Photoresponsive transformation from spherical to nanotubular assemblies: Anticancer drug delivery using macrocyclic cationic gemini amphiphiles

Article abstract of DOI:10.1039/d1cc01468d

We developed NIR-light-responsive macrocyclic cationic gemini amphiphiles, one of which displayed various favorable properties of lipids. The NIR-light-mediated cleavage of the strained dioxacycloundecine ring led to the conversion of the spherical to a n

Full text of DOI:10.1039/d1cc01468d

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Number 38
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1 May 2021
Pages 4595–4740
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COMMUNICATION
Debasis Manna et al.
Photoresponsive transformation from spherical to
nanotubular assemblies: anticancer drug delivery
using macrocyclic cationic gemini amphiphiles

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Photoresponsive transformation from spherical to
nanotubular assemblies: anticancer drug delivery
using macrocyclic cationic gemini amphiphiles†
Cite this: Chem. Commun., 2021,
7, 4646
5
Received 18th March 2021,
Accepted 12th April 2021
a
b
c
a
Subhasis Dey, Soumya Chatterjee, Anjali Patel, Nirmalya Pradhan,
d
d
b
Diship Srivastava,
Debasis Manna
Niladri Patra, Arindam Bhattacharyya and
DOI: 10.1039/d1cc01468d
ac
*
rsc.li/chemcomm
We developed NIR-light-responsive macrocyclic cationic gemini
Cationic lipids have attracted significant attention as effi-
amphiphiles, one of which displayed various favorable properties of cient DDSs due to the higher abundance of negatively charged
7
,8
lipids. The NIR-light-mediated cleavage of the strained dioxacycloun- phospholipids in the outer membrane of cancer cells.
decine ring led to the conversion of the spherical to a nanotubular Furthermore, if stimuli-responsive linkers are covalently
self-assembly in the aqueous medium. This photo-mediated transfor- attached to the lipid molecules that can be cleaved following
mation from the spherical to nanotubular self-assembly resulted in delivery to the cells, this provides a controllable tool for
the release of encapsulated hydrophobic anticancer drug molecule triggering the release of encapsulated drug molecules. The
doxorubicin (Dox) in a controlled manner. The potent cationic gemini endogenous stimuli are unique for diseased cells or tissue,
amphiphile also displayed lower cytotoxicity and efficient NIR-light- and they can be exploited for augmenting the specificity of the
6
mediated Dox release efficacy to cancerous cells.
drug molecules. However, most of the cleavages mediated by
these endogenous stimuli are restricted by the limiting condi-
Currently, significant efforts are being dedicated to early diagnosis tions obligatory for the release, as they rely on differences in
and developing various biocompatible drug delivery strategies to properties between cells. The use of a near-infrared (NIR;
1,2
fight against cancer. Lipid-based nanocarriers represent effec- exogenous stimulus) light-responsive DDS is one of the attrac-
tive agents for efficiently delivering appropriate therapeutic doses tive strategies as it can be used over long distances with
5
of various chemotherapeutic drugs to targeted cancer cells or excellent spatial and temporal resolutions. Meanwhile, the
2–5
tissues. Besides lower toxicity, biodegradability, prolonged cir- poor biocompatibility of several synthetic cationic lipids
culation properties, and non-immunogenicity are also the remark- restricted their clinical applications. Therefore, it is imperative
6
able advantages of lipid-based drug delivery system (DDSs).
to develop easily synthesizable and biocompatible photo-
However, the slow-drug release profiles of various lipid-based responsive lipids.
DDSs at disease sites significantly compromise their clinical Recently, a photoresponsive lipid–drug conjugate was developed
9
efficiency. It is challenging to control the delivery of appropriate to deliver drug molecules to cancer cells. However, the use of
9
doses of the drug at the right time. Moreover, precisely controlled UV-light (365 nm) restricted its further clinical applications.
release of drug molecules into deep tumors is a prerequisite for Several liposome-based DDSs were also developed where cocktail
enhancing the therapeutic efficiency of lipid-based DDSs. Hence, mixtures of drug molecules and NIR-light-sensitive dyes were used
3–5,10–13
the development of facile methods to synthesize novel lipid-based for NIR-light-mediated drug delivery applications.
