Synthesis, characterization and in vitro evaluation of novel vitamin D3 nanoparticles as a versatile platform for drug delivery in cancer therapy

Article abstract of DOI:10.1039/c3tb21176b

Development of novel nanotechnology based platforms can impact cancer therapeutics in a paradigm shifting manner. The major concerns in drug delivery in cancer therapy are the biocompatibility, biodegradability, non-toxic nature, easy and short synthesis and versatility of the nanovectors. Herein we report the engineering of versatile nanoparticles from biocompatible, biodegradable and non-toxic lipid soluble vitamin D3. We have conjugated different clinically used cytotoxic drugs (paclitaxel and doxorubicin) as well as PI3 kinase inhibitor (PI103) with vitamin D3 using a succinic acid linker. Sub-200 nm, monodispersed nanoparticles with high drug loading were engineered from the vitamin D3-succinic acid-drug conjugates. These nanoparticles released the active drugs at pH 5.5 in a slow and sustained manner over 100 h. Furthermore, these nanoparticles were taken up by HeLa cells into the low pH lysosomal compartments through an endocytosis mechanism in 6 h. Finally, these drug loaded vitamin D3 nanoparticles induced HeLa cervical cancer cell death in a dose dependent manner at 48 h to show their potential in cancer therapeutics.

Full text of DOI:10.1039/c3tb21176b

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Materials Chemistry B
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COMMUNICATION
Synthesis, characterization and in vitro evaluation of
novel vitamin D3 nanoparticles as a versatile platform
for drug delivery in cancer therapy†
Cite this: J. Mater. Chem. B, 2013, 1,
5742
a
a
a
Sumersing Patil,ab Suhas Gawali,‡ Sohan Patil‡ and Sudipta Basu
Received 23rd August 2013
*
Accepted 4th September 2013
DOI: 10.1039/c3tb21176b
www.rsc.org/MaterialsB
Development of novel nanotechnology based platforms can unique enhanced permeability and retention (EPR) effect.2
impact cancer therapeutics in a paradigm shifting manner. The The most effective size of the nanovectors for extravasation
major concerns in drug delivery in cancer therapy are the into tumors by the EPR effect is <200 nm.3 Several nano-
biocompatibility, biodegradability, non-toxic nature, easy and vectors including polymer–drug conjugates, liposomes,
short synthesis and versatility of the nanovectors. Herein we polymeric nanoparticles, polymerosomes, micelles, gold–
report the engineering of versatile nanoparticles from biocom- silica nanoshells, gold nanoparticles, nanocages and
patible, biodegradable and non-toxic lipid soluble vitamin D3. dendrimers are currently in pre-clinical studies or already in
We have conjugated different clinically used cytotoxic drugs clinics.4 However, major concerns in current cancer thera-
(paclitaxel and doxorubicin) as well as PI3 kinase inhibitor (PI103) peutics are the biocompatibility and biodegradability of the
with vitamin D3 using a succinic acid linker. Sub-200 nm, mono- nanovectors.5 Furthermore, for successful translation into
dispersed nanoparticles with high drug loading were engineered the clinics, the nanovectors should be easily synthesized in
from the vitamin D3–succinic acid–drug conjugates. These nano- high yield and purity in few steps, capable of incorporating
particles released the active drugs at pH 5.5 in a slow and sus- diverse drugs, releasing the drugs in a controlled manner
tained manner over 100 h. Furthermore, these nanoparticles were and nally degrading into non-toxic entities to minimize
taken up by HeLa cells into the low pH lysosomal compartments adverse toxicity. To address this, we hypothesize that natu-
through an endocytosis mechanism in 6 h. Finally, these drug rally occurring molecules would be the right choice for
loaded vitamin D3 nanoparticles induced HeLa cervical cancer cell developing novel drug delivery vehicles. Vitamins are a class
death in a dose dependent manner at 48 h to show their potential of naturally occurring biocompatible, biodegradable and
in cancer therapeutics.
non-toxic molecules, which are completely unexplored for
drug delivery purposes. Vitamin D3 (cholecalciferol) is an
essential lipid soluble vitamin which is biosynthesized in the
human body from its precursor 7-dehydrocholesterol using
sunlight and metabolized in the liver and kidney.6 Moreover,
vitamin D3 is one of the important ingredients in our daily
diet. Inspired by its biocompatibility, biodegradability and
structural similarity with cholesterol (an important compo-
nent of cellular membranes) we report herein the rst
development of novel vitamin D3 nanoparticles. These
nanoparticles are mono-dispersed in nature with a sub-200
nm size, ideal for the EPR effect. Moreover these vitamin D3
nanoparticles can incorporate diverse drugs in a short two
step synthesis with high loading capability and release
the drugs in a controlled manner at acidic pH. Finally,
these drug loaded nanoparticles induce cell death in
cervical cancer cell line by internalization through low pH
lysosomal compartments to show their potential in cancer
therapeutics.
1. Introduction
Cancer is the second leading cause of death worldwide with
more than 10 million new cases each year.1 Current tradi-
tional cancer treatments involve surgical intervention,
radiation therapy and chemotherapy. These strategies oen
kill healthy cells leading to toxicity to the patients as well as
development of drug resistance. To address this, nano-
vectors have emerged as outstanding tools in cancer therapy
by improving the pharmacokinetics and pharmacodynamics
of small molecule drugs and proteins by exploiting the
aDepartment of Chemistry, Indian Institute of Science Education and Research
(IISER)-Pune, Dr Homi Bhabha Road, Pashan, Pune, 411008, India
bInstitute of Bioinformatics and Biotechnology, University of Pune, Pune, 411007,
Maharashtra, India. E-mail: sudipta.basu@iiserpune.ac.in
† Electronic supplementary information (ESI) available: Further characterization
data. See DOI: 10.1039/c3tb21176b
‡ These authors contributed equally to this work.
