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Curcumin Releasing Eggshell Derived Carbonated Apatite Nanocarriers for

Combined Anti-Cancer, Anti-Inflammatory and Bone Regenerative Therapy

Article in Journal of Nanoscience and Nanotechnology · November 2019

DOI: 10.1166/jnn.2019.16640

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 Article
Journal of
Copyright © 2019 American Scientific Publishers
Nanoscience and Nanotechnology
All rights reserved Vol. 19, 6872–6880, 2019
Printed in the United States of America www.aspbs.com/jnn

Curcumin Releasing Eggshell Derived Carbonated

Apatite Nanocarriers for Combined Anti-Cancer,
Anti-Inflammatory and Bone Regenerative Therapy
Anish H. Verma1 , T. S. Sampath Kumar1 ∗ , K. Madhumathi1, Y. Rubaiya2 ,
Murugan Ramalingan3 , and Mukesh Doble2
1
Medical Materials Laboratory, Department of Metallurgical and Materials Engineering,
Indian Institute of Technology Madras, Chennai 600036, India
2
Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India
3
Biomaterials and Stem Cell Engineering Lab, Centre for Biomaterials, Cellular and Molecular Theranostics,
School of Mechanical Engineering, Vellore Institute of Technology (VIT) (Deemed to be University), Vellore 632014, India

Bone cancer or osteosarcoma is an aggressive cancer affecting the long bones and is treated by
a combination of surgery and chemotherapy. Local drug delivery directly to the site of bone cancer
and the use of plant-based drugs has been explored towards improving the efficacy and decreasing
the toxicity of the anti-cancer drugs. Curcumin, derived from turmeric is highly effective against can-
cer cells and shows very low toxicity against
IP: 14.139.160.231 On:normal
Thu, cells.
02 MayBone repair
2019 is facilitated by use of bone
05:39:19
substitutes such as bioceramics,
Copyright: American Scientific Publishers (CA) nanocarriers closely
amongst which the carbonated apatite
mimic the natural bone mineral. In the current work,
Delivered we have developed CA nanocarriers based
by Ingenta
local delivery of curcumin as an adjunct treatment for bone cancer. CA nanocarriers with 6 wt.%
carbonate were prepared by wet chemical synthesis using synthetic derived (6SWCA) and eggshell
derived (6EWCA) precursors along with hydroxyapatite (WHA) as a control. The X-ray diffraction
(XRD) patterns showed the CAs to be phase pure with a mean crystallite size of 17 nm. The Fourier-
transform infrared spectroscopy (FTIR) analysis of both CAs indicated the carbonate substitution
as B-Type. The amount of carbonate substitution was observed to be around 6 wt.% using FTIR
and CHNO elemental analyzer. The 6EWCA showed a greater loading (36%) and release (66%) of
curcumin than 6SWCA and WHA nanocarriers. The bovine serum albumin (BSA) protein denatura-
tion assay showed the curcumin loaded CAs to be highly anti-inflammatory while their anti-cancer
activity was confirmed by the high cytotoxic activity against MG-63 human osteosarcoma cells. Con-
clusively, an eggshell derived apatite drug delivery system was found to be very suitable to cure
osteosarcoma, prevent post-cancer inflammation and modulate bone repair and regeneration.
Keywords: Osteosarcoma, Eggshell, Carbonated Apatite, Curcumin, Local Drug Delivery,
Anti-Inflammatory, Anti-Cancer.

1. INTRODUCTION
Osteosarcoma (OS) is defined as the most common type of
cancer, which starts in the bone, mainly affecting children
and young adults. It is an aggressive malignant neoplasm
that exhibits uncontrolled osteoblastic differentiation and is
characterized by spindle cells of mesenchymal origin pro-
ducing malignant osteoid. It exhibits high affinity towards
the metaphysis of long bones, with 43% in the distal
∗
Author to whom correspondence should be addressed.
femur, 23% in the proximal tibia and 10% in the humerus.1
These cancer cells often show a metastatic growth in the
pulmonary area leading to failure of the respiratory system
and subsequent death.2 The conventional treatment for OS
is a combination of neoadjuvant chemotherapy, followed
by surgery and then adjuvant chemotherapy.3 Neoadjuvant
chemotherapy is defined as a chemotherapy given before
any surgical treatment whereas adjuvant chemotherapy
involves therapy post-surgery. Chemotherapy involves the
use of synthetic anti-cancer drugs such as methotrexate,

