Novel self-assembled lithocholic acid nanoparticles for drug delivery in cancer

Article abstract of DOI:10.1039/c3ra42994f

Novel versatile self-assembled nanoparticles were developed from biocompatible, biodegradable lithocholic acid derivatives. These nanoparticles can incorporate different cytotoxic drugs (paclitaxel and doxorubicin) and PI3K signalling inhibitor (PI103). D

Full text of DOI:10.1039/c3ra42994f

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Novel self-assembled lithocholic acid nanoparticles for
drug delivery in cancer†
Cite this: DOI: 10.1039/c3ra42994f
ab
a
a
a
Sumersing Patil, Sohan Patil,‡ Suhas Gawali,‡ Shrikant Shende,
a
a
Shraddha Jadhav and Sudipta Basu*
Received 15th June 2013
Accepted 15th August 2013
DOI: 10.1039/c3ra42994f
www.rsc.org/advances
Novel versatile self-assembled nanoparticles were developed from biocompatible, biodegradable, well characterized and easily
biocompatible, biodegradable lithocholic acid derivatives. These functionalized materials. Moreover, the nanovectors should
nanoparticles can incorporate different cytotoxic drugs (paclitaxel self-assemble into sub 200 nm particles with diverse drug
5
and doxorubicin) and PI3K signalling inhibitor (PI103). Drugs were loading to accumulate into tumor by EPR effect. Finally, the
released from the nanoparticles in a slow, sustained manner at acidic nanovectors should release the active drugs in a controlled, slow
pH. The drug loaded nanoparticles were internalized through and sustained manner. To address this we focus on developing
lysosomal compartments and induced cell death in HeLa cervical novel nanovectors from lithocholic acid (LA). LA is one of the
cancer cells.
most common naturally occurring secondary bile acids bio-
synthesized from cholesterol by bacterial modication of
intermediate chenodeoxycholic acid in colon and stored in liver
and gall bladder for lipid digestion, absorption, transport and
Cancer is a devastating disease killing almost 10 million people
1
per year. Current cancer treatment is relying on using toxic
6
excretion. Due to their versatile structural properties, bile acids
chemotherapeutic drugs, which oen kill healthy cells causing
off-target toxic side effects to the patients. To address this,
nanotechnology based platforms have emerged as revolution-
and their derivatives have shown great potential for host–guest
chemistry, biomimetics, molecular recognition, drug delivery
7
and nanotechnology. Moreover, lithocholic acid has structural
2
izing strategies in cancer therapeutics. Nanovectors can offer
similarity with cholesterol (an important component of cell
membrane) (Fig. 1c) and can self-assemble to develop supra-
many advantages over free drugs by (a) protecting the drugs
from premature degradation and interaction with the biological
systems, (b) enhancing accumulation of drugs in specic
tissues by active targeting or enhanced permeability and
retention effect (EPR), (c) improving the pharmacokinetic/
pharmacodynamics of the drugs and (d) increasing intracellular
7c
molecular nanostructures. Inspired by the unique amphiphi-
licity, high structural rigidity, biocompatibility and
biodegradability, herein we report the engineering of novel, self-
assembled lithocholic acid nanoparticles. These nanoparticles
are mono dispersed having sub 200 nm size ideal for tumor
homing by EPR effect. Furthermore, these nanoparticles can
incorporate diverse clinically approved cytotoxic drugs (doxo-
rubicin and paclitaxel) and PI3-kinase inhibitor (PI103) and
release the active drugs effectively in a controlled manner at pH
3
penetration. Several nanovectors including polymer–drug
conjugates, carbon nanotubes, micelles, dendrimers, lipo-
somes, nanoshells and polymeric nanoparticles have exten-
3,4
sively explored for drug delivery and diagnosis in cancer.
However, very few nanovector-based drugs are currently on the
¼
5.5. Finally, these drug loaded nanoparticles internalized into
3
market. So there is clearly a need to develop novel nanovectors
the lysosomal compartments by endocytosis and induced cell
death in a cervical cancer cell line to show their potential in
cancer therapeutics. To the best of our knowledge, this is the
for drug delivery in cancer. For rapid and successful clinical
translation, the nanovectors should be developed from

rst example of lithocholic acid based self-assembled nano-
a
Department of Chemistry, Indian Institute of Science Education and Research particle synthesis as a versatile platform for drug delivery in
IISER)-Pune, Pune, 411021, Maharashtra, India. E-mail: sudipta.basu@iiserpune. cancer.
