Fabrication of nanostructures through self-assembly of non-ionic amphiphiles for biomedical applications

Article abstract of DOI:10.1039/c6ra28654b

Fabrication of self-assembled nanostructures with defined size and morphology represents a formidable challenge and thus, has gained tremendous momentum in research because of their potential applications in various biological systems. Herein, we report o

Full text of DOI:10.1039/c6ra28654b

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Fabrication of nanostructures through self-
assembly of non-ionic amphiphiles for biomedical
Cite this: RSC Adv., 2017, 7, 22121
applications†
b
c
b
Suchita Prasad,a Katharina Achazi,
Christoph Bottcher, Rainer Haag
*
¨
a
*
and Sunil K. Sharma
Fabrication of self-assembled nanostructures with defined size and morphology represents a formidable
challenge and thus, has gained tremendous momentum in research because of their potential
applications in various biological systems. Herein, we report on the synthesis of novel non-ionic
amphiphiles using 2,2-di(prop-2-yn-1-yl)propane-1,3-diol as
a core further functionalized with
poly(ethylene glycol) monomethyl ether and alkyl chains employing a chemo-enzymatic approach.
Surface tension and fluorescence measurements along with dynamic light scattering studies revealed
that all of the amphiphilic systems spontaneously self-assemble in aqueous solution, which is further
supplemented by cryogenic transmission electron microscopy. The solubilization behavior of these
systems as evidenced from UV-Vis and fluorescence spectroscopy and high performance liquid
chromatography suggested the effective encapsulation of hydrophobic entities like Nile red, nimodipine,
curcumin and dexamethasone. A comparative study with a standard excipient, Cremophor® ELP
demonstrated that our nanocarriers exhibited superior/equivalent solubilization behavior for curcumin.
Confocal laser scanning microscopy revealed efficient uptake of encapsulated dye in the cytosol of lung
cancer cells, thus suggesting, that the reported amphiphilic systems can transport drugs into cells. A
study of cytotoxicity showed that the synthesized amphiphilic systems are non-cytotoxic at the
concentrations studied. The release profile of encapsulated Nile red incubated with/without a hydrolase
enzyme Candida antarctica lipase demonstrated that the dye is stable in the amphiphilic nanostructures
in the absence of enzyme for up to 12 days, however, more than 90% release of the dye occurred in 12
days when incubated with lipase. The results advocate the potential of these nanostructures as
prospective drug delivery vehicles.
Received 24th December 2016
Accepted 4th April 2017
DOI: 10.1039/c6ra28654b
rsc.li/rsc-advances
Among the diverse and myriad promising applications offered
by these architectures, the area of drug delivery is of colossal
1. Introduction
importance as more than 80% of drugs are small molecules that
diffuse rapidly all over the body without selectivity because of
their tendency to interact through multiple binding sites,
leading to undesired side-effects.11 Confronted with the chal-
lenges of drug delivery such as low aqueous solubility of drugs,
short half-life in the bloodstream and high drug dosage,
concomitant with the adverse effects associated with the
current solubilizers used,12–14 research on biocompatible nano-
scale drug delivery vehicles which are indispensable to mediate
target specic deployment of drugs, holds substantial promise.
Among the many nano-structured drug delivery vehicles
being studied, amphiphilic architectures have garnered
considerable attention, which stems from the extensive
research in this eld. Amphiphiles, delineated by dual binding
affinity, self-assemble as a consequence of the interplay of
hydrophobic and hydrophilic molecular domains in an aqueous
environment to achieve a state of minimum free energy, leading
to the formation of wide range of morphologies.15–17 Harnessing
The self-assembling behavior of molecular building blocks
(lipids, carbohydrates, proteins and nucleic acids) to generate
structurally precise and functional supramolecular architec-
tures, driven by various non-covalent interactions such as
hydrophobic, ionic, p–p stacking and hydrogen bonding,
outlines the essence of life on earth.1–5 This has prompted the
sophisticated fabrication of an array of structurally diverse and
functional nano-structured materials to open up newer avenues
for biomedical and bio-nanotechnological applications.6–10
aDepartment of Chemistry, University of Delhi, Delhi 110 007, India. E-mail:
sksharma@chemistry.du.ac.in; Tel: +91-11-27666646
b
¨
¨
Institut fur Chemie und Biochemie, Freie Universitat Berlin, Takustraße 3, 14195
Berlin, Germany. E-mail: haag@zedat.fu-berlin.de; Fax: +49-30-838-452633; Tel:
+49-30-838-52633
c
¨
¨
Forschungszentrum fur Elektronenmikroskopie, Institut fur Chemie und Biochemie,
¨
Freie Universitat Berlin, Fabeckstraße 36a, 14195 Berlin, Germany
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra28654b
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the potential of supramolecular assemblies of amphiphiles cytotoxicity prole of all the resulting amphiphilic architectures
requires playing with the experimental variables together with was evaluated using A549 cells (adenocarcinomic human alve-
engineering over their chemical, structural and functional olar basal epithelial cells). Furthermore, the amphiphiles
properties.17 Endowed with an internal cavity, these can be synthesized were explored for their transport potential using
successfully exploited for the delivery of active components.18,19 ‘Nile red, nimodipine, curcumin and dexamethasone’ as model
Though the amphiphilic nanocarriers can be either ionic or dyes/drugs. Cellular uptake and enzyme-triggered release
non-ionic, however, the latter have emanated as potential studies were also performed using Nile red as a model dye.