6872 J. Nanosci. Nanotechnol. 2019, Vol. 19, No. 11 1533-4880/2019/19/6872/009 doi:10.1166/jnn.2019.16640

Verma et al. Curcumin Releasing Eggshell Derived Carbonated Apatite Nanocarriers
doxorubicin, cisplatin and ifosfamide to kill the cancer
cells by interfering with their cell division cycle.3 Despite
the chemotherapy, OS has one of the lowest survival rates
of any pediatric cancer.4 This is because of the adverse
effects of the chemo-drugs such as nausea, fatigue, hair
loss, anaemia, diarrhoea, constipation, nerve and muscle
ailments, appetite and weight reduction, urinary infections,
5 apatite has the carbonate ion substituting the hydroxyl sites
and fertility problems.
Natural products have been an overwhelming success in
our society and are the upcoming trend for the replace-
ment of the synthetic chemo drugs due to the adverse
effects. Amongst these products, curcumin has gained spe-
cial attention for the treatment of cancer due to its anti-
6
cancer potency. Curcumin is a phytochemical derived
from the rhizomes of a very popular Indian spice, turmeric
(Curcuma Longa L.). Curcumin has already been used
in medical applications as an Alzheimer’s therapeutic,7
contraceptive extract,8 and infertility cure.9 Curcumin is
reported to act as a powerful anti-oxidant with strong
anti-carcinomic and anti-inflammatory abilities due to the
presence of numerous reactive functional groups.10 These
groups can interact with various molecular targets such as
transcription and growth factors, protein kinases, cytokines
and enzymes leading to multiple cell signalling resulting
in apoptosis of the cancer cell.10 Moreover, it is highly
biocompatible to normal cells and even at high dosage,
ranging from 1.12 to 2.5 g per day of curcumin, has been
IP: 14.139.160.231
11 On: Thu, 02
reported to be non-toxic to normal tissues. Hence,
Copyright: cur- Scientific Publishers
American
cumin is as an ideal choice as a potential anti-cancer
drug. by Ingenta by
Delivered
However, owing to its poor solubility (11 ng/mL), stability
and rapid metabolism, efficient delivery of curcumin still
remains a challenge in achieving therapeutic outcomes. 12
A localized nano-drug delivery system (NDDS) can
deliver the hydrophobic drug in high payload with
enhanced efficacy to the cancer cells directly at the
target site, thereby minimizing the drug plasma con-
centration in the bloodstream or any other organs and
ultimately aiding patient’s comfort.13 Many materials
have been reportedly used as an NDDS with the lat-
est including hybrid polymer-drug conjugates,14 meso-
porous biosilica,15 nano-lipids,16 magnetic nanoparticles,17
electrospun nanofibers,18 hydrogels19 and graphene-based
nanomaterials.20 However, the ideal carrier for any local
bone therapeutics will be calcium phosphate (CaP)
nanocarriers due to their composition mimicking the
bone mineral, availability of various binding sites, high
biocompatibility and biodegradability.21 Additionally, the
localized CaP based delivery system provides an added
advantage of bone repair and regeneration due to its osteo-
genesis ability, which is very much desirable for post-
cancer healing.22
Amongst the various CaPs, carbonated apatite is a suit-
able choice since its composition very closely mimics the
biological composition of human bone and teeth. Both
bone and teeth typically consist of an inorganic phase
J. Nanosci. Nanotechnol. 19, 6872–6880, 2019
comprising carbonated apatite nanocrystals (∼3–6 wt.%
CO2−3  of platelet-shaped morphology embedded in an
organic matrix composed largely of type-1 collagen with
minor amounts of other collagens, glycoproteins, proteo-
glycans and sialoproteins.23 The carbonated apatite (CA)
can be classified depending on the location of carbon-
ate substitution as A-type and B-type.23 A-type carbonated
whereas in B-type, carbonate ion substitutes at the phos-
phate site. It has been reported that only the B-type carbon-
ated apatite is present in bones and teeth.23 Nanocrystalline
carbonate-apatites have been studied for the release of anti-
cancer platinum bisphosphonates.24 But the carbonate was
on the surface of the materials by adsorption during the
storage and was mainly retained to mimic the biological
apatite. Recently, carbonated apatite (CA) nanoparticles
with a molecular formula of Ca10 (PO4 6−X (CO3 X (OH)2
loaded with paclitaxel showed enhanced efficacy against
cancer cells both in a cell culture and an animal model
compared to free paclitaxel.25 Surface modification with
poly(ethylene glycol) or citrate has also shown more effec-
tive uptake of drug-loaded CA nanoparticles by the tumor
cells compared to the uncoated particles and the free
drug.26 27
Eggshells are mainly composed of calcium carbonate
with traces of strontium, magnesium, fluorine, silica and
28
sodium. The synthesis of CAs from eggshells by par-
tially May 2019its05:39:19
retaining carbonate content has been demonstrated
28
earlier
our group. Inspired by the advantages of CA
for cancer drug delivery applications, we have studied the
potential of curcumin delivery by eggshell derived CA
(6EWCA) nanoparticles for the first time to the best of
available literature in comparison with synthetic derived
CAs. The curcumin loaded CAs was also evaluated for
their anti-inflammatory activity and anti-cancer activity by
in vitro cell culture studies. As bone substitutes are nec-
essary after the surgical removal of the bone tumor, the
curcumin-loaded 6EWCA is anticipated to perform both
bone regeneration and anticancer drug release features to
inhibit tumor re-growth.
2. MATERIALS AND METHODS
2.1. Materials
Precursors for preparation of CAs like diammonium
hydrogen phosphate [(NH4 2 HPO4 ], calcium hydroxide
[Ca(OH)2 ] and calcium carbonate (CaCO3  were obtained
from Merck, Mumbai. The drug curcumin (from Curcuma
longa) was purchased from Sigma Aldrich, India. Ethanol
(99.9%), used as a drug loading media, was obtained
from Changshu Hong sheng Fine Chemical Co. Ltd.,
China. Chemicals for preparation of phosphate buffer solu-
tion (PBS) like sodium biphosphate dihydrate (NaH2 PO4 ·
2H2 O) and disodium hydrogen phosphate anhydrous
(Na2 HPO4  were obtained from SD Fine Chemicals,
Mumbai and Merck, Mumbai respectively. Chemicals used
6873
Curcumin Releasing Eggshell Derived Carbonated Apatite Nanocarriers
for anti-inflammatory assay like bovine serum albumin
(BSA) and glacial acetic acid (C2 H4 O2  were purchased
from SRL Pvt. Ltd., Mumbai. Chemicals for cell culture
studies like thiazolyl blue tetrazolium bromide or MTT
(C18 H16 BrN5 S), dimethyl sulphoxide or DMSO (C2 H6 OS),
trypan blue dye and minimum essential medium (MEM)
were purchased from Himedia Lab, Mumbai.
2.2. Synthesis of CAs
CAs were prepared from synthetic sources as well as natu-
ral eggshell sources as reported28 using wet chemical syn-
thesis method. The carbonate substitution in CAs was kept
at 6 wt.% since it mimics the natural bone composition.
The synthetic precursors used were Ca(OH)2 , CaCO3 and
(NH4 2 HPO4 . 0.24 M (NH4 2 HPO4 was added dropwise
to a 0.4 M Ca(OH)2 and 6 wt.% CaCO3 solution to obtain
calcium- to-phosphate ratio (Ca/P) of 1.67. The resultant
mixture was kept for 2 days for proper aging and then
filtered. The precipitate obtained was then washed with
distilled (DI) water to remove any unwanted ions and oven
dried at 100  C. Finally, the precipitate was grinded using
a mortar and pestle to obtain a fine powder.
Eggshell derived CAs were synthesized using hen’s
eggshells as a calcium source. Hen’s eggshells were col-
lected and their surface was manually cleaned by strip-
ping the inner surface membrane. They were then washed

