Research Article Aggregation Behavior of Some Asymmetric ...downloads.hindawi.com/journals/ijp/2015/302587.pdfResearch Article Aggregation Behavior of Some Asymmetric Porphyrins versus

Research ArticleAggregation Behavior of Some Asymmetric Porphyrins versusBasic Biological Tests Response

Radu Socoteanu1 Mihai Anastasescu1 Anabela Oliveira23 Gianina Dobrescu1

Rica Boscencu4 and Carolina Constantin5

1Romanian Academy ldquoIlie Murgulescurdquo Institute of Physical Chemistry 202 Splaiul Independentei 77208 Bucharest Romania2Escola Superior de Tecnologia e Gestao Lugar da Abadessa 7301-901 Portalegre Portugal3Centro de Quımica-Fısica Molecular e Instituto de Nanociencias e Nanotecnologias Instituto Superior TecnicoUniversidade de Lisboa Avenida Rovisco Pais 1049-001 Lisboa Portugal4Faculty of Pharmacy ldquoCarol Davilardquo University of Medicine and Pharmacy Faculty of Pharmacy 6 Traian Vuia Street020956 Bucharest Romania5ldquoVictor Babesrdquo National Institute for Pathology and Biomedical Sciences 99-101 Splaiul Independentei 050096 Bucharest Romania

Correspondence should be addressed to Mihai Anastasescu manastasescuicfro and Anabela Oliveira asoliveiraestgppt

Received 12 August 2015 Accepted 29 September 2015

Academic Editor Maxim P Evstigneev

Copyright copy 2015 Radu Socoteanu et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Fractal analysis of free bases porphyrins was computed on atomic force microscopy (AFM) micrographs using two differentmethods the correlation function method and the variable length scale method The correlation function method provides fractaldimension only for short scale range results indicate that only few images have fractal properties for short ranges for the rest ofthem no fractal dimension was found using the correlation function methodThe variable length scale method occur informationfor long range scaling All samples have fractal properties at higher scaling range For three samples the correlation functionmethodleads to the same fractal dimension as the variable length scale method and scaling ranges for both methods overlap Results showthe necessity to use bothmethods to describe the fractal properties ofAB

3meso-porphyrins thatmay be used to predict their relative

cell localization In order to emphasize the influence of fractal and textural properties the results regarding their self-similarity andtexturemorphology were further compared with their behavior in biological assessment that is functionality of some Jurkat celllines

1 Introduction

Porphyrins are more and more involved in ldquolong-chainrdquostudies with multiple different approaches of the subjectmainly in diagnosis and therapy of cancer the emergingconcept of theranostics targeting the third generation ofphotosensitizers The use of the porphyrin derivatives inbiomedical field is a consequence of their ability to generatereactive species [1]The application domains are continuouslyexpanding [2 3] their use as sensitizers in photodynamictherapy of cancer being today the object of many interdis-ciplinary studies [4] Although a large number of meso-substituted porphyrins were synthesized since the approvalof the first photosensitizer for clinical photodynamic ther-apy [5] there are still improvements to be made in their

synthesis [6] and there is still no clear correlation between themesosubstituents of porphyrins and their selective accumu-lation and phototoxicity in tumor cells Recently it has beenestablished that subcellular localization plays a major role inphotodynamic efficacy [7]

We consider the balance between symmetry and asym-metry important regarding the influence of peripheral sub-stituents especially in tissue uptake This view is supportedby the fact that certain changes in the functional groupson a tetrapyrrolic macrocycle including those attached inmesopositions influence in significant way the reactivity andmechanism of the porphyrins involved in environmentalreactions Due to the extended electronic system the por-phyrinic behavior is marked by strong tendencies to formlarge aggregates [8] Most used methods to evaluate the

Hindawi Publishing CorporationInternational Journal of PhotoenergyVolume 2015 Article ID 302587 11 pageshttpdxdoiorg1011552015302587

2 International Journal of Photoenergy

(a)

(b)

Figure 1 Face-to-face (a) versus side-by-side (b) aggregates displayed on 5101520-meso-tetrakis-(34-methylenedyoxy)-phenyl-2123Hporphine

capacity of self-association of this type of molecules areset around spectral analysis as liquid samples [9] Severalparameters as pH and solvent polarity play a major rolein aggregation capacity in the liquid state of the sampleBy using AFM technique these influential parameters areexcluded The binomial frame formed by AFM studies andporphyrin structures seem to be lately point of interest mostof them focusing on symmetric structures free bases [10]from simple compounds [11] to advanced materials [12 13]From the behavior of porphyrins bind with peptides [14]to potential sensing devices [15] aspects as supramolecularchirality attract the attention via AFM studies The variationof peripheral substituents of porphyrins has direct conse-quences on the formation of porphyrin aggregates creatinghigh supramolecular chirality targeting supramolecular net-works that emulate the biological systems [16 17] One of themost detectable features of the porphyrins is the capacity toaggregate through several types of interactions where mostof them are governed by the 120587-120587 interactions or hydrogenbonding This characteristic is desirable for applications intechnology especially in nanotechnology [18] H-aggregates(face-to-face arrangement) and J-aggregates (side-by-side)are the well-known assemblies as shown in Figure 1 withstructures optimized by HyperChem [19] Instead for thebiological applications as it theranostics is the capacity toconglomerate is an obvious constraint of the membranecell penetration process AFM coupled with some cell lines

tests proved to be a useful tool to evaluate the capacity ofcertain porphyrin to interact with the cell membrane In thisexperiment the correlation is made between the scales ofmagnitude and is not related with the kind of aggregation

ldquoScalingrdquo is one of the modern concepts used to char-acterize the morphology of various complex surfaces whichcan be smooth when the ldquoobserverrdquo (ie the tip in scanningprobe microscopy) is far enough and rough when it isclose enough Scaling relations characterize self-similar orself-affine objects and lead to direct computation of animportant parameter fractal dimension [20] Not all roughsurfaces are fractals but if a surface can be described interms of fractal geometry this means that it is self-affinein a statistical way and can be characterized by its fractaldimension Fractal dimension gives information about thesurface roughness a higher fractal dimension meaning thatthe surface is very rough but tells us something about surfacegeometry that is the surface is self-affine This is a veryimportant thing that when something seems to be disorderedand hard to be described self-affinity and fractal dimensiongive quantitative information about the surface and describeit Fractal dimension can be measured in different waysthe direct method implies analysis of topographic imagesobtained from scanning tunneling microscopy [21ndash24] scan-ning probe microscopy [25] or atomic force microscopy[26ndash28] but there are a lot of other indirect methods lightscattering neutron scattering adsorption and so on

