Tunable Aggregation-Induced Multicolor Emission of Organic

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Tunable Aggregation Induced Multicolour Emission of Organic Nanoparticles by Varying Substituent in Naphthalene Diimide Pritam Choudhury, Saheli Sarkar, and Prasanta Kumar Das Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02996 • Publication Date (Web): 02 Nov 2018 Downloaded from http://pubs.acs.org on November 4, 2018

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Tunable Aggregation Induced Multicolour Emission of Organic Nanoparticles by Varying Substituent in Naphthalene Diimide Pritam Choudhury, Saheli Sarkar and Prasanta Kumar Das* School of Biological Sciences, Indian Association for the Cultivation of Science Jadavpur, Kolkata – 700032, India.

*To whom correspondence should be addressed: [email protected]

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ABSTRACT In this article, we have designed L-aspartic acid linked naphthalene diimide (NDI) based amphiphilic molecules having benzyl ester group at the both terminals with varying substituents (NAB-1-5). The substituent was judiciously modified from electron withdrawing group (EWG) like nitrobenzene to electron donating group (EDG), methoxybenzene and finally to extended aromatic residue (naphthalene) to regulate the -electron density at the terminal of NDI derivatives. All the synthesized NDI derivatives are molecularly dissolved in DMSO and with increase in water content within DMSO solution, NDI derivative starts to get self-assemble through J-aggregation at and above 40% water content. Self-assembled spherical organic nanoparticles formed 99% water in DMSO (fw = 99%) was characterized by microscopic studies. All the NDI derivatives showed very weak emission in molecularly dissolved state (DMSO). Aggregation induced emission (AIE) was observed for the NDI derivatives (except NAB-1) at the self-assembled state through excimer formation. Upon excitation at 350 nm, the emission maxima of these NDI based AIE luminogens (AIE-gens) (NAB-2-5) get red shifted from 463 nm to 588 nm upon altering the substitution from EWG to EDG at the donor site. Inclusion of proper donor-acceptor moieties in the molecular backbone of self-assembling unit can govern the AIE in combination with intramolecular charge transfer process. Consequently, the emission colour of these AIE-gens (NAB-2-5) gets tuned from cyan blue to faint green to strong green and finally to bright orange. The tunable aggregation induced multicolour emission was investigated by different spectroscopic techniques. These cytocompatible, multicolour emitting fluorescent organic nanoparticles (FONPs) were utilized for bioimaging application.


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INTRODUCTION Luminescence is an important light-emitting phenomenon having broad domain of relevance from decoding the fundamentals of physical/chemical processes to analytical sensing and also in bioimaging.1-8 Over the past few decades, photoluminescent materials including organic dyes, quantum dots, metallic as well as non-metallic nanoparticles have been widely explored in sensing, imaging, light-emitting diodes, optoelectronic devices, monitoring biogenic processes and so on.9-12 In addition, the recently emerged organic nanoparticles derived from -conjugated oligomers or chromophores having diameter in the range of 10 nm to 1 m, are reported to be more advantageous in biological applications considering its better dispersibility in water and considerable cytocompatibility.13,14 In contrast, aggregation caused quenching (ACQ) is known to weaken

emission behaviour of most of the organic

luminophores in the self-assembled state, which limits their applications particularly in cellular imaging and biosensing.15 Encouragingly, aggregation induced emission (AIE) are reported to be exhibited by few self-assembled luminogens , which is originated due to restriction of intramolecular rotation of the rotor like organic dyes such as tetraphenylethene, siloles, cyano-substituted diarylethene and distyrylanthracene derivatives.16-19 Tuning the emission behaviour of organic dyes is one of the contemporary topics in wide scientific domain.20-22 Extensive efforts have been devoted mostly to regulate the light emission of organic dyes, however, report on multicolour tuning of AIE luminogens (AIEgens) in the self-assembled state is scarce. Different chemical and physical approaches are generally adopted for the preparation of fluorescent organic nanoparticle (FONPs) with tunable optical properties and also to minimize non-radiative decay.16,23-26 In recent past, development of self-aggregated FONPs from naphthalene diimide (NDI) derivatives are also receiving notable importance because of the π-π stacking of its aromatic core and ability to form adduct with electron rich chromophoric units.27-31 NDIs, one of the versatile and 3 ACS Paragon Plus Environment

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fascinating sub-class of the rylene family, are neutral, planar, chemically robust, electrondeficient class of aromatic compounds.32-34 NDI derivatives have been explored in the development of different light emitting and light harvesting systems.35-39 However, unlike perylene bisimide (PBI), NDI derivatives often suffer from poor aggregation and low quantum yield, which may be improved by N-substitution or core substitution at the aromatic ring with non-covalently interacting structural moieties.28,30, 40-45 AIE of NDI derivatives are generally observed in the green region due to the formation of excimer band in the selfassembled state.28,30 However, multicolour tuning of AIE for core un-substituted NDI scaffold using a single wavelength excitation is yet to be investigated. With the aim of developing aggregation induced multicolour emission, we have synthesized naphthalene diimide based L-aspartic acid linked amphiphilic molecules having benzyl ester group at the both terminals with varying substituents (NAB-1-5, Figure 1). The -electron density at donor sites has been systematically tuned by modulating the substituent from an electron withdrawing group (EWG) to electron donating group (EDG) and finally to an extended aromatic residue. All amphiphiles were non-emissive/weakly emissive in molecular state dissolved in dimethyl sulfoxide (DMSO). Upon increase in the water content, the amphiphiles started to get self-assembled through J-type of aggregation. The NDI derivatives (NAB-2-5) showed aggregation induced multicolour (cyan blue to bright orange) emission through excimer formation (except NAB-1) at and above 40% water content in DMSO upon excitation at 350 nm. Formation of aggregated spherical shaped multicolour FONPs from NDI derivatives in 99% water in DMSO (fw = 99%) was characterized by microscopic study. Origin of the tunable multicolour AIE was investigated by different spectroscopic techniques. These multicolour emitting, water dispersible and cytocompatible, novel AIE-gens (NAB-2-5) were aptly utilized for bioimaging of mammalian cells.


