Super LCST thermo-responsive nanoparticle assembly for ATP binding through the Hofmeister effect

Article abstract of DOI:10.1039/c4tb01844c

The present investigation reports the development of a super LCST thermo-responsive amphiphilic nanoparticle assembly for the detection of adenosine triphosphate (ATP) through the Hofmeister effect. For this purpose, a new diblock molecule was designed based on hydrophilic polyethylene glycol and the renewable resource 3-pendadecylphenol as the hydrophobic unit. The amphiphile self-assembled as a 150 nm micellar nanoparticle and showed a super lower critical solution temperature (LCST) above 90 °C. The amphiphile followed the Hofmeister effect for the anion series and exhibited high selectivity for the recognition of ATP over its adenosine precursors such as ADP, AMP and inorganic phosphate (Pi). The preferential binding for ATP is attributed to the encapsulation in the hydrophobic pocket and modification of the hydration shell at the periphery of the amphiphilic nanoparticles. Electron and atomic force microscopes and dynamic light scattering techniques confirmed the size and shape of the amphiphilic assembly and its ATP complexes. Isothermal calorimetric experiments were carried out to determine the binding constants for the amphiphilic nanoparticle binding to ATP. The binding of the amphiphilic nanoparticle to ATP was found to be an endothermic process with a binding constant three times higher compared to its precursor Pi. This investigation provides the first insight into the development of a thermo-responsive scaffold for recognition of ATP. This journal is

Full text of DOI:10.1039/c4tb01844c

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Materials Chemistry B
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Super LCST thermo-responsive nanoparticle
assembly for ATP binding through the Hofmeister
Cite this: J. Mater. Chem. B, 2015, 3,
effect†
1957
*
Smita Kashyap‡ and Manickam Jayakannan‡
The present investigation reports the development of a super LCST thermo-responsive amphiphilic
nanoparticle assembly for the detection of adenosine triphosphate (ATP) through the Hofmeister effect.
For this purpose, a new diblock molecule was designed based on hydrophilic polyethylene glycol and
the renewable resource 3-pendadecylphenol as the hydrophobic unit. The amphiphile self-assembled as
a 150 nm micellar nanoparticle and showed a super lower critical solution temperature (LCST) above
90 ꢀC. The amphiphile followed the Hofmeister effect for the anion series and exhibited high selectivity
for the recognition of ATP over its adenosine precursors such as ADP, AMP and inorganic phosphate (Pi).
The preferential binding for ATP is attributed to the encapsulation in the hydrophobic pocket and
modification of the hydration shell at the periphery of the amphiphilic nanoparticles. Electron and atomic
force microscopes and dynamic light scattering techniques confirmed the size and shape of the
amphiphilic assembly and its ATP complexes. Isothermal calorimetric experiments were carried out to
determine the binding constants for the amphiphilic nanoparticle binding to ATP. The binding of the
amphiphilic nanoparticle to ATP was found to be an endothermic process with a binding constant three
times higher compared to its precursor Pi. This investigation provides the first insight into the
development of a thermo-responsive scaffold for recognition of ATP.
Received 8th November 2014
Accepted 12th January 2015
DOI: 10.1039/c4tb01844c
www.rsc.org/MaterialsB
(ELPs)11–13 are some of the most extensively studied thermo-
responsive commercial polymers for the Hofmeister effect.8 Wei
Introduction
et al. recently reviewed the anticancer drug loading and delivering
capabilities of PNIPAM to cancer cells.14 The Hofmeister effect was
also employed to understand enzymatic activity,15 peptide self-
assembly,16 uorescent polyacrylates,17 polyacrylate hydrogels,18,19
guest–host interactions,20 cationic polythiophenes,21 O-acylcho-
lines,22 cationic gemini surfactants23 and PEO–PPO–PEO tri-
blocks.24 These studies emphasized the importance of the
Hofmeister effect in the self-assemblies of organic materials and
polymers. However, no efforts have been made to study the role of
the Hofmeister effect in the binding (or sensing) of biologically
important anionic species such as adenosine phosphates. It is
very important to study the Hofmeister effect of biological phos-
phate anions since they play signicant roles in intra- and extra-
cellular signaling and ion transport.25 Adenosine triphosphate
(ATP) is a multifunctional nucleotide of immense importance in
biology as it serves as the molecular currency for intracellular
energy generation. Extracellular ATP plays vital roles in several
biochemical pathways such as platelet function, neurotransmis-
sion, signal transduction, vasodilatation, and muscle contrac-
tion.26 Recent studies have revealed that under physiological
conditions, the concentration of extracellular ATP was found to be
much higher in cancer tissues compared to normal tissues.27,28
For extracellular ATP administration studies at tumor sites, the
concentrations of ATP typically vary from 1 mM to 5 mM to trigger
The capability of metal salts for ‘salting-in’ and ‘salting-out’
proteins1 or macromolecules2 from aqueous solution was found to
be driven by the anions of the metal salts rather than the cations.3
This effect is known as the “Hofmeister effect” and the Hof-
2ꢁ
meister series for the anions is given as:4 CO32ꢁ > SO42ꢁ > S2O3
>
H2PO4ꢁ > Fꢁ > Clꢁ > Brꢁ z NO3ꢁ > Iꢁ > ClO4ꢁ > SCNꢁ. The anions
at the le end of the series are termed as water “structure makers”
(or kosmotropes) and those on the right end of the series are
termed as water “structure breakers” (or chaotropes).5 Theories
and experimental methods have been developed to address the
mechanism of the Hofmeister effect.6,7 In macromolecular
systems, the inuence of anions on thermo-responsive polymers
has been found to be of immense importance regarding
the fundamental understanding of polymer chain folding and
crystallization in the aqueous environment.8 Poly(N-iso-
propylacrylamide) (PNIPAM)9,10 and elastin-like polypeptides
Department of Chemistry, Indian Institute of Science Education and Research (IISER),
Dr Homi Bhabha Road, Pune 411008, Maharashtra, India. E-mail: jayakannan@
iiserpune.ac.in; Fax: +91-20-2590-8187
† Electronic supplementary information (ESI) available: Additional
%
transmittance plot for AMP, ADP and Pi binding towards the scaffold, DLS data
and NMR and MALDI-TOF data. See DOI: 10.1039/c4tb01844c
‡ All authors contributed equally.
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apoptosis or programmed cell death.29,30 The enhancement of the ATP binding shows interference with its precursors such as
extracellular ATP concentration in cancer tissues is generally ADP, AMP and Pi (inorganic phosphate); thus, the new scaffold
promoted by the release of ATP from the cytoplasm mediated via should have preferential selectivity for the detection of ATP
several plasma membrane receptors such as ABC transporters, among its other anionic precursors. Thus, the new material
P2X7 receptors, and connexins.31 Several optical chromophores design requires the following criteria: (i) super LCST charac-
have been developed to recognize ATP through colorimetric teristics for recognizing ATP through the Hofmeister effect; (ii)
changes32,33 or uorescence techniques34–37 and this topic was it should show selective binding towards ATP over its other
recently reviewed by Zhou et al.38 However, the sensing or binding precursors in aqueous medium; and (iii) it should have the
of ATP through the Hofmeister effect by thermo-responsive ability to self-organize into nano-size objects so that it can
amphiphiles has not been attempted to date .
penetrate through cancer tissues via the EPR effect. This
The uncontrolled cell division and fast growth make the concept is schematically shown in Fig. 1.
