Reduction-Triggered Doxorubicin Delivery by Self-Assembled Nanospheres of Lipoylated Caffeine

Article abstract of DOI:10.1002/cmdc.202000070

This study reports a new amphiphilic bioconjugate (CAFF-LA) derived from the lipoylation of a hydroxyethyl derivative of caffeine. In water, CAFF-LA self-assembles into nanospheres with an average size of 155 nm, as evidenced from dynamic light scattering and electron microscopy studies. The nanospheres are stable in serum and could be disintegrated upon exposure to the reducing environment of dithiothreitol (DTT; 10 mM) and glutathione (GSH; 10 mM). These nanospheres easily encapsulate the chemotherapy medication, doxorubicin (DOX), and demonstrate an efficacious transport into doxorubicin-resistant cervical cancer (HeLa) cells, wherein a marked induction in apoptosis and significantly lower IC₅₀ have been observed when compared to that of free drug. The in vitro assessment of cell viability and hemocompatibility present these nanospheres as potentially safe and efficient intracellular reduction stimulus-responsive drug-delivery vehicles.

Full text of DOI:10.1002/cmdc.202000070

Communications
doi.org/10.1002/cmdc.202000070
ChemMedChem
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Reduction-Triggered Doxorubicin Delivery by Self-
Assembled Nanospheres of Lipoylated Caffeine
Krishan Kumar+,[a] Bharti Rajesh Kumar Shyamlal+,[a] Rajbala Verma,[b] Paturu Kondaiah,*[c] and
Sandeep Chaudhary*[a]
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of reliable response in clinical development due to their toxicity
and drug interaction, limits their practical application.[3,4] The
prevention of MDR development by small molecules has also
been looked at as strategy to enhance efficacy of chemotherapy
treatment.[4b] On the other hand, advances in nanotechnology
have greatly influenced the design and development of drug-
delivery systems.[5] These nanomedicines aim at improving the
therapeutic index of drugs and deriving thereby maximum
therapeutic benefits. The relative clinical success of nano-
medicine platform fosters further development of safe and
efficacious nanomedicines in order to achieve desirable ther-
apeutic response. The stimuli responsive nanosystems have
emerged as powerful and potential tools to raise the intra-
cellular accumulation of drugs.[6] These are sensitive to specific
endogenous (e.g., pH and redox potential) and/or exogenous
(e.g., temperature and light) stimuli that trigger the release of
the encapsulated drug.[6] Amongst these stimuli is the exploita-
tion of relatively higher intracellular concentration (~2–10 mM)
of glutathione (GSH) to that of extracellular environment (~2–
10 μM), which can easily trigger the disorganization of nano-
carriers involving disulfide linkages.[6c] In addition, the tumor
cells are categorized with elevated levels of GSH which may
serve as a gateway to tumor-specific drug delivery.[7] This
understanding has been employed in developing various types
of reduction responsive nanosystems such as liposomes,[8]
polymeric nanoparticles/micelles,[9] niosomes,[10] dendrimers,[11]
nanogels,[12] and metal-organic frameworks,[13] to effectively
deliver the drug into cell cytoplasm. Scientific efforts are
constantly on the rise to develop more biocompatible delivery
systems to achieve desirable clinical success.[5b,14] The self-
assembling conjugates derived from molecules of biological
origin and which can respond to biological stimuli would
certainly serve as a basis for developing biocompatible systems
for efficacious drug delivery.[10,15] Therefore, we herein have
This study reports a new amphiphilic bioconjugate (CAFF-LA)
derived from the lipoylation of a hydroxyethyl derivative of
caffeine. In water, CAFF-LA self-assembles into nanospheres
with an average size of 155 nm, as evidenced from dynamic
light scattering and electron microscopy studies. The nano-
spheres are stable in serum and could be disintegrated upon
exposure to the reducing environment of dithiothreitol (DTT;
10 mM) and glutathione (GSH; 10 mM). These nanospheres
easily encapsulate the chemotherapy medication, doxorubicin
(DOX), and demonstrate an efficacious transport into doxorubi-
cin-resistant cervical cancer (HeLa) cells, wherein a marked
induction in apoptosis and significantly lower IC50 have been
observed when compared to that of free drug. The in vitro
assessment of cell viability and hemocompatibility present
these nanospheres as potentially safe and efficient intracellular
reduction stimulus-responsive drug-delivery vehicles.
