Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 351–356
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Disaggregation induced solvatochromic switch: A study of dansylated polyglycerol dendrons in binary solvent mixture Usharani Subuddhi a, Prasanna K. Vuram b, Anju Chadha c,⇑, Ashok K. Mishra b,⇑ a
Department of Chemistry, National Institute of Technology Rourkela, India Department of Chemistry, Indian Institute of Technology Madras, Chennai 600036, India c Laboratory of Bioorganic Chemistry, Department of Biotechnology and National Center for Catalysis Research, Indian Institute of Technology Madras, Chennai 600036, India b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Interesting solvatochromism in
Reversal in solvatochromic behaviour of dendrons in aqueous medium on addition of a nonaqueous solvent. Negative-solvatochromism is attributed to the progressive disaggregation of dendron aggregates and the positive-solvatochromism is exhibited by the dendron monomers.
polyether dendron aggregates in mixed aqueous media. Reversal in solvatochromic behaviour depending on percentage of nonaqueous solvent. Negative-solvatochromism due to progressive disaggregation of dendron aggregates. Positive-solvatochromism is shown by the dendron monomers. Higher dendron hydrophobicity requires more of second solvent for disaggregation.
a r t i c l e
i n f o
Article history: Received 16 November 2013 Received in revised form 18 February 2014 Accepted 21 February 2014 Available online 12 March 2014 Keywords: Dansylated polyether dendrons Solvatochromism Binary aqueous-nonaqueous solvent mixtures Fluorescence Aggregation–disaggregation
a b s t r a c t A reversal in solvatochromic behaviour was observed in second and third generation glycerol based dansylated polyether dendrons in water on addition of a second solvent like methanol or acetonitrile. Below a certain percentage of the nonaqueous solvent there is a negative-solvatochromism observed and above that there is a switch to positive-solvatochromism. The negative-solvatochromism is attributed to the progressive disaggregation of the dendron aggregates by the nonaqueous solvent component. Once the disaggregation process is complete, positive-solvatochromism is exhibited by the dendron monomers. Higher the hydrophobicity of the dendron more is the amount of the second solvent required for disaggregation. Ó 2014 Elsevier B.V. All rights reserved.
Introduction
⇑ Corresponding authors. Tel.: +91 44 22574207; fax: +91 44 22574202 (A.K. Mishra). Tel.: +91 44 22574106; fax: +91 44 22574102 (A. Chadha). E-mail addresses: [email protected] (A. Chadha), [email protected] (A.K. Mishra). http://dx.doi.org/10.1016/j.saa.2014.02.150 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
Polyethers, hyperbranched polyglycerols [1–3] and polyglycerol dendrimers [4–6] have been the focus of recent studies due to their excellent water solubility and biocompatibility. The microenvironment created in the core of dendrimers resembles that of biological
352
U. Subuddhi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 351–356
systems such as proteins, primarily because of the branching architecture of dendrimers [7,8], which in turn modulates their physical properties [9]. Aggregation of the primary units of dendrimers and dendrons into larger and more complex forms leads to a wide range of potential applications in the fields of drug delivery [10–12], biomedical applications [13–15] and nanotechnology [16–18,13]. Polyglycerol dendrimers have shown good transport capacity for poorly water soluble drugs [5,13,19,20]. Hence, it is important that their physical and chemical properties are understood under various conditions and a significant effort has been directed towards this [10–18,21–24]. Synthesis of glycerol based dansylated polyether dendrons ((Gn)-PGL-ipd, where Gn specifies the generation number of the Dendron (Scheme 1 in electronic supplementary material)), their photophysical properties and aggregation behaviour have been recently reported by us [25,26]. The emission maxima for all generations of dendrons showed strong dependence on solvent polarity in nonaqueous solvents, indicating the dominant importance of general solvent effect. In general they showed positive solvatochromism i.e. with increasing solvent polarity a red-shift in the emission maximum is exhibited. However, in water the most polar among all the solvents studied, the higher generation dendrons from second generation onwards exhibited unusual blue-shift in their emission maximum representing more or less dioxane like polarity. When compared with the lower generation dendrons like G0-PGL-ipd and G1-PGL-ipd, they showed almost 60 nm hypsochromic shift in the emission maximum accompanied by a remarkable enhancement in the fluorescence intensity, a ten-fold increase in fluorescence anisotropy and four-fold increase in the average fluorescence lifetime. Based on these observations it was suggested that there is a hydrophobicity induced aggregation taking place in these higher generation dendrons in aqueous medium even at concentrations as low as 1 108 M. If aggregation of these dendrons in aqueous medium is induced by hydrophobicity, it is expected that on addition of a second solvent like methanol or acetonitrile, the disaggregation process will set in, which possibly would result in interesting solvatochromism in the mixed solvents. An understanding of the process of disaggregation of these dendrons and its effect on the fluorescence behaviour of dansyl moiety is the objective of this work. The present work illustrates the interesting photophysical features of assembling and disassembling of dansylated polyglycerol dendrons, with isopropylidene end groups, depending on dendron generation and solvent polarity in binary aqueous–nonaqueous solvent mixtures (water–methanol and water–acetonitrile).