ꢀ
environment in cancer tissues (42–44 C; pH ¼ 6.5 to 5.5) very
The present investigation reports one of the rst examples of
different to normal tissues (37 ꢀC and pH ¼ 7.4).39 Thermo- super LCST thermo-responsive amphiphilic nano-scaffolds as a
chemotherapy is a recently developed drug administration tool to recognize and bind ATP. A new di-block amphiphile was
concept to enhance the cytotoxicity of anticancer drugs at least designed with a hydrogen bonded amide linkage that connects
three times by applying temperatures between 40.5 ꢀC and 43.5 both sides, with the hydrophobic unit based on the renewable
ꢀC.40 Thus, developing thermo-responsive drug carriers that resource 3-pentadecyl phenol (PDP) and polyethylene glycol
could disassemble either at abnormal tissue temperatures (40– chains as the hydrophilic part. PDP was chosen in the present
44 ꢀC) or due to external heat stimuli of in thermochemotherapy investigation since it was found to be an efficient hydrophobic
would enhance the efficacy of the cancer treatment.41 Addi- unit for producing polysaccharide vesicles,44–46 multi-vesicular
tionally, the porous network and defective lymphatic drainage bodies47 and shape transformable thermo-responsive core–shell
in cancer tissues allow the accumulation of larger size macro- nanoparticles48 in our earlier work. Thus, the selection of the
molecular or self-assembled polymer particles (150–250 nm) hydrophobic PDP unit with the appropriate PEG chain length
through the enhanced permeability and retention (EPR) has facilitated the designing of a thermo-responsive amphiphile
effect.42,43 Existing thermo-responsive materials such as PNI- that showed the highest LCST > 90 ꢀC (named “super LCST
PAM would support the EPR effect; however, they cannot be amphiphile”). This thermo-responsive scaffold followed the
used for ATP binding since their LCST is lower than 37 ꢀC.14 typical Hofmeister series and exhibited preferential binding
Hence, the above multi-task problem could be addressed only towards ATP at 42–44 ꢀC. Dynamic light scattering, electron and
by the design of a new thermo-responsive scaffold having atomic force microscopes provided direct evidence for the
“super LCST characteristics” to withstand the Hofmeister effect mechanism of nanoparticle driven ATP detection. Further,
ꢀ
of anions at cancer tissue temperatures (42–44 C). In general, isothermal calorimetric experiments were also carried out to
Fig. 1 Thermo-responsive amphiphilic nanoparticle entry in normal tissue (a) and cancer tissue (b) environments. The expanded part in b
demonstrates the ATP pathways for hydrolysis and binding to the nanoparticle. (c) A di-block molecular design for the super LCST amphiphile 1
and the self-assembly of the amphiphilic nanoparticle towards ATP.
1958 | J. Mater. Chem. B, 2015, 3, 1957–1967
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determine the association constants for the selective binding of
the thermo-responsive nanoparticle to ATP.
Isothermal titration calorimetry (ITC)
The titrations of ATP and the inorganic phosphate anion with
the scaffold (PDP-PEG-750) were performed at 25 C using an
ꢀ
iTC200 microcalorimeter (MicroCal Inc.). In these experiments,
ATP and the inorganic phosphate anion were titrated into a
scaffold solution. Both the solution in the injection syringe and
the one in the ITC cell were prepared in water. Two blank
titrations were also performed: (i) titration of water with the
same ATP and Pi solution; and (ii) titration of the amphiphile
with water. The stoichiometries of substrate binding (n) and the
binding constants (Ka) of amphiphile 1 with ATP and Pi were
obtained aer subtracting the blank titrations. The stirring
speed used was 1000 rpm and the reference power was set at 0.5
mcal sꢁ1 for all the titrations. The volume of the ITC200 cell was
0.200 mL while that of syringe was 0.040 mL.
Experimental methods
Materials
3-Pentadecylphenol, 2-ethanolamine, succinicanhydride, Boc-
anhydride triethylamine (Et3N), polyethylene glycol mono-
methylether (MW ¼ 750), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC), diisopropyl ethylamine (DIPEA), and dii-
sopropyl azodicarboxylate (DIAD) were purchased from Aldrich
chemicals. All other reagents and solvents were purchased
locally and puried following standard procedures. The tert-
butyl (2-hydroxyethyl) carbamate was synthesized using our
earlier report.44
Cell viability assay (MTT assay)
General procedures
To test the cytotoxicity of the amphiphile 1, a cell viability assay
was performed in HeLa cell lines using the tetrazolium salt 3-
(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT).
HeLa cell lines (1 ꢂ 103) were seeded in each well in a 96 well
plate (Corning, USA) in 100 mL of DMEM with 10% FBS (fetal
bovine serum) and allowed to adhere for 12 h and 24 h. Prior to
drug treatment, the medium from the cells was aspirated and
various concentration of amphiphile 1 were prepared. These
were added to 100 mL of DMEM with FBS in which the cells were
incubated. Blank controls, DMEM with FBS in the absence of
cells, were used in each experiment. All control and treated
experiment wells were done in triplicate. Cells were incubated
for 12 h and 24 h without a change in medium, and aer 12 h
and 24 h, the amphiphile 1 medium was aspirated. Freshly
prepared stock of MTT in sterile PBS (5 mg mLꢁ1) was diluted to
50 mg mLꢁ1 in 100 mL of DMEM with FBS and was added to the
cells. The cells were then incubated with MTT for 4 h at 37 ꢀC.
The medium with MTT was then aspirated from the wells and
the purple formazan crystals formed as a result of the reduction
of MTT by mitochondrial dehydrogenase enzyme from the cells
dissolved in 100 mL of 100% N,N-dimethylsulfoxide (DMSO)
(added per well). The absorbance from the formazan crystals
was immediately measured using a microplate reader at 570 nm
(Varioskan Flash) and is representative of the number of viable
cells per well. Values from the triplicates for each control and
treated set were noted and their means used for calculations.
The mean of the absorbance values for the blank control
samples was subtracted from the average of the treated
samples. The values thus obtained for the control samples were
equated to 100% and relative percentage values for amphiphile
1 were calculated accordingly.