with water followed by overnight oven drying at 100 C
IP: 14.139.160.231 On: Thu,the 02ratio
and then powdered using a mortar andCopyright:
pestle. These pow-
American Scientific Publishers
ders were then heated to 805  C for 1 h in theDelivered
furnace, bycurcumin
followed by immediate air quenching to obtain Ca(OH)2
with 6 wt.% arrested residues of carbonate content.28 The
product was then washed and powdered similar to syn-
thetic CAs. Additionally, a batch of synthetic hydroxya-
patite (WHA) sample was also prepared using the wet
chemical as a reference. The synthetic CA and eggshell
derived CA samples were coded as 6SWCA and 6EWCA
respectively.
2.3. Material Characterization
The X-ray powder diffraction (XRD, Bruker D8 Discover,
USA) was carried out to analyze the crystallographic
phase of CA samples, using CuK radiation ( = 1.54 Å).
The diffraction spectra were recorded with a step size of
0.1 /step and at a scanning rate of 1 step/sec between the
2 range of 20–80. The functional groups present in CA
and HA nanocarriers were analyzed in the range of 3500–
500 cm−1 using attenuated total reflectance (ATR) mode
in Fourier transform infrared spectrometer (Spectrum Two,
PerkinElmer, USA). Carbon content present in CA sam-
ples were measured by CHNSO elemental analyzer (LECO
TruSpec Micro and 6280 O analyzers, USA), where a sam-
ple of 1 gram was combusted in a stream of oxygen using
RF induction and carbonate % was calculated. Particle size
and zeta-potential analysis were done using a Zetasizer
analyzer (Nano-ZS90, Malvern, UK). The particles were
6874
Verma et al.
dispersed and sonicated in water media, followed by anal-
ysis at 25  C.
2.4. In Vitro Curcumin Loading and Release Studies
The in vitro curcumin loading and release studies were per-
formed similar to the method as reported earlier.29 50 mL
of 1 mg/mL solution of curcumin-ethanol was prepared to
which 50 mg of nanocarriers were added. The resultant
solution was left undisturbed for 24 h at 4  C. After 24 h,
2 mL of the supernatant was removed for the estimation
of the residual drug concentration using a UV-Vis spec-
trophotometer (Lambda 35, PerkinElmer, USA). The peak
at 424 nm was used to determine the curcumin concentra-
tion, and thereby calculating the drug loading percentage.
The remaining solution was then filtered and the drug-
loaded nanocarriers were obtained. The amount of drug
loaded onto the nanocarriers is calculated by the below
equation:
% Drug Loading = Ic − Fc /Ic × 100 (1)
where Ic and Fc are the initial and the final concentration
of curcumin in the solvent.
The release studies were performed by dispersing the
drug-loaded nanocarriers in a specially designed release
media of pH 7.4 in a constant temperature water bath at
37  C. The release media consisted of PBS and ethanol in
Mayof 4:1. 05:39:19
2019 A standard calibration curve was plotted
between the absorbance
and the various concentration of
Ingenta (g/mL) in the release media. About 2 mL of
the supernatant was removed from the release system for
UV-Vis characterization and was replaced by 2 mL freshly
prepared release media. The readings were noted at a reg-
ular interval of 2 h up to 12 h and then at 24th and 48th h.
The experiment was done in triplicates. The drug release
was calculated by:
% Drug release = Fr /Ir × 100 (2)
where Ir is the initial amount of drug loaded and Fr is the
final amount of drug released at regular intervals obtained
using the standard graph equation.
2.5. Release Kinetics
The kinetics of the release mechanism from the nanocar-
riers were evaluated using Korsmeyer-Peppas (Power law)
and Higuchi’s mathematical model which are shown in
Eqs. (3) and (4) respectively.
Mt
= kt n (3)
M
where Mt and M are defined as cumulative amounts of
drugs released at time ‘t’ and at infinite time (i.e., the
maximum released amount), k is defined as the release rate
constant and n is the diffusion coefficient.
Qt = kt 0 5 (4)
J. Nanosci. Nanotechnol. 19, 6872–6880, 2019
Verma et al. Curcumin Releasing Eggshell Derived Carbonated Apatite Nanocarriers
where Qt is a fraction of drug released at a time ‘t’, k is
the release rate constant (or Higuchi’s constant).
2.6. In Vitro Anti-Inflammatory Assay
The anti-inflammatory studies of curcumin loaded CAs
were carried out by inhibition of albumin denaturation
assay.30 1% aqueous solution of BSA was prepared and the
pH was adjusted to 6.5 using the glacial acetic acid. 10 mg
of the curcumin-loaded nanocarriers (WHA, 6SWCA and
6EWCA) were suspended in 10 ml of release media and
incubated at 37  C for 24 h. 1 mL of the supernatant from