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EXPERIMENTAL SECTION Materials. 1,4,5,8-Naphthalenetetracarboxylic dianhydride, 1-naphthaleneethanol, thiazolyl blue tetrazolium bromide (MTT), fluorescein isothiocyanate (FITC), 4-chlorobenzyl alcohol, 4-methoxybenzyl alcohol and all deuteriated NMR solvents experiments were purchased from Sigma-Aldrich. Benzyl alcohol, 4-nitrobenzyl alcohol, N,N-dicyclohexylcarbodiimide (DCC), 4-N,N-(dimethylamino) pyridine (DMAP), N-hydroxybenzotriazole (HOBT), trifluoroacetic acid (TFA), solvents, silica gel (60-120 mesh and 100-200 mesh) for column chromatography, other reagents were acquired from SRL, India. Milli-Q water was used for all experiments. Dulbecco’s Modified Eagle’s Medium (DMEM), fatal bovine serum (FBS), trypsin (origin: procine pancreas) were procured from Hi-Media, India. NMR spectroscopy was performed in AVANCE (Bruker) spectrometer. MALDI-TOF spectra were recorded on Bruker Ultraflex MALDI mass spectrometer with 2,5-dihydroxy benzoic (DHB) acid as matrix. Synthesis of NAB-1-5. Different benzyl ester protected L-aspartic acid linked naphthalene diimide (NDI) derivatives were synthesized by following methods (Scheme S1, Supporting Information). In case of NAB-1, N-(tert-butoxycarbonyl)-L-aspartic acid 4-benzyl ester (1.2 eqv) and 4-nitrobenzyl alcohol (1.0 eqv) were coupled together using DCC (1.1 eqv), DMAP (1.1 eqv) and HOBT (1.1 eqv). Dry dichloromethane (DCM) was used as solvent and the reaction was stirred for 12 h under nitrogen atmosphere. 1(N) HCl was used to wash the organic layer and the organic solvent was dried over anhydrous sodium sulfate. After evaporation of the organic part, the coupled product (Scheme S1, Supporting Information) was purified by column chromatography using 60-120 mesh silica gel using 1% methanol in chloroform as eluent. TFA (1.5 eqv) was used to deprotect the BOC (tert-butoxycarbonyl) group from the coupled product and the reaction was carried out in dry DCM. After 4h of stirring, the organic solvent was removed and the concentrated mass was dried. The obtained 5 ACS Paragon Plus Environment

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mass was solubilized in EtOAc. Aqueous Na2CO3 solution (10%) and water was used to wash the organic layer for two times and the organic part was dried over anhydrous Na2SO4. The organic part was evaporated to obtain free amine derivative. Finally, 1,4,5,8naphthalenetetracarboxylic dianhydride (1.0 eqv) and free amine derivative (2.2 eqv) were heated (90 °C,12 h) in dry DMF. The reaction mixture was cooled to room temperature and DMF was removed by vacuum distillation. The crude mass was solubilized in DCM and washed with water. The expected product was collected from DCM part. Purification of the condensed product (NAB-1) was performed by column chromatography (100-200 mesh silica gel, 2% MeOH/CHCl3 as an eluent) and washed with diethyl ether to obtain the precipitation of solid mass. Similar protocol was applied to synthesize NAB-2, NAB-3 (structure has been reported earlier)46, NAB-4 and NAB-5 by protecting one acid group of L-aspartic acid residue by 4-chlorobenzyl alcohol, benzyl alcohol, 4-methoxybenzyl alcohol and 1naphthaleneethanol, respectively. All the NDI derivatives (NAB-1-5) were characterized by 1H-NMR, 13C-NMR

and MALDI-TOF mass spectrometry (Supporting Information).

Preparation of samples. All the synthesized NDI derivatives (NAB-1-5) were highly soluble in DMSO. So, stock solution of all the NDI derivatives (1 mM) was prepared in DMSO. From this stock solution of DMSO, desired amount aliquot was added to the DMSO-water solvent system of different ratios to achieve

10-20 M concentration for different

experiments. Transmission electron microscopy (TEM) study. The solution (4 L) of NDI derivatives, NAB-1-5 (20 M) in 99% water in DMSO (fw = 99%) was deposited separately on copper grid (carbon coated, 300 mesh) and dried to obtain transmission electron microscopic images. 1 L (1% w/v) freshly prepared uranyl acetate solution was used to negatively stain the copper grid and the excess solution was immediately removed. Before taking the TEM


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images, the samples were kept for drying for 4 h under vacuum.. JEOL JEM 2010 microscope was used to take the TEM images. Field-emission scanning electron microscopy (FESEM) study. JEOL-6700F microscope was used to obtain FESEM images. 6 L solution of NAB-1-5 (20 M) taken in 99% water in DMSO (fw = 99%) was drop cast separately on glass cover slips followed by drying and kept for 3 h under vacuum before imaging. Dynamic light scattering (DLS) study. Mean hydrodynamic diameter (Dh) of selfaggregates of NAB-1-5 (20 M) in 99% water in DMSO (fw = 99%) were measured using Zen 3690 Zetasizer Nano ZS instrument (Malvern Instrument Ltd.). Scattering intensity was determined at 175° angle and data was analyzed by Cumulant Fit in Malvern Zetasizer software. UV-visible study. Perkin Elmer Lambda 25 spectrophotometer was utilized to record UVvisible spectra. We have monitored solvent dependent UV-visible spectra of all the amphiphilic molecules, NAB-1-5 (20 M) by varying solvent compositions of DMSO-water (100% to 1% DMSO) to investigate the self-aggregation pattern. UV-visible spectra of NAB1-5 (20 M) by varying temperature were also recorded in 1:3 v/v, DMSO-water from 25 °C to 95 °C. UV-visible spectra of NAB-5 (10 M) were also recorded in different organic solvents (benzene, chlorobenzene, chloroform, dichloromethane) of varying polarities. 1H-NMR

study. Avance 300 MHz (Bruker) spectrometer was used to record solvent

dependent 1H-NMR spectra of NAB-1-5 (1 mM) in DMSO-d6 and in various solvent mixture of DMSO-d6-D2O (3:1 (v/v) and 1:3 (v/v) DMSO-d6-D2O). Similarly, 1H-NMR spectra of the above mentioned NDI derivatives (NAB-1-5) were also obtained at varying concentrations (from 0.5 mM to 5 mM) in 2:1 v/v, DMSO-d6-D2O. Fluorescence microscopy study. Solution (10 L) of NAB-2-5 (50 μM) in 99% water in DMSO (fw = 99%) was drop cast on different glass slide. The solutions were air-dried and 7 ACS Paragon Plus Environment

