A stimuli-responsive anticancer drug delivery system with inherent antibacterial activities

Article abstract of DOI:10.1039/c9cc08834b

We describe a novel class of stimuli-sensitive sulfonium-based synthetic lipids, which exhibit several favorable biophysical properties of phospholipids. The potent sulfonium-based lipid was successfully disassembled by glutathione to release the encapsul

Full text of DOI:10.1039/c9cc08834b

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DOI: 10.1039/C9CC08834B
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Stimuli-Responsive Anticancer Drug Delivery System with
Inherent Antibacterial Activities
Subhasis Dey,a Anjali Patel,b Khyati Raina,c Nirmalya Pradhan,a Oindrila Biswas,a Rajkumar P.
Received 00th January 20xx,
Accepted 00th January 20xx
Thummer*,c and Debasis Manna*,a,b
DOI: 10.1039/x0xx00000x
We describe a novel class of stimuli-sensitive synthetic lipids, which potential, protein/enzyme levels, and others of the tumour
possesses several favorable biophysical properties of microenvironment, havebeenusedasstimuliforchemotherapeutic
phospholipids. The sulfonium-based lipid was successfully drug delivery applications. These stimuli cause a significant change
disassembled by glutathione to release the encapsulated drug in the chemical structure of the drug carriers due to the cleavage of
molecules in a controlled manner. The cationic lipid also showed specific functional groups or changes in physicochemical properties,
lower cytotoxicity against mammalian cells and displayed which instigate the release of encapsulated drug molecules
12
moderate antibacterial activities.
in‐or‐around the diseased cells.4,
The glutathione (GSH)-
sensitive lipids may provide a practical approach to develop
potential drug carriers. Using the thiol-disulfide exchange reaction
between GSH and disulfide bond of the carrier and the irregularity
of GSH concentration between normal cells and cancer cells, a large
number of complex polymeric materials have been developed as
drug carriers.4, 8, 13 Unfortunately, GSH sensitive vesicle formulation
has not been well explored. Recently, the GSH-sensitive disulfide
containing phosphatidylcholines (SS-PCs) has been demonstrated.14
However, this redox-sensitive disulfide-based nanocarrier showed a
quick response to S-S bond cleavage in the cellular
microenvironment. This high reactivity of GSH towards the S-S bond
limits its intracellular uptake and increases the risk of cytotoxic
effect.14, 15
To curtail the problems related to the high reactivity and low cellular
uptake abilities of the disulfide-based liposomal drug delivery
system, we introduced the sulfonium-based lipids. The sulfonium
moieties are naturally occurring and present in several therapeutic
agents including, bleomycin, and adenosylmethionine. It is
reported that the sulfonium containing compounds are less
toxic to the mammalian cells in comparison with the other
onium analogous.16 The higher concentration of GSH is only known
to cleave the sulfonium moieties in a controlled manner.15 We
postulate that the presence of the sulfonium moiety could
improve the cellular uptake efficiency and drug release profile of
these lipids in comparison to disulfide-containing lipids.
The delivery of drug molecules to the specific cells or tissues
through the blood circulation system is one of the major
impediments in pharmaceutical research.1-3 Advanced drug
delivery strategies including the lipid-based nanoparticles are
considered as a promising approach to effectively transport a wide
range of therapeutic agents including the hydrophobic drug
molecules to the targeted cells or tissues with apposite therapeutic
doses.1-5 The biodegradability, non-immunogenicity, andothersare
the unique felicitous properties of the lipid-based drug delivery
7
system.6, The growing number of FDA approved lipid-based
formulations, including Doxil®, Ambisome®, and DepoDur™ provide
evidence of their enormous potential as a capable drug carrier.8 The
structure of the natural phospholipids has been extensively
modified to optimize its self-assembly, serum-stability, circulation
time, targeted delivery, circumventing side effects, and others. The
modification of lipid head-group through the installation of
phosphate bioisosteres, polyethylenes, cationic moieties, cell-
specific ligands, and others have been exploited to augment its
stability, circulation times, cell-specific drug delivery ability.4, 6-11
Currently, the stimuli‐responsive drug delivery strategy is
considered as one of the practical approaches in terms of drug
efficacy, especially for cancer treatment.4, 12, 13 Significant changes
in internal physiological conditions, including the pH, redox
Herein, we synthesized a series of novel sulfonium-based lipids,
which showed a wide range of favourable biophysical properties
such as the formation of aqueous soluble spherical aggregates, high
phase transition temperature, stimuli-responsive system, and
others. Additionally, the potent lipid has lower cytotoxicity, higher
efficiency to encapsulate, and release hydrophobic drug,
aIndian Institute of Technology Guwahati, Chemistry, Guwahati, Assam, India.
bIndian Institute of Technology Guwahati, Centre for the Environment, Guwahati,
Assam, India.
cIndian Institute of Technology Guwahati, Biosciences and Bioengineering,
Guwahati, Assam, India.