1H-NMR and 13C-NMR spectra were recorded using a 400 MHz
Jeol NMR spectrophotometer in CDCl3 containing TMS as the
internal standard. Infra-red spectra were recorded using a
Thermo-Scientic Nicolet 6700FT-IR spectrometer with the
solid state in KBr. The masses of the amphiphiles and all the
intermediates were conrmed using the Applied Biosystems
4800 PLUS MALDI TOF/TOF analyzer. The purity of the
amphiphile was determined by gel permeation chromatography
(GPC) using a Viscotek VE 1122 pump, Viscotek VE 3580 RI
detector, and Viscotek VE 3210 UV/Vis detector in tetrahydro-
furan (THF) using polystyrene as the standard. The optical
transmittance measurement was done using a PerkinElmer
Lambda 45 UV-Visible spectrophotometer. The size determi-
nation of the aqueous solution of the amphiphile was carried
out by dynamic light scattering (DLS) using a Nano ZS-90
apparatus utilizing a 633 nm red laser (at 90ꢀ angle) from
Malvern instruments. The reproducibility of the data was
checked for at least three independent solutions. FE-SEM
images were recorded using a Zeiss Ultra Plus scanning electron
microscope. For FE-SEM analysis, the samples were prepared by
drop casting on silicon wafers and then coating with gold. TEM
images were recorded using a Technai-300 instrument by drop
casting the sample on a Formvar-coated copper grid. The ITC
ꢀ
measurement was carried at 25 C using an iTC200 microcalo-
rimeter (MicroCal Inc.). Atomic force microscope images were
recorded for drop cast samples using JPK instruments attached
with Nano wizard-II setup.
Optical transmittance measurement
The optical transmittance of PDP-PEG750 was measured at 500
nm using a quartz cell (path length: 1 cm) with a PerkinElmer
Lambda 45 UV-Visible spectrophotometer equipped with a
Synthesis of PEG750-COOH (PEG-Acid)
temperature-controller. The heating cycle was recorded by Poly(ethylene glycol) MW 750 monomethylether (10.0 g, 13.0
heating the sample continuously from 30 ꢀC to 90 ꢀC. Similarly, mmol) and succinic anhydride (1.6 g, 16.0 mmol) were dis-
the cooling cycle was recorded from 90 ꢀC to 30 ꢀC. The heating solved in dry dichloromethane (60 mL) and purged under N2 for
and cooling cycles were maintained at a rate of 10ꢀ minꢁ1 using 10 min. To this reaction mixture, Et3N (1.85 mL, 13.0 mmol) was
a Peltier heating/cooling source.
added drop wise. Immediately aer the addition of Et3N the
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reaction mixture started to boil vigorously. The reaction pro- mL, 7.8 mmol) were added under N2 atmosphere and the
ceeded at 25 ꢀC for 48 h with continuous stirring under N2 reaction was then le at 25 ꢀC for 48 h with continuous stirring.
atmosphere. The reaction mixture was poured into water (100 It was then poured into water (30.0 mL) and the product was
mL) and was neutralized with 2 N concentrated HCl (2.0 mL). extracted into chloroform. The organic layer was washed with
The organic layer was washed with brine solution, dried over 10% NaHCO3 (30.0 mL), neutralized with 2 N HCl (6.0 mL),
anhydrous sodium sulfate and concentrated to obtain a yellow dried over anhydrous sodium sulfate and concentrated to
liquid as the product. It was puried by passing through a silica obtain a yellow liquid as the product. It was further puried by
gel column of 60–120 mesh using 50% methanol in chloroform passing through a silica gel column of 60–120 mesh using 5%
as the eluent. Yield ¼ 6.0 g (66%). 1H NMR (CDCl3, 400 MHz) d: methanol in chloroform as the eluent. Yield ¼ 2.27 g (54.0%).
4.14 ppm (t, 2H, CO–OCH2), 3.56–3.50 ppm (s, 66H, OCH2- 1H NMR (CDCl3, 400 MHz) d: 7.18 ppm (t, 1H, Ar–H), 6.80–6.70
CH2O), 3.23 ppm (s, 3H, CH2–OCH3), 2.52 ppm (s, 4H, CO–CH2– ppm (m, 3H, Ar–H), 6.24 (CO–NH), 4.24 ppm (t, 2H, COO–CH2),
CH2). 13C NMR (CDCl3, 100 MHz) d: 177.05 (COOH), 171.88 4.02 ppm (t, 2H, Ar–OCH2), 3.69–3.66 ppm (m, 75H, O–CH2–
(COO–CH2), 70.56 (OCH2–CH2–O), 69.93 (COO–CH2), 58.51 (C– CH2), 3.56 ppm (t, 2H, CH2–N), 3.38 ppm (s, 3H, CH2–OCH3),
OCH3), 28.45 (CO–CH2–CH2). FT-IR (cmꢁ1): 3448, 2875, 1727, 2.70 ppm (t, 2H, Ar–CH2), 2.57 ppm (t, 2H, NH–CO–CH2), 2.51
1649, 1453, 1349, 1286, 1247, 1202, 1087, 947, 842, 677, 622. ppm (t, 2H, CH2–COO), 1.6–0.88 ppm (m, 29H, aliphatic H). 13
C
MALDI-TOF-MS: m/z calculated for C39H76O21: 880 and found: NMR (CDCl3, 100 MHz) d: 172.87 (NH–CO), 171.60 (CO–O),
903 (M+ + Na+).
158.54, 144.86, 129.32, 121.36, 114.69, 111.47 (Ar–C), 72.00
(CH2–OCH3), 70.64 (O–CH2–CH2), 69.07, 66.65 (Ar–OCH2), 63.83
(COO–CH2), 59.11 (O–CH3), 39.16 (CH2–N), 36.09, 31.99, 31.49,
29.75, 29.43, 22.76, 14.19. FT-IR (cmꢁ1): 3374, 2916, 2878, 1735,
1651, 1599, 1551, 1460, 1347, 1278, 1244, 1103, 955, 846, 784,
751, 697. MALDI-TOF-MS: m/z calculated for C62H115NO21: 1121
and found: 1144 (M+ + Na+).
Synthesis of tert-butyl-2-(3-pentadecylphenoxy)ethyl
carbamate (PDP-NH-Boc)
tert-Butyl(2-hydroxyethyl) carbamate (3.7 g, 23.0 mmol), 3-pen-
tadecylphenol (7.0 g, 23.0 mmol) and triphenylphosphine (6.6 g,
25.0 mmol) were dissolved in dry tetrahydrofuran (20 mL). The
contents were then placed in an ice-cooled bath for 10 min with
continuous N2 purge. To this ice-cooled reaction mixture, dii-
sopropyl azodicarboxylate (4.47 mL, 23.0 mmol) was added drop
wise under N2 atmosphere. The reaction mixture was further
Results and discussion
Synthesis and thermo-responsiveness of amphiphile 1
An amphiphilic molecule was synthesized from polyethylene
glycol monomethyl ether MW ¼ 750 g molꢁ1 as the hydrophilic
part and the renewable resource pentadecyl phenol (PDP) as the
hydrophobic unit, as shown in Scheme 1. Succinic anhydride
was ring opened with PEG in the presence of Et3N, which acts as
a base to give PEG-Acid. tert-Butyl (2-hydroxyethyl) carbamate
was coupled with PDP in the presence of diisopropyl azodi-
carboxylate and triphenylphosphine to give tert-butyl (2-(3-
pentadecy-8-en-1-yl) phenoxy) ethyl carbamate (PDP-NH-Boc).
