ARTICLE IN PRESS

Journal of Luminescence 129 (2009) 506–513

Contents lists available at ScienceDirect

Journal of Luminescence

0022-23

doi:10.1

� Corr

E-m

journal homepage: www.elsevier.com/locate/jlumin

Effect of solvent-controlled aggregation on the intrinsic emission propertiesof PAMAM dendrimers

Maria J. Jasmine, Manniledam Kavitha, Edamana Prasad �

Department of Chemistry, Indian Institute of Technology Madras (IIT M), Chennai, Tamil Nadu 600 036, India

a r t i c l e i n f o

Article history:

Received 8 July 2008

Received in revised form

24 November 2008

Accepted 1 December 2008Available online 9 December 2008

Keywords:

PAMAM dendrimer

Intrinsic emission

Solvation effect

Aggregation

13/$ - see front matter & 2008 Elsevier B.V. A

016/j.jlumin.2008.12.005

esponding author. Tel.: +9144 22574232; fax

ail address: pre@iitm.ac.in (E. Prasad).

a b s t r a c t

Solvent-induced aggregation and its effect on the intrinsic emission properties of amine, hydroxy and

carboxylate terminated, poly(amidoamine) (PAMAM) dendrimers have been investigated in glycerol,

ethylene glycol, methanol, ethylene diamine and water. Altering the solvent medium induces

remarkable changes in the intrinsic emission properties of the PAMAM dendrimers at identical

concentration. Upon excitation at 370 nm, amine terminated PAMAM dendrimer exhibits an intense

emission at 470 nm in glycerol, ethylene glycol as well as glycerol–water mixtures. Conversely, weak

luminescence is observed for hydroxy and carboxylate terminated PAMAM dendrimers in the same

solvent systems. When the solvent is changed to ethylene diamine, hydroxy terminated PAMAM

exhibits intense blue emission at 425 nm. While the emission intensity is varied when the solvent

milieu is changed, excited state lifetime values of PAMAM dendrimers remain independent of the

solvent used. UV–visible absorption and dynamic light scattering (DLS) experiments confirm the

formation of solvent-controlled dendrimer aggregates in the systems. Comparison of the fluorescence

and DLS data reveals that the size distribution of the dendrimer aggregates in each solvent system is

distinct, which control the intrinsic emission intensity from PAMAM dendrimers. The experimental

results suggest that intrinsic emission intensity from PAMAM dendrimers can be regulated by proper

selection of solvents at neutral conditions and room temperature.

& 2008 Elsevier B.V. All rights reserved.

1. Introduction

Dendrimers are hyperbranched macromolecules that havegained considerable attention in numerous fields starting fromfunctional nanomaterials to biomedical devices [1–5]. Since thepioneering works of Vogtle and Tomalia about three decades back,large numbers of synthetic strategies have been introduced forpreparing dendrimers with different structural scaffolds [6–9].Recent research focuses more on effective ways of tuning theproperties of dendrimers for practical applications [10,11].Modification in structure, size and shape of the dendritic designprovides an attractive pathway for controlling the properties ofdendrimers [2,12–14]. While dendrimers possess unambiguousstructural features compared to polymers due to differences inmorphological architecture, external factors such as pH [15,16],concentration [17] and solvation [18–20] can bring considerablestructural flexibility to dendrimers. It is critical to understandthe basic principles through which the external factors controlthe size and shape of dendrimers in order to have a desiredproperty.

ll rights reserved.

: +9144 22574200.

Poly(amidoamine) (PAMAM) dendrimers have been identifiedas one of the promising candidates for biomedical applications[21,22]. Theoretical and experimental works have confirmed thatPAMAM dendrimers exhibit conformational flexibility inducedby solvents, pH and salt concentration [15–20,23]. Among them,role of solvents in altering the structural features of PAMAMdendrimers has been investigated in detail [24–27]. For example,Stechemesser and Eimer [24] have shown that the change in thesolvent media leads to significant swelling in PAMAM dendrimers,which in turn can affect the host–guest chemistry of thedendrimer. In another elegant work, Goddard and co-workers[26] have shown through molecular dynamic simulations that thepresence of solvent leads to considerable size alterations inPAMAM dendrimers. A pioneering work by Luo and Imae [27] hasrecently shown that PAMAM dendrimers aggregate in aqueoussolutions, resulting alterations in their normal size, which assistthe template-based synthesis of platinum nanoparticles.

