• Ahmed A Abd-rabou Department of Hormones, Medical Research Division, National Research Centre, Dokki, Giza, Egypt and Stem Cell Lab., Centre of Excellence for Advanced Sciences, National Research Centre, Dokki, Giza, Egypt.
  • Hanaa H Ahmed Department of Hormones, Medical Research Division, National Research Centre, Dokki, Giza, Egypt and Stem Cell Lab., Centre of Excellence for Advanced Sciences, National Research Centre, Dokki, Giza, Egypt.
  • Mohamed S Kishta Department of Hormones, Medical Research Division, National Research Centre, Dokki, Giza, Egypt and Stem Cell Lab., Centre of Excellence for Advanced Sciences, National Research Centre, Dokki, Giza, Egypt.


Objective: Chemokine (C-C motif) ligand 2 (CCL2), a candidate of cytokines, orchestrates immune cell recruitment to inflamed organs. CCL2 has been shown to have direct angiogenic effects, so providing an anti-angiogenic agent, Avastin (AV), to be combined with the CCR2 antagonist (concentration ratio [CR]) plays an essential role in the hemostatic strategy for immunomodulation. Lack of targetability and the adverse effects of chemical treatments are the main obstacles led scientists to develop novel strategies using nano-delivery approaches such as pegylated nanoparticles (NPs) which exhibits reduced drug clearance rates. The rationale of the current study is to test the in vivo immunomodulatory effects of AV and/or CR in their NPs or free counterparts.

Methods: These NPs were synthesized and characterized using different physicochemical techniques. Males Wistar rats (n=114) were used and divided into 7 groups treated with vehicle, AV, AVNP, CCR2 antagonist (CR), CCR2 antagonist NPs (CRNP), AV-CCR2 antagonist (AVCR), and AV-CCR2 antagonist NPs (AVCRNP). Groups were subdivided into three subgroups according to the administrated dose. Blood was taken from rats for differential leukocyte and platelet profile measurements. Sera were collected to test vascular endothelial growth factor (VEGF) levels. Autopsy samples from liver were taken for histopathological investigation.

Results: The morphology of the NPs was spherical and had sizes ranging from 89.89 nm to 146 nm. Monocytes and lymphocytes accumulated in the blood circulation and VEGF levels were inhibited after AV and CR administrations. In addition, large platelets concentration ratio was elevated in the blood circulation.

Conclusion: We concluded that AV ad CR therapeutic regimens have an immunomodulatory role through induction of monocyte-platelet aggregation and inhibition of VEGF.

Keywords: Immunomodulation, Avastin, CCR2 antagonist, Monocyte-platelet connection, Vascular endothelial growth factor.


1. Rollins BJ. Chemokines. Blood 1997;90:909-28.
2. Deshmane SL, Kremlev S, Amini S, Sawaya BE. Monocyte chemoattractant protein-1 (MCP-1): An overview. J Interferon Cytokine Res 2009;29:313-26.
3. Bachelerie F, Ben-Baruch A, Burkhardt AM, Combadiere C, Farber JM, Graham GJ, et al. International union of basic and clinical pharmacology. [corrected]. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors. Pharmacol Rev 2014;66:1-79.
4. Charo IF, Myers SJ, Herman A, Franci C, Connolly AJ, Coughlin SR, et al. Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails. Proc Natl Acad Sci U S A 1994;91:2752-6.
5. Zhang T, Somasundaram R, Berencsi K, Caputo L, Gimotty P, Rani P, et al. Migration of cytotoxic T lymphocytes toward melanoma cells in three-dimensional organotypic culture is dependent on CCL2 and CCR4. Eur J Immunol 2006;36:457-67.
6. Rossi D, Zlotnik A. The biology of chemokines and their receptors. Annu Rev Immunol 2000;18:217-42.
7. Shi C, Pamer EG. Monocyte recruitment during infection and inflammation. Nat Rev Immunol 2011;11:762-74.
8. Torres S, Bartolomé RA, Mendes M, Barderas R, Fernandez-Aceñero MJ, Peláez-García A, et al. Proteome profiling of cancer-associated fibroblasts identifies novel proinflammatory signatures and prognostic markers for colorectal cancer. Clin Cancer Res 2013;19:6006-19.
