Newer Non-ionic A2B2-Type Enzyme-Responsive Amphiphiles for Drug Delivery

Article abstract of DOI:10.1002/cmdc.202100031

A new series of nonionic gemini amphiphiles have been synthesized in a multi-step chemoenzymatic approach by using a novel A₂B₂-type central core consisting of conjugating glycerol and propargyl bromide on 5-hydroxy isophthalic acid. A pair of hydrophilic monomethoxy poly(ethylene glycol) (mPEG) and hydrophobic linear alkyl chains (C₁₂/C₁₅) were then added to the core to obtain amphiphilic architectures. The aggregation tendency in aqueous media was studied by dynamic light scattering, fluorescence spectroscopy and cryogenic transmission electron microscopy. The nanotransport potential of the amphiphiles was studied for model hydrophobic guests, that is, the dye Nile Red and the drug Nimodipine by using UV/Vis and fluorescence spectroscopy. Evaluation of the viability of amphiphile-treated A549 cells showed them to be well tolerated up to the concentrations studied. Being ester based, these amphiphiles exhibit stimuli-responsive sensitivity towards esterases, and a rupture of amphiphilic architecture was observed in the presence of immobilized Candida antarctica lipase (Novozym 435), thus facilitating release of the encapsulated guest from the aggregate.

Full text of DOI:10.1002/cmdc.202100031

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Newer Non-ionic A2B2-Type Enzyme-Responsive
Amphiphiles for Drug Delivery
Krishna,[a] Badri Parshad,[a, b] Katharina Achazi,[c] Christoph Böttcher,[d] Rainer Haag,[c] and
Sunil K. Sharma*[a]
A new series of nonionic gemini amphiphiles have been
synthesized in a multi-step chemoenzymatic approach by using
a novel A2B2-type central core consisting of conjugating glycerol
and propargyl bromide on 5-hydroxy isophthalic acid. A pair of
hydrophilic monomethoxy poly(ethylene glycol) (mPEG) and
hydrophobic linear alkyl chains (C12/C15) were then added to the
core to obtain amphiphilic architectures. The aggregation
tendency in aqueous media was studied by dynamic light
scattering, fluorescence spectroscopy and cryogenic transmis-
sion electron microscopy. The nanotransport potential of the
amphiphiles was studied for model hydrophobic guests, that is,
the dye Nile Red and the drug Nimodipine by using UV/Vis and
fluorescence spectroscopy. Evaluation of the viability of
amphiphile-treated A549 cells showed them to be well
tolerated up to the concentrations studied. Being ester based,
these amphiphiles exhibit stimuli-responsive sensitivity towards
esterases, and a rupture of amphiphilic architecture was
observed in the presence of immobilized Candida antarctica
lipase (Novozym 435), thus facilitating release of the encapsu-
lated guest from the aggregate.
Introduction
reactions in aqueous solution, artificial enzyme-mimicking,[9,10]
stabilizers for emulsions used for cleaning and green organic
reactions.[11,12] Moreover, amphiphiles show unique and newer
opportunities for designing novel material for advanced
applications in biomedicine and bio-nanotechnology.[13–15] The
hydrophilic and hydrophobic groups of amphiphiles enable
them to self-assemble at an interface or in solution to form
diverse molecular assemblies. The size and morphology of these
assemblies such as micelles, toroids, monolayers, vesicles, rods
and sheet-like structures are dictated by the nature of
substituents and hydrophilic-lipophilic balance (HLB).[16–20] The
self-assembly is accompanied by the creation of a hydrophobic
space surrounded by hydrophilic groups and this arrangement
provides an attractive opportunity to address the challenges of
drug delivery, such as insufficient aqueous solubility of drug,
shorter half-life in the bloodstream, lack of selectivity, and high
overall clearance rate. Nanocarriers are known to provide the
drug a protective lipophilic environment, enhance the circula-
tion period in blood, and facilitate active site targeting, and
thus minimizing drug degradation and loss upon administra-
tion, and prevent harmful or undesired side-effects.[21–24]
Amphiphiles can be broadly divided into two major classes
each having its own advantages, although, polymeric amphi-
philes form relatively stable nanostructures, small molecule
amphiphiles (SMAs) on the other hand are easy to design and
their self-assemblies display more similarity with the natural
analogs.[25–30] Furthermore, small amphiphilic systems can be
either ionic or nonionic whereas, ionic, particularly cationic
systems are known to be cytotoxic to varying extent.[31–33]
Among SMAs, gemini and dimeric amphiphiles, character-
ized by the presence of two hydrophilic head groups and two
hydrophobic tails, linked by a rigid or flexible spacer, received
considerable attention, due to their unique properties such as
self-assembly behavior,[34,35] enhanced micellar stability,[36–38]
high wetting ability, multiple aggregate morphologies, low
Self-assembly and self-organization are the key tools in
supramolecular chemistry and are being intensively used by
researchers to build amazingly complex architectures.[1] The
advent of supramolecular chemistry in the last decades of the
20th century has provided chemists with a wealth of new
routes toward constructing molecular structures and materials
that exhibit self-assembly phenomenon due to relatively weak,
non-covalent interactions, such as hydrogen bonding, π-π
stacking, electrostatic and van der Waals interactions.[1,2] Such
assemblies are usually stabilized by thermodynamics (relative
free energies, enthalpy and entropy of binding) and/or kinetic
aspect,[3] and provide several hierarchical levels of molecular
organization. The self-assembly of amphiphiles has been shown
to be of significant importance in many research fields, such as
building blocks for the fabrication of novel organic nanofibers
or nanotubes for electrical and medical devices,[4–6] candidates
