7.3: (a) PC pure liposomes in darkness and (b) irradiated(1 h). The bar is 55 �m.


Fig. 5. Aggregate distribution visible under an optical microscope at pH 7.3: (a) PC liposomes doped by palmitic acid (molar ratiolipid to acid being 200:1) in darkness and (b) irradiated (1 h). The bar is 55 �m.

Fig. 6. Aggregate distribution aggregates visible under an optical microscope at pH 4.8: (a) PC pure liposomes in darkness and (b)irradiated (1 h). The bar is 55 �m.

Fig. 7. Aggregate distribution visible under an optical microscope at pH 4.8: (a) PC liposomes doped by palmitic acid (molar ratiolipid to acid being 200:1) in darkness and (b) irradiated (1 h). The bar is 55 �m.

4. Discussion

The aggregating behavior of colloidal particles

is described by the Derjaguin–Landau–Verwey–Overbeek theory [21,22]. According to this theory,total free energy consists of the repulsive electrical


double layer and the attractive van der Waalsenergies. At short distances, bilayer dispersion isrestricted by additional repulsive short-rangeinteractions namely hydration repulsion. Thisfactor is not involved in the theory underconsideration but it is adequately discussed byRand and Parsegian [23]. When surface potentialor surface charge diminishes or approaches zero,liposomes attract each other. As the van der Waalsattraction of membranes is very weak, aggregationoccurs only if a large contact area is provided [24].

The surface of liposomes formed from PC at pH7.3 is uncharged because at this pH PC phosphategroups (pK�2.581 for PO4

− in egg yolk PC [25])are fully deprotonated and the negative charge iscompensated by positive charged ammoniumgroups [26]. This explains the image observed undermicroscope: uncharged pristine PC liposomesassociate easily. Subsequently, liposome separationobserved under microscope indicates that theliposome surface becomes charged after exposureto radiation.

In a more acidic environment (pH 4.8) theconcentration of protonated PC phosphate groupsincreases, and surface charge is slightly positive[26].

For comparison with the agglomerating abilitiesof charged liposomes, we introduced a lowconcentration of palmitic acid to the bilayer. Thismodified surface is negatively charged (at pH 7.3)due to deprotonated carboxylic groups (pK 7.3 forRCOOH in PC) [26,27]. This charge separatesliposomes, as is visible under microscope (Fig. 6aand b). When liposomes doped by PA liposomes(pH 7.3) are exposed to NIR, one can observe animage identical with that of pristine PC at pH 7.3before irradiation. NIR probably neutralizes theadditional negative charge of the carboxylicgroups, hence the uncharged membranes associateeasily. At pH 4.8, PA is fully protonated [27]. Forthis reason, microscope images at pH 4.8 forliposomes containing PA should be nearly identicalwith the undoped liposomes, which is apparentlytrue (Fig. 7a and b). Also no meaningful change inaggregation is visible after irradiation. The resultsmentioned corroborate our explanation of themicroscope observations for pure PC: NIRradiation introduces charge to the liposome

membrane surface.On an average, 4.5 water molecules are hydrogen

bonded to each PC molecule, to its non-esterphosphate oxygen and carbonyl oxygen of its estergroup. Both are negatively charged. Only two ofthese hydrogen bonds simultaneously link twodifferent lipid molecules. About 70% of all lipidmolecules are hydrogen bonded and form clustersfrom two to seven molecules, mostly via phosphategroups. The presence of lipid molecules in thezwitterionic state suggests that between theirnegatively and positively charged groups, theadditional ionic interaction occurs similar to thatof ionic pairs in the protein molecule [28,29].Around the hydrophobic choline group, a clathratewater structure is experimentally observed [30].Molecular simulation showed that the perturbationof the global water hydrogen network extendsroughly two solvation layers from the hydrophobicside chain surface and is characterized by adistribution of hydrogen bonded ring sizes that ismore planar and dominated by pentagons inparticular than those near the hydrophilic sidechain [30].

