View Article Online View Journal
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: G. Lv, B. Cui, H. Lan, Y. Wen, A. Sun and T. Yi, Chem. Commun., 2014, DOI: 10.1039/C4CC07656G.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/chemcomm
Page 1 of 4
ChemComm View Article Online
DOI: 10.1039/C4CC07656G
Journal Name
RSCPublishing
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012, Accepted 00th January 2012
Diarylethene Based Fluorescent Switchable Probes for the Detection of Amyloid-β Pathology in Alzheimer’s Disease Guanglei Lv a, Baiping Cui b, Haichuang Lan a, Ying Wen a, Anyang Sun*b and Tao Yi*a
DOI: 10.1039/x0xx00000x www.rsc.org/
Two fluorescent switchable diarylethene derivatives which exhibit high affinity toward amyloid-β aggregates with the increase of fluorescence intensity were reported. Moreover, the probes show excellent photochromic and antiphotobleaching properties both in vitro and in vivo. Reactive fluorescent labeling technology has been becoming increasingly popular for the monitoring of internal cellular processes due to several advantages including low cost, no exposure to radioactivity, availability and ease of operation. 1-4 Fluorescent probes that can detect simple cations and anions have been widely developed. 5-7 However, rational design of a probe with high specificity towards macro biomolecules, such as proteins and DNA, remains a major challenge due to the complexity and lack of specific active sites of those macro biomolecules. Amyloid-β (Aβ) is the major component of senile plaques, stemming from the consecutive cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase.8, 9 It is well established that Aβ plays an important role in the pathophysiology and progression of Alzheimer’s Disease (AD), so it is of immediate and practical importance to design imaging probes to detect Aβ. To date, the most widely applied Aβ imaging probes utilize positron emission tomography (PET) and single photon emission computed tomography (SPECT) using Congo Red (CR) and Thioflavin T (ThT), 10-13 respectively. However these imaging modalities are hindered by their intrinsic high cost and time-consuming nature, and also result in patient exposure to radiation. Fluorescence-based imaging of Aβ has emerged as a potential alternative to other techniques. 14-17 Some fluorescent probes showing specific binding to Aβ have been reported. Theodorakis et al. developed a new family of fluorescent probes containing an aminonaphthalenyl-2-
This journal is © The Royal Society of Chemistry 2012
cyano-acrylate (ANCA) motif, which exhibited great specificity and affinity for Aβ (Kd = 1.4 ± 0.2 µM) deposits in tissue. 18 Ran et al. synthesized curcumin-based near-infrared (NIR) fluorescent probes for detecting Aβ species, which showed significant fluorescence changes upon mixing with Aβ species in vitro and in vivo. 19 However, clinical application of these fluorescent probes for Aβ detection is hampered by poor anti-photobleaching. Conventional probes for fluorescent imaging are easily photobleached and therefore can only respond irreversibly to one event. In contrast, fluorophores that can respond reversibly constitute a simple and powerful tool for regional optical marking and tracing dynamic process of a bio-event and are thus potentially more valuable for use as fluorescent labels. 20 Diarylethene (DAE) derivatives, which can reversibly photocyclise upon irradiation with UV and visible light, are the most promising candidate fluorescent switch systems because of their fast response, excellent thermal stability and outstanding fatigue resistance. 21-23 Those outstanding characters of DAE make it possible for long time and real time operation in bioamaging. Compared with always-on probes, switchable DAE readily avoids background interference. More importantly, fluorescent switchable diarylethene has potential application in super resolution imaging.24 However, far fewer reports on the biological applications of DAEs have been published.23 Irie reported a fluorescent photochromic DAE for labeling biomolecules. 25 Branda first demonstrated that light-induced reactions of a photoresponsive DAE could be reversibly triggered in a living organism. 26 Very recently, we reported a thiazole orange linked DAE for detection of DNA and the intracellular nucleus. 27 To the best of our knowledge, there is no report of photochromic compounds for fluorescent imaging of Aβ. Herein, by connecting the targeting unit (ANCA) of an Aβ deposit to a photochromic DAE unit on one and two
J. Name., 2012, 00, 1-3 | 1
ChemComm Accepted Manuscript
Published on 04 November 2014. Downloaded by The University of Auckland Library on 04/11/2014 21:34:35.
COMMUNICATION
ChemComm
Page 2 of 4 View Article Online
sides (T1 and T2, respectively, in Scheme 1), we have developed a fluorescent switch for detecting Aβ deposits for the first time. The probes showed high affinity towards Aβ aggregates in both solution and brain section with both a remarkable increase in the fluorescence intensity and a slight blue shift. The probes exhibited excellent photochromic properties with the fluorescent intensity undergoing reversible changes with alternating irradiation of UV and visible light both in solution and in brain tissue, further highlighting the remarkable anti-photobleaching capability of the probes in tissue.
Journal Name DOI: 10.1039/C4CC07656G confirms that T1 and T2 exhibit high affinity toward Aβ aggregates. Therefore these probes are potentially capable of tracing the Aβ aggregation process. The specificity of T1 and T2 toward Aβ aggregates was studied by comparing with prion 106-126 peptides (an aggregation-prone peptide containing 21 amino acid residues from prion protein associated with prion disease).29 No significant increase in the fluorescence intensity of both probes was observed when the probes were incubated with prion, indicating the excellent selectivity of T1 and T2 toward Aβ aggregates (Fig. 1, a and b).
2 | J. Name., 2012, 00, 1-3
(%)
71
20.6
λab (nm) PSS 645
21.9
643
2.1
14.2
687
0.4
15.8
679
2.6
T1
586 550
T2
592
T2+Aβ
563
Φo-c
63
ΦF (%)
∆F/FTb
1.1 8.2
12.7
1.0
a
T1 T1-Aβ T1-prion
0.8 0.6 0.4 0.2 0.0 450
1.0
500
550 600 Wavelength (nm)
650
0.8 24 µΜ
0.4
0 µΜ
0.2 0.0 450
500
550 600 Wavelength (nm)
650
1.0
700
b
T2 T2-Aβ T2-prion
0.8 0.6 0.4 0.2 0.0
700
c
0.6
Fluorescence Intensity (a.u.)