the curcumin-loaded nanocarriers was added to 3 mL of
BSA solution. The solution was then heated to 52  C for
20 min, and the solution’s absorbance was then measured
at 660 nm by UV-Vis spectroscopy. Pure samples (no drug)
were taken as control. The experiment was performed in
triplicates. The percentage inhibition of protein denatura-
tion was calculated as follows:
% inhibition = Ac − As /Ac × 100 (5)
where Ac is the absorbance of control and As is the
absorbance of samples.
2.7. In Vitro Cell Viability Assay
The cell viability studies of the curcumin-loaded nanocar-
riers was tested against human osteosarcoma MG-63
cell lines by MTT (thiazolyl blue tetrazolium bromide
IP: 14.139.160.231 On: Thu,carbonated
dye) calorimetric test for three days. Copyright: American
The cell lines Scientific Publishers
were obtained from the National Centre for cellDelivered
science by Ingenta
(NCCS), Pune, India.
These cells were grown to confluence in a T25 flask
in a minimum essential medium (MEM) and incubated at
37  C with 5% carbon dioxide in a CO2 incubator (Astec,
Japan). These cells were then trypsinized to detach the
cells from the surface of the T25 flask. Then a small
amount of MEM was added to stop the action of trypsin
and was followed by centrifugation at 2000 rpm for 5 min.
A cell pellet was formed in the bottom of the centrifuge
tube and the supernatant consisting of trypsin and MEM
was discarded. Fresh amount of MEM was added and the
pellet was mixed again to disperse the cells uniformly.
These were then dyed using trypan blue to differenti-
ate between live and dead cells. The number of cells
was counted with the help of a hemocytometer (Marien-
feld, Germany). They were then diluted (3000 cells/well)
and seeded in a 96-well plates and cultured for 24 h.
5 mg/mL of curcumin loaded 6EWCA and 6SWCA, were
suspended in MEM solution and incubated at 37  C for
24 h. The media in the 96-well plates was then replaced
with 100 L of the supernatant from the samples and incu-
bated for 24 h. 20 L of the supernatant was removed
from the 96-well plates and was replaced by 20 L of
MTT (5 mg/mL) and incubated for 4 h. All the media from
the 96 well-plate was removed for the reading. 100 L of
DMSO was added to the wells. The formazan precipitates
J. Nanosci. Nanotechnol. 19, 6872–6880, 2019
were solubilized in DMSO and the absorbance was mea-
sured at 570 nm using a multimode plate reader (EnSpire,
PerkinElmer, Singapore).
The percentage of viable cells was calculated as the per-
centage relative to the control using the equation:
% Cell viability = Sample OD/Control OD×100 (6)
where OD signifies optical density measured by the mul-
timode plate reader.
2.8. Cellular Uptake Studies
1 mg of CA nanocarriers were added to 1 mg/mL of rho-
damine B and sonicated for 30 min and then stirred at
50 rpm for 24 h. The samples were then centrifuged and
reconstituted with MEM and sonicated for 30 min in a
bath sonicator. MG-63 cells were seeded in a petri dish
with a density of 106 cells and incubated at 37  C for 24 h.
The rhodamine tagged samples were added and incubated
for 24 h after which the medium was removed and the
cells were washed three times with PBS to remove the
excess dye. The cells were then imaged using fluorescence
microscopy (Olympus IX51-URFLT, Japan).
3. RESULTS AND DISCUSSION
All the samples, including hydroxyapatite (WHA), syn-
thetic carbonated apatite (6SWCA) and eggshell derived
02 May 2019 05:39:19
apatite (6EWCA), were synthesized using a
wet chemical process. This method of preparing apatites
uses the conventional chemical co-precipitation reaction as
shown below:
10 CaOH2 + 6NH4 2 HPO4
→ Ca10 PO4 6 OH2 + 12NH3 + 18H2 O (7)
For CA synthesis, the reaction involves the decompo-
sition of CaCO3 source to CO2− 3 ion, which eventually
substitutes in place of the PO3−4 group in apatites to form
B-type CAs, as shown below:
CaCO3 → Ca 2+ + CO2− 3 (8)
Ca10 PO4 6 OH2 + CO2−3
→ Ca10 PO4 6−x CO3 x OH2 B − typeCA (9)
For 6SWCA, the carbonate substitution was controlled
using CaCO3 by calculation using stoichiometric meth-
ods. In the case of 6EWCA, controlled decomposition
of CaCO3 was carried out until 6 wt.% of carbonate
remained and then immediately air-quenched to arrest the
further decomposition and to obtain a uniform carbonate
distribution along the particles. The added advantage of
6EWCA is that it is a multi-ion substituted carbonated
apatite consisting of traces of Sr2+ , K+ , Mg2+ , Na+ and
F− ions, which have displayed an enhanced bone regener-
ation ability.31
6875
Curcumin Releasing Eggshell Derived Carbonated Apatite Nanocarriers Verma et al.

