Incorporation of Coumarin 6 in cyclodextrins: microcrystals to lamellar composites

Abstract

Coumarin 6 precipitates in water as microcrystals resulting in a considerable loss in fluorescence yield. Differential interaction of the microcrystals with cyclodextrins of different cavity size enhances the fluorescence yield of the dye by 160 fold in some cases. Highly fluorescent ultrathin lamellar entities form through hydrogen bonding.

---

Unique spectroscopic and photophysical properties of fluorescent organic nano and microcrystals make them important candidates for future technologies. They are potent materials for chemical and biological sensing,^1–3 photocatalysis,⁴ optics,^5–7 optoelectronics,^8,9 etc. Chemical structures of the constituting molecules of the nano/microcrystals as well as the various intermolecular interactions in solid state decide their inherent characteristics. Coumarin 6 (C6) (Fig. S1 in ESI†) and molecules of this family were recently studied in aspects of formation of nano/microcrystals. Some Coumarin derivatives form flat microcrystals while others develop short and long nanofibers.^10,11 By definition, these fluorescent nano-composites emit light after absorption of UV or visible radiations. Formation of microcrystals makes C6 and related compounds useful for various practical purposes, e.g., colouring in textile fibers,¹² biomedical imaging,¹³ lasers,^14,15 photonic devices,¹⁶ optical light emitting diode,¹⁷ etc. Crystal structures show that the individual molecules in C6 microcrystals are almost planar and centrosymmetrically arranged.¹⁸ Although microcrystals of C6 were previously obtained from re-precipitation method¹¹ and high dye concentration,¹⁹ our experiment shows that C6 readily forms microcrystals in water (co-solvated with 1% methanol) with time on incubation at room temperature (Fig. S2, ESI†). It is well known that intramolecular charge transfer (ICT) is the source of the strong emission from Coumarins.^20,21 However, spontaneous microcrystal formation even at relatively low dye concentration considerably reduces the fluorescence yield of C6 in water due to the non-radiative channels.

C6 is insoluble in water but soluble in methanol and ethanol at room temperature. Property of C6 in different heterogeneous systems has been studied before. C6 can be effectively loaded in solid lipid nanoparticles and liposomes for delivery to targeted biological locations.^22,23 Changed physicochemical properties of C6 in protein aggregates help in distinguishing amorphous and fibrillar aggregates.²⁴ In a recent report it is shown that cucurbit[7]uril (CB7) can uptake C6 both in 1 : 1 and 2 : 1 (CB7/C6) stoichiometries facilitating protonation of C6 at neutral pH.²⁵ However, aggregation of C6 in water with time and its subsequent loss of fluorescence yield because of microcrystal formation hardly drew any attention. It is well established that N,N′-dialkyl substituted Coumarin derivatives favour planar ICT states leading to partially charged separated ground state. Hence, probability of formation of parallel stacks through van der Waals¹⁸ as well as electrostatic interactions increases. As a consequence, probability of ICT in excited state decreases and results into reduction in fluorescence quantum yield of C6 in water.

Initially we have provided a critical report on microcrystal formation of C6 in water at low molecular concentration. The observations appreciably differ from previous reports. Later, host–guest interaction of C6 with cyclodextrins (CDs) of different cavity size has been presented where we have explained the changes in physicochemical properties of the fluorophore. Furthermore, we showed that the guest–host nanocapsules produce distinct aggregates depending on the cavity size of the CDs. These nanostructures are found to have intense bright green emission. It is also proved that on changing cavity size of the host, the mode of molecular interaction changes and in turn alters the quantum yield.