E = photocyclic transition efficiency; Φo-c = cyclization quantum yield; ΦF = absolute fluorescence quantum yields measured by using calibrated integrating sphere; amaximum emission wavelength with and without Aβ aggregates; bfold change of the fluorescence intensity with Aβ aggregates, ∆F = FT+Aβ-FT.
450
Fluorescence intensity (a.u.)
The binding properties of T1 and T2 towards Aβ were studied by fluorescence spectroscopy in buffer solution. T1 and T2 show very weak fluorescence in PBS buffer solution (pH = 7.3) at corresponding wavelengths of 586 and 592 nm with absolute fluorescence quantum yields (ΦF) of 1.1% and 0.4%, respectively, under excitation with 430 nm light. This fluorescence intensity was sharply increased by corresponding factors of 8.2 and 12.7 for T1 and T2 (Fig. 1, a and b), while the ΦF values increased to 2.1% and 2.6%, respectively, in the presence of Aβ aggregates (Table 1). In addition, the fluorescence emission maxima of T1 and T2 blue shifted from 586 to 550 nm and 592 to 563 nm in the presence of Aβ aggregates, respectively. T2 exhibited a longer emission wavelength and a larger fluorescence shift before and after the addition of Aβ aggregates, showing superior probing properties towards Aβ aggregates than T1. The concentrationdependent fluorescent spectral changes of T1 and T2 showed that the intensity gradually increased along with increasing concentration of Aβ aggregates, accompanied with a slight blue-shift in λmax (Fig. 1c and Fig. S1). A linear relationship between Aβ aggregate concentration and fluorescence intensity of the probes was observed in the concentration range of 1–13 µM (R2 = 0.98 for both T1 and T2, Fig. S2), indicating that the concentration of Aβ aggregates may be quantitatively estimated by the probes. The dissociation constant (Kd) of T1 and T2 to Aβ aggregates was measured as 1.3 µM and 1.1 µM, respectively, 28 which was comparative or smaller than that of ANCA.18 The better binding affinity of T2 towards Aβ aggregates compared with that of T1 may be due to its two binding sites. The dynamic aggregation process of Aβ monomer was followed by changes in fluorescence intensity of T1 or T2 with different incubation times up to 72 h. There was no detectable fluorescence change of T1 and T2 in PBS solution after addition of Aβ monomer, however both T1 and T2 showed fluorescence enhancement upon prolonged incubation times (Fig. 1d and Fig. S3). As Aβ aggregation increases with monomer incubation time, this fluorescence enhancement further
E (%)
T1+Aβ
Fluorescence Intensity (a.u.)
Scheme 1 The structure of T1, T2 and the photochromism of T2
λem (nm)a
1.0
500
550 600 Wavelength (nm)
650
700
T2 T2- 2 h T2-12 h T2- 36 h T2- 60 h T2- 72 h
d
0.8 0.6 0.4 0.2 0.0 450
500
550 600 650 Wavelength (nm)
700
Fig. 1 Normalized fluorescent spectra of (a) T1, T1 with Aβ aggregates (5.0 µM), T1 with prion aggregates (5.0 µM), and (b) T2, T2 with Aβ aggregates (5.0 µM), T2 with prion aggregates (5.0 µM); fluorescent spectral change of T2 with (c) different concentration of Aβ aggregates from 0 to 24 µM and (d) different incubation time of Aβ monomer over 72 h. The concentration of T1 and T2 is 2.0 µM (5% DMSO in PBS), λex = 430 nm
To investigate the binding mechanism of T1 and T2 toward Aβ aggregates (T2 taken here as an example), the fluorescent spectra of T2 with excitation at different wavelengths before and after addition of Aβ aggregates were studied. When exciting T2 with a wavelength shorter than 350 nm light, T2 only show weak fluorescence at about 440 nm belonging to DAE,23 whereas the fluorescence of ANCA was not observed (Fig. S4). This indicates that the effective energy transfer between the DAE and ANCA groups was not happened. When redshifting the excitation wavelength to 370 nm, the fluorescence of ANCA in T2 at about 560 nm was clearly observed. Both of the fluorescence intensity at 440 nm and 560 nm was sharply increased
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
Table 1. Photophysical and photochemical properties of T1 and T2
Fluorescent Intensity (a.u.)
Published on 04 November 2014. Downloaded by The University of Auckland Library on 04/11/2014 21:34:35.
COMMUNICATION
Page 3 of 4
ChemComm View Article Online
Absorbance
T1 T1+Aβ T1-PSS T1+Aβ-PSS
326 328 377
0.2 643 645
0.1
0.4 0.3
T2 T2-PSS T2+Aβ T2+Aβ-PSS
337
0.2
332
0.1
679 687
0.0
0.0 300
1.0
b 389
Absorbance
a 0.3
accompanied by significant fluorescence quenching (> 85%) in the PSS due to the energy transfer between ANCA and closed form of DAE (Fig. 2c). The Φo-c of both T1 and T2 were increased after binding to Aβ aggregates. The reverse process was performed by irradiation of T2+Aβ aggregates with visible light in its PSS (Fig. S11), demonstrating typical photoswitching characteristics of DAE. This reversible photochromic cycle can be repeated many times with a low rate of fatigue (Fig. 2d). The solution containing T1 and Aβ aggregates showed similar reversible changes in absorption and fluorescence spectra to those of T2 under the same conditions (Fig. S13 and S13). To further evaluate whether these fluorescent probes could stain amyloid deposits in brain tissue, brain sections of AD mice (12-monthold APP/PS1 transgenic mice) were stained with T1 and T2. Both T1 and T2 can be practically applied for the detection of Aβ aggregates in brain tissues by employing confocal laser scanning microscopy (CLSM). As expected, the fluorescent probes could specifically highlight Aβ deposits in brain sections, whereas the brain sections without probes did not specifically highlight aggregates (Fig. 3A and S14). T2 exhibited a longer emission wavelength and higher local fluorescent intensity than T1, rendering T2 more suitable for in vivo detection of Aβ aggregates in brain tissue. To determine the location and specificity of the probes, the T2-loaded brain tissue (red channel, Fig. 3A) was further incubated with Aβ antibody (green channel, Fig. 3B) and tau protein antibody (green channel, Fig. S15), respectively. The images of the brain section showed good colocalization of T2 and Aβ antibody (Fig. 3C), but different localization of T2 with tau protein, indicating that T2 specifically accumulated in Aβ deposits. These experiments clearly demonstrate that the probe can selective mark the location of amyloid deposits in brain tissues.