(
ac.in
b
To show the versatility of LA based nanoparticles, we chose
two cytotoxic drugs (doxorubicin and paclitaxel) which are
extensively used in clinics for the treatment of different types of
malignancies including breast, ovarian, prostate, cervical and
lung cancer. Doxorubicin is a highly potent anti-cancer drug
Institute of Bioinformatics and Biotechnology (IBB), University of Pune, Pune, 411007,
Maharashtra, India
†
Electronic supplementary information (ESI) available: Experimental details and
further characterization data are available. See DOI: 10.1039/c3ra42994f
These authors contributed equally to this work.
‡
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formulation of paclitaxel (Abraxaneꢀ) has been approved by
FDA for metastatic breast cancer treatment, although it leads to
several side effects like sensory neuropathy, heart attack,
14
nausea, infection, hair loss and so on. To overcome the off-
target side effects of cytotoxic drugs, kinase inhibitors have
15
emerged as novel personalized cancer therapeutics. Phos-
phatidyl-inositol-3-kinase (PI3K) signalling is one of the most
frequently mutated signalling pathways in almost 30% of
16
human cancers. We chose PI103, a PI3K inhibitor, currently in
17
pre-clinical study. PI103 is a dual inhibitor for Akt and
18
mammalian target of rapamycin (mTOR). However, PI103 is
poorly water soluble, metabolizes quickly and shows dose
19
dependent insulin resistance.
To address the above
mentioned drawbacks of cytotoxic drugs and PI3K inhibitors in
clinics, there is clearly a need to develop novel versatile nano-
vector to deliver different cytotoxic drugs as well as kinase
inhibitors specically to the tumor to avoid off-target toxicities
leading to side effects to the patients.
Doxorubicin was conjugated directly with carboxylic acid
end of LA (1, Fig. 1a) by amide linkage using HBTU as coupling
reagent to obtain LA–doxorubicin conjugate (2) in 75% yield. To
conjugate PI103, secondary alcohol of LA (1) was oxidized by
3 2 4
Jones oxidation using CrO –H SO in acetone to obtain corre-
sponding keto-lithocholic acid (3) in 53% yield. PI103 was then
conjugated with 3 by phenolic ester linkage by using EDC as
coupling reagent in presence of catalytic amount of DMAP at
ꢀ
0
C to room temperature for 24 h to obtain keto-lithocholic
Fig. 1 Synthesis of drug loaded self-assembled lithocholic acid nanoparticles. (a)
Synthesis of lithocholic acid–drug conjugates. (b) Schematic representation of
acid-PI103 conjugate (4) in 96% yield. Finally, to conjugate
self-assembled nanoparticle synthesis from lithocholic acid–drug conjugates. (c) paclitaxel, the free secondary –OH group in LA was rst pro-
Structure of lithocholic acid and cholesterol. Structural similarity is shown in red. tected by using acetic anhydride in presence of pyridine as base
to obtain acetyl protected LA (5) in 75% yield. Paclitaxel was
0
then conjugated with acetyl LA (5) by ester linkage at 2 OH
works by intercalating with the DNA as well as inhibiting the position to afford 6 in 77% yields.
8
action of topoisomerase II enzyme. However, doxorubicin's
Self-assembled nanoparticles were engineered by solvent
clinical use is limited as it may induce cardiotoxicity including evaporation–hydration–extrusion method from LA–drug conju-
9
congestive heart failure, dilated cardiomyopathy and death. To gates (2, 4 or 6), phosphatidylcholine (PC) and DSPE-PEG in
20
reduce its toxic side effects, several doxorubicin nano- 1 : 2 : 0.2 weight ratio (Fig. 1b). PC is a naturally occurring
formulations including antibody-targeted liposome, polymeric component of cellular membrane, hence biocompatible,
nanoparticles, gold nanoparticles and fullerene have been biodegradable and non-toxic. DSPE-PEG was used to pegylate
10
studied extensively. Currently, two FDA approved doxorubicin the surface of the nanoparticle to avoid clearance by reticulo-
nanoformulations (Myocetꢀ and Doxilꢀ) are available in the endothelial system (RES) and ensure the long circulation half-
21
market. However, both of them demonstrated side effects life in blood. Drug loading in the nanoparticle was determined
including congestive heart failure, myelosuppression, throm- by UV-Vis spectroscopy at characteristics lmax ¼ 480 nm,
bocytopenia and palmer-planter erythrodysesthesia (PPE) or 292 nm and 277 nm for doxorubicin, PI103 and paclitaxel
hand-foot syndrome due to their premature fast burst release of respectively (Fig. S1, ESI†). The drug loading was found to
1
1
ꢂ1
the physically encapsulated chemotherapeutic drugs. More- be ¼66.20 ꢁ 7.8 mg mL
(121.96 ꢁ 14.4 mM, loading
ꢂ1
over, due to non-pegylated formulation, Myocetꢀ rapidly efficiency ¼ 22%), 306.9 ꢁ 32.5 mg mL (359.40 ꢁ 38.2 mM,
3
ꢂ1
cleared from the body leading to it being less effective. On the loading efficiency ¼ 90%) and 34.15 ꢁ 2.3 mg mL (98.17 ꢁ 6.5
other hand, paclitaxel is a widely used chemotherapeutic drug, mM, loading efficiency ¼ 14%) (mean ꢁ SEM, n ¼ 3) for doxo-
exhibits its activity by binding and stabilizing microtubules, rubicin, paclitaxel and PI103 respectively (Fig. S2a and b, ESI†).