9. Lu B, Rutledge BJ, Gu L, Fiorillo J, Lukacs NW, Kunkel SL, et al. Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice. J Exp Med 1998;187:601-8.
10. Boring L, Gosling J, Chensue SW, Kunkel SL, Farese RV Jr., Broxmeyer HE, et al. Impaired monocyte migration and reduced Type 1 (Th1) cytokine responses in C-C chemokine receptor 2 knockout mice. J Clin Invest 1997;100:2552-61.
11. Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 2003;19:71-82.
12. Luther SA, Cyster JG. Chemokines as regulators of T cell differentiation. Nat Immunol 2001;2:102-7.
13. Roca H, Varsos Z, Pienta KJ. CCL2 protects prostate cancer PC3 cells from autophagic death via phosphatidylinositol 3-kinase/AKT-dependent survivin up-regulation. J Biol Chem 2008;283:25057-73.
14. Loberg RD, Ying C, Craig M, Yan L, Snyder LA, Pienta KJ, et al. CCL2 as an important mediator of prostate cancer growth in vivo through the regulation of macrophage infiltration. Neoplasia 2007;9:556-62.
15. Lim SY, Raftery MJ, Goyette J, Hsu K, Geczy CL. Oxidative modifications of S100 proteins: Functional regulation by redox. J Leukoc Biol 2009;86:577-87.
16. Tong RT, Boucher Y, Kozin SV, Winkler F, Hicklin DJ, Jain RK, et al. Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors. Cancer Res 2004;64:3731-6.
17. Hijaz M, Das S, Mert I, Gupta A, Al-Wahab Z, Tebbe C, et al. Folic acid tagged nanoceria as a novel therapeutic agent in ovarian cancer. BMC Cancer 2016;16:220.
18. Abd-Rabou AA, Bharali DJ, Mousa SA. Taribavirin and 5-fluorouracil-loaded pegylated-lipid nanoparticle synthesis, p38 docking, and antiproliferative effects on MCF-7 breast cancer. Pharm Res 2018;35:76.
19. Abd-Rabou AA, Ahmed HH. CS-PEG decorated PLGA nano-prototype for delivery of bioactive compounds: A novel approach for induction of apoptosis in hepG2 cell line. Adv Med Sci 2017;62:357-67.
20. Abd-Rabou AA, Abdalla AM, Ali NA, Zoheir KM. Moringa oleifera root induces cancer apoptosis more effectively than leave nanocomposites and its free counterpart. Asian Pac J Cancer Prev 2017;18:2141-9.
21. Shalby AB, Abd-Rabou AA, Ahmed HH. Nano-Se cross talks with nano-DOX/FU to selectively hack hepatic cancer cells and spare normal cells healthy: A mechanism-based study. J Appl Pharm Sci 2017;7:3-12.
22. Chae SY, Kim TH, Park K, Jin CH, Son S, Lee S, et al. Improved antitumor activity and tumor targeting of NH(2)-terminal-specific PEGylated tumor necrosis factor-related apoptosis-inducing ligand. Mol Cancer Ther 2010;9:1719-29.
23. Panyam J, Labhasetwar V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev 2003;55:329-47.
24. Lim SM, Kim TH, Jiang HH, Park CW, Lee S, Chen X, et al. Improved biological half-life and anti-tumor activity of TNF-related apoptosis-inducing ligand (TRAIL) using PEG-exposed nanoparticles. Biomaterials 2011;32:3538-46.
25. A.O.A.C. Official Methods of Analysis. 16th ed. Washington, DC: Association of Official Analysis; 1995.
26. Banchroft JD, Stevens A, Turner DR. Theory and Practice of Histological Techniques. 4th ed. New York, London, San Francisco, Tokyo: Churchill Livingstone; 1996.
27. Ueda Y, Yang K, Foster SJ, Kondo M, Kelsoe G. Inflammation controls B lymphopoiesis by regulating chemokine CXCL12 expression. J Exp Med 2004;199:47-58.
28. Rot A, von Andrian UH. Chemokines in innate and adaptive host defense: Basic chemokinese grammar for immune cells. Annu Rev Immunol 2004;22:891-928.
29. Kurihara T, Warr G, Loy J, Bravo R. Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor. J Exp Med 1997;186:1757-62.
30. Serbina NV, Salazar-Mather TP, Biron CA, Kuziel WA, Pamer EG. TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection. Immunity 2003;19:59-70.