The photophysical properties of PAMAM dendrimers, periph-erally modified by organic fluorophores, were reported by Jia andWei [28]. Attaching fluorophores for generating luminescentdendrimers has attracted wide attention because of their potentialuse in practical applications. PAMAM dendrimers are effectivedrug delivery agents, and the drug releasing rates can beeasily monitored by fluorescence-based techniques [29]. Intrinsic

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fluorescence (i.e., fluorescence in the absence of conventionalfluorophores) from carboxylate terminated PAMAM dendrimerswas initially reported by Larson and Tucker [30]. This was furthersubstantiated by the work of Wang and Imae, who reported theblue emission from amine terminated, structurally un-modifiedPAMAM dendrimers at highly acidic conditions. They have alsopointed out that the intrinsic emission properties of PAMAMdendrimers depend on the generation of the dendrimer as thefourth generation emits with enhanced intensity than the corres-ponding second generation. In a separate attempt, Bard and co-workers [33] reported blue luminescence from oxidized hydroxyterminated PAMAM dendrimer in the absence of fluorophores.High-luminescent quantum yield for structurally un-modifiedPAMAM dendrimers was also reported in the presence of goldnanoclusters. However, the origin of the emission was notexplicitly established [34,35]. Recently, Dai and co-workers [36]reported luminescent hydrophobic Bronsted acidic ionic liquidsbased on PAMAM dendrimer. Developing intrinsically fluorescentPAMAM dendrimers at mild conditions is highly desirable sincelarge number of fluorescence based applications in biologicalstudies require emission from dendrimers at neutral conditions.While the solvent-induced structural alterations and aggregationsin PAMAM dendrimers were identified previously, comprehensivestudy of its effect on the intrinsic emission properties of PAMAMdendrimers is absent in the literature. Herein, we describe theeffect of solvent-induced aggregation of PAMAM dendrimers ontheir intrinsic emission properties at neutral conditions.

We observed that the proper choice of solvents leads to narrowsize distribution in amine (–NH2), hydroxy (–OH) and carboxylate(–COO–) terminated PAMAM dendrimer aggregates at neutralconditions, which subsequently results in intense intrinsicemission. The role of aggregate size distribution in controllingthe intrinsic emission from PAMAM dendrimers was furtherdemonstrated by mixtures of dendrimers, which form extremelynarrow size distribution of PAMAM dendrimer aggregates withenhanced emission intensity. The data described here demon-strate two important features: (a) judicious choice of solvents/additives leads to controlled aggregation in PAMAM dendrimers,which in turn regulates their intrinsic emission properties; and (b)intense fluorescence emission from structurally un-modifiedPAMAM dendrimers is feasible at mild conditions.

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3

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Fig. 1. UV–vis absorption spectra of PAMAM (G-4, NH2) dendrimer (4.3�10�5 M)

in water (dot) and glycerol (solid). Inset shows the same in glycerol at a higher

concentration (8.6�10�5 M).

2. Experimental

Materials: The dendrimers were purchased from AldrichChemical Co. as 10 wt% solution in methanol and evaporatedunder inert atmosphere prior to dissolving in other solvents.Experiments are done with distilled deionized water (Milliporefiltration system). Glycerol (499.5%), acrolein free sample, waspurchased from Aldrich. Ethylene glycol and ethylene diaminewere doubly distilled before used. All dendrimer solutionsprepared were kept at room temperature for 24 h and de-aeratedprior to measurements, except for the time-dependant DLS andfluorescence experiments, where spectra were started recordingimmediately after mixing. Blank experiments for fluorescencespectra were carried out using solvents alone and subtracted fromthe respective sample’s spectra.

Instruments: UV–vis absorption experiments were performedon a Perkin Elmer Lambda 25 UV–visible spectrophotometer. Allthe luminescence experiments reported in the study were carriedout using a Hitachi F4500 Fluorescence spectrophotometer. Thefluorescence decay measurements were carried out using thetime-correlated single-photon counting technique (TCSPC) withmicrochannel plate photomultiplier tube (MCP-PMT) as detectorand picosecond laser as the excitation source (model 5000 U, IBH,

UK). Light scattering experiments were done on DLS 90 plus/BI-Mas, Brookhaven Instruments, NY.

3. Results and discussion

3.1. Results

The present study investigates the intrinsic fluorescenceproperties of –NH2, –OH and –COO– terminated PAMAM den-drimers (henceforth represented by PAMAM (G-n, –X), where nand X represent generation and the terminal group, respectively)in selected solvents such as glycerol, ethylene glycol, methanol,ethylene diamine and water. Fig. 1 shows the UV–vis absorptionspectra of PAMAM (G-4, –NH2) (4.3�10�5 M) in glycerol andwater. As evident from the spectra, a broad absorption bandbetween 300 and 400 nm is formed in glycerol. It is also notedthat the initial absorbance at 278 nm is bathochromically shiftedby �7 nm. Inset of Fig. 1 shows the absorption spectrum ofPAMAM (G-4, –NH2) in glycerol at a higher concentration(8.6�10�5 M), where the broad peak exhibits a clear maximumaround 360 nm. The absorption spectra of PAMAM (G-4, –NH2) inethylene glycol and methanol have shown broad shoulder peaksat 360 nm.

