Received: 27 June 2020 Revised: 18 November 2020 Accepted: 28 November 2020

DOI: 10.1002/jbm.a.37135

ORIGINAL ARTICLE

Meloxicam encapsulated nanostructured colloidal

self-assembly for evaluating antitumor and anti-inflammatory
efficacy in 3D printed scaffolds

Nilesh Rarokar1 | Ravikumar C2 | Shailendra Gurav3 | Pramod Khedekar1

1
Department of Pharmaceutical Sciences,
Rashtrasant Tukadoji Maharaj Nagpur Abstract
University, Nagpur, Maharashtra, India Nanostructured colloidal self-assembly (NCS) is one of the most promising drug
2
Department of Chemical Engineering,
delivery carriers in cancer treatment. The present research work aimed towards syn-
Visvesvaraya National Institute of Technology
(VNIT), Nagpur, Maharashtra, India thesizing meloxicam (MLX) loaded NCS for its improved circulation half-life and
3
Department of Pharmacognosy, Goa College increased cellular internalization. NCS was formulated using glyceryl monoolein,
of Pharmacy, Goa University, Panaji, Goa, India
Pluronic® F127, and MLX. Quality by Design experiments with a quadratic model
Correspondence was subjected to optimization of the formulation. The optimized NCS with an aver-
Dr. Nilesh R. Rarokar, Department of
Pharmaceutical Sciences, Rashtrasant Tukadoji age particle size of 185.5 ± 3.02 nm showed higher MLX encapsulation (94.74
Maharaj Nagpur University, Mahatma Jyotiba ± 3.41%) and sustained release behavior of MLX up to 24 hr. in vitro cytotoxicity of
Fuley Shaikshanik Parisar, University Campus,
Nagpur, Maharashtra, India. the developed NCS with MCF-7 and MDA-MB-231 cell lines confirmed lower cell
Email: nileshrarokar@outlook.com viability and a higher rate of cell growth inhibition. This MLX loaded NCS showed

Funding information
Department of Science and Technology,
Science and Engineering Research Board, India,
Grant/Award Number: DST-SERB/
EMR/2016/003320; University Grants
Commission, Grant/Award Number:
201415-RGNF-2014-15-SC-MAH-72973
dual activity as an antitumor and anti-inflammatory in highly invasive estrogen-
dependent MDA-MB-231 cells due to the high expression of cyclooxygenase-2
(COX-2). Besides, an activity of the MLX-NCS was also observed in 3D printed
MCF-7 cells. This investigation shows the possible use of MLX-NCS as an efficient
cancer drug delivery system with excellent colloidal stability, sustained release of
MLX, enhanced antitumor and anti-inflammatory efficacy in 3D printed scaffolds. In
contrast to toxicity study in 2D culture, the 3D constructs revealed the activity of
the MLX via COX-2 independent mechanism and demonstrated that the relationship
between COX-2 expression and antitumor activity of inhibitors is limited. In conclu-
sion, the overall observations and results of this study strengthen the hypothesized
development of NCS as a next-generation therapeutics regimen for cancer therapy.

KEYWORDS

3D printing, colloids, meloxicam, self-assembly, sustained release

1 | I N T RO DU CT I O N delivery carriers offer significant advantages over the conventional

Systemic chemotherapy is an emerging technique used to treat can-
cer. In the last two decades, US-FDA-approved numerous chemother-
apeutic drugs have been used alone or in the combinatorial form.1
The available anti-cancer drugs have their mechanism of action, which
may vary at different drug concentrations and affect different types
of normal and cancerous cells.2 Recently, various nanoparticulate drug
approaches in treating cancer, especially in reducing the toxicity-
related side effects of the chemotherapeutic agents.3,4 The small par-
ticles (<500 nm) can easily penetrate or cross the epithelial cells mem-
brane via the endocytosis mechanism. The large particles with a size
greater than 5 mm enter the cell through the lymphatics.5 Further, it
is reported that the therapeutic agent's passive targeting efficacy
depends on the nanoparticulate carrier size.6

J Biomed Mater Res. 2020;1–16. wileyonlinelibrary.com/journal/jbma © 2020 Wiley Periodicals LLC 1