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images of dried samples were taken under Olympus BX-61. Different excitation filters (BP 330-385 nm, BP 460-495, BP 530-550 nm for blue, green and red images, respectively) and band absorbance filters (covering wavelength below 405 nm, 505 nm and 570 nm for blue, green and red images, respectively) were used to take images. Photoluminescence study. We have recorded the emission spectra of the solutions of NAB1-5 (excitation wavelength (ex) = 350 nm) in a Varian Cary Eclipse luminescence spectrometer. From the stock solutions (1 mM in DMSO) of all the amphiphilic molecules (NAB-1-5), requisite amounts of sample solutions were added to different compositions of DMSO-water mixture to achieve experimental concentration of each amphiphilic molecules of 10 M. Excitation dependent emission spectra of NAB-5 (10 M) in (1:99 v/v) DMSOwater were also recorded with excitation range 350-400 nm. Emission spectra of NAB-5 (10 M) were also recorded in different organic solvents (benzene, chlorobenzene, chloroform, dichloromethane) of varying polarities to monitor the intramolecular charge transfer process. Quantum yield (QY) measurement. QYs are estimated with respect to a fluorophore taken as reference having known QY (s). The QYs of the unknown fluorophore (NAB-2-5) (u) were measured by following equation, ∅𝑢 =

( ) 𝐴𝑠𝐹𝑢𝑛2𝑢 𝐴𝑢𝐹𝑠𝑛2𝑠

∅𝑠

(1)

Here, Au and As are absorbance of unknown sample and reference sample at corresponding

ex, respectively. Fu and Fs are integrated fluorescence intensity for the unknown and known sample excited at the ex, respectively.nu and ns are refractive indices of solvents in which the unknown sample and known sample was prepared, respectively. Solutions having similar absorbance (< 0.01) were used for determination of QY. Herein, we had taken quinine sulfate in sulphuric acid (0.1 M) as reference (QY (s) = 54.0%).


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Time resolved study. Picosecond diode laser IBH-405 was used to determine time correlation single photon count (TCSPC) measurement. From the stock solutions of NAB-2-5 (1 mM) in DMSO, required volume of sample solution was added to 1 mL of DMSO, (1:1 v/v) and (1:99 v/v) DMSO-water solutions having the absorbance less than 0.1. All amphiphilic solutions were excited (ex) at 375 nm followed by monitoring of the emission (mon) at 400 nm in only in DMSO and at 463 nm, 470 nm, 480 nm and 588 nm for different ratio of DMSO-water binary solvent mixtures for NAB-2, NAB-3, NAB-4 and NAB-5, respectively. IBH DAS6 software was used to analyze the fluorescence decays. Experimental time resolved fluorescence decay p(t) was analyzed by following equation (2).

( ) 𝑡

𝑛

𝑝(𝑡) = 𝑏 + ∑𝑖 𝛼𝑖 exp ― 𝜏𝑖

(2)

Where, n = number of discrete emissive species, b = baseline correction (“dc” offset), and i = pre-exponential factors and i = excited state fluorescence lifetimes associated with the ith component, respectively. For multi-exponential decays, equation (3) was used to calculate the average life-time. 𝑛

=

∑𝛼 𝜏

𝑖 𝑖

𝑖=1

Where 𝛼𝑖 =

𝛼𝑖 ∑𝛼𝑖

(3)

which indicates the contribution of a decay component.

Cell culture. Both cancer cells (HeLa) and healthy cells (NIH3T3) were procured from NCCS, Pune and grown in FBS-DMEM (10%) media having streptomycin and penicillin . Cells were grown in culture flask at 37 °C. Sub-culture of the cells was carried out in every 23 days. FBS supplemented media was replaced after 48-72 h. The adherent cells were detached from the culture flask surface by trypsinization. Both HeLa and NIH3T3 cells were utilized to perform cytotoxicity and only HeLa cells were used for bioimaging experiments. Since FONPs derived from NAB-2-5 in 99% water in DMSO (fw = 99%) were found to be 9 ACS Paragon Plus Environment

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most fluorescent among the other ratio of DMSO-water, all the biological experiments were performed using NAB-2-5 FONPs in fw = 99%. MTT assay. Cytocompatibility of FONPs derived from NAB-2-5 in 99% water in DMSO (fw = 99%), was investigated by microculture MTT reduction assay. In this assay, soluble tetrazolium salt is reduced to an insoluble coloured formazan product by mitochondrial dehydrogenase liberated from viable cells. Amount of produced formazan was estimated spectrophotometrically by dissolving it in DMSO. Amount of produced formazan is proportional to number of live cells. The decrease in absorbance corresponds to the killing of the cells NAB-2-5. HeLa cells were cultured (20,000 cells per well) in a 96-well plate for 1824 h before the assay. MTT assay with NAB-2-5 FONPs (1:99 v/v DMSO-water) was performed over a concentration range of 5-50 M in the microtitre plate. Both the HeLa and NIH3T3 cells were incubated with NAB-2-5 for 12 h (37 °C, 5% CO2 atmosphere). 10 L from MTT stock solution (5 mg mL-1) in PBS was added to the above mixture and the cells were incubated for another 4 h. Precipitated formazan was dissolved in DMSO, and absorbance was recorded at 570 nm using BioTek1 Elisa Reader. The number of live cells was expressed as % viability by following the equation: A570(treated cells) ― background

% viability = A570(untreated cells) ―

background

× 100

(4)

Bioimaging. HeLa cells were grown (10,000 cells/well) within a chamber slide for 18-24 h . Cells were treated with NAB-2-5 FONPs (10 M and 50 M of each infw = 99%) for 6 h. The incubated cells were washed with PBS buffer for three times, followed by fixed with paraformaldehyde (4%) for 30 min. Then, glycerol (50%) was used to mount the cells on slide, which was covered using cover slip and kept for 24 h. Bioimaging was performed in Olympus BX-61 microscope at 40× magnification using different filters for the respective colours. Bioimaging of HeLa cells was also performed with free FITC as control experiment. 10 ACS Paragon Plus Environment