The long wavelength absorption band observed for PAMAM (G-4, –NH2) in glycerol was found to be absent for PAMAM (G-4, –OH)in water and glycerol. Conversely, this broad band appeared whenthe solvent was changed to ethylene diamine. PAMAM (G-3.5,–COO–) also exhibited broad absorption at 360 nm in glycerol atidentical concentration, but with less optical density [37].

Fig. 2 shows the comparison of absorption spectra of mixturesof PAMAM (G-4, –NH2) and PAMAM (G-3.5, –COO–) in glycerol atdifferent mole ratios. Three different mixtures were prepared inwhich the mole ratios of PAMAM (G-4, –NH2) to PAMAM (G-3.5,–COO–) were 1:1 (M1), 1:5 (M2) and 1:10 (M3).

The concentration of PAMAM (G-4, NH2) was kept constant inthe three dendrimer mixtures and the effect on the change ofconcentration of PAMAM (G-3.5, –COO–) on the low-energyabsorption peak at 360 nm was monitored. The dendrimermixtures M1, M2 and M3 showed different absorbance at360 nm, with optical density following the order, M14M24M3 [38].

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0300 400 500 600

1

2

Wavelength (nm)

Abs

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M1

M3

M2

Fig. 2. UV–vis absorption spectra for PAMAM dendrimer mixtures M1, M2 and M3

in glycerol. Absorption spectrum of PAMAM (G-3.5, –COO–) alone (8.6�10�5 M) in

glycerol (dot).

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Fig. 3. Fluorescence spectra of PAMAM (G-4, –NH2) (8.6�10�5 M) in glycerol

(solid), water at pH 2 (dot) and at pH 7 (dash). Excitation wavelength was 370 nm.

(Inset) The excitation and emission spectra of PAMAM (G-4, –NH2) in glycerol

collected at 470 nm.

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Fig. 4. Emission from –NH2 (solid), –OH (dot) and COO– (dash) terminated

PAMAM dendrimers (8.6�10�5 M) in glycerol. (Inset) Variation in the corrected

emission intensity for PAMAM dendrimer mixtures M1, M2 and M3.

M.J. Jasmine et al. / Journal of Luminescence 129 (2009) 506–513508

Since absorption experiments clearly indicate the formationof solvent-dependent, low-energy solution state structures inPAMAM (G-4, –NH2), PAMAM (G-4, –OH) and mixtures of PAMAM(G-4, –NH2) and PAMAM (G-3.5, –COO–), fluorescence experi-ments were performed to understand whether these speciesexhibit intrinsic emission properties. Steady-state fluorescencespectrum of PAMAM (G-4, –NH2) (8.6�10�5 M) was monitored inglycerol at room temperature and compared to that in water atdifferent pH. The excitation wavelength was kept close to 360 nm,where the solvent-dependent absorption peak appears. Fig. 3shows the comparison spectra of emission from PAMAM (G-4,–NH2) in glycerol, water at pH 7 and 2. As previously mentioned,blue luminescence from PAMAM (G-4, –NH2) in water at pH 2 wasreported earlier [31,32]. The emission spectrum of PAMAM (G-4,–NH2) in glycerol exhibited the characteristic features of theemission spectrum in water at pH 2, but with an order ofmagnitude higher in intensity. It was also noted that the emissionmaximum was bathochromically shifted (�20 nm) in glycerol.PAMAM (G-4, –NH2) exhibited emission in ethylene glycol as wellas in methanol and the emission intensity was higher in bothsolvents compared to that in water, at equal concentrations.

The steady-state fluorescence spectra of PAMAM (G-4, –OH)and PAMAM (G-3.5, –COO–) in glycerol show only marginalintensity, compared to that of PAMAM (G-4, –NH2), at identicalconditions. Fig. 4 shows the steady-state emission spectra of allthe three dendrimers in glycerol, showing intense emission onlyfrom amine terminated PAMAM dendrimer. The emission max-imum of amine terminated PAMAM in glycerol is red shiftedcompared to hydroxy and carboxylate terminated PAMAM.Interestingly, when the solvent was changed to ethylene diamine,PAMAM (G-4, –OH) exhibited an intense emission at 425 nm (seethe Supporting Information).

The steady-state fluorescence from PAMAM dendrimer mix-tures M1, M2 and M3 was monitored in glycerol under steady-state conditions. The emission from PAMAM (G-3.5, –COO–) inglycerol (lmax ¼ 441 nm) was not enhanced when its concentra-tion was increased in the dendrimer mixtures. On the contrary,emission from PAMAM (G-4, –NH2) (lmax ¼ 470 nm) was found tobe enhanced (inset of Fig. 4).