2 RAROKAR ET AL.
More than a dozen nanocarriers have developed using biodegrad-
able materials for cancer therapeutics, including those made up of
lipids, di-block and tri-block polymers, inorganic metals, and proteins
7,8
and peptides. Among the different drug nanocarriers, lyotropic liq-
uid crystalline lipid nanoparticles (LCNPs), that is, self-assembled
nanocarriers, can encapsulate both hydrophobic and hydrophilic
9
drugs. LCNPs consist of biocompatible food-grade glyceryl
10,11
monooleate, formed by internally well-organized nanostructured
self-assembly of hydrophilic and lipophilic molecules. The emulsifica-
tion of these lipids in excess of water results in lipids' self-assembly in
the dispersion; hence, it is also called nanostructured colloidal self-
assembly (NCS).12-14 However, in this technique, the use of an excess
of water may be one reason for its poor physical instability, which can
be enhanced by adding steric and tri-block copolymers like Pluronic®
15,16
F127 during the emulsification process. Further, the pharmacoki-
netic properties of drugs loaded in the NCS can be fine-tuned by vary-
ing the lipid to surfactant ratio, which in turn alters the size of the
nanocarrier (thus alters the rate of diffusion of NCS in biological
systems), controlled or sustained release of the drugs, and stimuli
responsiveness of the carrier.17,18 Moreover, NCS could be a better
drug delivery carrier than the existed dosage form because of many
reasons. One of those is the mechanism by which it targets the cancer
cells. Self-assembly can be act by two mechanisms, first by pericellular
and second intracellular. The pericellular assembly is responsible for
the blockage of mass transform of cancer cells by cellular apoptosis.
At the same time, intracellular assembly causes cell death or prevent
the drug from cytosolic elimination.
Previously, it has been shown that the oral bioavailability of sim-
vastatin (Biopharmaceutical Classification System [BCS]-class II drug)
could be improved by encapsulating the drug in the lyotropic liquid
crystalline type of bulk cubic phases using glyceryl monoolein (GMO)/
poloxamer 407.19 GMO is generally recognized as safe (GRAS) and
approved by US-Food and Drug Administration. GMO is biocompati-
ble and used to protect sensitive drug molecules from degradation,
for solubility purpose, and to control the release of pharmaceutically
active compounds.20-23
Meloxicam (MLX) is a non-steroidal anti-inflammatory (NSAID)
BCS Class-II drug which selectively inhibits the inducible isoform of
the COX-2 enzyme and proven to treat cancer. COX-2 enzyme acts
as a catalyst for prostaglandin synthesis, and its role in the develop-
ment of cancer has been well studied. It influences apoptosis, angio-
genesis and also involved in the production of carcinogens. Hence, a
24
high concentration of COX-2 is generally found in cancerous cells.
The expression of the COX-2 enzyme in the breast could be one rea-
son for the identification and breast cancer existence. For instance,
breast cancer cell lines such as MDA-MB-231 and Hs578T are highly
expressed with COX-2. On the other hand, the estrogen-dependent
breast cancer (MCF-7) cell line does not express COX-2. The anti-
cancer activity of MLX has studied in HCA-7 colonies for inhibition
and its cytotoxic effect in osteosarcoma (MG-63) and esophageal
25,26
squamous cell carcinoma cell lines. MLX showed improved sur-
vival of lung cancer patients when given in combination with car-
boplatin and paclitaxel. Further, it showed an anti-proliferative effect
in both in vitro and in vivo against prostate cancer (PC3 and DU-145
cell lines), prophylaxis of colorectal cancer, and urinary bladder cancer
(HT1376, T24 and 5637).27
Different formulations of MLX, such as multiple-component crys-
tal formation, recyclable poly D, L-lactic-co-glycolic acid/ polyethylene
glycol (PLGA/PEG)-derivative microspheres with control release have
been prepared so far for the anti-cancer treatment.28-31 However, the
work on developing NCS loaded MLX drug delivery system with
improved pharmacokinetic parameters and better colloidal stability
was not achieved until now. Our objective of the present study is to
engineer and optimize NCS formulation containing MLX for its
improved antitumor and anti-inflammatory activity in 3D printed scaf-
folds. The optimized NCS were characterized to determine the parti-
cle size, polydispersity index, colloidal stability, morphological
structure (by scanning electron microscopy [SEM]), encapsulation effi-
ciency of MLX, in vitro drug release. Further it was subjected to
in vitro cytotoxicity in MCF-7 and MDA-MB-231 cell lines, and its
efficacy in 3D printed MCF-7 cell lines, which has not been performed
previously with MLX drug. The 3D model cultures are important as
they reflect the in vivo environment and further allow the impact of
the extracellular matrix (ECM) to be assessed.32 3D cancer model cell
cultures can demonstrate nanoparticles real behavior in the actual
biological systems.33-35
2 | M A T E R I A L S A N D M ET H O D S
2.1 | Chemical and reagents
Meloxicam (MLX) was received as a gift from Ramdev Chemicals Pvt.
Ltd., Thane, (Maharashtra) India, glyceryl monooleate (GMO) was pur-
chased from Otto Chemie., Mumbai (Maharashtra) India. Pluronic®
F127 (PF127) was procured from Himedia, India. Milli-Q purified water
was used throughout the experiments, previously purified by Millipore®
synergy system (Millipore, Billerica, MA). All other chemicals and
reagents used were of high standard analytical grade.
2.2 | High-performance liquid chromatography
analysis
MLX concentration was measured using high-performance liquid
chromatography (HPLC) method developed in our laboratory on
SPD-M20A Shimadzu pump, LC-20AD Shimadzu with UV–vis detec-
tor at 362 nm (see Supporting Information). The samples were
chromatographed on a 150 × 4.6 mm reverse phase stainless steel
column packed with 5 μm particles (C-8, LiCrosphore® 100, Germany).
A mobile phase consisting of acetonitrile/phosphate buffer 20 mM,
(45:55, vol/vol), pH 3.5 adjusted with o-phosphoric acid was used for
elution at a flow rate of 1 ml/min. The column temperature was
maintained at room temperature. The samples were appropriately
diluted with mobile phase and spiked (20 μl) directly into the injector
of HPLC system with the help of syringe without further treatment.
RAROKAR ET AL.
2.3 | Drug solubility and compatibility study
The solubility of MLX was determined at pH 7.4 in phosphate buffer
saline (PBS) solution, Milli-Q purified water, and different organic sol-
vents as per the modified procedure previously described in the litera-
ture.36 Briefly, about 15 mg as received, MLX was weighed and
transferred to a container containing 5.0 ml water, PBS, or organic sol-
vent and then subject to sonication for 1 hr. The mixture was stirred
using a magnetic stirrer for 48 hr at room temperature. Then, the mix-
ture was filtered (using 0.45 μm Millipore filter) and diluted with the
respective solvent for spectrophotometric analysis at 362 nm. The
average of all three readings was taken.
The interaction between drug and excipient's was determined
using differential scanning calorimetry (DSC) and Fourier transform
infrared (FTIR) spectroscopy to confirm drug and excipients' compatibil-
ity. The drug sample (MLX), polymer (Pluronic® F127), and their physical
mixtures were analyzed by open pan DSC measurements using a DSC
Q20 (TA Instruments Inc., New Castle, DE). The analysis of results was
done using the Universal Analysis software version 4.5A, builds 4.5.0.5
(TA Instruments, Inc.), and then interpreted for confirmation of compati-
bility.37 FTIR spectra of MLX, polymer, and their physical mixtures were
obtained using IRAffinity-1S (Shimadzu Serial Number A219652,
Shimadzu Corporation 00904, Japan). For this, the dried samples of
MLX and polymer were uniformly mixed with FTIR grade potassium
bromide (KBr) in 1:100 proportions and analyzed for 45 scans; the
resolution was set at 4 cm−1 and analyzed from 4,500 to 400 cm−1.
2.4 | Synthesis of NCS
The MLX-loaded NCS was prepared following our previously reported
method38 with a slight modification. Briefly, a mixture of GMO and
PF127 (18:2%wt/wt) was heated to 60
C to get a homogenous mixture.
MLX (0.036–0.636%wt/vol) was added to this mixture in a solution form
by previously dissolving the drug in water and DMSO (1:1). The mixture
was heated at 60
C for 30 min to completely remove DMSO, followed
by the addition of ethanol (3.0%vol/vol). Ethanol, a hydrotropic solvent,
binds the hydrophilic and hydrophobic components and forms a
bicontinuous lipid bilayer. The entire mixture was then continuously
stirred for 2–3 hr at 100 rpm, and the viscous homogenous melt was
injected into excess water to get MLX loaded NCS. The final volume was
made up to 50 ml with Milli-Q water to get NCS colloidal dispersion and
kept overnight at room temperature. Further, the colloidal dispersion was
homogenized using a high-pressure homogenizer (Nano DeBEE, BEE
International, South Easton, MA) at 6000 psi for three cycles of 3 min to
get uniform size NCS and stored in glass bottles for further analysis.
2.5 | Quality by Design for optimization of NCS
formulation
The Quality by Design (QbD) serves as an ideal tool for the design of
experiments and optimization. We used this technique to optimize
3
the NCS formulation following the individual response properties
using DOE software (Design-Expert®, Version 10.0.4.0., Stat-Ease
Inc., Minneapolis, MN). The central composite design of experiment
(CCD) is known for the optimization of several processing parameters
like time, temperature, and rpm.39-41 The formulation variables like
concentration of GMO lipid (X1, %vol/vol), Pluronic® F127 surfactant
(X2, %wt/vol), and MLX drug (X3, %wt/vol) were taken into consider-
ation to study their influence on the final particle size. As suggested
by QbD design with various combinations of GMO, 20 batches of
experiments, Pluronic® F127 and MLX were carried out to optimize
the formulation with the smallest particle NCS size.42,43
Interactions between the polynomial terms were incorporated to
examine the response using a suitable statistical model. The below
Equation (1) was employed to study different parameters and their
effects on the responses.
Y = b0 + b1 X1 + b2 X2 + b3 X3 + b11 X1 2 + b22 X2 2 + b33 X3 2 + b12 X1 X2
ð1Þ
+ b23 X2 X3 + b13 X1 X3
Equation (1) comprises the value of the dependent variable (Y),
the arithmetic mean response of all the 20 runs represented by b0,
and the estimated coefficient for the factor Xi is represented by bi.
The X1, X2, and X3 represent the effect on the responses when one-
factor changes from lower to higher values. X1X2, X2X3, and X1X3 are
the interaction terms that denote the changes in the response with
the change in factors. Non-linearity was investigated using the poly-
nomial terms X12, X22, and X32. Real values of the independent vari-
ables at five levels (−1.681, −1, 0, +1, +1.681) were considered to
study their influence on the particle size. The data depicted in Tables 1
and 2 represents the coded level with real values, and the different
composition of batches was obtained by employing central composite
design, respectively.
2.6 | Physicochemical and functional analysis
of NCS
2.6.1 | Particle size, polydispersity index, and
colloidal stability
The particle size and colloidal stability of blank NCS and MLX-loaded
NCS were measured using dynamic light scattering (DLS, Model:
PSS-NICOMP® 380ZLS, Particle Sizing System, Santa Barbara, CA),
equipped with a 5-mW helium-neon laser with a wavelength of
633 nm. Colloidal stability of the NCS was studied by measuring zeta
potential using Smoluchowski's equation and electrophoretic mobility
of self-assembly was taken into consideration.44 Briefly, about 10 μl
of MLX-loaded NCS was taken in a disposable polystyrene cuvette,
and the final volume was made up to 1 ml using Milli-Q purified water.
The measurements were taken (10–15 runs) at 25
C, at an angle of
90
, and a run time of 2 min 8 s with the count rate of about 498 KHz.
The procedure was repeated thrice for each formulation, and the
mean and SD were determined.
4 RAROKAR ET AL.