31. Serbina NV, Pamer EG. Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2. Nat Immunol 2006;7:311-7.
32. Ansel KM, Ngo VN, Hyman PL, Luther SA, Förster R, Sedgwick JD, et al. A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature 2000;406:309-14.
33. Reif K, Ekland EH, Ohl L, Nakano H, Lipp M, Förster R, et al. Balanced responsiveness to chemoattractants from adjacent zones determines B-cell position. Nature 2002;416:94-9.
34. Wood S, Jayaraman V, Huelsmann EJ, Bonish B, Burgad D, Sivaramakrishnan G, et al. Pro-Inflammatory Chemokine CCL2 (MCP-1) Promotes Healing in Diabetic Wounds by Restoring the Macrophage Response. PLoS ONE 2014; 9(3): Avaliable from. https://
35. Auerbuch V, Brockstedt DG, Meyer-Morse N, O’Riordan M, Portnoy DA. Mice lacking the Type I interferon receptor are resistant to listeria monocytogenes. J Exp Med 2004;200:527-33.
36. Korniluk A, Koper O, Kemona H, Dymicka-Piekarska V. From inflammation to cancer. Ir J Med Sci 2017;186:57-62.
37. Qian BZ, Li J, Zhang H, Kitamura T, Zhang J, Campion LR, et al. CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis. Nature 2011;475:222-5.
38. Bottsford-Miller JN, Coleman RL, Sood AK. Resistance and escape from antiangiogenesis therapy: Clinical implications and future strategies. J Clin Oncol 2012;30:4026-34.
39. Verhoef JJF, Anchordoquy TJ. Questioning the Use of PEGylation for Drug Delivery. Drug Deliv Transl Res 2013;3:499-503.
40. Knop K, Hoogenboom R, Fischer D, Schubert US. Poly (ethylene glycol) in drug delivery: pros and cons as well as potential alternatives. Angew. Chem. Int. Ed 2010;49:6288-6308.
41. Petros RA, DeSimone JM. Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov 2010;9:615-27.
42. Geels P, Eisenhauer E, Bezjak A, Zee B, Day A. Palliative effect of chemotherapy: Objective tumor response is associated with symptom improvement in patients with metastatic breast cancer. J Clin Oncol 2000;18:2395-405.
43. Umano M, Uechi K, Uriuda T, Murayama S, Azuma H, Shinohara A, et al. Tumor accumulation of ε-poly-lysines-based polyamines conjugated with boron clusters. Appl Radiat Isot 2011;69:1765-7.
44. Tahara K, Furukawa S, Yamamoto H, Kawashima Y. Hybrid-modified poly(D,L-lactide-co-glycolide) nanospheres for a novel cellular drug delivery system. Int J Pharm 2010;392:311-3.
45. Li F, Hurley B, Liu Y, Leonard B, Griffith M. Controlled release of bevacizumab through nanospheres for extended treatment of age-related macular degeneration. Open Ophthalmol J 2012;6:54-8.
46. Abrishami M, Zarei-Ghanavati S, Soroush D, Rouhbakhsh M, Jaafari MR, Malaekeh-Nikouei B, et al. Preparation, characterization, and in vivo evaluation of nanoliposomes-encapsulated bevacizumab (avastin) for intravitreal administration. Retina 2009;29:699-703.
47. Hao Y, Patel A, Liu W, Krishna R, Sabates NR, Mitra AK. Preparation and Characterization of Bevacizumab (Avastin) Nanoparticles for the Treatment of Age Related Macular Degeneration. Los Angeles, California, U.S.A: American Association of Pharmaceutical Scientists (AAPS) Annual Meeting and Exposition; 2009. Available from:
48. Cao X, Schoichet MS. Delivering neuroactive molecules from biodegradable microspheres for application in central nervous system disorders. Biomaterials 1999;20:329-39.
49. Malhotra M, Lane C, Tomaro-Duchesneau C, Saha S, Prakash S. A novel method for synthesizing PEGylated chitosan nanoparticles: Strategy, preparation, and in vitro analysis. Int J Nanomedicine 2011;6:485-94.
50. Brown Z, Robson RL, Westwick J. Regulation and expression of chemokines: Potential role in glomerulonephritis. J Leukoc Biol 1996;59:75-80.