The excitation spectra of amine, hydroxy and carboxylateterminated PAMAM dendrimers as well as the dendrimermixtures M1, M2 and M3 were taken and compared to the

absorption spectra in respective solvents. The excitation spectracontain two main peaks (�260 and 390 nm) and one relativelysmall shoulder peak in between the main peaks, consistent withthe reported data [31]. The excitation spectrum of PAMAM (G4,–NH2) in glycerol is given in the inset of Fig. 3. The relativeintensity of the two peaks exhibits opposite behavior in theexcitation and absorption spectra for samples that emit intenseintrinsic emission (see Fig. 1 and inset of Fig. 3).

Steady-state analysis was followed by fluorescence lifetimeexperiments utilizing time-correlated single-photon countingtechnique for the following systems: PAMAM (G-4, –NH2),PAMAM (G-4, –OH), PAMAM (G-3.5, –COO–) and PAMAMdendrimer mixtures in glycerol, ethylene glycol, methanol,ethylene diamine and water. A short lifetime component followedby two discrete excited state decays was found for PAMAMdendrimers, irrespective of the terminal groups. The shortcomponent decay observed was identified earlier as backgroundfluorescence from solvents [30,31]. While the intensity of thesteady-state fluorescence from PAMAM dendrimers in different

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solvents differed considerably, the excited state lifetime valuesremained identical within the experimental error in all the solventsystems examined. The two excited state lifetime values foramine, hydroxy and carboxylate terminated PAMAM dendrimersand mixtures of PAMAM dendrimers were approximately same inall the solvent systems (�t1 ¼ 2 and t2 ¼ 7.5 ns). Also, the relativeamplitudes of the lifetime components did not show solventdependency. Fig. 5 shows a comparison of the excited state decaytraces of PAMAM (G-4, –NH2) in water and glycerol. Table 1summarizes the steady-state and time-resolved data for theintrinsic fluorescence from PAMAM dendrimers in differentsolvents.

While the results from UV–vis absorption experiments in-dicated structural variations in PAMAM dendrimers induced bysolvents, they do not provide insightful data regarding thesolution state structures of PAMAM dendrimers responsible forthe fluorescence. Absorption experiments clearly suggest non-covalent interactions between the dendrimers and solvents,which could presumably lead to dendrimer aggregate formation.

1

10

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10000

Cou

nts

Time (ns)

Fig. 5. Fluorescence decays of PAMAM (G-4, –NH2) (8.6�10�5 M) in glycerol (W)

and water at pH 7 (J). The emission was collected at 470 nm.

Table 1Excited state lifetimes of –NH2, –OH, –COO– terminated PAMAM dendrimers in differe

System Solvent lma

PAMAM (G-4, –NH2) Water 456

Glycerol 477

Ethylene glycol 440

Methanol 420

PAMAM (G-4, –OH) Glycerol 423

Ethylene diamine 425

PAMAM (G-3.5, –COO–) Glycerol 441

PAMAM (G-4, –NH2)+PAMAM (G-3.5, –COO–) Glycerol

1:1 ratio (M1) 469

1:5 ratio (M2) 468

1:10 ratio (M3) 467

a The excited state lifetime (ns) of PAMAM dendrimers was measured utilizing a tim

excitation source.b The remaining fraction [100�(t1%+t2%)] corresponds to the component due to ba

Extensive studies utilizing dynamic light scattering (DLS)technique were carried out to investigate the dendrimer aggrega-tion. Dynamic light scattering is a useful tool to investigate thesize and structure of aggregates in solution state and it has beenwidely used to assess the particle size of polymeric materials [39].Since mixtures of PAMAM (G-4, NH2) and PAMAM (G-3.5, –COO–)in glycerol emit light with enhanced intensity compared withother systems, initial DLS experiments were performed for themixtures of amine and carboxylate terminated PAMAM dendri-mers at various mole ratios in glycerol. DLS experiments forPAMAM (G-4, NH2) in water and glycerol were also carried out forcomparison. The DLS results suggested that PAMAM dendrimersaggregate both in water and in glycerol and the aggregate size aswell as size distribution differs significantly in the two solventsystems. PAMAM (G-4, NH2) in water (8.6�10�5 M) shows thepresence of aggregates having a wide range (10–500 nm) of sizes.Conversely, the aggregate size variation of PAMAM (G-4, NH2) inglycerol became significantly less (10–14 and 60–80 nm) at thesame concentration.

nt solvents..

x (nm) emission Excited state lifetimesa (relative amplitude %)b

t1 t2

2.25 (32) 7.16 (31)

2.30 (32) 7.16 (31)

1.90 (31) 6.70 (54)

2.30 (32) 7.20 (31)

2.03 (43) 7.53 (38)

2.30 (31) 8.80 (60)

2.30 (31) 8.50 (60)

2.25 (45) 8.52 (40)

1.95 (31) 7.19 (60)

2.40 (31) 8.80 (60)

e-correlated single-photon counting technique (TCSPC) and picosecond laser as the

ckground fluorescence from solvent.