T A B L E 1 Coded levels and “real”

Levels
values for each factor under study
Variables −1.681 −1 0 +1 +1.681
Independent Real values
Conc. of lipid (X1, %vol/vol) 12.954 15 18 21 23.045
Conc. of surfactant (X2, %wt/vol) 0.318 1 2 3 3.681
Conc. of drug (X3, %wt/vol) 0.036 0.1 0.3 0.5 0.636
Dependent
Particle size (Y, nm)

TABLE 2 Central composite design formulation batches
Formulation
Sr. no. batches
1 F1
2 F2
3 F3
4 F4
5 F5
6 F6
7 F7
8 F8
9 F9
10 F10
11 F11
equivalent to 10 mg of MLX in 100 ml of methanol by vigorous shak-
ing for 20 min, followed by sonication for 5 min. The solution was fil-
X1: Lipid X2: Surfactant X3: Drug
tered through a membrane filter (pore size 0.45 μ). About 1 ml of
filtrate was diluted up to 10 ml by adding methanol, spiked into HPLC
0 1.681 0
and analyzed spectrophotometrically at 362 nm.
−1 1 −1
1 −1 −1
0 0 0
2.7 | Scanning electron microscopy
0 −1.681 0
0 0 0 Microscopic images of MLX loaded NCS were taken using a SEM
−1 1 1 (Model: JSM-6390LV, Jeol Ltd., Tokyo, Japan). Briefly, a drop of MLX-
−1.681 0 0 loaded NCS formulation was poured onto the glass slide and dried to
1.681 0 0 form a smear. Then, the glass slide was coated with a thin layer of pal-
0 0 0 ladium by auto fine coater (Model: JFC1600, Jeol Ltd.). The prepared
0 0 0 slide was then observed under SEM equipped with a digital camera, at

12 F12 1 −1 1 10 kV accelerating voltage.

13 F13 0 0 1.681
14 F14 1 1 −1
2.8 | In vitro MLX release study
15 F15 −1 −1 1
16 F16 1 1 1
The diffusion of the drug across a cellophane membrane using Franz
17 F17 −1 −1 −1
diffusion cell was measured to estimate the in vitro release of MLX
18 F18 0 0 0 from the MLX-NCS dispersion and study the diffusion mechanism.
19 F19 0 0 −1.681 The formulation was added in the donor compartment separated by
20 F20 0 0 0 previously activated semi-permeable membrane from the receptor
Note: X1, X2 and X3 are independent variables presented in coded value. compartment containing 18 ml pH 7.4 PBS as a release medium. The
temperature was maintained at 37
C ± 0.5
C with continuous stirring
2.6.2 | Entrapment efficiency at 100 rpm on a magnetic stirrer. At pre-decided time intervals, the
aliquots were withdrawn and replenished with the equal volume of
The % entrapment efficiency of MLX-loaded NCS was determined fresh medium. The samples' drug concentrations were then analyzed
using the previously described method.45-47 For this, the HPLC method using HPLC to determine the drug's cumulative % release.
was used to measure the total concentration of MLX (Ct) and the con-
centration of free MLX in the filtrate obtained after centrifugation (Cf).
Entrapment efficiency (%) of MLX-loaded NCS was calculated as below; 2.9 | Cytotoxicity study (MTT assay)