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by using 20% ethyl acetate in petroleum ether to obtain
compound 2 as a colorless viscous liquid (46 mg) (yield ¼ 75%).
2.2.2 Synthesis of the vitamin D3–succinic acid–paclitaxel
conjugate (3). The vitamin D3–succinic acid conjugate (2) (5 mg,
0.0103 mmol, 1 equiv.) was dissolved in 1 mL dry DCM and
cooled to 0 ꢀC. Subsequently, EDC (3.95 mg, 0.020 mmol, 2
equiv.) and DMAP (1.25 mg, 0.0103 mmol, 1 equiv.) were added
to the reaction mixture with continuous stirring under dark
conditions. Aer 15 min, paclitaxel (9.67 mg, 0.01132 mmol, 1.1
equiv.) was added into the reaction mixture. The reaction was
monitored by TLC. Aer 24 h, the reaction was quenched with
dd water (5 mL) and diluted with DCM (10 mL). The organic
layer was extracted with DCM (20 mL ꢁ 2) and washed with
brine solution (10 mL). The organic layer was dried over anhy-
drous Na2SO4. The organic solvent was then evaporated using a
rotary evaporator and the crude product was puried using
silica gel (100–200 mesh size) column chromatography with
25% ethyl acetate in petroleum ether to obtain 10.2 mg of pure
compound 3 (yield ¼ 73%).
2. Experimental section
2.1 Materials
All reactions were performed under a nitrogen atmosphere
unless otherwise indicated. All commercially obtained
compounds were used without further purication. Ethyl
acetate, dimethyl sulfoxide (DMSO), petroleum ether, dry
dichloromethane (DCM), methanol, dry dimethylformamide
(DMF), cholecalciferol, succinic anhydride, sodium sulphate,
pyridine, N,N-diisopropylethylamine (DIPEA), 4-(dimethylamino)
pyridine
(DMAP),
N-(3-dimethylaminopropyl)-N0-ethyl-
carbodiimide hydrochloride (EDC), O-(benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium hexauorophosphate (HBTU),
L-a-phosphatidylcholine (PC), Sephadex G-25, uranyl acetate
dehydrate, mica for AFM, copper grids for TEM and silicon
wafers for FE-SEM were bought from Sigma-Aldrich. Paclitaxel,
PI103 and doxorubicin were bought from Selleck Chemicals.
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino-
(polyethylene glycol)2000] (DSPE-PEG) and the mini handheld
Extruder kit (including a 0.2 mm Whatman Nucleopore Track-
Etch Membrane, Whatman lter supports and 1.0 mL Hamil-
tonian syringes) were bought from Avanti Polar Lipids Inc.
Analytical thin-layer chromatography (TLC) was performed
using precoated silica gel aluminium sheets 60 F254 bought
from EMD Laboratories. DMEM media and 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were
purchased from HiMedia and HeLa cells were obtained from
the National Centre for Cell Science (NCCS), Pune, India. Spots
on the TLC plates were visualized using alkaline permanganate
or phosphomolybdic acid hydrate in methanol. 1H and 13C
NMR (400 MHz) spectra were recorded on a Jeol-400 spec-
trometer. The chemical shis are expressed in parts per million
(ppm) using suitable deuterated NMR solvents with reference to
TMS at 0 ppm. The release kinetic data, drug loading, nano-
particle size and cell viability assay were plotted using Graph-
Pad Prism soware. Each sample was done in triplicate.
LysoSensorꢀ Green DND-153 was purchased from Invitrogen
and Hoechst 33342 was purchased from Cell Signaling Tech-
nology. N-Propyl gallate was purchased from Sigma-Aldrich.
Confocal laser scanning microscopy was performed using a
Zeiss LSM 710 machine.
2.2.3 Synthesis of the vitamin D3–succinic acid–PI103
conjugate (4). The vitamin D3–succinic acid conjugate (2) (10
mg, 0.0206 mmol, 1 equiv.) was dissolved in 1 mL dry DCM and
cooled to 0 ꢀC. Subsequently, EDC (7.91 mg, 0.041 mmol, 2
equiv.) and DMAP (2.5 mg, 0.0206 mmol, 1 equiv.) were added
to the reaction mixture with continuous stirring under dark
conditions. Aer 15 min, PI103 (8.60 mg, 0.024 mmol, 1.1
equiv.) was added into the reaction mixture. The reaction was
monitored by TLC. Aer 24 h, the reaction was quenched with
0.1 N HCl (5 mL) and diluted with DCM (10 mL). The organic
layer was extracted with DCM (20 mL ꢁ 2) and washed with
brine solution (10 mL). The organic layer was dried over anhy-
drous Na2SO4. The organic solvent was then evaporated using a
rotary evaporator and the crude product was puried using
silica gel (100–200 mesh size) column chromatography with
25% ethyl acetate in petroleum ether to obtain 8.9 mg of pure
compound 4 as white solid powder (yield ¼ 53%).