International Journal of Photoenergy 3

NH N

HNN

A

B

B

B

for TMHAPP

OH

A = B =

OO

for TMDPP

CH2

A = B =

OO

for TRMDOPP

CH2

A = B =

OO

for TMDOPP

CH2

A = B =

OO

for MHTPP

CH2

A = B =

OH

OH

for THAPPA = B =

OCH3

OCH3

OCH3

Figure 2 Chemical structures of investigated porphyrins (1) TMHAPP (2) THAPP (3) MHTPP (4) TMDOPP (5) TMDPP and (6)TRMDOPP

The aim of this paper is to analyze AFM topographicimages of asymmetric porphyrins (AB

3meso-porphyrinic

type structures) in order to analyze their fractal behav-ior and textural properties and to identify the connectionwith some results of biological tests Two mathematicalmethods were used to compute fractal dimensions firstthe correlation function method [29ndash31] and second thevariable length scale method [32] Our work emphasizes theinterdependence between molecular architecture complexstructure analysis and biological studies especially AFMtechnique and porphyrin-cell interaction in the quest forphotosensitizers with improved properties The order foundin their aggregation tendencies is projected in the behaviorobserved in the cell interaction revealed by cytotoxicity tests

2 Experimental Section

21 Materials and Methods

211 Materials Several asymmetrically substituted porphy-rins (Figure 2) 5-(34-methylenedyoxy)-phenyl-101520-tris-phenyl-2123H-porphine (TMDPP) 5-(3-hydroxy-4-methoxy)-phenyl-101520-tris-phenyl-2123H-porphine(TMHAPP) 5-(3-hydroxy-4-methoxy)-phenyl-101520-tris-(34-methylenedyoxy)-2123H-porphine (MHTPP) and5-phenyl-101520-tris-(34-methylenedyoxy)-phenyl-2123H-

porphine (TRMDOPP) and symmetrical compounds5101520-meso-tetrakis-(34-methylenedyoxy)-phenyl-2123H porphine (TMDOPP) and 5101520-meso-tetrakis-(3-hydroxy-4-methoxy)-phenyl-2123H-porphine (THAPP) wereinvolved in the present studies

For the series of AB3asymmetrical mesoporphyrinic

structures details about synthetic technique are reportedelsewhere [33ndash37]The substituents attached to the porphyri-nic tetrapyrrolic core were chosen so as to balance solubilityaggregationtextural tendencies high singlet oxygen quan-tum yield and cell interaction two levels of asymmetry occurin the same individual structure in the macrocyclic core andof the external substituents complementary to several well-known ldquoclassicalrdquo macrocyclic porphyrin-type compounds

212 Methods

(a) Characterization Methods Atomic force microscopy(AFM) measurements were carried in true noncontact moderecommended for nondestructive sample scan with a XE-100apparatus from Park Systems equipped with flexure-guidedcross talk eliminated scanners using ultrasharp tips (lt8 nmtip radius NCHR type from NanosensorsTM) of 125 120583mlength 30 120583m width and 42Nm spring constantsim330 kHzresonance frequency Different imaging scales were usedfrom 8 times 8 120583m2 to 1 times 1 120583m2 In order to prepare the


specimens for AFM investigations a small quantity of pow-der was ultrasonically dispersed in ultrapure water (Millie-Q gt18MΩcm) thus using the same concentration of thecompounds as used in the biological investigations despitethe differences between the nature of solvents used formicroscopy and cytotoxicity tests this part of study chasesonly the tendencies of aggregation A drop from this sus-pension was deposited on atomically flat Highly OrientedPyrolytic Graphite (HOPG) and dried at room temperatureHOPG was used as to avoid any influence of the substrate onroughness and texture of the investigated samples The PhaseContrast AFM images were processed by Scanning ProbeImage Processor software (SPIPTM v 4600) (SPIP manualavailable at httpwwwimagemetcom)

(b) Correlation Function Method A fractal is an objectwith an observed volume that depends on the resolution(length scale) and follows a power law behavior with anontrivial exponent over several orders of magnitude Themost important property of fractals is self-similarity which isthe property of a part to look like the whole Isotropic fractalsare self-similar they are invariant under isotropic scaletransformationWhen the object scales differently in differentspace directions we call it a self-affine fractal From this pointof view rough surfaces are usually self-affine structures [20]In our work we shall use to compute fractal dimensions twomethods the height correlation functionmethod (119862) and thevariable length scale method (119871) [32] Different parameterscan be used to characterize the surface roughness Oneof these parameters that describe self-affine surfaces is theroughness exponent120572 In addition to the roughness exponent120572 it is possible to associate a fractal dimension119863 with a self-affine function The fractal dimension of a self-affine surfacecan be computed from the height correlation function [29ndash31]

119866 (119903) equiv ⟨119862 ( 119903)⟩119909 (1)

where the symbol ⟨sdot sdot sdot ⟩ denotes an average over 119909 and119862(x 119903)is defined as

119862 ( 119903) = [ℎ () minus ℎ ( +997888119903 )]

2

(2)

the surface being described by the function ℎ(x) which givesthe maximum height of the interface at a position given by xThus the height correlation function119866(119903) obeys the followingscaling relation [29]

119866 (119903) sim 1199032120572 119903 ≪ 119871 (3)

where for a surface embedded in a 3-dimensional Euclideanspace

120572 = 3 minus 119863 (4)

with119863 being the fractal dimensionThe scaling range in which (3) is obeyed is called the

ldquocut-offrdquo limits and it indicates the range of self-affinity inother words the range where there are correlations betweenthe surface points Correlation function method is suitable

for small scaling range because it requires enough points foraverage in (1) meaning that points computed for low scalingrange have low errors and points computed for high scalingrange have high errors

