Thermo-responsive and shape transformable amphiphilic scaffolds for loading and delivering anticancer drugs

Article abstract of DOI:10.1039/c4tb00134f

Shape transformable carriers are an important class of biomaterials for selective drug delivery in a cancer tissue physiological environment. Here, we report the first example of an in situ shape transformable thermo-responsive amphiphilic scaffold for loading and delivering anticancer drugs at the cancer tissue temperature. New amphiphiles having a hydrogen bonded amide linkage that connects hydrophilic oligoethylene glycol with the hydrophobic renewable resource 3-pendadecylphenol were tailor made through multi-step organic synthesis. These amphiphiles underwent reversible self-assembly from three dimensional core-shell to rod-like structures in water (or PBS at pH = 7.4). The temperature-induced shape transformation was attributed to the lower critical solution temperature (LCST) and the process was confirmed by light scattering studies, electron microscopy, atomic force microscopy, variable temperature NMR and single crystal structure study. Anticancer drugs such as doxorubicin (DOX) and camptothecin (CPT) were successfully loaded in the core-shell structure without altering the shape transformation ability of the scaffold. In vitro drug release studies revealed that the DOX loaded scaffolds showed a selective release of more than 90% of the drug at the cancer tissue temperature (40-43 °C) compared to normal body temperature (37 °C, <10%). The drug kinetics study revealed that the release of DOX at the cancer tissue temperature followed a non-Fickian diffusion process. Thus, the present investigation provides the first insight into the development of in situ shape transforming thermo-responsive scaffolds and also establishes the proof-of-concept of their loading and delivering capabilities at the cancer tissue temperature. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4tb00134f

Journal of
Materials Chemistry B
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PAPER
Thermo-responsive and shape transformable
amphiphilic scaffolds for loading and delivering
anticancer drugs†
Cite this: J. Mater. Chem. B, 2014, 2,
4142
*
Smita Kashyap and Manickam Jayakannan
Shape transformable carriers are an important class of biomaterials for selective drug delivery in a cancer
tissue physiological environment. Here, we report the first example of an in situ shape transformable
thermo-responsive amphiphilic scaffold for loading and delivering anticancer drugs at the cancer tissue
temperature. New amphiphiles having a hydrogen bonded amide linkage that connects hydrophilic
oligoethylene glycol with the hydrophobic renewable resource 3-pendadecylphenol were tailor made
through multi-step organic synthesis. These amphiphiles underwent reversible self-assembly from three
dimensional core–shell to rod-like structures in water (or PBS at pH ¼ 7.4). The temperature-induced
shape transformation was attributed to the lower critical solution temperature (LCST) and the process
was confirmed by light scattering studies, electron microscopy, atomic force microscopy, variable
temperature NMR and single crystal structure study. Anticancer drugs such as doxorubicin (DOX) and
camptothecin (CPT) were successfully loaded in the core–shell structure without altering the shape
transformation ability of the scaffold. In vitro drug release studies revealed that the DOX loaded scaffolds
showed a selective release of more than 90% of the drug at the cancer tissue temperature (40–43 ꢀC)
compared to normal body temperature (37 ꢀC, <10%). The drug kinetics study revealed that the release
of DOX at the cancer tissue temperature followed a non-Fickian diffusion process. Thus, the present
investigation provides the first insight into the development of in situ shape transforming thermo-
responsive scaffolds and also establishes the proof-of-concept of their loading and delivering capabilities
at the cancer tissue temperature.
Received 23rd January 2014
Accepted 8th April 2014
DOI: 10.1039/c4tb00134f
www.rsc.org/MaterialsB
internalization, biodistribution, phagocytosis, and so on.12,13 It
was reported that non-spherical objects like rod-like particles,14
lo-micelles15 and elliptical disks16 with pointed ends were
Introduction
Stimuli-responsive carriers are important biomaterials for
delivering anticancer drugs or genes in a controlled manner to a
specic site or organ.1,2 The abnormal cell growth and imperfect
lymphatic drainage of cancer tissues accumulates larger sized
nano-assemblies (or aggregates of >100 nm size) through an
enhanced permeability and the retention (EPR) effect.3–5
Further, the unusual physiochemical environments of cancer
tissues such as a high temperature (40–43 ꢀC)6,7 and a more
acidic environment (pH ¼ 6.1 to 6.8)8 compared to normal
tissues (37 ꢀC and pH ¼ 7.4) has facilitated the development of
temperature and pH stimuli drug carriers.9–11
readily internalized by cells compared to spherical objects like
micelles and vesicles. Szoka and Frechet proposed a membrane
model for the entry of various polymer architectures and
concluded that rod-shape carries have a better ability to pene-
trate the cell membrane.17 Additionally, the retention of the
spherical shape of nano-carriers is very important since they
possess a uniform ow behaviour in all three dimensions,
which is required for long circulation times, for example in
blood plasma during intravenous delivery.18 This would allow
the drug carriers to have both an enhanced transportation and
membrane penetration in a single system for maximizing the
treatment efficacy. Hence, hypothetically the ideal polymeric or
small molecular carriers should retain their three dimensional
spherical shape under the normal tissue conditions and should
be capable of undergoing in situ shape transformation into one
dimensional structures in the cancer tissue environment
(high temperature or low pH). This concept is schematically
shown in Fig. 1.
Recently, it was understood that apart from size and stimuli
the shape of the nano-carriers plays a crucial role in cellular
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
† Electronic supplementary information (ESI) available: Guinier plot,
crystallographic images, unit cells, DLS data, TCSPC life time table, and
1H-NMR, 13C-NMR, MALDI spectra are provided. CCDC 968254. For ESI and
crystallographic data in CIF or other electronic format see DOI:
10.1039/c4tb00134f
Thermo-responsive polymers have attracted signicant
interest in recent years due to their potential application in the
4142 | J. Mater. Chem. B, 2014, 2, 4142–4152
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tissues (see Fig. 1). Thus, shape transformable drug carriers
with respect to the cancer tissue environment are an important
class of biomaterials yet to be developed for the administration
of anticancer drugs.
Here, we report the rst example of shape tunable thermo-
responsive amphiphilic drug carriers and demonstrate the
proof-of-concept of their loading and delivering capabilities
under the conditions similar to that of cancer tissues. New
amide functionalized amphiphiles have been synthesized based
on 3-pentadecylphenol, a renewable hydrophobic unit resource,
along with oligoethylene glycols as the hydrophilic unit. The
amphiphile self-organized into a spherical core–shell nano-
particle at ambient conditions and underwent a morphological
transformation into rod-like structures at higher temperatures
(above LCST). Single crystal structure, variable temperature
NMR studies, light scattering techniques, electron and atomic
force microscopies provided evidence for the reversible
morphological transformation. Anticancer drugs such as
doxorubicin and camptothecin were successfully encapsulated
in these thermo-responsive scaffolds. In vitro release kinetic
Fig. 1 Schematic representation of shape transformable and thermo-
responsive nano-scaffolds in a cancer tissue.
ꢀ
studies revealed that the scaffolds were stable at 37 C in PBS
buffer and that they selectively underwent phase trans-
ꢀ
formations at temperatures higher than 42 C in PBS buffer to
release >90% of the loaded cargoes. Thus, the present investi-
gation opens a new area of shape transformable thermo-
responsive nano-carriers for loading and delivering anticancer
drugs.
eld of drug delivery. These thermo-responsive polymers
generally undergo conformational changes in response to a
variation in temperature which results in terms of a difference
in their solubility in the aqueous medium. The temperature at
which this phenomenon occurs is known as the lower critical
solution temperature (LCST). Thermo-responsive polymers are
ideally expected to retain the loaded cargoes (drugs) at the body
temperature (37 ꢀC) and expected to rupture to deliver their load
at the cancer tissue temperature (40–43 ꢀC).19,20 Poly(N-isopropyl
acrylamide) (PNIPAM) is one the most important thermo-
responsive commercial polymers with an LCST atꢁ32 ꢀC.
