Self-Assembly of Spherical Organic Molecules to Form Hollow Vesicular Structures in Water for Encapsulation of an Anticancer Drug and Its Release

Article abstract of DOI:10.1002/asia.201900211

Developing hierarchical supramolecular structures is important for better understanding of various biological functions and possibly generating new materials for biomedical applications. Herein, we report the first examples of functional vesicles derived from cationic spherical organic molecules (C₁-C₃) which were readily synthesized by reacting a C₃-symmetric tris-benzimmidazole derivative (possessing a 1,3,5-ethyl substituted aromatic core) with 1,3,5-substituted tris-bromomethyl benzene derivatives. Vesicle formation by C₁-C₃ was probed by high-resolution microscopy (TEM and AFM), dynamic light scattering (DLS) and fluorescence microscopic imaging of calcein-loaded vesicles. One of the vesicles [Vesicle(C₃)] displayed the ability to load the anticancer drug doxorubicin (DOX). The drug was subsequently released from DOX@Vesicle(C₃) in a stimuli-responsive manner in presence of the well-known vesicle destroyer Triton X-100, as revealed by in vitro cell migration assay carried out on a highly aggressive human breast cancer cell line (MDA-MB-231).

Full text of DOI:10.1002/asia.201900211

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Self-assembly of Spherical Organic Molecules to form Hollow
Vesicular Structure in Water for Encapsulation of an Anti-cancer
Drug and Its Release
Authors: Parthasarathi Dastidar, Koushik Sarkar, and Sabir Ahmed
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To be cited as: Chem. Asian J. 10.1002/asia.201900211
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Self-assembly of Spherical Organic Molecules to form Hollow
Vesicular Structure in Water for Encapsulation of an Anti-cancer
Drug and Its Release
Koushik Sarkar, Sabir Ahmed and Parthasarathi Dastidar*[a]
Abstract: Developing hierarchical supramolecular structures is
important for better understanding of various biological functions and
possibly generating new materials for biomedical applications. In
here, we report the first examples of functional vesicles derived from
cationic spherical organic molecules (C1-C3) which were readily
phenomenon and possibly help to develop materials for various
biomedical applications.[1-11] For example, self-assembly of small
molecules leading to prion-like nanofibrils proved effective in
killing cancer cells by impending cytoskeleton dynamics of
cancer cells;[12] capsid protein of infectious cowpea chlorotic
mosaic virus was reported to form vesicles without the viral
genome.[13] The structure and functions of cell membranes were
better understood from the contribution of Kunitake et al. who
reported, for the first time, that a small organic molecule namely
didodecyldimethylammonium bromide could form bilayer
lamellar structure akin to cell membrane that subsequently
resulted in multi-layered vesicles in polar solvent (water) upon
sonication.[14] Since then, innumerous examples of vesicles
derived from synthetic molecules have been reported.[15-20] Liu
and Fujita[21,22] reported single layered vesicles having ‘black
berry’ structure obtained through the self-assembly of cationic
cage molecules such as metal organic polyhedra (MOP) in
solution. Recently, we reported that single layered vesicle
capable of loading and releasing an anti-cancer drug in a pH
responsive fashion was obtained through the self-assembly of a
Cu(II) based MOP nanocage molecule.[23] Research on organic
cage molecules[24-29], on the other hand, gained an impetus in
recent years due to their extraordinary permanent porosity with
surface areas that rival even extended metal-organic
frameworks.[30-32] However, literature survey indicates that
vesicle derived from purely organic spherical molecule is hitherto
unknown. It is easy to recognize the
synthesized by reacting
a
C3-symmetric tris-benzimmidazole
derivative (possessing a 1,3,5-ethyl substituted aromatic core) with
1,3,5-substituted tris-bromomethyl benzene derivatives. Vesicle
formation by C1-C3 was probed by high resolution microscopy (TEM
and AFM) dynamic light scattering (DLS) and fluorescence
microscopic images of calcein loaded vesicles. One of the vesicles
[Vesicle(C3)] displayed the ability to load an anti-cancer drug
doxorubicin (DOX). The drug was subsequently released from
DOX@Vesicle(C3) in a stimuli responsive manner in presence of a
well-known vesicle destroyer Triton X–100 as revealed by in vitro
cell migration assay carried out on a highly aggressive human breast
cancer cell line (MDA-MB-231).