2.2.4 Synthesis of the vitamin D3–succinic acid–doxoru-
bicin conjugate (5). The vitamin D3–succinic acid conjugate (2)
(4.17 mg, 0.008 mmol, 1 equiv.) was dissolved in 1 mL dry DMF
and cooled to 0 ꢀC. HBTU (5 mg, 0.0129 mmol, 1.5 equiv.) and
dry DIPEA (4.48 mL, 0.0257 mmol, 3 equiv.) were added to the
reaction mixture with continuous stirring under dark condi-
tions. Aer 15 min, doxorubicin$HCl (5 mg, 0.0086 mmol, 1
equiv.) was added. The reaction was monitored by TLC. Aer
2.2 Methods
2.2.1 Synthesis of the vitamin D3–succinic acid conjugate 24 h, the reaction was diluted with 10 mL DCM and quenched
(2) (Fig. 1). Vitamin D3 (50 mg, 0.129 mmol, 1 equiv.) was dis- with 10 mL 0.1 N HCl. The organic layer was extracted with DCM
solved in 2 mL anhydrous pyridine and cooled at 0 ꢀC under an (20 mL ꢁ 2), washed with water (20 mL ꢁ 2) and brine (10 mL).
inert atmosphere. Succinic anhydride (129.99 mg, 1.29 mmol, The organic layer was dried over anhydrous Na2SO4. The
10 equiv.) and DMAP (7.93 mg, 0.065 mmol, 0.5 equiv.) were organic layer was then concentrated using a rotary evaporator
added into the reaction mixture. The reaction mixture was and puried by silica gel column chromatography using 2%
stirred for 24 h at room temperature. The reaction was moni- methanol in DCM to obtain 5.18 mg of compound 5 as a red
tored by TLC. Aer the reaction was over, the reaction mixture colored solid (yield ¼ 60%).
was diluted with 40 mL of DCM and quenched with 10 mL of
2.2.5 General procedure for synthesizing drug loaded
0.1 N HCl solutions. The organic layer was washed with 0.1 M vitamin D3 nanoparticles. 2.0 mg of PC, 1.0 mg of vitamin D3–
aq. HCl (40 mL ꢁ 2) & brine (40 mL) and dried over anhydrous succinic acid–drug conjugates (3, 4 or 5) and 0.2 mg of DSPE-
Na2SO4. The solvent was removed under vacuum and the crude PEG were dissolved in 5.0 mL DCM. The solvent was evaporated
product was puried by silica gel ash column chromatography into a thin and uniform lipid-drug lm with the help of a rotary
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evaporator. Aer thorough drying with a vacuum pump the was wicked off by using lter paper and then 15 mL of freshly
lipid-drug lm was hydrated with 1.0 mL H2O for 2 h at 60 ꢀC (or prepared 0.25% uranyl acetate (2.5 mg uranyl acetate in 1 mL dd
ꢀ
at 37 C). It was passed though a Sephadex G-25 column and water) solution was placed on the TEM copper grid. Aer one
extruded through a 200 nm (or 100 nm) Whatman poly- min uranyl acetate solution was wicked off and the sample was
carbonate membrane at 65 ꢀC (or at 37 ꢀC) to obtain sub-200 nm washed three times with 15 mL dd water each time. The sample
particles. The nanoparticles were stored at 4 ꢀC for further use. was dried overnight on a clean dust free surface under a funnel.
For drug encapsulated vitamin D3-NP synthesis the same The nanoparticles were imaged using Tecnai T300 HR-TEM and
method was followed using PC : vitamin D3 : DSPE-PEG ¼ 2 Tecnai G2 20-Twin LR-TEM instruments.
mg : 1 mg : 0.2 mg weight ratios with either 0.5 mg or 1 mg
drug.
2.2.12 Cell viability assay. 5000 HeLa cells per well (100 mL)
in DMEM were seeded in a 96-well microtitre plate and allowed
ꢀ
2.2.6 General procedure for the quantication of drug to attach overnight in a 5% CO2 incubator at 37 C. Cells were
loading. A calibration curve was plotted in the concentration then treated with 100 mL of either the free drug or drug conju-
range of 2.5–25 mM (for PI103), 10–40 mM (for paclitaxel) and 10– gated nanoparticles with different concentrations (25, 12.5,
100 mM (for doxorubicin) by diluting 1 mM standard stock 6.25, 3.2, 1.6, 0.8 0.4 and 0.2 mM) and incubated for 48 h in 5%
solution of drugs in DMSO. The absorbance was measured at CO2 at 37 ꢀC. Aer 48 h, media from all the wells were aspirated
293 nm, 273 nm and 480 nm for PI103, paclitaxel and doxoru- and 20 mL of MTT reagent from a stock solution of 5 mg mLꢂ1 in
bicin respectively against the corresponding solvent blank. The PBS was added to each well. Aer incubation for 3 h in the
linearity was plotted for absorbance (A) against concentration incubator, the purple formazan crystals formed were solubi-
(C). For drug loading in nanoparticles, the prepared nano- lized using acidied isopropanol (62.5 mL conc. HCl in 100 mL
particles were dissolved in spectroscopic grade DMSO in 5%, isopropanol) and the absorbance was measured at 540 nm. The
10% and 15% dilution. The absorbance was measured at the amount of purple formazan produced by cells treated with free
characteristic wavelength against the corresponding solvent drugs and drug loaded nanoparticles was compared with the
blank in a 400 mL quartz cuvette and from the calibration curve amount of formazan produced by untreated control cells to
drug loading was measured in triplicate.
calculate the effectiveness of the test samples.
2.2.7 General procedure for determining the drug release
2.2.13 Cell internalization observed by confocal laser
prole. Concentrated 250 mL drug loaded nanoparticles were scanning microscopy (CLSM). HeLa cells (5 ꢁ 104) were seeded
suspended in 250 mL pH 5.5 buffer and sealed in a dialysis on a cover slip in a 24 well plate and incubated overnight at
membrane (MWCO ¼ 500 Dalton for PI103 release and 1000 37 ꢀC in a CO2 incubator. The cells were then washed with PBS
Dalton for paclitaxel and doxorubicin release). The dialysis bags (pH ¼ 7.4) and treated with free doxorubicin and doxorubicin-
were incubated in 30.0 mL PBS buffer at room temperature with NPs with the content of doxorubicin being equivalent to that of
gentle shaking. A 500 mL portion of the aliquot was collected free doxorubicin at 2 mg mLꢂ1 for 1 h, 3 h and 6 h. Aer specied
from the incubation medium at predetermined time intervals, time intervals cells were washed twice with PBS and xed with
and the released drug was quantied using
spectrophotometer.