broadening at (002) was used to quantify the mean crystal-

lite size of the nanocarriers using the Scherrer’s equation:

D = 0 9 / Cos (11)

where D is the mean crystallite size,  is the wavelength

Figure 1. XRD pattern of WHA, 6SWCA and 6EWCA samples.
3.1. Material Characterization
The XRD pattern of 6EWCA, 6SWCA and WHA nanocar-
riers is shown in Figure 1. The patterns were compared
to standard apatite (JCPDS #01-072-1243). All the peaks
were found to correspond to that of the JCPDS data
file, indicating the formation of apatites. The CA samples
showed a minor peak shift i.e., (002) peak shifted towards
lower 2 angle and (300) peak shifting towards higher 2
angle when compared to that of HA. A shift to lower 2
angle corresponds to tensile microstrain whereas a shift to
IP: 14.139.160.231
higher 2 angle signifies a compressive On: Thu,are
microstrain. Thus,
Copyright: American Scientific Publishers−1
the change in the lattice parameters i.e., increase Delivered
in c-axis byatIngenta
and decrease in a-axis was established from the XRD pat-
tern, indicating B-type carbonate substitution. This was
further evaluated by quantifying the lattice parameters, as
shown in Table I, from the (002) peak for c-axis and (310)
peak for a-axis using the interplanar spacing formula for
a hexagonal crystal structure:
1 4 2
2
= h + h · k + k /a  + l /c
2 2 2 2
(10)
d 3
where d corresponds to inter-planar spacing and h, k, l are
the Miller indices representing the orientation of an atomic
plane along the crystallographic axis.
The quantified values of the lattice parameters as shown
in Table I suggest that the substitution is of B-type.
Additionally, peak broadening was observed in CA sam-
ples compared to WHA, which suggests that the car-
bonate addition decreases the crystallite size.28 The peak
of the X-rays ( = 1.5405 Å for Cu K radiation), cor-
responds to line broadening at half the maximum intensity
(FWHM) and  is the Bragg’s angle.
The crystallinity index (CI) is defined as the volume
fraction of the crystallinity of a given phase and is a quan-
titative indicator of crystallinity of the given sample. It
has been reported that apatites having a reduced crys-
tallinity has increased solubility and higher protein and
drug absorption capacity.32
Therefore, CI quantification was done using the
formula28 as shown below:
CI = 0 24/ 002 3 (12)
where 002 represents the full-width half maximum
(FWHM) of (002) reflection.
It was found that the CI of the CAs, i.e., 0.12 for
6SWCA and 0.09 for 6EWCA, was less than that of was
WHA sample, i.e., 0.29, suggesting a possible higher drug
absorption capacity.
The FT-IR spectra of WHA, 6EWCA and 6SWCA are
shown in Figure 2. All the characteristic bands of HA
02present
May 2019 05:39:19
in the WHA spectra such as PO3−
4 vibrations
602 and 562 cm ( 4 bending), 962 cm−1 ( 1 sym-
metrical stretching) and 1027 and 1091 cm−1 ( 3 anti-
symmetric stretching mode), OH− vibrations at 632 cm−1
(scissoring) and 1630 cm−1 (bending). The absence of
intense OH− peak around 3500 cm−1 indicates that no
residual water molecules are present in the lattice of the
apatites. For CA samples, the presence of characteristic
−1
B-type CO2− 3 peaks at 1418 cm ( 3b asymmetric stretch-
ing), 1490 cm −1
( asymmetric stretching) and 874 cm−1
3a
( 2 out-of-plane bending mode) confirmed the formation