It is well known that CDs are capable of forming complexes with various compounds through host–guest interaction that affects the molecular properties of the incorporated guest.^26–28 Cavity size of CDs increases in the order α-CD < β-CD < γ-CD. Interaction between CDs and guest molecules is largely dependent on the size and shape of the guest. Depending on the relative size of the CDs and the guest molecules, more than one guest can be accommodated inside a single CD cavity. Polarity and hydrophobicity of the guest molecule is also important to form inclusion complex. Guest–host interaction with CDs and formation of small molecule induced nanostructures has been the topic of interest in our group from long back.^29–31 In the present study we have shown that C6 can form ultrathin two dimensional micrometer sized nanostructures with β- and γ-CDs.

C6 shows structured absorption spectra in solvents of different polarity (Fig. 1a). A distinct new band is observed at 500 nm in water. This new peak develops with time and hence can be attributed to the formation of microaggregates of C6 that was confirmed from atomic force microscopy (AFM) and scanning electron microscopy (SEM) using aliquot from the same sample (Fig. S2, ESI†). Gradual addition of α-CD to the solution of C6 in water does not impart any appreciable change in the spectrum (Fig. 1b) indicating non-interference of α-CDs in changing the physical characteristics of the C6 microcrystals. There is a difference in absorbance in the blank C6 solutions that may be due to slight variation in settling time leading to formation of different extents of C6 microcrystals (Fig. S3†). An increase in absorbance on gradual addition of α-CD to the C6 solution indicates accumulation of α-CD around the microcrystals. In case of β- and γ-CDs, we observed noticeable structural change in the absorption spectra (Fig. 1c and d). The 500 nm peak due to the microaggregates of C6 disappeared with appearance of a new shoulder at ∼550 nm. Disappearance of the 500 nm peak indicates possible breakage of the microaggregates by the CDs and the 550 nm band reflects CD-C6 host–guest complex formation. Correlating with the spectral changes, SEM, confocal and epifluorescence micrographs show formation of interesting structural features as shown in Fig. 2.

Fig. 1 Absorption spectra of C6 (1 μM): (a) in solvents of different polarity (the brown arrow shows the band due to the microaggregates in water) and in (b) α-, (c) β- and (d) γ-CDs in aqueous environment.

Fig. 2 SEM images of C6 in (a) α-, (b) β-, and (c) γ-CDs; the height profiles of the aggregates in (d) α-, (e) β-, and (f) γ-CDs were taken from AFM images of the corresponding aggregates. The bottom panel shows the epifluorescence images of C6 in (left) β-, and (right) γ-CDs.

Fragmented microtubular structures were observed when α-CD was added to the aqueous solution of C6 (Fig. 2a). The tubules are nearly of the same length as the microcrystals of C6. Hence, it is presumable that the small cavities (∼6 Å inner diameter) of α-CD are not sufficient to incorporate the C6 molecules and thus could not break the microcrystals. Instead, they accumulated around the micro-aggregates providing tubular structures. Whereas, the inner cavity dimensions of β-CD (∼8 Å) and γ-CD (∼9.5 Å) suggest encapsulation of one and two C6 molecule/s, respectively. In both the cases we observed formation of lamellar microstructures (Fig. 2b and c) having lengths in the order of 2–3 μm and thickness in the range of 1.5–3 nm (Fig. 2d–f). Although it is known that after a certain critical concentration, β-CD forms self-aggregates,^32,33 bright green epifluorescent images in Fig. 2 confirm that the micro-structures obtained from β- and γ-CDs are impregnated with C6. The brightness, however, is much higher in case of β-CD compared to that in γ-CD.

As discussed before, the fluorescence quantum yield of C6 in water gradually decreases with time (∼0.007), although it is very high in methanol. This excessive decrease in quantum yield can be attributed to the formation of micro-aggregates. Due to addition of CDs, the fluorescence intensity of C6 in water increases with a concomitant blue shift of the emission maximum (Fig. 3a and b and S4 in ESI†). The extent of enhancement in intensity and hypsochromic shift are different in the three CDs. β-CD interacts with C6 resulting into highest enhancement in emission intensity whereas γ-CD produces largest hypsochromic shift. α-CD increases the fluorescence intensity very feebly with a small blue shift (6 nm). This indicates weak interaction of α-CD with C6 that may not be strong enough to break the microaggregates. Since the microcrystals of C6 are hydrophobic in nature, α-CD molecules may gather along the surface of the microaggregates and remove some of the accumulated water molecules thus lowering the polarity at the interface resulting into the small blue shift of C6 emission maximum. Formation of tubular aggregates of α-CD-C6 composites and effect of temperature on the C6 microcrystals before and after addition of α-CD explains the observation (Fig. 3d).