for drug delivery,[7,8] nano-/microreactors for carrying out
[a] Krishna, Dr. B. Parshad, Prof. S. K. Sharma
Department of Chemistry, University of Delhi
Delhi, 110007 (India)
E-mail: sksharma@chemistry.du.ac.in
[b] Dr. B. Parshad
Department of Chemical Engineering and Biotechnology
University of Cambridge
Cambridge, CB3 0AS (UK)
[c] Dr. K. Achazi, Prof. Dr. R. Haag
Institut für Chemie und Biochemie
Freie Universität Berlin
Takustraße 3, 14195 Berlin (Germany)
[d] Dr. C. Böttcher
Forschungszentrum für Elektronenmikroskopie
Institut für Chemie und Biochemie
Freie Universität Berlin
Fabeckstraße 36a, 14195 Berlin (Germany)
Supporting information for this article is available on the WWW under
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Krafft temperature and excellent lime-soap dispersing proper-
ties as compared with the monomeric single-chain amphiphilic
analogues.[39–42] The above factors bring focus on gemini
amphiphiles for use in biomedical applications. Our interest is
to explore the drug delivery potential of newer types of
amphiphiles. Considering various possible structures among
gemini amphiphiles, the nonionic one is advantageous for the
drug delivery applications due to reduced cytotoxicity.[43–45]
Furthermore, the presence of two hydrophobic alkyl tails in
gemini amphiphiles allow sharp and uniform decrease in CAC
value than their monomeric counterparts and thus making a
valuable contribution for therapeutic applications. Nonionic
gemini amphiphiles based on carbohydrates are known in the
literature.[46,47] As a part of our ongoing efforts to explore the
use of other type of competing novel moieties,[3,48–52] we aim to
explore the use of gold-standard poly(ethylene glycol) (PEG)
towards the synthesis of a wide variety of amphiphiles for drug
delivery applications, PEG is known for its unique properties,
Herein, we report the design, synthesis, and characterization
of a newer family of glycerol based nonionic gemini amphi-
philes and study of their supramolecular aggregation into
different nanostructures in an aqueous medium. Firstly, suitably
protected glycerol was conjugated via its secondary hydroxy
group with the 5-hydroxy isophthalic acid via Mitsunobu
reaction and the resulting diacid then undergoes esterification
with propargyl alcohol to yield an A2B2 central core in a multiple
step chemoenzymatic approach by making use of immobilized
C. antarctica lipase (Novozym 435). The primary alcohols of the
glyceryl moiety in the A2B2 core were first reacted with the
long-chain aliphatic acid, it was followed by the reaction of
mPEG-azide to couple with the two acetylenic groups of the
core by click coupling (Figure 1). The amphiphiles thus
generated by conferring hydrophobic and hydrophilic groups
were studied for their transport potential.
such as low toxicity, excellent solubility in aqueous solution, Results and Discussion
good chemical stability, ion-transporting ability, decreased
interaction with blood components, etc.[23,53,54] PEGylation is an
established method for increasing protein stability or solubiliz-
ing hydrophobic biomolecules as well as reduce immunogenic-
ity and antigenicity. PEG’s hydrophilic surface allows prolonged
circulation of polymeric micelles in the bloodstream.[55–57] The
other building block, that is, 5-hydroxyisophthalate was consid-
ered on the basis that its PEG based block copolymer reported
earlier by Kumar et al. is nontoxic and highly efficient drug
delivery vehicles for hydrophobic and partially hydrophilic
drugs.[58] The synthesis of stimuli responsive delivery systems is
another desirable feature for controlled drug delivery. Our
interest is to develop enzyme responsive drug delivery system
(DDS). Cells contain a variety of enzymes, such as esterase,
Camacho, lipase, phospholipase and nuclease, etc., but in the
tumor tissues some specific enzymes, such as matrix metal-
loproteinase, phospholipase, esterase are present in higher
amount than the other normal tissues. Esterase-responsive
nanoparticles could thus be sensitive for tumor cells due to the
overexpressed esterase in tumor cells. Esterase-responsive
micellar nanoparticle is thus considered as one of the best
strategies for drug delivery.[59]
The gemini amphiphiles were synthesized by following the
Novozym 435 catalyzed chemoenzymatic approach and their
supramolecular organization were characterized physicochemi-
cally by using infrared spectroscopy (IR), elemental analysis, gel
permeation chromatography (GPC), dynamic light scattering
(DLS), cryogenic transmission electron microscopy (cryo-TEM),
high-resolution transmission electron microscopy (HRTEM),
critical aggregation concentration (CAC) and 1H, 13C NMR
spectroscopy. Nile Red and Nimodipine were used as a model
dye/drug in order to explore the solubilization tendency of the
amphiphiles.
Synthesis and characterization
A2B2 type central core (7) of gemini amphiphiles were
synthesized from commercially available glycerol (1) and
dimethyl 5-hydroxyisophthalate (3) following the strategy as
outlined in Scheme 1.
Glycerol (1) was first converted to 2-hydroxypropane-1,3-
diyl diacetate (2) using vinyl acetate and Novozym 435 as a
Figure 1. Representation of the synthetic approach and self-assembly of the amphiphiles in aqueous media.
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confer hydrophilicity, mPEG-azides (14 and 15) were first
synthesized from commercially available mPEG-550 (10) and
mPEG-1000 (11) by following the literature report,[62] that is,
they were first reacted with methanesulfonyl chloride to get the
corresponding mesyl derivatives 12 and 13, respectively. The
mesylated product was then subjected to azidation using
sodium azide in DMF. The disappearance of the broad peak of
OH group at 3319 cmÀ 1 and the appearance of one sharp peak
for azide at 2100 cmÀ 1 in the IR spectrum confirmed the
completion of azidation reaction (Scheme 2).