appropriate volume of the solvent of interest. All the solvents used were of UV-spectroscopic grade (SRL Pvt. Ltd., India) and used without further purification. The water used was distilled three times with addition of alkaline permanganate. In order to check the effect of order of addition of the solvents on the properties of dendrimers, the binary aqueous–nonaqueous solutions were prepared in three different ways, (i) by adding different volumes of the nonaqueous solvent (methanol or acetonitrile) to the aqueous solution of the dendrimer, (ii) by adding different volumes of water to the nonaqueous solution of the dendrimer and (iii) by mixing the aqueous and nonaqueous solutions of dendrimer at different ratios so as to achieve the desired ratio (v/v) of the two solvents. The solutions were shaken well and left for about an hour for equilibration before carrying out the studies. The results were found to be very similar in all three cases irrespective of the method of preparation. The dendrimer concentration was kept constant at 1 105 M in all the studies. Instrumentation Fluorescence was recorded on Hitachi F-4500 and Jobin–Yvon Fluoromax-4 spectrofluorimeters. The steady state fluorescence anisotropy (rss) values were obtained by using the expression
rss ¼ ðIk GI? Þ=ðIk þ 2GI? Þ where I|| and I\ are fluorescence intensities when the emission polarizer is parallel and perpendicular, respectively to the direction of polarization of the excitation beam and G is the factor that corrects for unequal transmission by the diffraction gratings of the instrument for vertically and horizontally polarized light. Fluorescence lifetime (sf) measurements were carried out using Horiba Jobin–Yvon TCSPC lifetime instrument in a time-correlated single-photon counting (TCSPC) arrangement. A 340 nm nano LED was used as the light source. The pulse repetition rate was set to 1 MHz and the instrumental full width half maximum of the 340 nm LED including the detector response is 40% for G2-PGL-ipd and >60% for G3-PGL-ipd) is very similar to that observed in case of G0-PGL-ipd and G1-PGL-ipd. Earlier studies have suggested that higher order dendrons, from second generation onwards, show self-association behaviour in aqueous medium resulting in almost 60 nm blue-shift in the emission maximum in comparison to that of G0-PGL-ipd and G1-PGL-ipd [25]. This means in the aggregated form of dendrons, the dansyl moiety experiences a relatively more nonpolar microenvironment than in the non-aggregated monomeric form. In view of this, the unusual negative solvatochomism exhibited by G2-PGLipd and G3-PGL-ipd (red-shift in emission maximum by reducing
0
20
40
60
80
100
% of Methanol Fig. 2. Plots of (A) emission energy (B) normalised fluorescence intensity of G2PGL-ipd and G3-PGL-ipd with the percentage of methanol in water–methanol binary mixture.
the overall solvent polarity) at lower percentages of added CH3OH (60% for G3-PGL-ipd) the anisotropy value of both G2-PGL-ipd and G3-PGL-ipd is similar to that of G0-PGL-ipd and G1-PGL-ipd (Fig. A1 in electronic supplementary material). The anisotropy value for G0-PGL-ipd and G1-PGL-ipd is found to be very low in aqueous medium and remains invariant with the solvent composition in the binary mixture. Thus, fluorescence anisotropy study supports the fact that there is a progressive disaggregation of the aggregates taking place with addition of CH3OH and by 40% and 60% of CH3OH there is complete disaggregation of G2-PGL-ipd and G3-PGL-ipd dendrons, respectively. Fluorescence lifetime studies
Fluorescence anisotropy studies Fluorescence anisotropy provides information about the rotational diffusibility of a fluorophore. The dansyl moiety experiences relatively more viscous and rigid microenvironment at the core in G2-PGL-ipd and G3-PGL-ipd in aqueous medium due the aggregation of these dendrons that results in a ten-fold increase in fluorescence anisotropy as compared to that in G0-PGL-ipd and G1-PGL-ipd [25]. With addition of methanol, a gradual decrease in
Tables 1 and 2 summarise the fluorescence lifetime values for G2-PGL-ipd and G3-PGL-ipd, respectively in water–methanol mixture over the entire composition. In water both G2-PGL-ipd and G3-PGL-ipd show biexponential decays with a short (5–6 ns) and a long (18–19 ns) lifetime component. Up to 30% CH3OH for G2-PGL-ipd and 50% CH3OH for G3-PGL-ipd, both dendrons show biexponential fluorescence decay similar to that in pure water. The average fluorescence lifetime decreases with addition
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U. Subuddhi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 351–356 Table 1 Fluorescence lifetime (s) data for G2-PGL-ipd in water–methanol mixture.