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RESULTS AND DISCUSSION Designing of NDI based amphiphilic molecules NAB-1-5. Aggregation induced emission (AIE) phenomenon is often governed by restricted intramolecular rotation of the luminogens.16 Presence of an electron-rich (donor) and electron-deficient (acceptor) aromatic -moieties in the molecular scaffold may have the influence in the AIE phenomena through the charge-transfer process.16,26,47 Hence, inclusion of proper donor-acceptor (D-A) moieties in the molecular backbone of self-assembling unit can regulate the AIE. Considering the electron-deficient character of NDI aromatic core, intramolecular charge-transfer from electron-donating π-conjugated units to NDI core could play a key role in tuning its AIE. However, design and development of colour tunable AIE active materials using core unsubstituted NDI scaffold has not been attempted mainly because of its poor quantum yield and low association constant. Herein, we have designed and synthesized the NDI bearing amino acid linked amphiphilic benzyl ester derivatives (NAB-1-5, Figure 1 and Scheme S1, Supporting Information). Aromatic ring at NDI core can act as hydrophobic domain and it may assist the self-assembling process through its extended π-moieties. Chirality of the Laspartic acid residue may also induce the twisted nature in the molecular backbone to restrict the intramolecular rotation in the self-aggregated state, which is a prerequisite of AIE.16,30,31 In addition, the presence of unsubstituted benzyl residue in the molecular backbone may also have the influence on self-aggregation process through - stacking interaction. The donating residue at the terminal position was judiciously modulated from an electronwithdrawing group (EWG) to electron-donating group (EDG) and finally to an extended conjugated moiety (NAB-1-5, Figure 1). Enhancement in -electron density at the donor site may have favourable effect on intramolecular charge-transfer. Characterizations of all the synthesized NDI derivatives are given in the Supporting Information.


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Self-assembly of NDI derivatives: Microscopic and DLS study. All the synthesized NDI derivatives (NAB-1-5) were freely soluble in DMSO and upon gradual addition of water in DMSO, transparent solutions slightly turned to translucent at around 40% water content (for NAB-1, it was ~80% water content) indicating the initiation of self-assembly by NDI derivatives (Figure S1a, S1c, S1e, S1g, S1i, Supporting Information). Further increase in the water content up to 99%, steadily enhanced the macroscopic translucency of the solution possibly due to the formation of self-assembled structures. We have used different microscopic techniques to investigate the structure of self-aggregates formed by NDI derivates. TEM images of NAB-1-5 (20 M, taken in 99% water in DMSO (fw = 99%)) showed spherical shaped particles having dimension of 30-50 nm for NAB-1-4, while comparatively larger sized self-aggregates of 80-150 nm were observed for NAB-5 (Figure 2a-e). Notably, the organic nanoparticles formed by NAB-5 were found to be fused in nature (Figure 2e). In concurrence with the TEM images, FESEM images also confirmed the formation of spherical shaped organic nanoparticles by NAB-1-5 (20 M, taken in99% water in DMSO (fw = 99%)) having diameter of 70-100 nm for NAB-1-4 and 100-150 nm for NAB5 (Figure 2f-j). In few instances, larger particles were observed in FESEM images possibly due to the clustering of multiple particles in the sample slides. In addition, size of the organic nanoparticles was determined by dynamic light scattering (DLS) study. Each NDI derivative (20 M) taken in fw = 99% was used to determine the size of the organic nanoparticles. Mean hydrodynamic diameter (Dh) of organic nanoparticles of NAB-1-4 was observed in the range 20-90 nm with an average diameter of ~35 nm (Figure 2k-n). In case of NAB-5, Dhof the organic nanoparticles was considerably large (75-350 nm) and had an average diameter of ~100 nm (Figure 2o). This wide range of aggregate's size for NAB-5 indicates the possible fusion between the organic nanoparticles as observed in microscopic image (Figure 2e,j). Corresponding correlograms with good Cumulant Fit for the respective NDI derivatives were 12 ACS Paragon Plus Environment

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provided in the Supporting Information (Figure S2a-e). Importantly, the observed size of the self-aggregated particles in solution phase is mostly in concurrence with that of the microscopic investigations for all the NDI derivatives. Spectroscopic study on the self-assembly of NDI derivatives. Organic nanoparticle formation was initially studied by UV-visible (UV-vis) spectroscopy for all the NDI derivatives in various compositions of DMSO-water solvent system. Sharp absorption bands in the range of 330-400 nm was observed in the UV-vis spectra of NAB-1-5 (25 M) in DMSO (molecularly dissolved state) (Figure S3a, Supporting Information and Figure 3a-d). The absorption maxima (max) were observed at 381 nm (NAB-1, NAB-2), 380 nm (NAB-3), 379 nm (NAB-4) and 382 nm (NAB-5), respectively possibly due to -* transition polarized along the long axis of NDI chromophore.43 Initially, when water was added to the DMSO solution of NAB-1-5, the absorbance was immediately dropped.The optical density further decreased slightly with increasing water percent. Alongside, max was gradually red shifted (around 2 to 6 nm shift for NAB-1-5) in the range of 383-386 nm with the increased amount of water up to 99% in DMSO (Figure S3a, Supporting Information and Figure 3a-d). These characteristics bathochromic shifts in the absorption maxima in presence of water indicate that the formation of self-assembled organic nanoparticles took place through J-type aggregation by stair like stacking of the NDI chromophore (Scheme 1).28 In addition, we have also monitored UV-vis spectra of the synthesized naphthalene diimide derivatives, NAB-1-5 (20 M) in 1:3 v/v, DMSO-water at different temperature. With increase in temperature from 25 °C to 95 °C, the max of the respective NDI derivatives gradually blue shifted ~2-4 nm at and above 45 °C (Figure S3b, S4a-d, Supporting Information). This blue shifted change in the

max with temperature delineates that at and above 45 °C, the self-assembly possibly starts to get dissociated. Hence, the aggregated organic nanoparticles formed by NDI derivatives are stable at least up to 45 °C. 13 ACS Paragon Plus Environment

