of drug substances is currently a hot topic in the pharmaceutical field, in
which new therapeutic opportunities are being discovered for already existing
drugs [1,2], to minimize the time required for drug discovery and development,
in addition to reduction of the associated costs. Meloxicam is a well-known non
steroidal anti-inflammatory drug, categorized as selective COX-2 inhibitor. It
was recently discovered that meloxicam could act as an antitumor agent,
especially in colon cancer [3,4], owing to the involvement of COX-2 in the
regulation of colon tumor growth. Therefore, meloxicam was reported to be an
effective anticancer drug against colon cancer cells expressing COX-2 such as
HCA-7 and Moser-S cells, but was rather ineffective against COX-2 negative
cells as HCT-116 . Therefore, in an attempt to increase the effectiveness of
meloxicam against non COX-2 expressing cells, its formulation in a
nanoparticulate carrier was attempted in the current work.
nanoparticles were reported to increase the anticancer activity of drugs [5-7],
owing to their ability to enhance cellular internalization by virtue of their
small size . Among the promising nanoparticles are nanoemulsions, which are
composed of oil, water and surfactant/cosurfactant mixture [9-11], and can be
prepared as an isotropic extremely small sized (from 10-100 nm) delivery system
using the water dilution method (termed microemulsions) , and hence can be
better uptaken by cancer cells. A water dilutable nanoemulsion based on oleic
acid as the oily phase and tween 20 as surfactant was prepared by Deng et al.,
2015 , and to the authors’ knowledge, its efficacy was only tested when
loaded with antibiotics . Therefore, in the current manuscript, a very
small sized nanoemulsion was prepared and loaded with meloxicam, in order to
test its ability to potentiate the cytotoxic activity of meloxicam against non
responsive cycloxygenase negative cell line HCT 116.
Oleic acid, tween 20,
absolute ethanol, disodium hydrogen phosphate, potassium dihydrogen phosphate,
dimethylsulfoxide, MTT dye and dialysis membrane (molecular weight cut off
12000-14000) were purchased from Sigma Aldrich Co., USA. Meloxicam was kindly
obtained by Hikma pharmaceutical company, Jordan. Fetal bovine serum, DMEM, RPMI-1640, HEPES
buffer solution, L-glutamine, gentamycin and 0.25% trypsin-EDTA were purchased
from Lonza, Belgium. HCT-116 cancer cells were obtained from the American type
culture collection (ATCC, USA).
Preparation of meloxicam
meloxicam-loaded nanoemulsion was prepared using the water titration method
[9,10], in which 10 mg meloxicam was dispersed in a mixture composed of 4.1 ml
tween 20, 0.28 ml oleic acid and 0.32 ml ethanol, and stirred using a magnetic
stirrer. The mixture was titrated up to 10 gram water dropwise till the
formation of an oil in water nanoemulsion loaded with meloxicam.
Determination of the particle size, polydispersity index and zeta
potential of meloxicam nanoemulsion
particle size, zeta potential and polydispersity index (PDI) of the prepared
meloxicam nanoemulsion was measured using the Zetasizer device (model ZS3600,
using transmission electron microscopy
The prepared meloxicam nanoemulsion was
visualized using transmission electron microscopy without staining, after being
dried on a carbon-coated grid (VERSA 3D, USA).
In vitro release
of meloxicam from the prepared nanoemulsion
The release of meloxicam from the nanoemulsion
was performed using a dialysis-based method [15-17]. One ml of meloxicam
nanoemulsion was placed in a cylinder of a length eight cm and radius five cm,
and attached to the shaft of USP dissolution apparatus (Pharma Test, Germany).
The shaft was rotated at 50 rpm and 37°C, and the release medium was 200 ml
phosphate buffer pH 7.4 containing 2% tween 20 to ensure sink conditions for
meloxicam. Two ml samples were taken from the release medium at definite time
intervals (0.25, 0.5, 1, 2, 3, 4, 6, 24 hours), and the amount of meloxicam
released was measured spectrophotometrically at wavelength 269 nm (SPUV UV/VIS
double beam spectrophotometer, SCO TECH, Germany)
Assessment of the stability
of meloxicam nanoemulsion
The properties of the nanoemulsion (particle size,
PDI and zeta potential) were re-measured after 3 months storage at room
temperature, to assess the stability of the prepared formulation .