400 500 600 Wavelength (nm)
700
300
800
c
0.05 Absorbance at 680 nm
0s
0.8 0.6 100 s
0.4 0.2 0.0 450
500
550 600 Wavelength (nm)
650
700
400 500 600 Wavelength (nm)
700
800
d
0.04 0.03 0.02 0.01 0.00
0
1
2 3 4 Cycle Number (n)
5
6
Fig. 2 Absorption spectra of (a) T1 and (b) T2 in open form and PSS with and without Aβ aggregates (CT = 1.0 × 10-5 M, 5% DMSO in PBS); (c) fluorescent spectral change of T2 (2.0 × 10-6 M, 5% DMSO in PBS) in the presence of Aβ -6 aggregates (5.0 × 10 M) upon irradiation with UV light; (d) absorbance changes at 680 nm of T2+Aβ upon alternating irradiation of UV and visible light
The photochromic behaviours of T1 and T2 in the presence of Aβ aggregates were subsequently investigated (T2 taken here as an example). The wavelength of the absorption of the open form of T2 at 389 nm clearly decreased and the absorption band at 337 nm blueshifted to 332 nm after addition of Aβ aggregates (Fig. 2b). Upon irradiation of a solution containing T2 and Aβ aggregates with UV light, a new absorption band (687 nm) in the visible range emerged,
This journal is © The Royal Society of Chemistry 2012
Fig. 3 CLSM images of brain sections; (A) staining with 60 µM T2 (red channel: 575–625 nm); (B) staining with β-Amyloid antibody (green channel: 495–540 nm); (C) the merged image of A and B; scale bar: 20 µm. (D) Photochromic process of T2 (60 µM) in brain section, left: original state; middle: irradiated by 405 nm light in the assigned circled region for 150 s; right: irradiated with 633 nm light over the entire region. (E) Contrast experiment stained with ThT (60 µM), left: original state; middle: irradiated by 405 nm light in the oval region for 30 s; right: irradiated with 633 nm light over the entire region
J. Name., 2012, 00, 1-3 | 3
ChemComm Accepted Manuscript
COMMUNICATION DOI: 10.1039/C4CC07656G
with the addition of Aβ aggregates, indicating that both DAE and ANCA may contribute to the binding between T2 and Aβ aggregates. This was further proved by the study of the binding properties between Aβ aggregates and the reference compounds 5 (ANCA with short polyether chain) and 7 (DAE with short polyether chain) (Scheme S1, ESI). With addition of Aβ aggregates to 7 or 5, both the fluorescence intensity was drastically increased (Fig. S5 and S6), which further confirmed the synergistic binding effect of DAE and ANCA to the hydrophobic pockets of Aβ aggregates and restrict the mobility of T2, leading to fluorescence increase. The photochromic behaviour of T1 and T2 was investigated before and after addition of Aβ aggregates. The maximum absorption of T1 in its open-ring form centered at 328 nm (ε = 24,000 L.mol-1.cm-1) is ascribed to a π-π* transition. Upon irradiation with 365 nm UV light for 3 min, a new absorption band appeared at 645 nm, accompanied with a change of colour from colourless to green in the photo-stationary state (PSS) caused by an increase in π-electron delocalization (Fig. 2a and Fig. S7). The photochromic behaviour of T2 was similar to that of T1 with the absorption at 380 nm decreasing and a concommitant increase in absorption at 687 nm upon irradiation with UV light, slightly redshifted compared with that of T1 (Fig. 2b and Fig. S8). The reverse process was performed by irradiating the PSS of T1 or T2 with visible light (λ > 550 nm). The photocyclization efficiency of T1 was 71% in the PSS as measured by 1H NMR (Fig. S9) and the photocyclization quantum yield (Φo-c) 30 was determined to be 20.6% with 365 nm light irradiation. The Φo-c and transformation efficiency of T2 was 14.2% and 63% (Fig. S10), respectively, comparatively lower than that of T1.
Fluorescence intensity (a. u.)
Published on 04 November 2014. Downloaded by The University of Auckland Library on 04/11/2014 21:34:35.
Journal Name
ChemComm
Importantly, the fluorescence of T2 in brain sections also could be tuned by varying the irradiation wavelength. Micro-fluorescence switching in brain sections was achieved by alternating between UV and visible light illumination. The selected T2-stained Aβ deposits (circular region in Fig. 3D) were irradiated with 405 nm light for 150 s, and the fluorescence of the irradiated deposits was turned off, due to T2 in this area changing from an open state to a closed state. Interestingly, the fluorescence of the Aβ deposits could be recovered by 633 nm light irradiation from the CLSM lamp for 10 min (Fig. 3D). The fluorescence intensity of T2 showed reversible changes with alternating irradiation of UV and visible light, validating the photochromic properties of T2 and indicating that the probes show remarkable anti-photobleaching in tissues. As evident in Fig. S16, the optical switching of fluorescence can be repeated many times with little “fatigue” effects or photobleaching, that were thought to be fatal disadvantages of the general fluorophore. As a contrast, ThT (a common standard stain for Aβ plaques) stained brain sections were also irradiated with UV and visible light. The fluorescence intensity of ThT was rapidly quenched by UV light and did not recover under visible light (Fig. 3E). In addition, both T1 and T2 show low cytotoxicity by means of an MTT assay. The cellular viabilities were estimated to be greater than 85% after 2 h in the presence of 1–100 µM T1 or T2 (Fig. S17). Thus, we concluded that T2 is a novel and superior fluorescence dye for use as a marker of Aβ deposits. In summary, we designed and synthesized diarylethene-based fluorescent probes capable of specifically detecting Aβ aggregates. The fluorescent intensity dramatically increased in the presence of Aβ aggregates, accompanied with a blue shift. The fluorescence can be readily tuned under alternating irradiating with UV and visible light, exhibiting excellent fatigue resistance and anti-photobleaching. These probes can specifically target Aβ deposits in brain sections. The synergistic binding effect of DAE and ANCA to the hydrophobic pockets of Aβ aggregates contribute to the binding efficiency of the probes. The switchable fluorescence image operated by light in brain tissues may provide a new window for in vivo high resolution imaging. Further studies of these probes are ongoing in our laboratory. The authors thanks for the financially support by 973 (2013CB733700), NNSFC (21125104, 51373039), PIRTU (IRT1117) and Shanghai Sci. Tech. Comm. (12XD1405900).