As a rule, a thinner water-bound surface causesthe surface values of electrostatic membranepotential [26] and facilitates proton transfer [31].This leads to a decrease in interfacial area permolecule [32]. The protonation of non-esterphosphate oxygen, the only ones that can beprotonated, dramatically changes the existinghydrogen bond network in the liposome membraneinterface. Increasing values of TP-spin labelcorrelation time (incorporated into liposomes andsuspended in phosphate solution at pH 7.3) afterexposure to NIR illustrates this phenomenon. Theprotonation of polar heads leads to dehydration[32] thus �c increases slightly at lower pH and forliposomes doped by PA (control samples) ascompared to the pristine ones (Table 1). Never-theless, the influence of NIR on these liposomes ismanifested only by the increasing mobility of spinlabel. At such acidic pH and in the presence ofpalmitic acid, spin label protonation andsubsequent reduction induced by NIR occur [33].

The discontinuity that is visible in thetemperature dependence of spin label motionalcorrelation time does not reflect chain-melting main


phase transition. In contrast to synthetic lipids,main transition for fully hydrated egg PC is verybroad and begins around 258 K [34]. Effectssimilar to phase transition were reported for mul-tilamellar liposomes produced from bulk erythro-cyte lipids [35,36]. Thermally induced structuralchanges occur in the lipid membrane polar regionand at the depth of the fifth carbon of the hydro-carbon chain. Break temperature, determined forspin label mobility temperature characteristicswas 313 K for the hydrophobic region and 290 Kfor the polar heads region. These results confirmthe presence of such a temperature break, foundfor spherical lipid membranes. The transition tem-perature of this lipid structure, determined fromthe temperature dependence of resistance, capaci-tance and the water filtration coefficient was inthe range 307–311 K. The activation energy ofion conductivity below this temperature reachedthe value of 14.7�2.1 kJ/M and above it reached25.5�2.5 kJ/M [35,37,38]. The similarity of thethermal properties of membranes formed fromnatural lipids suggests that this phenomenon isassociated with the same factor.

Main transition from gel to fluid phase origi-nates from the reorientation and flexibility ofhydrocarbon chains within the bilayer. As a con-sequence of chain melting, polar head area in-creases and membranes become fully hydrated.However, these effects do not take bonds betweenwater and phosphate or carbonyl groups on themembrane surface into account. At elevated tem-peratures, the hydrogen bonds existing betweenphosphate oxygen and water in fully hydratedbilayers weaken, enhancing membrane surface hy-drophobicity and the protonation of these groups[26,32,39]. Decreasing spin label mobility activa-tion energy above the temperature break illus-trates the mobility of the nitroxide group whenthe hydrogen bond network within the interface isbroken. After exposure to NIR, water is partlyexpelled from the surface, having only that whichis strongly linked. The calculated isotropic hy-perfine splitting constant Aiso decreases, showingdecreasing polarity in the vicinity of spin label.Both head group area per molecule and spin label

mobility also decrease. Thus, the activation en-ergy of spin label mobility should increase. Thepresence of sodium ions, known to be strongmodifiers of water structure [40], increases theenergy value by about 10 kJ/M. The same energyvalue shift is visible after exposure to NIR.

The activation energy obtained for nitroxidesrotational motion is nearly the same as that calcu-lated using dielectric spectroscopy for the mobilityof free water—21 kJ/M and water bonded withDOPC (dioleoylphosphatidylcholine) and DMPC(dimyristoylphosphatidylcholine) in the liquidcrystalline state—30 kJ/M [41].

The action of NIR is most effective at neutralpH. The results presented fully agree with ourearlier studies [18]. The explanation of such adependence is not easy. We suggest that theseexperiments illustrate the existing equilibrium be-tween structure of global and hydrogen bondedwater with liposome surface. Moreover watermolecules do absorb NIR wavelengths [12].

The presence of excess protons or OH− groups,at pH lower or higher than neutral, dramaticallychanges global water structure, hydrogen bondsstrength and ion concentration near the liposomesurface. All these phenomena must have influenceon the action of NIR, if the primary targets ofradiation are hydrogen bonds.

Discussing our results, we cannot exclude thepossibility of lipid oxidation during irradiation.The products of egg yolk phosphatidylcholineoxidation may have influence on the parametersmeasured. However, the consecutive argumentsallow us to neglect this process:� liposomes were irradiated under an argon

atmosphere;� spin label is known to be an inhibitor of lipid

peroxidation [42–44];� peroxidation does not influence the thermal

properties of liposomes obtained from egg yolkPC [45];

� peroxidation is not restricted to neutral pH[46].Thus the phenomena presented can be dis-

cussed as dehydration processes that are due toweakened hydrogen bonds after the absorption ofnear-infrared radiation.


Acknowledgements

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

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