biomedical candidates and have witnessed tremendous
research due to the limitations experienced by the ionic coun-
2. Results and discussion
terparts, particularly the cationic one.11,20 Gemini surfactants,
characterized by the presence of two hydrophilic head groups
and two hydrophobic tails, linked by a rigid or exible spacer,
holds unique properties and rich self-assembly behaviour, as
a result of which they have gained considerable attention.21,22
Fabricating these dimeric or twin amphiphiles confers them
superior physico-chemical properties compared to the conven-
tional monomeric surfactants e.g. low critical micellar concen-
tration (CMC) as a consequence of the presence of two
hydrophobic tails, high surface activity, high solubilization
capacity, low Kra temperature, unusual rheological proper-
ties, multiple aggregate morphologies, high wetting ability
etc.23,24 The micellar assembly formed by Gemini surfactants
seem adequate as long circulating drug carriers, owing to the
much lower CMC values and longer micellar lifetimes.25 The
above factors make Gemini amphiphiles of special interest for
biomedical applications.
The amphiphilic architectures graed with mPEG (Mn: 550/
1000 g molꢀ1) as hydrophilic moiety and n-alkyl chains (C-10/C-
18) conjugated with aromatic moiety as hydrophobic units, were
synthesized by following the Novozym 435 catalyzed chemo-
enzymatic approach followed by copper catalyzed “1,3-dipolar
cycloaddition reaction” (Schemes 1–3, Fig. 1). The physico-
chemical characterization and solubilization behavior of all
the resulting amphiphiles were studied using various spectro-
scopic and analytical techniques, i.e., NMR, IR, UV, uores-
cence, gel permeation chromatography (GPC), dynamic light
scattering (DLS), and surface tension measurements. The
synthesized amphiphiles were also explored for their cytotox-
icity prole using A549 cell lines.
2.1. Synthesis and characterization
In an endeavor to create novel non-ionic Gemini amphiphilic
constructs having tailored structure, fashioned with suitable
functionalities, we used 2,2-di(prop-2-yn-1-yl)propane-1,3-diol
as the core unit. The diol was successfully graed with alkyl
chains and biocompatible poly(ethylene glycol) monomethyl
ether (mPEG) of variable lengths to confer hydrophobicity and
hydrophilicity, respectively and studied their effect on transport
capacities. A bunch of literature reports reveals that 2,2-di(prop-
2-yn-1-yl)propane-1,3-diol has been successfully used as
The hydrophobic components bearing dialkyne (9/10) were
synthesized following the strategy as outlined in Scheme 1
a
component for metathesis cyclopolymerization.26–33 In
contrast, there are only two reports wherein the diol has been
utilized to afford low-molecular weight amphiphilic molecules.
Das et al. initiated the synthesis of thiophene-capped cyclo-
penta[c]thiophene, further functionalized with carbohydrate
using the diol for Concanavalin A (protein isolated from jack
bean) recognition.34 Further, the diol has been exploited for the
synthesis of Y-shaped sugar-based surfactants by Ali and co-
workers and have been studied for their lyotropic and surface
properties.35 However, this is for the rst time that 2,2-di(prop-
2-yn-1-yl)propane-1,3-diol has been incorporated as a core along
with mPEG as hydrophilic moiety to accomplish the synthesis of
Gemini amphiphiles, which have been further investigated for
their transport behavior using model dyes/drugs. In pursuit of
enhanced stability of supramolecular assemblies and solubility
of hydrophobic guest molecules,36 aromatic units have been
introduced. The synthesized amphiphiles were studied for their
supramolecular aggregation behavior by dynamic light scat-
tering (DLS), surface tension and uorescence measurements
using ‘pyrene’ and ‘Nile red’ as probes. The aggregation
tendency of the amphiphilic molecules was further augmented
Scheme 1 Synthesis of 2,2-di(prop-2-yn-1-yl)propane-1,3-diyl bis(4-
(n-alkyloxy)benzoate); reagents and conditions: (i) NaH, propargyl
bromide, THF, 25 ꢁC, 12 h; (ii) LiAlH4, THF, 25 ꢁC, 12 h; (iii) K2CO3, 1-
bromoalkane, DMF, 40 ꢁC, 6 h; (iv) KOH, ethanol, reflux, 4 h; (v) EDC,
by cryogenic transmission electron microscopy (cryo-TEM). The DMAP, DCM : DMF (4 : 1), 30 ꢁC, 48 h.