Notes and references a
Department of Chemistry & Innovation Center of Chemistry for Energy Materials, Fudan University, 220 Handan Road, Shanghai 200433, P. R. China. Fax: +86-21-55664621; E-mail: [email protected]. b Laboratory of Neurodegenerative Diseases and Repair, Yancheng Institute of Health Sciences, 263 South Jiefang Road, Yancheng 224005, Jiangsu, P. R. China. E-mail：[email protected]. † Electronic Supplementary Information (ESI) available: [Synthesis detail and additional spectral]. See DOI: 10.1039/c000000x/
Journal Name DOI: 10.1039/C4CC07656G 5. M. Royzen, A. Durandin, V. G. Young, N. E. Geacintov and J. W. Canary, J. Am. Chem. Soc., 2006, 128, 3854. 6. J. Rosenthal and S. J. Lippard, J. Am. Chem. Soc., 2010, 132, 5536. 7. V. Amendola, G. Bergamaschi, M. Boiocchi, L. Fabbrizzi and L. Mosca, J. Am. Chem. Soc., 2013, 135, 6345. 8. D. J. Selkoe, Ann. N. Y. Acad. Sci., 2000, 924, 17-25. 9. W. Annaert and B. De Strooper, Annu. Rev. Cell Dev. Biol., 2002, 18, 25. 10. L. Cai, R. B. Innis and V. W. Pike, Curr. Med. Chem., 2007, 14, 19. 11. H. Quigley, S. J. Colloby and J. T. O'Brien, Int J Geriatr Psychiatry, 2011, 26, 991. 12. C. C. Rowe and V. L. Villemagne, J. Nucl. Med., 2011, 52, 1733. 13. C. A. Mathis, N. S. Mason, B. J. Lopresti and W. E. Klunk, Semin Nucl Med, 2012, 42, 423. 14. Y. O. Lee, J. W. Shin, C. Yi, Y. H. Lee, N. W. Sohn, C. Kang and J. S. Kim, Chem Commun, 2014, 50, 5741. 15. K. Liu, T. L. Guo, J. Chojnacki, H.-G. Lee, X. Wang, S. L. Siedlak, W. Rao, X. Zhu and S. Zhang, ACS Chem. Neurosci., 2012, 3, 141. 16. A. Aaslund, C. J. Sigurdson, T. Klingstedt, S. Grathwohl, T. Bolmont, D. L. Dickstein, E. Glimsdal, S. Prokop, M. Lindgren, P. Konradsson, D. M. Holtzman, P. R. Hof, F. L. Heppner, S. Gandy, M. Jucker, A. Aguzzi, P. Hammarstroem and K. P. R. Nilsson, ACS Chem. Biol., 2009, 4, 673. 17. E. E. Nesterov, J. Skoch, B. T. Hyman, W. E. Klunk, B. J. Bacskai and T. M. Swager, Angew. Chem., Int. Ed., 2005, 44, 5452. 18. K. Cao, M. Farahi, M. Dakanali, W. M. Chang, C. J. Sigurdson, E. A. Theodorakis and J. Yang, J. Am. Chem. Soc., 2012, 134, 17338. 19. X. Zhang, Y. Tian, Z. Li, X. Tian, H. Sun, H. Liu, A. Moore and C. Ran, J. Am. Chem. Soc., 2013, 135, 16397. 20. L. Zhu, W. Wu, M.-Q. Zhu, J. J. Han, J. K. Hurst and A. D. Q. Li, J. Am. Chem. Soc., 2007, 129, 3524. 21. M. Irie, Chem. Rev., 2000, 100, 1683. 22. W. Li, C. Jiao, X. Li, Y. Xie, K. Nakatani, H. Tian and W. Zhu, Angew. Chem., Int. Ed., 2014, 53, 4603. 23. Y. Zou, T. Yi, S. Xiao, F. Li, C. Li, X. Gao, J. Wu, M. Yu and C. Huang, J. Am. Chem. Soc., 2008, 130, 15750. 24. M. Bates, B. Huang, G. T. Dempsey and X. Zhuang, Science, 2007, 317, 1749-1753 25. N. Soh, K. Yoshida, H. Nakajima, K. Nakano, T. Imato, T. Fukaminato and M. Irie, Chem. Commun., 2007, 5206. 26. U. Al-Atar, R. Fernandes, B. Johnsen, D. Baillie and N. R. Branda, J. Am. Chem. Soc., 2009, 131, 15966. 27. K. Liu, Y. Wen, T. Shi, Y. Li, F. Li, Y. L. Zhao, C. Huang and T. Yi, Chem Commun., 2014, 50, 9141. 28. J. Sutharsan, M. Dakanali, C. C. Capule, M. A. Haidekker, J. Yang and E. A. Theodorakis, ChemMedChem, 2010, 5, 56. 29. B. Kurganov, M. Doh and N. Arispe, Peptides, 2004, 25, 217. 30. M. Irie, T. Lifka, K. Uchida, S. Kobatake and Y. Shindo, Chem. Commun., 1999, 747.
1. V. Ntziachristos, C. Bremer and R. Weissleder, Eur Radiol, 2003, 13, 195. 2. V. Ntziachristos, Annu. Rev. Biomed. Eng., 2006, 8, 1-33. 3. J. V. Frangioni, Curr. Opin. Chem. Biol., 2003, 7, 626. 4. K. Licha and C. Olbrich, Adv. Drug Delivery Rev., 2005, 57, 1087.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
Published on 04 November 2014. Downloaded by The University of Auckland Library on 04/11/2014 21:34:35.