This
nanocarriers with excellent drug release profiile has been of approach is time-consuming and an economic burden for both
significant interest.
pre-clinical and clinical validation of each molecule associated with
the DDSs and the cocktail mixtures of agents. However, the use of
only NIR-light-responsive lipids requires the clinical validation of a
lower number of molecules.
a
Indian Institute of Technology Guwahati, Chemistry, Guwahati, Assam, India.
E-mail: dmanna@iitg.ac.in
b
Calcutta University, Zoology, Kolkata, West Bengal, India
Most of the reported NIR-light responsive drug carriers are
designed with the ester or carbamate moiety as the linker with
the photon capturing 2-nitrobenzyl alcohol group. However, the
direct attachment of ester, carbamate, and oxime functional
groups to the 2-nitrobenzyl moiety makes the linker more
c
Indian Institute of Technology Guwahati, Centre for the Environment, Guwahati,
Assam, India
d
Indian Institute of Technology (Indian School of Mines) Dhanbad, Chemistry,
Dhanbad, Jharkhand, India
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
5
,12
d1cc01468d
susceptible to chemical and enzymatic degradation.
To
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curtail the aforementioned problems of structural complexity nIR16, respectively. The synthesized macrocyclic compounds are
and linker instability, in this study, we demonstrated the unconventional and structurally quite different from naturally
synthesis of 2-nitrobenzyl containing 11-membered strained occurring phospholipids.
macrocyclic amphiphiles. We hypothesized that the installation
The hydration of amphiphilic nIR12 resulted in the sponta-
of the UV-active, 2-nitrobenzyl moiety within the highly neous formation of colloidal aggregates. The hydrophobic
strained macrocyclic moiety would make it NIR sensitive and effect could be the major driving force for its self-assembly in
endow it with substantial structural change, aggregation behavior, the aqueous medium. However, the other synthesized amphi-
and efficient drug release behavior. Macrocyclic compounds such philes failed to self-assemble in the aqueous medium, which
as calixarenes, cyclodextrins, and others have been used in drug could be due to either the high hydration energy (nIR8) or the
14
and gene delivery, but NIR-light-responsive macrocyclic amphi- very low hydration energy (nIR16). The pyrene-based fluores-
philes have never been applied in drug delivery and represent a cence assay revealed that the critical aggregation constant
comprehensive strategy for smart DDSs. One of the synthesized (CAC) of nIR12 was 28.7 mM (Fig. 1B and Fig. S1, ESI†). The
gemini cationic amphiphile showed successful encapsulation and lower CAC value suggests the tighter packing of nIR12 mole-
delivery of the most widely used hydrophobic chemotherapeutic cules within the spherical NAs, which could be due to the
drug Dox to cancer cells.
Modular amphiphiles were synthesized from 3-bromoaniline headgroup region. The field-emission transmission electron
Fig. 1A and Scheme S1, ESI†). The reductive methylation of microscope (FETEM) analysis revealed the formation of sphe-
presence of a conformationally restricted cyclic ring in the
(
3
-bromoaniline provided 3-bromo-N,N-dimethylaniline (2). The rical nanoaggregates (NAs) with diameters of 150–200 nm
0
condensation of 2 with formaldehyde provided 4,4 -methylenebis (Fig. 1C). The pH-dependent dynamic light scattering (DLS)
15
(3-bromo-N,N-dimethylaniline) (3).
Subsequent ring-closure measurements showed that the hydrodynamic diameter of
reaction via Ullmann-type C–O coupling reaction between 3 and nIR12 varies within 225–275 nm, suggesting its stability in
8
(
3-nitro-1,2-phenylene)dimethanol (4) resulted in the macrocyclic different environments (Fig. S2 and S3, ESI†). The smaller
3
3
14 14
compound N ,N ,N ,N -tetramethyl-7-nitro-6,11-dihydro-17H- hydrodynamic diameter and pH-dependent stability of nIR12
tribenzo[c,g,j][1,6]dioxacycloundecine-3,14-diamine (5). Finally, could be useful for its effective use in clinical applications due
N-alkylation of compound 5 with iodooctane, 1-iodododecane, to the enhanced permeation and retention (EPR) effect. The
6
and 1-iodohexadodecane yielded compounds nIR8, nIR12, and surface potential of the NAs was around +13.0 mV at physiolo-
gical pH, which could be useful for selective targeting of cancer
cells over normal cells (Fig. S2, ESI†). Meanwhile, the pH-dependent
DLS and surface potential measurements of a DPPC/DPPS/nIR12
(2 : 2 : 1) lipid mixture also showed that the hydrodynamic diameter
varies within 225–250 nm and the surface potential of the NAs was
around +4.8 mV at physiological pH (Fig. S4 and S5, ESI†). The
higher abundance of PS and other negatively charged lipids makes
the outer membrane of cancer cells negatively charged. Therefore,
these cationic NAs could show preferential uptake efficiency by
8
cancer cells. The decrease in surface potential with the increase in
pH could be due to the increased solvation number.