The carbamate was then treated with triuoroacetic acid (TFA)
to give 2-(3-pentadecy-8-en-1-yl) phenoxy ethanamine, which
was further coupled with PEG-Acid in the presence of EDC and
diisopropylethyl amine (DIPEA) to give amphiphile 1.
ꢀ
stirred at 25 C for 24 h. It was puried by passing through a
silica gel column of 60–120 mesh using 1% ethyl acetate in
hexane as the eluent. Yield ¼ 8.0 g (66%). 1H NMR (CDCl3, 400
MHz) d: 7.19 ppm (t, 1H, Ar–H), 6.80–6.70 ppm (m, 3H, Ar–H),
5.02 ppm (s, 1H, NH), 4.02 ppm (t, 2H, Ar–OCH2), 3.54 ppm (t,
2H, CH2–N), 2.58 ppm (t, 2H, Ar–CH2), 1.46 ppm (s, 9H, OC–
C(CH3))3, 1.6–0.88 ppm (m, 29H, aliphatic H). 13C NMR (CDCl3,
100 MHz) d: 158.62 (Ar–C), 155.98 (CO–O), 144.85, 129.29,
121.32, 114.75, 111.40 (Ar–C), 79.57 (OC(CH3))3, 67.08 (Ar–
OCH2), 40.26 (CH2–N), 36.09, 32.00, 29.76, 26.47, 22.77, 14.20.
FT-IR (cmꢁ1): 3396, 2916, 2850, 1690, 1590, 1512, 1453, 1362,
1250, 1157, 1060, 95, 866, 778, 690. MALDI-TOF-MS: m/z
calculated for C28H49NO3: 447 and found: 470 (M+ + Na+).
1
The structure of amphiphile 1 was characterized by H and
13C-NMR (see ESI SS-1†). The MALDI-TOF spectrum of amphi-
phile 1 showed the distribution of mass peaks in the range of
the range of m/z 900 to 1500 (see Fig. 2(a)). The mass differences
Synthesis of 2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-oxo-4-((2-(3-
pentadecylphenoxy)ethyl)amino)butanoate (amphiphile 1)
PDP-NH-Boc (2.0 g, 44.0 mmol) was dissolved in dichloro- between the adjacent peaks were obtained as 44 amu with
methane (5.0 mL) and to this mixture triuoroacetic acid (10.0 respect to the distribution in the PEG chains in the diblock
mL, 129.7 mmol) was added drop wise. The contents were amphiphile 1. The mass of each peak was calculated using the
ꢀ
stirred at 25 C for 1 h and then the solvent was evaporated by formula: (44.05)n + 461 + 23, where 44.05 is the repeating unit
Rotavapor. The TFA was removed by adding fresh DCM (10.0 mL mass of PEG units and 461 and 23 correspond to the mass of the
ꢂ 3 times) and was evaporated by Rotavapor. The content was hydrophobic unit and the sodium ion, respectively. The mass of
further poured into ice-cooled diethyl ether (15.0 mL) and the peak corresponding to n ¼ 15 is shown by an arrow. The
evaporated by Rotavapor to obtain a white solid as the product. thermo-responsive behavior of amphiphile 1 was studied by
The white solid product (1.34 g, 3.8 mmol) was dissolved in dry optical transmittance measurement as a function of tempera-
dichloromethane (20.0 mL) and was purged with N2 for 15 ture using an absorbance spectrophotometer. The plots of
minutes. To it, PEG-Acid (3.0 g, 3.5 mmol) was added and N2 optical transmittance in the heating and cooling cycles are
purging was continued for the next 15 minutes. To this reaction shown in Fig. 2(b). The plots reveal that the transmittance of the
mixture, EDC (0.81 g, 4.2 mmol) and diisopropylethylamine (1.4 aqueous solution of amphiphile 1 decreases from 97% to 10%
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Scheme 1 Synthesis of the thermo-responsive amphiphile 1.
ꢀ
at 92 C (LCST). From the plots it is also evident that both the the formation of larger aggregates above the LCST. The revers-
heating and cooling cycle follow the same kinetic path, indi- ibility of the thermo-responsive self-assembly process was
cating that changes in the aggregates with temperature occur at further inferred from the cooling cycle (see Fig. 2(d)). It is
similar rates. In other words, the assembly and disassembly of important to mention that the DLS histograms are broad at low
the aggregates do not exhibit hysteresis.
temperature; however, they become very narrow while
The temperature-driven self-assembly was further studied by approaching the LCST temperature (see Fig. 2(c)). On the other
subjecting the aqueous solution of amphiphile 1 (10ꢁ4 M) to hand, in the subsequent cooling cycle (see Fig. 2(d)), the DLS
variable temperature dynamic light scattering (DLS). The DLS histograms also retained the narrow distribution at room
histograms recorded for amphiphile 1 in the heating and temperature. The reversibility of the amphiphilic self-assembly
cooling cycles are shown in Fig. 2(c) and (d), respectively. was further conrmed by repetitive scanning in the continuous
Amphiphile 1 showed a mono-modal distribution indicating heating and cooling cycles (see Fig. SF-1†). The size of the
the formation of uniform size aggregates in water. Further, it aggregates varied from 140 ꢃ 20 nm to 600 ꢃ 35 nm with perfect
showed a sharp increase in the hydrodynamic diameter from reproducibility. This implies that the temperature induced self-
140 ꢃ 30 nm (below the LCST) to 600 ꢃ 30 nm with respect to assembly process in amphiphile 1 is completely reversible;
Fig. 2 (a) MALDI-TOF spectrum of amphiphile 1 and the repeating unit mass for the peak at n ¼ 15 is shown by arrow. (b) Temperature
dependent optical transmittance of amphiphile 1 in water in consecutive heating and cooling cycles. DLS histogram of amphiphile 1 in the
heating (c) and cooling (d) cycles. The concentration of amphiphile 1 is 10ꢁ4 M.
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however, the dis-assembly occurred more uniformly as
compared to the self-assembling process in the cooling and
heating cycles, respectively.