Finally, four different amphiphiles were synthesized by
using the click chemistry approach, that is, The dipropargylated
hydrophobic intermediate (8/9) and hydrophilic mPEG-azide
moiety (14/15) were coupled together using tris
(triphenylphosphine) copper(I) bromide and DIPEA in the
presence of DMF to yield the desired nonionic gemini
amphiphiles 16–19 (Scheme 3).
The formation of amphiphiles 16–19 was confirmed by the
disappearance of azide peak at around 2100 cmÀ 1 in the IR
spectrum. The formation of the coupled product, for example
compound 16, was further ascertained by the appearance of
aromatic protons of triazole moieties at around δ 7.86 ppm in
Scheme 1. Synthesis of the A2B2 core and its functionalization with hydro-
°
phobic moieties. i) vinyl acetate, Novozym 435, THF, 40 C, 3 h; ii) DIAD,
°
°
triphenylphosphine, toluene, 25 C, 12 h; iii) K2CO3, ethanol, 25 C, 12 h; iv)
°
°
KOH, ethanol, 80 C, 24 h; v) K2CO3, propargyl bromide, DMF, 50 C, 12 h; vi)
°
n-alkyl carboxylic acid, EDC·HCl, DMAP, CH2Cl2, 25 C, 12 h.
1
the H NMR spectrum. The observance of peaks in the aromatic
region corresponding to triazolyl ring carbons at around δ 125
and 142 ppm in the 13C NMR spectrum further support the
formation of the desired product. Furthermore, triazole ring
formation also results in the observance of characteristic
downfield shift for the methylene protons of propargyl moiety
from δ 4.93 ppm to 5.43 ppm in the 1H NMR spectrum.
biocatalyst.[60,61] Dimethyl 5-hydroxyisophthalate (3) was sub-
jected to undergo Mitsunobu reaction with 1,3-diacetoxyglycer-
ol (2) to yield dimethyl 5-((1,3-diacetoxypropan-2-yl)oxy)
isophthalate (4). The observance of a peak at 1720 cmÀ 1 in the
IR spectrum confirms the presence of ester carbonyls in the
molecule. The appearance of two singlets (six protons each) at
δ 2.07 and 3.94 ppm, accounting for two acetoxy and two
methyl ester protons supports the formation of product. The
peaks for other protons and carbons were also observed
ascertaining the formation of product. Furthermore, the
coupling reaction resulted in a downfield shift of the methine
protons of the glyceryl moiety from δ 4.05–4.03 ppm[60] to δ
1
4.80–4.75 ppm and from δ 67.78 ppm to δ 74.52 ppm in the H
and 13C NMR spectra, respectively. Subsequently compound 4
was subjected to deacetylation in the presence of potassium
carbonate followed by methyl ester hydrolysis using KOH to
afford 5-((1,3-dihydroxypropan-2-yl)oxy)isophthalic acid (6). The
disappearance of the two singlets at δ 2.07 and 3.94 ppm in the
1H NMR spectrum along with the appearance of peaks at 3464
and 3319 cmÀ 1 corresponding to COOH and OH in the IR
spectrum confirmed the complete deprotection in compound
4. The propargylation of the resulting compound (6) yielded the
desired A2B2 core (7), its structure was established by the
observance of a triplet (2H) and doublet (4H) at δ 2.53 and
Scheme 2. Synthesis of hydrophilic moieties, mPEG-azide. i) mesyl chloride,
°
°
triethyl amine, CH2Cl2, 0–25 C, 2 h; ii) sodium azide, DMF, 80 C, 12 h.
1
4.93 ppm, respectively in the H NMR spectrum. Compound 7
was then subjected to coupling with dodecanoic/pentadeca-
noic acid using EDC·HCl and DMAP. The compounds 8 and 9 so
obtained were completely characterized by their IR, 1H, 13C NMR
and HRMS spectra. The presence of hydrophobic chain was
ascertained by the appearance of a triplet at 0.81 ppm (J=
6.7 Hz) integrating for six protons and other methylene protons
in 1.17–1.24 ppm range in 1H NMR spectrum. mPEG was used to
Scheme 3. Synthesis of nonionic gemini amphiphiles. i) [Cu (PPh3)3]Br, DIPEA,
°
DMF, 60 C, 24 h.
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Physicochemical characterization of amphiphiles and
self-assembly study in aqueous medium
Table 1. Physicochemical properties of amphiphiles 16–19.
Amphiphile
DLS size (d) [nm]
CAC [M]
HLB
C-15/mPEG-550 (16)
C-12/mPEG-550 (17)
C-15/mPEG-1000 (18)
C-12/mPEG-1000 (19)
12.05
10.31
13.70
13.36
7.84×10À 5
7.70×10À 5
1.06×10À 4
9.33×10À 5
11.13
11.64
13.69
14.10
DLS, cryo-TEM, HRTEM, UV/Vis and fluorescence spectroscopy
were used to study the physicochemical, transport and stimuli-
responsive behavior of the mPEG based amphiphiles. GPC was
used to determine the molecular weight and polydispersity
index (PDI) of the synthesized gemini amphiphiles. The aqueous
solution of these amphiphilic architectures displays
supramolecular aggregation behavior leading to the formation
of nanostructures which were characterized by CAC using Nile
Red as a fluorescent probe,[63] and DLS measurement was used
for determining the particle size and also the cryo-TEM and
HRTEM image were recorded to study the morphology of
amphiphilic architectures.
dye’s fluorescence intensity versus log[amphiphile] gives the
CAC value. The CAC of the amphiphilic system 16–19 evaluated
by fluorescence spectrophotometer was found to be of the
order of 10À 4 to 10À 5 M (Figure 2 and Table 1), and for the
comparison purpose the CAC value of
a representative
amphiphile 18 was also evaluated by plotting the its con-
ductivity versus its concentration, and the obtained value
(2.50×10À 4) shows a good correlation with the data from
fluorescence spectroscopy (Figure S16b). The hydrophilic-lip-
ophilic balance (HLB) of amphiphiles was calculated by Griffin’s
equation; that is, HLB=20×Mh/Mw, where Mw is molecular
weight of amphiphiles determined by GPC, and Mh is molecular
weight of hydrophilic part.[64] The CAC and the HLB values of all
the nanocarriers are shown in Table 1.