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Participation of intermolecular non-covalent interaction in self-assembly. The presence of non-covalent interactions to form self-assembled organic nanoparticles by NDI derivatives at higher water content in DMSO was monitored by solvent dependent and concentration dependent 1H-NMR spectroscopy. In solvent dependent 1H-NMR study, the aromatic protons of NAB-2 exhibited sharp NMR signal in the region  = 8.70 ppm (aromatic core of NDI) and  = 7.15-7.38 ppm (substituted phenyl rings at the terminal) in DMSO-d6 suggesting its characteristics molecular proton peak at non-aggregated state (Figure S5b, Supporting Information). With gradual increase in D2O percent (3:1 v/v and 1:3 v/v, DMSO-d6-D2O), the NMR signals of the aromatic protons got broadened along with an upfield shift in  value. At (1:3, v/v) DMSO-d6-D2O, the aromatic protons shifted to  = 8.44 ppm (NDI aromatic core) and  = 6.94-7.38 ppm (other phenyl rings) with reduced NMR signal intensity (Figure S5b, Supporting Information). Similar changes in the 1H-NMR spectral pattern were observed for other NDI derivatives (NAB-1/3/4/5) (Figure S5a and S5c-e, Supporting Information). In DMSO-d6, the sharp peak intensity was the characteristics of molecular protons in absence of participation of non-covalent interactions. Addition of D2O facilitates the self-assembly process through - stacking between NDI chromophore as well as the terminal aromatic residues of the amphiphiles. Consequently, upfield shifting of the aromatic protons took place along with the broadening of peak intensity due to the restricted motion of spinning nuclei in the aggregated state. The - interaction between the NDI chromophore and substituted phenyl rings was also monitored by 1H-NMR study using varying concentration of the amphiphiles in 2:1 (v/v) DMSO-d6-D2O (Figure S6a-e, Supporting Information). At 0.5 mM of NAB-1, moderately sharp 1H-NMR signals with low intensity around  = 8.60 ppm (for NDI core), 8.14 ppm and  = 7.0-7.2 ppm (for -NO2 substituted and un-substituted phenyl ring) were observed (Figure S6a, Supporting Information). With steady increase in the concentration of NAB-1 up to 5.0 mM, these characteristics peaks got slightly upfield shifted 14 ACS Paragon Plus Environment

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and suppressed with very poor intensity. Similar, spectral pattern were observed for rest of the NDI derivatives (NAB-2-5, Figure S6b-e, Supporting Information). This upfield shifting and broadening in the nature of aromatic protons reiterates that with increase in the concentration of the amphiphiles, self-aggregated structure formation takes place through - stacking between the aromatic residues of the NDI derivatives. Investigation of emission behaviour of self-aggregated organic nanoparticles. Presence of electron donor-acceptor moieties in the molecular framework and formation of the selfaggregated organic nanoparticles by the NDI derivatives may induce the chromophoric molecules to exhibit the AIE. To this end, we investigated the emission behaviour of the synthesized NDI derivatives in its non-self-assembled state to self-aggregated state in varying DMSO-H2O binary mixtures in the process of formation of organic nanoparticles. All the NDI derivatives (NAB-1-5) taken in DMSO were non-emissive upon UV lamp exposure at 365 nm (Figure 4a,c,e,g,i). Upon UV-light irradiation (ex = 365 nm), NAB-1 (-NO2 group containing NDI derivative) did not show any colour emission (Figure S1b, Supporting Information) despite its self-aggregation with gradual increase in water content. Even at the highest water content (fraction of water (fw) = 99%) and also for the solid powder lyophilized from fw = 99%, no significant emission colour was found for NAB-1 (Figure 4b,k). In case of NAB-2, no emission colour was observed up to fw = 30% (Figure S1d, Supporting Information). Interestingly, at 40% water content (fw = 40%), NAB-2 first time showed the feeble cyan blue emission upon UV-light irradiation at ex = 365 nm (Figure S1d, Supporting Information). Further addition of water gradually enhanced the intensity of the cyan blue colour (Figure S1d, Supporting Information). Strong cyan blue emission was found at the fw = 99% (Figure 4d). Similarly, for NAB-3/4/5 different fluorescent colours (faint green, strong green and orange, respectively) were found to appear at and above fw = 40% (Figure S1f, S1h, S1j, Supporting Information). Further increase in the water content resulted in more 15 ACS Paragon Plus Environment

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strong emission for respective NDI derivatives (Figure S1f, S1h, S1j, Supporting Information), which reached its highest intensity at fw = 99% (Figure 4f,h,j). Notably, the lyophilized mass obtained from fw = 99%, also showed characteristics emission colour for the respective NDI derivatives upon excitation at 365 nm (Figure 4k-o). Hence, depending on the nature of the substituent at the terminal position of the NDI derivatives (NAB-2-5), the emission colour gets tuned from cyan blue to faint green to strong green and finally to bright orange, respectively (Figure 4d,f,h,j). The preceding observations confirm the formation of the colour tuned fluorescent organic nanoparticles (FONPs) by self-aggregation of NAB-2-5. Fluorescence microscopic images of self-aggregated organic particles derived from the respective NDI derivatives at fw = 99% further ascertained the formation of multiple coloured FONPs (blue dots, faint green dots, strong green dots and red dots for NAB-2/3/4/5, respectively, Figure 5a-d). Photoluminescence for aggregation induced multicolour emission of NDI derivatives. The observed multicolour emission of the self-aggregated NDI derivatives depending on the aromatic substitution at the terminal of the NDI derivatives was investigated by fluorescence spectroscopy in DMSO-water solvent system. Emission spectra of NAB-1-5 (10 M) in DMSO exhibited weak fluorescence band having emission maximum (em) at 398 nm upon excitation at 350 nm (Figure S3c, Supporting Information and Figure 6a,c,e,g). In case of NAB-1 (-NO2 benzene substitution), the weak emission intensity of the monomeric band further reduced with increase in water content without the formation of any excimer peak (Figure S3c, Supporting Information). Gradual drop in the fluorescence intensity of the monomeric band was also noted for other NDI derivatives (NAB-2-5) with increasing water content up to around 30% in DMSO solution (Figure 6a-h). However, an interesting change in the emission behaviour was noted for NAB-2-5, with the development of a new excimer band beyond fw = 30%. In case of NAB-2 (chlorobenzene substitution), the excimer band was 16 ACS Paragon Plus Environment

