Preparation and Characterization of Celecoxib-Conjugated Polyethylenimine as a Potential Nanocarrier for Gene Delivery

Document Type : Research(Original) Article



The objective of the present investigation was to conjugate celecoxib as a cyclooxygenase-2 (COX-2) inhibitor onto polyethylenimine (PEI) in order to prepare nanoparticles for tissue targeting. Since celecoxib binds to COX-2 and this enzyme is over expressed in several pathological conditions including cancer, the final goal of the study was to direct the nanoparticles into specific tissues. Celecoxib was conjugated on PEI structure at two substitution degrees of 5 and 10 % and the new conjugates were characterized in terms of size, zeta potential, buffering capacity, plasmid DNA binding affinity and protection against enzymatic degradation as well as cytotoxicity. The results demonstrated the ability of the PEI conjugates in the formation of nanoparticles with the size of around 200 nm with buffering capacity comparable with unmodified PEI. The celecoxib conjugated PEI derivatives demonstrated high binding affinity to pDNA and protection effect against degradation by DNase I. The conjugation of celecoxib onto PEI structure slightly reduced the toxic effects of unmodified PEI especially at the conjugation degree of 10%. However, this result showed that the significant decrease of PEI cytotoxic effects could not be achieved by the shielding of surface amines even at the conjugation degree of 10%. Therefore, it is suggested to investigate the effects of higher degrees of amine substitution to produce less toxic PEI -based nanocarriers.


  1. Verdoodt F, Friis S, Dehlendorff C, Albieri V, Kjaer SK. Non-steroidal anti-inflammatory drug use and risk of endometrial cancer: A systematic review and meta-analysis of observational studies. Gynecologic Oncology. 2016;140(2):352-8.
  2. Cuzick J, Thorat MA, Bosetti C, Brown PH, Burn J, Cook NR, et al. Estimates of benefits and harms of prophylactic use of aspirin in the general population. Annals of Oncology. 2015;26(1):47-57.
  3. Cuzick J, Otto F, Baron JA, Brown PH, Burn J, Greenwald P, et al. Aspirin and non-steroidal anti-inflammatory drugs for cancer prevention: an international consensus statement. The Lancet Oncology. 2009;10(5):501-7.
  4. Ulrich CM, Bigler J, Potter JD. Non-steroidal anti-inflammatory drugs for cancer prevention: promise, perils and pharmacogenetics. Nat Rev Cancer. 2006;6(2):130-40.
  5. Kilic G, Gurates B, Garon J, Kang H, Arun B, Lampley CE, et al. Expression of cyclooxygenase-2 in endometrial adenocarcinoma. European Journal of Gynaecological Oncology. 2005;26(3):271-4.
  6. Knapp P, Chabowski A, Błachnio-Zabielska A, Walentowicz-Sadłecka M, Grabiec M, Knapp PA. Expression of Estrogen Receptors (α, β), Cyclooxygenase-2 and Aromatase in normal endometrium and endometrioid cancer of uterus. Advances in Medical Sciences. 2013;58(1):96-103.
  7. Ferrandina G, Legge F, Ranelletti FO, Zannoni GF, Maggiano N, Evangelisti A, et al. Cyclooxygenase‐2 expression in endometrial carcinoma. Cancer. 2002;95(4):801-7.
  8. Cao QJ, Einstein MH, Anderson PS, Runowicz CD, Balan R, Jones JG. Expression of COX-2, Ki-67, cyclin D1, and p21 in endometrial endometrioid carcinomas. International Journal of Gynecological Pathology. 2002;21(2):147-54.
  9. Roos J, Grösch S, Werz O, Schröder P, Ziegler S, Fulda S, et al. Regulation of tumorigenic Wnt signaling by cyclooxygenase-2, 5-lipoxygenase and their pharmacological inhibitors: A basis for novel drugs targeting cancer cells? Pharmacology & therapeutics. 2016;157:43-64.
  10. Chandrasekharan N, Dai H, Roos KLT, Evanson NK, Tomsik J, Elton TS, et al. COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proceedings of the National Academy of Sciences. 2002;99(21):13926-31.
  11. Kurumbail RG, Stevens AM, Gierse JK, McDonald JJ, Stegeman RA, Pak JY, et al. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nature. 1996;384(6610):644-8.
  12. Picot D, Loll PJ, Garavito RM. The X-ray crystal structure of the membrane protein prostaglandin H2 synthase-1. 1994.