Calculation of critical aggregation concentration using
fluorescence measurements
The minimum concentration at which the synthesized amphi-
philes (16–19) begin to form the aggregates was determined by
using poorly water-soluble dye, Nile Red, as a fluorescent
probe.[63] A fixed amount of the dye was used for encapsulation
in different concentration of amphiphiles. In the absence of
supramolecular aggregates, that is at concentrations below the
CAC, the fluorescence intensity of the dye remains low, while
gradually increasing the amphiphile concentration, it was
observed that at a specific concentration, a considerable
increment in the fluorescent intensity occurs because of the
surrounding of the dye within the hydrophobic core of the self-
assembled nanostructures. A break in the plot of encapsulated
Dynamic light scattering and HRTEM measurements
The DLS measurement at a concentration of 5 mg/mL was used
to obtain the particle size of the resulting amphiphiles (Table 1).
A mostly bimodal size distribution profile in intensity, referring
to micelles and micellar aggregates, whereas monomodal in
volume and number corresponding to micelles was observed
°
Figure 2. Plots of fluorescence intensity versus log of the concentration of amphiphiles a) 16, b) 17, c) 18, and d) 19 in aqueous solution at 25 C.
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(Figure 3), which suggested that there was a small proportion
of micellar aggregates and micelles were the predominant
species in aqueous solutions. On comparing the intensity
distribution of amphiphiles with mPEG-550/1000 chains in
corresponding ratios, the amphiphile with mPEG-1000 hydro-
philic chain were observed to be larger than the corresponding
analogues containing mPEG-550. Corresponding volume distri-
bution plots display values in the range of 11–13 nm for
amphiphiles 16–19. In order to know the actual morphology of
the nanostructured architectures formed within the aqueous
solution of dendritic mPEG based amphiphiles, cryo-TEM micro-
graph of the aggregates formed from amphiphile 17 shows the
formation of small micelles as well as micellar aggregates with a
uniform diameter of 7 nm (Figure S15). The study was extended
further by recording the HRTEM micrographs of amphiphiles
16–19 at a concentration of 5 mg/mL. The HRTEM data
uncovered the formation of small micelles as well as micellar
aggregates with a uniform diameter of 7–10 nm, which is in
good agreement with the corresponding DLS profile as shown
in Figure 4.
Transport potential of amphiphiles for Nile Red and
Nimodipine
Figure 4. HRTEM micrographs of amphiphiles a) 16, b) 17, c) 18 and d) 19
showing small uniform micelles of a diameter 7–10 nm. Scale bars: 50 nm.
The applicability of the synthesized nonionic gemini amphi-
philes as nanocarriers for hydrophobic guests was investigated
using the fluorescent dye, Nile Red and the calcium channel
blocker drug, Nimodipine, both of them interact with the
nanocarriers through non-covalent interactions such as hydro-
phobic, π-π stacking and hydrogen bonding. The encapsulation
of the compounds was carried by the thin film method.[65] The
resulting data of the transport efficiency, transport capacity and
encapsulation efficiency are shown in Table 2.
Figure 3. DLS profiles for the amphiphiles 16–19 in water. a) intensity graph; b) volume graph.
Table 2. Transport efficiency, transport capacity and encapsulation efficiency of amphiphiles 16–19 for Nile Red and Nimodipine
Amphiphile
Transport efficiency [mg/g]
Transport capacity [mmo/mol]
Encapsulation efficiency [%]
Nile Red
Nimodipine
Nile Red
Nimodipine
Nile Red
Nimodipine
16
17
18
19
1.13
1.76
1.16
0.64
24.10
23.68
20.66
17.40
6.95
112.50
105.00
144.44
118.18
2.83
4.42
2.90
1.61
7.53
7.37
6.45
5.43
10.03
10.69
5.73
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Nile Red encapsulation
amphiphile is required to formulate 50 mg of Nimodipine.
Amphiphile 17 consisting of mPEG-550 and C12 alkyl chain
shows highest encapsulation efficiency and highest transport
capacity (Figure 5), it was observed that increasing the size of
the mPEG head group from Mn 550 (in 16, 17) to 1000 (in 18,
19) leads to a considerable decrease in transport capacity for
either of the guest molecules. However, increasing the length
of the alkyl chain from C12 to C15 like in the case of amphiphile
16 led to an enhancement of the transport capacity (Figure 5
and Table 2). Qualitative analysis of the data shows that the
transport capacity depends on the size of both alkyl and mPEG
chains and hydrophobic-lipophilic balance of the amphiphiles.
Nimodipine consists of a 3-nitrophenyl moiety attached to
the dihydropyridine unit, a specific EDA-π interactions between
the drugs’ electron-deficient aromatic ring and the isophthalate
moiety of the amphiphile (16–19) may be the origin for the
transport capacity,[70] besides the common hydrophobic inter-
actions (Figure 5). This fact is strengthened further by the
observance of stronger absorption peak in the UV–visible
spectrum of the solution of nimodipine encapsulated amphi-
phile (16–19) in comparison to blank amphiphile (Figure S17).