Several attempts were made to modify PNIPAM to adjust the
LCST close to the cancer tissue temperature (40–43 ꢀC) so that it
could be employed as a drug delivery vehicle. The PNIPAAm-b-
PMMA diblock copolymer,21 PMMA-b-PNIPAM-b-PMMA tri-
block,22 PNIPAM–poly(lactic acid) copolymer,23 PNIPAM-co-
acrylamide-b-poly(caprolactone) random block copolymer24 and
PNIPAM-octadecyl acrylate copolymer25 are some of the impor-
tant examples of modied PNIPAMs with an LCST close to or
higher than the body or cancer tissue temperature (43 ꢀC).
These polymer based micelles were further utilised to encap-
sulate anticancer drugs like doxorubicin (DOX), anti-inam-
matory drugs, and so on. Zhuang and co-workers reported
another class of PLG-g-PMEOiMA polypeptide with tuneable
LCST in the range of 19 to 40 ꢀC for delivering DOX at a low
temperature and pH. The same group has also reported pH and
reduction responsive PEGylated polypeptide nanogels to
successfully deliver doxorubicin (DOX) in the intracellular
microenvironment.26 Though the above literature reports
emphasised the need for the thermo-responsive polymers for
drug delivery applications, unfortunately, to date there is no
reported nano-carrier which could also undergo shape trans-
formations at physiological conditions similar to that of cancer
Experimental section
Materials
3-Pentadecylphenol, 2-ethanolamine, succinic anhydride, Boc-
anhydride triethylamine (Et3N), triethyleneglycol mono-
methylether, ethylene glycol monomethylether, diethylene
glycol monomethyl ether, 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC), dicyclohexylcarbodiimide, DIAD (diiso-
propyl azodicarboxylate), diisopropyl ethylamine (DIPEA),
4-dimethylamino pyridine were purchased from Aldrich chem-
icals. And all other reagents and solvents were purchased locally
and puried following the standard procedure.
General procedures
1H-NMR and 13C-NMR spectra were recorded using a 400 MHz
Jeol NMR spectrophotometer in CDCl3 containing a small
amount of TMS as the internal standard. Infra-red spectra were
recorded using a Thermo-Scientic Nicolet 6700 FT-IR spec-
trometer in KBr. The mass of all the amphiphiles as well as the
intermediate compounds was analysed by an Applied Bio-
systems 4800 PLUS MALDI TOF/TOF analyser. DLS of the
amphiphile was carried out using a Nano ZS-90 apparatus
utilizing a 633 nm red laser (at 90ꢀ angle) from Malvern
instruments. The static light scattering experiment (SLS) was
carried out using a 3D-DLS spectrometer, from LS Instruments,
Switzerland, utilizing toluene as a reference. The measurement
was performed in autocorrelation mode from 30 to 100ꢀ by steps
of 5ꢀ. FE-SEM images were recorded using a Zeiss Ultra Plus
This journal is © The Royal Society of Chemistry 2014
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scanning electron microscope. For FE-SEM analysis, the (CDCl3, 100 MHz) d: 177.05 (COOH), 171.88 (COO–CH2), 69.93
samples were prepared by drop casting on silicon wafers and (OCH2–CH2–O), 58.51 (C–OCH3), 28.45 (COOH–CH2–CH2–CO).
coated with gold. TEM images were recorded using a Technai- FT-IR (cmꢂ1): 2923, 1728, 1450, 1351, 1244, 1201, 1162, 1134,
300 instrument by drop casting the sample on a Formvar-coated 1101, 1028, 934, 839, 625. MALDI-TOF-MS: m/z calculated for
copper grid. Atomic force microscope images were recorded for C7H12O5: 220.22, and found: 259.93 (M+ + K+).
drop caste samples using JPK instruments attached with Nano
wizard-II setup. The reproducibility of the data was checked for Synthesis of 12-oxo-2,5,8,11-tetraoxapentadecan-15-oic acid
at least three independent amphiphile solutions. Single crystals (1c)
were subjected to data collection at 100 K on a Bruker APEX duo
CCD-X-ray diffractometer equipped with a graphite mono-
Triethyleneglycol monomethylether (10.00 g, 60.0 mmol), suc-
cinic anhydride (7.30 g, 73.0 mmol) and Et3N (8.5 mL, 60.0
mmol) were used as given in the procedure for compound 1a.
˚
chromator Mo Ka radiation (l ¼ 0.71073 A). The frames were
integrated with the Bruker APEX soware package. The struc-
The product was puried by passing through a silica gel column
tures were solved by direct methods and rened using SHELX S
with a 60–120 mesh using 10% methanol in chloroform as the
v97 programs. The absorption and emission studies were done
eluent. Yield ¼ 10.0 g (62%). 1H-NMR (CDCl3, 400 MHz) d: 4.14
by a Perkin-Elmer Lambda 45 UV-Visible spectrophotometer
ppm (t, 2H, CO–OCH2), 3.60 ppm (t, 2H, CH2–O), 3.56–3.46 ppm
and a SPEX Flurolog HORIBA JOBIN YVON uorescence spec-
(t, 8H, OCH2CH2O), 3.27 ppm (s, 3H, CH2–OCH3), 2.54 ppm
trophotometer with a 450 W Xe lamp as the excitation source at
(s, 4H, CO–CH2–CH2). 13C-NMR (CDCl3, 100 MHz) d: 177.05
room temperature. Fluorescence intensity decays were collected
by the time correlated single photon counting technique
(TCSPC) setup from Horiba Jobin Yvon, using NanoLED-460 for
DOX and NanoLED-374 for CPT as the sample excitation
(COOH), 171.88 (COO–CH2), 70.56 (OCH2–CH2–O), 69.93 (COO–
CH2), 58.51 (C–OCH3), 28.45 (CO–CH2–CH2). FT-IR (cmꢂ1):
3471, 2881, 2933, 1792, 1453, 1392, 1349, 1244, 1199, 1094,
1026, 941, 846, 751, 666, 622. MALDI-TOF-MS: m/z calculated for
sources.
C7H15NO3: 264.12, and found: 287.04 (M+ + Na+).
Synthesis of 4-(2-methoxyethoxy)-4-oxobutanoic acid (1a)
Synthesis of tert-butyl (2-hydroxyethyl)carbamate (2)
Ethylene glycol monomethylether (5.00 g, 65.0 mmol) and suc-
cinic anhydride (7.89 g, 78.0 mmol) were dissolved in dry
dichloromethane (25 mL) under a N2 atmosphere. To this
reaction mixture, Et3N (9.15 mL, 65.0 mmol) was added drop
wise (Caution: the mixtures start to boil vigorously just aer the
addition of Et3N). The reaction mixture was then stirred at 25 ꢀC
for 24 h under nitrogen. It was poured into water (60 mL) and
neutralized with 2 N concentrated HCl (2.0 mL). The organic
layer was then washed with brine solution, dried over anhy-
drous sodium sulphate and was concentrated to obtain a pale
yellow liquid as the product. It was puried by passing through
a silica gel column with a 60–120 mesh using 5% methanol in
chloroform as the eluent. Yield ¼ 8.0 g (69%). 1H-NMR (CDCl3,
400 MHz) d: 4.24 ppm (t, 2H, CO–OCH2), 3.60 ppm (t, 2H, CH2–
O), 3.38 ppm (s, 3H, CH2–OCH3), 2.66 ppm (s, 4H, CO–CH2–
CH2). 13C-NMR (CDCl3, 100 MHz) d: 177.05 (COOH), 171.88
(COO–CH2), 69.93 (COO–CH2), 58.51 (C–OCH3), 28.45 (CO–
CH2–CH2). FT-IR (cmꢂ1): 3495, 2926, 2852, 1714, 1453, 1406,
1381, 1351, 1198, 1161, 1125, 1096, 1028, 982, 957, 906, 863,
833, 638. MALDI-TOF-MS: m/z calculated for C7H12O5: 176.07;
and found: 215.03 (M+ + K+).
