POTENTIATION OF THE CYTOTOXIC ACTIVITY OF MELOXICAM AGAINST COX-2 NEGATIVE COLON CANCER CELLS BY NANOPARTICULATE ENCAPSULATION
Areeg Awadallah1, Rawan Al-Karaki1, Maha Nasr2*
1. Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Mutah University, Jordan 2. Department of Pharmaceutics and Industrial pharmacy , Faculty of Pharmacy, Ain Shams University, Cairo, Egypt
Keywords: Meloxicam, nanoemulsion, colon cancer, HCT-116 cells, COX-2.
Abstract

Objective: The aim of the current work was to elucidate whether the nanoencapsulation of meloxicam would allow its exertion of anticancer activity on colon cancer cells despite lacking COX-2 expression. Methods: Meloxicam nanoemulsion was prepared using the water dilution method, in which oleic acid was chosen as the oily phase, tween 20 as surfactant and ethanol as cosurfactant. The nanoemulsion was characterized in terms of particle size, zeta potential, polydispersity index, morphology, in vitro drug release, stability and anticancer activity on HCT-116 colon cancer cells. Results: It was shown that meloxicam nanoemulsion was successfully prepared with a particle size of 11 nm, polydispersity index of 0.278 and a neutral charge, and it was able to sustain the release of drug for 24 hours with a cumulative percent released of 86%. Its spherical morphology was confirmed using transmission microscopy. No significant changes in particle size, polydispersity or charge were observed upon storage, suggesting the stability of the nanosystem. Moreover, the nanoemulsion exhibit significant cytotoxicity on HCT-116 compared to non-encapsulated meloxicam, suggesting that the prepared nanoemulsion was a successful delivery carrier for meloxicam, which is capable of potentiating its anticancer activity in a non COX-2 dependant manner.

Article Information

Identifiers and Pagination:
Year:2019
Volume:11
First Page:9
Last Page:17
Publisher Id:JAppPharm (2019 ). 11. 9-17
Article History:
Received:March 10, 2019
Accepted:May 17, 2019
Collection year:2019
First Published:May 20, 2019

INTRODUCTION

Re-profiling 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 [3]. 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.

Several 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 [8]. 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) [12], 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 [13], and to the authors’ knowledge, its efficacy was only tested when loaded with antibiotics [14]. 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.

 

MATERIALS AND METHODS

Materials

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 nanoemulsion

The 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

The particle size, zeta potential and polydispersity index (PDI) of the prepared meloxicam nanoemulsion was measured using the Zetasizer device (model ZS3600, Malvern, UK).

Morphological examination 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 [18].

 

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 [17]:

Viability % =   X 100%

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 calculated.

Statistical analysis

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 [14]. 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 meloxicam nanoemulsion.

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 nanoemulsion

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 %

for meloxicam

500

2.75±0.13

74.95±1.74

250

5.63±0.45

90.73±0.51

125

11.29±0.97

98.14±0.28

62.5

20.78±0.36

100

31.25

28.47±0.51

100

15.6

35.61±0.83

100

7.8

42.85±0.79

100

3.9

50.34±1.42

100

2

62.96±.64

100

1

75.13±0.35

100

0.5

82.97±0.11

100

0.25

89.56±0.28

100

0

100

100

 

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 [3], 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 [19]. The superiority of nanoemulsions could be related to their small size, which allow better cellular uptake, and hence enhanced anticancer activity [8]. 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 [20], suggesting that the proper selection of nanocarrier is crucial to achieve good anticancer activity of the drug.

 

CONCLUSION

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 nanoemulsion form.

Declaration of interest

The authors report no conflict of interest

 

REFERENCES

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 2017; 14:426-32.

10. Nasr M, Abdel-Hamid S. Optimizing the dermal accumulation of a tazarotene microemulsion using skin deposition modeling. Drug Dev Ind Pharm 2016; 42:636-43.

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; 10:e0127291.

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; 2018:7658238.

15. Nasr M, Mansour S, Mortada ND, Elshamy AA. Vesicular aceclofenac systems: a comparative study between liposomes and niosomes. J Microencapsul 2008; 25:499-512.

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.


© 2016 The Author(s). This open access article is distributed under a Creative Commons Attribution (CC-BY) 4.0 license. You are free to: Share — copy and redistribute the material in any medium or format Adapt — remix, transform, and build upon the material for any purpose, even commercially. The licensor cannot revoke these freedoms as long as you follow the license terms. Under the following terms: Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. No additional restrictions You may not apply legal terms or technological measures that legally restrict others from doing anything the license permits
Editor in Chief
Prof. Dr. Cornelia M. Keck (Philipps-Universität Marburg)
Marburg, Germany

Bibliography

Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

Journal Highlights
Abbreviation: J App Pharm
doi: http://dx.doi.org/10.21065/19204159
Frequency: Annual 
Current Volume: 9 (2017)
Next scheduled volume: December, 2018 (Volume 10)
Back volumes: 1-9
Starting year: 2009
Nature: Online 
Submission: Online  
Language: English

Subject & Scope
  • Pharmaceutics
  • Physical Pharmacy 
  • Dosage Forms Science 
  • Pharmaceutical Microbiology & Immunology 
  • Industrial Pharmacy 
  • Bio-Pharmaceutics 
  • Pharmaceutical Chemistry 
  • Pharmaceutical Instrumentation 
  • Medicinal Chemistry 
  • Pharmacognosy 
  • Physiology &Histology 
  • Anatomy & Pathology 
  • Pharmacology & Therapeutics 
  • Pharmacy Practice 
  • Pharmaceutical Mathematics   
  • Biostatistics 
  • Dispensing 
  • Community Social & Administrative Pharmacy 
  • Hospital Pharmacy 
  • Clinical Pharmacy 
  • Pharmaceutical Quality Management 
  • Forensic Pharmacy 
  • Pharmaceutical Technology 
  • Pharmaceutical Management & Marketing

Consortium Publisher is an online publisher that enjoys global presence with International Journals

Follow Us

©2009 - 2019 Consortium Publisher Canada

Contact Info

6252 Lisgar Dr Mississauga Ontario L5N7V2 Canada
+1 (647) 526-0885
office@consortiumpublisher.ca
http://www.consortiumpublisher.ca/