Ct −Cf
Entrapment efficiency ð%Þ = × 100 ð2Þ Human breast adenocarcinoma MCF-7 and MDA-MB-231 cell lines
Ct
were cultured in tissue culture flasks (75 ml) containing Dulbecco's
Modified Eagle's Medium (DMEM) and kept in a CO2 incubator under
2.6.3 | Estimation of MLX drug content 37
C (5% CO2, 95% humidity). Culture media were regularly replaced
with fresh media. The secondary culture was performed with fresh
The MLX content in NCS was determined using by HPLC method. media by culturing more confluated (up to 70–80%) cells. The MTT
The HPLC analysis sample was prepared by dissolving MLX-NCS [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay
RAROKAR ET AL.
is generally used to study cytotoxicity of drug molecules against the
cancer cell lines. Here, we have done a cytotoxicity test to evaluate
the toxicity of MLX-loaded NCS formulations.
The in vitro cell toxicity of MLX-NCS and MLX- DMSO solution
(0.5 mM) was studied with MCF-7 and MDA-MB-231 cell lines for
72 hr. IC50 was determined according to the detailed procedure
39
reported in a previous study using the MTT assay. The cells were
cultured, and about 10,000 cells/well were seeded in 96-well plates
and incubated for 24 hr. The MLX-NCS formulation and MLX-DMSO
solution were diluted with phenol red-free DMEM at different con-
centrations of 0.01, 0.1, 1, 10, 100, 1,000 μg/ml. The culture medium
was replaced with 200 μl of sample solutions at 37
C in an incubator
supplied with CO2 and incubated for 72 hr. After incubation, a previ-
ously added medium was replaced with a fresh medium followed by
50 μl of MTT reagent (1 mg/ml in PBS) to each well. The MTT reagent
containing cells was again kept for incubation for 3 hr at 37
C. Then
the intracellular formazan was solubilized using 160 μl DMSO, and the
media was removed. The absorbance was measured spectroscopically
at 570 nm (SpectraMax M2 with SoftMax Pro; Molecular Devices
Corporation Sunnyvale, CA). Only medium treated cells were used as
controls, and the percentage of cell viability was determined based on
the measurement of comparative absorbance.48
2.10 | In vitro study in 3D printed cells
3D printing technology was used to develop the scaffolds following
the stepwise procedure shown in Figure 1. 3D cell culture model was
developed by spheroid generation by transferring the hydrogel
F I G U R E 1 Schematic representation of the stepwise process of 3D printing showing (1) filling of separate syringes with cells and ink,
(2) mixing of cells and ink to get homogeneous cell-containing hydrogel, and (3) printing of cells on the 3D printer to form 3D printed scaffold
5
printed on a 3D printer by plating 1 × 104 cells/well. The preparation
of hydrogel using acrylamide and bis-acrylamide was followed
according to the standard protocol.49-51 Briefly, the acrylamide and
bis-acrylamide were taken from a stock of 29:1 (wt%) acrylamide:bis-
acrylamide mixture and diluted (1:1) with water or glycerol, tartrazine
(≈0.027 wt%), and 0.1 wt% lithium phenyl-2,4,6 trimethyl benzoyl
phosphinate (LAP). Then, the solution was stored in a light-tight bottle
at 4
C. The solution was first brought to room temperature and
loaded into the extrusion syringe, and then subject to 3D printing.
2.10.1 | 3D printing of MCF-7 cells
The MCF-7 cells were printed using a 3D bioprinter following a step-
wise procedure of cell mixing, bioprinting, and cross-linking to check
the cell viability and cytotoxicity of the prepared MLX-NCS in 3D
scaffolds (Figure 1). Briefly, the 1 ml of MCF-7 cell suspension was
filled in a syringe and mixed with the 3 ml of bioprinting ink (poly
(ε-caprolactone), 25 kDa) by using a cell mixer. The mixture was then
transferred to an empty cartridge and further used for the bioprinting
using a suitable nozzle. The acrylamide-bis-acrylamide hydrogel
loaded extrusion syringes and the cell loaded syringe with nozzles
(0.33 mm ID) were placed and connected to the air system of the bio-
printer. Then, the cells were printed at a speed of 60 mm/min. The
mesh type rectangular design was selected from the CADD design
software, and the cells were printed on to the petri dish. Bioprinted
cells were then transferred to the container containing 2%wt/vol
CaCl2 solution (cross-linking agent) and kept for a few seconds to
allow them to cross-linked with the hydrogel.
6 RAROKAR ET AL.

2.10.2 | Toxicity study
After 72 hr, the relative potency of MLX-NCS was determined against
the cell growth inhibition of the 3D printed MCF-7 cells. Briefly, the
3D printed MCF-7 cells were transferred to a 12-well plate (about
10,000 cells/well) with the fresh medium and incubated 24 hr. Then,
the MLX-NCS formulation was diluted with the phenol red-free
DMEM culture medium to the concentrations of 0.01, 0.1, 1, 10,
100, and 1,000 μM and then added to the plate. The above procedure
was repeated by adding MLX-DMSO solution to the plate as used as
control.
existing peak positions implicates only physical interaction between
MLX and Pluronic® F127 (see Figure 2c).
The interaction between MLX and Pluronic® F127 was further
studied by FTIR spectroscopy. The IR spectrum of standard MLX
exhibits bands at or near (±1) wave numbers of 3,289.76 cm−1 (sec-
ondary amine stretching), 1,619 cm−1 (NH2 scissoring vibrations),
1,550.04 and 1,530.36 cm−1 (C N stretching), 1,346.73, 1,265.88
and 1,183 cm−1 (S O stretching).52 Figure 3a of pure MLX depicts

2.11 | Stability study

The stability of a dosage form is the parameter used to quantify the

shelf-life of the formulations. It gives information about how the
quality of a drug substance or drug product varies at different storage
conditions such as temperature, humidity, and light. The stability
study of selected MLX-NCS formulation was performed as per the
International Council for Harmonisation of Technical Requirements
for Pharmaceuticals for Human Use (ICH) guidelines at room tempera-
ture, elevated temperature (40 ± 2
C) with 75 ± 5% relative humidity
(RH) and freezing temperature. The test parameters, such as phase
separation, particle size, and drug content were conducted over a
period of 3 months at different time intervals.

2.12 | Statistical analysis

All the data were expressed as mean ± SD. The statistical analysis was
carried out by a two-way analysis of variance (ANOVA) followed by
Bonferroni post-test using GraphPad® Prism® software version 5.03
(SNP Diego, CA). The differences between means were considered to
be significant if the p value was <.05.