(c) Variable Length ScaleMethodThemodel was proposed byChauvy et al [32] and consists of several steps (i) defining aninterval of length 120576 (or a box of size 120576 times 120576) (ii) performinga linear (or planar) least square fit on the data within theinterval and calculating the roughness (iii) moving theinterval (box) along the profile (surface) and repeating step(ii) (iv) computing Rms deviation for multiple intervals and(v) repeating steps (ii)ndash(iv) for increasing lengths (box sizes)The smallest size for an interval corresponds to 10 data points(10 times 10 points for 3-dimensional embedded objects) and itsmaximum size is the total length of the profile (size of thesurface) Rms deviation 119877

119902120576 averaged over 119899

120576 the number of

intervals of length 120576 is defined by

119877119902120576=

1

119899120576

119899120576

sum

119894=1

radic

1

119901120576

119901120576

sum

119895=1

1199111198952 (5)

where 119911119895is the 119895th height variation from the best fit line

within the interval 119894 and 119901120576is the number of points in the

interval 120576The log-log plot of 119877

119902120576versus 120576 gives the Hurst or

roughening exponent119867 and the fractal dimension119863 can becalculated as

119863 = 119863119879minus 119867 (6)

where 119863119879is the topological dimension of the embedding

Euclidean space (119863119879= 2 for profiles and119863

119879= 3 for surfaces)

Variable length scale method is suitable for higher scalingrange compared to correlation function method because thenecessity to have enough points in an interval 120576times120576 to computeRms deviation 119877

119902120576 averaged over 119899

120576 means that 120576 must be

high enough for a good statistic Both methods correlationfunction method (119862) and variable length scale (119871) methodwill be used to compute fractal dimension of topographicAFM images

22 Preparation Method of Biological Samples Preliminaryin vitro cytotoxicity studies were performed on the Jurkatcell line human T cell lymphoblast-like cell line 10mMstock solutions of the test porphyrinic compounds wereprepared in DMSO by sonication at 22000Hz for 30 secondsFor cellular tests stock solutions were further diluted inRPMI 1640 culture medium in the concentration range 125ndash40 120583M Solutions were handled in sterile conditions Cell linewas cultivated at 37∘C in 5 CO

2humid atmosphere in

RPMI 1640medium supplemented with 100UmL penicillin01mgmL streptomycin 025120583gmL amphotericin 2mMglutamine and 10 fetal bovine serum For cellular viabilityand proliferation assays 5 times 10

minus4 Jurkat cellsmL was incu-bated for 3 h with various concentrations of compounds Thecellular control consisted in unloadeduntreated Jurkat cellsCellular viability was assessed bymeasuring the bioreduction


of a tetrazolium salt (MTS) to a formazan product usingCellTiter 96 AQeous One Solution Cell Proliferation Assaykit (Promega) The reaction performed by dehydrogenaseenzymes takes place in metabolically active cells [38] andtherefore MTS reduction is proportional to the number ofviable cells

Membrane integrity wasmeasured bymeans of the lactatedehydrogenase (LDH) release assay [39] using CytoTox 96Non-Radioactive Cytotoxicity Test (Promega) Briefly cellculture supernatants were collected for the LDH release assaywhilst the rest of the cell suspension was used for the MTSreduction test Optical densities (OD) were measured fortriplicate samples on a Jasco V630 spectrophotometer in asingle beammode at 490 nmwithout any reference (for LDHrelease) and at 490 nm with reference at 640 nm (for MTSreduction) The mean value of triplicates was calculated andresults were further expressed as percent effect relative tocontrol

3 Results and Discussion

31 Topography Typical AFM images for the investigatedporphyrins are presented in Figure 3 at the scale of 2times2 120583m2for samples (1)ndash(6) in which the first column representsthe 2D topographic images presented in Enhanced Colorview mode and the second column the images recorded inPhase Contrast mode and below them are the characteristicsurface profiles (line scans) It was used the Enhanced Colorview mode for the topographic 2D AFM images in orderto increase the morphological details of the samples This isbecause Enhanced Color uses the change of a pixel relativeto its neighbors instead of its absolute value Phase Contrastworking mode was also registered in AFM experimentsas to check for possible chemical inhomogeneities of theinvestigated compounds

Some topographical characteristics that were consistentlyobserved by AFM are discussed further (1) TMHAPP ischaracterized by grains with height up to 10 nm and typicaldiameters in the range of 60ndash100 nm (2) THAPP shows largegrains (agglomerated particles) with diameters from 150 to400 nm and height of around 20 nm (up to 30ndash40 of thelarger ones) Between them some small surface particles arevisible (better in Amplitude or Phase Contrast images) withdiameters in the range of 30ndash40 nmThere is already a visibletendency towards ldquoself-assemblingrdquo for sample (3) MHTPPis in terms of creating large ldquoparcelsrdquo with mean height ofaround 16 nm and length in the microns range (aspect ratioof up to 1100 ndash heightlength) (4) TMDOPP exhibit thehighest tendency towards agglomeration as could be seen inthe corresponding 2D AFM image (topography) 4th row inFigure 3 which could be due to the presence of trace amountsof impurities (5) TMDPP has also a strong tendency to formlarge ldquo2Drdquo islands ofmaterials (the height of the largest islandin the 5th row being 5 nm) instead of stacks as visible forsample (4) TMDOPP (6) For sample TRMDOPP there arevisible some hills beside the formation of large 2D sheets (fewnm height and fewmicrons lengthwidth) with heights up to30 nm

32 Fractal Analysis Fractal dimensions of the porphyrins-based samples were analyzed (Table 1) using AFM micro-graphs and (1)ndash(4) for correlation function method and (5)and (6) for variable length scale method