Table I. List of cell parameters, crystallite size and crystallinity index

of WHA, 6SWCA and 6EWCA samples.

a-axis c-axis Cell volume Mean crystallite Crystallinity

Samples (Å) (Å) (Å3 ) size (nm) index (CI)

WHA 9.42 6.89 530 24 0.29

6SWCA 9.39 6.90 527 18 0.12
6EWCA 9.36 6.92 525 16 0.09
Figure 2. FTIR spectra of WHA, 6SWCA and 6EWCA samples.

6876 J. Nanosci. Nanotechnol. 19, 6872–6880, 2019

Verma et al. Curcumin Releasing Eggshell Derived Carbonated Apatite Nanocarriers

of B-type carbonated apatites. Additionally, the absence of Table III. Mean particle size and zeta potential calculations of WHA,
−1 6SWCA and 6EWCA samples.
CO2−3 at 2349 cm indicates that no residual carbonate is
present and all the CO2−3 have been substituted in place of Samples Size of particles (dia. nm) Zeta potential (mV)
PO3− . On further analysis, it was found out that the relative
4 WHA 208 ± 14.7 −17.5 ± 3.4
intensities at 962 and 1091 cm−1 for 6EWCA and 6SWCA 6SWCA 156 ± 10.6 −21.8 ± 7.9
samples were reduced when compared to WHA sample as 6EWCA 106.2 ± 2.8 −21.5 ± 4.6
3−
a result of CO2−3 replacing PO4 in the hexagonal lattice
structure. Moreover, for CA samples, the OH− vibration
peak at 632 cm−1 was found to reduce in intensity signify- 3.2. Curcumin Loading and Release Studies
3− −
ing the CO2− 3 substituting for PO4 and formation of OH
The loading of curcumin on CAs and HA are shown in
vacancies for charge compensation. Figure 3. The WHA has a loading of 20.8%, 6SWCA has
The CO2− 3 substitution in the B-type carbonated apatite
32.7% and 6EWCA has 35.6%. Both the CA samples have
was also quantified by FT-IR spectra33 and CHNO- higher curcumin loading than HA. As discussed earlier,
elemental analyzer, as shown in Table II. Both confirmed a the carriers with lower crystallinity index (CI) will have
6 wt.% substitution in the 6EWCA and 6SWCA samples. high drug absorption capacity. Hence, CAs with lower
The formula used to calculate the wt.% of carbonate from CI exhibits higher loading, as expected. Amongst the two
the FTIR spectra is shown below:33 CA samples, 6EWCA has a slightly higher loading than
6SWCA. It may be due to the presence of ions in 6EWCA
wt % CO3 = 28 62 × rc/p  + 0 0843 (13) inherited from the eggshell.
2− The cumulative release profile of curcumin-loaded
where rc/p is the area ratio between the 3 CO3
−1 3− 6EWCA, 6SWCA and WHA were plotted as shown in
band (1550–1330 cm  and the 1 3 PO4 band
Figure 4. The curcumin-release from WHA, 6SWCA and
(1230–900 cm−1 .
6EWCA was found out to be 45.48%, 58.57% and 66.24%
The particle size and zeta potential of the apatites are
respectively, confirming the role of CO2− 3 in the release
listed in Table III. It was found that both the CA samples
of the curcumin from the nanocarriers. Moreover, it was
were significantly smaller than the HA sample. The WHA
found that 6EWCA had a greater release than 6SWCA due
sample had a mean size of 208 nm whereas 6SWCA and
to the inherited trace elements present in 6EWCA.
6EWCA had a mean size of 156IP: and14.139.160.231
106 nm respectively.
On: Thu, 02TheMay 2019kinetics
release 05:39:19 of curcumin-loaded nanocarriers
The particle size obtained from DLS Copyright:
are typicallyAmerican
higher Scientific Publishers
were studied using both Korsmeyer-Peppas and Higuchi
since it reports hydrodynamic radius as an intensity dis- by Ingenta
Delivered
models. The release rate constants (K) were calculated
tribution. The 6EWCA had a hydrodynamic diameter less
from the slope of the plots using regression analysis. The
than that of 6SWCA. The particle size is said to play a
parameters of release kinetics are listed in Table IV. The
key role in drug circulation and absorption into tissue and
34 coefficient of correlation (R2  ranged in the domain of 0.97
cellular membranes. It is reported that the smaller are the
to 0.99, depicting a very good fit of the data with both
particles, higher will be the particle uptake by the cells.34
the mathematical models. The diffusion coefficient (n) is
Zeta potential is a term used to quantify the electroki-
an important entity that gives the drug release mechanism.
netic potential in a colloidal system. The zeta potential is
As per the model, if n < 0 5, it indicates Fick’s diffusion,
said to affect the pharma kinetics as well as the phagocy-
35
tosis of the drug-loaded nanocarriers in the bloodstream.
It has been reported that the highest cellular uptake was
found out to be in the samples having higher negative zeta
potential value.35 As shown in Table III, 6EWCA tends to
have a higher negative zeta potential value of −21.5 mV
whereas WHA shows the least, i.e., −17.5 mV. As the
smaller particles and higher negative zeta potential tend to
show a higher affinity for cellular membrane due to elec-
trostatic interactions,35 it is expected that 6EWCA will be
having a higher affinity towards osteosarcoma cell uptake
than any other sample.