51. Haberstroh U, Stilo K, Pocock J, Wolf G, Helmchen U, Wenzel U, et al. L-arginine suppresses lipopolysaccharide-induced expression of RANTES in glomeruli. J Am Soc Nephrol 1998;9:203-10.
52. Handel TM, Domaille PJ. Heteronuclear (1H, 13C, 15N) NMR assignments and solution structure of the monocyte chemoattractant protein-1 (MCP-1) dimer. Biochemistry 1996;35:6569-84.
53. Karpus WJ, Lukacs NW, Kennedy KJ, Smith WS, Hurst SD, Barrett TA, et al. Differential CC chemokine-induced enhancement of T helper cell cytokine production. J Immunol 1997;158:4129-36.
54. Gonzalo JA, Lloyd CM, Wen D, Albar JP, Wells TN, Proudfoot A, et al. The coordinated action of CC chemokines in the lung orchestrates allergic inflammation and airway hyperresponsiveness. J Exp Med 1998;188:157-67.
55. Sallusto F, Lenig D, Mackay CR, Lanzavecchia A. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med 1998;187:875-83.
56. Ohta M, Kitadai Y, Tanaka S, Yoshihara M, Yasui W, Mukaida N, et al. Monocyte chemoattractant protein-1 expression correlates with macrophage infiltration and tumor vascularity in human gastric carcinomas. Int J Oncol 2003;22:773-8.
57. Sato K, Kuratsu J, Takeshima H, Yoshimura T, Ushio Y. Expression of monocyte chemoattractant protein-1 in meningioma. J Neurosurg 1995;82:874-8.
58. Valković T, Dobrila F, Melato M, Sasso F, Rizzardi C, Jonjić N, et al. Correlation between vascular endothelial growth factor, angiogenesis, and tumor-associated macrophages in invasive ductal breast carcinoma. Virchows Arch 2002;440:583-8.
59. Dixon DA, Tolley ND, Bemis-Standoli K, Martinez ML, Weyrich AS, Morrow JD, et al. Expression of COX-2 in platelet-monocyte interactions occurs via combinatorial regulation involving adhesion and cytokine signaling. J Clin Invest 2006;116:2727-38.
60. Weyrich AS, Elstad MR, McEver RP, McIntyre TM, Moore KL, Morrissey JH, et al. Activated platelets signal chemokine synthesis by human monocytes. J Clin Invest 1996;97:1525-34.
61. Juliano RL, Haskill S. Signal transduction from the extracellular matrix. J Cell Biol 1993;120:577-85.
62. FitzGerald GA. COX-2 and beyond: Approaches to prostaglandin inhibition in human disease. Nat Rev Drug Discov 2003;2:879-90.
63. Mickelson JK, Lakkis NM, Villarreal-Levy G, Hughes BJ, Smith CW. Leukocyte activation with platelet adhesion after coronary angioplasty: A mechanism for recurrent disease? J Am Coll Cardiol 1996;28:345-53.
64. Irving PM, Macey MG, Shah U, Webb L, Langmead L, Rampton DS, et al. Formation of platelet-leukocyte aggregates in inflammatory bowel disease. Inflamm Bowel Dis 2004;10:361-72.
65. Miladiyah I, Jumina J, Haryana SM, Mustofa M. In silico molecular docking of xanthone derivatives as cyclooxygenase-2 inhibitor agents. Int J Pharm Pharm Sci 2017;9:98.
66. Chaithanya KK, Gopalakrishnan VK, Hagos Z, Rao DG. Mesuaferrin a-bioactive flavonoid isolated from the bark of Mesua ferrea l. against phospholipase a2, cyclooxygenase and lipoxygenase: An in vitro, in vivo and in silico approach. Int J Pharm Pharm Sci 2018;10:102.
122 Views | 125 Downloads
How to Cite
Abd-rabou, A. A., H. H. Ahmed, and M. S. Kishta. “SYNTHESIS, CHARACTERIZATION, AND IN VIVO IMMUNOMODULATION OF CCR2 AND VASCULAR ENDOTHELIAL GROWTH FACTOR ANTAGONISTS-LOADED PEGYLATED NANOPARTICLES”. Asian Journal of Pharmaceutical and Clinical Research, Vol. 12, no. 1, Jan. 2019, pp. 275-8, doi:10.22159/ajpcr.2019.v12i1.29337.
Original Article(s)