Ethanolamine (10.00 g, 164.0 mmol) was added to the mixture
of 10% Na2CO3 (60 mL) and THF (5 mL) and stirred at 25 ꢀC for
10 minutes. Boc-anhydride (42.0 g, 196.0 mmol) in THF (40 mL)
was added drop wise in the reaction mixture. Aer the addition,
the content was stirred at 25 ꢀC for 12 h. At the end of the
reaction, a white color precipitate was observed. THF was
removed by a rota evaporator and the content was extracted with
ethyl acetate (60 mL). The organic layer was neutralized with 5%
HCl (40 mL), dried over anhydrous sodium sulphate and the
solvent was removed to obtain a colorless liquid as the product.
It was puried by passing through a silica gel column with a 60–
120 mesh using 10% methanol in chloroform as the eluent.
1
Yield ¼ 23.0 g (88%). H-NMR (CDCl3, 400 MHz) d: 3.64 ppm
(t, 2H, CH2–OH), 3.23 ppm (t, 2H, CH2–NH), 1.41 ppm (s, 9H,
OC–(CH3)3, 5.25 ppm (s, 1H, CH2–NH). 13C-NMR (CDCl3, 100
MHz) d: 156.84 (COO), 79.65 (OC–CH3), 62.48 (CH2–OH), 43.09
(CH2–NH), 28.33 (OC–CH3). FT-IR (cmꢂ1): 3352, 2976, 2933,
2881, 1683, 1518, 1453, 1393, 1365, 1274, 1249, 1164, 1064, 999,
972, 900, 862, 781, 650. MALDI-TOF-MS: m/z calculated for
C7H15NO3: 161.11, and found: 184.03 (M+ + Na+).
Synthesis of tert-butyl (2-(3-pentadecylphenoxy)ethyl)
carbamate (3) PDP–NH–Boc
Synthesis of 4-(2-(2-methoxyethoxy)ethoxy)-4-oxobutanoic
acid (1b)
Compound 2 (2.64 g, 16.0 mmol), 3-pentadecylphenol (5.00 g,
Diethyleneglycol monomethylether (5.00 g, 42.0 mmol), suc- 16.0 mmol) and triphenylphosphine (4.30 g, 16.0 mmol) were
cinic anhydride (5.00 g, 50.0 mmol) and Et3N (7.00 mL, 50.0 dissolved in dry tetrahydrofuran (20 mL). The reaction mixture
mmol) were used as given in the procedure for compound 1a. was then kept in an ice-cooled bath for 10 minutes under a N2
The product was puried by passing through a silica gel column purge. Diisopropyl azodicarboxylate (3.65 mL, 18.0 mmol) was
ꢀ
with a 60–120 mesh using 5% methanol in chloroform as the added drop wise and the reaction mixture was stirred at 25 C
eluent. Yield ¼ 2.50 g (30%). 1H-NMR (CDCl3, 400 MHz) d: 4.24 for 24 h. The mixture was directly loaded in a silica gel column
ppm (t, 2H, CO–OCH2), 3.60 ppm (t, 2H, CH2–O), 3.38 ppm with a 60–120 mesh and was eluted using 1% ethyl acetate in
(s, 3H, CH2–OCH3), 2.66 ppm (s, 4H, CO–CH2–CH2). 13C-NMR hexane as the eluent. Yield ¼ 4.2 g (58%). 1H-NMR (CDCl3,
4144 | J. Mater. Chem. B, 2014, 2, 4142–4152
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400 MHz) d: 7.19 ppm (t, 1H, Ar-H), 6.80–6.70 ppm (m, 3H, Ar- eluent. Yield ¼ 0.6 g (42.0%). 1H-NMR (CDCl3, 400 MHz) d: 7.19
H), 5.02 ppm (s, 1H, NH), 4.02 ppm (t, 2H, Ar-OCH2), 3.54 ppm ppm (t, 1H, Ar-H), 6.80–6.69 ppm (m, 3H, Ar-H), 6.13 ppm (CO–
(t, 2H, CH2–N), 2.58 ppm (t, 2H, Ar-CH2), 1.46 ppm (s, 9H, OC– NH), 4.24 ppm (t, 2H, COO–CH2), 4.02 ppm (t, 2H, Ar-OCH2),
C(CH3)3, 1.6–0.88 ppm (m, 29H, aliphatic H). 13C-NMR (CDCl3, 3.67 ppm (t, 2H, CH2–OCH3), 3.56 ppm (t, 2H, CH2–N), 3.38
100 MHz) d: 158.62 (Ar-C), 155.98 (CO–O), 144.85, 129.29, ppm (s, 3H, CH2–OCH3), 2.73 ppm (t, 2H, Ar-CH2), 2.57 ppm (t,
121.32, 114.75, 111.40 (Ar-C), 79.57 OC(CH3)3, 67.08 (Ar-OCH2), 2H, NH–CO–CH2), 2.52 ppm (t, 2H, CH2–COO), 1.6–0.88 ppm
40.26 (CH2–N), 36.09, 32.00, 29.76, 26.47, 22.77, 14.20. FT-IR (m, 29H, aliphatic H). 13C-NMR (CDCl3, 100 MHz) d: 172.95
(cmꢂ1): 3396, 2916, 2850, 1690, 1590, 1512, 1453, 1362, 1250, (NH–CO), 171.52 (CO–O), 158.52, 144.89, 129.33, 121.43, 114.71,
1157, 1060, 959, 866, 778, 690. MALDI-TOF-MS: m/z calculated 111.45 (Ar-C), 70.42 (Ar-OCH2), 66.66 (CH2–OCH3), 63.79 (COO–
for C28H49NO3: 447.37, and found: 470.29 (M+ + Na+).
CH2), 59.07 (O–CH3), 39.16 (CH2–N), 36.10, 32.00, 31.51, 31.07,
29.64, 29.77, 22.77, 14.21 (aliphatic C). FT-IR (cmꢂ1): 3318, 2845,
2915, 1731, 1647, 1542, 1449, 1406, 1351, 1256, 1179, 1124,
1042, 982, 925, 761, 724, 690. MALDI-TOF-MS: m/z calculated for
Synthesis of 2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-oxo-4-((2-(3-
pentadecylphenoxy)ethyl)amino)butanoate (PDP–TEG)
C
30H51NO5: 505.38 and found: 528.37 (M+ + Na+).
Triuoroacetic acid (10 mL, 134.2 mmol) was added drop wise
to compound 3 (2.00 g, 4.5 mmol) in dichloromethane (5 mL).