Table II. Amount of carbonate content in 6SWCA and 6EWCA

samples.

Samples % CO2−
3 (FTIR) % CO2−
3 (CHNO-analyzer)

6SWCA 5.73 ± 0.5 5.97 ± 0.4

Figure 3. Curcumin loading profiles for WHA, 6SWCA and 6EWCA
6EWCA 6.02 ± 0.5 6.09 ± 0.4
nanocarriers (n = 3; values shown as mean ± SD).

J. Nanosci. Nanotechnol. 19, 6872–6880, 2019 6877

Curcumin Releasing Eggshell Derived Carbonated Apatite Nanocarriers Verma et al.

Figure 4. Curcumin release profiles for WHA, 6SWCA and 6EWCA

nanocarriers (n = 3; values shown as mean ± SD).

if 0 5 < n < 0 85 drug release follows non-Fickian diffu- Figure 5. Anti-BSA denaturation studies of curcumin loaded HA (Cur-
WHA) and CAs (Cur6SWCA and Cur6EWCA) nanocarriers (n = 3; val-
sion and if n > 0 85, it indicates a zero-order release.36 The ues shown as mean ± SD).
WHA sample followed two-step kinetics, i.e., non-Fickian
mechanism to 10 h, followed by a steady Fickian release
up to 48 h. On the contrary, the CAs followed three-step induces a greater anti-inflammatory activity. Amongst
kinetics. Till 4 h, it was non-Fickian and then followed by CAs, the 6EWCA showed a higher inhibition of 71.1%.
Fickian release to 48 h but with different release constants The significant difference between 6EWCA (71.1%) and
for 4 to 12 h and 12 to 48 h. 6SWCA (52.3%) may be due to the presence of trace ions,
which might themselves be involved in suppressing protein
IP: 14.139.160.231
3.3. In Vitro Anti-Inflammatory, Cell Viability and On: Thu, 02 May 2019 05:39:19
denaturation.
Copyright: American Scientific Publishers
The results of the MTT assay on MG-63 human
Uptake Studies Delivered byosteosarcoma
Ingenta cell lines performed at 5 mg/mL nanocar-
Figure 5 shows the result of anti-BSA denaturation study
by the curcumin-loaded nanocarriers. Denaturation of pro- rier concentration for 3 days are shown in Figure 6.
teins is a well-documented cause of inflammation26 and The nanocarriers (6SWCA and 6EWCA) were found to
it has been reported that even though if cancer cells are be highly biocompatible (>80%) and showed a greater
destroyed, prolonged chronic inflammation can lead to cell viability for all three days. On curcumin load-
regression.37 Therefore, the inhibition of protein denat- ing, the nanocarriers displayed a reduction in cell via-
uration was studied as a mean of evaluating the anti- bility. After the 3rd day, only 6.97% (in the case of
inflammatory activity of curcumin loaded nanocarriers.
The percentage of inhibition from WHA was found to
be 22.3%. The drug-loaded CA samples (6EWCA and
6SWCA) showed a higher inhibition than WHA sam-
ple. It may be concluded that the presence of carbonate

Table IV. Release kinetic profile displaying the diffusion coefficient (n),
release constant (K) and time-dependent release mechanism for curcumin
release from WHA, 6SWCA and 6EWCA nanocarriers.