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Fig. 1 Representation of synthetic approach and self-assembly of the amphiphiles in aqueous media.
using 2,2-di(prop-2-yn-1-yl)propane-1,3-diol (3) as the core. Di- by the appearance of triplet at d 0.88 ppm (J ¼ 6.9 Hz) inte-
propargylation of diethyl malonate (1) using sodium hydride grating for six protons and other methylenes in d 1.2–1.8 ppm
1
as base afforded diethyl 2,2-di(prop-2-yn-1-yl)malonate (2),37 range in H NMR spectrum.
which on reduction with lithium aluminium hydride led to the
The synthesis of mPEG based hydrophilic motif functional-
formation of compound 3.38 Steglich esterication was ized with azide group (18/19) was successfully achieved from
successfully carried out using the diol 3 and 4-(n-alkyloxy)ben- azido-glycerol (17) and mPEG ethyl ester (15/16) using a Novo-
zoic acid (7/8) employing N-(3-dimethylaminopropyl)-N-ethyl- zym 435-catalyzed biocatalytic route under solvent-free condi-
carbodiimide hydrochloride (EDC) and 4-dimethylaminopyr- tions (Scheme 2). mPEG ethyl esters (15/16) were synthesized
idine (DMAP) to yield 2,2-di(prop-2-yn-1-yl)propane-1,3-diyl from commercially available mPEG-550 (11) and mPEG-1000
bis(4-(n-alkyloxy)benzoate) (9/10). 4-(n-Alkyloxy)benzoic acid (7/ (12) in two steps. mPEG (11/12) was rst oxidized to its corre-
8) in turn, was synthesized from ethyl 4-hydroxybenzoate (4) by sponding acids (13/14) using alkaline KMnO4 in aqueous media
O-alkylation using 1-bromoalkane followed by de-esterication in almost quantitative yield without further purication. The
in the presence of potassium hydroxide as a base and ethanol mPEG-550/1000 acids (13/14) were then converted to their ethyl
as solvent under reux conditions (Scheme 1).39
ester derivatives (15/16) by using a standard esterication
bis(4-(n-alkyloxy) protocol (Scheme 2).40 Azido glycerol (17), used to introduce
2,2-Di(prop-2-yn-1-yl)propane-1,3-diyl
benzoate) (9/10) synthesized following Scheme 1 was completely azide functionality to the hydrophilic part, was synthesized
characterized by IR, 1H, 13C NMR and HRMS. In the IR spectra from commercially available glycerol in four steps following the
of compound 9/10, peak around 3280 cmꢀ1 conrmed the protocol as reported earlier by our group.40
1
presence of terminal alkyne group and in H NMR spectrum,
IR, 1H, and 13C NMR spectra were used to conrm the
the terminal acetylenic proton appeared as a triplet at structure of mPEG acids (13 and 14). In the IR spectra of
d 2.07 ppm due to coupling with the methylene adjacent to the compounds 13 and 14, appearance of an extra peak around 1700
triple bond. The peak for the carbon at the acetylenic end was cmꢀ1 for the carboxyl group and a singlet at d 4.14 ppm, inte-
observed at around d 72 ppm in 13C NMR spectrum, however, grating for two methylene protons adjacent to carboxyl func-
1
the adjacent carbon (–C^CH) appeared relatively downeld (d tionality in H NMR conrmed the oxidation of the alcoholic
79 ppm). The presence of hydrophobic chains was ascertained group. The integration ratio for the peaks of corresponding
Scheme 2 Synthesis of 2-azidopropane-1,3-diyl bis(methoxypoly(oxyethylene)oate); reagents and conditions: (i) KMnO4, NaOH, H2O, 0–80 ꢁC,
24 h; (ii) ethanol, conc. H2SO4 (cat.), 90 ꢁC, 12 h; (iii) Novozym 435, 70 ꢁC, 60 h, vacuum.
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Scheme 3 Synthesis of amphiphiles; reagents and conditions: (i) [Cu(PPh3)3]Br, DIPEA, DCM, 30 ꢁC, 72 h.
methylene protons and PEG main chain established the aromatic region corresponding to (n-alkyloxy)benzoates (d 113,
complete oxidation of mPEG (11/12) to mPEG acid (13/14). The 121, 131 & 162 ppm) and triazolyl ring carbons (d 124 & 142
peak for carboxylic carbon was also observed at around ppm). Furthermore, triazole ring formation also resulted in the
d 171 ppm in 13C NMR. Further, the formation of mPEG ethyl characteristic downeld shi for the methine protons of glyc-
1
ester (15/16) was conrmed by the triplet around d 1.20 ppm erol from d 3.86–3.94 ppm to d 5.37–5.48 ppm in the H NMR
and quartet around d 4.10 ppm in 1H NMR spectrum integrating spectrum, which was further established from 2D heteronuclear
for three and two protons of the methyl and methylene groups correlation (HETCOR) and correlation spectroscopy (COSY)
of ethyl moiety, respectively. Novozym 435-catalyzed synthesis NMR spectra (Fig. S13, ESI†). The integration ratio for the
of hydrophilic scaffolds (18/19) was also characterized utilizing terminal methyl protons of hydrophobic chain and PEG main
the spectroscopic techniques. The peak for azide group at chain protons further conrmed the formation of the desired
around 2100 cmꢀ1 in IR spectrum and multiplets in the range of products.
d 4.11–4.44 and 3.87–3.93 ppm corresponding to methylene
groups of azido glycerol and the ones adjacent to the carboxyl
group of mPEG, and methine of azido glycerol in the H NMR
1
2.2. Physico-chemical characterization of supramolecular
nanostructures
spectrum, respectively conrmed the formation of compounds
18 and 19. Furthermore, in the 13C NMR spectrum, the methy-
All the amphiphilic systems displayed supramolecular aggre-
lene groups next to the carboxyl functionality and those of azido
glycerol appeared at around d 68 and 63 ppm, respectively, and
the methine carbon of azido glycerol appeared at d 58 ppm. The
methoxy carbon of mPEG was observed at d 58 ppm, relatively
deshielded to the methine carbon of azido glycerol.