Synthesis of 2-(2-methoxyethoxy)ethyl 4-oxo-4-((2-(3-
pentadecylphenoxy)ethyl)amino)butanoate (PDP–DEG)
ꢀ
Aer stirring the reaction mixture for 1 h at 25 C the solvent
was removed by a rotavapour. Fresh dichloromethane (5 mL)
was added to the product and washing was repeated for 3 times
to remove TFA. The content was poured in ice-cooled diethyl
ether (15 mL). The white solid mass (2.00 g, 5.7 mmol) obtained
was dissolved in dry dichloromethane (15 mL) and purged with
nitrogen for 15 minutes. To this reaction mixture, 1c (1.52 g,
5.7 mmol) was added and the content was purged under
nitrogen for 15 minutes. EDC (1.32 g, 6.9 mmol) and diisopro-
pylethylamine (2.0 mL, 11.5 mmol) was added to the reaction
mixture under nitrogen atmosphere and the reaction was
continued for 24 h at 25 ꢀC. The mixture was poured into water
(30 mL) and extracted with chloroform (20 mL). The organic
layer obtained was neutralized with 2 N HCl (2 mL), washed
with aqueous 5% NaHCO3 (50 mL) and brine. Aer drying over
anhydrous sodium sulphate, the solvent was removed to obtain
a yellow liquid as the product. It was further puried by passing
through a silica gel column with a 60–120 mesh using 25%
methanol in chloroform as the eluent. Yield ¼ 2.5 g (78.0%). 1H-
NMR (CDCl3, 400 MHz) d: 7.18 ppm (t, 1H, Ar-H), 6.80–6.69 ppm
(m, 3H, Ar-H), 6.26 (CO–NH), 4.24 ppm (t, 2H, COO–CH2), 4.02
ppm (t, 2H, Ar-OCH2), 3.69–3.63 ppm (m, 10H, O–CH2–CH2),
3.56 ppm (t, 2H, CH2–N), 2.70 ppm (t, 2H, Ar-CH2), 3.38 ppm
(s, 3H, CH2–OCH3), 2.57 ppm (t, 2H, NH–CO–CH2), 2.51 ppm
(t, 2H, CH2–COO), 1.6–0.88 ppm (m, 29H, aliphatic H). 13C-NMR
(CDCl3, 100 MHz) d: 172.75 (NH–CO), 171.52 (CO–O), 158.42,
144.74, 129.20, 121.27, 114.58, 111.32 (Ar-C), 71.85 (CH2–OCH3),
70.48 (O–CH2–CH2), 68.92, 66.52 (Ar-OCH2), 63.70 (COO–CH2),
58.96 (O–CH3), 39.05 (CH2–N), 35.97, 31.87, 31.38, 29.64, 29.32,
22.64, 14.08. FT-IR (cmꢂ1): 3309, 2848, 2915, 1741, 1640, 1552,
1454, 1405, 1351, 1293, 1249, 1203, 1166, 1106, 1045, 952, 857,
777, 696. MALDI-TOF-MS: m/z calculated for C34H59NO7: 593.43,
and found: 616.35 (M+ + Na+).
Compound 3 (2.00 g, 4.5 mmol) and triuoroacetic acid (10 mL,
134.2 mmol) was added drop wise and 1b (1.20 g, 5.5 mmol),
DCC (1.35 g, 6.5 mmol) and DMAP (0.07 g, 0.6 mmol) were used.
The product was puried by passing through a silica gel column
with a 60–120 mesh using 50% ethyl acetate in pet ether as the
eluent. Yield ¼ 1.2 g (40.0%). 1H-NMR (CDCl3, 400 MHz) d: 7.19
ppm (t, 1H, Ar-H), 6.81–6.70 ppm (m, 3H, Ar-H), 6.18 ppm (CO–
NH), 4.24 ppm (t, 2H, COO–CH2), 4.03 ppm (t, 2H, Ar-OCH2),
3.70–3.63 ppm (m, 6H, OCH2CH2O), 3.56 ppm (t, 2H, CH2–N),
3.39 ppm (s, 3H, CH2–OCH3), 2.70 ppm (t, 2H, Ar-CH2), 2.57
ppm (t, 2H, NH–CO–CH2), 2.51 ppm (t, 2H, CH2–COO), 1.6–0.88
ppm (m, 29H, aliphatic H). 13C-NMR (CDCl3, 100 MHz) d: 172.88
(NH–CO), 171.61 (CO–O), 158.54, 144.86, 129.31, 121.40, 114.71,
111.46 (Ar-C), 71.93 (CH2–OCH3), 70.50 (Ar-OCH2), 69.10, 66.65
(CH2–OCH2), 63.81 (COO–CH2), 59.11 (O–CH3), 39.16 (CH2–N),
36.09, 31.99, 31.49, 29.75, 29.67, 22.76, 14.19 (aliphatic C). FT-IR
(cmꢂ1): 3306, 2848, 2913, 1739, 1639, 1551, 1454, 1402, 1348,
1250, 1203, 1134, 1047, 961, 863, 757, 713. MALDI-TOF-MS: m/z
calculated for C30H51NO5: 549.40 and found: 572.32 (M+ + Na+).
Similarly, two other amphiphiles, DD–TEG and CAR–TEG, were
also synthesized and their details are given in the ESI.†
Optical transmittance measurement
Optical transmittance of the amphiphile and drug loaded
nanoparticles was measured using a quartz cell (path length:
1 cm) with s Perkin-Elmer Lambda 45 UV-Visible spectropho-
tometer which was equipped with a temperature-controller. The
ꢀ
ꢀ
sample was heated from 30 C to 80 C in a stepwise manner
with an interval of 5 ꢀC. Similarly, a cooling cycle was recorded
from 80 C to 30 ꢀC with an interval of 5 C.
ꢀ
ꢀ
Doxorubicin and CPT encapsulation
Synthesis of 2-methoxyethyl 4-oxo-4-((2-(3-pentadecylphenoxy)
ethyl)amino)butanoate (PDP–EG)
The ability of these core–shell nanoparticles to encapsulate
hydrophobic molecules in the hydrophobic inner core was
Compound 3 (2.00 g, 4.5 mmol), triuoroacetic acid (10 mL, determined by using DOX. DOX$HCl (0.5 mg) was neutralized
134.2 mmol), 1a (0.50 g, 2.8 mmol), EDC (0.59 g, 2.2 mmol) and with triethylamine prior to the encapsulation. DOX (0.5 mg) and
diisopropylethylamine (0.98 mL, 5.7 mmol) were used. The PDP–TEG (5.0 mg) were added to DMSO (1.0 mL). To it trieth-
product was puried by passing through a silica gel column ylamine (1.5 equivalents to DOX) and water (3.0 mL) were added
ꢀ
with a 60–120 mesh using 70% ethyl acetate in pet ether as the and stirred at 25 C for 12 h. It was then extensively dialyzed
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(SPECTRA/POR, MWCO-500-1000) against deionized water (200 phenoxy)ethyl carbamate (3). Compound 3 was further hydro-
mL) for 48 h. The DOX encapsulated solution was ltered lysed by triuoroacetic acid (TFA) and coupled with 1a–1c to
through a 0.45 mm lter and the sample was freeze-dried in a give the amphiphiles. These amphiphiles are named PDP–X,
lyophilizer.
where X ¼ EG, DEG, and TEG with respect to the number of
CPT was encapsulated as described. PDP–TEG (10.0 mg) and (CH2CH2)xO units x ¼ 1, 2, and 3, respectively. Two more
CPT (1.0 mg) were dissolved in 2.0 mL of DMSO and stirred for amphiphiles based on cardanol (CAR, have unsaturated double
15 minutes at 25 ꢀC. Water (5.0 mL) was added to the above bonds in the C15 alkyl tail) and dodecyl amine (DD)
solution drop wise and the mixture was further stirred for 12 h. (see structures in Scheme 1) were also prepared and their details
It was transferred to a dialysis bag (SPECTRA/POR, MWCO-500- are given in the ESI (see SS-1).†
1000) and dialyzed against deionized water (200 mL) for 48 h.