12
thus inhibiting cell division. Paclitaxel is highly hydrophobic The hydrodynamic diameter of the drug loaded nanoparticles
in nature, causing poor water solubility and leading to its was determined by dynamic light scattering (DLS) (Fig. 2a–c and
clinical formulation using toxic Cremophor EL and ethanol. To S2c, ESI†) and found to be 99.3 ꢁ 0.2 nm, 130.2 ꢁ 1.8 nm and
address this, several nanovectors have been explored to deliver 116.4 ꢁ 1.2 nm (mean ꢁ SEM, n ¼ 3) for PI103-NP, paclitaxel-NP
13
paclitaxel specically into tumors. However, the nano- and dox-NP respectively. Size, shape and morphology of the
formulations lead to limited improvement in water solubility, nanoparticles were also determined by atomic force microscopy
poor release of drugs. Recently, albumin bound nanoparticle (AFM) (Fig. 2d–f and S3, ESI†) and eld-emission scanning
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in pH 7.4 buffer, which mimics the physiological pH. PI103-NP
exhibited 76.5 ꢁ 7.4% (mean ꢁ SEM, n ¼ 3) PI103 release at pH
5
.5, whereas, 63.7 ꢁ 9.8% (mean ꢁ SEM, n ¼ 3) PI103 was
release at pH 7.4 at 112 h (Fig. 3a). On the other hand, doxo-
rubicin-NP demonstrated 84.5 ꢁ 7.2% (mean ꢁ SEM, n ¼ 3)
release of doxorubicin at 96 h (Fig. 3b). However, only 34.5 ꢁ
1
.7% (mean ꢁ SEM, n ¼ 3) doxorubicin was released from the
nanoparticles even aer 120 h. Finally, 83.0 ꢁ 7.7% (mean ꢁ
SEM, n ¼ 3) paclitaxel was released from self-assembled nano-
particles at pH 5.5, whereas, only 49.8 ꢁ 1.1% (mean ꢁ SEM, n
¼
3) paclitaxel was released at pH 7.4 aer 120 h (Fig. 3c). We
rationalized that amide linkage in conjugate 2 is more prone to
hydrolysis in acidic pH (pH ¼ 5.5) than neutral pH (pH ¼ 7.4)
leading to increased release of active doxorubicin at pH ¼ 5.5.
Fig. 2 Characterization of lithocholic acid NPs. (a–c) Size distribution of PI103-
NP, doxorubicin-NP and paclitaxel-NP respectively by dynamic light scattering
(
DLS). (d–f) AFM images of PI103-NP, doxorubicin-NP and paclitaxel-NP On the other hand, phenolic ester in conjugate 4 is labile in
respectively.
neutral pH leading to comparable release of PI103 in acid and
neutral pH. Finally, the aliphatic ester bond in conjugate 6 is
more labile in pH ¼ 5.5 compared to pH ¼ 7.4 leading to
electron microscopy (FE-SEM) (Fig. S4, ESI†). DLS, AFM and FE- increased paclitaxel release in acidic pH. We also observed that
SEM data provided convincing evidence of self-assembly of the lithocholic acid–drug conjugates 2, 4 and 6 are completely
different LA–drug conjugates into sub 200 nm particles for converted to the free drugs by using MALDI-TOF (Fig. S7–S9,
homing into tumor by EPR effect. To understand the role of ESI†). The release prole data clearly showed that different
lithocholic acid in nanoparticle formation, we synthesized drug drugs were released from the nanoparticles in a slow and sus-
encapsulated nanoparticles in the same solvent evaporation– tained manner at pH 5.5, which would lead the constant drug
hydration–extrusion method using only PC and DSPE-PEG in level in the body while the drug loaded nanoparticles being
2
: 0.2 ratio without any lithocholic acid. The nanoparticles administered. Furthermore, higher amount of drug released in
formed were evaluated using DLS for size distribution. Unfor- pH ¼ 5.5 compared to pH ¼ 7.4 clearly indicates that
tunately, this formulation produced particles having mean more drugs will be released in acidic lysosomal compartments
hydrodynamic diameter ¼ 377.34 ꢁ 3.3 nm, 286.88 ꢁ 9.9 nm of the tumor cells than premature drug release in the blood
and 270. 30 ꢁ 2.7 nm (mean ꢁ SEM, n ¼ 3) for PI103-NP, dox-NP circulation.