Fig. 3 (a) Change in fluorescence intensity of C6 (1 μM) on addition of 0–15 mM α-, 0–10 mM β-, and 0–16 mM γ-CD; (b) normalized emission spectra of free (saturated emission) and CD encapsulated C6; (c) comparison of fluorescence quantum yield of C6 in β- and γ-CDs measured using Coumarin 153 as standard; and (d) effect of temperature on C6 and its composites with the CDs. λ_ex = 430 nm.

A 160 fold enhancement in fluorescence and 20 nm blue shift of the C6 emission maximum were observed upon addition of about 10 mM of β-CD to C6 (Fig. 3a and b). The molecular dimension of C6 suggests encapsulation of one dye molecule inside β-CD cavity leading to initial formation of 1 : 2 guest–host complex.^29,30 The C6 microaggregates break due to the approach of the relatively hydrophobic nanocavities of β-CD and results into formation of guest–host nanocapsules through primary interactions. Such units unite though hydrogen bonding between the hydroxyl groups on the rims of the CDs to form microstructures.^29–31 The guest–host assemblies and mutual hydrogen bonding leads to crystallization of the lamellar entities.^34,35 Formation of flat quasi-two dimensional colloidal quantum dot nanoplatelets having electronic properties of two dimensional quantum wells were reported earlier.³⁶ We propose formation of similar structures from C6 encapsulated β-CD that remarkably enhances the fluorescence yield of the dye. Fluorescence intensity of C6 increases ∼11 fold on interaction with γ-CD until saturation with a blue shift of 26 nm (Fig. 3b and c). Larger interior of γ-CD is capable to encapsulate multiple planar probe molecules.³⁷ Coumarins can form stacked dimers³⁸ and hence get encapsulated inside the γ-CDs resulting into much lower enhancement in fluorescence intensity compared to that in case of β-CD. This could also be the reason behind the highest blue shift in the emission maximum for γ-CD. Fluorescence quantum yield of C6 did not change appreciably upon addition of α-CD and changed only moderately in case of γ-CD. A double reciprocal Benesi–Hildebrand plot for β-CD shows a linear fit according to the equation: 1/(I − I₀) = [1/(I_m − I₀)] + [1/K[CD]²(I_m − I₀)]. Here, I₀ and I_m are the fluorescence intensities at zero and maximum concentrations of β-CD, I denotes the fluorescence intensities at different concentrations of CD, [CD] is the total β-CD concentration, and K is the binding constant. This indicates 1 : 2 encapsulation of one C6 by two β-CD molecules at very low CD concentration (∼2.5 μM). The binding constant for C6 with β-CD is calculated to be 2.57 × 10⁵ M⁻². However, the plot deviates from linearity at higher concentration indicating formation of β-CD nanotubular suprastructures. The nature of interaction of C6 with α- and γ-CDs does not provide specific binding constants.

On increasing the solution temperature from 25 to 65 °C we observed that the relative emissions from the low emitting microcrystals of C6 increased considerably indicating partial cleavage of the larger aggregates to smaller fragments (Fig. 3d). The enhancement in fluorescence is much less on application of α-CD to C6 aggregates. On addition of β- and γ-CDs we witnessed a lowering in fluorescence intensity. In the previous two cases, the molecular C6 entities were absent and the change in fluorescence was due to alteration in non-radiative pathways for the C6 aggregates, whereas, for the latter two instances, molecular C6 was encapsulated inside the CD cavities and on enhancement of temperature got exposed to water and suffered lowering in fluorescence yield.