Hofmeister effect of amphiphile 1
Three anion sources were chosen to investigate the Hofmeister
effect on the LCST of amphiphile 1: NaSCN, a strong chaotrope
(water “structure breaker”), NaCl and Na2SO4, a strong kosmo-
trope (water “structure maker”). The plot of transmittance (%)
as a function of temperature at various concentrations of
Na2SO4 is shown in Fig. 4(a) (for NaCl and NaSCN, see SF-3 and
SF-4†). The transitions appeared in two phases and this
behavior is attributed to: (i) the initial fast binding of the anions
to the nanoparticles; (ii) followed by the diffusion controlled
slow binding process due to the precipitation of the amphi-
phile. At the initial stage, the homogeneous environment
facilitates the fast binding whereas the heterogeneous precipi-
tation at the secondary stage became a diffusion process. The
onset of the cloud point in the heating cycle is termed as the
turbidity ‘turn-on temperature’ while the temperature at which
turbidity gets cleared completely is termed as the ‘turn-off
temperature’. The ‘turn-on temperatures’ for Na2SO4, NaCl and
To visualize the size and shape of the nano-aggregates,
amphiphile 1 was subjected to eld emission-scanning electron
microscopy (FE-SEM), high resolution transmission electron
microscopy (HR-TEM) and atomic force microscopy (AFM)
analysis. In Fig. 3(a), the FE-SEM image of amphiphile 1 showed
the formation of spherical shape particles of 170 ꢃ 22 nm
diameter. The HR-TEM image of these particles conrmed that
these spherical aggregates (150 ꢃ 20 nm in size) are hard
spheres with respect to micellar nanoparticles (see Fig. 3(b)).
The AFM image of amphiphile 1 also conrmed the existence of
spherical nanoparticles of 130 ꢃ 25 nm (see Fig. 3(c)). The HR-
TEM image of the nano-aggregates above the LCST is shown in
Fig. 3(d). Above the LCST, the nanoparticles of size 140 ꢃ 20 nm
were found to form clusters together as larger aggregates. The
sizes of the nano-particulate assemblies below and above the
LCST from the microscope images matched very well with their
solution aggregates in DLS (see Fig. 2(c) and (d)). The critical
micellar concentration (CMC) of the amphiphile was deter-
mined using Nile red as a uorescent probe. Nile red is a
hydrophobic dye that is insoluble in water and displays no
uorescence in aqueous medium. Once it has been seques-
trated in the hydrophobic core, the uorescence is generated
and can be used as an indicator of the formation of the micellar
aggregates (see SF-2†). The concentration of Nile red was kept
constant (0.2 mM) while the concentration of amphiphile 1 was
varied from 1.0 mM from 1.0 mM (see SF-2†). The emission
intensity of the Nile red was plotted against the concentration
(see SF-2†) . The plot showed break point at 1.0 ꢂ 10ꢁ5 M with
respect to its CMC. The Nile red encapsulation study also
suggests that the thermo-responsive micellar assemblies could
be useful for loading diverse hydrophobic molecules in their
inner core, including anticancer drug molecules.
2ꢁ
NaSCN (see Fig. 4(b)) revealed that the SO4 anion showed
higher salting out compared to Clꢁ ions while the SCNꢁ ions
did not induce any change in the LCST of the amphiphile. At
very high concentrations (above 500 mM), a large amount of
precipitation occurred and the error became very high. This is
one of the main reasons that the Hofmeister effect could not be
studied for more than 600 mM salt concentration. The lowering
of the LCST from 90 ꢀC to 55 ꢀC in the presence of the sulfate ion
was attributed to the binding of anions towards the amide and
PEG-chains, which altered the hydration shell in the micellar
assemblies. The reduction in the hydration induces the
precipitation of the PEG chains on the periphery of the nano-
particulate assemblies which results in the turbidity. During
precipitation, many nanoparticles come together as a result of
nucleation followed by growth. This process is evident from the
DLS and HR-TEM images, as shown in Fig. 3. This experiment
conrmed that the newly designed thermo-responsive amphi-
phile 1 showed binding towards anions and follows the Hof-
meister series.
To further validate the Hofmeister effect of the present
molecular design, another thermo-responsive diblock with tri-
ethylene glycol (TEG) as the hydrophilic unit (in the place of
PEG chains) was chosen and subjected to the anion studies (see
the structure PDP-TEG in SF-5†). This short amphiphile has a
ꢀ
LCST of 42 C, which is much lower compared to the LCST of
ꢀ
amphiphile 1 (90 C) (see SF-5†). This indicates that the super
LCST nature of the amphiphile arose from the long PEGylated
chains in the diblocks design strategy (see Scheme 1). Surpris-
ingly, the short amphiphile PDP-TEG lost its LCST behaviors
upon binding to the anions. This clearly supports the structure
of the thermo-responsive material being very crucial for
studying the Hofmeister effect towards anions. In the present
case, the super LCST amphiphile 1 provided an appropriate
hydrophilic and hydrophobic balance for the above purpose.
Recently, Cremer and co-workers reported that the LCST of the
PNIPAM and elastic-like peptide decreased upon complexation
Fig. 3 (a) FE-SEM image, (b) HR-TEM image and (c) AFM image of
amphiphile 1 below the LCST (25 ꢀC). (d) HR-TEM of amphiphile 1
above the LCST at 90 ꢀC. The concentration of amphiphile 1 is 10ꢁ4 M
for the imaging.
2ꢁ
with the anions by 10–11 ꢀC in the presence of the SO4 anion.9
In the present case, the LCST of amphiphile 1 decreased by
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Fig. 4 (a) Temperature dependent transmittance of amphiphile 1 with various concentrations of Na2SO4. (b) Turn-on temperature at various
concentrations of NaSCN, NaCl and Na2SO4.
2ꢁ
35 ꢀC upon binding to the SO4
anion. It suggests that not change much and retained the size in the micrometer
amphiphile 1 experienced the Hofmeister effect as equivalent to range. This indicates that the complex formation between ATP
that of the high molecular weight thermo-responsive PNIPAM. and the nanoparticle produced larger size aggregates in the
Thus, amphiphile 1 has an appropriate molecular design with heating cycle; however, they did not di-assemble into the indi-
super LCST characteristics for binding or sensing anions in a vidual nanoparticle and ATP in the subsequent cooling. This
ꢀ
large temperature window from 90 C to 30 ꢀC.
implies that once the nanoparticles bind to ATP, they would not
disassociate or release the ATP molecule immediately (see
Fig. 1(b)). It is also important to note that the scaffold alone did
not show any larger change in size in the heating/cooling cycles
(see Fig. 2(c) and (d)); thus, the non-occurrence of disassembly
observed in Fig. 5(d)–(f) corresponds to the formation of a stable
ATP + scaffold.