and paclitaxel-NP respectively (Fig. S5, ESI†) with broad distri-
To ensure the effect of our novel nanoparticles in cancer, we
butions. This clearly demonstrated that lithocholic acid is evaluated the in vitro efficacy of drug loaded lithocholic acid-
essential to self-assemble the particles in sub 200 nm size. NPs in HeLa cervical cancer cell line by cell viability assay using
To deliver the drugs successfully in cancer, the nanoparticles (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
should be stable enough in blood circulation over a period of (MTT) at 24 h and 48 h post-incubation. At 24 h, PI103-NP
time to home into the tumor using EPR effect. To evaluate the showed HeLa cell death with IC50 ¼ 9.4 mM compared to free
stability in physiological conditions, we incubated different drug PI103 with IC50 ¼ 4.78 mM (Fig. S10, ESI†). On the other hand,
ꢀ
loaded nanoparticles in fetal bovine serum (FBS) at 37 C for 5 dox-NP induced HeLa cell death having IC50 ¼ 7.5 mM compared
days and monitored their properties by size and polydispersity to free doxorubicin with IC50 ¼ 4.0 mM. Finally, paclitaxel-NP
index (PDI) using DLS. The size of the paclitaxel-NP changed demonstrated IC50 ¼ 19.2 mM whereas free paclitaxel induced
from 129.63 ꢁ 0.08 nm to 132.67 ꢁ 0.32 nm whereas PDI value cell death in IC50 ¼ 0.1 mM. Interestingly, at 48 h, PI103-NP
changed from 0.193 ꢁ 0.0 to 0.166 ꢁ 0.01 over 5 days (Fig. S6, induced HeLa cell death having IC50 ¼ 2.54 mM compared to
ESI†). On the other hand, size of dox-NP increased from 122.36 ꢁ IC50 ¼ 2.39 mM for free PI103 (Fig. 4a). PI103-NP induced 73.33
0
0
.67 nm to 158.90 ꢁ 0.5 nm, whereas PDI value changed from
.180 ꢁ 0.0 to 0.411 ꢁ 0.0 over 5 days. However, PI103-NP showed
huge increase in size from 88.17 ꢁ 0.69 nm to 416.00 ꢁ 18.66 nm
and PDI value from 0.219 ꢁ 0.0 to 1.0 ꢁ 0.0 in 4 days (mean ꢁ
SEM, n ¼ 3). From this stability experiment it is clear that
paclitaxel-NP and dox-NP are stable over 5 days, however
PI103-NP is stable over 3 days in serum, which is sufficient
enough time to accumulate into the tumor by EPR effect.
To be successful in clinics, the nanoparticles should release
the drugs in a slow and sustained manner over a long period of
time. To evaluate the release prole of different drugs, we used
2
0b
dialysis method by incubating the drug loaded NPs at pH 5.5
ꢀ
Fig. 3 Release profile of drugs from lithocholic acid-NPs at 37 C at different pH.
a) Release profile of PI103 over 112 h. (b) Release profile of doxorubicin over 120
ꢀ
buffer at 37 C which mimics the lysosomal compartment
(
inside the cells. As a control we incubated the drug loaded NPs h (c) release profile of paclitaxel over 120 h.
