Efficacious anticancer drug delivery mediated by a pH-sensitive self-assembly of a conserved tripeptide derived from tyrosine kinase NGF receptor

Article abstract of DOI:10.1002/anie.201307247

We present herein a short tripeptide sequence (Lys-Phe-Gly or KFG) that is situated in the juxtamembrane region of the tyrosine kinase nerve growth factor (Trk NGF) receptors. KFG self-assembles in water and shows a reversible and concentration-dependent switching of nanostructures from nanospheres (vesicles) to nanotubes, as evidenced by dynamic light scattering, transmission electron microscopy, and atomic force microscopy. The morphology change was associated with a transition in the secondary structure. The tripeptide vesicles have inner aqueous compartments and are stable at pH 7.4 but rupture rapidly at pH≈6. The pH-sensitive response of the vesicles was exploited for the delivery of a chemotherapeutic anticancer drug, doxorubicin, which resulted in enhanced cytotoxicity for both drug-sensitive and drug-resistant cells. Efficient intracellular release of the drug was confirmed by fluorescence-activated cell sorting analysis, fluorescence microscopy, and confocal microscopy. Package for special delivery: A biologically active tripeptide self-assembles to produce nanovesicles at lower concentrations and nanotubes at higher concentrations (see scheme). The nanovesicles rupture at pH≈6 and are highly efficient in doxorubicin delivery to both drug-sensitive and drug-resistant cancer cells. This system is highly promising as a stimulus-responsive biocompatible nanovehicle. Copyright

Full text of DOI:10.1002/anie.201307247

Angewandte
Chemie
DOI: 10.1002/anie.201307247
Drug Delivery
Efficacious Anticancer Drug Delivery Mediated by a pH-Sensitive Self-
Assembly of a Conserved Tripeptide Derived from Tyrosine Kinase
NGF Receptor**
Parikshit Moitra, Krishan Kumar, Paturu Kondaiah, and Santanu Bhattacharya*
Abstract: We present herein a short tripeptide sequence (Lys–
Phe–Gly or KFG) that is situated in the juxtamembrane region
of the tyrosine kinase nerve growth factor (Trk NGF)
receptors. KFG self-assembles in water and shows a reversible
and concentration-dependent switching of nanostructures from
nanospheres (vesicles) to nanotubes, as evidenced by dynamic
light scattering, transmission electron microscopy, and atomic
force microscopy. The morphology change was associated with
a transition in the secondary structure. The tripeptide vesicles
have inner aqueous compartments and are stable at pH 7.4 but
rupture rapidly at pH ꢀ 6. The pH-sensitive response of the
vesicles was exploited for the delivery of a chemotherapeutic
anticancer drug, doxorubicin, which resulted in enhanced
cytotoxicity for both drug-sensitive and drug-resistant cells.
Efficient intracellular release of the drug was confirmed by
fluorescence-activated cell sorting analysis, fluorescence mi-
croscopy, and confocal microscopy.
Recently, stimuli-responsive morphological transforma-
tions of short peptide sequences in water have also been
reported.[12,14] We considered a short tripeptide sequence
(Lys–Phe–Gly or KFG) that is situated in the juxtamembrane
region of the tyrosine kinase nerve growth factor (Trk NGF)
receptors, which are involved in neuronal growth and differ-
entiation. The absence of the KFG sequence in the receptor
polypeptide chain seriously affects the activation of signaling
cascades including the Ras superfamily, phosphoinositide 3-
kinase, and the Suc1-associated neurotrophic factor target
(SNT) protein.[15] This also results in impaired neurite out-
growth and sometic hypertrophy. This small but critically
conserved entity, which plays an important role in the growth
factor signal transduction process, was chosen for the present
study. We investigated this biologically important tripeptide,
KFG (Figure 1), which spontaneously self-assembled into
T
he design and development of different nanobiomaterials
mimicking natural processes is among the leading approaches
in nanobiotechnology.[1] Toward therapeutic goals, one of the
emerging interests is the development of biocompatible
nanocarriers for efficient drug delivery.[2] The main aim is to
overcome barriers that stand against the therapeutic potential
of drugs, like overload side-effects,[3] specific cell targeting,[4]
the development of multidrug resistance (MDR),[5] and the
blood–brain barrier in gliomas.[6] In order to solve such
outstanding problems, “decorated” liposomes,[7] multifunc-
tional micelles,[8] polymeric hydrogels,[9] functionalized
organic[10] and inorganic nanomaterials,[11] etc. have been
employed. Among these, building blocks that are organized
from short peptide sequences have become promising tools
for biomedical applications.[12,13]
Figure 1. Molecular structure of the synthesized tripeptide, KFG.