Time resolved fluorescence signals provide valuable information about the microenvironment of the fluorophore. There are hardly any report on the excited state lifetime of C6 in aqueous environment since this fluorophore is insoluble in water. In our experiment we have used 1% methanol as co-solvent to prepare the aqueous solutions. Excited C6 decays in about 2.5 ns in pure ethanol or methanol.³⁹ C6 shows biexponential emission decay in water on excitation with 402 nm radiation, the faster component shows a lifetime of ∼700 ps and the slower one of ∼2.4 ns (Table 1 and Fig. S5 in ESI†). Since water is a highly polar solvent, hence the faster component of decay can be easily attributed to the intramolecular charge transfer (ICT) in C6. The longer lifetime is for the C6 aggregates in water.

Table 1 Fitting parameters for the decay of C6 in absence and presence of β- and γ-CDs monitoring the 525 nm emission. Values in parentheses are percentage contributions to the fits for each component and the χ² values measure the fitting accuracy

CD	Concentration (mM)	τ1 (ps)	τ2 (ns)	τ3 (ns)	χ^2
β-	0	730 (60)	2.40 (40)	—	1.02
	5	137 (−3)	2.05 (103)	—	1.07
	9	148 (−3)	2.08 (103)	—	1.03
γ-	0	730 (60)	2.40 (40)	—	1.02
	10	116 (9)	1.34 (43)	3.99 (49)	1.13
	20	50 (8)	1.01 (26)	3.60 (66)	1.19

---

On addition of β-CD to C6 we observed a growth component having lifetime ∼150 ps and a longer decaying species. Normally the growth appears due to occurrence of any excited state phenomenon in the time regime of the detecting instrument, for example, electron transfer, charge transfer, proton transfer, slow solvation, etc.^40–42 In the present case, the rise time evokes two possibilities: (i) slow solvation of C6 by water, and (ii) excited state ICT in the probe. However, occurrence of slow solvation accompanies gradual increase in contribution and rise time on moving towards red edge of the emission spectrum of the fluorophore. No such change was observed in our case indicating excited state ICT inside the β-CD cavity.

In γ-CD, the time resolved decay of C6 emission provides different information. The raw data could be fitted with a three exponential function indicating a difference in guest–host interaction compared to that for β-CD (Table 1). A species with relatively long lifetime of ∼4 ns is observed. As mentioned earlier that bigger interior of γ-CD is capable of encapsulating multiple guests, we find concurrence with the time resolved data. The longer decaying species can be attributed to emission from the C6 dimer in γ-CD. The second decaying species (expressed by τ₂) could be due to the 1 : 2 guest–host complexes as usual. The fastest component is due to ICT as observed in aqueous bulk. Less availability of water molecules reduces the ICT time of C6 inside γ-CD.²¹ The lamellar structures formed due to interaction of C6 with β- and γ-CDs can thus be represented by Fig. 4.

Fig. 4 Representation of probable lamellar aggregation of (a) β- and (b) γ-CD encapsulated C6. The view is inspired by the SEM image taken from the samples as shown alongside.

In summary, we have shown that C6 can form microcrystals in water (co-solvated with 1% methanol) with time even at μM concentration. This results into an enormous loss in its fluorescence quantum yield that reaches 0.7%. Hence, the dye becomes unsuitable to be used as laser dye or biological marker. Previous studies did not take this property or problem with C6 into consideration as there is hardly any report on C6 in aqueous environment. We found that CD molecules of different sizes affect the C6 micro-aggregates differently. The smallest of the CDs (α-CD) could not break the C6 aggregates applying hydrophobic force and accumulated around the crystal surface. The larger CDs (β- and γ-) could, on the other hand, break the micro-aggregates of C6 and encapsulate the dye molecules. Encapsulation of C6 by β-CD results into enormous increase in C6 fluorescence (160 fold) and the encapsulated species were observed to form lamellar microcrystals with surface area of ∼4 μM². This, on one hand, considerably uplifts the use of C6 as an efficient laser dye and on the other makes them suitably protected by bio-compatible CD molecules for biological applications. γ-CDs have larger interiors and can accommodate two C6 molecules together that results into a fluorescence enhancement by 11 fold. The γ-CD-C6 micro-aggregation also leads to formation of lamellar microcrystals similar to the β-CDs but with lower brightness. The findings were supported by spectroscopy and microscopy and we suppose that the results will add up to the applications of brightly fluorescent lamellar guest–host composites toward usage in liquid crystal science and beyond.