Time Diffusion Release Mechanism

period coefficient constant of drug
Sample (h) (n) K, hours−n R2 release

WHA 0 to 10 0 768 0.131 0.992 Non-Fickian

10 to 48 05 0.050 0.973 Fickian (Higuchi’s)
6SWCA 0 to 4 0 77 0.236 0.999 Non-Fickian
4 to 12 05 0.154 0.997 Fickian (Higuchi’s)
12 to 48 05 0.016 0.999 Fickian (Higuchi’s)
6EWCA 0 to 4 0 53 0.317 0.999 Non-Fickian
Figure 6. Percentage of cell viability for 6SWCA and 6EWCA and
4 to 12 05 0.168 0.997 Fickian (Higuchi’s)
curcumin loaded CAs (Cur6SWCA and Cur6EWCA) nanocarriers (n = 3;
12 to 48 05 0.011 0.999 Fickian (Higuchi’s)
values shown as mean ± SD).

6878 J. Nanosci. Nanotechnol. 19, 6872–6880, 2019

Verma et al.
Cur6SWCA) and 5.55% (in the case of Cur6EWCA) of
the osteosarcoma cells were found alive. The decrease
in cell viability in case of curcumin loaded nanocar-
riers (Cur6SWCA and Cur6EWCA) suggests that they
have an excellent anti-cancerous property. Additionally,
the 6EWCA sample shows an increase in viability with
increasing days increase, suggesting a greater biocompati-
bility than 6SWCA.
The bright field and the fluorescence images of rho-
damine tagged 6EWCA and 6WSCA are shown in
Figure 7. It was observed that the uptake of CA nanocarri-
ers into the cells were very high. As discussed earlier, it is
due to their small size and high negative zeta potential val-
ues. No significant changes in morphology were noticed
with 24 h treatment of rhodamine with CA nanocarriers.
Typically, an ideal drug delivery system for osteosar-
coma treatment should not only kill the cancer cells
but also prevent any future recurrence and contribute
in repairing the damage done to the bones by the
tumor. As already discussed, curcumin is responsible for
osteosarcoma apoptosis.12 Additionally, it removes the
inflammation associated with the affected region to avoid
any recurrence of the cancer cells.11 Moreover, the CA
has shown to inhibit osteoclast cell activity leading to
bone healing.38 The 6EWCA showed a greater loading
and release of curcumin compared to synthetic derived
CA and HA nanocarriers. Eggshells also contain a trace
IP: 14.139.160.231
amount of elements such as Mg, Na, Si etc., essential On:
31 Copyright: American Scientific
for biomineralization. Hence, 6EWCA seem toDeliveredhave an by Ingenta W. Winkelmann, A. Zoubek, H. Jürgens, and K. Winkler, J. Clin.
optimal composition for bone regeneration and delivery of
curcumin in bone cancer applications. Therefore, bioac-
tive curcumin loaded EWCA could perform bone grafting
and drug release material to inhibit tumor re-growth. Addi-
tionally; being nanoparticles, it can be also used to form
injectable bone substitute materials for the treatment of
bone tumors.
Figure 7. Cell uptake studies depicting bright field and fluorescence
images (rhodamine tagged) of 6EWCA and 6SWCA nanocarriers in MG-
63 cells.
J. Nanosci. Nanotechnol. 19, 6872–6880, 2019
Curcumin Releasing Eggshell Derived Carbonated Apatite Nanocarriers
4. CONCLUSIONS
Local delivery of the anti-cancer drug curcumin from
6 wt.% carbonated apatite nanocarriers, synthesized from
synthetic and eggshell precursors, was evaluated for the
treatment of osteosarcoma in the present study. The car-
bonated apatite nanocarriers showed higher curcumin load-
ing and release compared to hydroxyapatite nanocarriers.
The higher suitability of carbonated apatites as curcumin
carriers can be due to its lower crystallinity, higher reactiv-
ity and greater solubility. Among the carbonated apatites,
eggshell derived apatites showed greater curcumin loading
and release. Curcumin loaded samples also exhibited high
anti-inflammatory activity and anti-cancer efficacy in vitro.
These results suggest that carbonated apatite nanocarriers
are a promising choice for local curcumin delivery for
combined anti-cancer, anti-inflammatory and bone repair
activity and can be used as an adjunct treatment for bone
cancer.
Acknowledgment: The authors thank Sophisticated
analytical instruments facility (SAIF), IIT Madras and
Department of Metallurgical and Materials Engineering,
IIT Madras for CHNO-elemental analytical and diffrac-
tometry services respectively.
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Received: 18 August 2018. Accepted: 25 September 2018.
J. Nanosci. Nanotechnol. 19, 6872–6880, 2019