defined nanostructures in aqueous media. The tripeptide
showed an exciting phenomenon of reversible and concen-
tration-dependent switching of nanostructures between nano-
vesicles and nanotubes. This change in morphology was
associated with a transition in the secondary structure at the
peptide level. The vesicles that resulted from the self-
assembly of KFG were extremely sensitive toward the pH
value of the medium. They were stable at pH 7.4 but ruptured
rapidly at pH ꢀ 6. This property is often exploited for
improved cellular internalizations in anticancer drug deliv-
ery.[11c,16] The pH-sensitive nature of the vesicles was accord-
ingly tested for the delivery of one of the most widely used
drugs for many types of cancer, doxorubicin (DOX). Cellular
uptake of DOX dramatically increased after the drug was
loaded into the vesicles. Overall, we report for the first time
a smart stimulus-responsive (pH-induced) tripeptide-based
self-assembled nanocarrier that induces significant therapeu-
tic delivery of a chemotherapeutic drug, DOX, to cancer cells.
To discern the nature of the supramolecular structures
that were formed upon self-assembly of Lys–Phe–Gly,
dynamic light scattering (DLS) measurements were per-
[*] P. Moitra,[+] K. Kumar,[+] Prof. S. Bhattacharya
Department of Organic Chemistry
Indian Institute of Science, Bangalore 560 012 (India)
E-mail: sb@orgchem.iisc.ernet.in
Prof. P. Kondaiah
Department of Molecular Reproduction, Development and Genetics
Indian Institute of Science, Bangalore 560 012 (India)
[+] These authors contributed equally to this work.
[**] NGF = Nerve growth factor. This work was supported by the J. C.
Bose Fellowship grant of the Department of Science and Technology
to S.B. Doxorubicin-resistant HeLa cells were a kind gift from Dr.
Annapoorni Rangarajan. We thank the Chemical Sciences Division
for the transmission electron microscopy facility.
formed. At
a
low concentration of the tripeptide
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201307247.
(0.5 mgmLÀ1), nanostructures of approximately 50–70 nm
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were observed, whereas aggregates of about 190–200 nm were
formed at a higher concentration (5 mgmLÀ1; Figure S5 in the
Supporting Information). Interestingly, we found that this
transition in the sizes of the nanostructures is reversible in
nature (Figure S6). These observations prompted us to
investigate the KFG aggregates at various concentrations by
using transmission electron microscopy (TEM). The tripep-
tide formed spherical structures with diameters of (50 Æ
10) nm at the low concentration, whereas it formed tubular
aggregates with diameters of (190 Æ 10) nm at the higher
concentration (Figure S7). Atomic force microscopy (AFM;
Figure 2) showed morphological features of the tripeptide
aggregates similar to those observed in the TEM observa-
tions.
Figure 2. AFM images showing the topography of the KFG tripeptide
at different concentrations: a) 0.5 mgmLÀ1 and b) 5 mgmLÀ1. The
double arrow between (a) and (b) indicates that the transition from
nanovesicles to nanotubes is reversible as a function of concentration.
Figure 3. a) CD spectra of KFG at two different concentrations.
b) Schematic illustration of the concentration-dependent self-assembly
process of the tripeptide.
The thermotropic properties of the tripeptide self-assem-
blies at both concentrations in aqueous media were inves-
tigated by high-sensitivity differential scanning calorimetry
(DSC).[17] The suspension of KFG at low concentration
(vesicles) did not show any transition in the temperature
range 20–708C, but at the higher concentration (nanotubes),
the tripeptide showed a quasireversible transition during
heating and cooling scans. Two peaks appeared at approx-
imately 47 and 608C (Figure S8). The DSC results were
corroborated by using temperature-dependent AFM studies
at low and high concentrations (Figure S9). There was no
change in the morphology of the vesicles even after the
sample had been heated up to 858C, but the nanotubes
changed topology upon heating. The transition from tubular
to spherical nanostructures (vesicles) started occurring from
458C and ended at 608C. Above 908C, the vesicular structures
collapsed, probably due to water evaporation. Interestingly,
such a temperature-induced transition from tubular-to-vesic-
ular morphology is quite unprecedented.
of the tripeptide, which is also associated with the morpho-
logical transition. Again, such a reversible transition from
vesicles to nanotubes along with changes in the secondary
structure is rarely observed, to the best of our knowledge.
Furthermore, we examined the self-assembly by molecular
modeling studies (Figure S10), which evidenced that the
peptide assemblies are formed mainly due to the interplay of
electrostatic, p-stacking, and H-bonding interactions.