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observed beyond 30% water content with em at 463 nm upon excitation at 350 nm (Figure 6a). Intensity of em steadily enhanced with increment in water content and at fw = 99%, the

em showed highest intensity (almost 9-fold higher than that was observed in DMSO) (Figure 6a,b). In case of NAB-3 (un-substituted benzene at the terminal), the excimer band was observed at em = 470 nm in presence of 40% water content (Figure 6c). Here also, the emission intensity steadily increased with enhanced water content and at fw = 99%, the observed intensity is around 13-fold higher than that was observed in only DMSO (Figure 6c, d). Similarly, NAB-4 (methoxybenzene substitution) and NAB-5 (naphthyl substitution) showed excimer peaks at em = 480 nm and 588 nm, respectively at and above fw = 40%. In both cases, excimer peak achieved highest intensity at fw = 99%, which is ~4-fold and 6-fold higher than that was observed in DMSO for NAB-4 and NAB-5, respectively (Figure 6e-h). The notable enhancement in the emission intensity of the newly generated excimer peaks (for NAB-2-5) with increase in water content is primarily due to the AIE of the NDI derivatives. The fine tuned multicolour emission at self-aggregated state (AIE phenomena) of NDI derivatives may be explained considering the electronic factor i.e., resonance effect (±Reffect) as well as the inductive effect (±I-effect) of the substituted moieties at the terminal. The electron withdrawing nitro group (-NO2) possesses both strong R and I effects, which inhibit intramolecular charge transfer from the donor site to the acceptor NDI core. Consequently, no AIE was observed in the self-aggregated state of NAB-1. Replacement of nitro group by chloro group having moderate +R-effect and strong

I effects (moderately

weak EWG than -NO2) initiated a “push-pull” interaction (intramolecular charge transfer between electron donor-acceptor residues) that exhibited the excimer band in the cyan blue region (em = 463 nm) for NAB-2. The excimer peak in the self-assembled state steadily gets red shifted due to the inclusion of much strong +R-effect containing substitution from NAB-3 to NAB-5 (Figure 7). In case of NAB-3 (devoid of any EWG/EDG) having only moderate 17 ACS Paragon Plus Environment

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+R-effect, faint green fluorescence (em = 470 nm) was observed while methoxy group (strong +R-effect and moderate I-effect) containing NAB-4 exhibited strong green emission at 480 nm. Among all the NDI derivatives, NAB-5 exhibited the maximum shift in its emission band having em = 588 nm (bright orange) presumably due to the presence of more conjugated -system (naphthyl group) at the donor site. These findings suggest that the presence of resonance effect in the electron donating site has the predominant role over the inductive effect for systematic red shifting of emission wavelength. Variation in the aromatic substitution at the terminal of NDI derivatives from EWG to EDG facilitated the intramolecular charge transfer to the NDI core resulting in change in the em of excimer band as well as the emission colour from cyan blue to bright orange. To further investigate the origin of charge transfer emission, excitation dependent emission spectra were recorded for NAB-5 in fw = 99% as NAB-5 showed strong red-shifted AIE (Figure S7a, Supporting Information). Excitation dependent charge transfer band (ex = 360-400 nm) of NAB-5 showed emission spectra having similar features to that obtained at ex = 350 nm (Figure S7a, Supporting Information). This suggests that the emission has been raised due to chargetransfer.29 In addition, the intramolecular charge transfer (ICT) process was envisaged by examining the absorption and emission properties of NAB-5 in varying solvents of different polarities. No significant changes in absorbance values was found for NAB-5 at different solvents (from less polar benzene to moderately polar chloroform and finally to chlorobenzene and dichloromethane) (Figure S7b and Table S1, Supporting Information). In case of fluorescence study, as the solvent polarity increased, the emission peak were gradually red-shifted (Figure S7c and Table S1, Supporting Information). The Stokes shift () of NAB-5 in varying solvents was calculated, and their solvatochromic behaviours were quantitatively described by the Lippert-Mataga equation48,49:


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∆𝜈 = 𝜈𝑎 ― 𝜈𝑒 =

2∆𝑓 ℎ𝑐𝑎

3

(𝜇𝐸 ― 𝜇𝐺)2 + 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡

(5)

where νa and νe represent the maximum absorbance and emission wavenumbers, μG and μE are the dipole moments in the ground and excited states. h, c, a, and n are the Planck constant, the speed of light, the Onsager solvent cavity radius and the refractive index of the solvent, respectively. The orientational polarizability or solvent polarity parameter Δf, described in equation 5, is chosen as the measure of polarity. 𝜀―1 𝑛2 ― 1 ∆𝑓 = ― 2𝜀 + 1 2𝑛2 + 1

(6)

where, ε is the static dielectric constant and n is the optical refractive index of the solvent. The linear dependence and positive solvatochroism was found for NAB-5, indicating that the involvement solvent polarity dependent intramolecular charge transfer (ICT) emissive states (Figure 7d, Supporting Information). The relative fluorescence quantum yield of NAB2/3/4/5 in fw = 99% was also found to be 4.43%, 2.04%, 2.35% and 3.70%, respectively (in reference quinine sulfate hydrate), which is superior to that of previously reported AIE of naphthalene diimide derivatives in aqueous domain.28,30 Time resolved study. To further understand the fluorescence behaviour as well as multicolour emission of the NDI derivatives, TCSPC experiment was performed with a picosecond diode laser (ex = 375 nm) for NAB-2-5 (10 M) in DMSO and DMSO-water mixture (Figure 8). In each case, the decay curves were fitted by a tri-exponential function. In molecularly dissolved state (DMSO) of NAB-2-5, emission was monitored at wavelength (mon) 400 nm and the estimated short average lifetimes were ˂˃NAB-2 = 1.02 ns, ˂˃NAB-3 = 1.07 ns, ˂˃NAB-4 = 0.42 ns, and ˂˃NAB-5 = 0.16 ns (Table 1). With gradual increment of water content in DMSO solution, long-lived components was monitored at the emission 19 ACS Paragon Plus Environment