From the correlation function method combined withvariable length scale method the following fractal char-acteristics could be summarized For sample (1) the baresubstrate was evidenced (119863 = 216 220) the porphyrinpowder being organized in fractal structures of differentvalues 245ndash246 for the correlations between the muchclosed particles (close ldquoneighborsrdquo) and 260ndash281 for longdistance correlations between porphyrin particles (grains)The structure is self-similar on a rather large domain of self-similarity Regarding sample (2) it is also revealed that thebare substrate is visible in fractal analysis at the scale of 1 times 1

and 2 times 2 microns (119863 = 200 and 207 resp) The porphyrinpowder exhibits low distance correlations between particlesand low fractal dimensions (222ndash230) The image at thescale of 8 times 8 microns shows a diffuse structure with fractaldimension of 222 Medium and long distance correlationsare characterized by fractal dimensions on the range of 245ndash257 Sample (3) has a fractal dimension 119863 = 207 and206 for the bare substrate at the scale of 2 times 2 micronswhile the porphyrin is characterized by self-similar structuresof low fractal dimension 222ndash237 at low scales of 2 times 2

microns (not well agglomerated) A fractal dimension of 232is kept at larger scales onwhich is superimposed a self-similarcolumnar structure with 119863 = 253ndash260 In sample (4) self-similar agglomerations (aggregates-stacks) with low fractaldimensions of 223ndash230 on a large domain of self-similarityare obtained on which a structure with medium fractaldimension (251) is superimposed but with a low domainof self-similarity at middle scales Self-similar columnarstructures were obtained for sample (5) with different fractaldimensions without correlations between structures thoughthere is a structure of low fractal dimension (226) on alarge domain of self-similarity In sample (6) besides thebare substrate (119863 = 218) there is an agglomerated self-similar structure with low fractal dimension 229ndash237 on alarge domain of similarity A diffuse but self-similar structurescattered was also observed (119863 = 251ndash254) The agglom-erations are self-correlated with big fractal dimensions270

From all the collected data a profile for the aggregationtendencies could be set as follow TMHAPP lt THAPP lt

MHTPP lt TMDOPP lt TMDPP lt TRMDOPP the last com-pound being the most susceptible to form larger aggregates

33 Cytotoxicity Tests The investigated porphyrinic sampleswere further tested for their biological response in two cellsinteraction tests eventually targeting the cancer therapyThe results are different but complementary as followssignificant influence of aggregation process in the MTS assayresults (Figure 4) and constant low impact behavior in LDHrelease tests (Figure 5)

331 MTS Assay The short-term dark toxicity of theporphyrinic compounds was evaluated on standard T lym-phocytes cell lines Jurkat type For these biosamples was


0 04 08 12 16 2

6420

(nm

)

(1)(120583m)

500nm

(nm

)

10

75

5

25

0500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

0 04 08 12 16 2

(2)

1612

840

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus10

0

10

20

30

40

(nm

)

500nm

0 04 08 12 16 2

(3)

840(n

m)

(120583m)

minus4

(nm

)

10

0

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

minus10

Figure 3 Continued


0 04 08 12 16 2

302010

0

(nm

)

(120583m)

500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

50

75

(nm

)

(4)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nm 0

25

5

10

75(n

m)

(5)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

5

75

10

(nm

)

(6)

Figure 3 Typical AFM recorded images at the scale of (2 times 2) 120583m2 for the investigated porphyrins in which the first column represents the2D topographic images presented in Enhanced Color view mode and the second column the images recorded in Phase Contrast mode andbelow them are the characteristic surface profiles (line scans) (1) TMHAPP (2) THAPP (3) MHTPP (4) TMDOPP (5) TMDPP and (6)TRMDOPP


Table 1 Fractal dimensions and self-similarity domains for samples (1)ndash(6) (1) TMHAPP (2) THAPP (3) MHTPP (4) TMDOPP (5)TMDPP and (6) TRMDOPP

Sample Size (120583mtimes120583m) Fractal dimension Self-similar domain (nm) Linear correlation coefficient

(1)

1 times 1 246 plusmn 001(119862)216 plusmn 002(119871)

19ndash3225ndash103

09970998

2 times 2245 plusmn 001(119862)220 plusmn 002(119871)281 plusmn 001(119871)

23ndash4652ndash129207ndash467

099709980966

8 times 8

245 plusmn 003(119862)271 plusmn 001(119871)266 plusmn 001(119871)260 plusmn 002(119871)

20ndash882182ndash25973012ndash39485714ndash7272

0972099909930947

(2)



099809990991



099709960996



099709970980

(3)

1 times 1



0995099309980995


22ndash15452ndash363

09960998



099609910996

(4)2 times 2 230 plusmn 001(119862)

226 plusmn 002(119871)47ndash28752ndash260

09970997



09930980

(5)



099809950999

8 times 8



0996099509790997

(6)


13ndash3552ndash182

09990996



099809930996

8 times 8



0997099709860997

119862 is correlation function method and 119871 is variable length scale method


06

05

04

03

02

01

00 2 4 6 8 10 12

(120583gmL)

(1)(2)(3)

(4)(5)(6)

MTS reduction-3h incubation

OD490

nm

Figure 4 MTS assay on Jurkat cell line after 3 hours incubation at37∘C for samples (1)ndash(6) (1) TMHAPP (2) THAPP (3)MHTPP (4)TMDOPP (5) TMDPP and (6) TRMDOPP

assessed the cell proliferation from the test of [3-(4 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction the cells beingcultured in 96-well plates in RPMI medium for 3 hoursat 37∘C in an atmosphere containing 5 CO

2with the

porphyrins in DMSO added in different concentrations Thetargeted characteristics of porphyrins act invariably for thechange of the solvent from water to DMSO

Also because the aggregation behavior is largely relatedto the solvents the incompatible ones with biological experi-ments were from start eliminated Water and DMSO provedto be suitable for this type of studies

A direct linear relation between AFM and biologicalexperiments is revealed by the MTS assay studies estab-lishing a perfect match with the aggregation tendencies inporphyrins

TMHAPP lt THAPP lt MHTPP lt TMDOPP

lt TMDPP lt TRMDOPP(7)

It is observed that at incipient values of compoundconcentration the response in terms of the cell viabilityremains between restraint borders Increasing the concentra-tion of added porphyrin solution the influence in the cellresponse scattered the compounds behavior at maximumused concentration the order in response is the same as in thetendency of aggregation scaleThe differences in the values ofoptical density for the MTS release after 3 hours are 5 timesbigger for the TRMDOPP than for the TMHAPPThis impliesthat the porphyrin with lower aggregation tendency behaveless aggressive in bio environment

332 LDH Release The cell viabilitymembrane integrityevaluation was deduced from the lactate dehydrogenase(LDH) release test

We highlighted that the investigated porphyrinic struc-tures might interfere with cellular LDH particularly after24 h incubation but this issue has to be further investigatedanyway from cytotoxicity point of view the different aggre-gation tendencies are not a significant factor in this kind

016

014

012

01

008

006

004

002

0

LDH release-3h incubation

0 05 1 15 2 25 3

(120583gmL)

(1)(2)(3)

(4)(5)(6)

