Tunable Aggregation-Induced Multicolor Emission of Organic Nanoparticles by Varying the Substituent in Naphthalene Diimide

Article abstract of DOI:10.1021/acs.langmuir.8b02996

In this article, we have designed l-aspartic acid-linked naphthalene diimide (NDI)-based amphiphilic molecules having a benzyl ester group at both the terminals with varying substituents (NAB-1-5). The substituent was judiciously modified from an electron-withdrawing group (EWG) like nitrobenzene to an electron-donating group (EDG), methoxybenzene, and finally to an extended aromatic residue (naphthalene) to regulate the π-electron density at the terminal of NDI derivatives. All of the synthesized NDI derivatives were molecularly dissolved in dimethyl sulfoxide (DMSO), and with an increase in the water content within the DMSO solution, the NDI derivative starts to get self-assembled through J-aggregation at and above 40% water content. Self-assembled spherical organic nanoparticles formed in 99% water in DMSO (f_w = 99%) were characterized by microscopic studies. All of the NDI derivatives showed very weak emission in the 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 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 the self-assembling unit can govern the AIE in combination with the intramolecular charge-transfer process. Consequently, the emission color 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 multicolor emission was investigated by different spectroscopic techniques. These cytocompatible, multicolor-emitting fluorescent organic nanoparticles were utilized for bioimaging applications.

Full text of DOI:10.1021/acs.langmuir.8b02996

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins,
Polymers
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
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Pritam Choudhury, Saheli Sarkar and Prasanta Kumar Das*
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School of Biological Sciences, Indian Association for the Cultivation of Science
Jadavpur, Kolkata – 700032, India.
*To whom correspondence should be addressed: bcpkd@iacs.res.in
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ABSTRACT
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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
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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
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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 (AIE-
gens) 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
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fascinating sub-class of the rylene family, are neutral, planar, chemically robust, electron-
deficient 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 self-
assembled state.28,30 However, multicolour tuning of AIE for core un-substituted NDI
scaffold using a single wavelength excitation is yet to be investigated.
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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
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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.
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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
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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, 1,4,5,8-
naphthalenetetracarboxylic 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 1-
naphthaleneethanol, 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.
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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.
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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.
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Dynamic light scattering (DLS) study. Mean hydrodynamic diameter (Dh) of self-
aggregates 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 UV-
visible 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 NAB-
1-5 (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
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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.
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Photoluminescence study. We have recorded the emission spectra of the solutions of NAB-
1-5 (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) DMSO-
water 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,
퐴푠퐹푢푛2푢
∅푢 =
∅
푠
(1)
2
(
)
퐴푢퐹푠푛푠
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
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(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).
푡
푝(푡) = 푏 + 푛훼 exp ―
(2)
∑
(
)
푖
휏푖
푖
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.
푛
< 휏 > =
훼푖휏푖
∑
푖 = 1
(3)
훼푖
Where
which indicates the contribution of a decay component.
훼푖 =
∑
훼푖
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 2-
3 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
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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%.
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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 18-
24 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
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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 = A (untreated cells) ― background × 100
(4)
570
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.
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RESULTS AND DISCUSSION
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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 un-
substituted 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 L-
aspartic 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 electron-
withdrawing 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 NAB-
5 (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).
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Corresponding correlograms with good Cumulant Fit for the respective NDI derivatives were
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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.
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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.
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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)
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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,
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Supporting Information). Similar changes in the H-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
1
phenyl rings was also monitored by H-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
1
of NAB-1, moderately sharp H-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
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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.
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Investigation of emission behaviour of self-aggregated organic nanoparticles. Presence of
electron donor-acceptor moieties in the molecular framework and formation of the self-
aggregated 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
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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).
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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
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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
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em showed highest intensity (almost 9-fold higher than that was observed in DMSO) (Figure
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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 (±R-
effect) 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
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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
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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 charge-
transfer.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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2∆푓
ℎ푐푎3
∆휈 = 휈푎 ― 휈푒 =
(휇퐸 ― 휇퐺)2 + 푐표푛푠푡푎푛푡
(5)
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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.
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휀 ― 1
푛2 ― 1
2푛2 + 1
∆푓 =
―
(6)
2휀 + 1
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 NAB-
2/3/4/5 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
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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 life-
time 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
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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 self-
aggregated 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.
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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-5-
isothiocyanate (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.
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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 self-
assembled 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
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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.
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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.
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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)
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Compound
fraction of
Average lifetime
monitored
(nm)
1 (ns)
(a1)
2 (ns)
(a2)
3 (ns)
(a3)
water (fw)
<> ns
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NAB-2
0%
50%
99%
0%
400
0.38
1.55
4.23
1.02
(0.435)
2.03
(0.546)
0.43
(0.019)
8.86
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
(0.262)
4.02
(0.713)
15.57
(0.023)
0.94
(0.025)
1.03
(0.163)
0.21
(0.814)
3.02
NAB-3
NAB-4
NAB-5
(0.415)
0.69
(0.375)
2.01
(0.210)
7.75
50%
99%
0%
(0.525)
1.21
(0.378)
4.46
(0.097)
20.33
(0.043)
5.05
(0.538)
0.06
(0.419)
0.94
(0.635)
0.45
(0.356)
2.68
(0.009)
11.50
(0.148)
17.61
(0.155)
8.84
50%
99%
0%
(0.482)
1.04
(0.370)
5.20
(0.485)
0.003
(0.972)
5.93
(0.360)
3.66
(0.018)
0.64
(0.010)
14.93
(0.329)
16.82
(0.299)
50%
99%
(0.433)
1.38
(0.238)
7.31
(0.201)
(0.500)
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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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