Evaluation of the
cytotoxicity of meloxicam and the nanoemulsion in HCT-116 cancer cell line
HCT-116 cells were grown on RPMI-1640
medium containing 10% inactivated fetal calf serum and 50µg/ml gentamycin at
37ºC in a humidified atmosphere with 5% CO2, and were subcultured twice to
three times every week. When properly grown, cells were placed in culture medium
at a concentration of 50000 cells per well (Corning® 96-well tissue culture
plates) then incubated for twenty four hours. Either meloxicam alone dissolved
in 0.5% dimethyl sulfoxide, or meloxicam nanoemulsion was added to the cells at
different concentrations, in addition to vehicle controls with media or 0.5 %
DMSO. The cell viability was assessed after 24 hour incubation, in which the
media was substituted with another fresh media containing MTT dye followed by
incubation for 4 hours and further addition of dimethyl sulfoxide. The optical density was measured at 590 nm
with a microplate reader (SunRise, TECAN Inc, USA) to calculate cellular viability according to the
following equation :
Viability % =
in which ODt and
ODc is the optical density of the treated and untreated cells respectively. The
IC50 value (the concentration causing 50% cellular death) was
Measurements were done in triplicate
and reported as mean±S.D. T-test was performed using Graphpad® Instat software,
at significance of P=0.05. The IC50 values were calculated using
Graphpad Prism software (San Diego, CA. USA)
RESULTS AND DISCUSSION
Measurement of particle size,
PDI and zeta potential of meloxicam nanoemulsion
As shown in Figure 1, the meloxicam
nanoemulsion displayed a particle size of 11.01±0.25 nm, a PDI value of
0.278±0.01 and a zeta potential value of 0.08 mV. Owing to the surfactant and
cosurfactant content of nanoemulsions prepared by the water dilution method,
they are known to exhibit very small particle size . The low polydispersity
of the nanoemulsion indicates its homogeneity, and the effective solubilization
of meloxicam within the oily phase of the nanoemulsion. The almost neutral charge
on the particles of the nanoemulsion is attributed to the non-ionic nature of
the surfactant constituting the majority of the formulation.
Figure 1: The particle size, PDI and zeta potential values of
meloxicam nanoemulsion either freshly prepared or after storage.
Figure 2: Transmission electron microscopy picture of the prepared
Figure 3: Cumulative percent released of meloxicam for 24 hours from
the nanoemulsion formulation.
Morphological examination of
the nanoemulsion using transmission electron microscopy
As shown in Figure 2, the meloxicam
nanoemulsion displayed homogenous non-aggregated spherical droplets, confirming
the small size obtained with the Zetasizer.
In vitro release
of meloxicam from the nanoemulsion
As seen from Figure 3, a sustained release of
meloxicam was achieved over 24 hours from the nanoemulsion, reaching a
percentage of 86% after 24 hours. The sustained release
property of the nanoemulsion is advantageous for cancer treatment, since after
internalization of the particles in the cancer cells, meloxicam is expected to
be released in a continuous manner over time.
Stability of meloxicam
As shown in Figure 1, no significant changes
occurred to the particle size, zeta potential or PDI of the meloxicam
nanoemulsion after storage for 3 months (P>0.05), suggesting the stable
nature of the prepared nanoemulsion.
Table 1: HCT-116 cancer cell viability percentage obtained as a function of
concentration for meloxicam and meloxicam nanoemulsion
Sample conc. (µg/ml)
Cell viability %
for meloxicam nanoemulsion
Cell viability %
Evaluation of cytotoxicity of
meloxicam nanoemulsion in HCT-116 cancer cell line
The cytotoxicity of meloxicam nanoemulsion in
comparison with meloxicam drug was compared. As shown in Table 1, meloxicam did
not exhibit any cytotoxic action on the cells at all tested concentrations, and
its IC50 value could not be determined. On the other hand, its
inclusion in the nanoemulsion form resulted in significant decrease in the
cellular viability, resulting in an IC50 value of 4.08±0.4 µg/ml.
The non-cytotoxic effect of meloxicam as free form on the cells came in
accordance with other authors , and they attributed this to their non
expression of COX-2. This suggests the suitability of our prepared system in
enhancing cellular uptake of drugs especially NSAIDs and allowing them to
function as anticancer molecules in a non-COX dependant pathway such as
apoptosis induction, as similarly encountered by other authors working on
meloxicam . The superiority of nanoemulsions could be related to their
small size, which allow better cellular uptake, and hence enhanced anticancer
activity . Interestingly, when meloxicam was loaded in chitosan
nanoparticles, an IC50 value could not be obtained for neither
meloxicam nor the prepared nanoparticles on HT29 cancer cells , suggesting
that the proper selection of nanocarrier is crucial to achieve good anticancer
activity of the drug.
New indications are being discovered
for both drugs and nanoparticles all the time. In the current work, it was
proven that the nanoencapsulation of a drug as meloxicam would result in
alteration of its mechanistic therapeutic effect. More futuristic studies are
required to delineate the exact anticancer mechanisms of meloxicam in the
Declaration of interest
The authors report no conflict of
1. Doan TL, Pollastri M,
Walters MA, Georg GI. The future of drug repositioning. Old drugs, new
opportunities. Annu Rep Med Chem 2011; 46:385-401.