COMMUNICATION
Page 4 of 4 View Article Online
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: G. Lv, B. Cui, H. Lan, Y. Wen, A. Sun and T. Yi, Chem. Commun., 2014, DOI: 10.1039/C4CC07656G.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
www.rsc.org/chemcomm
Page 1 of 4
ChemComm View Article Online
DOI: 10.1039/C4CC07656G
Journal Name
RSCPublishing
Cite this: DOI: 10.1039/x0xx00000x
Received 00th January 2012, Accepted 00th January 2012
Diarylethene Based Fluorescent Switchable Probes for the Detection of Amyloid-β Pathology in Alzheimer’s Disease Guanglei Lv a, Baiping Cui b, Haichuang Lan a, Ying Wen a, Anyang Sun*b and Tao Yi*a
DOI: 10.1039/x0xx00000x www.rsc.org/
Two fluorescent switchable diarylethene derivatives which exhibit high affinity toward amyloid-β aggregates with the increase of fluorescence intensity were reported. Moreover, the probes show excellent photochromic and antiphotobleaching properties both in vitro and in vivo. Reactive fluorescent labeling technology has been becoming increasingly popular for the monitoring of internal cellular processes due to several advantages including low cost, no exposure to radioactivity, availability and ease of operation. 1-4 Fluorescent probes that can detect simple cations and anions have been widely developed. 5-7 However, rational design of a probe with high specificity towards macro biomolecules, such as proteins and DNA, remains a major challenge due to the complexity and lack of specific active sites of those macro biomolecules. Amyloid-β (Aβ) is the major component of senile plaques, stemming from the consecutive cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase.8, 9 It is well established that Aβ plays an important role in the pathophysiology and progression of Alzheimer’s Disease (AD), so it is of immediate and practical importance to design imaging probes to detect Aβ. To date, the most widely applied Aβ imaging probes utilize positron emission tomography (PET) and single photon emission computed tomography (SPECT) using Congo Red (CR) and Thioflavin T (ThT), 10-13 respectively. However these imaging modalities are hindered by their intrinsic high cost and time-consuming nature, and also result in patient exposure to radiation. Fluorescence-based imaging of Aβ has emerged as a potential alternative to other techniques. 14-17 Some fluorescent probes showing specific binding to Aβ have been reported. Theodorakis et al. developed a new family of fluorescent probes containing an aminonaphthalenyl-2-
This journal is © The Royal Society of Chemistry 2012
cyano-acrylate (ANCA) motif, which exhibited great specificity and affinity for Aβ (Kd = 1.4 ± 0.2 µM) deposits in tissue. 18 Ran et al. synthesized curcumin-based near-infrared (NIR) fluorescent probes for detecting Aβ species, which showed significant fluorescence changes upon mixing with Aβ species in vitro and in vivo. 19 However, clinical application of these fluorescent probes for Aβ detection is hampered by poor anti-photobleaching. Conventional probes for fluorescent imaging are easily photobleached and therefore can only respond irreversibly to one event. In contrast, fluorophores that can respond reversibly constitute a simple and powerful tool for regional optical marking and tracing dynamic process of a bio-event and are thus potentially more valuable for use as fluorescent labels. 20 Diarylethene (DAE) derivatives, which can reversibly photocyclise upon irradiation with UV and visible light, are the most promising candidate fluorescent switch systems because of their fast response, excellent thermal stability and outstanding fatigue resistance. 21-23 Those outstanding characters of DAE make it possible for long time and real time operation in bioamaging. Compared with always-on probes, switchable DAE readily avoids background interference. More importantly, fluorescent switchable diarylethene has potential application in super resolution imaging.24 However, far fewer reports on the biological applications of DAEs have been published.23 Irie reported a fluorescent photochromic DAE for labeling biomolecules. 25 Branda first demonstrated that light-induced reactions of a photoresponsive DAE could be reversibly triggered in a living organism. 26 Very recently, we reported a thiazole orange linked DAE for detection of DNA and the intracellular nucleus. 27 To the best of our knowledge, there is no report of photochromic compounds for fluorescent imaging of Aβ. Herein, by connecting the targeting unit (ANCA) of an Aβ deposit to a photochromic DAE unit on one and two
J. Name., 2012, 00, 1-3 | 1
ChemComm Accepted Manuscript
Published on 04 November 2014. Downloaded by The University of Auckland Library on 04/11/2014 21:34:35.
COMMUNICATION
ChemComm
Page 2 of 4 View Article Online
sides (T1 and T2, respectively, in Scheme 1), we have developed a fluorescent switch for detecting Aβ deposits for the first time. The probes showed high affinity towards Aβ aggregates in both solution and brain section with both a remarkable increase in the fluorescence intensity and a slight blue shift. The probes exhibited excellent photochromic properties with the fluorescent intensity undergoing reversible changes with alternating irradiation of UV and visible light both in solution and in brain tissue, further highlighting the remarkable anti-photobleaching capability of the probes in tissue.
Journal Name DOI: 10.1039/C4CC07656G confirms that T1 and T2 exhibit high affinity toward Aβ aggregates. Therefore these probes are potentially capable of tracing the Aβ aggregation process. The specificity of T1 and T2 toward Aβ aggregates was studied by comparing with prion 106-126 peptides (an aggregation-prone peptide containing 21 amino acid residues from prion protein associated with prion disease).29 No significant increase in the fluorescence intensity of both probes was observed when the probes were incubated with prion, indicating the excellent selectivity of T1 and T2 toward Aβ aggregates (Fig. 1, a and b).
2 | J. Name., 2012, 00, 1-3
(%)
71
20.6
λab (nm) PSS 645
21.9
643
2.1
14.2
687
0.4
15.8
679
2.6
T1
586 550
T2
592
T2+Aβ
563
Φo-c
63
ΦF (%)
∆F/FTb
1.1 8.2
12.7
1.0
a
T1 T1-Aβ T1-prion
0.8 0.6 0.4 0.2 0.0 450
1.0
500
550 600 Wavelength (nm)
650
0.8 24 µΜ
0.4
0 µΜ
0.2 0.0 450
500
550 600 Wavelength (nm)
650
1.0
700
b
T2 T2-Aβ T2-prion
0.8 0.6 0.4 0.2 0.0
700
c
0.6
Fluorescence Intensity (a.u.)