5.60 5.34 4.93 4.90 – – – – – – –
a1 (%)
s2 (ns)
8 10 13 29 – – – – – – –
a2 (%)
18.20 17.90 17.60 16.98 09.35 10.49 11.62 12.20 12.79 13.53 13.01
92 90 88 81 100 100 100 100 100 100 100
12
Fl. Lifetime (ns)
0 10 20 30 40 50 60 70 80 90 100
s1 (ns)
v2
savg (ns) 17.19 16.64 16.13 15.17 09.35 10.49 11.62 12.20 12.79 13.53 13.01
18
1.07 1.07 1.04 1.11 1.12 1.12 1.12 1.08 0.99 1.07 1.13
Average Fl. Lifetime (ns)
% Methanol
20 10 8 6 4
16
G1 G0
2 0
20
40
12
G3 G2 0
a1 (%)
s2 (ns)
a2 (%)
savg (ns)
v2
0 10 20 30 40 50 60 70 80 90 100
5.70 5.39 5.50 5.47 5.52 5.40 – – – – –
6 7 9 11 12 15 – – – – –
19.00 18.95 18.90 18.73 18.41 18.01 12.52 12.98 13.35 14.03 13.52
94 93 91 89 88 85 100 100 100 100 100
18.20 18.00 17.69 17.27 16.86 16.12 12.52 12.98 13.35 14.03 13.52
1.07 1.12 1.08 1.13 1.14 1.01 1.02 1.13 1.12 1.05 1.07
G2-PGL-ipd G3-PGL-ipd
20000 19600 19200 18800
A 20
40
60
80
0.8
0.6
0.4
B 0
0.06
0.03
C 60
% of Acetonitrile
80
100
Average Fluorescence Lifetime (ns)
Fluorescence Anisotropy
0.09
40
100
20
40
60
80
100
% of Acetonitrile G2-PGL-ipd G3-PGL-ipd
20
80
G2-PGL-ipd G3-PGL-ipd
1.0
100
0.12
0
60
dendron fluorescence shows monoexponential decay with a gradual increase in the fluorescence lifetime. The inset in Fig. 5 shows more or less a linear dependence of fluorescence lifetime for G0PGL-ipd and G1-PGL-ipd on the percentage of CH3OH added, which is very similar to that of G2-PGL-ipd and G3-PGL-ipd at higher percentage of CH3OH. The biexponential nature of fluorescence and the large amplitudes associated with the longer lifetime
% of Acetonitrile
0.00
40
Fig. 5. Variation of average fluorescence lifetime of G2-PGL-ipd and G3-PGL-ipd with the percentage of methanol in water–methanol binary mixture. Inset: Variation of fluorescence lifetime of G1-PGL-ipd and G0-PGL-ipd with the percentage of methanol in water–methanol binary mixture.
Normalised Fluorescence Intensity
−1
Emission Energy (cm )
20400
0
20
% of Methanol
of methanol up to 40% for G2-PGL-ipd and 60% for G3-PGL-ipd (Fig. 5). On further increase in the methanol percentage the
18400
100
% of Methanol
10
s1 (ns)
80
14
Table 2 Fluorescence lifetime (s) data for G3-PGL-ipd in water–methanol mixture. % Methanol
60
G2-PGL-ipd G3-PGL-ipd
18 16 14 12 10 8
D 0
20
40
60
80
100
% of Acetonitrile
Fig. 6. Variation of (A) emission energy (cm1), (B) fluorescence intensity, (C) fluorescence anisotropy and (D) average fluorescence lifetime (ns) with the acetonitrile percentage for G2-PGL-ipd and G3-PGL-ipd dendrons in water–acetonitrile mixture.
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component at lower percentage of CH3OH (up to 30% CH3OH for G2-PGL-ipd and 50% CH3OH for G3-PGL-ipd), implies significant aggregation-induced shielding and motional restriction experienced by the dansyl moiety. However, at higher percentage of CH3OH once the disaggregation process is complete, the dansyl moiety experiences the continuous dielectric change due to the binary solvent mixture and show monoexponential fluorescence decay just like that in case of G0-PGL-ipd and G1-PGL-ipd.
photophysical behaviour of these dendrons in water–methanol and water–acetonitrile systems supports the fact that the fluorescence properties of dansyl in these dendrons are indicative of the polarity of the solvent mixtures rather than their micro-heterogeneous structure. Higher the hydrophobicity of the dendron more is the amount of the second solvent required for disaggregation. These findings can contribute significantly to the general understanding of the dendron systems and eventually also for biomedical applications such as delivery processes.