The transportation study also revealed that for both nonpolar
guest molecules (Nile Red and Nimodipine) the amphiphile 17
exhibits higher transport potential (Table 2). The solutions of
drug encapsulated amphiphiles prepared by simple stirring
Nile Red is a neutral and environmentally sensitive fluorescent
dye with limited aqueous solubility. It exhibits highly solvato-
chromic fluorescence with strong emission in a lipophilic
environment. It is very commonly used to predict the position
of encapsulated guest and for evaluating the transport
potential of nanocarriers.[65] Nile Red is known to form H- and J-
type aggregates, beyond a certain concentration which shows a
shoulder additional peak in the absorption spectra.[66] To avoid
aggregate formation, we have taken 0.12 mg of the dye with
5 mg/mL of the nanocarrier solution, which is already stand-
ardized earlier by our group.[52] For quantification of the amount
of Nile Red in various encapsulated samples, the samples were
first lyophilized and re-dissolved in a known quantity of
methanol for recording their UV/Vis absorbance and
fluorescence emission spectra. The transport efficiency and
transport capacity of the encapsulated dye or concentration of
Nile Red in methanol were then calculated by applying the
Beer–Lambert law and using the molar extinction coefficient (ɛ)
of 45000 MÀ 1 cmÀ 1 at 552 nm.[52,67] The amphiphile 17 consti-
tuted from mPEG-550 and C12 alkyl chain exhibit the maximum
encapsulation followed by amphiphiles 16, 18 and 19 (Fig-
ure 5).
°
showed long-term stability up to 115 h at 37 C that shown by
fluorescence spectrometry in Figure 8d, below.
Nimodipine encapsulation
Nimodipine, a 1,4-dihydropyridine derivative is a well-known
calcium channel blocker. It is used to increase the cerebral
blood flow in humans as well as in animals.[68] Hydrophobic
character of Nimodipine limits its aqueous solubility (0.4 mg/L).
For 50 mg of Nimodipine, PEG (62.5 g) and ethanol (37.5 g) are
currently used to yield the commercial formulation.[69] In this
regard, we synthesized PEG based amphiphilic architectures
that enhance the solubility of Nimodipine besides facilitating its
encapsulation and circulation in the biological systems. The
quantification of Nimodipine per gram of amphiphile was
measured using UV/Vis spectrophotometer following the Beer–
Lambert law and extinction coefficient of the drug as
7200 MÀ 1 cmÀ 1 at 365 nm in ethanol. 2.5 g of synthesized
Cell viability study
Good cellular viability is one of the desirable criteria for the
synthesis of efficient nanocarriers for biomedical applications.
Therefore, the effect of the synthesized amphiphiles (16–19) on
the viability of A549 lung cancer cells was assessed using the
CCK-8 Kit after amphiphilic treatment for 24 h using three
different test concentrations: 2.0, 1.0 and 0.5 mg/mL (Figure 6).
For all tested systems a concentration dependent effect is seen
as the cell viability decreases at higher concentration. However,
for the amphiphiles 16, 17 and 19, the cell’s viability is above
80% up to a concentration of 1 mg/mL. On further increasing
Figure 5. a) Transport capacity and b) transport efficiency of amphiphiles 16–19 for Nile Red and Nimodipine.
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72 h, thus we confined the study for 24 h at different
concentrations.
Cellular uptake study
Amphiphiles 16 and 17 exhibit the highest encapsulation for
Nile Red, therefore, the cellular uptake potential of Nile Red
encapsulated in these two amphiphiles was studied by confocal
laser scanning microscopy (CLSM) using A549 lung cancer cells.
The Nile Red encapsulated supramolecular architectures were
prepared by incubating the dye in an aqueous solution of
amphiphile using the film method at a concentration of 5 mg/
mL. The CLSM image (Figures 7 and S14) reveal that the dye
encapsulated in amphiphiles 16 and 17 can be internalized into
cells and after 4 h, a strong signal is seen inside the cell’s
cytosol (Figure S14). After 24 h, the fluorescence intensity of the
Nile Red inside the cells increased, indicating a continuous
uptake of the encapsulated dye and accumulating over time. In
order to establish a proof-of-concept to use these amphiphiles
as nanocarriers, their general tolerability in cells was studied by
using A549 cell lines.[49,71]
Figure 6. Effect of amphiphiles 16–19 on cell viability after 24 h. The viability
of the cells treated with the nanocarriers was determined in a CCK-8 assay
on A549 cells. Each bar represents the mean value of three independent
experiments (n=3) with the standard deviation.
the concentration to 2 mg/mL the cell viability of all of these
amphiphiles (16, 17 and 19) reduce to values below 80%.
Amphiphile 18 on the other hand is not so well tolerated and
even at a concentration of 0.5 mg/mL the cell viability is less
than 80%. Interestingly, amphiphiles 16, 17 and 18 show
promotion of cell growth most probably due to better cell
adhesion that could influence the cytotoxicity of the carrier
systems. In summary it can be concluded that the amphiphiles
constructed from a C15/C12 alkyl chain with mPEG-550, that is,
16 and 17, are better tolerated than the analogues 18 and 19
having mPEG-1000 moiety. In cell viability test, all of the
amphiphiles were found to be safe for 24 h at each tested
concentration. Similar types of amphiphiles were earlier meas-
ured for cytotoxic studies by our group,[49] but no significant
difference was observed on cell viability on going from 24 to
Enzyme triggered release study
In order to develop an efficient drug delivery system, the
release of encapsulated therapeutic from the nanocarrier in a
controlled manner is very essential. As the amphiphiles
reported herein are constituted from ester linkages that might
offer sensitivity to pH/enzyme mediated hydrolytic conditions,
we decided to explore the ester hydrolysis of amphiphiles using
immobilized C. antarctica lipase (Novozym 435). Our group has
reported earlier the selective hydrolysis of aliphatic acid ester
group over the aromatic acid ester in the presence of Novozym
Figure 7. Images taken by confocal laser scanning fluorescence microscopy images from A549 cells after 24 h of incubation with Nile Red encapsulated in
amphiphiles 16 (top) and 17 (bottom). In the images, fluorescence from the Nile Red dye is shown in red colour, the fluorescence of DNA stained with
Hoechst 33342 indicating the cell nucleus is shown in blue colour, and transmitted light images are in greyscale. Scale bar: 50 μm.