  13. Qiu H-Y, Wang P-F, Li Z, Ma J-T, Wang X-M, Yang Y-H, et al. Synthesis of dihydropyrazole sulphonamide derivatives that act as anti-cancer agents through COX-2 inhibition. Pharmacological research. 2016;104:86-96.
  14. Lamy S, Ben Saad A, Zgheib A, Annabi B. Olive oil compounds inhibit the paracrine regulation of TNF-α-induced endothelial cell migration through reduced glioblastoma cell cyclooxygenase-2 expression. The Journal of Nutritional Biochemistry. 2016;27:136-45.
  15. Williams CS, Mann M, DuBois RN. The role of cyclooxygenases in inflammation, cancer, and development. Oncogene. 1999;18(55):7908-16.
  16. Cho S-J, Kim N, Kim JS, Jung HC, Song IS. The Anti-Cancer Effect of COX-2 Inhibitors on Gastric Cancer Cells. Digestive Diseases and Sciences. 2007;52(7):1713-21.
  17. Kirane A, Toombs JE, Ostapoff K, Carbon JG, Zaknoen S, Braunfeld J, et al. Apricoxib, a Novel Inhibitor of COX-2, Markedly Improves Standard Therapy Response in Molecularly Defined Models of Pancreatic Cancer. Clinical Cancer Research. 2012;18(18):5031-42.
  18. Vosooghi M, Amini M. The discovery and development of cyclooxygenase-2 inhibitors as potential anticancer therapies. Expert opinion on drug discovery. 2014;9(3):255-67.
  19. Ng K, Meyerhardt JA, Chan AT, Sato K, Chan JA, Niedzwiecki D, et al. Aspirin and COX-2 Inhibitor Use in Patients With Stage III Colon Cancer. Journal of the National Cancer Institute. 2015;107(1).
  20. Roos J, Grösch S, Werz O, Schröder P, Ziegler S, Fulda S, et al. Regulation of tumorigenic Wnt signaling by cyclooxygenase-2, 5-lipoxygenase and their pharmacological inhibitors: A basis for novel drugs targeting cancer cells? Pharmacology & therapeutics. 2016;157:43-64.
  21. Simmons DL, Botting RM, Hla T. Cyclooxygenase Isozymes: The Biology of Prostaglandin Synthesis and Inhibition. Pharmacological Reviews. 2004;56(3):387-437.
  22. Eberhart CE, Coffey RJ, Radhika A, Giardiello FM, Ferrenbach S, Dubois RN. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology-Orlando. 1994;107(4):1183-8.
  23. Hida T, Yatabe Y, Achiwa H, Muramatsu H, Kozaki K-i, Nakamura S, et al. Increased expression of cyclooxygenase 2 occurs frequently in human lung cancers, specifically in adenocarcinomas. Cancer research. 1998;58(17):3761-4.
  24. Hosomi Y, Yokose T, Hirose Y, Nakajima R, Nagai K, Nishiwaki Y, et al. Increased cyclooxygenase 2 (COX-2) expression occurs frequently in precursor lesions of human adenocarcinoma of the lung. Lung Cancer. 2000;30(2):73-81.
  25. Chan G, Boyle JO, Yang EK, Zhang F, Sacks PG, Shah JP, et al. Cyclooxygenase-2 expression is up-regulated in squamous cell carcinoma of the head and neck. Cancer research. 1999;59(5):991-4.
  26. Molina MA, Sitja-Arnau M, Lemoine MG, Frazier ML, Sinicrope FA. Increased cyclooxygenase-2 expression in human pancreatic carcinomas and cell lines growth inhibition by nonsteroidal anti-inflammatory drugs. Cancer research. 1999;59(17):4356-62.
  27. Kulkarni S, Rader JS, Zhang F, Liapis H, Koki AT, Masferrer JL, et al. Cyclooxygenase-2 is overexpressed in human cervical cancer. Clinical Cancer Research. 2001;7(2):429-34.
  28. Singh TD, Gupta S, Shrivastav BR, Tiwari PK. Epigenetic profiling of gallbladder cancer and gall stone diseases: Evaluation of role of tumour associated genes. Gene. 2016;576(2, Part 2):743-52.
  29. Chikman B, Vasyanovich S, Lavy R, Habler L, Tolstov G, Kapiev A, et al. COX2 expression in high-grade breast cancer: evidence for prognostic significance in the subset of triple-negative breast cancer patients. Medical Oncology. 2014;31(6):989.