E = photocyclic transition efficiency; Φo-c = cyclization quantum yield; ΦF = absolute fluorescence quantum yields measured by using calibrated integrating sphere; amaximum emission wavelength with and without Aβ aggregates; bfold change of the fluorescence intensity with Aβ aggregates, ∆F = FT+Aβ-FT.
450
Fluorescence intensity (a.u.)
The binding properties of T1 and T2 towards Aβ were studied by fluorescence spectroscopy in buffer solution. T1 and T2 show very weak fluorescence in PBS buffer solution (pH = 7.3) at corresponding wavelengths of 586 and 592 nm with absolute fluorescence quantum yields (ΦF) of 1.1% and 0.4%, respectively, under excitation with 430 nm light. This fluorescence intensity was sharply increased by corresponding factors of 8.2 and 12.7 for T1 and T2 (Fig. 1, a and b), while the ΦF values increased to 2.1% and 2.6%, respectively, in the presence of Aβ aggregates (Table 1). In addition, the fluorescence emission maxima of T1 and T2 blue shifted from 586 to 550 nm and 592 to 563 nm in the presence of Aβ aggregates, respectively. T2 exhibited a longer emission wavelength and a larger fluorescence shift before and after the addition of Aβ aggregates, showing superior probing properties towards Aβ aggregates than T1. The concentrationdependent fluorescent spectral changes of T1 and T2 showed that the intensity gradually increased along with increasing concentration of Aβ aggregates, accompanied with a slight blue-shift in λmax (Fig. 1c and Fig. S1). A linear relationship between Aβ aggregate concentration and fluorescence intensity of the probes was observed in the concentration range of 1–13 µM (R2 = 0.98 for both T1 and T2, Fig. S2), indicating that the concentration of Aβ aggregates may be quantitatively estimated by the probes. The dissociation constant (Kd) of T1 and T2 to Aβ aggregates was measured as 1.3 µM and 1.1 µM, respectively, 28 which was comparative or smaller than that of ANCA.18 The better binding affinity of T2 towards Aβ aggregates compared with that of T1 may be due to its two binding sites. The dynamic aggregation process of Aβ monomer was followed by changes in fluorescence intensity of T1 or T2 with different incubation times up to 72 h. There was no detectable fluorescence change of T1 and T2 in PBS solution after addition of Aβ monomer, however both T1 and T2 showed fluorescence enhancement upon prolonged incubation times (Fig. 1d and Fig. S3). As Aβ aggregation increases with monomer incubation time, this fluorescence enhancement further
E (%)
T1+Aβ
Fluorescence Intensity (a.u.)
Scheme 1 The structure of T1, T2 and the photochromism of T2
λem (nm)a
1.0
500
550 600 Wavelength (nm)
650
700
T2 T2- 2 h T2-12 h T2- 36 h T2- 60 h T2- 72 h
d
0.8 0.6 0.4 0.2 0.0 450
500
550 600 650 Wavelength (nm)
700
Fig. 1 Normalized fluorescent spectra of (a) T1, T1 with Aβ aggregates (5.0 µM), T1 with prion aggregates (5.0 µM), and (b) T2, T2 with Aβ aggregates (5.0 µM), T2 with prion aggregates (5.0 µM); fluorescent spectral change of T2 with (c) different concentration of Aβ aggregates from 0 to 24 µM and (d) different incubation time of Aβ monomer over 72 h. The concentration of T1 and T2 is 2.0 µM (5% DMSO in PBS), λex = 430 nm
To investigate the binding mechanism of T1 and T2 toward Aβ aggregates (T2 taken here as an example), the fluorescent spectra of T2 with excitation at different wavelengths before and after addition of Aβ aggregates were studied. When exciting T2 with a wavelength shorter than 350 nm light, T2 only show weak fluorescence at about 440 nm belonging to DAE,23 whereas the fluorescence of ANCA was not observed (Fig. S4). This indicates that the effective energy transfer between the DAE and ANCA groups was not happened. When redshifting the excitation wavelength to 370 nm, the fluorescence of ANCA in T2 at about 560 nm was clearly observed. Both of the fluorescence intensity at 440 nm and 560 nm was sharply increased
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
Table 1. Photophysical and photochemical properties of T1 and T2
Fluorescent Intensity (a.u.)
Published on 04 November 2014. Downloaded by The University of Auckland Library on 04/11/2014 21:34:35.
COMMUNICATION
Page 3 of 4
ChemComm View Article Online
Absorbance
T1 T1+Aβ T1-PSS T1+Aβ-PSS
326 328 377
0.2 643 645
0.1
0.4 0.3
T2 T2-PSS T2+Aβ T2+Aβ-PSS
337
0.2
332
0.1
679 687
0.0
0.0 300
1.0
b 389
Absorbance
a 0.3
accompanied by significant fluorescence quenching (> 85%) in the PSS due to the energy transfer between ANCA and closed form of DAE (Fig. 2c). The Φo-c of both T1 and T2 were increased after binding to Aβ aggregates. The reverse process was performed by irradiation of T2+Aβ aggregates with visible light in its PSS (Fig. S11), demonstrating typical photoswitching characteristics of DAE. This reversible photochromic cycle can be repeated many times with a low rate of fatigue (Fig. 2d). The solution containing T1 and Aβ aggregates showed similar reversible changes in absorption and fluorescence spectra to those of T2 under the same conditions (Fig. S13 and S13). To further evaluate whether these fluorescent probes could stain amyloid deposits in brain tissue, brain sections of AD mice (12-monthold APP/PS1 transgenic mice) were stained with T1 and T2. Both T1 and T2 can be practically applied for the detection of Aβ aggregates in brain tissues by employing confocal laser scanning microscopy (CLSM). As expected, the fluorescent probes could specifically highlight Aβ deposits in brain sections, whereas the brain sections without probes did not specifically highlight aggregates (Fig. 3A and S14). T2 exhibited a longer emission wavelength and higher local fluorescent intensity than T1, rendering T2 more suitable for in vivo detection of Aβ aggregates in brain tissue. To determine the location and specificity of the probes, the T2-loaded brain tissue (red channel, Fig. 3A) was further incubated with Aβ antibody (green channel, Fig. 3B) and tau protein antibody (green channel, Fig. S15), respectively. The images of the brain section showed good colocalization of T2 and Aβ antibody (Fig. 3C), but different localization of T2 with tau protein, indicating that T2 specifically accumulated in Aβ deposits. These experiments clearly demonstrate that the probe can selective mark the location of amyloid deposits in brain tissues.