3 | RESULTS

3.1 | Drug solubility and compatibility

The solubility of MLX in Milli-Q purified water, ethanol, dimethyl

sulfoxide (DMSO), and phosphate buffer saline (pH 7.4) was studied.
MLX was practically insoluble in the Milli-Q purified water, whereas it
is slightly soluble in ethanol and pH 7.4 PBS (0.8 mg/ml). It was
found to be soluble in DMSO (25 mg/ml).
DSC thermogram of pure MLX (Figure 2a) reveals a sharp endo-
thermic peak at 251.94
C, the melting point of MLX. Figure 2b illus-
trates the DSC thermograms of Pluronic® F127, and the appearance F I G U R E 2 Represents differential scanning calorimetry (DSC)
thermogram of MLX (a), Pluronic® F127 (b), and physical mixture of
of a sharp endothermic peak at 58.13
C, is the melting point of
MLX and Pluronic® F127 (c). Thermal behavior of MLX was found to
Pluronic® F127. The thermogram of the physical mixture of MLX and
be unaltered in the presence of Pluronic® F127, which reveals the
Pluronic® F127 (1:1) shows peaks at 247.66 and 57.73
C, respec- compatibility of drug and polymer with devoid of interaction
tively. The absence of any new peak or any large deviations in the between them
RAROKAR ET AL. 7

that all the bands exist as standard MLX bands, as mentioned above.
IR spectra of triblock copolymer (Pluronic® F127) (Figure 3b) show
−1
the identifiable peaks at 840.96, 1,240.23, 1,278.8, 2,235.50 cm .
The IR spectra of the physical mixture (MLX and Pluronic® F127)
show that all the bands of pure MLX and Pluronic® F127 are retained
in the spectrum (see Figure 3c). Further, no shifting in the band posi-
tions or the absence of any new bands in the spectrum 3C confirms
only physical interaction between the MLX and Pluronic® F127 for-
mulation. Thus, both the DSC and FTIR results indicate drug and
excipient's compatibility with only physical interaction, which is a pre-
requisite. Otherwise, the MLX release behavior from NCS would be
altered.

F I G U R E 3 Fourier transform
infrared (FTIR) spectra for MLX (a),
Pluronic® F127 (b), and physical
mixture of MLX and polymers
(c) showing characteristic peaks at
various wavenumbers. Spectrum for
the physical mixture of MLX and
polymers (c) confirms the absence of
significant interaction with retention of
characteristic peaks for MLX and
polymer
8 RAROKAR ET AL.

TABLE 3 Central composite design formulation batches with respect to particle size

Factor 1 Factor 2 Factor 3 Response 1

Sr. no. Batches XA: Conc. of XB: Conc. of XC: Conc. of Particle size (nm)
GMO (%vol/vol) PF127 (%wt/vol) MLX (%wt/vol)
1 F1 15 3 0.5 171.9 ± 2
2 F2 23.04 2 0.3 248.1 ± 5
3 F3 15 1 0.5 189.5 ± 2
4 F4 18 2 0.3 199.3 ± 2
5 F5 18 2 0.3 190.2 ± 3
6 F6 18 2 0.3 192.6 ± 4
7 F7 18 2 0.036 175.7 ± 2
8 F8 21 1 0.5 231 ± 3
9 F9 18 2 0.63 210.4 ± 3
10 F10 18 3.68 0.3 188 ± 3
11 F11 21 1 0.1 216 ± 4
12 F12 15 3 0.1 165.2 ± 2
13 F13 12.95 2 0.3 149.1 ± 3
14 F14 18 2 0.3 183.7 ± 4
15 F15 21 3 0.1 239.4 ± 4
16 F16 18 2 0.3 185.5 ± 3
17 F17 21 3 0.5 241.3 ± 3
18 F18 15 1 0.1 179.6 ± 2
19 F19 18 2 0.3 193.5 ± 3
20 F20 18 0.31 0.3 207.8 ± 3

Note: Values are mean ± SD (n = 3).

XA represents the concentration of GMO (%vol/vol), XB is the concen-

tration of tri-block copolymer Pluronic® F127 (%wt/vol) and XC denotes
the concentration of MLX (%wt/vol) in actual values. Here, we have
taken particle size (Y) as an experimental response represented in the
quadratic equation. The results of particle size (see Figure 4) obtained by
employing experimental design for all the 20 experimental batches were
analyzed with the Design of Experiment software (Design-Expert®, Ver-
sion 10.0.4.0., Stat-Ease, Inc.). It provides useful information on the influ-
ence of formulation parameters on the response (particle size). The
ANOVA results and regression coefficients for each response based on
the 20 runs are summarized in Table 4. The response Y determined from
a quadratic model, written in terms of coded factors, is shown below

Particle Size ðY Þ = + 190:56 + 28:41A− 2:31B + 6:73C + 8:21AB

F I G U R E 4 Illustration of particle size (nm) of 20 different batches
obtained by employing Quality by Design (QbD) with all possible
combination of lipid, surfactant, and drug while bar shown in green
color represents the smallest particle size (≤149 nm) obtained for
nanostructured colloidal self-assembly (NCS) prepared with
formula F13
3.2 | Optimization of MLX-NCS formulation
The quantification of responses against the various MLX-NCS formula-
tion variables for its optimization employing CCD is depicted in Table 3.
+ 0:038AC + 2:04BC + 4:31A2 + 4:07B2 + 2:35C2
ð3Þ
The above equation consists of statistically significant (p < .05)
coefficients, and the positive values represent the positive effect of
parameters on the responses considered in the study design. In con-
trast, the negative values correspond to the inverse relationship
between them. As per the above Equation (3), an increase in the con-
centration of GMO represented by XA (+28.41A) positively influences
the particle size. It is responsible for the proportional increase in the
RAROKAR ET AL. 9

TABLE 4 Analysis of variance (ANOVA) for response surface quadratic model (response particle size)

Source Sum of squares Degrees of freedom Mean square Fcritical p* value R2 value Adjusted R2 value
Regression 12,785.07 9 1,420.56 23.47 .0001 .9548 .9141
Residual 605.39 10 60.54
Lack of fit 443.75 5 88.75 2.75 .0232
Total 13,390.46 19

p ≤ .05.
*

F I G U R E 5 The response surface

plot (a, c) and contour plots (b, d)
based on the particle size (Y) showing
the relationship between the
concentration of GMO and PF127 (a,
b); GMO (X1) and MLX (X2, C, D) and
their effect on particle size (Y1). The
response surface plot (e) and contour
plots (f) based on the particle size (Y)
showing the relationship between the
concentration of MLX and PF127 (a,
b) and their effect on particle size (Y)

particle size of MLX-NCS. However, the concentration of Pluronic®
F127 surfactant represented by XB (−2.31B) shows an inverse rela-
tion and hence the negative effect on the particle size. It means the
®
gradual increase in surfactant concentration, that is, Pluronic
leads to a decrease in particle size MLX-NCS. The ANOVA results of
response (particle size) given in Table 4 shows that the regression
model corresponds to above Equation (3) is found to be significant
(F = 23.47, p < .0001), with a coefficient of regression (R2) .9548 and
F127 the adjusted R2 value was about .9141. The p and F value for lack of
fit was found to be .0232 (p ≤ .05) and 2.75, respectively.
10 RAROKAR ET AL.