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wavelength (mon = 463 nm, 470 nm, 480 nm and 588 nm for NAB-2/3/4/5, respectively), which indicate that the aggregates contain excimer species of the corresponding NDI derivatives (Table 1). Average life time notably increased with increase in water content (fw = 50%, Table 1) and at fw = 99%, NAB-2/3/4/5 showed average lifetime of ˂˃ = 1.53 ns, 3.39 ns, 5.11 ns and 8.96 ns, respectively (Figure 8a-d, Table 1). Presumably, this long decay lifetime arises due to the presence of associated chromophores within the self-aggregates formed by the respective amphiphile at higher water content. Moreover, regulation of -electron density at the donor site by varying the substituent from EWG to EDG has been reflected in the enhanced average lifetime. Among these NDI derivatives, NAB-5 comprising of most delocalized -electron cloud (naphthyl substitution) exhibited highest average lifetime (8.96 ns) in the self-assembled state with maximum red shifted excimer band (em = 588 nm). TCSPC experiment further confirmed the AIE through excimer formation in the selfaggregated state of NDI derivatives. It also delineates the influence of peripheral substituent towards tuning the aggregation induced multicolour emission. Cytocompatibility of NDI derivatives-based FONPs. We intend to explore these novel multicolour emitting NDI-derived AIE-gens in bioimaging where biocompatibility of FONPs against eukaryotic cells is an important prerequisite. Cytocompatibility of FONPs (5-50 M) derived from NDI derivatives (NAB-2-5) in fw = 99% was investigated by MTT assay in HeLa cancer cells and NIH3T3 healthy cells. In all instances ~80-85% eukaryotic cells were found to be alive after 12 h incubation even at the highest experimental concentration (50 M) of FONPs (Figure S8 and S9, Supporting Information). Hence, these NDI-based FONPs are found to be sufficiently biocompatible and eligible to be utilized in cellular imaging study. Multicolour bioimaging by NDI-based FONPs. Labelling of the mammalian cells by NDI derived cytocompatible multicolour FONPs was monitored by fluorescence microscopy. 20 ACS Paragon Plus Environment

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HeLa cells were incubated separately with 50 M of each FONP derived from NAB-2-5 ( fw = 99%) for 6 h. Thereafter, the fluorescence microscopic images showed bright multicolour fluorescence (blue, faint green, strong green and red for NAB-2/3/4/5, respectively) inside HeLa cells which indicated significant internalization of the FONPs within the mammalian cells (Figure 9a-h). Bioimaging was also carried out upon incubation of cancer cells with FONPs even at lower concentration (10 M) and the fluorescence microscopic images also showed bright multicolour fluorescence aforesaid (Figure S10a-h, Supporting Information). Imaging of HeLa cells was also performed with conventional dye fluorescein-5isothiocyanate (FITC) as control experiment, which also showed green fluorescence inside the cells (Figure S11, Supporting Information). Hence, this “light-up” approach of these NDI based colour tunable FONPs affirms its cell staining ability in visible region (from blue to green to red), which can be potentially used in diagnostic as bio-probes. CONCLUSION In conclusion, we have designed L-aspartic acid appended naphthalene diimide (NDI) based amphiphilic molecules comprise of benzyl ester at the both terminals with varying substituents (EWG to EDG and finally to extended aromatic residue, NAB-1-5). These NDI derivatives were soluble in DMSO and formed self-aggregated organic nanoparticles in 1:99 v/v, DMSO-water. The NDI core as well as aromatic residues at the terminals facilitated the self-assembly through J-aggregation in binary solvent mixture at and above 40% water content.

All the NDI derivatives were non-fluorescent/weakly fluorescent in non self-

assembled state (in DMSO) and showed strong emission (except NAB-1) of different colours (cyan blue, faint green, strong green and bright orange for NAB-2-5, respectively) in the selfassembled state (above fw = 40%). Spectroscopic studies revealed the AIE of NDI derivatives (NAB-2-5) took place through excimer formation in the self-assembled state at different emission maxima (463-588 nm) upon single wavelength excitation at 350 nm. Moreover, the 21 ACS Paragon Plus Environment

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AIE colours of the NDI derivatives get regulated along with the combination of intramolecular charge transfer by varying the substituent. This unique emission feature made these newly developed cytocompatible, multicoloured FONPs (NAB-2-5) potentially useful in cellular imaging. Supporting Information Available: Synthetic scheme of NAB-1-5 and characterization data, Table S1 for photophysical property of NAB-5, photographs of formation of translucent solutions and change in emission colour with increasing water content of NAB-1-5, raw correlograms of NAB-1-5, UV-visible spectra of NAB-1, UV-visible spectra of NAB-1-5 (varying temperature), fluorescence spectra of NAB-1, solvent and concentration dependent 1H-NMR

spectra, excitation dependent emission spectra , solvent dependent absorption and

emission spectra, plot of the Stokes shift vs solvent polarity parameter of NAB-5, MTT assay of NAB-2-5 in HeLa cells, NIH3T3 cells, bioimaging of HeLa cells by NAB-2-5 FONPs at lower concentration (10 M), fluorescence microscopic images of HeLa cells incubated with NAB-4 and conventional dye FITC. ACKNOWLEDGEMENTS P.K.D. is thankful to Science and Engineering Research Board (SERB), Department of Science and Technology (DST), India for financial assistance (No. EMR/2017/000656). P.C. acknowledges CSIR, India and S.S. acknowledges DST, India for their Research Fellowships. REFERENCES (1) Schuster, G. B. Chemiluminescence of organic peroxides. conversion of ground-state reactants to excited-state products by the chemically initiated electron-exchange luminescence mechanism. Chemiluminescence 1979, 12, 366-373. (2) Gorris, H. H.; Wolfbeis, O. S. Photon-upconverting nanoparticles for optical encoding and multiplexing of cells, biomolecules, and microspheres. Angew. Chem. Int. Ed. 2013, 52, 3584-3600. 22 ACS Paragon Plus Environment