Studies in water–acetonitrile binary solvent mixture Acknowledgements In order to verify for any possible role of the micro-heterogeneity of the water–methanol system in the above observed phenomenon, studies were carried out in water–acetonitrile system. The behaviour of G2-PGL-ipd and G3-PGL-ipd in water– acetonitrile mixture (Fig. 6) was found to be very similar to that in water–methanol mixture in terms of all the parameters studied i.e. fluorescence emission energy, fluorescence intensity, anisotropy and fluorescence lifetime, although these two solvent pairs differ remarkably in their molecular level interactions and structural properties [30,31]. The only difference that was observed between the two binary mixtures is in the volume percentage of the nonaqueous solvent that is needed for the complete disaggregation of the two dendrons in binary systems. In water–methanol case the percentages are 40% for G2-PGL-ipd and 60% for G3-PGLipd but in water–acetonitrile mixture the percentages are 30% for G2-PGL-ipd and 50% for G3-PGL-ipd i.e. a slightly lower percentage of acetonitrile is needed for complete disaggregation of the dendrons as compared to that of methanol, which can be due to the more polar nature of acetonitrile compared to methanol (dielectric constant of acetonitrile = 37.5 and methanol = 32.7). The close resemblance of photophysical behaviour of these dendrons in water–methanol and water-acetonitrile supports the fact that the fluorescence properties of dansyl in these dendrons are indicative of the solvent polarity effect on dendron aggregates and not the micro-heterogeneous structures of the solvent mixtures. Conclusion The photophysical behaviour up to third generations of polyglycerol dendrons with isopropylidene end groups and dansyl moiety attached at the focal point in binary aqueous–nonaqueous solvent mixtures were investigated. For the studies two binary mixtures were chosen (i) water with methanol, a polar protic solvent, and (ii) water with acetonitrile, a polar aprotic solvent. Four fluorescence parameters namely fluorescence intensity, emission energy, fluorescence anisotropy and fluorescence lifetime were employed. Interesting solvatochromism was observed in second and third generation dendrons in water, on addition of the second solvent, methanol or acetonitrile. A reversal in solvatochromic behaviour was observed i.e. below a certain percentage of the nonaqueous solvent there is a negative-solvatochromism and above that a positive-solvatochromism. The negative-solvatochromism is attributed to the progressive disaggregation of the dendron aggregates by the nonaqueous solvent component. Once the disaggregation process is complete, positive-solvatochromism is shown by the dendron monomers similar to that of lower order dendrons. The dansyl moiety in the dendron monomer in all generations of dendrons experiences a relatively more polar microenvironment as compared to that in the bulk, which is due to the preferential solvation of the fluorophore as a result of dielectric enrichment in the two binary solvent mixtures. The close resemblance of
Prasanna Kumar thanks the Council of Scientific and Industrial Research (CSIR), Govt. of India for financial support. We thank DBT for the Jobin-Yvon Fluoromax 4 spectrofluorimeter and DST for the TCSPC lifetime instrument.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.02.150. References [1] E. Burakowska, J.R. Quinn, S.C. Zimmerman, R. Haag, J. Am. Chem. Soc. 131 (2009) 10574–10580. [2] A. Sunder, M. Kramer, R. Hanselmann, R. Mulhaupt, H. Frey, Angew. Chem. Int. Ed. 38 (1999) 3552–3555. [3] M. Adeli, N. Mirab, M.S. Alavidjeh, Z. Sobhani, F. Atyabi, Polymer 50 (2009) 3528–3536. [4] M.A. Carnahan, M.W. Grinstaff, Macromolecules 39 (2006) 609–616. [5] M.T. Morgan, M.A. Carnahan, S. Finkelstein, C.A.H. Prata, L. Degoricija, S.J. Lee, M.W. Grinstaff, Chem. Commun. 34 (2005) 4309–4311. [6] M. Calderon, M.A. Quadir, S.K.. Sharma, R. Haag, Adv. Mater 22 (2010) 190–218. [7] S. Hecht, J.M.J. Fre´chet, Angew Chem. Int. Ed. 40 (2001) 74–91. [8] D.K. Smith, L. Muller, Chem. Commun. 18 (1999) 1915–1916. [9] D.K. Smith, F. Diederich, Chem. Eur. J. 4 (1998) 1353–1361. [10] A. Agarwal, A. Asthana, U. Gupta, N.K. Jain, J. Pharm. Pharmacol. 