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435.[72] Subjecting the synthesized nanoaggregates under such
hydrolytic conditions it is anticipated that the nanoarchitecture
will eventually start collapsing and the encapsulated hydro-
phobic guest will be released in a controlled manner. We
in fact, insignificant amount of Nile Red was released from the
system adopting such conditions.
investigated the time dependent release of encapsulated Nile Conclusion
Red from the core shell of nanocarriers by means of
fluorescence spectroscopy. To the solution of dye encapsulated
in the amphiphile was added 200 wt% of Novozym 435 and a
few drops of n-butanol for trapping the acid liberated from
ester hydrolysis of the amphiphile.[73] The solution was
Herein, we have reported a “greener” chemoenzymatic method
to synthesize nonionic gemini amphiphiles from easily available
and biocompatible starting materials by incorporating different
alkyl chains and mPEGs on a newer type of A2B2 core
synthesized by conjugation of glycerol and propargyl bromide
on 5-hydroxy isophthalic acid in a multiple step chemo-
enzymatic approach. All of the synthesized amphiphiles were
well characterized from their spectroscopic and physical data,
°
incubated at 37 C under dark conditions and the progress of
dye release was monitored by drawing aliquots of reaction
mixture at regular intervals and measuring their fluorescence
spectrum, Figure 8a and b represent the time-dependent decay
of fluorescence intensity of Nile Red loaded in amphiphile 17
under physiological conditions in the absence, and presence of
the enzyme, respectively. Approximately 50% decay in the
intensity occurred after 72 h and it was possible to have more
than 90% dye release only after 115 h (Figure 8b). In Table 2,
4.42% Nile Red encapsulate in amphiphile (17). The encapsu-
lated Nile Red was found to release within 115 h, interestingly,
the dye encapsulated nanocarrier under physiological condi-
tions in the absence of the enzyme was observed to be stable;
1
for example, H NMR, 13C NMR, HRMS, Elemental analysis and
GPC techniques. The amphiphilic systems have the tendency to
form nano-sized aggregates in aqueous medium with CAC in
the range of 10À 4 – 10À 5 M and micellar size in the range of 10–
14 nm. Cryo-TEM and HRTEM data of the amphiphiles further
support the morphology of amphiphiles. The nanotransport
potential of amphiphiles was investigated using model hydro-
phobic guests, that is, the fluorescent dye Nile Red and drug
Nimodipine. The results show that the dye was more efficiently
Figure 8. Release profile of Nile Red encapsulated in nanocarrier 17 under physiological conditions. Variation of fluorescence intensity with time of
°
amphiphile-encapsulated Nile Red in the a) absence and b) presence of enzyme at 37 C. c) Comparison of fluorescence intensity variation of the dye with
°
time in the presence (red) and absence (black) of enzyme at 37 C. d) Time-dependent release of Nile Red from amphiphile with/without enzyme after 115 h.
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[7] A. V. Kabanov, V. A. Kabanov, Adv. Drug Delivery Rev. 1998, 30, 49–60.
[8] A. Rosler, G. W. M. Vandermeulen, H.-A. Klok, Adv. Drug Delivery Rev.
2001, 53, 95–108.
[9] D. M. Vriezema, M. C. Aragonès, J. A. A. W. Elemans, J. J. L. M. Cornelis-
sen, A. E. Rowan, R. J. M. Nolte, Chem. Rev. 2005, 105, 1445–1489.
[10] Y. Wang, H. Xu, N. Ma, Z. Wang, X. Zhang, J. Liu, J. Shen, Langmuir 2006,
22, 5552–5555.
[11] M. Häger, F. Currie, K. Holmberg, Top. Curr. Chem. 2003, 227, 53–74.
[12] M. Morikawa, M. Yoshihara, T. Endo, N. Kimizuka, Chem. Eur. J. 2005, 11,
1574–1578.
[13] S. I. Stupp, L. C. Palmer, Chem. Mater. 2014, 26, 507–518.
[14] Y. Wang, H. Xu, X. Zhang, Adv. Mater. 2009, 21, 2849–2864.
[15] Y.-b. Lim, K.-S. Moon, M. Lee, Chem. Soc. Rev. 2009, 38, 925–934.
[16] J. N. Isrealachvili, D. J. Mitchell, B. W. Ninham, J. Chem. Soc. Faraday
Trans. 2, 1976, 72, 1525–1568.
encapsulated by amphiphiles having a lower HLB value when
compared with Nimodipine. Efficient uptake of encapsulated
dye in the cytosol of lung cancer cells was shown by confocal
microscopy indicating that the amphiphilic systems can trans-
port drugs into cells. The performed cell viability study showed
that the synthesized amphiphiles are relevant as a drug delivery
system. The enzyme triggered release profile of the encapsu-
lated dye revealed that approximately 50% decay in the
fluorescence intensity occurred after 72 h in the presence of the
enzyme with more than 90% release taking place in 115 h.
[17] J. N. Israelachvili, Intermolecular and Surface Forces, Academic Press,
Experimental Section
New York, 1985.
[18] H. V. Berlepsch, K. Ludwig, B. Schade, R. Haag, C. Böttcher, Adv. Colloid
Interface Sci. 2014, 208, 279–292.
[19] H.-J. Kim, T. Kim, M. Lee, Acc. Chem. Res. 2011, 44, 72–82.