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Fig. 6. Histogram plot of the dendrimer mixture M2 in glycerol on DLS. Inset

shows the same for mixture M3. [PAMAM (G-4, –NH2)] ¼ 8.6�10�5 M.

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M.J. Jasmine et al. / Journal of Luminescence 129 (2009) 506–513510

DLS studies of the dendrimer mixtures M2 and M3 exhibitedexceptionally narrow size distribution of dendrimer aggregates(Fig. 6). While the aggregate size was varied for the dendrimermixture M1, the aggregate size distribution remained monodis-perse (see the Supporting Information).

DLS studies of PAMAM (G-4, –NH2) in water and glycerol werecarried out at a higher concentration (1.72�10�4 M) to estimatethe critical aggregation concentration. Nonetheless, similar resultswere obtained, suggesting that the solvent-induced PAMAMaggregate formation is independent of the monomer concentra-tion. The diffusion coefficients (D) of the aggregates of PAMAMdendrimers were calculated using the Stokes–Einstein equation

D ¼kBT

6pZRh(1)

where kB is the Boltzmann constant, T is the temperature inKelvin, Z is the viscosity of the solvent and Rh is the hydrodynamicradius of the dendrimer aggregate. The diffusion coefficient of theaggregates for the dendrimer mixture M1 was 0.14�10�9 cm2 s�1.The other two mixtures M2 and M3 exhibited lower and identicalvalues (0.04�10�9 cm2 s�1) of diffusion coefficients. PAMAM(G-4, NH2) in glycerol leads to two sets of dendrimer aggregateswith crucial size difference, and the diffusion coefficients valueswere differed by an order of magnitude (0.40�10�10 and0.20�10�9cm2 s�1). Analysis of DLS data for PAMAM in varioussolvents suggests that intrinsic emission intensity and aggregatesize distribution are related in a linear fashion. When theaggregate size distribution is narrow, the emission is very intense.

Next, the dependence of glycerol concentration on the intrinsicemission intensity from PAMAM (G-4, –NH2) was determined bymeasuring the emission intensity in the presence of increasingamount of glycerol in water (Fig. 7). Detectable emission fromPAMAM (G-4, –NH2) was observed in the presence of 25equivalents of glycerol in bulk water.

It has been recently reported that ageing has an important rolein enhancing the emission intensity from PAMAM dendrimer atpH 2 [32]. We have also observed similar behavior in the case ofPAMAM (G-4, –NH2) in glycerol at neutral conditions, where 9 h ofstoring has resulted in four times enhanced emission intensitycompared to that of a freshly prepared sample (inset, Fig. 7). Theenhancement in emission intensity of PAMAM (G-4, –NH2) in

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Fig. 7. Steady-state fluorescence spectra of PAMAM (G-4, –NH2) dendrimer

(8.6�10�5 M) in water in the presence of varying [glycerol]. From bottom to

top; 25, 75, 150 and 250 equivalents of glycerol with respect to [dendrimer]. Inset:

the ageing effect on emission intensity.

glycerol came to a plateau after 10 h. The ageing effect was morepronounced in ethylene glycol and the system took a period of 2weeks to achieve the identical status of luminescence intensityshown in glycerol.

In order to test whether the ageing effect observed in thefluorescence spectra is related to the solvent-induced aggregationor aggregate distribution of PAMAM dendrimers, DLS experi-ments were performed for PAMAM (G4, –NH2) over a period oftime (Fig. 8).

As evident from the figure, the size distribution has changed asa function of time. While there was no definite aggregateformation immediately after the mixing, the system exhibitedan aggregate size of �22.5 nm after 2 h from preparation. Theaggregate size increased over time and reached a stable value

8 hour

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Fig. 8. Histogram plot of PAMAM (G4, –NH2) in glycerol on DLS as a function of

time. [PAMAM (G-4, –NH2)] ¼ 8.6�10�5 M.

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(�70 nm) after 8 h, which does not exhibit further ageing effect. Itis worth noting that PAMAM (G4, NH2) in glycerol took identicaltime period for fluorescence enhancement due to ageing. Whileprevious studies have demonstrated air oxidation as the mainreason for the ageing effect [32], our experimental results clearlysuggest that slow solvent-induced dynamics also play a crucialrole in the ageing effect of PAMAM dendrimers, especially inviscous solvents.