†
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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doxorubicin (Dox) to the cancer cells. The cellular uptake efficiency formation of any soluble aggregates, which couVldiewbAerticdleuOenlitnoe
intheabsenceandpresenceofGSHinhibitordemonstratedthatthe their higher hydration energies. HenceD,OIf:u10rt.1h0e3r9/Cb9ioCpCh08y8si3c4aBl
intracellular GSH level acts as a stimulus to release the drug characterizations were performed only with the SL1a lipid. The
molecules in cancer cells. Additional studies revealed that the dynamic light scattering (DLS) measurement revealed that the
sulfonium-based lipid itself has moderate antibacterial activity hydrodynamic diameters (dH) of the vesicles were within 200-
against both gram-positive and gram-negative bacteria.
450 nm, and the pH of the solution has a negligible effect on its
7
The novelty and recent applications of the GSH-responsive drug dH values (Fig. S2).6, The zeta‐potential measurement at
delivery approach motivated us to design sulfonium-based lipids various pH values revealed that the vesicles form positive
(Fig. 1A). These modular lipids were synthesized using 1,3,5- surface potential, which could be due to the presence of
tris(bromomethyl)benzene as the core unit. The mono-modification sulfonium moieties (Fig. S3). The decrease in surface potential
with azide and trimethylamine resulted in the formation of values with the increase in pH suggest that the reduction of
compounds 2 and 3, respectively. The nucleophilic addition of sulfonium positive charge could be due to the increase in the
aliphatic thiols with compounds 2 and 3 yielded compounds 4 and solvation number of the negatively charged hydroxyl group.
5, respectively. The azide-alkyne click reaction of compound 4 with The temperature-dependent steady-state fluorescence
propynesulfonic acid produced compound 6. Finally, the S- anisotropy measurements revealed that the phase transition
methylation of compounds 4, 5, and 6 yielded the target temperature (Tm) of SL1a lipid was 64 °C (Fig. 1C).6, 7 The higher
compounds SL1, SL2, and SL3, respectively, with good yields Tm values demonstrate higher thermal stability of the vesicles.
(Scheme S1).15 For all these SL-lipids, the long alkyl chains (tail Whereas, the Tm value of co-liposome (SL1a: DOPE of 1:4) was
lengths of 12 and 16) were associated with sulfonium moieties. The 54 °C, which is much higher than phospholipid, DOPE (Tm = -16
presence of azide group provides access to install various °C), suggesting that the presence of sulfonium moieties plays a
headgroups. We hypothesize that the nature and inherent crucial role in their molecular arrangements. The higher Tm
properties of these synthetic sulfonium lipids would be quite value of the SL1a vesicle and co-liposome than normal human
different from that of natural glycerophospholipids. Hence, it is body temperature (37 °C) also suggests their better drug
vital to explore its physicochemical properties.
retention ability in comparison with only DOPE, which indicates
their prospective application in thermally triggered drug
delivery systems. It is important to mention that the increase in
kinetic energy of the vesicles with the increase in temperature
could induce the rearrangement of their aggregation pattern
prior to or post to their phase transition.
The presence of sulfonium groups encouraged us to investigate
whether the vesicles can be deformed in a controlled manner in
the presence of stimuli like GSH and others. To confirm the
dealkylation of SL1a lipid, we performed the HPLC analysis in
the absence and presence of GSH (10 mM) in PBS (pH 7.4) at 37
°C (Fig. 2A). The time-dependent HPLC analysis revealed that
the formation of mono- and di-dealkylated intermediates start
after 36 and 72 h of incubation (Fig. 2A and B). The dealkylations
of SL1a lipid were scrutinized by mass spectrometry (Fig. S4 and
S5). The control experiment showed that the SL1a lipid is very
much stable in the absence of GSH under similar experimental
conditions (Fig. S6). The TEM and TEM-EDX analysis of the SL1a
lipid in the presence of GSH also showed a significant change in
their aggregation arrangements, indicating its instability under
the reduced environment (Fig. 2C and S7).