Introduction
Complex supramolecular machineries in biological systems that
perform crucial biological functions include DNA double helices,
cell membranes, viral capsids, ribosomes, cytoskeleton and
many others. Studying such model supramolecular systems
fact that vesicle obtained from
spherical organic molecule is
expected to be free from the
plausible toxicity induced by metal
ion (a major concern in biomedical
applications) compared to its metal-
organic counterpart (MOP). Studies revealed that both
counterions and hydrophobic interactions played crucial roles in
the self-assembly process of MOPs generating vesicles.[22] We,
therefore, considered synthesizing cationic spherical organic
molecules having large -surface and studying their self-
assembly properties leading to vesicles that might be useful in
drug delivery applications (Scheme 1).
Scheme 1: Schematic overview of the work reported herein.
from synthetic molecules in the laboratory has been quite
rewarding as it provides better understanding of such
[a]
K. Sarkar, S. Ahmed and Prof. P. Dastidar
School of Chemical Sciences
Results and Discussion
Indian Association for the Cultivation of Science (IACS)
2A & 2B, Raja S. C. Mullick Road
Synthesis and characterization
Jadavpur, Kolkata, 700032, West Bengal (India)
Fax: (+91) 33-2473-2805
E-mail: ocpd@iacs.res.in
Supporting information for this article is given via a link at the end of
the document.
Straight forward synthesis starting from commercially available
1,3,5-triethyl benzene afforded C1-C3 in good yield; H and 13C
1
NMR supported the formation of C1-C3 . The HRMS spectra
showed prominent peaks which were attributed to m/z [M-H+]2+
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376.0916 and [M]3+ 251.0886 for C1, [M-H+]2+ 355.1704 and
TEM (Figure S7, supporting information). The data presented
thus far clearly indicated that C1-C3 could produce vesicles
namely vesicle(C1), vesicle(C2) and vesicle(C3) in water,
respectively.
+ 2+
[M]3+ 237.1185 for C2 and [M-CH3 ] 372.1395 and [M]3+
253.1035 for C3; the experimental isotope distribution patterns
also matched very well with that of the calculated patterns
supporting the formation of C1-C3 (Figure S4, supporting
information).
Scheme 2: Chemical and energy minimized structures (DFT) of C1-C3.
Figure 1: TEM [a)-c)] and AFM [d)-f)] images of dried samples of C1, C2 and
C3 in water, respectively; g)-i) the corresponding height profile of AFM images
d)-f).
Further support came from DOSY NMR spectra in D2O at room
temperature. Peaks corresponding to all the protons of C1-C3
were located on the same horizontal line in each case and
therefore, had same diffusion coefficient (D = 7.19×10-10
,
4.613×10-10 and 9.09×10-10 m2/s for C1, C2 and C3, respectively)
indicating the existence of discrete molecules in solution. The
hydrodynamic radius (Rh = 0.34, 0.53 and 0.27 nm for C1, C2
and C3, respectively) obtained from the corresponding diffusion
coefficients corroborated well with that of the single special
molecule estimated from DFT energy minimized structures
(Scheme 2) (Figure S5 and Table S1, supporting information).
Vesicle formation
The compounds were then tested for vesicle formation in water.
In a typical procedure, measured amount of a compound (3 mg)
was taken in milli-Q water (4 mL) in a vial and was sonicated for
15 min followed by vigorous shaking. Turbidity with frothing was
observed in each case. It was then subjected to high resolution
microscopy (TEM and AFM). Spherical objects with wide range
of size (40-99, 44-195 and 39-287 nm for C1, C2 and C3,
respectively) were observed in TEM images. Interestingly,
similar spherical objects were also seen in AFM images and the
height profile analyses indicated that they were most likely the
flattened vesicles formed due to drying of the samples (Figure 1).