[20] K. G. Goswami, S. Mete, S. S. Chaudhury, P. Sar, E. Ksendzov, C. D.
Mukhopadhyay, S. V. Kostjuk, P. De, ACS Appl. Polym. Mater. 2020, 2,
2035–2045.
[21] M. Mammen, S.-K. Choi, G. M. Whitesides, Angew. Chem. Int. Ed. 1998,
37, 2754–2794; Angew. Chem. 1998, 110, 2908–2953.
[22] A. Mulder, J. Huskens, D. N. Reinhoudt, Org. Biomol. Chem. 2004, 2,
3409–3424.
[23] S. Gupta, R. Tyagi, V. S. Parmar, S. K. Sharma, R. Haag, Polymer 2012, 53,
3053–3078.
IR, NMR spectroscopy, HRMS and GPC analysis
Infrared spectra (IR) of neat samples were recorded using a
PerkinElmer FTIR model 9 spectrometer. The 1H and 13C NMR
spectra were recorded on JEOL 400 MHz spectrometer. The 1H NMR
spectra were calibrated using the residual solvent peak, where the
chemical shift values are on a δ scale and the coupling constant (J)
values are in Hertz. High-resolution mass spectrometry (HRMS) data
were recorded on Q-TOF LCMS-Agilent Technology-6530 and HPLC/
MS-Agilent 6210 (Agilent Technologies). A Water’s GPC system
equipped with a Waters 515 HPLC pump and refractive index (RI)
detector was used to determine the molecular weight Mw, Mn and
polydispersity index (PDI) of the amphiphiles using Styragel HR
column with THF as an eluent at a flow rate of 1.2 mLminÀ 1 and
polystyrene standards were used for molecular weight calibration.
[24] A. J. ten Tije, J. Verweji, W. J. Loos, A. Sparreboom, Clin. Pharmacokinet.
2003, 42, 665–833.
[25] C. Wang, Z. Wang, X. Zhang, Small 2011, 7, 1379–1383.
[26] S. Pal, S. G. Roy, P. De, Polym. Chem. 2014, 5, 1275–1284.
[27] C. Wang, Z. Wang, X. Zhang, Acc. Chem. Res. 2012, 45, 608–618.
[28] A. Sorrenti, O. Illa, R. M. Ortuño, Chem. Soc. Rev. 2013, 42, 8200–8219.
[29] T. Shimizu, M. Masuda, H. Minamikawa, Chem. Rev. 2005, 105, 1401–
1443.
[30] L. C. Palmer, S. I. Stupp, Acc. Chem. Res. 2008, 41, 1674–1684.
[31] N. Singh, L. Sharma, Lett. Org. Chem. 2019, 16, 607–614.
[32] R. S. G. Krishnan, S. Thennarasu, A. B. Mandal, J. Phys. Chem. B 2004, 108,
8806–8816.
[33] B. Trappmann, K. Ludwig, M. R. Radowski, A. Shukla, A. Mohr, H. Rehage,
C. Böttcher, R. Haag, J. Am. Chem. Soc. 2010, 132, 11119–11124.
[34] F. Bordi, G. Cerichelli, N. D. Berardinis, M. Diociaiuti, L. Giansanti, G.
Mancini, S. Sennato, Langmuir 2010, 26, 6177–6183.
Acknowledgements
We gratefully acknowledge the Department of Science and
Technology (DST), Government of India and DFG, Germany for
financial support and University of Delhi to partially support the
research under Faculty Research Program (FRP-2020-21) and also
thankful to the CRF IIT Delhi for providing assistance in the
analysis of the samples. The helpful suggestions of Dr. Abhishek K.
Singh are gratefully acknowledged.
[35] L. Shi, F. Chen, N. Sun, L. Zheng, Soft Matter 2015, 11, 4075–4080.
[36] G. Paddon-Jones, S. Regismond, K. Kwetkat, R. Zana, J. Colloid Interface
Sci. 2001, 243, 496–502.
[37] A. R. Tehrani-Bagha, R. G. Singh, K. Holmberg, J. Colloid Interface Sci.
2012, 376, 112–118.
[38] M. Dreja, W. Pyckhout-Hintzen, H. Mays, B. Tieke, Langmuir 1999, 15,
391–399.
[39] W. Zhao, Y. Wang, Adv. Colloid Interface Sci. 2017, 239, 199–212.
[40] R. Zana, Adv. Colloid Interface Sci. 2002, 97, 205–253.
[41] Y. Han, Y. Wang, Phys. Chem. Chem. Phys. 2011, 13, 1939–1956.
[42] L. D. Song, M. J. Rosen, Langmuir 1996, 12, 1149–1153.
[43] H. Lv, S. Zhang, B. Wang, S. Cui, J. Yan, J. Controlled Release 2006, 114,
100–109.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: amphiphiles · cellular uptake · controlled release ·
drug delivery · nanostructures · self-assembly
[44] M. A. Rub, A. M. Asiri, A. Z. Naqvi, M. M. Rahman, S. B. Khan, K. -ud-Din, J.
Mol. Liq. 2013, 177, 19–25.
[45] D. R. Sikwal, R. S. Kalhapure, M. Jadhav, S. Rambharose, C. Mocktar, T.
Govender, RSC Adv. 2017, 7, 14233–14246.
[46] S. Liu, R. Sang, S. Hong, Y. Cai, H. Wang, Langmuir 2013, 29, 8511–8516.
[47] M. J. L. Castro, J. Kovensky, A. F. Cirelli, Langmuir 2002, 18, 2477–2482.
[48] B. Parshad, P. Yadav, Y. Kerkhoff, A. Mittal, K. Achazi, R. Haag, S. K.
Sharma, New J. Chem. 2019, 43, 11984–11993.