3.2. Discussion

Solvents play an important role in altering the solution statestructure of PAMAM dendrimers [24]. The non-covalent interac-tions between the peripheral groups of PAMAM dendrimer andthe solvents or additives can potentially lead to unique solutionstate structures for PAMAM dendrimers. It is thus intriguing toinvestigate how the energetics and the photophysical propertiesof the solution state structures can be tuned by altering thesolvation parameters. In order to test this, UV–vis spectroscopicstudies of amine, hydroxy and carboxylate terminated PAMAMdendrimers were carried out in glycerol, ethylene glycol, metha-nol, ethylene diamine and water. These solvents were takenbecause of their potential hydrogen-bonding capability with theterminal groups (64 numbers in the case of fourth-generationdendrimer) in the PAMAM dendrimers used in the study. Uponchanging the solvent from water to glycerol, methanol andethylene glycol a new absorption band is generated for PAMAM(G-4, –NH2) at a lower energy region (360 nm). Identical behaviorwas observed for PAMAM (G-4, –OH) when dissolved in ethylenediamine, but not in other solvents. PAMAM (G-3.5, –COO–) alsoexhibited a broad absorption band in glycerol, with less opticaldensity. This clearly suggests that the absorption peak formed at360 nm is due to the non-covalent interaction between the solventand the dendrimers.

The fact that intrinsic emission intensity from PAMAMdendrimers is high when excitation wavelength is close to thesolvent-induced, lower energy absorption peak and drasticallylow at other wavelengths suggests that the newly formed, solvent-controlled solution state structures are responsible for theintrinsic emission in PAMAM dendrimers. This was furtherconfirmed by examining the excitation spectra of the sampleswhere intense emission is observed around 360 nm. DLS studieshave shown that the low-energy solution state structures are dueto the formation of dendrimer aggregates, with a unique size ineach solvent system.

Dendrimer aggregations in solutions and related studies arereceiving wide attention. A detailed study of spherical aggregatesof PAMAM dendrimers, peripherally modified by different organicfluorophores, was recently reported [28]. J-type and H-typeaggregates are reported for anionic porphyrin derivatives in thepresence of amine terminated PAMAM dendrimers [40]. Recentstudy of Luo and Imae [27] has shown that fourth-generation,amine terminated PAMAM dendrimers aggregate in aqueoussolution to form templates for platinum nanoparticles. They havesuggested that the motive force for the formation of aggregates isintermolecular hydrogen bonding. They have also found that theaggregate size of PAMAM (G-4, –NH2) in water differs in a widerange. In another elegant work, Orberg et al. [39] have shownthrough DLS analysis that salmon sperm DNA molecule andPAMAM dendrimers form discrete aggregate in dilute solutionwith mean apparent hydrodynamic radius of 50 nm. In a separateattempt, Masuhara and co-workers [41] have described theformation of p-stacked dendrimeric aggregates from wire typedendrimers. Aggregation and fluorescence from PAMAM dendri-mers, containing peripheral phenyl groups, were recently reported

by Wei and co-workers [42] and p-stacking tendency of phenylgroups was attributed as the driving force for the aggregation.

Even though dendrimer aggregation has been widely studied,solvent-induced aggregation and its effect on intrinsic emissionproperties of structurally un-modified PAMAM dendrimers havenot been reported [43–48]. The present study suggests thatsolvents control the size of PAMAM dendrimer aggregates in aremarkable way. For example, DLS studies have shown that amineterminated PAMAM dendrimer exhibited a range of aggregatesizes, from 10 to 500 nm in water. Conversely, the same dendrimerdisplayed only two types of aggregates of close size range (10–14and 60–80 nm) in glycerol. Among them, most of the scatteredlight has come from the aggregate of diameter range close to69–82 nm (see Supporting Information). DLS data obtained forPAMAM (G4, –NH2) at different time indicate that solvent-induced aggregate distribution in glycerol requires 8–9 h toachieve the aggregate size of 69–82 nm. Mixtures of PAMAM(G-4, –NH2) and PAMAM (G-3.5, –COO–) in glycerol exhibited verynarrow size distribution of the dendrimer aggregates. While theaggregate size in the dendrimer mixture M1 was 2175 nm, thesize becomes 7171 and 6773 nm for M2 and M3, respectively. Ingeneral, amine, hydroxy and carboxylate terminated PAMAMdendrimers display aggregates of wide size distribution in waterand narrow size distribution in glycerol. Control DLS experimentswere performed with solvents alone to check the purity of themedium.

The role of viscosity of the medium on the unique emissionfrom PAMAM dendrimers is ruled out since PAMAM (G-4, –NH2)exhibited intense emission in ethylene glycol as well. In additionto that, hydroxy and carboxylate terminated PAMAM dendrimersshowed only marginal fluorescence in glycerol.