60 (2008) 671–688. [11] S.H. Medina, M.E.H. El-Sayed, Chem. Rev. 109 (2009) 3141–3157. [12] B.K. Nanjwade, H.M. Bechra, G.K. Derkar, F.V. Manvi, V.K. Nanjwade, Eur. J. Pharm. Sci. 38 (2009) 185–196. [13] J.B. Wolinsky, M.W. Grinstaff, Adv. Drug Delivery Rev. 60 (2008) 1037–1055. [14] S. Svenson, D.A. Tomalia, Drug Delivery Rev. 57 (2005) 2106–2129. [15] W.D. Jang, S.K.M. Kamruzzaman, C.H. Lee, I.K. Kang, Prog. Polym. Sci 34 (2009) 1–23. [16] M.R. Radowski, A. Shukla, H. von Berlepsch, C. Bottcher, G. Pickaert, H. Rehage, R. Haag, Angew. Chem. Int. Ed. 46 (2007) 1265–1269. [17] U. Gupta, H.B. Agashe, A. Asthana, N.K. Jain, Dendrimers: Biomacromolecules 7 (2006) 649–658. [18] S. Svenson, A.S. Chauhan, Nanomedicine 3 (2008) 679–702. [19] H. Turk, A. Shukla, R.P.C. Alves, H. Rehage, R. Haag, Chem. Eur. J. 13 (2007) 4187–4196. [20] M.T. Morgan, M.A. Carnahan, C.E. Immoos, A.A. Ribeiro, S. Finkelstein, S.J. Lee, M.W. Grinstaff, J. Am. Chem. Soc. 125 (2003) 15485–15489. [21] S.K. Mohanty, S. Thirunavukarasu, S. Baskaran, A.K. Mishra, Macromolecules 37 (2004) 5364–5369. [22] S.K. Mohanty, U. Subuddhi, S. Baskaran, A.K. Mishra, Photochem. Photobiol. Sci. 6 (2007) 1164–1169. [23] C.M. Cardona, J. Alvarez, A.E. Kaifer, T.D. McCarley, S. Pandey, G.A. Baker, N.J. Bonzagni, F.V. Bright, J. Am. Chem. Soc. 122 (2000) 6139–6144. [24] C.M. Cardona, T. Wilkes, W. Ong, A.E. Kaifer, T.D. McCarley, S. Pandey, G.A. Baker, M.N. Kane, S.N. Baker, F. V. Bright. J. Phy. Chem. B 106 (2002) 8649– 8656. [25] P.K. Vuram, U. Subuddhi, S.T. Krishnaji, A. Chadha, A.K. Mishra, Eur. J. Org. Chem. 26 (2010) 5030–5040. [26] U. Subuddhi, P.K. Vuram, A.K. Mishra, A . Chadha, J. Photochem. Photobiol. A: Chem. 217 (2011) 411–416. [27] P.B. Leezenberg, C.W. Frank, Macromolecules 28 (1995) 7404–7415. [28] P. Suppan, J. Chem. Soc. Faraday Trans. 1: Phys. Chem. Condens. Phases 83 (1987) 495–509. [29] R.L. Reeves, M.S. Maggio, L.F. Costa, J. Am. Chem. Soc. 96 (1974) 5917–5925. [30] S. Nigam, A. de Juan, R.J. Stubbs, S.C. Rutan, Anal. Chem. 72 (2000) 1956–1963. [31] H. Kovacs, A. Laaksonen, J. Am. Chem. Soc. 113 (1991) 5596–5605.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Disaggregation induced solvatochromic switch: A study of dansylated polyglycerol dendrons in binary solvent mixture Usharani Subuddhi a, Prasanna K. Vuram b, Anju Chadha c,⇑, Ashok K. Mishra b,⇑ a
Department of Chemistry, National Institute of Technology Rourkela, India Department of Chemistry, Indian Institute of Technology Madras, Chennai 600036, India c Laboratory of Bioorganic Chemistry, Department of Biotechnology and National Center for Catalysis Research, Indian Institute of Technology Madras, Chennai 600036, India b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Interesting solvatochromism in
Reversal in solvatochromic behaviour of dendrons in aqueous medium on addition of a nonaqueous solvent. Negative-solvatochromism is attributed to the progressive disaggregation of dendron aggregates and the positive-solvatochromism is exhibited by the dendron monomers.
polyether dendron aggregates in mixed aqueous media. Reversal in solvatochromic behaviour depending on percentage of nonaqueous solvent. Negative-solvatochromism due to progressive disaggregation of dendron aggregates. Positive-solvatochromism is shown by the dendron monomers. Higher dendron hydrophobicity requires more of second solvent for disaggregation.
a r t i c l e
i n f o
Article history: Received 16 November 2013 Received in revised form 18 February 2014 Accepted 21 February 2014 Available online 12 March 2014 Keywords: Dansylated polyether dendrons Solvatochromism Binary aqueous-nonaqueous solvent mixtures Fluorescence Aggregation–disaggregation
a b s t r a c t A reversal in solvatochromic behaviour was observed in second and third generation glycerol based dansylated polyether dendrons in water on addition of a second solvent like methanol or acetonitrile. Below a certain percentage of the nonaqueous solvent there is a negative-solvatochromism observed and above that there is a switch to positive-solvatochromism. The negative-solvatochromism is attributed to the progressive disaggregation of the dendron aggregates by the nonaqueous solvent component. Once the disaggregation process is complete, positive-solvatochromism is exhibited by the dendron monomers. Higher the hydrophobicity of the dendron more is the amount of the second solvent required for disaggregation. Ó 2014 Elsevier B.V. All rights reserved.