Even if the particles appear to be nearly spherical, it may
not necessarily signify that they are vesicles and not solid
nanoparticles. It is important to know whether the nano-
spheres derived from the tripeptide assemblies possess inner
aqueous compartments. Therefore, we attempted to encap-
sulate a water-soluble solute or a drug in such particles.
Toward this end, we employed a cationic dye, methylene blue
(MB),[19] to check the entrapment capacities of the spherical
aggregates in aqueous media. Indeed, the vesicles could
encapsulate approximately 6.8% of the total dye inside their
inner aqueous compartments (Figure S11). In another experi-
ment, we found that these tripeptide vesicles were also able to
encapsulate the neutral, impermeant, water-soluble solute
glucose[20] (Figure S12), which has no p-stacking unit, unlike
the commonly available aromatic dyes.
At higher concentration (5 mgmLÀ1), the tripeptide
exhibited a maximum at 196 nm and a minimum at 218 nm
in the CD spectrum (Figure 3). This signature in the CD
spectrum is analogous to those of b sheets in polypeptides.
After dilution of this suspension to 0.5 mgmLÀ1, the CD
spectrum showed a negative band at 195 nm together with
a positive band at 212 nm, which suggests the formation of
a ꢀrandom-coilꢁ structure.[18] This change in the secondary
structure from a b sheet to a random coil occurs upon dilution
Furthermore, because the tripeptide formed nanovesicles
spontaneously at physiological pH values and the same
vesicles could be disrupted at pH 6, we thought of exploiting
this property for drug delivery.[16,21] Accordingly, we loaded
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Figure 4. Fluorescence microscopy images of a) DOX-loaded vesicles
at pH 7.4 and b) the disruption of the vesicles and drug release at
pH 6. Scale bar: 20 mm.
the vesicles with DOX at pH 7.4 (Figure 4a) and followed the
release of the drug at pH 6 upon rupture of the vesicles
(Figure 4b). The percentage of DOX encapsulation was
approximately 25.7%, and importantly, the resulting DOX-
loaded vesicles were also quite stable in buffer containing
serum (Figure S14). The kinetic profile obtained for the
system showed a zero-order drug release at pH 6, which could
be explained on the basis of a non-Fickian diffusion mech-
anism,[22] as fitted from the Peppas model (Figure S15). Also,
the drug release profile of the DOX-loaded vesicles showed
a bimodal release profile, with an initial burst release
followed by a sustained release (Figure S14). A plausible
reason for this burst release could be the disruption of the
vesicular self-assembly and the increased hydrophilicity of
DOX molecules at this lowered pH value. The remaining
amount of DOX, which followed a sustained release profile
thereafter, could be attributed to the partial adsorption
effects between the tripeptide assembly and the DOX
molecules, as reported in other nanocarrier systems.[11a]
The toxicity of the KFG nanovesicles was assayed in cell
lines of different origins, namely HeLa (human cervical
carcinoma), HEK 293T (human embryonic kidney, trans-
formed), U251 (glioblastoma), and A549 (human lung
adenocarcinoma) cells. The toxic effect of the empty vesicles
appeared to be insignificant toward each cell line, with > 95%
cells found to be viable (Figure 5a). The toxicity of the DOX-
loaded vesicles was then assessed in the same set of cell lines
and compared with that of free DOX. Notably, the cytotox-
icity due to the DOX-loaded vesicles was significantly greater
than that of free DOX at each of the examined concentrations
(10, 20, 30, and 40 mgmLÀ1) for the same period of incubation
in all of the cells studied. The differences in cell viabilities
between the treatment with DOX alone and the DOX-loaded
vesicles at a representative 20 mgmLÀ1 drug concentration in
the above cells are shown in Figure 5b.
Figure 5. a) Cytotoxicity of the tripeptide vesicles at different concen-
trations. b) Cytotoxicity of DOX-loaded vesicles in comparison with
free DOX at a concentration of 20 mgmLÀ1
.