The different hydrophobic (9/10) and hydrophilic units (18/
19) were coupled together under ‘Click regime’ using tris
(triphenylphosphine)copper(I) bromide and DIPEA with DCM
as a solvent to yield mPEG based non-ionic amphiphiles 20–22
(Scheme 3).
gation behavior in aqueous solution (Fig. 2). The CAC of the
resulting nano-structures was determined by the surface
tension20 (pendant drop method) and uorescence measure-
ments using pyrene41 and Nile red42 as probes. DLS measure-
ments were used for determining the particle size. In order to
supplement the results obtained from DLS measurements and
to have an idea about the morphology of amphiphilic archi-
tectures, cryo-TEM images were also recorded.
2.2.1. Calculation of critical aggregation concentration
using surface tension and uorescence measurements. Surface
tension, one of the most commonly used properties of the
amphiphiles which varies consistently with the concentration
until the aggregates are formed, was used for calculating the
CAC in aqueous media. Once the CAC is attained, surface
tension either becomes constant or changes with a lower slope.
The plot of surface tension values versus log[amphiphile conc.]
led to the observance of curve break and the amphiphile
concentration at this point was referred to CAC, as exemplied
in Fig. 3a and S15 (ESI†). The CAC value of the amphiphilic
The completion of coupling reaction of azide functional
group of hydrophilic scaffold with the alkyne functionalized
hydrophobic chains leading to the formation of amphiphiles
20–22 was conrmed by the disappearance of azide peak at
around 2100 cmꢀ1 in the IR spectra. The formation of product
was further ascertained by the observance of aromatic protons
1
of (n-alkyloxy)benzoates and triazole moieties in the H NMR
spectrum. The 13C NMR spectrum also supported the synthesis
of desired products by the appearance of additional peaks in the
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Fig. 2 Schematic representation of supramolecular aggregation of nanocarriers 20–22.
Fig. 3 Exemplary plots of (a) surface tension and (b) I3/I1 ratio of pyrene (c) Nile red fluorescence intensity versus log concentration of the
amphiphile 22 in aqueous solution at 25 ꢁC.
systems which formed stable micellar aggregates were calcu- in the range of 5.77 ꢂ 10ꢀ5 M to 6.32 ꢂ 10ꢀ5 for all the
lated and are given in Table 1. All the amphiphiles exhibited synthesized amphiphiles (Table 1).
a low CAC value ranging from 1.15 ꢂ 10ꢀ5 M to 1.96 ꢂ 10ꢀ5 M.
The plot of uorescence intensity of encapsulated Nile red
The CAC of an aqueous solution of self-assembled amphi- versus log[amphiphile conc.] gave the CAC value of the amphi-
philes was also determined by calculating the ratio of the philes ranging from 2.69 ꢂ 10ꢀ5 M to 3.01 ꢂ 10ꢀ5 M (Table 1,
uorescence intensity of I3 (385 nm) and I1 (374 nm) vibronic Fig. 3c and S17 (ESI†)). The amphiphile 21 bearing C-10 alkyl
bands of encapsulated pyrene at different amphiphile concen- chains as hydrophobic component and mPEG-1000 units as
trations (Fig. 3b and S16, ESI†). A correlation between the ratio hydrophilic part was found to have the lowest CAC value,
of the rst and third highest energy vibrational bands (I1/I3) of measured by surface tension as well as uorescence method,
pyrene has been established with the solvent polarity.43,44 Below however the amphiphile 22 having C-18 alkyl chains along with
the CAC, assemblies are not present, and the I3/I1 ratio is low, mPEG-1000 aggregated at a relatively higher concentration.
equivalent to that of pyrene in water (0.55).45 However, with the
2.2.2. Dynamic light scattering and cryo-TEM measure-
formation of the assemblies, pyrene gets partitioned into the ments. The aggregation phenomenon of synthesized amphi-
hydrophobic core and an increase in the I3/I1 ratio is observed philes was also studied in aqueous medium by DLS
(up to 1.66 in aliphatic hydrocarbon solvents such as hexane).45 measurements at a concentration of 5 mg mLꢀ1 (Table 1). The
The CAC calculated using uorescence method was found to be size distribution prole was monomodal in volume and
Table 1 Physico-chemical properties of amphiphiles 20–22
CAC (M)
DLS (Dh, nm)
Pyrene uorescence
measurement
Nile red uorescence
measurement
Amphiphile
Surface tension measurement
Ia
Vb
Nc
6.08
6.82
16.07
20
21
22
1.89 ꢂ 10ꢀ5
1.15 ꢂ 10ꢀ5
1.96 ꢂ 10ꢀ5
6.24 ꢂ 10ꢀ5
5.77 ꢂ 10ꢀ5
6.32 ꢂ 10ꢀ5
2.87 ꢂ 10ꢀ5
2.69 ꢂ 10ꢀ5
3.01 ꢂ 10ꢀ5
10.24
11.98
19.32
7.49
8.53
17.49
Intensity distribution. b Volume distribution. c Number distribution. DLS data was measured at 5 mg mLꢀ1 concentration for all the amphiphiles.