ATP binding
Biological anions such as adenosine triphosphate (ATP) and its
precursors ADP (adenosine diphosphate), AMP (adenosine
monophosphate) and inorganic phosphate (Pi) were chosen to
test the binding abilities of the thermo-responsive nano-scaf-
fold. Under physiological conditions, ATP ecto-nucleotidase
hydrolyzes ATP into ADP and Pi with the release of 30.5 kJ molꢁ1
of energy, which is further utilized for several biological
processes (see Fig. 5(a)). The amphiphile 1 was subjected to
optical transmittance measurement with variable concentra-
tions of nucleotides and their % transmittance in the heating
and cooling cycles are shown in Fig. 5(b) and (c), respectively
(for Pi, ADP, and AMP SF-6, SF-7 and SF-8†). From the heating
cycle (see Fig. 5(b)), it is evident that with an increase in
concentration of ATP, the LCST of the nano-particulate
assembly signicantly reduced. This is attributed to the
tendency of ATP to alter the hydration shell at the periphery of
the nanoparticle, which results in the precipitation of PEG
Further, the aggregation phenomenon of the nanoparticle in
the presence of ATP was validated by subjecting the scaffold +
ATP complex to HR-TEM analysis. The HR-TEM images of the
nano-aggregates showed the scaffold + ATP complex as indi-
vidual nanoparticles below the LCST (see Fig. 5(g)). Above the
LCST, the nanoparticles were assembled together to produce
micrometer sized aggregates (see Fig. 5(h)). This morphological
assembly was further evident from the observation in the %
transmittance and DLS (see Fig. 5(d) and (e)). The photographs
of vials containing the scaffold with ATP, ADP, AMP and Pi at
ꢀ
45 C are shown in Fig. 5(i). Only the ATP + scaffold complex
showed turbidity at 45 ꢀC indicating that the nanoparticle
assembly has preferential binding towards ATP compared to
other nucleotides. This is further supported by variable
temperature DLS analysis of the scaffold in the presence of Pi
(see SF-10†). The size of the aggregates increases from 250 nm
to 750 nm upon heating (see SF-11†) while in the cooling cycle
(see SF-11†) it completely reverts to its original size. This
suggests that the complex formed between the scaffold and Pi is
not stable, unlike its ATP complex. ADP and AMP did not show
any remarkable changes in the LCST of the nanoparticle (see SF-
7 and SF-8†). These control experiments conrmed that the
thermoresponsive nanoparticle has preferential binding
towards ATP.
2ꢁ
chains (as similar to SO4 ions). In the subsequent cooling
cycle (see Fig. 5(c)), the amphiphile + ATP complex showed a
slow disassembly process and the complex was stable up to 42–
44 C at the higher concentration of ATP. The non-reversibility
ꢀ
of the ATP aggregates sizes in the cooling cycles was checked in
repeat experiments (SF-9†). The reason for this stable assembly
is not clear at present; however, it implies that the large accu-
mulation of ATP in cancer tissues could preserve the amphi-
phile 1 + ATP complex in a stable form. The ATP binding studies
were further subjected to variable temperature dynamic light
scattering (DLS) measurements for xed concentrations of ATP
(300 mM) and scaffold (see Fig. 5(d) and (e)). The hydrodynamic
diameter from the heating and cooling cycle was plotted against
temperature and is shown in Fig. 5(f). From the heating cycle, it
is evident that the size of the aggregates increased gradually
from 300 nm to 1.7 mm with the increase in temperature. On the
other hand, in the cooling cycle the size of the aggregates did
In order to shed more light on the selectivity of amphiphile 1
towards ATP, the differences in the turn-on and turn-off
turbidity temperatures of the scaffold in the presence of ATP,
ADP, AMP and Pi were plotted and are shown in Fig. 6(a) and
(b). The plot of turn-on temperature as a function of concen-
tration reveals that the affinity of amphiphile 1 was signicantly
more towards ATP as compared to ADP, AMP and Pi. The ‘turn-
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Fig. 5 (a) Mechanism of ATP hydrolysis into ADP and inorganic phosphate (Pi). Temperature dependent optical transmittance of amphiphile 1 in
water at various concentrations of ATP in the heating (b) and (c) cooling cycles. Temperature dependent DLS histogram of amphiphile 1 in water
in presence of adenosine triphosphate (ATP) (300 mM) in the heating cycle (d) and cooling cycle (e). (f) Plot of hydrodynamic diameter against
temperature in the heating and cooling cycles. HR-TEM image of the ATP–amphiphile 1 complex in water below the LCST (g) aꢁn4d above the
LCST (h). (i) Photograph of amphiphile 1 with ATP AMP, ADP and Pi in water at 45 ꢀC. The concentration of amphiphile 1 is 10 M and the
concentrations of the analytes are 300 mM.
Fig. 6 (a) Plot of turn-on temperature of amphiphile 1 as a function of concentration of ATP, ADP, AMP and Pi. (b) Plot of turn-off temperature of
amphiphile 1 as a function of concentration of ATP and Pi. (c) Temperature-sensing scale of amphiphile 1 for the ATP, ADP, AMP and Pi.
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off temperature’ ploꢀt revealed that ATP could bring down the MTT assay. The cells were treated for 12 h and 24 h and the data
scaffold LCST to 45 C. On the other hand, in the case of Pi it are shown in Fig. 7. The cells showed more than 85% cell
only reaches 70 C. This suggests that the nanoparticle is effi- viability below 1 mg mLꢁ1. At higher amphiphile concentrations
ꢀ
cient to bind ATP over its precursors. The reason for the efficient (5 mg mLꢁ1 and 10 mg mLꢁ1), the cells showed 75% viability. For
binding of ATP towards the amphiphile was attributed to the most drug delivery research, the polymer or scaffold concen-
occupation of ATP in the micellar scaffold. ATP has three trations used are typically <1.0 mg mLꢁ1; thus, the new renew-
phosphate anionic groups that are hydrophilic and well sepa- able resource based LCST amphiphile may be a very useful
rated from the hydrophobic adenosine + sugar units (see biomaterial.
Fig. 5(a)). Recently, Wang et al.49 and Liu et al.50 independently
reported that the ATP anion binding to the polymer nano-
particle or liposomes were found to be driven by host and guest
Isothermal titration calorimetry (ITC) directly measures the
interactions through the hydrophobic inner core of the nano-
assemblies. Further, it was found that ADP and AMP were found
to lack perfect guest geometry to occupy the hydrophobic host
Isothermal calorimetry and binding constants
heat of the complexation (enthalpy, DH), the stoichiometry of
substrate binding (n), and the binding constant (Ka). The
binding efficiency of ATP and its close interfering anion Pi
(based on Fig. 6(b)) with the scaffold were further evaluated by
ITC and the data are provided in Fig. 8. The titration of ATP with
the nanoparticle displayed endothermic curves (see Fig. 8(a))
while exothermic curves were obtained for titration of the
phosphate anion (see Fig. 8(b)). Further, the binding constant
for the ATP and phosphate anion complexes with the nano-
particle were found to be 2230 Mꢁ1 and 719 Mꢁ1, respectively.
The three times higher binding constant for the ATP towards
the scaffold indicates that the ATP binding was stronger than
the inorganic phosphate anion.