These data strongly suggested vesicle formation due to the self-
assembly of the spherical organic molecules in water. To
provide further support, we then recorded DLS data of the
compounds in water. Normal distribution of particle size within
the range of 142–396, 190–295 and 396–825 nm for C1, C2 and
C3, respectively observed in DLS were expectedly larger than
the particle size observed in TEM as Rh of the particles in water
were expected to be larger than that of the dried particles in
To evaluate whether the vesicles, thus obtained, were suitable
as cargo to load and deliver drugs, we first loaded a hydrophilic
dye namely calcein in the vesicles. In a typical experiment, both
calcein and the spherical organic molecule were dissolved in
milli-Q water by sonication and shaking. The extra-vesicular
calcein was removed through dialysis following a standard
protocol.[33] UV-vis spectra of calcein@vesicle were found to
be red-shifted (13, 12 and 8 nm for C1, C2 and C3, respectively)
compared to that of the free calcein indicating its confined
environment. Significant quenching of the emission band ( =
~510 nm) of calcein (compared to that of free calcein) in each
case was observed when the calcein@vesicle were excited at
 = 450 nm suggesting that calcein molecules were indeed
encapsulated within the vesicles.[34] Furthermore, calcein
encapsulated vesicles were clearly observed through green
fluorescence of calcein@vesicle under
microscope (blue filter,  = 480 nm).
Drug loading and its release
a
fluorescence
Following similar protocol, an anti-cancer drug namely
doxorubicin hydrochloride (DOX) was loaded within the vesicles.
The data revealed that vesicle(C3) was found to be the most
efficient in loading DOX (10.5 %) (Figure 2). Since all the
vesicles reported herein were found to be stable up to pH 3
(Figure S8, supporting information), we explored a well-known
vesicle destroyer[35] (Triton-X-100) in order to deliver the
encapsulated drug in a stimuli responsive fashion. DLS data of
all the vesicles in presence of Triton-X-100 (5 L) showed
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of cancer cells reflected through hyperactivity in cell migration
leads to metastasis.[37,38]
Figure 3: Cell migration assay. Scale bar = 100 m.
Figure 2: Comparative UV-vis spectra of the encapsulated calcein within a)
vesicle(C1), d) vesicle(C2), g) vesicle(C3) with free calcein; comparative
photoluminescence spectra of encapsulated calcein within b) vesicle(C1), e)
vesicle(C2), h) vesicle(C3) with free calcein; fluorescence microscopic
images of encapsulated calcein within c) vesicle(C1), f) vesicle(C2), i)
vesicle(C3); j) comparative UV-vis and k) photoluminescence spectra of
DOX@vesicle(C3) with free DOX, l) the corresponding fluorescence
microscopic image. Scale bars for c), f), i), l) are 250, 250, 250, 500 nm,
respectively.
Cell migration assay allows us to evaluate the anti-cancer
property of a material in vitro by quantifying the speed with
which the cancer cells migrate in presence of the material under
study.[39,40] The slower the speed, the better is the anti-cancer
property. After achieving confluency of the cells (MDA-MB-231)
grown on a six well plate in an incubator, a gap was created on
the plates by a scratch using a sterile microtip. Each well was
then treated with the media (DMEM), vesicle(C3), DOX,
significantly smaller particle size (~10 nm) indicating disruption
of the vesicular structures (Figure S9, supporting information).
We decided to carry out further studies with vesicle(C3) since
its DOX loading capacity was the highest amongst all the
vesicles studied herein. Photoluminescence studies revealed
that Triton-X-100 was quite effective in releasing the
encapsulated DOX from the vesicle (Figure S10, supporting
information). The next task was to demonstrate that
DOX@vesicle(C3) could be utilized to deliver the drug in
presence of Triton-X-100 in vitro on a cancer cell line.
However, it was important to evaluate the cytotoxicity of C3 on a
normal cell before evaluating the possibility of delivery DOX from
DOX@vesicle(C3). MTT assay[36] on mouse macrophage cell
line (RAW 264.7) revealed that C3 was biocompatible up to 100
M which was far superior to that of the Cu(II) MOP (0.6 M)
reported by us[23] (Figure S11, supporting information). To
DOX@vesicle(C3),
containing Triton-X-100
Triton-X-100
and
DOX@vesicle(C3)
keeping the
–
all in DMEM –
concentration of the additives as listed in Table S2(supporting
information). The progress of cell migration was monitored under
an optical microscope for 24 h. The cells were completely
merged in the cases of control (on DMEM) and Triton-X-100
suggesting no anti-cancer activity arising due to these materials.