a
UV-VIS 500 mL paraformaldehyde (3.7% in PBS, pH ¼ 7.4) by incubating
for 10 min at 4 ꢀC. The paraformaldehyde was aspirated and
2.2.8 Determination of size distribution of nanoparticles cells were washed with PBS to remove the excess of para-
by dynamic light scattering (DLS). The mean particle size of the formaldehyde. Low pH lysosomes were stained with 1 mM
nanoparticles was measured by the DLS method using a Zeta- LysoSensorꢀ Green DND-153 (Invitrogen) by incubating the
sizer Nano 2590 (Malvern, UK). 10 mL of nanoparticle solution cells at 37 ꢀC in a CO2 incubator for 45 min. The cells were then
was diluted to 1 mL using deionized water and 3 sets of 10 washed three times to remove the unbound LysoSensorꢀ Green
measurements each were performed at a 90 degree scattering followed by staining the cells for nuclei with 2 mg mLꢂ1 Hoechst
angle to get the average particle size.
33342 (Cell Signaling Technology) by incubating at 37 ꢀC for 20
2.2.9 Field-Emission Scanning Electron Microscopy (FE- min. Then cells were washed three times with PBS buffer and
SEM) of nanoparticles. 5 mL of nanoparticles in water was mounted on a glass slide using 5 mL antifed-mounting medium.
placed on a silicon chip without any dopant and it was allowed The slides were subjected to uorescence imaging using a
to dry at room temperature in a desiccator for 2 h. The silicon CLSM (Zeiss LSM 710). The uorescence of LysoSensor Green,
chip was then gold coated (30–40 nm thickness) using a Hoechst 33342 and doxorubicin was excited with an argon laser
Quorum, Q150T-E5. The FE-SEM measurements were done at 458 nm, 405 nm and 488 nm, and the emission was collected
using a Carl Zeiss, Ultra plus, scanning electron microscope at through 470–509 nm, 403–452 nm and 560–590 nm lters,
an operating voltage of 4.0 kV.
respectively. Antifed mounting medium was prepared by dis-
2.2.10 Atomic Force Microscopy (AFM) of nanoparticles. solving 500 mg of n-propyl gallate in 10 mL of PBS and 90 mL of
5 mL of nanoparticles in water was placed on a mica sheet and glycerol.
dried under a light bulb for 20 min. The shape and size of the
nanoparticles were determined using a Nano Wizard Atomic
Force Microscope (AFM).
3. Results and discussion
3.1 Choosing the drugs for delivery in cancer therapy
2.2.11 High-Resolution Transmission Electron Microscopy
(HR-TEM) of nanoparticles. 15 mL of nanoparticles in water was To show the versatility of our vitamin D3 nanoparticle platform,
placed on a TEM copper grid. Aer 30 min, this sample drop we chose two cytotoxic anticancer drugs (paclitaxel and
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doxorubicin), which are used extensively in clinics and one
phosphatidyl-inositol-3-kinase (PI3K) inhibitor (PI103), which is
currently in clinical study.7 Paclitaxel is a natural product
having highly potent anti-tumor activity against different types
of cancers by stabilizing microtubules in cell division.8
However, its clinical use is limited due to its poor solubility in
water (0.25 mg mLꢂ1). Several polymeric nanovectors like poly-
mer conjugates, liposomes, polymeric micelles, emulsions and
nanospheres have been studied extensively for paclitaxel
delivery.9 However, these nanoformulations lead to low drug
loading and poor release of the active drug due to stable
conjugate formation with the polymers. On the other hand,
doxorubicin, an anticancer agent, extensively used in clinics,
shows its activity by binding with the DNA. But doxorubicin
induces non-specic cardiotoxicity and myelosuppression to
the patients.10 Currently, two FDA approved doxorubicin nano-
formulations (Myocetꢁ and Doxilꢁ) are available in the market.
However, both of them demonstrated side effects including
congestive heart failure, myelosuppression, thrombocytopenia
and palmer-planter erythrodysesthesia (PPE) or hand-foot
syndrome due to their premature burst release of drugs from their
encapsulated formulations.11 Moreover, being non-pegylated
formulation, Myocetꢁ is readily cleared from the body by the
reticuloendothelial system (RES), leading to its lower efficacy.4
Finally, we chose PI103, a potent PI3K inhibitor, as PI3K
signaling is one of the most frequently targeted pathways in all
sporadic human tumors, with mutations implicated in over
30% of all human cancers.12 PI103 shows anti-cancer activity by
inhibiting Akt and mammalian target of rapamycin (mTOR)
downstream of PI3K signalling.13 However, PI103 showed
limited aqueous solubility, extensive metabolism and dose
dependent insulin resistance.14 Hence it is extremely necessary
to develop novel biocompatible, biodegradable and non-toxic
nanotechnology based platforms for successful delivery of these
drugs in cancer therapy.
Fig. 1 Synthesis of drug loaded vitamin D3 nanoparticles. (a) Synthetic scheme
of vitamin D3–succinic acid–drug conjugation. (b) Schematic representation of
nanoparticle synthesis. (c) Schematic representation of vitamin D3-NP internali-
zation into lysosomal compartments by endocytosis.