a
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number, but exhibited bimodal distribution in intensity which
may be assigned to micelles and micellar aggregates (Fig. S18,
ESI†). This gave an indication that the smaller size particles
observable in volume and number, were the predominant
species in the aqueous solution. In the light of the literature
reports which state that the smaller nanoparticles in the range
of 8–20 nm not only can easily penetrate the deep regions of
interstitial tissue, but also circumvent the rapid renal clearance
of the drugs being delivered,46 it seemed that the size of the self-
assembled system was quite appropriate for transport purposes.
The estimated assembly dimensions of the dominating nano-
sized species in the aqueous solution ranged from 6–19 nm and
followed the order: mPEG-550; C-10 chain (20) < mPEG-1000; C-
10 chain (21) < mPEG-1000; C-18 chain (22).
Assuming that the morphology of the particles is spherical,
DLS is generally used to determine the hydrodynamic size of the
particles and their distribution prole in a sample. However,
the actual morphology of the nanostructures cannot be ascer-
tained by DLS. Therefore, cryo-TEM was utilized to directly
investigate the complementary morphology for compounds 20,
21 and 22. Fig. 4 presents a representative cryo-TEM micrograph
for amphiphile 22 showing a population of micellar assemblies
Fig. 4 Cryo-TEM image of amphiphile 22 showing micellar assembly
(arrows and white circles indicate micelles and surface contamination, with a diameter of 8 nm, with some of them having discoidal
respectively), some of them having slightly discoidal morphology.
morphology. In case of amphiphiles 20 and 21, particles with
diameter < 5 nm were observed (Fig. S19, ESI†), which is
reasonably in agreement with DLS data given that the hydro-
dynamic radius of the assemblies is generally larger than the
visible assembly structure, as the hydration shell does not
contribute density in the cryo-TEM images (Fig. 4).
2.3. Encapsulation potential
To explore the applicability of these new non-ionic amphiphilic
systems as nanocarriers, drug/dye solubilization experiments
were performed. The encapsulation potential of the amphi-
philes was determined for four different exemplary hydro-
phobic entities, the dye Nile red, and the three drugs
nimodipine, curcumin and dexamethasone, all of which might
interact with the nanocarriers through non-covalent interac-
tions such as hydrophobic, p–p stacking and hydrogen
bonding, employing either the lm method47 or solid disper-
sion method.48 The resulting calculations of the transport effi-
ciency, transport capacity and encapsulation efficiency are
presented in Table 2. Further, to get insight into the encapsu-
Fig. 5 Fluorescence intensity of encapsulated Nile red in the amphi-
lation process, drug/dye encapsulated samples were studied by
philes 20–22 in methanol.
DLS which revealed that there was no signicant change in size
Table 2 Transport efficiency, transport capacity and encapsulation efficiency of 20–22 for Nile red, nimodipine, curcumin and dexamethasone
Transport efficiency (mg gꢀ1 Transport capacity (mmol molꢀ1 Encapsulation efficiencye (%)
)
)
Amphiphile
NRa
NIMb
CURc
DXMd
NR
NIM
CUR
DXM
NR
NIM
CUR
DXM
20
21
22
1.12
0.97
5.79
21.03
8.00
22.86
37.89
26.45
41.66
17.33
12.67
12.67
10.86
14.83
93.03
155.28
93.63
279.33
317.87
351.22
578.16
136.47
157.83
165.06
4.66
4.02
23.33
21.03
8.00
22.86
18.95
13.22
20.83
8.67
6.33
6.33
a
NR ¼ Nile red. b NIM ¼ nimodipine. c CUR ¼ curcumin. d DXM ¼ dexamethasone. e Encapsulation efficiency (%) ¼ amount of dye encapsulated ꢂ
100/amount of dye added.
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Table 3 Size distribution profile (by volume) of 20–22 before and after encapsulation determined by DLS measurements
Aer encapsulation
Compound
Before encapsulation
Nile Red
Curcumin
Nimodipine
Dexamethasone
20
21
22
7.49
8.53
17.49
10.31
12.10
14.75
7.57
9.53
16.04
7.76
9.44
12.72
10.06
9.66
12.29
aer encapsulation of the hydrophobic guest molecules (Table 3
To quantify Nile red encapsulation, lyophilization of the
and Fig. S20 (ESI†)). In case of C-18 alkyl chain functionalized encapsulated samples followed by dissolution in methanol was
amphiphile 22, slight contraction in particle size was observed carried out with subsequent recording of their absorbance and
upon encapsulation of dye/drug molecules. This could be uorescence emission spectra. The transport efficiency (Fig. 6)
attributed to greater hydrophobic interactions of encapsulated and transport capacity (Table 2) of the encapsulated dye was
dye/drug with C-18 alkyl chain as compared to C-10 alkyl chain. calculated for all the amphiphiles employing the Beer–
Moreover, smaller assemblies formed by C-10 alkyl chain Lambert's law and using the molar extinction coefficient (3) of
bearing amphiphiles 20 and 21 might be responsible for the 45 000 Mꢀ1 cmꢀ1 at 552 nm.53 The amphiphile 22 consisting of
observance of slight increase and no contraction in size, when mPEG-1000 and C-18 alkyl chains showed the maximum
hydrophobic guest molecules were encapsulated.