Whereas, in other cases, some amount of gap still remained
after 24 h incubation. Nearly equal migration speed in presence
of vesicle(C3) and DOX@vesicle(C3) (~16.6 m/h) suggested
that the anti-cancer drug DOX was tightly held within vesicle(C3)
and therefore, did not have a chance to retard the speed of
migration of the cancer cells whereas, the cell migration speed
in presence of DOX and DOX@vesicle(C3) containing Triton-X-
100 were found to be comparable (10.2 and 13.2 m/h,
respectively) indicating delivery of the confined drug in presence
of vesicle destroyer Triton-X-100 (Figure 3).
demonstrate
that
DOX
could
be
released
from
DOX@vesicle(C3) on cancer cells in presence of Triton-X-100,
we carried out cell migration assay on a highly aggressive
human breast cancer cell line MDA-MB-231. Cell migration is an
essential activity in healthy cells. However, uncontrolled growth
To see whether DOX@vesicle(C3) was also internalized, we
carried out cell imaging studies with the same cell line.[41]
Fluorescence image of the cells incubated in presence of
DOX@vesicle(C3) showed intense red color of DOX suggesting
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AP-0100 in semi contact-mode. UV−Vis spectroscopic measurements
successful internalization (Figure 4). Control experiment wherein
the cells were incubated with DOX alone did not produce any
color indicating that DOX itself could not penetrate the cell
membrane (Figure S12). These data clearly established that
vesicle(C3) did act as a cargo to carry the payload (DOX) inside
the cells (Figure 4).
were carried out on
a
Hewlett-Packard 8453 diode array
spectrophotometer equipped with a Peltier temperature controller. NMR
spectra were recorded using 300, 400 and 500 MHz spectrometer
(Bruker Ultrasheild Plus- 300, 400 and 500). Emission spectra were
recorded with a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer.
Fluorescence images has been collected in light microscope (BX51,
Olympus) equipped with a 100 W mercury lamp housing for an exciter
and a excitation band filter covering wavelengths 420−440 nm. Dynamic
light scattering experiments were executed using Malvern Particle Size
Analyser (Model No. ZEN 3690 ZETASIZER NANO ZS 90 version 7.03).
MTT assay were conducted using a multiplate ELISA reader (Varioskan
Flash Elisa Reader, Thermo Fisher). Confocal microscopy was done in a
C1 Nikon confocal microscopy.
Figure 4: Fluorescence microscopic images of MDA-MB-231 cells incubated
with DOX@vesicle(C3): a) bright field, b) fluorescence and c) overlay; Scale
bar = 75 m.
Conclusions
Synthesis of C1-C3:
Thus, vesicle formation by the spherical organic molecules (C1-
C3) is reported for the first time. Vesicle(C3) could load an anti-
cancer drug DOX and release it to a cancer cell in presence of a
vesicle destroyer Triton-X-100 as revealed by cell migration
assay with a highly aggressive human breast cancer cell line
(MDA-MB-231). DOX@vesicle(C3) was also successfully
internalized as evident from cell imaging studies. The results
presented herein open up avenue to develop vesicles derived
from spherical organic molecules for drug delivery applications,
which are understandably advantageous compared to that of its
metal-organic counterpart such as MOPs because vesicles
derived from purely organic molecules are free from metal-
induced toxicity expected for MOP. The results presented herein
indicate that vesicles may be developed from cationic organic
cage molecules. Therefore, intriguing possibility of developing
multi-drug delivery system by encapsulating two different drugs
– one within the cage and the other within the vesicles is
perhaps feasible. Attempts are being made to synthesize
cationic orgnaic cage molecules following the same synthetic
strategy adopted herein.
Step 1:
Synthesis of 1:
1,3,5-triethylbenzene (2 mL, 10.6 mmol), paraformaldehyde (3.38 g,
112.5 mmol), zinc bromide (3.94 g, 17.5 mmol) were taken in a round
bottom (r.b) flask (250 mL) and HBr in glacial acetic acid (40 ml 33%)
was then added. The mixture was then heated to 95 °C with constant
stirring for 24 h. Precipitate was filtered, washed with water thoroughly
and dried. 1,3,5-trimethyl-2,4,6-tribromomethylbenzene and 1,3,5-
trimethoxy-2,4,6-tribromomethylbenzene were synthesized following
similar procedure. 1,3,5-trimethylbenzene (1.47 mL, 10.6 mmol),
paraformaldehyde (3.38 g, 112.5 mmol), zinc bromide (3.94 g, 17.5
mmol) were taken in a round bottom (r.b) flask (250 mL) and HBr in 50
glacial acetic acid (40 ml 33%) was added. The mixture was then heated
to 95 °C with constant stirring for 24 h. Precipitate was filtered, washed
with water thoroughly and dried. 1,3,5-trimethoxybenzene (1.78 g, 10.6
mmol), paraformaldehyde (3.38 g, 112.5 mmol) were taken in a round
bottom (r.b) flask (250 mL) along with glacial acetic acid (20 mL) and
stirred for 1 h. HBr in glacial acetic acid (40 ml 33%) was then added.