ure S16 and S17a, respectively. The fluorescence microscopy
results substantiated the observation that the fluorescence
intensity of the DOX was significantly higher for cells treated
with DOX-loaded peptide vesicles than for the free-DOX-
treated cells after 1 h of incubation (Figure S17). We also
assessed the intracellular localization of the drug by confocal
microscopy. Interestingly, cells treated with the DOX-loaded
vesicles for 4 h showed nuclear localization of the drug with
To explore the possible reasons for the enhanced toxicity,
the internalization of DOX before and after being loaded into
the KFG vesicles was examined, both by fluorescence-
activated cell sorting (FACS) analysis and fluorescence
microscopy. Based on the FACS data, the geometric mean
of fluorescence intensity (GMFI) values for cells treated with
DOX-loaded KFG vesicles were significantly higher than
those of the free-DOX-treated cells. This effect was more
prominent with increased exposure to and concentrations of
DOX (Figure 6a) and remained consistent among all of the
cell lines studied. Representative flow cytometry dot plots
and histograms for DOX internalization are shown in Fig-
Figure 6. a) GMFI of DOX internalizations in HeLa cells at two differ-
ent concentrations based on FACS analysis. The first two stacked bars
show the results after 1 h and the second two stacked bars show the
results after 4 h. b) Cytotoxicity of DOX-loaded vesicles in HeLa-DOXR
cells relative to the cytotoxicity with free DOX (20 mgmLÀ1), and GMFI
values in HeLa-DOXR cells after a 4 h incubation (10 mgmLÀ1).
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concentration (0.5 mgmLÀ1) and hollow nanotubes at higher
concentration (5 mgmLÀ1). The secondary structure of the
tripeptide changes from random-coil to b-sheet-like assem-
blies as the concentration is increased. The transition scheme
in the secondary structure is corroborated well with a molec-
ular modeling study. The vesicles rupture and release their
contents at pH ꢀ 6. A continuous drug release profile
consistent with zero-order kinetics is maintained so that the
concentration of drug remains constant throughout its
delivery period. The pH-targeted drug delivery results in an
enhanced cytotoxicity to both drug-sensitive and drug-resist-
ant cells in vitro. The efficient intracellular release of the drug
after loading into vesicles was confirmed by FACS analysis,
fluorescence microscopy, and confocal microscopy. These
tripeptide vesicles show the distinct advantage of pH-driven
translocation even in serum, which may be important because
it may bypass factors involved in drug resistance.
Such a system is therefore highly promising as a stimulus-
responsive biocompatible nanovehicle that translocates into
cells once exposed to the tumor pH regime. The fact that this
tripeptide could be either synthesized readily or obtained
commercially makes it particularly attractive for the delivery
of anticancer drugs selectively to the cancer site with relevant
functionalizations. We are now extending the utility of this by
exploring its therapeutic potential toward other MDR cell
lines with specific recognition sites in vivo. With the aim of
eradicating the root of cancer, the specialized cancer stem
cells that are responsible for the resistance to cancer therapies
and the recurrence of cancer, work is underway to achieve
targeting by a combination chemotherapeutic approach with
the design of appropriate aptamer-loaded tripeptide nano-
carriers.
Figure 7. Representative confocal microscopy images after DOX treat-
ment of HeLa cells at 5 mgmLÀ1 for 4 h (a,b) and HeLa-DOXR cells at
20 mgmLÀ1 for 24 h (c,d). Panels (a) and (c) represent treatment with
free DOX and panels b and d represent treatment with DOX-loaded
vesicles. Each panel, from left to right, shows the bright-field image,
DOX fluorescence (red), 4’,6-diamidino-2-phenylindole (DAPI) stained
nuclei (blue), and an overlay of the last two.
much brighter DOX fluorescence than that observed in cells
treated with DOX alone (Figure 7a,b). This observation was
tested by quantifying the increase in fluorescence intensity of
the internalized DOX by using confocal microscopic images
of several independent experiments (Figure S19). Scatter
diagrams (Figure S18) for the representative confocal mi-
croscopy images also substantiated the colocalization phe-
nomenon. This observation is striking because the nuclear
localization of drugs delivered by nanomaterials is known to
be challenging.[23]
This interesting phenomenon of accelerated drug release
and, thereby, increased cytotoxicity selectively toward the
cancer cells encouraged us to deal with drug-resistant cell
lines. We, therefore, performed cell viability experiments and
drug internalization studies in DOX-resistant HeLa cells
(HeLa-DOXR). In this case also, the DOX-loaded vesicles
were found to be more efficient in delivering a larger amount
of the drug at fixed concentrations and time points, which
imparted greater toxicity than the free DOX (Figure 6b).
Confocal microscopy was performed for HeLa-DOXR at
relatively longer time points and higher drug concentrations
because of the drug-efflux-related issues in drug-resistant cell
lines. At an optimized concentration and time point, cells
treated with DOX-loaded vesicles showed much higher DOX
fluorescence than those treated with free DOX, although the
drug was mainly localized in the cytoplasm this time (Fig-
ure 7c,d). Thus, the increased internalization of the drug
could be attributed to the pH-sensitive response of the
vesicles.
Received: August 18, 2013
Revised: November 4, 2013
Published online: December 11, 2013
Keywords: drug delivery · nanostructures · peptides ·
.
pH sensitivity · self-assembly
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