Earlier ITC reports for ATP binding to poly(dC) (double
stranded DNA made up of cytosine)51 and 3-phophoglycerate
kinase52 showed binding constants of 2.45 ꢂ 104 Mꢁ1 and 3.5 ꢂ
10ꢁ3 Mꢁ1, respectively. The binding constant of the ATP-nano-
particle complex in the present case has a value similar to the
above examples. Hence, it can be said that the affinity of the
thermo-responsive nanoparticles towards ATP was very good as
similar to that of DNA and proteins. Further, the ATP binding to
the amphiphilic scaffold is endothermic; thus it was a natural
choice for thermodynamically binding in the high temperature
environment of the cancer tissue compared to normal tissues.
Additionally, the amphiphilic nanoparticle has encapsulation
capabilities for hydrophobic dyes and this concept may be
expanded to load and deliver water insoluble anticancer drugs.
This may provide the additional advantages of simultaneous
binding of ATP and releasing the drugs at the cancer tissue
temperature. Based on the above studies, it may be summarized
pocket.48 Thus, in the present case, during the complexation
with the amphiphile, the hydrophobic part of ATP could pene-
trate into the hydrophobic core of the micellar nanoparticle
through like–like interactions. These geometrical arrangements
provide better contact between the ATP hydrophilic phosphate
groups towards the amide and PEG chains in the micellar
exterior shell (see Fig. 6(c)). This arrangement gives the ATP
aromatic core in the hydrophobic pocket of the nanoparticle
and the anion projecting towards the amide and PEG chains to
maximize the Hofmeister effect. As a result, the ATP anion
induced maximum inuence on the hydration of the amphi-
philic scaffold through the Hofmeister effect that reduces the
LCST of the ATP + scaffold complex. However, in the case of
inorganic Pi, the inorganic anion could easily diffuse as
compared to AMP and ADP in the hydroshell of the PEGylated
chains at the nanoparticle exterior to signicantly induce the
Hofmeister effect. Further, to estimate the dynamic range of the
ATP binding, the concentration of amphiphile was xed at 10ꢁ4
M and the ATP concentration was varied from 10 mM to 100
mM (see SF-12†). The plots showed that the change in the LCST
phenomena could be detected in transmittance mode at
minimum [ATP] ¼ 100 mM.
This mechanism is further supported by the large increase in
the size of amphiphilic scaffold in the ATP + scaffold complex as
compared to that of nascent scaffold. For example, the sizes of
the scaffold doubled from 140 nm to 300 nm upon binding to
ATP at 25 ꢀC (see Fig. 2(c) and 5(d) for 25 ꢀC). This large increase
in the size of the scaffold is attributed to the encapsulation of
ATP in the micellar core of the amphiphilic self-assembly. On
the other hand, both ADP and AMP have an inadequate
hydrophilic and hydrophobic balance. Thus, the shape specic
encapsulation towards the micellar nanoparticle is not effective
to show the Hofmeister effect. Inorganic phosphates affect the
scaffold like any other kosmotropes (like sulfate anions) and
this was supported by no change in the size of the micellar
assemblies aer binding to Pi (see SF-11†). Hence, the ATP
complexation towards the nanoparticle assembly is the natural
choice for maximizing the Hofmeister effect though host–guest
interactions. The temperature ranges at which ATP, ADP, AMP
and Pi are active on the amphiphilic scaffold are shown in the
scale with their possible self-assembled structures in Fig. 6(c).
The cytotoxicity of the super LCST amphiphile 1 towards the
Fig. 7 Cytotoxicity data of amphiphile 1 at various concentrations in
cancer tissue was checked in HeLa cell lines at 37 ꢀC using the MTT assay at 24 h and 12 h.
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Fig. 8 (a) Isothermal calorimetric titration curves for the binding of amphiphile 1 (0.1 mM) with ATP (10 mM) at 25 ꢀC. (b) Calorimetric curves for
the binding of amphiphile 1 (1 mM) with Pi (10 mM) at 25 ꢀC.
that the newly developed thermo-responsive amphiphilic could be expanded to a wide range of other amphiphiles for
nanoparticle could be useful for targeting ATP as a trigger and future cancer treatment.
also as a carrier for drugs to cancer tissues.
Conflicts of interest
The authors declare no competing nancial interests.
Conclusion
In conclusion, a new thermo-responsive molecular assembly
was developed to detect and bind to ATP in aqueous medium.
Acknowledgements
The authors thank research grant from Department of Science
and Technology (DST), New Delhi, INDIA, under nano-mission
initiative project SR/NM/NS-42/2009. Smita thanks CSIR, New
Delhi, India for research fellowship. The authors thank Dr
Amitava Das at NCL-Pune for extending the ITC facilities for the
present work, and Dr Harinath Chakrapani and Ms. Kavita
Sharma of IISER-Pune for helping us with the cell viability data.
The amphiphilic molecule was designed with an appropriate
hydrophilic polyethylene glycol (PEG 750) unit and the renew-
able resource 3-pentadecylphenol hydrophobic to produce the
super LCST amphiphile with an LCST of more than 90 ꢀC in
water. Amphiphile 1 self-assembled as 150 nm micellar nano-
particles in water at ambient conditions and its hydrophobic
pocket was capable of loading water insoluble dyes like Nile red.
Dynamic light scattering techniques, electron and atomic force
microscopes conrmed that the self-assembled structures exist
in the form of spherical nanoparticles. Amphiphile 1 showed
selective phase separation for anions such as sulfates and
chlorides (salting-out) following the Hofmeister series. The
Hofmeister effect of amphiphile 1 was explored for binding to
anionic biomolecules ATP and its precursors ADP, AMP and Pi.
Amphiphile 1 showed high preferential binding towards ATP
over its precursors ADP, AMP and Pi. Isothermal calorimetric
experiments further conrmed the ATP binding and it esti-
mated three times higher binding constant for ATP towards
amphiphile 1 as compared to Pi. Endothermic nature of the ATP
binding to amphiphile 1 could provide advantages for selective
binding at cancer tissue temperatures (42–44 ꢀC), which are
higher than normal tissues (37 ꢀC). The thermo-responsive
scaffold for ATP binding concept demonstrated here is not only
restricted to the present example; in principle, this approach
References
1 Y. Zhang and P. S. Cremer, Proc. Natl. Acad. Sci. U. S. A., 2009,
106, 15249–15253.
2 X. Chen, T. Yang, S. Kataoka and P. S. Cremer, J. Am. Chem.
Soc., 2007, 129, 12272–12279.
3 H. I. Okur, J. Kherb and P. S. Cremer, J. Am. Chem. Soc., 2013,
135, 5062–5067.
4 P. L. Nostro and B. W. Ninham, Chem. Rev., 2012, 112, 2286–
2322.
5 Y. Zhang and P. S. Cremer, Annu. Rev. Phys. Chem., 2010, 61,
63–83.
6 A. Salis and B. W. Ninham, Chem. Soc. Rev., 2014, 43, 7358–
7377.
7 D. F. Parsons, M. Bostrom, P. L. Nostro and B. W. Ninham,
Phys. Chem. Chem. Phys., 2011, 13, 12352–12367.