[1] J. A. A. W. Elemans, A. E. Rowan, R. J. M. Nolte, J. Mater. Chem. 2003, 13,
2661–2670.
[2] F. J. M. Hoeben, P. Jonkheijm, E. W. Meijer, A. P. H. J. Schenning, Chem.
Rev. 2005, 105, 1491–1546.
[3] S. Prasad, K. Achazi, C. Böttcher, R. Haag, S. K. Sharma, RSC Adv. 2017, 7,
22121–22132.
[49] A. K. Singh, B. N. S. Thota, B. Schade, A. Khan, C. Böttcher, S. K. Sharma,
R. Haag, Chem. Asian J. 2017, 12, 1796–1806.
[50] A. Mittal, A. K. Singh, A. Kumar, Parmanand, K. Achazi, R. Haag, S. K.
Sharma, Polym. Adv. Technol. 2020, 31, 1208–1217.
[4] R. C. Claussen, B. M. Rabatic, S. I. Stupp, J. Am. Chem. Soc. 2003, 125,
[51] Parmanand, A. Mittal, A. K. Singh, Aarti, K. Achazi, C. Nie, R. Haag, S. K.
12680–12681.
Sharma, RSC Adv. 2020, 10, 37555–37563.
[52] M. Kumari, S. Gupta, K. Achazi, C. Böttcher, J. Khandare, S. K. Sharma, R.
Haag, Macromol. Rapid Commun. 2014, 36, 254–261.
[5] J. P. Hill, W. Jin, A. Kosaka, T. Fukushima, H. Ichihara, T. Shimomura, K.
Ito, T. Hashizume, N. Ishii, T. Aida, Science 2004, 304, 1481–1483.
[6] P. K. Vemula, G. John, Acc. Chem. Res. 2008, 41, 769–782.
ChemMedChem 2021, 16, 1457–1466
www.chemmedchem.org
1465
© 2021 Wiley-VCH GmbH

Full Papers
doi.org/10.1002/cmdc.202100031
ChemMedChem
[53] J. M. Harris, Topics in Applied Chemistry: Poly(ethylene glycol) Chemistry:
Biotechnical and Biomedical Applications, Plenum, New York, 1992.
[54] N. Sadhukhan, T. Muraoka, D. Abe, Y. Sasanuma, D. R. G. Subekti, K.
Kinbara, Chem. Lett. 2014, 43, 1055–1057.
[55] K. Knop, R. Hoogenboom, D. Fischer, U. S. Schubert, Angew. Chem. Int.
Ed. 2010, 49, 6288–6308; Angew. Chem. 2010, 122, 6430–6452.
[56] S. Zalipsky, Adv. Drug Delivery Rev. 1995, 16, 157–182.
[57] B. Obermeier, F. Wurm, C. Mangold, H. Frey, Angew. Chem. Int. Ed. 2011,
50, 7988–7997; Angew. Chem. 2011, 123, 8136–8146.
[58] R. Kumar, M.-H. Chen, V. S. Parmar, L. A. Samuelson, J. Kumar, R. Nicolosi,
S. Yoganathan, A. C. Watterson, J. Am. Chem. Soc. 2004, 126, 10640–
10644.
[66] I. N. Kurniasih, H. Liang, S. Kumar, A. Mohr, S. K. Sharma, J. P. Rabe, R.
Haag, J. Mater. Chem. B 2013, 1, 3569–3577.
[67] R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals,
6th ed., Molecular Probes. Eugene, 1996.
[68] D. Tomassoni, A. Lanari, G. Silvestrelli, E. Traini, F. Amenta, Clin. Exp.
Hypertens. 2008, 30, 744–766.
[69] H. Türk, A. Shukla, P. C. A. Rodrigues, H. Rehage, R. Haag, Chem. Eur. J.
2007, 13, 4187–4196.
[70] S. K. Sharma, R. Kumar, S. Kumar, R. Mosurkal, V. S. Parmar, L. A.
Samuelson, A. C. Watterson, J. Kumar, Chem. Commun. 2004, 23, 2689–
2691.
[71] Rashmi, A. K. Singh, K. Achazi, S. Ehrmann, C. Böttcher, R. Haag, S. K.
Sharma, Polym. Chem. 2020, 11, 6772–6782.
[59] H. Donga, L. Panga, H. Conga, Y. Shena, B. Yua, Drug Delivery 2019, 26,
416–432.
[72] A. K. Singh, R. Nguyen, N. Galy, R. Haag, S. K. Sharma, C. Len, Molecules
2016, 21, 1038.
[73] B. Norjannah, H. C. Ong, H. H. Masjuki, J. C. Juan, W. T. Chong, RSC Adv.
2016, 6, 60034–60055.
[60] S. Gupta, B. Schade, S. Kumar, C. Böttcher, S. K. Sharma, R. Haag, Small
2013, 9, 894–904.
[61] A. Halldorsson, C. D. Magnusson, G. G. Haraldsson, Tetrahedron 2003, 59,
9101–9109.
[62] M. B. van Eldijk, F. C. M. Smits, N. Vermue, M. F. Debets, S. Schoffelen,
J. C. M. van Hest, Biomacromolecules 2014, 15, 2751–2759.
[63] A. Klaikherd, C. Nagamani, S. Thayumanavan, J. Am. Chem. Soc. 2009,
131, 4830–4838.
[64] W. C. Griffin, J. Soc. Cosmet. Chem. 1949, 1, 311–326.
[65] E. Fleige, B. Ziem, M. Grabolle, R. Haag, U. Resch-Genger, Macro-
molecules 2012, 45, 9452–9459.
Manuscript received: January 14, 2021
Revised manuscript received: February 5, 2021
Accepted manuscript online: February 8, 2021
Version of record online: March 8, 2021
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