400 500 600 Wavelength (nm)
700
300
800
c
0.05 Absorbance at 680 nm
0s
0.8 0.6 100 s
0.4 0.2 0.0 450
500
550 600 Wavelength (nm)
650
700
400 500 600 Wavelength (nm)
700
800
d
0.04 0.03 0.02 0.01 0.00
0
1
2 3 4 Cycle Number (n)
5
6
Fig. 2 Absorption spectra of (a) T1 and (b) T2 in open form and PSS with and without Aβ aggregates (CT = 1.0 × 10-5 M, 5% DMSO in PBS); (c) fluorescent spectral change of T2 (2.0 × 10-6 M, 5% DMSO in PBS) in the presence of Aβ -6 aggregates (5.0 × 10 M) upon irradiation with UV light; (d) absorbance changes at 680 nm of T2+Aβ upon alternating irradiation of UV and visible light
The photochromic behaviours of T1 and T2 in the presence of Aβ aggregates were subsequently investigated (T2 taken here as an example). The wavelength of the absorption of the open form of T2 at 389 nm clearly decreased and the absorption band at 337 nm blueshifted to 332 nm after addition of Aβ aggregates (Fig. 2b). Upon irradiation of a solution containing T2 and Aβ aggregates with UV light, a new absorption band (687 nm) in the visible range emerged,
This journal is © The Royal Society of Chemistry 2012
Fig. 3 CLSM images of brain sections; (A) staining with 60 µM T2 (red channel: 575–625 nm); (B) staining with β-Amyloid antibody (green channel: 495–540 nm); (C) the merged image of A and B; scale bar: 20 µm. (D) Photochromic process of T2 (60 µM) in brain section, left: original state; middle: irradiated by 405 nm light in the assigned circled region for 150 s; right: irradiated with 633 nm light over the entire region. (E) Contrast experiment stained with ThT (60 µM), left: original state; middle: irradiated by 405 nm light in the oval region for 30 s; right: irradiated with 633 nm light over the entire region
J. Name., 2012, 00, 1-3 | 3
ChemComm Accepted Manuscript
COMMUNICATION DOI: 10.1039/C4CC07656G
with the addition of Aβ aggregates, indicating that both DAE and ANCA may contribute to the binding between T2 and Aβ aggregates. This was further proved by the study of the binding properties between Aβ aggregates and the reference compounds 5 (ANCA with short polyether chain) and 7 (DAE with short polyether chain) (Scheme S1, ESI). With addition of Aβ aggregates to 7 or 5, both the fluorescence intensity was drastically increased (Fig. S5 and S6), which further confirmed the synergistic binding effect of DAE and ANCA to the hydrophobic pockets of Aβ aggregates and restrict the mobility of T2, leading to fluorescence increase. The photochromic behaviour of T1 and T2 was investigated before and after addition of Aβ aggregates. The maximum absorption of T1 in its open-ring form centered at 328 nm (ε = 24,000 L.mol-1.cm-1) is ascribed to a π-π* transition. Upon irradiation with 365 nm UV light for 3 min, a new absorption band appeared at 645 nm, accompanied with a change of colour from colourless to green in the photo-stationary state (PSS) caused by an increase in π-electron delocalization (Fig. 2a and Fig. S7). The photochromic behaviour of T2 was similar to that of T1 with the absorption at 380 nm decreasing and a concommitant increase in absorption at 687 nm upon irradiation with UV light, slightly redshifted compared with that of T1 (Fig. 2b and Fig. S8). The reverse process was performed by irradiating the PSS of T1 or T2 with visible light (λ > 550 nm). The photocyclization efficiency of T1 was 71% in the PSS as measured by 1H NMR (Fig. S9) and the photocyclization quantum yield (Φo-c) 30 was determined to be 20.6% with 365 nm light irradiation. The Φo-c and transformation efficiency of T2 was 14.2% and 63% (Fig. S10), respectively, comparatively lower than that of T1.
Fluorescence intensity (a. u.)
Published on 04 November 2014. Downloaded by The University of Auckland Library on 04/11/2014 21:34:35.
Journal Name
ChemComm
Importantly, the fluorescence of T2 in brain sections also could be tuned by varying the irradiation wavelength. Micro-fluorescence switching in brain sections was achieved by alternating between UV and visible light illumination. The selected T2-stained Aβ deposits (circular region in Fig. 3D) were irradiated with 405 nm light for 150 s, and the fluorescence of the irradiated deposits was turned off, due to T2 in this area changing from an open state to a closed state. Interestingly, the fluorescence of the Aβ deposits could be recovered by 633 nm light irradiation from the CLSM lamp for 10 min (Fig. 3D). The fluorescence intensity of T2 showed reversible changes with alternating irradiation of UV and visible light, validating the photochromic properties of T2 and indicating that the probes show remarkable anti-photobleaching in tissues. As evident in Fig. S16, the optical switching of fluorescence can be repeated many times with little “fatigue” effects or photobleaching, that were thought to be fatal disadvantages of the general fluorophore. As a contrast, ThT (a common standard stain for Aβ plaques) stained brain sections were also irradiated with UV and visible light. The fluorescence intensity of ThT was rapidly quenched by UV light and did not recover under visible light (Fig. 3E). In addition, both T1 and T2 show low cytotoxicity by means of an MTT assay. The cellular viabilities were estimated to be greater than 85% after 2 h in the presence of 1–100 µM T1 or T2 (Fig. S17). Thus, we concluded that T2 is a novel and superior fluorescence dye for use as a marker of Aβ deposits. In summary, we designed and synthesized diarylethene-based fluorescent probes capable of specifically detecting Aβ aggregates. The fluorescent intensity dramatically increased in the presence of Aβ aggregates, accompanied with a blue shift. The fluorescence can be readily tuned under alternating irradiating with UV and visible light, exhibiting excellent fatigue resistance and anti-photobleaching. These probes can specifically target Aβ deposits in brain sections. The synergistic binding effect of DAE and ANCA to the hydrophobic pockets of Aβ aggregates contribute to the binding efficiency of the probes. The switchable fluorescence image operated by light in brain tissues may provide a new window for in vivo high resolution imaging. Further studies of these probes are ongoing in our laboratory. The authors thanks for the financially support by 973 (2013CB733700), NNSFC (21125104, 51373039), PIRTU (IRT1117) and Shanghai Sci. Tech. Comm. (12XD1405900).