OD490

nm

Figure 5 LDH release on Jurkat cell line after 3 hours incubationat 37∘C for samples (1)ndash(6) (1) TMHAPP (2) THAPP (3) MHTPP(4) TMDOPP (5) TMDPP and (6) TRMDOPP

of tests all the registered values being concentrated in arestraint domain at lower and increased concentrations Asdisplayed in Figure 4 in spite of their significant differentagglomeration tendencies the influence on the integrity ofcellmembrane could be qualified as neutralThe evaluation ofthe amount of LDH release as key index for the permeabiliza-tion of plasma membrane in the presence of the porphyrinicsamples leads to the observation that they are nontoxicand more importantly independent of the aggregationdegree

Biology tests on these particular compounds are theessential departuremilestone in revealing that they localize inthemalignant cells and they have a reduced unwanted activityin the absence of laser irradiation

4 Conclusions

AB3asymmetric meso-porphyrins have been studied by

atomic force microscopy in order to evaluate their morphol-ogy and establish a direct visual pattern of their tendenciesof aggregation AFM images were further analyzed basedon fractal theory with two mathematical methods namelythe correlation function method and the variable lengthscale method From AFM and fractal investigations thefollowing order of the aggregation tendencies of the studiedcompounds has been established TMHAPP lt THAPP lt

MHTPP lt TMDOPP lt TMDPP lt TRMDOPP This order ismaintained in the basic biological experiment result involvedin the assessment of cell metabolic activity targeting cancertreatment the larger the aggregates the lower the power tointerfere in the cells activity Instead the aggregation degreeof porphyrins remains with no consequences when involvedin dark-cytotoxicity tests all proving the same low toxicbehavior against tested cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

This research was supported by Project MNT-7-0302010 ofthe Romanian Ministry of Education and Research Supportfrom the EU (ERDF) and Romanian Government infras-tructure POS-CCE O 221 Project INFRANANOCHEM no192009 is gratefully acknowledged (AFM equipment)

References

[1] K Lang J Mosinger and D M Wagnerova ldquoPhotophysicalproperties of porphyrinoid sensitizers non-covalently bound tohost molecules models for photodynamic therapyrdquo Coordina-tion Chemistry Reviews vol 248 no 3-4 pp 321ndash350 2004

[2] D M Guldi ldquoFullerene-porphyrin architectures photosyn-thetic antenna and reaction center modelsrdquo Chemical SocietyReviews vol 31 no 1 pp 22ndash36 2002

[3] CMDrain A Varotto and I Radivojevic ldquoSelf-organized por-phyrinic materialsrdquo Chemical Reviews vol 109 no 5 pp 1630ndash1658 2009

[4] M Ethirajan Y Chen P Joshi and R K Pandey ldquoThe roleof porphyrin chemistry in tumor imaging and photodynamictherapyrdquo Chemical Society Reviews vol 40 no 1 pp 340ndash3622011

[5] B E Hueger J R Lawter V HWaringrekar andM C CucoloUS Patent no 5059619 1991

[6] J S Lindsey ldquoSynthetic routes to meso-patterned porphyrinsrdquoAccounts of Chemical Research vol 43 no 2 pp 300ndash311 2010

[7] J S Modica-Napolitano M Kulawiec and K K Singh ldquoMito-chondria and human cancerrdquo Current Molecular Medicine vol7 no 1 pp 121ndash131 2007

[8] L Kelbauskas S Bagdonas W Dietel and R Rotomskis ldquoExci-tation relaxation and structure of TPPS

4J-aggregatesrdquo Journal of

Luminescence vol 101 no 4 pp 253ndash262 2003[9] A V Udalrsquotsov A V Bolshakova and J G Vos ldquoHighly ordered

surface structure of large-scale porphyrin aggregates assembledfromprotonated TPP andwaterrdquo Journal ofMolecular Structurevol 1065-1066 no 1 pp 170ndash178 2014

[10] Y Zhang P Chen andM Liu ldquoA general method for construct-ing optically active supramolecular assemblies from intrinsi-cally achiral water-insoluble free-base porphyrinsrdquoChemistrymdashA European Journal vol 14 no 6 pp 1793ndash1803 2008

[11] A V Udalrsquotsov M Tosaka and G Kaupp ldquoMicroscopy oflarge-scale porphyrin aggregates formed from protonated TPPdimers in water-organic solutionsrdquo Journal of Molecular Struc-ture vol 660 no 1ndash3 pp 15ndash23 2003

[12] J A A W Elemans R van Hameren R J M Nolte and AE Rowan ldquoMolecular materials by self-assembly of porphyrinsphthalocyanines and perylenesrdquo Advanced Materials vol 18no 10 pp 1251ndash1266 2006

[13] S Ogi K Sugiyasu S Manna S Samitsu and M TakeuchildquoLiving supramolecular polymerization realized through abiomimetic approachrdquo Nature Chemistry vol 6 no 3 pp 188ndash195 2014

[14] CNakamura S TakedaM Kageshima et al ldquoMechanical forceanalysis of peptide interactions using atomic force microscopyrdquoPeptide Science vol 76 no 1 pp 48ndash54 2004

[15] M Gilaki ldquoUVndashVis and AFM Study of tetrakis (4-sulfonato-phenyl) nano-porphyrin aggregationrdquo Trends in Applied Sci-ences Research vol 6 no 3 pp 304ndash308 2011

[16] D Monti S Nardis M Stefanelli R Paolesse C Di Nataleand A DrsquoAmico ldquoPorphyrin-based nanostructures for sensingapplicationsrdquo Journal of Sensors vol 2009 Article ID 856053 10pages 2009

[17] DMontiMDeRossi A Sorrenti et al ldquoSupramolecular chira-lity in solvent-promoted aggregation of amphiphilic porphyrinderivatives kinetic studies and comparison between solutionbehavior and solid-state morphology by AFM topographyrdquoChemistry vol 16 no 3 pp 860ndash870 2010

[18] Z Wang C J Medforth and J A Shelnutt ldquoPorphyrin nan-otubes by ionic self-assemblyrdquo Journal of the AmericanChemicalSociety vol 126 no 49 pp 15954ndash15955 2004

[19] Hypercube Inc HyperChem Professional 751 Hypercube IncGainesville Fla USA 2003