–OH as the reactive group, which was conjugated with vitamin
D3–succinic acid (2) using a similar EDC and DMAP coupling
reaction to obtain the vitamin D3–succinic acid–PI103 conju-
gate (4) in 53% yield. Finally, doxorubicin was also conjugated
with vitamin D3–succinic acid (2) by an amide linkage using
HBTU as a coupling reagent and DIPEA as a base at room
temperature to obtain the vitamin D3–doxorubicin conjugate
(5) in 60% yield. The vitamin D3–succinic acid conjugate (2) and
3.2 Synthesis and characterization of vitamin D3–succinic
acid–drug conjugates
The free hydroxyl group of vitamin D3 (1) (Fig. 1a) was rst
reacted with succinic anhydride in the presence of DMAP as a
catalyst to attach a succinic acid linker to obtain the vitamin
D3–succinic acid conjugate (2) in 75% yield. We chose succinic
acid due to its biocompatibility and biodegradability as it is one
of the important components in the tricarboxylic acid (TCA)
cycle converting acetyl CoA from glucose to CO2 and water in the
mitochondria in our body.15 We further used the vitamin D3–
1
all the drug conjugates (3, 4 and 5) were characterized by H
NMR, 13C NMR and HR-MS spectroscopy (Fig. S12–S23 in the
ESI†).
3.3 Synthesis and characterization of vitamin D3
nanoparticles
succinic acid conjugate (2) as a platform to tether different types We engineered the nanoparticles from vitamin D3–succinic
of drugs for cancer therapy. Paclitaxel has two reactive acid–drug conjugates (3, 4 or 5), PC and DSPE-PEG in a
secondary –OH groups at 20 and 70 positions. However, the 1 : 2 : 0.2 weight ratio using a solvent evaporation–lipid-lm
20 –OH group is very reactive compared to the 70 –OH group due hydration–extrusion method17 (Fig. 1b). We chose PC as it is one
to higher steric hindrance at the 70-position.16 Hence the 20 –OH of the components of biological cellular membranes. DSPE-PEG
group of Paclitaxel was conjugated with vitamin D3–succinic was incorporated to provide “stealth” capability to the nano-
acid (2) by an ester linkage using EDC and DMAP as coupling particles as surface modication with PEG reduces opsonisa-
reagents at 0 ꢀC to room temperature for 24 h to obtain the tion,18 and hence reduces clearance by the reticuloendothelial
vitamin D3–succinic acid–paclitaxel conjugate (3) in 73% yield. system (RES). The hydrodynamic diameter and the poly-
On the other hand, the PI3K inhibitor, PI-103, has a phenolic dispersity index (PDI) of the drug loaded vitamin D3
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480 nm and 293 nm for paclitaxel, doxorubicin and PI103
respectively (Fig. 2d and S1 in the ESI†). The shape, size and
morphology of the drug loaded vitamin D3 nanoparticles were
determined by FE-SEM (Fig. 3a–c and S2, upper panel, in the
ESI†), TEM (Fig. 3d–f and S2, lower panel, in the ESI†) and AFM
(Fig. 3g–i and S3 in the ESI†). From DLS, FE-SEM, TEM and AFM
data, it is clear that different vitamin D3–succinic acid–drug
conjugates formed mono-dispersed, sub-200 nm spherical
particles which are ideal for tumor targeting by the EPR effect.
To understand the role of vitamin D3 in improving the drug
solubility and drug loading, we physically encapsulated pacli-
taxel, PI103 and doxorubicin in vitamin D3 nanoparticles
without using any vitamin D3–drug conjugates. We synthesized
the drug encapsulated vitamin D3 nanoparticles using PC : vi-
tamin D3 : DSPE-PEG in a 2 mg : 1 mg : 0.2 mg ratio. To opti-
mize the drug loading we used 0.5 mg and 1 mg of each drug for
encapsulation into the nanoparticles using the same solvent
evaporation–lipid lm hydration–extrusion method. Paclitaxel
encapsulation leads to the formation of nanoparticles having
diameters 135.28 ꢃ 1.86 nm and 134.74 ꢃ 0.47 nm (mean ꢃ
s.e.m., n ¼ 3) for 0.5 mg and 1 mg encapsulation, respectively.
We evaluated the paclitaxel loading in the nanoparticles by UV-
VIS spectroscopy and it was found to be 30.33 ꢃ 4.3 mg mLꢂ1 (for
0.5 mg encapsulation) and 10.26 ꢃ 2.67 mg mLꢂ1 (for 1 mg
encapsulation) (mean ꢃ s.e.m., n ¼ 3) (Fig. S4, le panel, in the
ESI†). The loading of paclitaxel was reduced by 3.8 times (for
0.5 mg encapsulation) and 11.5 times (for 1 mg encapsulation)
compared to the loading of NPs synthesized from vitamin D3–
paclitaxel conjugate 3 (Fig. 2d). For PI103 encapsulated NPs, the
diameter was found to be 145. 89 ꢃ 0.9 nm (for 0.5 mg encap-
sulation) and 173.41 ꢃ 0.9 nm (for 1 mg encapsulation)
(mean ꢃ s.e.m., n ¼ 3). The loading of PI103 in encapsulated
nanoparticles was found to be 2.27 ꢃ 0.6 mg mLꢂ1 and 2.28 ꢃ
0.5 mg mLꢂ1 (mean ꢃ s.e.m., n ¼ 3) for 0.5 mg and 1 mg
encapsulation, respectively (Fig. S4, middle panel, in the ESI†).