The structures of the amphiphiles were characterized by 1H,
The CPT encapsulated solution was ltered through a 0.45 mm 13C-NMR, and MALDI-TOF (see the ESI†). The solubility of the
lter and the sample was freeze-dried in a lyophilizer. Drug amphiphiles in water was found to be dictated by the number of
loading efficiency (DLE) and drug loading content (DLC) were the ethyleneoxy units rather than the hydrophobic units (PDP,
calculated using the following equations:17
CAR or DD). For example, the amphiphiles with very short
(CH2CH2O)x units, PDP–EG and PDP–DEG, were found to be
completely insoluble in water (thus, these two short amphi-
philes were not included in any further studies). On the other
hand, the other three amphiphiles, PDP–TEG, CAR–TEG and
DD–TEG, were found to be dispersible in water or PBS buffer
DLE (%) ¼ {weight of encapsulated CPT/weight of CPT in feed}
ꢃ 100%.
DLC (%) ¼ {weight of CPT in nanoparticles/weight of CPT
loaded nanoparticles} ꢃ 100%.
ꢀ
(pH ¼ 7.4) at 25 C.
To study the thermo-responsive behaviours of the amphi-
philes, they were subjected to optical transmittance measure-
For the above purpose, approximately 1.5 mg of drug loaded
nanoparticles were dissolved in DMSO (2.0 mL) and their
absorbance was measured to determine the DLE and DLC using
their molar extinction coefficients {3CPT ¼ 10 500 (in PBS),
3CPT ¼ 11 250 (in DMSO) and 3DOx ¼ 4188 (in PBS), 3DOx ¼ 7035
(in DMF)}.
ments as
a function of temperature using absorption
spectroscopy. The plot of the optical transmittance of PDP–TEG
is shown in Fig. 2a. The plot consists of data from two consec-
utive heating and cooling cycles from 30 ꢀC to 80 ꢀC. The plots
reveal that the optical transmittance was 90% below 40 ꢀC
(solution was clear) and the sample became opaque and turbid
above 42 ꢀC (<50% transmittance). The vials in the photographs
clearly show the change in the transmittance in the heating and
cooling cycles. Further, the plots also revealed that the forma-
tion and clearance of turbidity in the heating and cooling cycles,
respectively, follow different kinetic paths. For example, in the
In vitro drug release studies
The release prole of DOX was studied using the dialysis
method. Briey, 3.0 mg of drug loaded sample was dispersed in
3.0 mL of PBS and the content was transferred into a dialysis
bag, which was ꢀthen immersed in 100 mL of PBS and was
incubated at 37 C. Periodically, 3.0 mL of solution was with-
drawn from the system and was replaced with 3.0 mL of fresh
PBS solution. The aliquots obtained were then subjected to
absorbance measurements and the amount of DOX released
was calculated. The release prole of DOX was also studied at
25 ꢀC and 44 ꢀC. Similarly, 3.0 mg of CPT loaded
nanoparticlesꢀwere subjected to in vitro release studies at 25 ꢀC,
ꢀ
heating cycle, the appearance of turbidity began at 40 C and
slowly completed itself at 70 ꢀC. On the other hand, in the
cooling cycle the change from a turbid to a clear solution
occurred sharply at 45 ꢀC. This suggested that self-assembly was
a slow process in the heating cycle whereas the reversibility was
very sharp in the subsequent cooling. Thus, the lower
critical solution temperature (LCST) of the PDP–TEG was
ꢀ
assigned as 42 C.
ꢀ
37 C and 55 C.
The thermo-responsive nature of PDP–TEG at a concentra-
tion ranging from 1 ꢃ 10ꢂ5 to 1 ꢃ 10ꢂ3 M was investigated and
the cooling cycle data are shown in Fig. 2b (see Fig. S1 for their
heating cycle data†). The LCST of PDP–TEG decreased with an
increasing amphiphile concentration. At very low concentra-
Results and discussions
Synthesis and thermo-responsiveness of amphiphiles
The synthesis of amphiphilic molecules from commercially tions, PDP–TEG did not show any phase-separation
available ethylene glycols as the hydrophilic part and the phenomena. This indicated that the LCST of the amphiphile
renewable resource 3-pentadecylphenol (PDP) as the hydro- was a concentration driven process. Further, the complete
phobic part is shown in Scheme 1. Briey, succinic anhydride reversibility of the thermo-responsive behaviour of the amphi-
was ring opened with oligoethylene glycol monomethyl ether philes was studied by measuring the optical transmittance of
derivatives CH3O(CH2CH2)xOH, where x ¼ 1, 2 and 3 in pres- the amphiphiles both at temperatures above and below LCST
ence of Et3N to give acids 1a–1c. Ethanolamine was reacted with for ten consecutive cycles. And it was observed that the thermal
Boc-anhydride to give tert-butyl (2-hydroxyethyl) carbamate (2). response of the amphiphiles was completely reversible
Compound 2 was reacted with PDP under Mitsunobu coupling (see Fig. S2†). The cloud points obtained for each concentration
reaction in the presence of triphenylphosphine and diisopropyl were plotted and are shown in Fig. 2c. The plot reveals that
azodicarboxylate to give tert-butyl (2-(3-pentadecy-8-en-1-yl)- LCST varied linearly with the concentration of the amphiphile
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Scheme 1 Synthesis of thermo-responsive amphiphiles.
also played a crucial role in the molecular self-assembly.
Further, the importance of the amide linkages in the amphi-
philic design was also investigated by checking the LCST
behaviour for structurally identical amphiphiles having an ester
linkage (ester linkage instead of amide, see Fig. S3†).28 This
ester molecule did not show signicant LCST behaviour. Thus,
an appropriate molecular design is essential to make the small
molecular derivatives such as PDP–TEG thermo-responsive
amphiphiles. In the present case, the combination of the
renewable hydrophobic PDP unit, amide linkages and hydro-
philic triethylene glycol monomethyl ether units, provided the
appropriate molecular geometry for thermo-responsiveness in
PDP–TEG amphiphiles.
Fig. 2 (a) Temperature dependent optical transmittance of PDP–TEG
(10ꢂ4 M) in water in consecutive heating and cooling cycles. (b) Optical
transmittance of PDP–TEG in the cooling cycle for concentrations
varying from 10ꢂ3 M to 10ꢂ5 M. (c) Plot of the cloud point of PDP–TEG
versus concentration. (d) Optical transmittance of CAR–TEG and DD–
TEG in the heating and cooling cycles (10ꢂ4 M) in water.
Shape and size of the amphiphile self-assembly
To study the thermo-responsive self-organization of the
amphiphile in water, PDP–TEG was subjected to variable
temperature dynamic light scattering (DLS) and static light
scattering (SLS) studies. The DLS histograms of the aggregates
(with an increasing concentration from 10ꢂ5 M to 10ꢂ3 M), the
cloud point decreased from 56 ꢀC to 32 ꢀC. The slope of the line
indicates that the cloud point changes by 13.10ꢀ molꢂ1 Lꢂ1 (or
0.022ꢀ gꢂ1 Lꢂ1) of the amphiphile concentration. The thermo-
responsive PNIPAM-copolymer was found to show a cloud point
over the concentration range 0.51ꢀ gꢂ1 Lꢂ1
.