encapsulation followed by amphiphiles 20 and 21 (transport
2.3.1. Nile red encapsulation. Nile red, a model hydro- efficiency; 20: 1.12 mg gꢀ1, 21: 0.97 mg gꢀ1, 22: 5.79 mg gꢀ1),
phobic and solvatochromic uorescent dye, is very commonly which was also supported by uorescence measurements (Fig.
used for evaluating the transport potential and local polarity of 5). Further, comparison of transport and encapsulation effi-
nanocarriers.49 H- and J-type aggregates which are known to be ciency of amphiphile 22 with aromatic and non-aromatic poly-
formed by Nile red beyond a certain concentration, is marked by meric systems synthesized earlier by our group revealed that
an additional shoulder peak in the absorption spectra and this system (22) possessed superior solubilization
a change in the uorescence intensity.50 As reported earlier by behavior.51,52,54
our group, 0.12 mg of the dye with 5 mg mLꢀ1 of nanocarrier
2.3.2. Nimodipine encapsulation. Nimodipine, a 1,4-dihy-
solution resulted in monomolecular and maximum encapsu- dropyridine calcium channel blocker with therapeutic indica-
lation of the chromophore determined by uorescence tions for cerebrovascular spasm, stroke, migraine, and ischemic
measurements.51,52 In order to further ascertain this for our neurological disorders, has restricted clinical usefulness due to
systems, uorescence intensity measurements were carried out low oral bioavailability and aqueous solubility.55 In this regard,
by varying the amount of Nile red and the amphiphile (20) nanoparticles hold substantial promise and thus, we attempted
concentration, which revealed that maximum uorescence was to entrap the drug in the amphiphilic architectures (20–22) at
observed for 0.12 mg of chromophore with 5 mg mLꢀ1 of an amphiphile concentration of 5 mg mLꢀ1. Absorbance
amphiphile (Fig. S21†). Thus, we decided to use 5 mg mLꢀ1 of measurements of the encapsulated samples at different drug
amphiphile concentration and 0.12 mg of Nile red for encap- concentration (0.2, 0.5 and 1 mg) (Fig. S23, ESI†) propelled us to
sulation to avoid the formation of aggregates. Further, the same use 0.5 mg of nimodipine with quantitative estimation achieved
concentration (5 mg mLꢀ1) of the amphiphile was used for all using a molar extinction coefficient of the drug in ethanol (7200
other encapsulation studies.
Mꢀ1 cmꢀ1 at 356 nm).20 Amphiphile 22 having greater hydro-
phobic core exhibited highest encapsulation efficiency as it
could efficiently transport 22.86 mg of nimodipine for 1 g of 22
(Fig. 6).
2.3.3. Curcumin encapsulation. Enriched with profound
therapeutic properties, curcumin nds myriad applications
such as anticancer, antimicrobial, antioxidant and anti-
inammatory agent.54 Curcumin having a biphenolic struc-
ture, has been a promising candidate for the treatment of Alz-
heimer's disease as it binds to amyloid plaques in in vivo and
prevents the aggregation of Ab into brils.56 However, low
aqueous solubility together with high chemical and photo-
chemical instability leading to poor bioavailability, connes its
use as drug.54 In an attempt to enhance its bioavailability, we
have successfully encapsulated curcumin using our amphi-
philic systems (20–22). Encapsulation and quantitative evalua-
tion was assessed from UV-Vis spectrophotometry at two
different drug concentrations (1 and 2.5 mg) and 5 mg mLꢀ1
Fig. 6 Guest transport efficiencies (mg gꢀ1) for amphiphiles 20–22.
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containing amphiphile 22 to encapsulate higher percentage of
curcumin as compared to the C-18 alkyl functionalized PEGylated
copolymers employing 1 : 2 curcumin/polymer ratio (Table 4).48
Moreover, the synthesized amphiphiles 20–22 using 1 : 5 and 1 : 2
formulation ratio were found to possess superior transport effi-
ciency, transport capacity and % encapsulation efficiency as
compared to the alkyl and peruoroalkyl-functionalized
dendronized polymers synthesized earlier by our group,58
wherein 1 : 3 curcumin/polymer ratio was used (Table 4).
Furthermore, Rabanel et al.59 also used 1 : 5 loading ratio for PEG–
poly lactic acid (PLA) polymers (PEG 8%-g-PLA, PEG 20%-g-PLA,
PEG-b-PLA and OH-g-PLA) but reported lower % transport and
encapsulation efficiency than our system, with the exception of
PEG 8%-g-PLA (Table 4). In addition, the % encapsulation effi-
ciency of amphiphile 22 was found to be comparable to the
mPEG2000–oleic acid (OA) diblock copolymer, however, the %
transport efficiency was ꢃ1.5 fold lower in our case (Table 4).60
2.3.4. Dexamethasone encapsulation. In order to further
investigate the suitability of these novel amphiphilic nano-
Fig.