Introduction
⇑ Corresponding authors. Tel.: +91 44 22574207; fax: +91 44 22574202 (A.K. Mishra). Tel.: +91 44 22574106; fax: +91 44 22574102 (A. Chadha). E-mail addresses: [email protected] (A. Chadha), [email protected] (A.K. Mishra). http://dx.doi.org/10.1016/j.saa.2014.02.150 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
Polyethers, hyperbranched polyglycerols [1–3] and polyglycerol dendrimers [4–6] have been the focus of recent studies due to their excellent water solubility and biocompatibility. The microenvironment created in the core of dendrimers resembles that of biological
352
U. Subuddhi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 128 (2014) 351–356
systems such as proteins, primarily because of the branching architecture of dendrimers [7,8], which in turn modulates their physical properties [9]. Aggregation of the primary units of dendrimers and dendrons into larger and more complex forms leads to a wide range of potential applications in the fields of drug delivery [10–12], biomedical applications [13–15] and nanotechnology [16–18,13]. Polyglycerol dendrimers have shown good transport capacity for poorly water soluble drugs [5,13,19,20]. Hence, it is important that their physical and chemical properties are understood under various conditions and a significant effort has been directed towards this [10–18,21–24]. Synthesis of glycerol based dansylated polyether dendrons ((Gn)-PGL-ipd, where Gn specifies the generation number of the Dendron (Scheme 1 in electronic supplementary material)), their photophysical properties and aggregation behaviour have been recently reported by us [25,26]. The emission maxima for all generations of dendrons showed strong dependence on solvent polarity in nonaqueous solvents, indicating the dominant importance of general solvent effect. In general they showed positive solvatochromism i.e. with increasing solvent polarity a red-shift in the emission maximum is exhibited. However, in water the most polar among all the solvents studied, the higher generation dendrons from second generation onwards exhibited unusual blue-shift in their emission maximum representing more or less dioxane like polarity. When compared with the lower generation dendrons like G0-PGL-ipd and G1-PGL-ipd, they showed almost 60 nm hypsochromic shift in the emission maximum accompanied by a remarkable enhancement in the fluorescence intensity, a ten-fold increase in fluorescence anisotropy and four-fold increase in the average fluorescence lifetime. Based on these observations it was suggested that there is a hydrophobicity induced aggregation taking place in these higher generation dendrons in aqueous medium even at concentrations as low as 1 108 M. If aggregation of these dendrons in aqueous medium is induced by hydrophobicity, it is expected that on addition of a second solvent like methanol or acetonitrile, the disaggregation process will set in, which possibly would result in interesting solvatochromism in the mixed solvents. An understanding of the process of disaggregation of these dendrons and its effect on the fluorescence behaviour of dansyl moiety is the objective of this work. The present work illustrates the interesting photophysical features of assembling and disassembling of dansylated polyglycerol dendrons, with isopropylidene end groups, depending on dendron generation and solvent polarity in binary aqueous–nonaqueous solvent mixtures (water–methanol and water–acetonitrile).
appropriate volume of the solvent of interest. All the solvents used were of UV-spectroscopic grade (SRL Pvt. Ltd., India) and used without further purification. The water used was distilled three times with addition of alkaline permanganate. In order to check the effect of order of addition of the solvents on the properties of dendrimers, the binary aqueous–nonaqueous solutions were prepared in three different ways, (i) by adding different volumes of the nonaqueous solvent (methanol or acetonitrile) to the aqueous solution of the dendrimer, (ii) by adding different volumes of water to the nonaqueous solution of the dendrimer and (iii) by mixing the aqueous and nonaqueous solutions of dendrimer at different ratios so as to achieve the desired ratio (v/v) of the two solvents. The solutions were shaken well and left for about an hour for equilibration before carrying out the studies. The results were found to be very similar in all three cases irrespective of the method of preparation. The dendrimer concentration was kept constant at 1 105 M in all the studies. Instrumentation Fluorescence was recorded on Hitachi F-4500 and Jobin–Yvon Fluoromax-4 spectrofluorimeters. The steady state fluorescence anisotropy (rss) values were obtained by using the expression
rss ¼ ðIk GI? Þ=ðIk þ 2GI? Þ where I|| and I\ are fluorescence intensities when the emission polarizer is parallel and perpendicular, respectively to the direction of polarization of the excitation beam and G is the factor that corrects for unequal transmission by the diffraction gratings of the instrument for vertically and horizontally polarized light. Fluorescence lifetime (sf) measurements were carried out using Horiba Jobin–Yvon TCSPC lifetime instrument in a time-correlated single-photon counting (TCSPC) arrangement. A 340 nm nano LED was used as the light source. The pulse repetition rate was set to 1 MHz and the instrumental full width half maximum of the 340 nm LED including the detector response is 40% for G2-PGL-ipd and >60% for G3-PGL-ipd) is very similar to that observed in case of G0-PGL-ipd and G1-PGL-ipd. Earlier studies have suggested that higher order dendrons, from second generation onwards, show self-association behaviour in aqueous medium resulting in almost 60 nm blue-shift in the emission maximum in comparison to that of G0-PGL-ipd and G1-PGL-ipd [25]. This means in the aggregated form of dendrons, the dansyl moiety experiences a relatively more nonpolar microenvironment than in the non-aggregated monomeric form. In view of this, the unusual negative solvatochomism exhibited by G2-PGLipd and G3-PGL-ipd (red-shift in emission maximum by reducing
0
20
40
60
80
100
% of Methanol Fig. 2. Plots of (A) emission energy (B) normalised fluorescence intensity of G2PGL-ipd and G3-PGL-ipd with the percentage of methanol in water–methanol binary mixture.