  30. Kim HS, Moon H-G, Han W, Yom CK, Kim WH, Kim JH, et al. COX2 overexpression is a prognostic marker for Stage III breast cancer. Breast Cancer Research and Treatment. 2012;132(1):51-9.
  31. Rao R, Redha R, Macias-Perez I, Su Y, Hao C, Zent R, et al. Prostaglandin E2-EP4 receptor promotes endothelial cell migration via ERK activation and angiogenesis in vivo. Journal of Biological Chemistry. 2007;282(23):16959-68.
  32. Xu L, Stevens J, Hilton MB, Seaman S, Conrads TP, Veenstra TD, et al. COX-2 inhibition potentiates antiangiogenic cancer therapy and prevents metastasis in preclinical models. Science translational medicine. 2014;6(242):242ra84-ra84.
  33. Ghosh N, Chaki R, Mandal V, Mandal SC. COX-2 as a target for cancer chemotherapy. Pharmacological Reports. 2010;62(2):233-44.
  34. Khan Z, Khan N, P Tiwari R, K Sah N, Prasad G, S Bisen P. Biology of Cox-2: an application in cancer therapeutics. Current drug targets. 2011;12(7):1082-93.
  35. Prescott SM, White RL. Self-promotion? Intimate connections between APC and prostaglandin H synthase-2. Cell. 1996;87(5):783-6.
  36. Tavolari S, Bonafè M, Marini M, Ferreri C, Bartolini G, Brighenti E, et al. Licofelone, a dual COX/5-LOX inhibitor, induces apoptosis in HCA-7 colon cancer cells through the mitochondrial pathway independently from its ability to affect the arachidonic acid cascade. Carcinogenesis. 2008;29(2):371-80.
  37. Cai H, Huang X, Xu S, Shen H, Zhang P, Huang Y, et al. Discovery of novel hybrids of diaryl-1,2,4-triazoles and caffeic acid as dual inhibitors of cyclooxygenase-2 and 5-lipoxygenase for cancer therapy. European Journal of Medicinal Chemistry. 2016;108:89-103.
  38. Tong X, Van Dross RT, Abu-Yousif A, Morrison AR, Pelling JC. Apigenin prevents UVB-induced cyclooxygenase 2 expression: coupled mRNA stabilization and translational inhibition. Molecular and cellular biology. 2007;27(1):283-96.
  39. Gupta RA, DuBois RN. Colorectal cancer prevention and treatment by inhibition of cyclooxygenase-2. Nature Reviews Cancer. 2001;1(1):11-21.
  40. Harris R. Cyclooxygenase-2 (cox-2) blockade in the chemoprevention of cancers of the colon, breast, prostate, and lung. Inflammopharmacology. 2009;17(2):55-67.
  41. Arber N. Cyclooxygenase-2 inhibitors in colorectal cancer prevention: point. Cancer Epidemiology Biomarkers & Prevention. 2008;17(8):1852-7.
  42. Gao M, Wang M, Miller KD, Hutchins GD, Zheng Q-H. Synthesis of carbon-11 labeled celecoxib derivatives as new candidate PET radioligands for imaging of inflammation. Applied Radiation and Isotopes. 2009;67(11):2019-24.
  43. Pasinetti GM, Aisen PS. Cyclooxygenase-2 expression is increased in frontal cortex of Alzheimer's disease brain. Neuroscience. 1998;87(2):319-24.
  44. Ho L, Pieroni C, Winger D, Purohit DP, Aisen PS, Pasinetti GM. Regional distribution of cyclooxygenase-2 in the hippocampal formation in Alzheimer's disease. Journal of Neuroscience Research. 1999;57(3):295-303.
  45. Pasinetti GM. Cyclooxygenase and inflammation in Alzheimer's disease: Experimental approaches and clinical interventions. Journal of Neuroscience Research. 1998;54(1):1-6.
  46. Kitamura Y, Shimohama S, Koike H, Kakimura J-i, Matsuoka Y, Nomura Y, et al. Increased Expression of Cyclooxygenases and Peroxisome Proliferator-Activated Receptor-γ in Alzheimer's Disease Brains. Biochemical and Biophysical Research Communications. 1999;254(3):582-6.
  47. Teismann P, Tieu K, Choi D-K, Wu D-C, Naini A, Hunot S, et al. Cyclooxygenase-2 is instrumental in Parkinson's disease neurodegeneration. Proceedings of the National Academy of Sciences. 2003;100(9):5473-8.