This suggested
27
that the custom designed amphiphile was capable of showing
thermo-responsiveness at much lower concentration
a
(ꢁ25 times lower concentration) compared to high molecular
weight polymers like PNIPAM. Hence, it may be concluded that
PDP–TEG is a potential amphiphilic molecule with a thermo-
responsive behaviour equivalent to that of high molecular
weight polymers for drug delivery applications.
To study the inuence of hydrophobic units on the thermo-
responsive behaviour, both DD–TEG and CAR–TEG were
investigated and their optical transmittances are shown in
Fig. 2d. DD–TEG did not show LCST phenomena while CAR–
TEG showed very weak phase-separation. This suggested that
the nature of the hydrophobic unit in the amphiphile structure
Fig. 3 (a) Variable temperature DLS histograms of PDP–TEG in water
at 10ꢂ4 M. (b) Plot of the hydrodynamic radius versus temperature of
PDP–TEG. (c) Average radius of gyration (Rg) of PDP–TEG obtained
from static light scattering. The inset shows the plot of Rg/Rh versus
temperature.
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at different temperatures were measured for a 10ꢂ4 M solution
and their plots are shown in Fig. 3a. PDP–TEG showed mono-
modal distributions at all the temperatures indicating the
formation of homogeneous size aggregates. The stability of
these nanoparticles was also investigated in different pH solu-
tions (see Fig. S4†) and it was found that these nanoparticles
were stable from the acidic to basic pH of 4.0 to 10.0. The
hydrodynamic radius of the aggregates (half of their hydrody-
namic diameter) from the DLS were plotted as a function of
temperature and shown in Fig. 3b. The gure signied that the
hydrodynamic radius of these aggregates decreased with
increasing temperature. Below LCST the hydrodynamic radius
of these aggregates was 110 ꢄ 10 nm while above LCST it
reduced to 45 ꢄ 4 nm. From the plot of Rh versus the tempera-
ture, the break point was obtained as 40 ꢀC, which is almost
identical to the onset temperature for the phase separation (see
Fig. 2a). The reversibility of the self-assembly process of the
amphiphiles was further investigated by dynamic light scat-
tering techniques (equipped with laser source, excitation 633
nm) for ten consecutive cycles. The amphiphiles showed
complete reversibility at temperatures both above and below
LCST (see Fig. S5†).
Fig. 4 (a) FE-SEM image of PDP–TEG at 30 ꢀC. (b) HR-TEM image at
30 ꢀC. (c) FE-SEM images of PDP–TEG at 45 ꢀC. (d) TEM images of
PDP–TEG at 45 ꢀC. (e) AFM image of PDP–TEG at 30 ꢀC. (f) AFM image
of PDP–TEG at 45 ꢀC.
SLS measurements were carried out on a 10ꢂ4 M solution by
heating the sample in a stepwise manner with an interval of 5
ꢀC. The intensity of the scattered light obtained at various
angles and at different temperature was then plotted against q2,
where q is the scattering vector magnitude and the plot
obtained is known as the Guinier plot (see Fig. S6†). The slope structures were found to be hollow, similarly to the core–shell
of the Guinier plot gives (Rg)2/3, from which the radius of gyra- nanoparticles. AFM images in Fig. 4f also support the formation
tion (Rg) was calculated. The plot of the radius of gyration of elongated structures above LCST. From all three images
against temperature is shown in Fig. 3c. The plot revealed that (Fig. 4c, d and f), it is very clear that the amphiphiles exist as
with increases in temperature, the radius of gyration decreased rod-like nanoparticles above LCST and as core–shell nano-
from 180 nm to 150 nm. Utilizing the Rg and Rh values obtained particles below LCST.
at various temperatures from the DLS and SLS measurements,
To prove the existence of the in situ shape transformation in
the Rg/Rh ratio was determined (see inset in Fig. 3c). The Rg/Rh the thermo-responsive scaffolds (see Fig. 5a) in the heating and
increase from 1.6 to ꢁ3.0 as an indication of the transformation cooling cycles, the dimensions of the core–shell structures were
from globular to rod-like structures.29,30
compared with the rod-like objects. For the above trans-
To visualize the shape and size of the aggregates formed by formation, one would expect the circumference (2pr, where r ¼
the amphiphile, the samples were subjected to eld emission radius of the core–shell) of the spherical core–shell structure to
scanning electron microscopy (FE-SEM), high resolution trans-
mission electron microscopy (HR-TEM), and atomic force
microscopy (AFM) analysis. FE-SEM, HR-TEM and AFM images
of PDP–TEG at 25 ꢀC (below LCST) are given in Fig. 4. In Fig. 4a,
the FE-SEM image of the PDP–TEG shows the existence of
spherical core–shell like aggregates with 250 ꢄ 17 nm diame-
ters. The formation of these core–shell structures 255 ꢄ 24 nm
in diameter was further conrmed by HR-TEM (see Fig. 4b). The
TEM image shows a hollow hydrophobic core surrounded by a
hydrophilic shell (dark layer). Further, the AFM image (see
Fig. 4e) also shows the existence of 220 ꢄ 20 nm spherical
particles. The images of the aggregates above LCST are shown in
Fig. 4c, d and f. In Fig. 4c, the aggregates show the formation of
clusters instead of isolated particles (as observed below LCST in
Fig. 4a). Further, the shape of the aggregates was also trans-
formed from spherical to rod-like structures. The formation of
these rod-like nanostructures was further conrmed by HR-
Fig. 5 (a) Mechanism of shape transformation in thermo-responsive
scaffolds. (b) Single crystal structure of molecule 3 in Scheme 1. (c)
Three dimensional packing of molecule 3 along a-axis showing the
inter-digitations of the hydrophobic tails and inter-chain hydrogen
TEM image (see Fig. 4d). The internal parts of the rod-like bonding.
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be equal to twice the lengths (L) plus the widths (W) of the nano- other via hydrophobic–hydrophobic interactions and were
rods.31 The average diameter of core–shell nanoparticles was inter-digitated. While the enlarged view of the molecular
254 ꢄ 35 nm, 225 ꢄ 40 nm and 183 ꢄ 25 nm from FE-SEM, TEM packing (see Fig. 5c) revealed that the molecules were inter-
and AFM, respectively. Thus, the average diameter of the core– locked via interchain hydrogen bonding present between the
shell nanoparticles based on all three techniques together was amide linkages of the amphiphiles. Based on the morphology
found to be 220 ꢄ 36 nm (average radius ¼ 110 ꢄ 17 nm). The (FE-SEM, HR-TEM and AFM), variable temperature NMR and
average diameter of the rod-like structure above LCST was 94 ꢄ single crystal structure study, it may be concluded that the
16 nm, 88 ꢄ 18 nm and 108 ꢄ 16 nm from FE-SEM, TEM and newly designed PDP–TEG was a very unique molecule to
AFM, respectively. Likewise, the length of these rod-like struc- undergo thermo-responsive phase transition from core–shell to
tures was 178 ꢄ 37 nm, 193 ꢄ 35 nm and 185 ꢄ 16 nm from FE- rod-like structures.
SEM, TEM and AFM, respectively. All three techniques together
gave a diameter and length of the rod-like structures equal to
97 ꢄ 10 nm and 185 ꢄ 8 nm, respectively. The average
Anticancer drug encapsulation
circumference of the core–shell structure (below LCST) was The thermo-responsive amphiphile was further utilized as a
calculated as 2pr ¼ 0.69 ꢄ 0.11 mm. This value matches the 2L + scaffold for loading and delivering anticancer drug molecules.