The mixture was then heated to 65 °C with constant stirring for 24 h.
Reaction mixture was then added to cold water taken in a beaker (500
mL) drop wise with constant stirring. Precipitate was filtered, washed with
excess water and purified by column chromatography (4% ethyl
acetate/petroleum ether).
Step 2:
Synthesis of 2:
Experimental Section
Benzimidazole (2.67 g, 22.6 mmol) and KOH pellet (1.26 g, 22.6 mmol)
were taken in a r.b flask (250 mL). Tetrahydrofuran (THF, 100 mL) was
added into it and heated with stirring for 30 minutes. 1,3,5-triethyl-2,4,6-
tribromomethylbenzene (2 g, 4.5 mmol) in THF (50 mL) was then added
drop wise and refluxed overnight. The mixture was then evaporated in a
rotary evaporator and the compound was extracted in dichloromethane
and water solvent mixture. The organic layer was thoroughly washed with
water and the compound was obtained by evaporating the
dichloromethane in rotary evaporator.
Materials and Physical measurements:
All the chemicals were commercially available and used without further
purification. FT-IR spectra were obtained on a FT-IR instrument (FTIR-
8300, Shimadzu). Melting point (m.p.) was measured by electronic
melting point apparatus. TEM images were recorded using a JEOL
instrument with 300 mesh copper TEM grid. Diameter of the vesicle from
TEM images was measured using ImageJ software (version- 1.41o/Java
1.8.0_45). AFM images were taken with an NTMDT instrument, model no.
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Step 3:
found: 237.1185; m/z: Calculated for [(C48H50N6)2+]: 710.4086/2
=
Synthesis of the C1:
355.2043; found: 355.1704; FT-IR: 3421.3, 3012.11, 2974, 1605.8,
1574.07, 1484.25, 1321.91, 1235.36, 1182.21, 1006.81, 761.33, 592.76,
507.84 cm-1.
A double necked round bottom flask (500 mL) was fitted with a reflux
condenser and pressure equalizing funnel. A solution of Compound 2
(750 mg, 1.35 mmol) in a 1:2 Acetone-Chloroform mixtures (i.e. 80 mL
Acetone + 160 mL CHCl3) was added to the round bottom flask and
stirred for 30 minutes at 50 - 60 oC. A solution of 1,3,5-triethyl-2,4,6-
tribromomethylbenzene (595 mg, 1.35 mmol) in acetone (80 mL) was
taken in the pressure equalizing funnel and added drop wise for a period
of 2-3 h with vigorous stirring. A white precipitate started forming after 1.5
h. After the addition was complete, the pressure equalizing funnel was
removed and closed the neck with a stopper. The mixture was stirred for
another three days under reflux. Temperature was maintained to 90 oC.
The white compound was obtained as a 3Br- salt and was collected by
evaporating the solvent in a rotary evaporator.
C3: (White solid, m.p. > 250 °C, Yield: 64%); 1H NMR (300 MHz, D2O,
25°C): δ 8.18-8.10 (dd, 6H), 7.89-7.83 (dd, 6H), 5.75 (s, 9H), 5.68 (s, 3H),
3.94 (s, 6H), 3.79 (s, 6H), 2.47-2.40 (q, 6H), 1.18-1.12 (t, 9H) ppm; 13C
NMR (126 MHz, D2O, 25°C): δ 148.82, 132.54, 128.63, 128.01, 118.95,
113.73, 113.47, 64.43, 63.69, 44.87, 41.84, 23.13, 15.07 ppm; HRMS
(CH3OH) m/z: Calculated for [(C48H51N6O3)3+]: 759.4006/3 = 253.1335;
found: 253.1035; m/z: Calculated for [(C47H48N6O3)2+]: 744.3777/2 =
372.1888; found: 372.1395; FT-IR: 3390.75, 2980.9, 1595.1, 1562.84,
1452.48, 1325.99, 1186.11, 1101.62, 982.25, 747.58, 504.34 cm-1.