This stimuli-responsive dealkylation-mediated deformation of
vesicles of SL1a lipid prompted us to investigate its drug
encapsulation and release profiles (Fig. S9). The SL1a lipid
showed adequate loading aptitude (33% at pH 7.4) for
hydrophobic anticancer drug Doxorubicin (Dox). This moderate
drug loading capacity of SL1a lipid suggests that the positively
charged hydrophobic Dox (pKa = 8.4) molecules could be
entrapped within the hydrophobic core of the positively
charged lipid assembly. Fluorescence studies revealed that
57%ꢀand 88% of Dox was released after 60 h in the absence and
presence of GSH, respectively at 37°C (Fig. 2D).6, 7 The release-
profile also revealed a drastic difference after 36 h, suggesting
the possibility of the destabilization of SL1a lipid assembly in the
Fig. 1. Synthetic routes to the sulfonium-based lipids (A). The TEM image of the soluble
aggregates generated from the 100% SL1a lipid (B). The steady-state fluorescence
anisotropy measurement of 1,6-diphenyl-1,3,5-hexatriene of the lipids within the range
of 5–90ꢀ°C. The Inset shows the Tm values of the lipids (C).
The hydration of the dried film of the SL1a lipid ensued in the
formation of colloidal aggregates. The analysis of transmission
electron microscopic (TEM) images revealed the formation of
spherical aggregates of the SL1a lipids (Fig. 1B).6, 7 The TEM
analysis also revealed that the mixture of SL1a and
dioleoylphosphatidylethanolamine (DOPE) lipids (1:4) form
excellent vesicles (Fig. S1), suggesting the competence of SL1a
lipid in forming vesicles with other phospholipids, which could
be beneficial in constructing size-tunable vesicles for specific
uses like bio-imaging and drug delivery. The transmission
electron microscopy-energy dispersive X-ray spectrometry
(TEM-EDX) elemental mappings of sulfur provided direct
evidence of the self-assembled structure of SL1a lipid (Fig. S1).
However, the other synthetic lipids were less efficient in the
2 | J. Name., 2012, 00, 1-3
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presence of GSH. This time-dependent release profile is in buthionine sulfoximine (BSO).15 The result showed that the cell
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accordance with the HPLC data. This controlled Dox release viability increased in the BSO treated celDlsO,Iw: 1h0i.1c0h39c/oCu9lCdCb0e88d3u4Be
profile of SL1a lipid is advantageous for its further biological to the inhibition of GSH activity (Fig. 3B and S11).15
applications, including a reduction in side effects due to The cellular uptake efficiency of SL1a lipid was further
nonspecific delivery and enhancement of the activity the Dox investigated using a confocal laser scanning microscope
and others.
(CLSM).6, 7 The microscopic images of the MDA-MB-231 cells
revealed a higher Dox delivery efficiency of the SL1a lipid in
comparison with the lipid 4a (Fig. 3C, S12-14). The long alkyl
chains of the neutral lipid 4a were associated with only thioether
moieties. To exclude the probability of fluorescence signal
originated due to the adherence of the Dox@SL1a or Dox@4a
to the cell surface, the flow cytometry analysis was performed
using the inherent fluorescence signal of Dox molecules (FL2-H
channel). The result showed that the Dox@SL1a has a higher
cellular uptake efficacy in comparison with that of Dox@4a in
MDA-MB-231 cells (Fig. S15), which could be due to the superior
electrostatic interaction of the positively charged sulfonium
moieties with the negatively charged phosphatidylserine (PS)
lipid present in higher abundance on the outer surface of the
cancer cells.18 This directed electrostatic interaction could also
help the cationic sulfonium lipids to reduce the off-target side
effects of the Dox itself. The higher cellular uptake of free Dox
could be due to its diffusion–mediated delivery to the cells, whereas
liposome-mediated Dox delivery generally follows the endocytosis
process (Fig. S15).2,7
Fig.2. The HPLC traces for the dealkylation of lipid SL1a in the presence of GSH (10 mM)
at different time intervals (A). Schematic representation of the dealkylation strategy (B).
The dealkylation of compound SL1a (1 mmol) in the presence of GSH (10 mmol) in PBS
(pH = 7.4) at 37 °C. The TEM image of the SL1a lipid in the presence of GSH (10 mmol)
after 36 h of incubation (C). The Dox release profile of the SL1a lipid at pH7.4 in the
absence and presence of GSH (10 mM).
The MTT-based viability assay showed that the SL1a lipid had
lower cytotoxic effect against MDA-MB-231 (human triple-
negative breast cancer; IC50 = 125 µM) and BJ (human foreskin
fibroblasts; IC50 = 166 µM) cell lines, suggesting its relevance in
drug delivery applications (Fig. S10A and S10C).2, 7 The MTT
assay in the presence of free Dox and Dox encapsulated SL1a
lipid (Dox@SL1a) revealed that the IC50 values were 0.49 and
0.36 μM (with respect to effective Dox concentration),
Fig.3. Time-dependent viabilities of MDA-MB-231 cells in the presence of free Dox and
respectively in MDA-MB-231 cell line, which support the Dox Dox@SL1a at 0.36 and 0.49 µM, respectively (A). The effective concentration of Dox in
Dox@SL1a was 0.49 µM (calculated using the loading capacity). Viabilities of the
Dox@SL1a (0.49 µM) treated MDA-MB-231 cells in the absence and presence of
glutathione inhibitor, BSO (1 mM) (B). CLSM images of the MDA-MB-231 cells treated
with Dox@SL1a (0.49 µM) for 8 h. Red channel (C),ꢁblue channel (D),ꢁmerge of red and
encapsulation and release efficiency of SL1a lipid (Fig. S10B).