Cancer incidence and mortality have become a global issue
with continuously increasing and frightening number of new
cases every year.[1] Chemotherapy is routinely recommended
treatment for cancer, but it can lead to adverse side effects due
to damage to healthy cells. Additionally, the emergence of
multi drug resistance (MDR) in cancer cells resists the
therapeutic effect of cytotoxic chemotherapy.[2] This, in turn,
leads to the requirement of high and frequent dose regimen,
further worsening the situation of many patients. The drug
efflux via ATP-binding cassette (ABC) transporters is a well-
known mechanism associated with the development of MDR in
cancer.[3] The anti-cancer research community has contributed
significantly in past decade which delivers hope to address this
issue. The use of MDR inhibitors in chemotherapy offers a
potential strategy for overcoming drug resistance. But, the lack
synthesized
a biocompatible amphiphilic conjugate from
[a] Dr. K. Kumar,+ B. R. Kumar Shyamlal,+ Dr. S. Chaudhary
Department of Chemistry, Malaviya National Institute of Technology
Jawaharlal Nehru Marg, Jaipur-302017, India
E-mail: schaudhary.chy@mnit.ac.in
[b] Dr. R. Verma
Department of Zoology, University of Rajasthan
Jaipur-302004, India
[c] Prof. P. Kondaiah
caffeine and lipoic acid that readily self-assembles into nano-
spheres in an aqueous solution. Caffeine is a globally consumed
alkaloid of pharmacological importance with unequivocal
antioxidant characteristics.[16] It is found in a variety of plant
species including coffee beans, tea leaves and cocoa beans.
Interestingly, caffeine has also been reported to possess dose-
dependent anticancer activity and also to potentiate the
cytotoxic effects of various anticancer drugs against different
tumor types including the drug-resistant ones.[17] Moreover,
caffeine is reported to inhibit drug efflux from tumor cells and
thereby increases their antitumor effects.[17b] On the other hand,
other moiety of the synthesized conjugate is universal antiox-
Department of Molecular Reproduction, Development and Genetics
Indian Institute of Science
Bangalore-560012, India.
E-mail: paturu@iisc.ac.in
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000070
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idant, lipoic acid which possesses remarkable therapeutic
potential and serves as an approved ingredient in many
pharmaceutical products.[18] The lipoic acid based self-assem-
blies are prone to disorganization within the reducing environ-
ment of cell cytoplasm associated with the cleavage of disulfide
linkage of its dithiolane ring which imparts redox responsive
revealed the self-assembled structures to be nanospheres in the
size range of 130–200 nm (Figure 2a). Very Interestingly, N-
functionalized theophylline based polyelectrolyte also has been
reported to produce similar spherical structures.[21] Few of these
CAFF-LA nanospheres manifested porous morphology with
different size and number of pores on their surface as shown in
group image of four individual spheres (Figure 2b). The
morphological analysis using transmission electron microscopy
(TEM) substantiated the presence of nanospheres of CAFF-LA
(Figure 2c). The atomic force microscopic (AFM) imaging further
evidenced the spherical nanostructures (Figure 2d). The surface
charge of nanospheres was determined by zeta potential
measurement and was observed to be 14.8�0.8 mV (Fig-
ure S7).