T A B L E 5 Comparison of the observed and predicted values in 3.2.1 | Response surface methodology
NCS under predicted optimum conditions

Predicted Observed The effect of various concentrations of GMO, Pluronic® F127, and
Response variable value valuea Bias (%) MLX on the MLX-NCS particle size was studied using response
Particle size (nm) 183 185.5 ± 3 −1.36 surface methodology and by employing QbD based central composite
design of experiments (shown as contour plots, Figure 5).
Abbreviation: NCS, Nanostructured colloidal self-assembly.
a
Value obtained from evaluation of optimized formula using predicted
composition. Relationship between concentrations of GMO and Pluronic® F127
The effect of independent variables (X1 and X2) on the MLX-NCS par-
ticle size (Y) shows that a gradual increase in the GMO concentration
from 15 to 21% leads to an increase in the MLX-NCS particle size.

F I G U R E 6 Particle size distribution for blank nanostructured colloidal self-assembly (NCS) (a) and MLX-loaded NCS (c) measured at 25
C, at
an angle of 90
with dynamic light scattering (DLS) on a PSS-NICOMP® 380ZLS, Particle Sizing System and Zeta potential with respect to total
counts also measured for blank NCS (b) and MLX-loaded NCS (d)

F I G U R E 7 Scanning electron
microscopic images of MLX-loaded
nanostructured colloidal self-assembly
(NCS) showing hexagonal to somewhat oval
shape phases (a), MLX surrounded with the
lipid bilayer, and spherical shape formation
structure (b) with entrapped MLX. The
arrows pointed in (a) and (b) mark the
(1) lipid bilayer of NCS which surrounds the
drug molecule, (2) outer shell of the internal
core where an encapsulated drug is
present, (3) drug molecule in the internal
core of NCS
RAROKAR ET AL. 11

However, an increase in Pluronic® F127 surfactant concentration
from 1 to 3% leads to a decrease in the particle size.39 An increase in
®
the particle size is probably due to the Pluronic F127 forming as a
multilayer on the outer surface of NCS.52 The contour plot (Figure 5b)
gives the optimized particle size at the specific concentration of GMO
and Pluronic® F127 of 18%vol/vol and 2%wt/vol, respectively. At this
optimized concentration, the MLX-NCS particle size obtained is
183 nm.
depicts that the concentration of MLX exerted a positive influence. In
contrast, the concentration of PF127 shows a negative influence on
the particle size. The contour plot (see Figure 5f) depicts that the con-
centration of the respective MLX and PF127 of 0.3%wt/vol and 2%
wt/vol the particle size is about 183 nm.
3.2.2 | Validation of the model

Relationship between concentrations of GMO and MLX
The effect of GMO concentration and MLX on the MLX-NCS particle
size (Y) is shown in Figure 5c. The results clearly show the positive
influence of GMO concentration on the particle size following Equa-
tion (3). The contour plot (Figure 5d) shows the optimized particle size
of 183 nm at a specific concentration of GMOs and MLX of 18%
vol/vol and 0.3%wt/vol, respectively.
Relationship between concentrations of MLX and Pluronic® F127
The effect of MLX and PF127 concentration on the MLX-NCS particle
size is shown as a 3D response surface plot (see Figure 5e), which
Model validation needs to be performed to evaluate the optimiza-
tion capability of the central composite design. MLX-loaded NCS
were prepared using optimized concentration of GMO, PF127 and
MLX of 18%vol/vol, 2%wt/vol and 0.3%wt/vol, respectively. The
model significance was predicted by calculating the % Bias using
Equation (4) shown below. The value of % Bias was found to be
−1.36, which is in the range of ±3.0%, as shown in Table 5. This
result confirms the significance and successful validation of the
model.

F I G U R E 8 Graphical representation of % release of MLX from F I G U R E 1 0 In vitro cytotoxicity of MLX-loaded nanostructured

formulated nanostructured colloidal self-assembly (NCS) and its colloidal self-assembly (NCS) and MLX solution on human breast
solution in DMSO. in vitro release pattern was studied by using the adenocarcinoma cells (MCF-7) after 72 hr in 3D scaffold-based
Franz diffusion cell for 24 hr spheroid culture

F I G U R E 9 Effect of DMSO, blank

nanostructured colloidal self-assembly
(NCS), MLX-loaded NCS, and MLX-
DMSO solution on human breast
adenocarcinoma cells (MCF-7).
(a) in vitro cytotoxicity of DMSO and
blank NCS and (b) MLX-loaded NCS and
MLX solution on human breast
adenocarcinoma cells (MCF-7) were
studied using MTT assay and then cells
were analyzed after 72 hr on
SpectraMax M2. The bars represent
mean ± SD (n = 3). The results are
expressed in percentage of relative
proliferation (a) and percentage of
relative inhibition (b)
12 RAROKAR ET AL.

T A B L E 6 Stability study data for

Sr. no. Stability study parameters Initial After 3 months
MLX-loaded NCS at 40 ± 2
C, 75 ± 5 %
Stability study at 40 ± 2
C, 75 ± 5 %RH RH, room temperature and freezing
1 Phase separation No phase separation No phase separation temperature
2 Drug content (%) 96.25 97.34
3 Particle size (nm) 185.5 ± 3 200.3 ± 4
Stability study at room temperature
4 Phase separation No phase separation No phase separation
5 Drug content (%) 96.25 96.86
6 Particle size (nm) 185.5 ± 3 195.0 ± 3
Stability study at freezing temperature
7 Phase separation No phase separation No phase separation
8 Drug content (%) 96.25 96.0
9 Particle size (nm) 185.5 ± 3 192.65 ± 3

Note: Values are mean ± SD (n = 3).

Predicted value− Observed value

Bias ð%Þ = × 100 ð4Þ about 34% of MLX was released from MLX-loaded NCS in the initial
Predicted value
first hour, then after, a slow release of MLX can be observed,
suggesting the sustained release of MLX from the MLX-NCS, which
continued up to 24 hr. The entire MLX release at the end of 24 hr is
3.3 | Particle size, polydispersity index, and 89.56%. Experiments conducted with pure MLX in the DMSO solvent
colloidal stability showed 98% of MLX diffused within 2 hr.