the overall solvent polarity) at lower percentages of added CH3OH (60% for G3-PGL-ipd) the anisotropy value of both G2-PGL-ipd and G3-PGL-ipd is similar to that of G0-PGL-ipd and G1-PGL-ipd (Fig. A1 in electronic supplementary material). The anisotropy value for G0-PGL-ipd and G1-PGL-ipd is found to be very low in aqueous medium and remains invariant with the solvent composition in the binary mixture. Thus, fluorescence anisotropy study supports the fact that there is a progressive disaggregation of the aggregates taking place with addition of CH3OH and by 40% and 60% of CH3OH there is complete disaggregation of G2-PGL-ipd and G3-PGL-ipd dendrons, respectively. Fluorescence lifetime studies
Fluorescence anisotropy studies Fluorescence anisotropy provides information about the rotational diffusibility of a fluorophore. The dansyl moiety experiences relatively more viscous and rigid microenvironment at the core in G2-PGL-ipd and G3-PGL-ipd in aqueous medium due the aggregation of these dendrons that results in a ten-fold increase in fluorescence anisotropy as compared to that in G0-PGL-ipd and G1-PGL-ipd [25]. With addition of methanol, a gradual decrease in
Tables 1 and 2 summarise the fluorescence lifetime values for G2-PGL-ipd and G3-PGL-ipd, respectively in water–methanol mixture over the entire composition. In water both G2-PGL-ipd and G3-PGL-ipd show biexponential decays with a short (5–6 ns) and a long (18–19 ns) lifetime component. Up to 30% CH3OH for G2-PGL-ipd and 50% CH3OH for G3-PGL-ipd, both dendrons show biexponential fluorescence decay similar to that in pure water. The average fluorescence lifetime decreases with addition
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5.60 5.34 4.93 4.90 – – – – – – –
a1 (%)
s2 (ns)
8 10 13 29 – – – – – – –
a2 (%)
18.20 17.90 17.60 16.98 09.35 10.49 11.62 12.20 12.79 13.53 13.01
92 90 88 81 100 100 100 100 100 100 100
12
Fl. Lifetime (ns)
0 10 20 30 40 50 60 70 80 90 100
s1 (ns)
v2
savg (ns) 17.19 16.64 16.13 15.17 09.35 10.49 11.62 12.20 12.79 13.53 13.01
18
1.07 1.07 1.04 1.11 1.12 1.12 1.12 1.08 0.99 1.07 1.13
Average Fl. Lifetime (ns)
% Methanol
20 10 8 6 4
16
G1 G0
2 0
20
40
12
G3 G2 0
a1 (%)
s2 (ns)
a2 (%)
savg (ns)
v2
0 10 20 30 40 50 60 70 80 90 100
5.70 5.39 5.50 5.47 5.52 5.40 – – – – –
6 7 9 11 12 15 – – – – –
19.00 18.95 18.90 18.73 18.41 18.01 12.52 12.98 13.35 14.03 13.52
94 93 91 89 88 85 100 100 100 100 100
18.20 18.00 17.69 17.27 16.86 16.12 12.52 12.98 13.35 14.03 13.52
1.07 1.12 1.08 1.13 1.14 1.01 1.02 1.13 1.12 1.05 1.07
G2-PGL-ipd G3-PGL-ipd
20000 19600 19200 18800
A 20
40
60
80
0.8
0.6
0.4
B 0
0.06
0.03
C 60
% of Acetonitrile
80
100
Average Fluorescence Lifetime (ns)
Fluorescence Anisotropy
0.09
40
100
20
40
60
80
100
% of Acetonitrile G2-PGL-ipd G3-PGL-ipd
20
80
G2-PGL-ipd G3-PGL-ipd
1.0
100
0.12
0
60
dendron fluorescence shows monoexponential decay with a gradual increase in the fluorescence lifetime. The inset in Fig. 5 shows more or less a linear dependence of fluorescence lifetime for G0PGL-ipd and G1-PGL-ipd on the percentage of CH3OH added, which is very similar to that of G2-PGL-ipd and G3-PGL-ipd at higher percentage of CH3OH. The biexponential nature of fluorescence and the large amplitudes associated with the longer lifetime
% of Acetonitrile
0.00
40
Fig. 5. Variation of average fluorescence lifetime of G2-PGL-ipd and G3-PGL-ipd with the percentage of methanol in water–methanol binary mixture. Inset: Variation of fluorescence lifetime of G1-PGL-ipd and G0-PGL-ipd with the percentage of methanol in water–methanol binary mixture.