2D ¼ 0.57 ꢄ 0.04 mm of the rod within the experimental error Two different hydrophobic anticancer drugs, doxorubicin
limit. Thus, the average sizes of the rod-like structure were in (DOX) and camptothecin (CPT), were chosen as drug candidates
close agreement with the average diameter of the core–shell to demonstrate the proof-of-concept of the shape transformable
nanoparticles. This conrmed that the core–shell nanoparticles thermo-responsive scaffold. The drugs were encapsulated in the
collapsed above LCST to produce rod-like structures. The hydrophobic interior of the core–shell particle by the dialysis
resultant rod-like structures aggregated together to produce a method. The drug loading content was estimated using absor-
turbid solution above LCST which was completely reversible in bance spectroscopy as 4.2 wt% and 1.6 wt% for DOX and CPT,
the subsequent cooling cycle (see Fig. 2).
respectively. The sizes of the DOX and CPT loaded scaffolds
The in situ shape transition from the hydration (below LCST) were determined by DLS and they were found to be 220 ꢄ 20 nm
to the dehydration (above LCST) state of PDP–TEG was further and 190 ꢄ 20 nm, respectively (see Fig. 6a and b). The sizes of
supported by variable-temperature 1H-NMR studies (see Fig. S7 the drug loaded particles was similar to that of the nascent ones
for more details†). The 1H-NMR spectra of PDP–TEG at various (see Fig. 3a) indicating that the scaffold retained its self-orga-
temperatures were recorded in D2O with an interval of 10 ꢀC nization even aer the encapsulation of the hydrophobic drugs.
from 30 to 70 ꢀC. Below LCST, the signals corresponding to the The temperature-dependent phase-transition of the DOX and
hydrophobic tail (1.6–0.88 ppm) and aromatic protons (7.18– CPT loaded core–shell nanoparticles was investigated and the
6.69 ppm) of PDP in the amphiphile appeared with less inten- data are given in Fig. 6c and d. The drug loaded scaffolds
sity. This was attributed to the lower exposure of the hydro- preserved the reversible self-organization in the heating cooling
phobic part of the amphiphile in the aqueous environment in cycles similarly to their un-loaded core–shells. The LCST of the
the core–shell state.32,33 With increase in temperature, the DOX encapsulated scaffold was found to be 40 ꢀC which was
intensity of the signals corresponding to the hydrophobic tail almost closer to the nascent scaffold (42 ꢀC). On the other hand,
(1.6–0.88 ppm) and aromatic protons (7.18–6.69 ppm) were the LCST of the CPT loaded scaffolds (see Fig. 6d) was found to
enhanced. This was attributed to the breaking of the hydrogen be 50 ꢀC which was 8–10ꢀ higher than that of the nascent
bonds of the amide-linkage with water molecules resulting in scaffold.
an increase in chain mobility.34 These signals showed a
complete reversibility in the subsequent cooling cycles. Thus,
below LCST, the PDP–TEG amphiphile exist in the form of core–
shell nanoparticles. As the temperature was increased above
LCST the hydrophilic segments collapsed on the top of the
hydrophobic core to produce rod-like assemblies. This type of
transition would occur only if the hydrophobic segments are
tightly held together in the inner core to facilitate the collapsing
or un-coiling of PEG chains at the periphery of the nano-
scaffolds.
To provide evidence for the strong packing of the hydro-
phobic PDP units, the single crystal for compound 3 (see
Scheme 1) was obtained in a dichloromethane–methanol
solvent mixture (2 : 3 v/v) (for more details see Fig. S8, S9 and
ST1 in the ESI†). As it can be seen in Fig. 5b and c, the terminal
Boc-protected amine group and the long alkyl tail were arranged
in a trans-conrmation with respect to each other. The three
Fig. 6 DLS histogram of DOX (a) and CPT (b) encapsulated scaffolds.
dimensional packing of the molecules along the a-axis showed
(see Fig. 5c) that the alkyl chains were extended towards each (d) CPT loaded PDP–TEG in water at 10ꢂ4 M.
Temperature dependence of the transmittance of (c) DOX loaded and
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The morphologies of the drug loaded scaffolds are shown in nanoparticles are shown in Fig. 7g and h, respectively. Absor-
Fig. 7. In the FE-SEM images DOX loaded scaffolds (see Fig. 7a) bance and emission spectra of free DOX as well as DOX loaded
appear as spherical particles with diameters of 200 ꢄ 10 nm. nanoparticles were recorded in water (see Fig. 7i). The absor-
Above LCST (at 45 ꢀC), the shape of the drug loaded amphiphile bance and emission spectra of DOX (see Fig. 7i) did not show
underwent morphological transformations from spherical to any variation upon encapsulation as compared to free DOX.
rod-like structures (see Fig. 7b). These rod-like structures were
Similarly, the absorbance and emission spectra of free CPT
aggregated together to produce bundles which were arranged in and CPT loaded scaffolds (see Fig. 7j) were found to be almost
a dendritic fashion (see larger area image Fig. 7c). In Fig. 7d, the identical, which indicated that the properties of CPT did not
TEM images of the DOX loaded scaffolds conrmed the exis- change upon encapsulation. This was further conrmed by the
tence of the dendritic nature of the rod-like structures. Though uorescence lifetime of the free drug as well as the drug loaded
the CPT loaded scaffolds appear as spherical particles having a nanoparticles by TCSPC techniques. The decay prole of DOX
diameter of 160 ꢄ 10 nm below LCST (see Fig. 6e), they and CPT in the absence and presence of nanoparticles was
underwent shape transformations into nano-brous structures collected at 558 nm and 448 nm respectively, using a nano-LED
at higher temperatures (see Fig. 7f). These brous structures are laser source with 460 nm (for DOX) and 347 nm (for CPT) as the
typically produced by the long range aggregation of the drug excitation wavelength (see Fig. S10 for more details†). The decay
plus scaffold. Thus, it may be concluded that the DOX loaded proles were tted by bi-exponential decay ts using the DAS6
core–shell structures retained the in situ phase transitions of program and their lifetime data are summarized in Table ST2 in
nascent scaffolds whereas a long nano-brous morphology was the ESI.† The TCSPC lifetime values (s1) of DOX upon encap-
obtained for the CPT loaded core–shells.
sulation were found to be 1.49 ns, which are in close agreement
As both DOX and CPT are uorescent in nature, the drug with the lifetime value of free DOX (0.95 ns). Similarly, lifetime
loaded nanoparticles were subjected to uorescence micros- (s1) of CPT in loaded as well as free form was 4.66 ns and
copy analysis as well as photophysical studies in order to 4.59 ns, respectively. The DOX and CPT loaded nanoparticles
elucidate their properties in free and encapsulated states. The retained their original structural features inside the scaffolds.