2. Hatem S, Nasr M, Moftah NH, Ragai MH, Geneidi AS, Elkheshen SA.
Clinical cosmeceutical repurposing of melatonin in androgenic alopecia using
nanostructured lipid carriers prepared with antioxidant oils. Expert Opin Drug
Deliv 2018; 15:927-35.
3. Goldman AP, Williams CS,
Sheng H, et al. Meloxicam inhibits the growth of colorectal cancer cells.
Carcinogenesis 1998; 19:2195-9.
4. Sengel-Turk CT, Hascicek C,
Dogan AL, Esendagli G, Guc D, Gonul N. Preparation and in vitro evaluation of
meloxicam loaded PLGA nanoparticles on HT-29 human colon adenocarcinoma cells.
Drug Dev Ind Pharm 2012; 38:1107-16.
5. Aldalaen S, El-Gogary RI, Nasr M. Fabrication of
rosuvastatin-loaded polymeric nanocapsules: a promising modality for treating
hepatic cancer delineated by apoptotic and cell cycle arrest assessment. Drug
Dev Ind Pharm 2019; 45:55-62.
6. Fadel M, Kassab K, Abd El Fadeel DA, Nasr M, El Ghoubary NM.
Comparative enhancement of curcumin cytotoxic photodynamic activity by
nanoliposomes and gold nanoparticles with pharmacological appraisal in Hep G2
cancer cells and Erlich solid tumor model. Drug Dev Ind Pharm 2018; 44:1809-16.
7. Said-Elbahr R, Nasr M, Alhnan MA, Taha I, Sammour O. Nebulizable
colloidal nanoparticles co-encapsulating a COX-2 inhibitor and a herbal
compound for treatment of lung cancer. Eur J Pharm Biopharm 2016; 103:1-12.
8. Ramzy L, Nasr M, Metwally AA, Awad GAS. Cancer nanotheranostics: A
review of the role of conjugated ligands for overexpressed receptors. Eur J
Pharm Sci 2017; 104:273-92.
9. Nasr M, Abdel-Hamid S, Moftah NH, Fadel M, Alyoussef AA. Jojoba oil
soft colloidal nanocarrier of a synthetic retinoid: preparation,
characterization and clinical efficacy in psoriatic patients. Curr Drug Deliv
10. Nasr M, Abdel-Hamid S. Optimizing the dermal accumulation of a
tazarotene microemulsion using skin deposition modeling. Drug Dev Ind Pharm
11. Nasr M. Development of an optimized hyaluronic acid-based lipidic
nanoemulsion co-encapsulating two polyphenols for nose to brain delivery. Drug
Deliv 2016; 23:1444-52.
12. Ramez SA, Soliman MM, Fadel M, et al. Novel methotrexate soft
nanocarrier/fractional erbium YAG laser combination for clinical treatment of
plaque psoriasis. Artif Cells Nanomed Biotechnol 2018; 46:996-1002.
13. Deng L, Que F, Wei H, Xu G, Dong X, Zhang H. Solubilization of tea
seed oil in a food-grade water-dilutable microemulsion. PLoS One 2015;
14. Mohamed MA, Nasr M, Elkhatib WF, Eltayeb WN. In vitro evaluation
of antimicrobial activity and cytotoxicity of different nanobiotics targeting
multidrug resistant and biofilm forming Staphylococci. Biomed Res Int 2018;
15. Nasr M, Mansour S, Mortada ND, Elshamy AA. Vesicular aceclofenac
systems: a comparative study between liposomes and niosomes. J Microencapsul
16. Nasr M, Mansour S, Mortada ND, El Shamy AA. Lipospheres as
carriers for topical delivery of aceclofenac: preparation, characterization and
in vivo evaluation. AAPS PharmSciTech 2008; 9:154-62.
17. Ashraf O, Nasr M, Nebsen M, Said AMA, Sammour O. In vitro
stabilization and in vivo improvement of ocular pharmacokinetics of the
multi-therapeutic agent baicalin: Delineating the most suitable vesicular
systems. Int J Pharm 2018; 539:83-94.
18. Mouez MA, Nasr M, Abdel-Mottaleb M, Geneidi AS, Mansour S.
Composite chitosan-transfersomal vesicles for improved transnasal permeation
and bioavailability of verapamil. Int J Biol Macromol 2016; 93:591-9.
19. Naruse T, Nishida Y, Hosono K, Ishiguro N. Meloxicam inhibits
osteosarcoma growth, invasiveness and metastasis by COX-2 dependent and
independent routes. Carcinogenesis 2006; 27:584-92
20. Venu K, Mondal S, Mondal P. Preparation and investigation of
cytotoxic activity of meloxicam loaded chitosan nanoparticles in HT29 colon.
Saudi J Med Pharm Sci 2018; 4:270-7.