Normalised Fluorescence Intensity
−1
Emission Energy (cm )
20400
0
20
% of Methanol
of methanol up to 40% for G2-PGL-ipd and 60% for G3-PGL-ipd (Fig. 5). On further increase in the methanol percentage the
18400
100
% of Methanol
10
s1 (ns)
80
14
Table 2 Fluorescence lifetime (s) data for G3-PGL-ipd in water–methanol mixture. % Methanol
60
G2-PGL-ipd G3-PGL-ipd
18 16 14 12 10 8
D 0
20
40
60
80
100
% of Acetonitrile
Fig. 6. Variation of (A) emission energy (cm1), (B) fluorescence intensity, (C) fluorescence anisotropy and (D) average fluorescence lifetime (ns) with the acetonitrile percentage for G2-PGL-ipd and G3-PGL-ipd dendrons in water–acetonitrile mixture.
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component at lower percentage of CH3OH (up to 30% CH3OH for G2-PGL-ipd and 50% CH3OH for G3-PGL-ipd), implies significant aggregation-induced shielding and motional restriction experienced by the dansyl moiety. However, at higher percentage of CH3OH once the disaggregation process is complete, the dansyl moiety experiences the continuous dielectric change due to the binary solvent mixture and show monoexponential fluorescence decay just like that in case of G0-PGL-ipd and G1-PGL-ipd.
photophysical behaviour of these dendrons in water–methanol and water–acetonitrile systems supports the fact that the fluorescence properties of dansyl in these dendrons are indicative of the polarity of the solvent mixtures rather than their micro-heterogeneous structure. Higher the hydrophobicity of the dendron more is the amount of the second solvent required for disaggregation. These findings can contribute significantly to the general understanding of the dendron systems and eventually also for biomedical applications such as delivery processes.
Studies in water–acetonitrile binary solvent mixture Acknowledgements In order to verify for any possible role of the micro-heterogeneity of the water–methanol system in the above observed phenomenon, studies were carried out in water–acetonitrile system. The behaviour of G2-PGL-ipd and G3-PGL-ipd in water– acetonitrile mixture (Fig. 6) was found to be very similar to that in water–methanol mixture in terms of all the parameters studied i.e. fluorescence emission energy, fluorescence intensity, anisotropy and fluorescence lifetime, although these two solvent pairs differ remarkably in their molecular level interactions and structural properties [30,31]. The only difference that was observed between the two binary mixtures is in the volume percentage of the nonaqueous solvent that is needed for the complete disaggregation of the two dendrons in binary systems. In water–methanol case the percentages are 40% for G2-PGL-ipd and 60% for G3-PGLipd but in water–acetonitrile mixture the percentages are 30% for G2-PGL-ipd and 50% for G3-PGL-ipd i.e. a slightly lower percentage of acetonitrile is needed for complete disaggregation of the dendrons as compared to that of methanol, which can be due to the more polar nature of acetonitrile compared to methanol (dielectric constant of acetonitrile = 37.5 and methanol = 32.7). The close resemblance of photophysical behaviour of these dendrons in water–methanol and water-acetonitrile supports the fact that the fluorescence properties of dansyl in these dendrons are indicative of the solvent polarity effect on dendron aggregates and not the micro-heterogeneous structures of the solvent mixtures. Conclusion The photophysical behaviour up to third generations of polyglycerol dendrons with isopropylidene end groups and dansyl moiety attached at the focal point in binary aqueous–nonaqueous solvent mixtures were investigated. For the studies two binary mixtures were chosen (i) water with methanol, a polar protic solvent, and (ii) water with acetonitrile, a polar aprotic solvent. Four fluorescence parameters namely fluorescence intensity, emission energy, fluorescence anisotropy and fluorescence lifetime were employed. Interesting solvatochromism was observed in second and third generation dendrons in water, on addition of the second solvent, methanol or acetonitrile. A reversal in solvatochromic behaviour was observed i.e. below a certain percentage of the nonaqueous solvent there is a negative-solvatochromism and above that a positive-solvatochromism. The negative-solvatochromism is attributed to the progressive disaggregation of the dendron aggregates by the nonaqueous solvent component. Once the disaggregation process is complete, positive-solvatochromism is shown by the dendron monomers similar to that of lower order dendrons. The dansyl moiety in the dendron monomer in all generations of dendrons experiences a relatively more polar microenvironment as compared to that in the bulk, which is due to the preferential solvation of the fluorophore as a result of dielectric enrichment in the two binary solvent mixtures. The close resemblance of
Prasanna Kumar thanks the Council of Scientific and Industrial Research (CSIR), Govt. of India for financial support. We thank DBT for the Jobin-Yvon Fluoromax 4 spectrofluorimeter and DST for the TCSPC lifetime instrument.
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