The temperature-dependent steady-state anisotropy mea-
surements revealed that the phase transition temperature
(T ) value of nIR12 (48 1C; Fig. 1D) is much higher than that
m
of the dipalmitoylphosphatidylcholine (DPPC) lipid (41 1C),
demonstrating its higher thermal stability and superior drug
8
,16
retention capability at normal body temperature.
The Tm
value for a mixture of lipids DPPC and dipalmitoylphosphati-
dylcholine (DPPS) (1 : 1) was 42 1C. In comparison, the Tm value
for the DPPC/DPPS/nIR12 (2 : 2 : 1) lipid mixture was 47 1C
m
(Fig. S6, ESI†). This increase of the T value could be due to
the interaction of the cationic ammonium moieties of nIR12
with the phosphate group of the phospholipids. The lipid
mixture also showed excellent liposome formation capability,
suggesting the ability of nIR12 to form liposomes with other
model lipids (Fig. S7, ESI†), which could be useful in drug
delivery, preparing size controllable liposomes, and others.
Meanwhile, the molecular dynamics (MD) simulation studies
also revealed the formation of a stable lipid bilayer of a mixture
of nIR12, DOPC, and DOPS and the interaction of the
Fig. 1 Synthetic routes to macrocyclic gemini cationic amphiphiles (A).
Measurement of the critical aggregation constant of nIR12 using a pyrene
probe (B). Representative FETEM image of the water-soluble aggregates
produced from only nIR12 (C). The temperature-dependent fluorescence
anisotropy measurement of environment-sensitive 1,6-diphenyl-1,3,
5-hexatriene dye incorporated within the water soluble aggregates (D).
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In addition, H
a
(d = 4.00 ppm) and H
c
(d = 5.22 ppm) proton
signals were also shifted to d = 3.78 and 5.01 ppm, respectively.
Overall, these changes in proton signals in the 1H NMR spectra
upon photoirradiation indicate the cleavage of the o-nitro-
benzyl moiety and the opening of the 11-membered strained
macrocyclic ring which was further confirmed by mass spectro-
metric analysis (Fig. S9, ESI†). The strained cyclic moiety of
nIR12 is linked with a photon absorbing 2-nitrobenzyl moiety.
On NIR-light absorption, the 2-nitrobenzyl group depletes the
energy by opening the constrained ring to an open-chain
conformation. Within the macrocyclic system, the molecular
angular strain leads to a drift from the tetrahedral geometry,
which is liable to easy cleavage of the C–O bond via the Norrish
1
7
type II mechanism. The FESEM and FETEM analysis of nIR12
in the presence of NIR-light also showed a significant change in
its aggregation patterns, indicating its instability upon photo-
irradiation. The spherical aggregation pattern of nIR12 in the
aqueous medium turned into a nanotubular self-assembly
(diameter around 3.7 nm) upon NIR irradiation (Fig. 2C and
Fig. S8, ESI†), which could allow the release of encapsulated
drug molecules. The photocleavage of the 2-nitrobenzyl con-
taining macrocyclic ring generated an open-chain conforma-
tion, which would self-aggregate in the aqueous medium to
form the nanotubular assembly. These organic nanotubes
could be beneficial for constructing building blocks for various
1
8
optoelectronic devices. The FETEM analysis of the DPPC/
DPPS/nIR12 lipid mixture showed complete disruption of the
spherical self-assembly after the NIR-light treatment (Fig. S10,
ESI†). The nIR12 and DPPC/DPPS/nIR12 lipid mixture showed
adequate loading capability of hydrophobic Dox molecules
(41 and 46% at pH 7.4; Fig. S10, ESI†) for the chemotherapeutic
drug, Dox, suggesting that the hydrophobic Dox molecules
could be encapsulated within the hydrophobic core of the
8
spherical assembly of nIR12. The Dox release profiles without
and with photoirradiation (10 min) showed 2% and 83%
release efficiencies after 2 h, respectively (Fig. 2D), suggesting
the conformational changes in the presence of NIR-light led to
the destabilization of the bilayer assembly of nIR12 and
induced Dox release capability. The Dox release efficiencies
Fig. 2 The schematic diagram represents the self-aggregation patterns of
nIR12 before and after NIR-light treatment (10 min) (A). H NMR spectra of
1
nIR12 without and with NIR-light treatment at different time intervals (B). from the lipid mixture without and with photoirradiation
Representative FESEM images of the water-soluble aggregates produced
(
10 min) were 2% and 74% after 2 h, respectively (Fig. S11,
from only nIR12 before and after NIR-light treatment (10 min) (C). The Dox
release profiles of nIR12 at pH 7.4 without and with NIR-light treatment
ESI†). This faster Dox release profile of nIR12 is beneficial for
its drug delivery applications, including minimizing adverse
effects because of the nonspecific delivery to the healthy cells
and augmentation of the local concentration of the Dox mole-
cules within the cancer cells and others (Fig. 2E).