C2 and C3 were synthesized following the same procedure by adding
1,3,5-trimethyl-2,4,6-tribromomethylbenzene (538 mg, 1.35 mmol) and
1,3,5-trimethoxy-2,4,6-tribromomethylbenzene (603 mg, 1.35 mmol)
instead of 1,3,5-triethyl-2,4,6-tribromomethylbenzene, respectively.
DOSY NMR spectra :
Diffusion-Ordered Spectroscopy (DOSY) 1H NMR was performed on a
Bruker 400 and 500 MHz spectrometer with the magnetic field gradient
() 4.258×103 Hz/G .The length of the gradient () for C1, C2 and C3 was
1 ms, and the two pulsed gradients () for C1, C2 and C3 was 200, 80 and
200 ms, respectively. All spectra were taken at room temperature. After
the data collection, FIDs were processed and analyzed with the NMR
software TopSpin 2.0 provided by Bruker.
Preparation of vesicles and characterization:
Compounds (3 mg) were taken in a separate vials and 4 mL milli-Q water
was added [concentration of C1 = 754.7 M, C2 = 788.1 M, C3 = 750.2
M]. The solutions were then sonicated for 15 minutes followed by
vigorous shaking. Turbidity and bubbles appeared on the vials after
sonication and shaking. The resulted dispersions were separately
subjected for various analyses like DLS, TEM and AFM etc.
TEM sample preparation:
The water dispersion of the corresponding compounds were drop casted
on a carbon-coated Cu (300 mesh) TEM grid. The grid was dried under
vacuum at room temperature for one day and used for recording TEM
images.
Atomic Force Microscopic (AFM) Study:
The water dispersions of the vesicles were drop-casted separately on
mica and air dried for 24 h. It was then subjected for AFM analysis.
Scheme 3: Schematic representation of the reaction procedure.
DLS analyses:
The dispersion of the vesicles were separately taken in a glass cuvette
and subjected to the DLS experiment.
Characterization data:
Calcein encapsulation within the vesicles:
C1: (White solid, m.p. > 250 °C, Yield: 79%); 1H NMR (300 MHz, D2O,
25°C): δ 8.26-8.23 (dd, 6H), 7.98-7.95 (dd, 6H), 6.42 (s, 3H), 5.82 (s,
12H), 2.54-2.47 (q, 12H), 1.26-1.21 (t, 18H) ppm; 13C NMR (75 MHz,
D2O, 25°C): δ 149.16, 133.49, 132.96, 128.72, 128.67, 113.85, 45.38,
22.97, 15.20 ppm; HRMS (CH3OH) m/z: Calculated for [(C51H57N6)3+]:
Calcein (1.24 mg) was taken in a vial and methanol (1 mL) was added.
The mixture was then heated and sonicated for several times to make a
nearly homogeneous solution. An aliquot (100 L) withdrawn from the
solution and taken in another vial. The methanol was then evaporated to
dryness by homogeneous gentle heating. The corresponding vesicles (3
mg) separately were added along with the addition of milli-Q water (4 mL).
The mixture was then subjected to sonication followed by vigorous
shaking. The resulting solution was then subjected to dialysis using
SnakeSkin dialysis tubing with molecular weight cut off 3500 for 72
hours following standard technique. Concentration of calcein inside the
vesicles was estimated from UV-Vis spectra.
753.4628/3
=
251.1542; found: 251.0886; m/z: Calculated for
[(C51H56N6)2+]: 752.4555/2 = 376.2277; found: 376.0916; FT-IR: 3404.08,
2970.2, 2931.4, 1615.6, 1572.95, 1483.64, 1453.29, 1322.08, 1230.61,
1183.62, 1133.8, 1001.3, 505.1 cm-1.