uorescence microscopy images of both DOX and CPT loaded
In vitro drug release studies
The thermo-responsive drug release of DOX and CPT loaded
nanoparticles was studied under physiological conditions (PBS,
pH ¼ 7.4) as well as for a cancer tissue environment (PBS, pH ¼
6.8 see Fig. S11†). It was observed that the release proles of the
drug at pH ¼ 7.4 or pH ¼ 6.8 were identical, indicating that the
scaffold was very stable and was also capable of preserving the
drug at both pH 6.8 and 7.4. The temperatures for these studies
were chosen based on the physiological temperature in cancer
ꢀ
ꢀ
tissues (40–43 C), normal body temperature (37 C) and drug
storage at ambient temperature (25 ꢀC) (see Fig. 1). The drug
loaded scaffolds were subjected to incubation at these three
ꢀ
ꢀ
ꢀ
different temperatures, i.e. 25 C, 37 C and 44 C. The cumu-
lative release proles of DOX are shown in Fig. 8a. The
Fig. 7 FE-SEM image of DOX encapsulated PDP–TEG at 30 ꢀC (a),
45 ꢀC (b), and large area image at 45 ꢀC (c) in water at 10ꢂ4 M. (d) TEM
image of DOX encapsulated PDP–TEG at 45 ꢀC in water at 10ꢂ4 M. FE-
SEM image of CPT encapsulated PDP–TEG at 30 ꢀC (e), and 45 ꢀC (f) in
water at 10ꢂ4 M. Fluorescence microscopy images of DOX loaded
scaffolds (g), and CPT loaded scaffolds (h) in water at 10ꢂ4 M. (i) Fig. 8 Cumulative release profile of (a) DOX and (b) CPT loaded
Absorbance and emission spectra of free DOX and DOX loaded scaffolds. (c) Kinetic plots of DOX and CPT loaded scaffolds. Table
scaffolds. (j) Absorbance and emission spectra of free CPT and CPT contains the values of the rate constant (k) and n of the DOX and CPT
loaded scaffolds.
releases.
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percentage release of DOX at temperatures below LCST, i.e. to the release of DOX in a controlled manner while the change
37 ꢀC and 25 ꢀC, was less than 20%. This suggested that the in the morphology (nan-brous structures) in the CPT loaded
DOX loaded scaffold is very stable both at ambient as well as scaffold results in a burst release. Though, the approach
normal body temperature. At the cancer tissue temperature demonstrated here only for two hydrophobic drugs (DOX and
(above LCST at 44 ꢀC), the DOX loaded scaffold showed a CPT), in principle, it is applicable to a wide range of hydro-
selective release of more than 95% of the drug within 5 h. At 25 phobic drugs which need to be explored. Thus, the custom
ꢀC, the CPT loaded particles (see Fig. 8b) were stable enough, designed thermo-responsive amphiphile is a potential candi-
however, at body temperature (37 ꢀC) more than 80% of the date for the loading and delivering of anticancer drugs like DOX
drug was released. At temperatures above LCST (55 ꢀC), almost selectively at the cancer tissue temperature. The current inves-
95% of the drug was released within 5 h. This suggested that the tigation provides for the rst time insight into the development
CPT loaded scaffold lost its ability to selectively release the drug of thermo-responsive shape-transformable amphiphilic drug-
at the cancer tissue temperature.
carriers. The concept was successfully demonstrated based on a
The release of the drug from the polymer matrix involved new molecular design as well as delivering the anti-cancer drug
several processes such as diffusion of the drug from the exclusively at the cancer tissue temperature. The cytotoxicity of
membrane, erosion of the polymeric scaffolds, and so on. the amphiphile and loaded nanoparticles and their cellular
Peppas and co-workers developed a semi-empirical model, as uptake mechanism are yet to be studied to conrm their bio-
given below, for the drug release:35
logical activity.
Mt
¼ ktn
(1)
(2)
MN
Conclusion
ꢀ
ꢁ
The present investigation demonstrated the design and devel-
opment of in situ shape transformable and thermo-responsive
core–shell scaffolds for the rst time and established their
ability to load and deliver anticancer drug molecules at the
cancer tissue temperature. For this purpose, a new amphiphilic
molecule consisting of oligoethylene glycols and the renewable
resource 3-pentadecyl phenol as the hydrophilic and hydro-
phobic units, respectively, was custom designed. The amide
linkage was used as the self-organization director to facilitate
the self-assembly in an aqueous medium with respect to the
lower critical solution temperature. The amphiphile self-
assembled to produce core–shell nanoparticles at ambient
temperature which underwent transformations into one
dimensional rod-like assemblies at temperatures closer to the
cancer tissue temperature. Dynamic and static light scattering
conrmed the occurrence of the in situ phase transition with
respect to the Rg/Rh ratio. Morphological analysis by FE-SEM,
HR-TEM and AFM provided direct evidence for the existence of
the amphiphilic core–shell spherical morphology below LCST
and rod-like structures above LCST. The shape transformation
was further conrmed by carrying out detailed calculations on
the circumference of the core–shell and rod-like assemblies.
Mt
log
¼ n log t þ log k
MN
where, Mt and MN are the cumulative amount of drug released
at time t and innite time, respectively, k is a constant that
depends on the structural and geometric characteristics of the
polymer, and n is the release exponent which indicates the drug
release mechanism. In case of spherical particles, the value of n
¼ 0.43 for Fickian diffusion and $0.85 for non-Fickian diffu-
sion. This equation generally holds for the rst 60% of the
fractional drug release or for values in the interval of 0.1 <
Mt/MN
< 0.7. This methodology was adopted by many
researchers to study the drug release kinetics for micelles,
vesicles and nanogels, and so on. Zhuang and co-workers used
the above mentioned equation to study the release mechanism
of drug from nanogels,36 Lecommandoux and co-workers have
used the above equation to analyse the release mechanism of
drug from polymersomes.37 The Peppas model was currently
employed by Surnar et al. from our research group and used the
expression to analyse the mode of drug release from poly-
caprolactone vesicular assemblies.11,38 In the present investiga-
tion, drug release from the thermo-responsive polymer matrix
was analysed by the Ritger and Peppas equation and the data
are summarized in Fig. 8. The drug release proles were tted to
the above equation and their kinetics plots log (Mt/MN) against
log t are shown in Fig. 8c. The rate constant k and n values are
reported in the table in Fig. 8. The DOX loaded particles fol-
lowed non-Fickian diffusion (n ¼ 1.052) for selective delivery at
the cancer tissue temperature. On the other hand, the CPT
loaded scaffold showed an unusual trend in which either
non-Fickian diffusion (n ¼ 0.824) or Fickian diffusion process (n
¼ 0.357) occurs with respect to the normal body and cancer
tissue temperatures.
1
Variable temperature H-NMR studies and single crystal struc-
tures established the existence of strong inter-digitations
among the hydrophobic units which facilitated the thermo-
responsive shape transformation. Anticancer drugs, doxoru-
bicin (DOX) and camptothecin (CPT), were successfully loaded
into the core–shell nanoparticles. These drug loaded nano-
scaffolds retained their thermo-responsive molecular self-
organization, similarly to that of their nascent amphiphiles.
In vitro studies revealed that the DOX loaded scaffolds were very
stable at normal body temperature (37 ꢀC) and exclusively
collapsed to release more than 90% of the drug at 44 ꢀC, which
is similar to that of cancer tissues under physiological condi-
tions. The drug release kinetics indicated that DOX underwent
non-Fickian diffusion. Nevertheless, the present investigation
provides for the rst time insight into the development of in situ
The difference in the release rate of DOX and CPT can be
attributed to the difference in their morphology obtained at
higher temperatures (T > LCST). The retention of the rod-like
structure in the DOX loaded scaffold (similar to nascent one) led
This journal is © The Royal Society of Chemistry 2014
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Journal of Materials Chemistry B
Paper
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19 Z. Ge and S. Liu, Chem. Soc. Rev., 2013, 42, 7289–7325.
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Acknowledgements
The authors thank the research grant from the Department of
Science and Technology (DST), New Delhi, India, under nano-
mission initiative project SR/NM/NS-42/2009 and the SERB-
scheme under the project SB/S1/OC-37/2013. Smita Kashyap
thanks CSIR, India for Ph.D. research fellowship. The authors
thank HR-TEM facilities and SLS facilities at NCL-Pune.
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