(10 min) (D). Cartoon diagram representing the NIR-light treated Dox
release from the Dox encapsulated NAs (E).
phosphate groups of the phospholipids with the cationic
moieties of nIR12 (Fig. S8 and Movie S1, ESI†).
The cell viability assay showed that nIR12 had a low or
negligible toxic effect (after 48 h of incubation) on human MDA-
The NIR-light-mediated ring-opening (Fig. 2A) of macro- MB-231 (triple-negative breast cancer) cells and human periph-
1
cyclic amphiphile nIR12 was confirmed by H NMR (Fig. 2B). eral blood mononuclear cells (PBMCs; healthy cells), indicating
1
6
3
A solution of nIR12 in CDCl solvent was irradiated with NIR its significance in drug delivery applications (Fig. S12, ESI†).
ꢀ
2
laser light (808 nm, 1 W cm ) at different time intervals (0, 3, The MTT assay showed that the IC50 value of free Dox was
1
5
, 7, and 10 minutes). The time-dependent H NMR analyses 0.23 ꢁ 0.02 mM. The MTT assay revealed that the IC value of
5
0
revealed the disappearance of characteristic H proton signals Dox encapsulated nIR12 (Dox@nIR12) with NIR-light treatment
b
(
benzyl moiety) at d = 5.56 ppm and the appearance of a new (10 min) was 0.72 ꢁ 0.05 mM (with respect to the effective Dox
signal at d = 9.69 ppm that corresponds to the aldehyde proton. concentration). However, when the cells were incubated with
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The amphiphile showed lower cytotoxicity and successful
entrapment and delivery of the hydrophobic anticancer drug,
Dox, to cancerous MDA-MB-231 cells. The NIR-light mediated
photocleavage of the amphiphile within the cancer cells pro-
motes cell death. Hence, this simple and innovative concept of
developing macrocyclic gemini cationic amphiphiles as
potential DDSs could be useful in photodynamic therapy,
which could be dissected in a controlled fashion using
NIR-light to liberate the enveloped hydrophobic chemothera-
peutic drug molecules in a spatially- and temporally-controlled
manner for surface and deep-tissue cancer treatment.
The authors acknowledge the Department of Biotechnology,
Government of India (BT/PR41335/NNT/28/1780/2021), for
Fig. 3 Viabilities of MDA-MB-231 cells in the presence of free Dox and financial support. NP acknowledges TEQIP-III for funding.
Dox@nIR12 after 48 h of incubation (A). Viabilities of MDA-MB-231 cells in
the presence of nIR12 (125 mM) and Dox@nIR12 (1.25 mM Dox and 125 mM
amphiphile) (B). Flow cytometry analysis to measure the Dox uptake
efficiencies of MDA-MB-231 cells treated with nIR12, free Dox (125 mM), Conflicts of interest
and Dox@nIR12 (1.25 mM Dox and 125 mM nIR12) (C). Representative CLSM
images of the MDA-MB-231 cells incubated with Dox@nIR12 (1.25 mM Dox There are no conflicts to declare.
and 125 mM amphiphile) for 8 h followed by NIR-light treatment for 10 min.
The blue channel (DAPI; D), red channel (Dox; E), overlay of the blue and
red channels (F), and bright-field (G) illustrate the Dox release efficacy to References
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