[20] B B Mandelbrot The Fractal Geometry of Nature FreemanNew York NY USA 1982

[21] M W Mitchell and D A Bonnell ldquoQuantitative topographicanalysis of fractal surfaces by scanning tunneling microscopyrdquoJournal ofMaterials Research vol 5 no 10 pp 2244ndash2254 1990

[22] J P Carrejo T Thundat L A Nagahara S M Lindsay andA Majumdar ldquoScanning tunneling microscopy investigationsof polysilicon films under solutionrdquo Journal of Vacuum Scienceamp Technology B vol 9 article 955 1991

[23] D R Denley ldquoScanning tunneling microscopy of rough sur-facesrdquo Journal of Vacuum Science amp Technology A VacuumSurfaces and Films vol 8 no 1 pp 603ndash607 1990

[24] J M Gomez-Rodrıguez A M Baro L Vazquez R C Sal-varezza J M Vara and A J Arvia ldquoFractal surfaces of gold andplatinum electrodeposits Dimensionality determination byscanning tunneling microscopyrdquo Journal of Physical Chemistryvol 96 no 1 pp 347ndash350 1992

[25] J M Williams and T P Beebe Jr ldquoAnalysis of fractal surfacesusing scanning probe microscopy and multiple-image variog-raphy 2 Results on fractal and nonfractal surfaces observationof fractal crossovers and comparison with other fractal analysistechniquesrdquo Journal of Physical Chemistry vol 97 no 23 pp6255ndash6260 1993

[26] G Dobrescu C Obreja and M Rusu ldquoAdhesion AFM appliedto lipid monolayers A fractal analysisrdquo in Fractals Theory andApplications in Engineering M Dekking J L Vehel E Luttonand C Tricot Eds pp 259ndash271 Springer Berlin Germany1999

[27] G Dobrescu C Obreja and M Rusu ldquoAdhesion AFM appliedto DMPE monolayers A fractal analysisrdquo in Proceedings of theConference Fractals in Engineering DelftTheNetherlands June1999

[28] G Dobrescu C Obreja and M Rusu ldquoFractal analysis ofadhesion atomic force microscopy applied to lipid monolayersrdquoRevue Roumaine de Chimie vol 43 no 5 pp 417ndash424 1998

[29] F Family ldquoDynamic scaling and phase transitions in interfacegrowthrdquo Physica A Statistical Mechanics and Its Applicationsvol 168 no 1 pp 561ndash580 1990

[30] A L Barabasi and H E Stanley Fractal Concepts in SurfaceGrowth Cambridge University Press Cambridge UK 1995

[31] G Dobrescu andM Rusu ldquoDynamic scalingmethod and inter-face growthrdquo Advances in Colloid and Interface Science vol 95no 1 pp 83ndash93 2002

[32] P F Chauvy C Madore and D Landolt ldquoVariable length scaleanalysis of surface topography characterization of titaniumsurfaces for biomedical applicationsrdquo Surface and CoatingsTechnology vol 110 no 1-2 pp 48ndash56 1998


[33] R Socoteanu ldquoPorphyrinic Compound Double Grafted Hete-rocyclicrdquo OSIM Patent No2008-122035

[34] R Socoteanu ldquoAsymmetrical Substituted Porphyrin Deriva-tiverdquo OSIM Patent No 2008-122036

[35] R Socoteanu ldquoAsymmetrical Free Base Porphyrinrdquo OSIMPatent No 2008-122037

[36] R Socoteanu ldquoPorphyrinic Compound as Singlet Oxygen Gen-eratorrdquo OSIM Patent No 2008-122038

[37] R Socoteanu ldquoAsymmetrical Tetrapyrrolic Compoundrdquo OSIMPatent no 2008-122039

[38] J A Barltrop T C Owen A H Cory and J G Cory ldquo5-(3-car-boxymethoxyphenyl)-2-(45-dimethylthiazolyl)-3-(4-sulfophe-nyl)tetrazolium inner salt (MTS) and related analogs of 3-(45-dimethylthiazolyl)-25-diphenyltetrazolium bromide (MTT)reducing to purple water-soluble formazans as cell-viabilityindicatorrdquo Bioorganic amp Medicinal Chemistry Letters vol 1 no11 pp 611ndash614 1991

[39] C Korzeniewski and D M Callewaert ldquoAn enzyme-releaseassay for natural cytotoxicityrdquo Journal of Immunological Meth-ods vol 64 no 3 pp 313ndash320 1983

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


(a)

(b)

Figure 1 Face-to-face (a) versus side-by-side (b) aggregates displayed on 5101520-meso-tetrakis-(34-methylenedyoxy)-phenyl-2123Hporphine

capacity of self-association of this type of molecules areset around spectral analysis as liquid samples [9] Severalparameters as pH and solvent polarity play a major rolein aggregation capacity in the liquid state of the sampleBy using AFM technique these influential parameters areexcluded The binomial frame formed by AFM studies andporphyrin structures seem to be lately point of interest mostof them focusing on symmetric structures free bases [10]from simple compounds [11] to advanced materials [12 13]From the behavior of porphyrins bind with peptides [14]to potential sensing devices [15] aspects as supramolecularchirality attract the attention via AFM studies The variationof peripheral substituents of porphyrins has direct conse-quences on the formation of porphyrin aggregates creatinghigh supramolecular chirality targeting supramolecular net-works that emulate the biological systems [16 17] One of themost detectable features of the porphyrins is the capacity toaggregate through several types of interactions where mostof them are governed by the 120587-120587 interactions or hydrogenbonding This characteristic is desirable for applications intechnology especially in nanotechnology [18] H-aggregates(face-to-face arrangement) and J-aggregates (side-by-side)are the well-known assemblies as shown in Figure 1 withstructures optimized by HyperChem [19] Instead for thebiological applications as it theranostics is the capacity toconglomerate is an obvious constraint of the membranecell penetration process AFM coupled with some cell lines

tests proved to be a useful tool to evaluate the capacity ofcertain porphyrin to interact with the cell membrane In thisexperiment the correlation is made between the scales ofmagnitude and is not related with the kind of aggregation

ldquoScalingrdquo is one of the modern concepts used to char-acterize the morphology of various complex surfaces whichcan be smooth when the ldquoobserverrdquo (ie the tip in scanningprobe microscopy) is far enough and rough when it isclose enough Scaling relations characterize self-similar orself-affine objects and lead to direct computation of animportant parameter fractal dimension [20] Not all roughsurfaces are fractals but if a surface can be described interms of fractal geometry this means that it is self-affinein a statistical way and can be characterized by its fractaldimension Fractal dimension gives information about thesurface roughness a higher fractal dimension meaning thatthe surface is very rough but tells us something about surfacegeometry that is the surface is self-affine This is a veryimportant thing that when something seems to be disorderedand hard to be described self-affinity and fractal dimensiongive quantitative information about the surface and describeit Fractal dimension can be measured in different waysthe direct method implies analysis of topographic imagesobtained from scanning tunneling microscopy [21ndash24] scan-ning probe microscopy [25] or atomic force microscopy[26ndash28] but there are a lot of other indirect methods lightscattering neutron scattering adsorption and so on