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5.1% (mean ꢁ SEM, n ¼ 3) cell death compared to 92.38 ꢁ
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ꢁ
2
.6% (mean ꢁ SEM, n ¼ 3) cell death induced by free PI103 at 20
mM concentration. On the other hand, doxorubicin-NP
demonstrated IC50 ¼ 1.32 mM by inducing 77.83 ꢁ 7.0% (mean
ꢁ
¼
SEM, n ¼ 3) cell death, whereas free doxorubicin showed IC50
0.17 mM by killing 87.55 ꢁ 3.9% (mean ꢁ SEM, n ¼ 3) cells
(
Fig. 4b) at highest concentration (20 mM). Finally, paclitaxel-NP
exhibited IC50 ¼ 8.88 mM by inducing 68.74 ꢁ 2.7% (mean ꢁ
SEM, n ¼ 3) cell death, whereas free paclitaxel showed IC50
¼
0
.11 mM by inducing 94.98 ꢁ 0.3% (mean ꢁ SEM, n ¼ 3) cell
death at 20 mM concentration (Fig. 4c). It is evident from this
time dependent cytotoxicity assay that different drug loaded
nanoparticles are more effective at 48 h compared to 24 h. It is
expected that drug loaded nanoparticles would exhibit lower
efficacy than the free drugs as nanoparticles would release the
active drugs in a slow and sustained manner over long period of
Fig. 5 Internalization of LA–doxorubicin-NPs in HeLa cells in 1 h, 3 h and 6 h time
time, whereas free drugs would induce toxicity very quickly. points. Lysosomal compartments and nucleus were stained by LysoSensorꢂ
Green DND-153 (green) and Hoechst 33342 (blue) respectively. The merged
However, different drug loaded nanoparticles showed dose
dependent cytotoxicity in HeLa cells at 24 h and 48 h which
showed their efficacy as cancer chemotherapy.
images are showing the co-localization of LA–doxorubicin-NPs in the lysosomal
compartments in a time dependent manner. Scale bar ¼ 30 mm.
We further investigated the mechanism of cellular internal-
ization of these novel lithocholic acid based NPs. We treated the
HeLa cells with LA–doxorubicin-NPs and visualized their time
Conclusions
dependent internalization by confocal laser scanning micros-
copy (CLSM). LysoSensorꢁ Green DND-153 and Hoechst 33342
dye were used to stain low pH lysosomal compartments (shown
In conclusion, we successfully developed novel, versatile, self-
assembled lithocholic acid nanoparticles which can hold
in green) and nucleus (shown in blue) respectively. We treated
diverse drugs (paclitaxel, doxorubicin and PI103) to deploy
the cells with red uorescent LA–dox-NPs or free doxorubicin in
them inside the tumor. These nanoparticles exhibited slow and
ꢂ1
2
mg mL concentrations for 1 h, 3 h and 6 h time points to
sustained drug release over a period of time at pH ¼ 5.5. Finally,
these nanoparticles internalized through lysosomal compart-
ments by endocytosis to induce cytotoxicity in HeLa cervical
cancer cells to show their future potential in cancer treatment.
These nanoparticles can also be surface decorated by different
tumor targeting moieties like antibodies, aptamers, cell pene-
trating peptides or cell surface receptor targeting peptides for
tissue specic delivery of cytotoxic drugs or signalling inhibi-
tors. We envision that lithocholic acid nanoparticles can serve
as non-toxic, non-immunogenic versatile platform to conjugate
diverse array of cytotoxic drugs or signalling inhibitors for
temporal targeting of cancer and will be successfully translated
to the clinics to provide better quality of life to the cancer
patients.
understand the temporal internalization of the nanoparticles
compared to free doxorubicin. As shown in Fig. 5, aer incu-
bation with LA–dox-NP, the red and green uorescence co-
localize (yellow regions) with each other in a time dependent
manner. At 6 h, it is clear from the CLSM images, that LA–dox-
NPs home into the low pH lysosomal compartments by endo-
cytosis mechanism. In contrast, aer incubation with free
doxorubicin, the red and green uorescence did not co-localize
(Fig. S11, ESI†) even aer 6 h. However, blue and red uores-
cence co-localize (purple regions) with each other within 3 h,
which clearly indicates that free doxorubicin internalized
through diffusion pathway and accumulated into the nucleus.
From this CLSM images, it is evident that LA–dox-NPs were
internalized into the HeLa cells through low pH lysosomal
compartments in a time dependent manner over 6 h.
Acknowledgements
We sincerely thank IISER-Pune, Department of Biotechnology
(DBT) (Ramalingaswami Fellowship) and Science and Engi-
neering Research Board (SERB) (Fast Track Scheme for Young
Scientist) for nancial support. We thank Department of
Science and Technology (DST) Nanoscience unit for providing
AFM facility. We thank Dr Shouvik Datta for capturing FE-SEM
images. We also thank Dr Geetanjali Tomar and Ms Aishwarya
Sivakumar from Institute of Bioinformatics and Biotechnology
(IBB), University of Pune for providing tissue culture facility for
doing MTT assay. Finally, we thank Mr Vijay Vittal from IISER
Pune for helping in capturing CLSM images.
Fig. 4 (a–c) In vitro dose-dependent cytotoxicity assay of PI103-NP, doxorubicin-
NP and paclitaxel-NP against HeLa cells at 48 h.
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