The luminescence from the mixtures of PAMAM (G-4, –NH2)and PAMAM (G-3.5, –COO–) suggests some interesting aspects. A1:1 mixture of PAMAM (G-4, –NH2) and PAMAM (G-3.5, –COO–)(8.6�10�5 M) exhibited slight turbidity in the system and keepingthe solution for 1 week resulted in a brownish yellow precipitate,reinforcing the hypothesis of aggregate formation in the system.Fig. 4 suggests that the dendrimer mixture at different mole ratiosdisplays emission spectrum identical to that of PAMAM (G-4,–NH2) (lmax of emission from amine and carboxylate terminatedPAMAM differs by 35 nm). This suggests that PAMAM (G-3.5,–COO–) acts as an additive, which assists PAMAM (G-4, –NH2) toaggregate, presumably through non-covalent interactions be-tween amine and carboxylate groups in the two dendrimers[49]. These results suggest that not only solvents but also suitableadditives can play a crucial role in enhancing the intrinsicemission from PAMAM dendrimer. Fluorescence from PAMAM(G-4, –NH2) in the presence of 25 equivalents of glycerol in bulkwater (Fig. 7) also indicates that suitable additives can enhancethe intrinsic emission in PAMAM dendrimers.

DLS studies have shown that the dendrimer mixture M2displays the narrowest size distribution. However, the correctedemission intensity is highest from dendrimer mixture M3 (inset,Fig. 4). This apparent discrepancy can be attributed to fluores-cence self-quenching. Higher concentration of PAMAM (G-4,–NH2) aggregates is present in M2 compared to that in M3 (thisis clear from the increase in absorbance at 360 nm for theaggregates in the order M14M24M3), which can effectivelyquench the excited state by self-quenching mechanism. The roleof the mole ratio dependence on the aggregate formation ispresumably related to the morphology of the dendrimer interac-tion between amine and carboxylate terminated PAMAM inglycerol.

Time-resolved fluorescence studies have clearly shown thatthe excited state decays from PAMAM dendrimers are double-exponential. It is known from DLS studies that PAMAM dendrimer

ARTICLE IN PRESS

hν

*

Fluorescence

*

Solvent 1 Solvent 2

+n

hν Fluorescence

*

n-x x

Scheme 1. Altering the solvent medium changes the intrinsic emission intensity

from PAMAM dendrimers by varying the size distribution of dendrimer aggregates.

M.J. Jasmine et al. / Journal of Luminescence 129 (2009) 506–513512

possesses distinct distribution of aggregate size in each solution.The biexponential decay can be due to the presence of either (a)two discrete dendrimer aggregates or (b) two different conforma-tions of same dendrimer aggregate. The latter hypothesis issupported by the fact that mixtures of PAMAM (G-4, –NH2) andPAMAM (G-3.5, –COO–) in glycerol exhibit biexponential decays,even though the size distribution of dendrimer aggregates isremarkably narrow [50]. Moreover, the fact that relative ampli-tude of the biexponential components remains unaffected by thesolvent milieu (Table 1) suggests that they are not originated fromdifferent sized aggregates. While steady-state fluorescence in-tensity from PAMAM dendrimers depends on the solvent medium,repeated analysis of time-resolved study suggests that excitedstate lifetime values remain the same irrespective of the solventsused. This indicates that the fluorescent species formed fromPAMAM dendrimers in different solvents possesses identicalexcited states.

Results taken together suggest that aggregation of size7071 nm is a preferred size range in PAMAM dendrimers (fourthgeneration) to have a stable, emissive state. Aggregates with lowersize than the optimum value might lose the excited state energy,presumably through non-radiative mechanism due to the in-creased degree of freedom (Scheme 1) [51]. The optimum size of7071 nm for the dendrimer aggregates suggests the possibility ofassembling approximately 15 PAMAM dendrimers, assuming thateach monomer is 4.5 nm in diameter [28,52].

The decrease in the steady-state fluorescence from PAMAM(G-4, –NH2) in glycerol upon altering the solvent medium wasexamined through the Stern–Volmer analysis (Eq. (2)) in thepresence of increasing concentrations of water, which acts as aquencher:

I0

I¼ 1þ Ksv½water� (2)

where I0 is the intensity of emission of PAMAM (G-4, –NH2) inglycerol, I is the intensity of the emission in presence of water andKsv is Stern–Volmer constant.