NH N

HNN

A

B

B

B

for TMHAPP

OH

A = B =

OO

for TMDPP

CH2

A = B =

OO

for TRMDOPP

CH2

A = B =

OO

for TMDOPP

CH2

A = B =

OO

for MHTPP

CH2

A = B =

OH

OH

for THAPPA = B =

OCH3

OCH3

OCH3



3meso-porphyrinic








212 Methods





119866 (119903) equiv ⟨119862 ( 119903)⟩119909 (1)


119862 ( 119903) = [ℎ () minus ℎ ( +997888119903 )]

2

(2)


119866 (119903) sim 1199032120572 119903 ≪ 119871 (3)


120572 = 3 minus 119863 (4)





119902120576 averaged over 119899



119877119902120576=

1

119899120576

119899120576

sum

119894=1

radic

1

119901120576

119901120576

sum

119895=1

1199111198952 (5)






119863 = 119863119879minus 119867 (6)





119902120576 averaged over 119899






















0 04 08 12 16 2

6420

(nm

)

(1)(120583m)

500nm

(nm

)

10

75

5

25

0500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

0 04 08 12 16 2

(2)

1612

840

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus10

0

10

20

30

40

(nm

)

500nm

0 04 08 12 16 2

(3)

840(n

m)

(120583m)

minus4

(nm

)

10

0

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

minus10

Figure 3 Continued


0 04 08 12 16 2

302010

0

(nm

)

(120583m)

500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

50

75

(nm

)

(4)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nm 0

25

5

10

75(n

m)

(5)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

5

75

10

(nm

)

(6)





(1)


19ndash3225ndash103

09970998



099709980966

8 times 8



0972099909930947

(2)



099809990991



099709960996



099709970980

(3)

1 times 1



0995099309980995



09960998



099609910996

(4)2 times 2 230 plusmn 001(119862)


09970997



09930980

(5)



099809950999

8 times 8



0996099509790997

(6)


13ndash3552ndash182

09990996



099809930996

8 times 8



0997099709860997



06

05

04

03

02

01

00 2 4 6 8 10 12

(120583gmL)

(1)(2)(3)

(4)(5)(6)


OD490

nm



2with the









016

014

012

01

008

006

004

002

0


0 05 1 15 2 25 3

(120583gmL)

(1)(2)(3)

(4)(5)(6)

OD490

nm




4 Conclusions







Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


NH N

HNN

A

B

B

B

for TMHAPP

OH

A = B =

OO

for TMDPP

CH2

A = B =

OO

for TRMDOPP

CH2

A = B =

OO

for TMDOPP

CH2

A = B =

OO

for MHTPP

CH2

A = B =

OH

OH

for THAPPA = B =

OCH3

OCH3

OCH3



3meso-porphyrinic








212 Methods





119866 (119903) equiv ⟨119862 ( 119903)⟩119909 (1)


119862 ( 119903) = [ℎ () minus ℎ ( +997888119903 )]

2

(2)


119866 (119903) sim 1199032120572 119903 ≪ 119871 (3)


120572 = 3 minus 119863 (4)





119902120576 averaged over 119899



119877119902120576=

1

119899120576

119899120576

sum

119894=1

radic

1

119901120576

119901120576

sum

119895=1

1199111198952 (5)






119863 = 119863119879minus 119867 (6)





119902120576 averaged over 119899






















0 04 08 12 16 2

6420

(nm

)

(1)(120583m)

500nm

(nm

)

10

75

5

25

0500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

0 04 08 12 16 2

(2)

1612

840

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus10

0

10

20

30

40

(nm

)

500nm

0 04 08 12 16 2

(3)

840(n

m)

(120583m)

minus4

(nm

)

10

0

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

minus10

Figure 3 Continued


0 04 08 12 16 2

302010

0

(nm

)

(120583m)

500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

50

75

(nm

)

(4)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nm 0

25

5

10

75(n

m)

(5)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

5

75

10

(nm

)

(6)





(1)


19ndash3225ndash103

09970998



099709980966

8 times 8



0972099909930947

(2)



099809990991



099709960996



099709970980

(3)

1 times 1



0995099309980995



09960998



099609910996

(4)2 times 2 230 plusmn 001(119862)


09970997



09930980

(5)



099809950999

8 times 8



0996099509790997

(6)


13ndash3552ndash182

09990996



099809930996

8 times 8



0997099709860997



06

05

04

03

02

01

00 2 4 6 8 10 12

(120583gmL)

(1)(2)(3)

(4)(5)(6)


OD490

nm



2with the









016

014

012

01

008

006

004

002

0


0 05 1 15 2 25 3

(120583gmL)

(1)(2)(3)

(4)(5)(6)

OD490

nm




4 Conclusions







Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




119866 (119903) equiv ⟨119862 ( 119903)⟩119909 (1)


119862 ( 119903) = [ℎ () minus ℎ ( +997888119903 )]

2

(2)


119866 (119903) sim 1199032120572 119903 ≪ 119871 (3)


120572 = 3 minus 119863 (4)





119902120576 averaged over 119899



119877119902120576=

1

119899120576

119899120576

sum

119894=1

radic

1

119901120576

119901120576

sum

119895=1

1199111198952 (5)






119863 = 119863119879minus 119867 (6)





119902120576 averaged over 119899






















0 04 08 12 16 2

6420

(nm

)

(1)(120583m)

500nm

(nm

)

10

75

5

25

0500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

0 04 08 12 16 2

(2)

1612

840

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus10

0

10

20

30

40

(nm

)