Acknowledgements

We thank Department of Science and Technology, Government of India for financial assistance (Project number: SR/S1/PC-35/2011). P. G., T. D., A. M. and S. M. acknowledge Council of Science and Industrial Research and University Grants Commission for their fellowships. We thank Mr Ayon Bir for his help in the experiments.

Notes and references

1. L. Wang, L. Wang, T. Xia, L. Dong, G. Bian and H. Chen, Anal. Sci., 2004, 20, 1013.
2. L. Jinshui, W. Lun, G. Feng, L. Yongxing and W. Yun, Anal. Bioanal. Chem., 2003, 377, 346.
3. E. Botzung-Appert, V. Monnier, T. Ha, T. Duong, R. Pansu and A. Ibanez, Chem. Mater., 2004, 16, 1609.
4. H. Y. Kim, T. G. Bjorklund, S. H. Lim and C. J. Bardeen, Langmuir, 2003, 19, 3941.
5. H. Oikawa, H. Kasai and H. Nakanishi, Anisotropic Organic Materials, ACS Symposium Series, American Chemical Society, 2001, ch. 11.
6. F. Bertorelle, F. Al-Ali and S. Fery-Forgues, Int. J. Photoenergy, 2004, 6, 221.
7. H. Katagi, H. Kasai, S. Okada, H. Oikawa, H. Matsuda and H. Nakanishi, Polym. Adv. Technol., 2000, 11, 778.
8. W. Li, C. Zhao, B. Zou, X. Zhang, J. Yu, X. Zhang and J. Jie, CrystEngComm, 2012, 14, 8124.
9. X. Zhang, C. Dong, J. A. Zapien, S. Ismathullakhan, Z. Kang, J. Jie, X. Zhang, J. C. Chang, C. S. Lee and S. T. Lee, Angew. Chem., Int. Ed., 2009, 48, 9121.
10. M. Mille, J. F. Lamère, F. Rodrigues and S. Fery-Forgues, Langmuir, 2008, 24, 2671.
11. S. Fery-Forgues, R. El-Ayoubi and J. F. Lamère, J. Fluoresc., 2008, 18, 619.
12. K. A. Ahmed, M. M. El-Molla, M. S. A. Abdel-Mottaleb, S. A. Mohamed and S. El-Saadany, Res. J. Chem. Sci., 2013, 3, 3.
13. H. Koo, M. S. Huh, J. H. Ryu, D. E. Lee, I. C. Sun, K. Choi, K. Kim and I. C. Kwon, Nano Today, 2011, 6, 204.
14. G. Jones II and M. A. Rahman, J. Phys. Chem., 1994, 98, 13028.
15. P. M. French, M. M. Opalinska and J. R. Taylor, Opt. Lett., 1989, 14, 217.
16. X. Gan, H. Clevenson, C. C. Tsai, L. Li and D. Englund, Sci. Rep., 2013, 3, 2145.
17. B. Wei, N. Kobayashi, M. Ichikawa, T. Koyama, Y. Taniguchi and T. Fukuda, Opt. Express, 2006, 14, 9436.
18. J. P. Jasinski and E. S. Paight, Acta Crystallogr., Sect. C: Cryst. Struct. Commun., 1995, 51, 531.
19. S. Corrent, P. Hahn, G. Pohlers, T. J. Connolly, J. C. Scaiano, V. Fornés and H. García, J. Phys. Chem. B, 1998, 102, 5852.
20. P. Hazra, D. Chakrabarty and N. Sarkar, Langmuir, 2002, 18, 7872.