The hydrodynamic diameters of these nanospheres were
also determined by means of dynamic light scattering (DLS)
measurements which revealed them to be ~155 nm in size
(Table 1 and Figure S8a). The low polydispersity index (PDI)
values (~0.21) denoted nearly monodisperse population of
nanospheres (Table 1).[22] In addition, no significant difference in
the size of nanospheres was observed in 0.9% NaCl and 5%
glucose solution (Table S1) which remarks on possible practical
utility of these nanospheres. Subsequently, the DLS study was
further extended to assess the redox sensitive behavior of the
CAFF-LA nanospheres in the presence of reducing agent,
dithiothreitol (DTT; 10 mM). A significant increase in mean
hydrodynamic diameters (~295 nm) and PDI values (~0.7) of
CAFF-LA was observed post DTT treatment (2 h) which signified
the disorganization of nanospheres resulting from the reduction
of disulfides of lipoic acid moieties (Table 1 and Fig-
ure S8b).[10,15c,19b,23]
This redox sensitive behavior of nanospheres was further
exploited in a drug-delivery endeavor against cancer cells. For
which, the well-known anticancer drug, doxorubicin (DOX) was
encapsulated in nanospheres and the observed encapsulation
efficiency (EE) was 52.23% as determined based on a calibration
curve of DOX. These DOX loaded CAFF-LA nanospheres (DOX-
CAFF-LA) were then examined for their stability against serum
(fetal bovine serum; FBS) treatment. An overnight incubation (~
12 h) of DOX-CAFF-LA with FBS revealed substantial retention
(>85%) of encapsulated drug (Table 1). This result indicated
that these DOX nanospheres do not show any detrimental drug
release and may thus show good stability against serum
proteins in circulation.[24] In contrast, the DTT (10 mM; 6 h)
treatment of DOX-CAFF-LA resulted in nearly 76% release of
DOX which evidenced the reducing environment induced
disorganization of nanospheres leading to the release of drug.
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behavior to such systems for
a favorable drug-delivery
application.[10,15c,19] The present study investigates the self-
assembly of this lipoylated caffeine bioconjugate for drug-
delivery application.
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The conjugate (Figure 1) was synthesized by using DCC
(N,N’-dicyclohexyl carbodiimide)-induced coupling between
lipoic acid and hydroxyethyl derivative of caffeine 1 as shown in
supporting information (Scheme S1) and fully characterized
using 1H NMR, 13C NMR, ESI-MS and FTIR. The caffeine derivative
1 mentioned herein was developed by appending hydroxyethyl
group to caffeine using cross-dehydrogenative coupling with
ethanol following CÀ H bond activation as reported previously
(Scheme S2).[20] The hydroxy functional group on caffeine made
in convenient to the coupling reaction with lipoic acid.
The thin-film hydration method was used to look into self-
assembling process of CAFF-LA and the sample was analyzed
by starting with scanning electron microscopic (SEM) study. It
Figure 1. Molecular structure of the lipoylated caffeine derivative.
Table 1. Evaluation of size of CAFF-LA nanospheres (DLS) and DOX release
from DOX-CAFF-LA nanospheres.
Size [nm]/PDI
DOX release [%]
À DTT
+DTT[a]
+DTT[b]
75.6�4.8
+GSH[c]
53.1�6.8
+FBS[d]
13.3�1.5
154.9�4.3/
0.21�0.01
294.7�11.1/
Figure 2. Morphological investigation of CAFF-LA self-assembly (0.50 mM).
SEM images of self-assembled nanospheres of CAFF-LA (a) and of a few of
those with porous surfaces (b). The CAFF-LA nanospheres as observed under
TEM (c) and 3D AFM (d) analysis. Scale bars: 200 nm.
0.71�0.07
[a] CAFF-LA+DTT (2 h). [b] DOX-CAFF-LA+DTT (6 h). [c] DOX-CAFF-LA+
GSH (6 h). [d] DOX-CAFF-LA+FBS (12 h).
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We also evaluated the redox sensitive release of DOX from
nanospheres in the presence of biologically relevant redox
active molecule, glutathione (GSH; 10 mM) which contributes to
the relatively higher reducing environment of cell cytosol.[8,9a,10]
The GSH induced ~53% release of DOX within 6 h (Table 1).
This information helped in understanding the intracellular
reduction responsive behavior of DOX-CAFF-LA. The reduction
triggered delivery strategy may be relatively beneficent in order
to elicit the specific antitumor therapeutic response as many
different tumor types have been shown to possess elevated
GSH levels and its association with chemotherapy resistance.[7a]
The intracellular internalization of DOX-CAFF-LA was then
studied in MDA-MB-231 (human breast cancer) and HeLa
(human cervical cancer) cells in comparison with that of free
DOX using confocal microscopy. The free DOX treatment of
both MDA-MB-231 and HeLa cells showed efficient internal-
ization and nuclear entry of drug which is because of the well-
known fact that free DOX enter cells via diffusion across cell
membrane (Figure 3b and d). Interestingly, DOX-CAFF-LA medi-
ated treatments also showed nuclear internalization of the drug
against both the cell lines which demonstrated a reduction
triggered release of drug (Figure 3a and c). The cellular internal-
ization of DOX was further quantified using flow cytometry
which revealed significant internalization of DOX by means of
DOX-CAFF-LA treatments, however, remained slightly less than
that of free DOX treatment (Figure S9).