7 Fluorescence intensity of encapsulated curcumin in the
amphiphiles 20–22 in methanol.
amphiphile solution using a molar extinction coefficient of the carriers (20–22) as possible drug delivery systems, we tried to
drug in methanol (55 306 Mꢀ1 cmꢀ1 at 425 nm), obtained from exploit the poor aqueous soluble corticoid, dexamethasone.
calibration curve (Fig. S25, ESI†). Highest encapsulation was Endowed with anti-inammatory properties, dexamethasone
observed when 1 mg of curcumin along with 5 mg mLꢀ1 of has been used for the treatment of diabetic macular edema.61
amphiphile was used (Fig. S26, ESI†). In a 5 mg mLꢀ1 solution Analysis of the encapsulated samples at two different drug
of nanocarriers 20–22, it was possible to solubilize 132.2– concentrations (1 and 2.5 mg) and 5 mg mLꢀ1 of nanocarrier
208.2 mg Lꢀ1 of curcumin; thus, enhancing its solubility in
solution (Fig. S27 and S28, ESI†) via HPLC using acetoni-
water at 25 ꢁC by approximately 104 times from ꢃ11 ng mLꢀ1 as trile : water mixture (40 : 60) as an eluent, led to the observance
reported in literature.57
of best transportation while using 1 mg of the drug. Since,
Furthermore, amphiphile 22 was found to be the best candi- dexamethasone has partial solubility in water, therefore,
date as 1 g of it could uptake 41.66 mg of curcumin, followed by a blank sample was prepared wherein the drug was stirred in
amphiphiles 20 and 21, which could transport 37.89 and 26.45 mg water without the amphiphile. From the results, it was inferred
of curcumin respectively (Fig. 6). This observation was further that unlike the outcomes for Nile red, nimodipine and curcu-
supplemented by uorescence measurements (Fig. 7). This min where compound 22 had the highest transport efficiency,
1 g of the nanocarrier having C-10 alkyl chain decorated with
suggests the attainment of an optimum balance between hydro-
phobic and hydrophilic units in the amphiphile 22 for curcumin mPEG-550 (20) successfully transported 17.33 mg of dexa-
encapsulation. It has been observed that the formulation of methasone. On the other hand, amphiphiles 21 and 22 exhibi-
curcumin/amphiphile in 1 : 5 ratio (w/w) led the C-18 alkyl ted lower transport efficiency of 12.67 mg gꢀ1 (Fig. 6). This
Table 4 Comparison of % transport efficiency, transport capacity and % encapsulation efficiency of 20–22 for curcumin with that of the
nanocarriers reported in literature
Curcumin/nanocarrier
ratio (w/w)
Transport
efficiency (%)
Transport capacity
Encapsulation
efficiency (%)
Nanocarrier
(mmol molꢀ1
)
20
21
22
20
21
22
1 : 2
1 : 2
1 : 2
1 : 5
1 : 5
1 : 5
1 : 2
1 : 2
1 : 3
1 : 3
1 : 3
1 : 3
1 : 5
1 : 5
1 : 5
2.28
2.11
2.29
3.79
2.65
4.17
—
279.92
191.69
317.97
317.87
351.22
578.16
—
4.57
4.21
4.58
18.95
13.22
20.83
15.41
9.26
1.6
Stearyl polymer48
Stearoyl polymer48
—
—
50% peruoroalkyl-functionalized dendronized polymers58
70% peruoroalkyl-functionalized dendronized polymers58
50% alkyl-functionalized dendronized polymers58
70% alkyl-functionalized dendronized polymers58
PEG–PLA polymer59
0.53
0.45
0.27
0.39
<2
5
ꢃ6
213.17
176.75
98.95
135.41
—
1.4
0.8
1.2
</¼ 10
PEG 8%-g-PLA polymer59
—
—
15
mPEG2000–OA polymer60
ꢃ20
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Fig. 8 Absorbance spectra of encapsulated curcumin in the amphiphiles 20–22 and Cremophor® ELP in (a) water and (b) methanol.
might be due to the presence of a carboxylic group in dexa- Absorbance spectra measurements were carried out at
methasone which renders it slight hydrophilic character. a concentration of 1 mg for curcumin and 5 mg mLꢀ1 for
Moreover, higher dexamethasone carrying ability of the compounds 20–22 and Cremophor® ELP. As presented in Fig. 8,
synthesized nanocarriers 20–22 was observed on comparison amphiphiles with relatively appropriate hydrophobic–hydro-
with those of peruoroalkyl-functionalized dendronized poly- philic balance (20/22) possessed a remarkably higher solubili-
mers reported earlier by our group.58
zation for curcumin compared with the standard excipient
It was of interest to note the correlation between the CAC of used. On the other hand, nanocarrier with mPEG-1000 and C-10
a nanotransporter with its dye/drug loading capacity; in fact, alkyl chain (21) had comparable drug loading tendency.
amphiphile 22 allowed to encapsulate approximately 1.6–6
times more dye/drug, in most cases, than the nanocarrier 21.