Notes and references a
Department of Chemistry & Innovation Center of Chemistry for Energy Materials, Fudan University, 220 Handan Road, Shanghai 200433, P. R. China. Fax: +86-21-55664621; E-mail: [email protected]. b Laboratory of Neurodegenerative Diseases and Repair, Yancheng Institute of Health Sciences, 263 South Jiefang Road, Yancheng 224005, Jiangsu, P. R. China. E-mail：[email protected]. † Electronic Supplementary Information (ESI) available: [Synthesis detail and additional spectral]. See DOI: 10.1039/c000000x/
Journal Name DOI: 10.1039/C4CC07656G 5. M. Royzen, A. Durandin, V. G. Young, N. E. Geacintov and J. W. Canary, J. Am. Chem. Soc., 2006, 128, 3854. 6. J. Rosenthal and S. J. Lippard, J. Am. Chem. Soc., 2010, 132, 5536. 7. V. Amendola, G. Bergamaschi, M. Boiocchi, L. Fabbrizzi and L. Mosca, J. Am. Chem. Soc., 2013, 135, 6345. 8. D. J. Selkoe, Ann. N. Y. Acad. Sci., 2000, 924, 17-25. 9. W. Annaert and B. De Strooper, Annu. Rev. Cell Dev. Biol., 2002, 18, 25. 10. L. Cai, R. B. Innis and V. W. Pike, Curr. Med. Chem., 2007, 14, 19. 11. H. Quigley, S. J. Colloby and J. T. O'Brien, Int J Geriatr Psychiatry, 2011, 26, 991. 12. C. C. Rowe and V. L. Villemagne, J. Nucl. Med., 2011, 52, 1733. 13. C. A. Mathis, N. S. Mason, B. J. Lopresti and W. E. Klunk, Semin Nucl Med, 2012, 42, 423. 14. Y. O. Lee, J. W. Shin, C. Yi, Y. H. Lee, N. W. Sohn, C. Kang and J. S. Kim, Chem Commun, 2014, 50, 5741. 15. K. Liu, T. L. Guo, J. Chojnacki, H.-G. Lee, X. Wang, S. L. Siedlak, W. Rao, X. Zhu and S. Zhang, ACS Chem. Neurosci., 2012, 3, 141. 16. A. Aaslund, C. J. Sigurdson, T. Klingstedt, S. Grathwohl, T. Bolmont, D. L. Dickstein, E. Glimsdal, S. Prokop, M. Lindgren, P. Konradsson, D. M. Holtzman, P. R. Hof, F. L. Heppner, S. Gandy, M. Jucker, A. Aguzzi, P. Hammarstroem and K. P. R. Nilsson, ACS Chem. Biol., 2009, 4, 673. 17. E. E. Nesterov, J. Skoch, B. T. Hyman, W. E. Klunk, B. J. Bacskai and T. M. Swager, Angew. Chem., Int. Ed., 2005, 44, 5452. 18. K. Cao, M. Farahi, M. Dakanali, W. M. Chang, C. J. Sigurdson, E. A. Theodorakis and J. Yang, J. Am. Chem. Soc., 2012, 134, 17338. 19. X. Zhang, Y. Tian, Z. Li, X. Tian, H. Sun, H. Liu, A. Moore and C. Ran, J. Am. Chem. Soc., 2013, 135, 16397. 20. L. Zhu, W. Wu, M.-Q. Zhu, J. J. Han, J. K. Hurst and A. D. Q. Li, J. Am. Chem. Soc., 2007, 129, 3524. 21. M. Irie, Chem. Rev., 2000, 100, 1683. 22. W. Li, C. Jiao, X. Li, Y. Xie, K. Nakatani, H. Tian and W. Zhu, Angew. Chem., Int. Ed., 2014, 53, 4603. 23. Y. Zou, T. Yi, S. Xiao, F. Li, C. Li, X. Gao, J. Wu, M. Yu and C. Huang, J. Am. Chem. Soc., 2008, 130, 15750. 24. M. Bates, B. Huang, G. T. Dempsey and X. Zhuang, Science, 2007, 317, 1749-1753 25. N. Soh, K. Yoshida, H. Nakajima, K. Nakano, T. Imato, T. Fukaminato and M. Irie, Chem. Commun., 2007, 5206. 26. U. Al-Atar, R. Fernandes, B. Johnsen, D. Baillie and N. R. Branda, J. Am. Chem. Soc., 2009, 131, 15966. 27. K. Liu, Y. Wen, T. Shi, Y. Li, F. Li, Y. L. Zhao, C. Huang and T. Yi, Chem Commun., 2014, 50, 9141. 28. J. Sutharsan, M. Dakanali, C. C. Capule, M. A. Haidekker, J. Yang and E. A. Theodorakis, ChemMedChem, 2010, 5, 56. 29. B. Kurganov, M. Doh and N. Arispe, Peptides, 2004, 25, 217. 30. M. Irie, T. Lifka, K. Uchida, S. Kobatake and Y. Shindo, Chem. Commun., 1999, 747.
1. V. Ntziachristos, C. Bremer and R. Weissleder, Eur Radiol, 2003, 13, 195. 2. V. Ntziachristos, Annu. Rev. Biomed. Eng., 2006, 8, 1-33. 3. J. V. Frangioni, Curr. Opin. Chem. Biol., 2003, 7, 626. 4. K. Licha and C. Olbrich, Adv. Drug Delivery Rev., 2005, 57, 1087.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 2012
ChemComm Accepted Manuscript
Published on 04 November 2014. Downloaded by The University of Auckland Library on 04/11/2014 21:34:35.
COMMUNICATION
Page 4 of 4 View Article Online