A plot of I0/I vs. [water] provided Ksv as the slope. TheStern–Volmer constant, Ksv, is related to the quenching constant kq

by the following equation:

kq ¼Ksv

t0(3)

where t0 is the excited state lifetime of PAMAM (G-4, –NH2) inglycerol in the absence of any quencher. Since PAMAM (G-4,–NH2) exhibits two discrete excited state lifetime values in theabsence of any quencher, the Stern–Volmer analysis provides twocorresponding quenching constants (5.7�108 and 1.8�108 M�1

s�1 for t1 and t2, respectively).Comparison of DLS data with steady-state fluorescence spectra

of PAMAM dendrimers in different solvents suggests that the

fluorescence intensity is high when the aggregate size distributionis narrow. The size of the dendrimer aggregates and its distribu-tion were not considerably altered at different PAMAM concen-trations. This suggests that the critical aggregation concentrationis independent of the initial concentration of the dendrimer inthese cases.

The mechanism of fluorescence quenching for PAMAM (G-4,–NH2) in glycerol is similar to that of static type, rather thandynamic quenching. This was evident by the fact that ratio ofsteady-state luminescence intensities (I0/I) of PAMAM (G-4, –NH2)in glycerol was increased in the presence of increasing amount ofa quencher (water), but ratio of excited state lifetime valuesremained unchanged (t0/t ¼ 1) [53]. The Stern–Volmer plotprovides a linear line with a correlation value of 0.99, againindicating the presence of static type fluorescence quenching.

The reasons of non-classical emission from PAMAM dendri-mers have been briefly discussed by Bard and Imae [31,33]. Themost striking observation here is that a conventional fluorophoreis absent in these cases. A number of factors including (a)oxidation by air or other oxidizing agents to generate luminescentmoieties, (b) rigidity of the backbone through electrostaticrepulsion at lower pH and (c) enhanced hydrogen-bonding abilityunder acidic conditions were proposed to explain the unusualemission from PAMAM dendrimers. The reported data of intrinsicfluorescence from PAMAM dendrimers in the literature concludedthat the backbone structure of the dendrimer plays a key role indendrimer emission. The invariable excited state lifetime valuesobserved for all the dendrimer–solvent system in the presentstudy support this finding. Nevertheless, the current studies alsosuggest that the role of peripheral groups is equally importantsince aggregate formation and its distribution are controlled bythe functional groups at the periphery of the PAMAM dendrimers.

Enhanced aggregation tendency in dendrimers at lower pHwas reported prior to the observation of pH-dependent intrinsicemission from PAMAM dendrimers [54]. This suggests that theenhanced intrinsic emission from PAMAM dendrimers in acidicconditions might be originated due to the aggregation of PAMAMdendrimers at lower pH.

Regardless of the complexities of the mechanism associatedwith the intrinsic emission from PAMAM dendrimers, the dataobtained here suggests that novel class of fluorescent materialscan be generated from structurally un-modified PAMAM dendri-mers by proper choice of solvents or additives. The experimentaldata have shown that solvent-assisted intrinsic fluorescence fromPAMAM dendrimers can be generated under mild conditions andlower concentration ranges than reported in the literature. Thefact that the emission intensity from PAMAM dendrimer caneffectively be controlled by glycerol, which is a bio-compatiblereagent, in a non-covalent fashion suggests that supramolecularapproach can be adopted to generate intrinsically fluorescentPAMAM dendrimers for biomedical applications.

4. Conclusion

The intrinsic fluorescence from fourth generation of amine,hydroxy and 3.5 generation of carboxylate terminated PAMAMdendrimers was examined in glycerol, ethylene glycol, methanol,ethylene diamine and water at neutral conditions and roomtemperature. The results obtained from steady-state fluorescenceand DLS studies suggest that solvent/additive-induced dendrimeraggregates play a pivotal role in regulating the intrinsic emissionfrom PAMAM dendrimers. The results also indicate that narrowsize distribution of dendrimer aggregates with an optimum size isa pre-requisite for enhanced emission from PAMAM dendrimers atmild conditions. The role of solvent-induced PAMAM dendrimer

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M.J. Jasmine et al. / Journal of Luminescence 129 (2009) 506–513 513

aggregates in regulating the host–guest properties of the system iscurrently being initiated in our laboratory and the results will bepublished in due course.

Acknowledgements

We are grateful to the Department of Science and Technology(DST), Govt. of India, for the financial support (SR/S1/PC-26/2007)for this work. We thank Professor P. Ramamurthy, Director, NationalCentre for Ultrafast Processes (NCUP), University of Madras, forproviding facilities for excited state lifetime studies. We also thankProfessor S. Ramanathan (Department of Chemical Engineering,IITM) for DLS experimental facilities. We thank Dr. P.K. SudhadeviAntharjanam, Scientist under Fast Track Scheme of DST, Departmentof chemistry, IITM, for her useful comments on the manuscript.

Appendix A. Supporting Information

Supplementary data associated with this article can be foundin the online version at doi:10.1016/j.jlumin.2008.12.005.

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