In the encapsulated NPs, PI103 loading was reduced by 25 times
compared to the loading of the NPs synthesized from vitamin
D3–PI103 conjugate 4 (Fig. 2d). Finally, we encapsulated doxo-
rubicin in vitamin D3 nanoparticles which led to the formation
of nanoparticles in the size of 165.82 ꢃ 0.9 nm (for 0.5 mg
encapsulation) and 170.30 ꢃ 1.2 nm (for 1 mg encapsulation)
(mean ꢃ s.e.m., n ¼ 3). However, the loading of doxorubicin in
encapsulated NPs was found to be 85.53 ꢃ 3.7 mg mLꢂ1 (0.5 mg
encapsulation) and 163.25 ꢃ 23.7 mg mLꢂ1 (1 mg encapsulation)
(mean ꢃ s.e.m., n ¼ 3) (Fig. S4, right panel, in the ESI†). The
loading of doxorubicin was increased in encapsulated NPs by
1.8 times and 2.8 times respectively compared to the loading
of NPs synthesized from vitamin D3–doxorubicin conjugate 5
(Fig. 2d). From this experiment we conclude that the high drug
loading achieved in vitamin D3–drug conjugated NPs is due to
the integration of conjugates into nanoparticles in the presence
of PC and DSPE-PEG. In our previous study,17 we have demon-
strated that PC : cholesterol–cisplatin conjugate : DSPE-PEG in
a 2 : 1 : 0.2 ratio formed sub-200 nm particles having a 6 nm
hydrophobic lipid layer with a hydrophilic core. We anticipate
that in the drug encapsulated vitamin D3-NPs, hydrophobic
Fig. 2 Size and drug loading in vitamin D3–drug-NPs. (a–c) Hydrodynamic
diameters of paclitaxel-NPs, PI103-NPs and doxorubicin-NPs measured by DLS. (d)
Graph showing the loading of different drugs in nanoparticles measured by UV-
VIS spectroscopy.
nanoparticles were evaluated by DLS and found to be 133.3 ꢃ
1.1 nm (PDI ¼ 0.255 ꢃ 0.01), 121.6 ꢃ 0.9 nm (PDI ¼ 0.241 ꢃ
0.01) and 101.1 ꢃ 0.4 nm (PDI ¼ 0.243 ꢃ 0.01) (mean ꢃ s.e.m.,
n ¼ 3) for paclitaxel-NPs, PI103-NPs and doxorubicin-NPs
respectively (Fig. 2a–c). Mean drug loadings in nanoparticles
were determined by UV-VIS spectroscopy to be 118.19 ꢃ 11.6
mg mLꢂ1 (loading efficiency ¼ 36.8%), 58.53 ꢃ 8.5 mg mLꢂ1
(loading efficiency ¼ 21.7%) and 57.56 ꢃ 3.7 mg mLꢂ1 (loading
efficiency ¼ 26.9%) (mean ꢃ s.e.m., n ¼ 3) for paclitaxel,
doxorubicin and PI103 respectively from a concentration vs.
absorbance calibration graph at characteristic lmax ¼ 277 nm,
Fig. 3 Characterization of vitamin D3–drug-NPs by FE-SEM, TEM and AFM. (a–c)
FE-SEM images of paclitaxel-NPs, PI103-NPs and doxorubicin-NPs. (d–f) TEM
images of paclitaxel-NPs, PI103-NPs and doxorubicin-NPs. (g–i) AFM images of
paclitaxel-NPs, PI103-NPs and doxorubicin-NPs. FE-SEM, TEM and AFM images
show that the vitamin D3–drug-NPs are spherical in shape and sub-200 nm in size. paclitaxel and PI103 entrapped in the thin lipid layer lead to
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very low drug loading, whereas hydrophilic doxorubicin D3 nanoparticle at pH 5.5 buffer at 37 ꢀC which mimics the
entrapped into the large hydrophilic core with increased drug acidic lysosomal compartment.20 We also evaluated the release
loading. However, in the vitamin D3–drug conjugated NPs, the prole of different drugs at physiological pH (pH ¼ 7.4) as a
conjugates themselves formed the lipid layer giving rise to control. For PI103, we used a 500 Dalton molecular weight cut-
improved loading for paclitaxel and PI103 and moderate off (MWCO) dialysis membrane to ensure that only free PI103
loading for doxorubicin.
(Mw ¼ 348 Dalton) comes out of the membrane, not vitamin D3–
Moreover, to make our vitamin D3 nanoparticle platform PI103 conjugate 4 (Mw ¼ 814 Dalton). Similarly, we used a 1000
more versatile for thermo-sensitive drugs, we performed the Dalton MWCO membrane to determine paclitaxel (Mw ¼ 853
nanoparticle synthesis at 37 ꢀC (temperature of our body) Dalton) and doxorubicin (Mw ¼ 543 Dalton) release proles
ꢀ
instead of 60 C. We evaluated the size and PDI of the nano- whereas the molecular weights of vitamin D3–paclitaxel conju-
ꢀ
particles by DLS experiments. At 37 C, the vitamin D3–pacli- gate 3 and vitamin D3–doxorubicin conjugate 5 are 1319 Dalton
taxel conjugate formed NPs having diameter 125.0 ꢃ 1.21 nm and 1009 Dalton respectively. Paclitaxel NPs demonstrated a
and PDI 0.31 ꢃ 0.02 (mean ꢃ s.e.m., n ¼ 3) (Fig. S5 in the ESI†). sustained release prole over 142 h, having 75.56 ꢃ 3.5%
On the other hand, the vitamin D3–PI103 conjugate formed NPs (mean ꢃ s.e.m., n ¼ 3) paclitaxel released at 48 h at pH 5.5,
having hydrodynamic diameter 177.2 ꢃ 0.36 nm and PDI 0.24 ꢃ whereas only 34.88 ꢃ 3.2% (mean ꢃ s.e.m., n ¼ 3) paclitaxel was
0.01 (mean ꢃ s.e.m., n ¼ 3). Finally, the vitamin D3–doxorubicin released at pH 7.4 at 130 h, which is 2 times less release than at
conjugate gave rise to NPs with diameter 138.2 ꢃ 0.6 nm and pH 5.5 (Fig. 4a). On the other hand, PI103-NPs also showed a
PDI 0.21 ꢃ 0.005 (mean ꢃ s.e.m., n ¼ 3). From the hydrody- sustained release of active PI103 over 93 h, having 77.74 ꢃ 9.2%
namic diameter and PDI values of different drug loaded nano- (mean ꢃ s.e.m., n ¼ 3) release of PI103 at 69 h, whereas at pH
particles, we concluded that our vitamin D3 nanoparticle 7.4 a similar amount 74.03 ꢃ 10.7% (mean ꢃ s.e.m., n ¼ 3)
platform can also be synthesized even at 37 ꢀC to deploy PI103 was released at 50 h (Fig. 4b). Finally, 66.35 ꢃ 4.82%
temperature sensitive drugs with sub-200 nm size to utilize EPR (mean ꢃ s.e.m., n ¼ 3) doxorubicin was released at 120 h at pH
effect in cancer therapy.