The mean particle size of blank NCS and MLX-loaded NCS were
found to be 149.1 ± 2.5 nm and 185.5 ± 3.0 nm, respectively
(Figure 6a,c). Moreover, the low polydispersity index values of 0.190
and 0.209 for blank NCS and MLX-loaded NCS indicates a narrow
particle size distribution. The zeta potential of blank NCS and MLX-
loaded NCS were found to be −39.48 ± 2.8 mV and −36.82
± 13.6 mV, respectively (Figure 6b,d).
3.4 | Entrapment efficiency and drug content
The entrapment efficiency of the MLX in NCS was determined to be
94.74 ± 3.4%, indicating a large amount of MLX was successfully
encapsulated in NCS.
3.7 | In vitro cytotoxicity study (MTT assay)
The cytotoxicity of pure MLX and MLX loaded NCS with MCF-7 cells
is shown in Figure 9. The relative cell proliferation (cell grows and
multiplies) in PBS (control), DMSO, and blank NCS (no MLX loaded) is
more than 90%, which suggests the non-toxic nature of these com-
pounds (Figure 9a). The results of Figure 9b shows that the % relative
inhibition of MCF-7 cells was significantly higher in the case of MLX-
loaded NCS than pure MLX dissolved in DMSO at the concentrations
of 0.01, 0.1, 1, 10, 100, 1,000 μg/ml. The IC50 (50% inhibition) values
of MLX-loaded NCS and pure MLX in DMSO are found to be 3.2 and
5.1 μM, respectively, after 72 hr.