500nm

0 04 08 12 16 2

(3)

840(n

m)

(120583m)

minus4

(nm

)

10

0

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

minus10

Figure 3 Continued


0 04 08 12 16 2

302010

0

(nm

)

(120583m)

500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

50

75

(nm

)

(4)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nm 0

25

5

10

75(n

m)

(5)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

5

75

10

(nm

)

(6)





(1)


19ndash3225ndash103

09970998



099709980966

8 times 8



0972099909930947

(2)



099809990991



099709960996



099709970980

(3)

1 times 1



0995099309980995



09960998



099609910996

(4)2 times 2 230 plusmn 001(119862)


09970997



09930980

(5)



099809950999

8 times 8



0996099509790997

(6)


13ndash3552ndash182

09990996



099809930996

8 times 8



0997099709860997



06

05

04

03

02

01

00 2 4 6 8 10 12

(120583gmL)

(1)(2)(3)

(4)(5)(6)


OD490

nm



2with the









016

014

012

01

008

006

004

002

0


0 05 1 15 2 25 3

(120583gmL)

(1)(2)(3)

(4)(5)(6)

OD490

nm




4 Conclusions







Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
















0 04 08 12 16 2

6420

(nm

)

(1)(120583m)

500nm

(nm

)

10

75

5

25

0500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

0 04 08 12 16 2

(2)

1612

840

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus10

0

10

20

30

40

(nm

)

500nm

0 04 08 12 16 2

(3)

840(n

m)

(120583m)

minus4

(nm

)

10

0

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

minus10

Figure 3 Continued


0 04 08 12 16 2

302010

0

(nm

)

(120583m)

500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

50

75

(nm

)

(4)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nm 0

25

5

10

75(n

m)

(5)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

5

75

10

(nm

)

(6)





(1)


19ndash3225ndash103

09970998



099709980966

8 times 8



0972099909930947

(2)



099809990991



099709960996



099709970980

(3)

1 times 1



0995099309980995



09960998



099609910996

(4)2 times 2 230 plusmn 001(119862)


09970997



09930980

(5)



099809950999

8 times 8



0996099509790997

(6)


13ndash3552ndash182

09990996



099809930996

8 times 8



0997099709860997



06

05

04

03

02

01

00 2 4 6 8 10 12

(120583gmL)

(1)(2)(3)

(4)(5)(6)


OD490

nm



2with the









016

014

012

01

008

006

004

002

0


0 05 1 15 2 25 3

(120583gmL)

(1)(2)(3)

(4)(5)(6)

OD490

nm




4 Conclusions







Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0 04 08 12 16 2

6420

(nm

)

(1)(120583m)

500nm

(nm

)

10

75

5

25

0500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

0 04 08 12 16 2

(2)

1612

840

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus10

0

10

20

30

40

(nm

)

500nm

0 04 08 12 16 2

(3)

840(n

m)

(120583m)

minus4

(nm

)

10

0

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

minus10

Figure 3 Continued


0 04 08 12 16 2

302010

0

(nm

)

(120583m)

500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

50

75

(nm

)

(4)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nm 0

25

5

10

75(n

m)

(5)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

5

75

10

(nm

)

(6)





(1)


19ndash3225ndash103

09970998



099709980966

8 times 8



0972099909930947

(2)



099809990991



099709960996



099709970980

(3)

1 times 1



0995099309980995



09960998



099609910996

(4)2 times 2 230 plusmn 001(119862)


09970997



09930980

(5)



099809950999

8 times 8



0996099509790997

(6)


13ndash3552ndash182

09990996



099809930996

8 times 8



0997099709860997



06

05

04

03

02

01

00 2 4 6 8 10 12

(120583gmL)

(1)(2)(3)

(4)(5)(6)


OD490

nm



2with the









016

014

012

01

008

006

004

002

0


0 05 1 15 2 25 3

(120583gmL)

(1)(2)(3)

(4)(5)(6)

OD490

nm




4 Conclusions







Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0 04 08 12 16 2

302010

0

(nm

)

(120583m)

500nm(120583

m)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

50

75

(nm

)

(4)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nm 0

25

5

10

75(n

m)

(5)

0 04 08 12 16 2

6420

(nm

)

(120583m)

500nm

(120583m

)

04 08 12 160 2(120583m)

0

04

08

12

16

2

500nmminus25

0

25

5

75

10

(nm

)

(6)





(1)


19ndash3225ndash103

09970998



099709980966

8 times 8



0972099909930947

(2)



099809990991



099709960996



099709970980

(3)

1 times 1



0995099309980995



09960998



099609910996

(4)2 times 2 230 plusmn 001(119862)


09970997



09930980

(5)



099809950999

8 times 8



0996099509790997

(6)


13ndash3552ndash182

09990996



099809930996

8 times 8



0997099709860997



06

05

04

03

02

01

00 2 4 6 8 10 12

(120583gmL)

(1)(2)(3)

(4)(5)(6)


OD490

nm



2with the









016

014

012

01

008

006

004

002

0


0 05 1 15 2 25 3

(120583gmL)

(1)(2)(3)

(4)(5)(6)

OD490

nm




4 Conclusions







Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




(1)


19ndash3225ndash103

09970998



099709980966

8 times 8



0972099909930947

(2)



099809990991



099709960996



099709970980

(3)

1 times 1



0995099309980995



09960998



099609910996

(4)2 times 2 230 plusmn 001(119862)


09970997



09930980

(5)



099809950999

8 times 8



0996099509790997

(6)


13ndash3552ndash182

09990996



099809930996

8 times 8



0997099709860997



06

05

04

03

02

01

00 2 4 6 8 10 12

(120583gmL)

(1)(2)(3)

(4)(5)(6)


OD490

nm



2with the









016

014

012

01

008

006

004

002

0


0 05 1 15 2 25 3

(120583gmL)

(1)(2)(3)

(4)(5)(6)

OD490

nm




4 Conclusions







Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


06

05

04

03

02

01

00 2 4 6 8 10 12

(120583gmL)

(1)(2)(3)

(4)(5)(6)


OD490

nm



2with the









016

014

012

01

008

006

004

002

0


0 05 1 15 2 25 3

(120583gmL)

(1)(2)(3)

(4)(5)(6)

OD490

nm




4 Conclusions







Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Acknowledgments


References




















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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