21. A. Nag, T. Chakrabarty and K. Bhattacharyya, J. Phys. Chem., 1990, 94, 4203.
22. I. Rivolta, A. Panariti, B. Lettiero, S. Sesana, P. Gasco, M. R. Gasco, M. Masserini and G. Miserocchi, J. Physiol. Pharmacol., 2011, 62, 45.
23. Y. Inokuchi, K. Hironaka, T. Fujisawa, Y. Tozuka, K. Tsuruma, M. Shimazawa, H. Takeuchi and H. Hara, Invest. Ophthalmol. Visual Sci., 2010, 51, 3162.
24. P. K. Makwana, P. N. Jethva and I. Roy, Analyst, 2011, 136, 2161.
25. N. Barooah, J. Mohanty, H. Pal and A. C. Bhasikuttan, J. Phys. Chem. B, 2012, 116, 3683.
26. S. Li and W. C. Purdy, Chem. Rev., 1992, 92, 1457.
27. J. Szejtli, Chem. Rev., 1998, 98, 1743.
28. L. Szente and J. Szemán, Anal. Chem., 2013, 85, 8024.
29. S. S. Jaffer, S. K. Saha, G. Eranna, A. K. Sharma and P. Purkayastha, J. Phys. Chem. C, 2008, 112, 11199.
30. S. S. Jaffer, S. K. Saha and P. Purkayastha, J. Colloid Interface Sci., 2009, 337, 294.
31. P. Ghosh, A. Maity, T. Das, J. Dash and P. Purkayastha, J. Phys. Chem. C, 2011, 115, 20970.
32. M. Bonini, S. Rossi, G. Karlsson and M. Almgren, Langmuir, 2006, 22, 1478–1484.
33. S. Rossi, M. Bonini, P. L. Nostro and P. Baglioni, Langmuir, 2007, 23, 10959–10967.
34. J. Carlstedt, A. Bilalov, E. Krivtsova, U. Olsson and B. Lindman, Langmuir, 2012, 28, 2387.
35. L. Jiang, Y. Peng, Y. Yan and J. Huang, Soft Matter, 2011, 7, 1726.
36. S. Ithurria, M. D. Tessier, B. Mahler, R. P. S. M. Lobo, B. Dubertret and A. L. Efros, Nat. Mater., 2011, 10, 936.
37. G. Li and L. B. McGown, Science, 1994, 264, 249.
38. K. Gnanaguru, N. Ramasubbu, K. Venkatesan and V. A. Ramamurthy, J. Org. Chem., 1985, 50, 2337.
39. G. Jones II, W. R. Jackson, C. Y. Choi and W. R. Bergmark, J. Phys. Chem., 1985, 89, 294.
40. K. Gavvala, A. Sengupta, R. K. Koninti and P. Hazra, Phys. Chem. Chem. Phys., 2014, 16, 933.
41. A. W. Schill, C. S. Gaddis, W. Qian, M. A. El-Sayed, Y. Cai, V. T. Milam and K. Sandhage, Nano Lett., 2006, 6, 1940.
42. D. Zhong, S. K. Pal, D. Zhang, S. I. Chan and A. H. Zewail, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 13.

---

Footnote

† Electronic supplementary information (ESI) available: Structure of C6, AFM and SEM images of C6 after incubation in water (with 1% methanol) for 30 minutes at room temperature, fluorescence spectra of C6 in α-, β- and γ-CDs in aqueous environment and time resolved fluorescence decay profile for C6 in presence of β- and γ-CDs. See DOI: 10.1039/c4ra13706j

---