5.5 from the nanoparticle, whereas at pH 7.4 the highest
Furthermore, it has been demonstrated that particles with amount of doxorubicin was released around 9.47 ꢃ 2.8%
<100 nm diameter show signicantly greater internalization (mean ꢃ s.e.m., n ¼ 3) at 72 h, which is 7 times less release than
compared with the particles with >100 nm diameter.19 To at pH 5.5 (Fig. 4c).
develop vitamin D3 particles with less than 100 nm size, we
To determine whether the free drugs or the vitamin D3–drug
extruded the nanoparticles using a 100 nm polycarbonate conjugates are released from the nanoparticles, we analyzed
membrane. However, we obtained paclitaxel-NPs, PI103-NPs the released compound by using MALDI-TOF. We found the
and doxorubicin-NPs having size 104.7 ꢃ 1.31 nm (PDI ¼ 0.35 ꢃ [M + K]+ peak for free PI103 [386.9369 Dalton], [M ꢂ H]+ peak for
0.01), 116.8 ꢃ 0.1 nm (PDI ¼ 0.34 ꢃ 0.03) and 101.1 ꢃ 0.4 nm free paclitaxel [852.5859 Dalton] and [M + Na]+ peak for free
(PDI ¼ 0.21 ꢃ 0.005) (mean ꢃ s.e.m., n ¼ 3) which are very close doxorubicin [567.3539 Dalton], which clearly revealed that only
to 100 nm (Fig. S6 in the ESI†). However, we used sub-200 nm free drugs are released from the nanoparticles and not the
particles for further studies.
vitamin D3–drug conjugates (Fig. S8, S9 and S10 in the ESI†).
We rationalized that the aliphatic ester bond in conjugate 3
To be successful in delivering the drugs into the tumor by the
EPR effect, the NPs must be stable in the blood circulation for a is more cleavable at acidic pH (pH ¼ 5.5) compared to the
considerable amount of time. To evaluate the stability of different neutral pH (pH ¼ 7.4), which leads to a higher amount of
drug-loaded NPs, we incubated the NPs in fetal bovine serum paclitaxel release in shorter time. But the phenolic ester linkage
(FBS) at 37 ꢀC for 72 h and checked their stability by measuring in conjugate 4 is more labile at neutral pH leading to compa-
the hydrodynamic diameter and PDI using DLS. Paclitaxel-NPs rable release of PI103 at pH 5.5 and 7.4. Finally, the amide
showed changes in diameter from 104.7 ꢃ 1.3 nm to 107.1 ꢃ linkage in conjugate 5 is more prone to hydrolysis at acidic pH
2.2 nm, whereas, PDI values changed from 0.255 ꢃ 0.01 to (pH ¼ 5.5) than at neutral pH (pH ¼ 7.4) leading to 7 times more
0.241 ꢃ 0.04 over 72 h (Fig. S7 in the ESI†). On the other hand, release of active doxorubicin at acidic pH than at neutral pH.
doxorubicin-NPs demonstrated a small increase in size from From these release proles, it is clear that paclitaxel-NPs and
128.3 ꢃ 0.1 nm to 137.0 ꢃ 2.5 nm and change in the PDI value
from 0.243 ꢃ 0.01 to 0.216 ꢃ 0.13 over 72 h. Finally, PI103-NPs
also showed an almost negligible change in size from 177.2 ꢃ
0.3 nm to 178.4 ꢃ 0.3 nm and change in PDI from 0.241 ꢃ 0.01 to
0.275 ꢃ 0.01 over 72 h. From the stability data, it is clear that all
the drug-loaded NPs are stable under physiological conditions for
3 days, which is long enough time to home into the tumor by the
EPR effect. All the data were evaluated as mean ꢃ s.e.m., n ¼ 3.
Fig. 4 Release profile of drugs from vitamin D3-NPs at pH 5.5 and pH 7.4 at 37
ꢀC. (a) Release profile of paclitaxel over 142 h. (b) Release profile of PI103 over 90
3.4 Release of drugs from vitamin D3 nanoparticles
To evaluate the release prole of the active drugs we used a
h. (c) Release profile of doxorubicin over 120 h. From the release profile it is clear
dialysis method17 where we incubated the drug loaded vitamin that the vitamin D3-NPs release drugs in a slow and sustained manner.
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Fig. 5 (a–c) In vitro dose-dependent cytotoxicity assay of doxorubicin-NPs,
paclitaxel-NPs and PI103-NPs in HeLa cells. All of the cells were incubated with
either free drugs or the nanoparticles for 48 h and the cell viability was deter-
mined by the MTT assay. Data are given as mean ꢃ s.e.m. (n ¼ 3).
doxorubicin-NPs released more active drugs at pH 5.5 in a slow
and sustained manner compared to pH 7.4, whereas PI103-NPs
demonstrated comparable drug release at both pH 5.5 and 7.4.
Moreover, this increased drug release at acidic pH indicates
that more active drugs will be released in acidic lysosomal
compartments compared to neutral blood circulation.