achievement of therapeutic benefits in clinical cancer
treatment.[2a] The P-glycoprotein (P-gp) and other transporters
are overexpressed on cancer cell membranes and are ad-
equately characterized to efflux different anticancer drugs out
of the cells which results in chemotherapy failure.[3a] Therefore,
we checked out the DOX accumulation inside DR-HeLa cells for
the treatment of DOX-CAFF-LA in comparison with that of free
DOX. Remarkably, a 24 h incubation of DOX-CAFF-LA with DR-
HeLa cells resulted in substantial intracellular accumulation of
DOX which was significantly greater than that observed for free
DOX treatment as shown in confocal micrographs of Figure 4a
and 4b. We then quantified the DOX fluorescence in DR-HeLa
cells which showed that it was nearly 2.3 fold higher for DOX-
CAFF-LA treatment (Figure S10). This higher intracellular DOX
accumulation was further substantiated by flow cytometry
analysis wherein cells treated with DOX-CAFF-LA showed
relatively large positive shift than those treated with DOX alone
as displayed in the histograms (Figure 4c) and thus, significantly
higher geometric means of fluorescence intensity (gMFI) were
recorded for DOX-CAFF-LA treatments (Figure S11a). The plau-
sible explanation of this phenomenon is that during free DOX
treatment, the drug molecules enter the cells through diffusion
and become substrate for efflux transporters and are thereby
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We also evaluated the cellular internalization of DOX-CAFF-
LA nanospheres against a DOX resistant version of HeLa cells
(DR-HeLa). The drug resistance renders anticancer chemother-
apeutics ineffective and remains a serious impediment to the
Figure 4. Confocal micrographs depicting cellular internalization of DOX-
CAFF-LA (a) and free DOX (b) in DR-HeLa cells (15 μg/mL; 24 h). Micrographs
(left to right) represent DOX fluorescence (red), DAPI fluorescence (blue,
nuclear stain), bright-field image and merged composite (scale bar: 20 μm).
Flow cytometry histograms for comparative c) DOX cellular internalization
(10 μg/mL; 4 h) and d) annexin V binding (20 μg/mL; 72 h). e) Cytotoxicity
data of DOX-CAFF-LA against DR-HeLa cells in comparison with free DOX
(96 h) based on triplicates of three independent experiments (mean�SEM;
n=3). Phase-contrast micrographs of f) untreated (normal saline) red blood
cells (RBCs) and g) those treated with CAFF-LA (1.0 mM) and PEI (positive
control; 1.0 mg/mL; scale bar: 50 μm).
Figure 3. Confocal micrographs depicting cellular internalization of DOX-
CAFF-LA (a and c) and free DOX (b and d) in MDA-MB-231 (a and b) and
HeLa (c and d) cells at a concentration of 5.0 μg/mL for 4 h. Micrographs (left
to right) represent DOX fluorescence (red), DAPI fluorescence (blue, nuclear
stain), bright-field image and merged composite (scale bar: 20 μm).
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cleared out. However, unlike free drug, the nanoencapsulated
drug enters the cells via endocytosis and thus escapes
recognition by these transporters.[25] Upon endocytosis, the
drug is released in cytosol under the influence of redox stimulus
which results in higher intracellular accumulation of drug.