1966 | J. Mater. Chem. B, 2015, 3, 1957–1967
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
Journal of Materials Chemistry B
8 Y. Zhang and P. S. Cremer, Curr. Opin. Chem. Biol., 2006, 10, 29 L. M. Zheng, A. Ztchlinsky, C.-C. Liu, D. M. Ojcius and
658–663. J. D.-E. Young, J. Cell Biol., 1991, 112, 279–288.
9 Y. Zhang, S. Furyk, D. E. Bergbreiter and P. S. Cremer, J. Am. 30 L. T. Wen and A. L. Knowles, Br. J. Pharmacol., 2003, 140,
Chem. Soc., 2005, 127, 14505–14510. 1009–1018.
10 Y. Zhang, S. Furyk, L. B. Sagle, Y. Cho, D. E. Bergbreiter and 31 F. D. Virgillio, Cancer Res., 2012, 72, 5441–5447.
P. S. Cremer, J. Phys. Chem. C, 2007, 111, 8916–8924.
11 Y. Cho, Y. Zhang, T. Christensen, L. B. Sagle, A. Chilkoti and
P. S. Cremer, J. Phys. Chem. B, 2008, 112, 13765–13771.
12 J. Kherb, S. C. Flores and P. S. Cremer, J. Phys. Chem. B, 2012,
116, 7389–7397.
32 P. P. Neelakandan, M. Hariharan and D. Ramaiah, Org. Lett.,
2005, 7, 5765–5768.
33 D. A. Jose, S. Mishra, A. Ghosh, A. Shrivastav, S. K. Mishra
and A. Das, Org. Lett., 2005, 9, 1979–1982.
34 J. Kaur and P. Singh, Chem. Commun., 2011, 47, 4472–4474.
13 K. B. Rembert, J. Paterova, J. Heyda, C. Hilty, P. Jungwirth 35 H. Jeon, S. Lee, Y. Li, S. Park and J. Yoon, J. Mater. Chem.,
and P. S. Cremer, J. Am. Chem. Soc., 2012, 134, 10039–10046. 2012, 22, 3795–3799.
14 H. Wei, S.-X. Cheng, X.-Z. Zhang and R.-X. Zhuo, Prog. Polym. 36 H. Wang and W.-H. Chan, Org. Biomol. Chem., 2008, 6, 162–
Sci., 2009, 34, 893–910. 168.
15 P. L. Nostro, B. W. Ninham, S. Milani, A. L. Nostro, 37 A. J. Moro, P. J. Cywinski, S. Korsten and G. J. Mohr, Chem.
G. Pesavento and P. Baglioni, Biophys. Chem., 2006, 124,
208–213.
Commun., 2010, 46, 1085–1087.
38 Y. Zhou, Z. Xu and J. Yoon, Chem. Soc. Rev., 2011, 40, 2222–
16 J. Paterova, K. B. Rembert, J. Heyda, Y. Kurra, H. I. Okur,
2235.
W. R. Liu, C. Hilty, P. S. Cremer and P. Jungwirth, J. Phys. 39 Z. Ge and S. Liu, Chem. Soc. Rev., 2013, 42, 7289–7325.
Chem. B, 2013, 117, 8150–8158. 40 R. D. Issels, Eur. J. Cancer, 2008, 44, 2546–2554.
17 J. P. Magnusson, A. Khan, G. Pasparakis, A. O. Saeed, 41 G. Kong, R. D. Barun and M. W. Dewhirst, Cancer Res., 2000,
W. Wang and C. Alexander, J. Am. Chem. Soc., 2008, 130,
10852–10853.
18 J. M. G. Swann, W. Bras, P. D. Topham, J. R. Howse and
A. J. Ryan, Langmuir, 2010, 26, 10191–10197.
60, 4440–4445.
42 J. Fang, H. Nakamura and H. Maeda, Adv. Drug Delivery Rev.,
2011, 63, 136–151.
43 K. Maruyama, Adv. Drug Delivery Rev., 2011, 63, 161–169.
19 L. Li, J.-H. Ryu and S. Thayumanavan, Langmuir, 2013, 29, 44 P. S. Pramod, C. Katamura, S. Chapaker, N. Balasubramaniam
50–55. and M. Jayakannan, Biomacromolecules, 2012, 13, 3627–3640.
20 C. L. D. Gibb and B. C. Gibb, J. Am. Chem. Soc., 2011, 133, 45 P. S. Pramod, R. Shah, C. Sonali, N. Balasubramanian and
7344–7347. M. Jayakannan, Nanoscale, 2014, 6, 11841–11855.
21 M. Qiu, S. Long, B. Li, L. Yan, W. Xie, Y. Niu, X. Wang, Q. Guo 46 U. Sridhar, P. S. Pramod and M. Jayakannan, RSC Adv., 2013,
and A. Xia, J. Phys. Chem. C, 2013, 117, 21870–21878. 3, 21237–21241.
22 P. K. Tarafdar, S. T. Reddy and M. J. Swamy, J. Phys. Chem. B, 47 S. Kashyap and M. Jayakannan, J. Phys. Chem. B, 2012, 116,
2013, 117, 9900–9909. 9820–9831.
23 S. Manet, Y. Karpichev, D. Dedovets and R. Oda, Langmuir, 48 S. Kashyap and M. Jayakannan, J. Mater. Chem. B, 2014, 2,
2013, 29, 3518–3526. 4142–4152.
24 B. A. Deyerle and Y. Zhang, Langmuir, 2011, 27, 9203–9210. 49 C. Wang, Q. Chen, Z. Wang and X. Wang, Angew. Chem., Int.
25 E. M. Schwiebert and A. Zsembery, Biochim. Biophys. Acta,
2003, 1615, 7–32.
26 W. G. Junger, Nat. Rev. Immunol., 2011, 11, 201–212.
Ed., 2010, 49, 8612–8615.
50 J. Liu, M.-a. Morikawa and N. Kimizuka, J. Am. Chem. Soc.,
2011, 133, 17370–17374.
27 A. Ohta, E. Gorelik, S. J. Prasad, F. Ronchese, D. Lukashev, 51 A. P. Borgan, W. R. Widger, D. Bensadek, I. R. Garcia,
M. K. K. Wong, X. Huang, S. Caldwell, K. Liu, P. Smith,
J.-F. Chen, E. K. Jackson, S. Apasov, S. Abrams and
S. J. Gaskell and H. Kohn, J. Am. Chem. Soc., 2005, 127,
2741–2751.
M. Sitkovsky, Proc. Natl. Acad. Sci. U. S. A., 2006, 103, 52 B. Flachner, Z. Kovari, A. Varga, Z. Gugolya, F. Vonderviszt,
13132–13137.
G. N. Szabo and M. Vas, Biochemistry, 2004, 473, 3436–3449.
28 P. Pellagatti, L. Raffaghello, G. Bianchi, F. Piccardi, V. Pistoia
and F. D. Virgillio, PLoS One, 2008, 3, e2599.
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