Fig. 6 Internalization of doxorubicin-NPs in HeLa cells in 1 h, 3 h and 6 h time
points. Lysosomal compartments and nuclei were stained with LysoSensorꢂ
Green DND-153 (green) and Hoechst 33342 (blue), respectively. The merged
images show the co-localization of doxorubicin-NPs in the lysosomal compart-
ment in a time dependent manner. Scale bar ¼ 30 mm.
is no previous report of cellular internalization of vitamin D3-
NPs. To understand the cellular uptake mechanism of vitamin
3.5 In vitro cytotoxicity assay
To evaluate the efficacy of vitamin D3–drug-NPs in vitro, we D3-NPs, we treated HeLa cells with vitamin D3–doxorubicin-
performed a cell viability assay using HeLa cervical cancer cell NPs and observed the time dependent internalization of the
line. The cell viability was quantied by the MTT assay at 48 h NPs by CLSM. To determine the precise localization of doxo-
post-incubation. Doxorubicin-NPs showed HeLa cell death with
rubicin-NPs inside the cells, we stained low pH lysosomal
IC50 ¼ 0.26 mM compared to free doxorubicin having IC50 ¼ 0.21
compartments and nuclei using LysoSensorꢀ Green DND-153
mM (Fig. 5a). Doxorubicin-NPs induced 7.87 ꢃ 3.3% (mean ꢃ (shown in green) and Hoechst 33342 (shown in blue) respec-
s.e.m., n ¼ 3) cell viability whereas free doxorubicin induced tively. To understand the temporal internalization, the cells
1.37 ꢃ 0.2% (mean ꢃ s.e.m., n ¼ 3) cell viability at 25 mM. On the were treated with either doxorubicin-NPs (red) or free doxoru-
other hand, paclitaxel-NPs showed cell death with IC50 ¼ 0.25 bicin (red) at 2 mg mLꢂ1 concentration for 1 h, 3 h and 6 h. As
mM inducing 17.78 ꢃ 1.1% (mean ꢃ s.e.m., n ¼ 3) cell viability, shown in Fig. 6, aer incubation with doxorubicin-NPs, the red
whereas free paclitaxel showed IC50 ¼ 0.092 mM inducing 5.03 ꢃ and green uorescence co-localized (yellow regions) with each
0.3% (mean ꢃ s.e.m., n ¼ 3) cell viability at 25 mM concentration
other in a time dependent manner. At 6 h, doxorubicin-NPs
home into the low pH lysosomal compartments by an endocy-
(Fig. 5b). Finally, PI103-NPs showed cell killing with IC50 ¼ 12.8
mM inducing 38.65 ꢃ 1.8% (mean ꢃ s.e.m., n ¼ 3) cell viability, tosis mechanism. In contrast, for free doxorubicin treatment,
whereas free PI103 showed cell death with IC50 ¼ 0.76 mM the red and green uorescence did not co-localize (Fig. S11 in
inducing 5.38 ꢃ 0.8% (mean ꢃ s.e.m., n ¼ 3) cell viability at the ESI†) even aer 6 h. However, blue and red uorescence co-
25 mM concentration at 48 h (Fig. 5c). It is anticipated that in the localized (purple regions) with each other in a time dependent
in vitro study free drugs will be more cytotoxic than the nano- manner. From these CLSM images, it is clear that doxorubicin-
particles as the nanoparticles showed slow and sustained NPs were internalized into the HeLa cells through a low pH
release of active drugs over a long period of time, whereas the
free drugs will induce cell death very quickly. Moreover, we
lysosomal compartment over 6 h, whereas, free doxorubicin
internalized through the diffusion pathway. Based on this
reasoned that PI103-NPs induced less cell death with a high observation, we anticipate that paclitaxel-NPs and PI103-NPs
IC50 value compared to doxorubicin-NPs and paclitaxel-NPs will also be internalized by a similar endocytosis mechanism
because PI103 inhibits PI3K survival signaling through inhib- through low pH lysosomal compartments.
iting Akt and mTOR, which can be overcome by other cross-
talking signaling pathways leading to kinase-inhibitor related
resistance mechanisms.21 Moreover, PI103-NPs were more
stable (leading to 77.74 ꢃ 9.2% of drug release at 69 h), which
required a greater amount of drugs to show comparable cell
death with paclitaxel-NPs and doxorubicin-NPs.
4. Conclusions
In this study, we have successfully developed versatile, novel,
mono-dispersed, sub-200 nm sized vitamin D3 particles which
can carry diverse drugs (doxorubicin, paclitaxel and PI103) for
cancer chemotherapy. These nanoparticles showed high drug
loading yet slow and sustained drug release at acidic pH to
3.6 In vitro internalization of vitamin D3-nanoparticles
To the best of our knowledge, this is the rst report of vitamin mimic lysosomal compartments inside the cancer cells.
D3-nanoparticles used for drug delivery in cancer therapy; there Furthermore, these drug-loaded vitamin D3 nanoparticles
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induced in vitro cytotoxicity in HeLa cervical cancer cell lines by
internalization through lysosomal compartments to show their
potential in cancer therapeutics. These nanoparticles can also
be surface decorated by different tumor targeting moieties like
peptides, antibodies or aptamers for tissue specic delivery of
drugs. We believe that these vitamin D3 nanoparticles can serve
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Acknowledgements
We sincerely thank the Indian Institute of Science Education
and Research (IISER) Pune for nancial and instrumental 11 (a) L. Harris, G. Batist, R. Belt, D. Rovira, R. Navari,
support. We also thank the Department of Biotechnology (DBT)
for “Ramalingaswami Fellowship”. We thank Dr Shouvik Datta
for capturing FE-SEM images. We thank Ms Usha Shete for
capturing the AFM images and the Department of Science and
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