C2: (White solid, m.p. > 250 °C, Yield: 72%); 1H NMR (300 MHz, D2O,
25°C): δ 8.24-8.21 (dd, 6H), 7.98-7.95 (dd, 6H), 6.40 (s, 3H), 5.92 (s, 6H),
5.82 (s, 6H), 2.54-2.47 (q, 6H), 2.30 (s, 9H), 1.28-1.23 (t, 9H) ppm; 13C
NMR (126 MHz, D2O, 25°C): δ 149.00, 142.59, 132.89, 132.74, 129.43,
128.55, 113.79, 113.72, 46.09, 45.02, 22.94, 15.16, 15.06 ppm; HRMS
(CH3OH) m/z: Calculated for [(C48H51N6)3+]: 711.4159/3 = 237.1386;
Sample preparation for fluorescence microscopy:
The aqueous solutions of the dye/drug encapsulated vesicles were
separately drop-casted on a glass slide. The slide was then dried under
ambient condition for one day and used for recording fluorescence
images.
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Doxorubicin encapsulation within the vesicle(C3):
Cell imaging:
C3 (4 mg) and solution of doxorubicin.HCl (DOX, 2 mL, 0.1 mg/mL) were
taken in a vial and subjected to sonication followed by vigorous shaking.
This mixture was then subjected to dialysis using SnakeSkin dialysis
tubing with molecular weight cut off 3500 for 72 hours following standard
technique. Concentration of DOX inside vesicle(C3) was estimated from
UV-Vis spectra.
For cell imaging, MDA-MB-231 cells were cultured by using DMEM
supplemented with 10% FBS and 1% penicillin–streptomycin on ethanol
etched cover slips kept in a 35 mm tissue culture dishes. The dishes
were then kept in a humidified incubator at 37 C overnight. The cells
were then washed with PBS and incubated in serum-free media (SFM)
for half an hour. DOX encapsulated vesicle(C3) was then taken in SFM
keeping the concentration of vesicle(C3) at IC50 concentration and DOX
= 0.9 M and added to the cells. It was then incubated for 30 min. After
incubation, SFM was discarded followed by washing with PBS. The cells
were fixed by using 4% paraformaldehyde for 10 min at room
temperature. After fixing, the cells were washed with PBS and mounted
on glass slides for microscopy. For control experiment, only DOX solution
was taken in SFM keeping the concentration of DOX = 0.9 M.
DOX release study:
The disruption of the vesicular assembly was studied in presence of two
different stimuli, i.e. pH and in presence of Triton X-100.
For pH dependent disruption study, vesicle(C3) (1 mL) was taken in a
vial and the respective buffer solutions (1 mL) of different pH value (i.e.
pH 7.4, 5.6 and 3.0) were added separately. The mixtures were then
incubated for 1 h and subjected to DLS analysis. From the DLS data, it
was quite clear that no disruption of the assembly of vesicle(C3) was
taking place with varying pH up to 3.0.
For Triton X-100 mediated disruption study, each of the vesicles (1 mL)
separately were taken in a vial and Triton X-100 (5 L) was added. The
mixtures were then sonicated for 5 minutes and shaken vigorously. The
mixtures were separately subjected to DLS analysis.
DOX release from the vesicle(C3) has been established by treating DOX
loaded vesicle(C3) (1 mL) with Triton X-100 (5 L) and sonicated
followed by vigorous shaking. On the other hand, DOX loaded
vesicle(C3) solution (1 mL) was separately treated with buffer solution of
pH 5.6 (1 mL) followed by sonication and vigorous shaking. These
solutions were separately subjected to photoluminescence studies.
Acknowledgements
K.S thanks CSIR, IACS and S.A thanks CSIR for research
fellowship. P.D thanks DST (EMR/2016/000894) for financial
support. We thank Mr. Shanti Gopal Patra, School of Chemical
Sciences, IACS for helping in DFT calculations.
Keywords: Spherical organic compounds • Vesicle • Drug
delivery • Cell migration • Supramolecular chemistry
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Entry for the Table of Contents
FULL PAPER
Vesicle derived from cationic spherical
organic molecules showed stimuli
responsive release of the drug.
Koushik Sarkar, Sabir Ahmed and
Parthasarathi Dastidar*
Page No. – Page No.
Self-assembly of Spherical Organic
Molecules to form Hollow Vesicular
Structure in Water for Encapsulation
of an Anti-cancer Drug and Its
Release
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