3.8 | Growth inhibition of COX-2 expressing

3.5 | Scanning electron microscopy breast cancer cells using MLX-NCS

The scanning electron micrographs of MLX loaded NCS were taken
using SEM (Model: JSM-6390LV, Jeol Ltd.). Figure 7a,b show that
MLX-loaded NCS is present in the dispersion in the form of both hex-
agonal and oval shapes.
3.6 | In vitro MLX release study
The percent release of MLX from MLX-loaded NCS was determined
using Franz diffusion cell, shown in Figure 8. We can observe that
Breast cancer MDA-MB-231 cells and MCF-7 cells were treated with
different concentrations (10 μM to 200 μM) of MLX-NCS for 24 hr,
and their effect on cell proliferation was observed. At the end of
24 hr only 20% of cells were viable after incubation of MLX-NCS
equivalent to 200 μM of meloxicam in MDA-MB-231 cells. Whereas
more than 50% of MCF-7 cells were found to be viable at the end of
24 hr after incubation with MLX-NCS equivalent to 200 μM of MLX.
The results show that MLX-NCS inhibited cell proliferation and
showed more relative inhibition in MDA-MB-231 cells than the
MCF-7 cell line.
RAROKAR ET AL.
3.9 | Estimation of antitumor efficacy in 3D
scaffold MCF-7 cell lines
Figure 10 illustrates the in vitro cytotoxicity of MLX-loaded NCS and
pure MLX in DMSO on 3D scaffold spheroid MCF-7 cells. Both forms
of MLX show increasing cell inhibition with respect to an increase in
drug concentration. However, comparatively, the % relative inhibition
efficiency of MLX-NCS in a 2D model containing MCF-7 cancerous
cell lines shows a decrease in inhibition. About 70% of cells were via-
ble at the end of 24 hr, which was reduced to 60% after 72 hr. It
means that the scaffolds formed by the cells after 3D printing has
been more resistant and less responsive to chemotherapeutic agents
than the 2D cultured cells (see Figures 9 and 10).
3.10 | Stability study
Stability analysis of MLX-NCS formulation is necessary to determine
the quality of the NCS under the influence of various environmental
factors. The stability study results of optimized MLX-loaded NCS for-
mulation were investigated for 3 months as per ICH guidelines at 40
± 2
C, 75 ± 5% RH, room temperature, and in freezing temperature as
quoted in Table 6. No appreciable change in the size of MLX-NCS at
all the above conditions suggests that MLX-NCS is stable and can be
used as a therapeutic agent for about 3 months.
4 | DISCUSSION
MLX-loaded NCS with different concentrations of MLX (0.3–0.6%wt/
vol) were successfully prepared using GMO (18%wt/wt), PF127 (2%
wt/wt) via high pressure homogenization method. The optimized
MLX-NCS formulation parameters were determined using employing
QBD based central composite design of experiments. For this, 20 dif-
ferent experimental batches with different possible combinations of
GMO, PF127, and MLX were prepared, and the responses were com-
pared with the experimental particle size. The results revealed a posi-
tive influence of GMO and MLX concentration on the MLX-NCS
particle size, whereas the negative effect of surfactant concentration
on the particle size. ANOVA test results confirmed that the model is
significant as the p value is less than .05.53
The results obtained in the solubility study of MLX confirm the
hydrophobic nature of the drug. The hydrophobicity of therapeutic
agents always restricts its efficacy by limiting its bioavailability due to
insufficient release. Hence, there is a need to develop a formulation
that overcomes these problems associated with chemotherapeutic
agents clinical use. DSC and FTIR studies indicated the compatibility
of drugs and excipient's without any interaction between MLX and
PF127. The nanocarrier system's particle size always has been an
essential parameter as it influences the release of the encapsulated
drug. The particle size of MLX-NCS with respect to MLX loading
showed only a marginal increase in the mean particle size. MLX-NCS
shows particle size in the nanometer range of less than 200 nm.54 The
13
zeta potential value of MLX-NCS showed more than ±30 mV, which
is a good sign of high colloidal stability. It will improve the application
of NCS due to self-assembly behavior and mutual interactions of
nanoparticles at fluid interfaces.55 The stability analysis of MLX-NCS
at 40 ± 2
C, 75 ± 5 %RH, room temperature, and freezing tempera-
ture showed that MLX-NCS were stable for about 3 months without
any appreciable change in particle size at these conditions. The use of
lipids and polymer/surfactants during the preparation of nanoparticles
could form the lipid bilayer and coating surfactant over the particles.
It will also lead to the formation of the electrical double layer when
two different surfaces come closer and try to acquire the space on
the surface of other particles that could be balanced by the oppositely
charged ions in the solution.56 The microscopical study revealed the
formation of hexagonal and somewhat spherical shape MLX-loaded
NCS formation with scale bar showing the formation of NCS with size
in the nanometer range.57
In this work, we have examined the mechanism of release of the
drug after incorporation into the complex internal domains of NCS,
which follows facilitate diffusion sustained release of the drug. in vitro
release study confirmed a burst release of MLX initially due to the
weakly bounded MLX on the surface of the NCS, to achieve the mini-
mum therapeutic concentration required for the desired therapeutic
effect, followed by sustained release of MLX up to 24 hr.
The lipid forms the lipid bilayer, which protects the encapsulated
drug molecule from metabolism and avoids the early elimination from
the body. Hence the drug retained during the circulation.58,59 They
can also be coated with polymers. Previous literature mentioned the
prolongation of circulation half-life of the drug due to the encapsula-
tion in lipid-based nanocarriers. In nanoparticle literature, circulation
half-life is the commonly used term for terminal half-life.60 Therefore
we have used the term circulation half-life. Moreover, many other
kinds of literature stated the longer circulation half-life due to charged
nanoparticles, which can be assessed by zeta potential analysis. Some
literature shows 2.6-folds longer apparent circulation half-life of drug-
loaded nanoparticles than the pure drug.61 Here in this study, we have
evaluated the NCS for zeta potential, which demonstrated the charge
on the NCS. Hence the overall observations and the basis of litera-
ture, we can say that the developed NCS may have the ability to
improve circulation half-life.
The anti-proliferative effect of MLX-loaded NCS at various con-
centrations on MCF-7 was assessed using the MTT assay. Phosphate
buffer saline solution, DMSO, and blank NCS showed no toxicity to
the MCF-7 cells upon the treatment. However, MLX in-DMSO and
MLX-loaded NCS show significant toxicity with increased relative
inhibition with an increase in MLX-NCS concentration. The cell toxic-
ity study results revealed that relative inhibition (%) of MCF-7 cells
was more significant for MLX-loaded NCS than pure MLX dissolved in
DMSO due to the sustained release of MLX62,63 from NCS.
The prepared MLX-NCS showed dual activity of antitumor and
anti-inflammatory of MLX in highly invasive estrogen-dependent
MDA-MB-231 cells because of the high expression of COX-2, and
also showed enhanced antitumor activity in MCF-7 cells. It could be
correlated with the previous findings.64-66 The 3D culture model is
14
the best way to evaluate the efficacy of drug-loaded formulation67
and the impact of the ECM in 3D scaffold cells.68 3D culture model is
a chemically defined, highly porous (90%) 3D scaffold, and recovery
from 3D alginate scaffold is achieved using dissolving buffer, a non-
enzymatic solution. It helps to dissolves the scaffold within a few
minutes but leaves the cellular aggregates intact for further processing
and/or analysis of long-term viability, cellular toxicity measurements,
and ultra-structural analysis. Here, 3D scaffold-based spheroid con-
taining MCF-7 cells incubated with MLX-loaded NCS for 5 days
showed higher % relative cell inhibition than cells incubated with pure
MLX dissolved in DMSO. The cell inhibition study in 2D and 3D cell
models revealed the extent of efficacy of MLX-NCS. In the 2D model,
cells are arranged in a single layer and are easily affected by the drug
molecules in the formulation and show more growth inhibition.
Whereas, a similar type of study in 3D scaffolds shows lower efficacy
of the same drug-loaded formulations. These three-dimensional con-
structs reflect the in vivo environment and allow the ECM, which
could not be considered in the cells' 2D model. Hence the study in 3D
in vitro models gives results that correspond to human clinical trials.
Our study's main objective was to demonstrate the antitumor effi-
cacy of MLX by developing the self-assembly as its antitumor activity
was already proven and reported in previous literature. In the present
study, an antitumor and toxicity study in both the cell lines (MCF-7 and
MDA-MB-231) using the 2D model showed lower toxicity and higher
cell growth inhibition and viability. The MDA-MB-231 cell line shows
promising results with an enhanced anti-proliferative effect than
MCF-7 cells. The expression of COX-2 inhibition in MDA-MB-231 may
be the reason for enhanced activity as MLX acts via the COX-2 inhibi-
tion mechanism. However, MCF-7 cells did not follow the expected
hypothesis when studied in the 2D model, and the reason behind the
lower activity was also un-cleared. Hence, there was a need to study
MCF-7 cells further to understand the mechanism of MLX and demon-
strate the behavior of MLX in the in vivo environment. The toxicity
study's overall findings in the 3D printed scaffolds using MCF-7 rev-
ealed lower activity and lower cell growth inhibition. It confirms the
effectiveness of MLX when it acts through the COX-2 inhibition mech-
anism. In contrast, other types of cells devoid of COX-2 may not be
suitable to get the desired antitumor activity of MLX.
On the other hand, the activity shown during the toxicity in 3D con-
structs revealed that the MLX might act via COX-2 independent mecha-
nism.69,70 Similar types of results were previously reported in the
literature,71 and these findings suggested the COX-2 independent mech-
anism of meloxicam.72,73 Although the COX-2 expression is important
for antitumor activity, and it was reported in the literature, but many of
the studies published stated that COX-2 inhibitors do not require the
presence of COX-2 for antitumor activity. Hence the relationship
between COX-2 expression and antitumor activity of inhibitors is limited.
5 | C O N CL U S I O N
The present study results proved that MLX-loaded NCS has the poten-
tial for its use in sustained delivery of drug improved the circulation
RAROKAR ET AL.
half-life. Further, the approach present here can be used to engineer
the other chemotherapeutic agents by encapsulating in NCS, which can
serve as a next-generation therapeutics regimen for cancer therapy.
ACKNOWLEDG MENTS
We thank Dr. Vivek Dave, associate professor, St. John Fischer Col-
lege, Wegmans School of Pharmacy, United States, to carry out DSC
analysis.
CONFLIC T OF INT ER E ST
The authors declared no conflict of interest and financial interest
related to any product come out of this study.
DATA AVAILABILITY STAT EMEN T
The data that supports the findings of this study are available in the
supplementary material of this article.
OR CID
Nilesh Rarokar https://orcid.org/0000-0003-3401-7997
Ravikumar C https://orcid.org/0000-0003-4572-8997
Shailendra Gurav https://orcid.org/0000-0001-5564-2121
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SUPPORTING INF ORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Rarokar N, C R, Gurav S, Khedekar P.
Meloxicam encapsulated nanostructured colloidal self-
assembly for evaluating antitumor and anti-inflammatory
efficacy in 3D printed scaffolds. J Biomed Mater Res. 2020;
1–16. https://doi.org/10.1002/jbm.a.37135