Therefore, such nano based delivery systems are of utmost
importance for deriving clinically applicable outcomes in treat-
ments against drug-resistant tumors especially those which lack
therapeutic targets such as triple negative breast cancer where-
in chemotherapy is a standard clinical practice.[26]
The in vitro cytotoxicity assessment against MDA-MB-231
cells demonstrated that toxicity of DOX-CAFF-LA was nearly
comparable to that of free DOX (Figure S12a), whereas against Acknowledgements
HeLa, it was free DOX which was more cytotoxic than DOX-
CAFF-LA (Figure S12b). In contrast to the cytotoxicity noted
against drug sensitive cancer cells, significantly enhanced
cytotoxicity was observed for DOX-CAFF-LA (IC50: 31.13 μg/mL)
treatment against DR-HeLa cells than DOX alone treatment
(IC50: 62.52 μg/mL; Figure 4e). The observed cytotoxicity profile
was in line with the results of intracellular internalization of
drug obtained from flow cytometry and confocal microscopy
experimentations. In addition, no notable loss in cell viability
was observed for the treatment of blank CAFF-LA nanospheres
consumed antioxidant, caffeine with another powerful antiox-
idant of pharmacological importance, lipoic acid. The doxorubi-
cin encapsulated nanospheres demonstrate adequate extracel-
lular stability and prompt intracellular release of drug resulting
in significant cytotoxic effects, particularly against the drug-
resistant cells where drug alone treatment turns out to be
ineffective. The hemocompatibility experiments sufficiently
convince their safe interaction with blood cells which is
suggestive of the practical utility of such systems in drug
delivery.
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S.C. acknowledges DST, New Delhi for DST-ARRS Indo-Slovenian
Joint Research Project (DST/INT/Slovenia/P-14/2014). K.K. acknowl-
edges CSIR, New Delhi for Research Associate (RA) fellowship
(award no: 09/964(0009)2K17). B.R.K.S. thanks UGC, New Delhi for
SRF. P.K. is supported by INSA fellowship and DBT-IISc partnership
funding.Material Research Centre, MNIT Jaipur is acknowledged
for characterization studies.
(Figure S13) which demonstrates promising practical utility of Conflict of Interest
such bioresponsive aggregated nanostructures derived from
small biomolecules. The pronounced enhancement in cytotox-
icity against DR-HeLa cells by means of DOX-CAFF-LA treatment
was also evaluated in terms of quantifying apoptosis induced
by DOX.[10,27] The flow cytometric analysis of annexin binding to
DR-HeLa cells treated with DOX-CAFF-LA revealed significantly
higher number of apoptotic cells whereas no marked apoptosis
was seen for free DOX treatments (Figures 4d and S11b). The
observation could easily be attributed to rapid and enhanced
intracellular accumulation of DOX by the treatment of DOX-
CAFF-LA.
Lastly, hemocompatibility of these nanospheres was as-
sessed following their incubation with human red blood cells
(RBCs). Any untoward interaction of nanocarriers with RBCs
leads to their lysis and the released amount of hemoglobin is
quantified by recording UV/Vis spectra at a wavelength of
540 nm which in turn profiles the hemolytic action of
nanocarriers.[15c,28] The CAFF-LA treatment did not evidence any
detrimental hemolytic effect on RBCs at different concentrations
studied (0.1 mM–2.0 mM) as shown in Figure S13. In addition,
the visual investigation of RBC aggregation phenomenon post
CAFF-LA treatment revealed no evident aggregation of RBCs
which was comparable to untreated control and was in contrast
to that observed for PEI treated (positive control) cells with
clear sign of aggregation (Figure 4f–h). Therefore, it can be
stated that these nanospheres which are derived from self-
assembly of biological small molecules caffeine and lipoic acid
demonstrate a safe interaction with blood cells and could serve
as promising drug-delivery vectors for in vivo application.
In conclusion, we herein have attempted to develop
biocompatible redox sensitive drug-delivery vector (nano-
spheres) originating from the self-assembly of an amphiphile
derived from coupling of hydroxyethyl derivative of globally
The authors declare no conflict of interest.
Keywords: Bioconjugate · caffeine · drug delivery · lipoic acid ·
self-assembly
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COMMUNICATIONS
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Taken in by caffeine: A lipoylated
caffeine (CAFF-LA) derivative self-
assembles in water into nanospheres
that can be used as biocompatible
vectors for the cellular delivery of
doxorubicin (DOX). The DOX-loaded
nanospheres (DOX-CAFF-LA) are
redox-responsive in nature and show
significantly higher intracellular accu-
mulation of DOX against drug-
Dr. K. Kumar, B. R. Kumar Shyamlal,
Dr. R. Verma, Prof. P. Kondaiah*, Dr. S.
Chaudhary*
1 – 6
Reduction-Triggered Doxorubicin
Delivery by Self-Assembled Nano-
spheres of Lipoylated Caffeine
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resistant cancer cells than free DOX