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(43) Bell, T. D. M.; Bhosale, S. V.; Forsyth, C. M.; Hayne, D.; Ghiggino, K. P.; Hutchison, J. A.; Jani, C. H.; Langford, S. J.; Lee, M. A. P.; Woodward, C. P. Melt-induced fluorescent signature in a simple naphthalenediimide. Chem. Commun. 2010, 46, 4881-4883. (44) Shao, H.; Nguyen, T.; Romano, N. C.; Modarelli, D. A.; Parquette, J. R. Self-assembly of 1-D n-type nanostructures based on naphthalene diimide-appended dipeptides. J. Am. Chem. Soc. 2009, 131, 16374-16376. (45) Kar, H.; Ghosh, S. J-aggregation of a sulfur-substituted naphthalenediimide (NDI) with remarkably bright fluorescence. Chem. Commun. 2016, 52, 8818-8821. (46) Tambara, K.; Ponnuswamy, N.; Hennrich, G.; Pantos, G. D. Microwave-assisted synthesis of naphthalenemonoimides and N-desymmetrized naphthalenediimides. J. Org. Chem. 2011, 76, 3338-3347. (47) Yuan, W. Z.; Gong, Y.; Chen, S.; Shen, X. Y.; Lam, J. W. Y.; Lu, P.; Lu, Y.; Wang, Z.; Hu, R.; Xie, N.; Kwok, H. S.; Zhang, Y.; Sun, J. Z.; Tang, B. Z. Efficient solid emitters with aggregation-induced emission and intramolecular charge transfer characteristics: molecular design, synthesis, photophysical behaviors, and OLED application. Chem. Mater. 2012, 24, 1518-1528. (48) Lippert, V. E. Dipolmoment und elektronenstruktur von angeregten molekülen. Z. Naturforschg. 1955, 10a, 541-545. (49) Mataga, N.; Kaifu, Y.; Koizumi, M. Solvent effects upon fluorescence spectra and the dipolemoments of excited molecules. Bull. Chem. Soc. Jpn. 1956, 29, 465-470.


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Table 1. Lifetime values of NAB-2-5 in the presence of different fractions of water in DMSO (λex = 375 nm) Compound

NAB-2

fraction of

monitored

1 (ns)

2 (ns)

3 (ns)

Average lifetime

water (fw)

(nm)

(a1)

(a2)

(a3)

ns

0%

400

0.38

1.55

4.23

1.02

(0.435)

(0.546)

(0.019)

2.03

0.43

8.86

(0.262)

(0.713)

(0.025)

4.02

15.57

1.03

(0.163)

(0.023)

(0.814)

0.21

0.94

3.02

(0.415)

(0.375)

(0.210)

0.69

2.01

7.75

(0.525)

(0.378)

(0.097)

1.21

4.46

20.33

(0.538)

(0.419)

(0.043)

0.06

0.94

5.05

(0.635)

(0.356)

(0.009)

0.45

2.68

11.50

(0.482)

(0.370)

(0.148)

1.04

5.20

17.61

(0.485)

(0.360)

(0.155)

0.003

3.66

8.84

(0.972)

(0.018)

(0.010)

5.93

0.64

14.93

(0.433)

(0.238)

(0.329)

1.38

7.31

16.82

(0.201)

(0.500)

(0.299)

50% 99% NAB-3

0% 50% 99%

NAB-4

0% 50% 99%

NAB-5

0% 50% 99%

463 463 400 470 470 400 480 480 400 588 588


1.06 1.53 1.07 1.87 3.39 0.42 2.91 5.11 0.16 7.63 8.96

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Scheme 1. Schematic representation of the self-aggregation of the NDI derivatives in presence of water to exhibit multicolour aggregation induced emission.


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Figure 1. Structures of naphthalene diimide (NDI) derivatives, NAB-1-5.


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Figure 2. (a-e) TEM images; (f-j) FESEM images of NAB-1-5 (20 M), respectively in 1:99 v/v, DMSO-water; (k-o) corresponding DLS studies of NAB-1-5 (20 M), respectively in 1:99 v/v, DMSO-water. 31 ACS Paragon Plus Environment

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Figure 3. UV-visible spectra of (a) NAB-2, (b) NAB-3, (c) NAB-4, (d) NAB-5 in varying compositons of DMSO-water solvent mixture at 20 M.


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Figure 4. (a-j) Photographs of emission colour of NDI derivatives: (a, c, e, g, i) in DMSO and (b, d, f, h, j) in 1:99 v/v, DMSO-water for NAB-1/2/3/4/5, respectively at 50 M. (k-o) Multicolour emission of solid lyophilized powder of NDI derivatives (NAB-1-5) from 1:99 v/v, DMSO-water. In each case, photographs were taken under illumination of a UV lamp excited at 365 nm wavelength.


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Figure 5. Fluorescence microscopic images of (a) NAB-2, (b) NAB-3, (c) NAB-4 and (d) NAB-5 in 1:99 v/v, DMSO-water at 50 M.


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Figure 6. Fluorescence spectra of (a) NAB-2, (c) NAB-3, (e) NAB-4, (g) NAB-5 in DMSOwater solvent mixture at 10 M. Plot of relative emission intensity (I/I0) vs the fraction of water in DMSO of (b) NAB-2, (d) NAB-3, (f) NAB-4, (h) NAB-5. I0 = Emission intensity in pure DMSO solution.


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Figure 7. (a) Photographs of multicolour emission of substituent modified NDI derivatives (NAB-1-5) in 1:99 v/v, DMSO-water irradiated with UV-lamp (ex = 365 nm). (b) Corresponding tunable emission maxima of the NDI derivatives in 1:99 v/v, DMSO-water (ex = 350 nm).


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Figure 8. TCSPC decay profiles of (a) NAB-2, (b) NAB-3, (c) NAB-4 and (d) NAB-5 with a 375 nm excitation in different solvent compositions of DMSO-water.


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Figure 9. Bright field and fluorescence microscopic images of HeLa cells after 6 h incubation with (a, b) NAB-2, (c, d) NAB-3, (e, f) NAB-4 and (g, h) NAB-5 FONPs at concentration of 50 M of each. Scale bars correspond to 20 μm.


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For Table of Contents Use Only

Tunable Aggregation Induced Multicolour Emission of Organic Nanoparticles by Varying Substituent in Naphthalene Diimide Pritam Choudhury, Saheli Sarkar and Prasanta Kumar Das* School of Biological Sciences, Indian Association for the Cultivation of Science Jadavpur, Kolkata – 700032, India.


Tunable Aggregation-Induced Multicolor Emission of Organic

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