2.4. Cellular uptake study
Nevertheless, it was possible to conrm the fundamental
importance of the hydrophobicity of the micellar core from the
results collected, that allowed the formation of relatively larger
aggregates (amphiphile 22), and ultimately enabling high dye/
drug solubilization.
The cellular internalization of Nile red encapsulated in two
selected amphiphiles 20 and 22 having different hydrophilic
and hydrophobic ratios in A549 lung cancer cells was studied by
confocal laser scanning microscopy (cLSM).
2.3.5. Comparison with Cremophor® ELP. Among all the
dyes/drugs screened for their encapsulation behavior, curcumin
was found to be the best candidate in terms of its solubilization
efficiency and capacity (Fig. 6). Therefore, in pursuit of
a comparative study of our developed amphiphilic systems (20–
22) with a standard excipient, Cremophor® ELP was used.
The Nile red encapsulated supramolecular architectures
were prepared by incubating the dye in an aqueous solution of
amphiphile using the lm method at a concentration of 5 mg
mLꢀ1. The cLSM images (Fig. 9) revealed that the dye encap-
sulated in amphiphiles 20 and 22 could be internalized into the
cells. Aer 5 h, a strong signal was seen in the cytosol and in
Fig. 9 Confocal laser scanning fluorescence microscopy images from A549 cells after 5 h incubation with Nile red encapsulated by amphiphiles
20 and 22. In the images, Nile red is shown in red color, labeled early endosomes and lysosomes in green. Yellow color indicates a merge of green
and red color and therefore shows co-localization of Nile red and early endosomes or lysosomes. The bright field channel is shown in grey scale.
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some lysosomes. Further, it was observed that the uores-
cence intensity was increasing over time indicating a contin-
uous uptake of the encapsulated dye and accumulation
(Fig. S29, ESI†). The labeled and non-labeled control cells
showed no uorescence in the Nile red channel (Fig. S29,
ESI†).
2.5. Cytotoxicity
In order to develop efficient nanocarriers for biomedical
applications such as drug delivery, the cytotoxicity is one of the
major areas of concern that needs to be addressed. Therefore, to
examine the in vitro cytotoxicity of the synthesized amphiphilic
systems, A549 cells were treated with the compounds 20–22 at
a concentration of 2, 1, 0.5 and 0.1 mg mLꢀ1 and the viability
was assayed using MTS assay aer 24 h.
The cytotoxicity prole unveiled that all the developed
systems exhibited nearly no cytotoxicity even up to the
highest concentration tested. Though, amphiphiles 20 and
Fig. 10 Cytotoxicity profile of amphiphiles (20–22) after 24 h. Cyto-
toxicity of the nano-carriers was determined by an MTS assay in A549
cells 24 h post-treatment. Each bar represents the mean value of three
independent experiments (n ¼ 3) measured in triplicates with SEM.
21 exhibited marginal toxicity of ꢃ10% at 2 mg mLꢀ1
,
however, nanocarrier 22 containing C-18 alkyl chain was
found to be non-cytotoxic at this concentration even up to
24 h post-treatment (Fig. 10).
Fig. 11 (a) Release profile of Nile red encapsulated nanocarrier 22 under physiological conditions, in the presence and absence of enzyme at
37 ꢁC; (b) time dependent release of Nile red from amphiphile 22 with/without enzyme; (c) fluorescence intensity of encapsulated Nile red in the
presence of enzyme; (d) fluorescence intensity of encapsulated Nile red in the absence of enzyme.
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no enzyme showed insignicant Nile red release. Thus, it could
be concluded from the results obtained here that a balance
between hydrophobicity and hydrophilicity plays a key role for
the amphiphilic architectures to act as good nanotransporters.
This study may facilitate further development of optimized
amphiphiles as nanocarriers for biomedical applications.
2.6. Enzyme-triggered release study
The release of encapsulated drug in a controlled manner to
maintain its therapeutic concentration holds great relevance
apart from its encapsulation. Since the amphiphiles reported
herein contain ester linkages that might be sensitive to enzyme
catalyzed hydrolytic conditions, Novozym 435 mediated
cleavage of Nile red encapsulated nanocarrier 22 was performed
under dark conditions. Fig. 11 presents the time-dependent
decay of uorescence intensity maximum of Nile red-loaded
amphiphile 22 under physiological conditions in the presence
and absence of the enzyme.
The intensity of the pink colour of the Nile red encapsulated
solution diminished with time when the nanocarrier was placed
in an environment similar to the human body, in the presence
of the enzyme. Approximately 50% decay in the intensity
occurred aer 54 h and it was possible to have more than 80%
release only aer 120 h (Fig. 11). Interestingly, the dye encap-
sulated nanocarrier under physiological conditions in the
absence of the enzyme was observed to be fairly stable; in fact
insignicant amount of Nile red was released from the system
adopting such conditions.
Acknowledgements
We gratefully acknowledge the nancial assistance from SERB-
DST, Government of India and University of Delhi. Also, we are
thankful to CSIR, New Delhi, for providing fellowship to Suchita
Prasad.
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