The IC50 value of Dox@SL1a treated BJ cells was 1.47 μM (with
respect to effective Dox concentration; Fig. S10D). The higher
concentration of GSH levels in MDA-MB-231 cells attributed to
the cleavage of sulfonium lipids, resulting in much lower IC50
value of the Dox@SL1a.17 The final concentration of Dox was
kept same for both the MTT assay in the presence of free Dox
and Dox@SL1a. The time-dependent MTT assay showed that
the rate of cell death was faster for Dox@SL1a, indicating its
superior uptake and release of Dox molecules, which is one of
the rudiments of a potential drug carrier (Fig. 3A). To further
investigate that the GSH promotes the Dox release efficiency
from Dox@SL1a under the cellular environment, the MTT assay
was performed in the absence and presence of GSH inhibitor,
blue channels (E) illustrate the Dox delivery. Scale bar 25ꢁμm.
The presence of sulfonium moieties and lower cytotoxicity
against mammalian cell lines endorsed us to investigate the
antibacterial activities of SL1a lipid.16 The antibacterial activity
of the SL1a lipid was tested against both gram-negative (E. coli;
MTCC-1687) and gram-positive (S. aureus; MTCC-96) bacteria and
the minimum inhibitory concentration (MIC) values were 32 ± 2
and 14 ± 1 µM, respectively (Fig. 4A, S16 and Table S1). For
further investigation, the antibacterial activities were also
performed in the presence of free Dox and Dox@SL1a.
This journal is © The Royal Society of Chemistry 20xx
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Interestingly, the free Dox has much lower antibacterial activities under the reductive tumour environment to VireewlAeraticslee Ontlhinee
DOI: 10.1039/C9CC08834B
(MIC values >72 and 36 µM for E. coli and S. aureus, encapsulated drug molecules.
respectively.19 Whereas, the MIC values were 0.19 and 0.10 µM for
The authors gratefully acknowledge the Department of
Biotechnology, Government of India (MED/2015/04) and the
Science and Engineering Research Board, Govt. of India
(EMR/2016/005008) for financial support.
E. coli and S. aureus, respectively (with respect to effective Dox
concentration; Table S1), in the presence of Dox@SL1a. Thefield
emission scanning electron microscope (FESEM) analysis showed
a noticeable loss of cell shape and integrity in the presence of SL1a
and Dox@SL1a (Fig. S17 and 4B). Overall, the antimicrobial studies
revealed that the ammonium-free drug carrier itself possesses
antibacterial activities. Recent studies revealed that the
peptidoglycan of bacterial cell wall promotes the invasiveness of
breast cancer cells by upregulating the Toll-like receptor 2.20 Gram-
negative bacteria like E. coli and B. fragilis are also known to
produce colorectal cancer-promoting toxin.21 Therefore, the
inherent antibacterial activity of the sulfonium lipid, along with its
anticancer drug delivery efficacy, represents a highly efficient
stimuli-responsive delivery system.
Conflicts of interest
There are no conflicts to declare.
Notes and references
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Fig. 4. The 96 well plate bacterial assay of Dox@SL1a (first and third row; with respect to
SL1a concentration) against E. coli (MTCC-1687S). The clear wells specify inhibition of
bacterial growth, while the cloudy wells indicate unconstrained bacterial growth. FESEM
images of E. coli (MTCC-1687S) treated with Dox@SL1a (B) and only Dox (C).
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Conclusions
The sulfonium-based modular lipids were successfully
synthesized under mild reaction conditions. The potent cationic
lipid self-assembled in the aqueous medium to form soluble
spherical aggregates, which showed high phase transition
temperature, GSH-responsive cleavage, and other favorable
biophysical properties of drug delivery agents. The sulfonium
lipid displayed successful encapsulation and delivery of the
hydrophobic anticancer drug, Dox to the MDA-MB-231 cells.
Interestingly, the sulfonium lipid itself possesses antimicrobial
activities against both gram-positive and gram-negative
bacteria. Hence, the sulfonium lipid could be a potential
alternative to the natural phospholipids, which can fight against
bacterial infection and can be cleaved in a controlled fashion
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