  48. Hunot S, Vila M, Teismann P, Davis RJ, Hirsch EC, Przedborski S, et al. JNK-mediated induction of cyclooxygenase 2 is required for neurodegeneration in a mouse model of Parkinson's disease. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(2):665-70.
  49. Uddin MJ, Crews BC, Ghebreselasie K, Huda I, Kingsley PJ, Ansari MS, et al. Fluorinated COX-2 Inhibitors as Agents in PET Imaging of Inflammation and Cancer. Cancer Prevention Research. 2011;4(10):1536-45.
  50. McCarthy TJ, Sheriff AU, Graneto MJ, Talley JJ, Welch MJ. Radiosynthesis, In Vitro Validation, and In Vivo Evaluation of 18F-Labeled COX-1 and COX-2 Inhibitors. Journal of Nuclear Medicine. 2002;43(1):117-24.
  51. El-Azony KM. Preparation of 125I-celecoxib with high purity as a possible tumor agent. Journal of Radioanalytical and Nuclear Chemistry. 2010;285(2):315-20.
  52. Kuge Y, Katada Y, Shimonaka S, Temma T, Kimura H, Kiyono Y, et al. Synthesis and evaluation of radioiodinated cyclooxygenase-2 inhibitors as potential SPECT tracers for cyclooxygenase-2 expression. Nuclear Medicine and Biology. 2006;33(1):21-7.
  53. Desai D, Kaushal N, Gandhi UH, Arner RJ, D'Souza C, Chen G, et al. Synthesis and evaluation of the anti-inflammatory properties of selenium-derivatives of celecoxib. Chemico-biological interactions. 2010;188(3):446-56.
  54. Dehshahri A, Sadeghpour H, Keykhaee M, Khalvati B, Sheikhsaran F. Enhanced Delivery of Plasmid Encoding Interleukin-12 Gene by Diethylene Triamine Penta-Acetic Acid (DTPA)-Conjugated PEI Nanoparticles. Applied biochemistry and biotechnology. 2016:1-19.
  55. Dehshahri A, Sadeghpour H, Oskuee RK, Fadaei M, Sabahi Z, Alhashemi SH, et al. Interleukin-12 plasmid DNA delivery using l-thyroxine-conjugated polyethylenimine nanocarriers. Journal of Nanoparticle Research. 2014;16(5):1-14.
  56. Dehshahri A, Oskuee RK, Ramezani M. Plasmid DNA delivery into hepatocytes using a multifunctional nanocarrier based on sugar-conjugated polyethylenimine. Gene Ther Mol Biol. 2012;14:62-71.
  57. Sabahi Z, Samani SM, Dehshahri A. Conjugation of poly (amidoamine) dendrimers with various acrylates for improved delivery of plasmid encoding interleukin-12 gene. Journal of biomaterials applications. 2015;29(7):941-53.
  58. OSKOUEI R, Dehshahri A, Shier W, Ramezani M. Modified polyethylenimine: Self assemble nanoparticle forming polymer for pDNA delivery. 2008.
  59. Dehshahri A, Alhashemi SH, Jamshidzadeh A, Sabahi Z, Samani SM, Sadeghpour H, et al. Comparison of the effectiveness of polyethylenimine, polyamidoamine and chitosan in transferring plasmid encoding interleukin-12 gene into hepatocytes. Macromolecular Research. 2013;21(12):1322-30.
  60. Khalvati B, Sheikhsaran F, Sharifzadeh S, Kalantari T, Behzad Behbahani A, Jamshidzadeh A, et al. Delivery of plasmid encoding interleukin-12 gene into hepatocytes by conjugated polyethylenimine-based nanoparticles. Artificial cells, nanomedicine, and biotechnology. 2017;45(5):1036-44.
  61. Nouri F, Sadeghpour H, Heidari R, Dehshahri A. Preparation, characterization, and transfection efficiency of low molecular weight polyethylenimine-based nanoparticles for delivery of the plasmid encoding CD200 gene. International journal of nanomedicine. 2017;12:5557.
  62. Sheikhsaran F, Sadeghpour H, Khalvati B, Entezar-Almahdi E, Dehshahri A. Tetraiodothyroacetic acid-conjugated polyethylenimine for integrin receptor mediated delivery of the plasmid encoding IL-12 gene. Colloids and Surfaces B: Biointerfaces. 2017;150:426-36.