Pharmaceutical Research

, Volume 31, Issue 12, pp 3361–3370 | Cite as

In Vitro and In Vivo Biological Evaluation of O-Carboxymethyl Chitosan Encapsulated Metformin Nanoparticles for Pancreatic Cancer Therapy

  • K. S. Snima
  • R. Jayakumar
  • Vinoth-Kumar Lakshmanan
Research Paper



In vitro anticancer effect and in vivo biodistribution and biocompatibility of metformin encapsulated O-Carboxymethyl chitosan nanoparticles were evaluated for its application as pancreatic cancer therapy.


In vitro studies such as cell migration assay, clonogenic assay, cell cycle analysis and qRT-PCR analysis were done in pancreatic cancer cells (MiaPaCa-2) treated with O-CMC-metformin NPs for evaluating its anticancer potential. In vivo biodistribution studies were carried out by NIR imaging of O-CMC-metformin NPs after tagging it with ICG. In vivo biocompatibility of the NPs was assessed by histopathology analysis of organs from mice administered with the NPs.


In vitro cell migration assay showed marginal effect of NPs on migration property of pancreatic cancer cells (MiaPaCa-2). In vitro clonogenic assay established that the O-CMC-metformin NPs reduced colony formation ability of the cancer cells. While cell cycle analysis showed that the O-CMC-metformin NPs had only minor effect on progression of cell cycle in the cancer cells. qRT-PCR analysis exhibited reduced mRNA expression of p21, vanin 1 and MMP9 in pancreatic cancer cells treated with the nanoparticles. In vivo NIR imaging study showed normal biodistribution pattern of the intravenously injected O-CMC-metformin NPs suggesting normal clearance rate of nanoparticles and no adverse toxicity to the organs.


The biocompatible O-CMC-metformin NPs with anticancer potential and capability for normal biodistribution can be beneficial for the treatment of pancreatic cancer.

Key Words

cancer therapy clonogenecity drug delivery gene expression metformin migration O-CMC nanoparticles pancreatic cancer 


Acknowledgments and Disclosures

The authors are thankful to Department of Science and Technology (DST), Government of India, for their financial support under Fast Track SERC project (Ref.No.: SERC 0558/2009/LS). K. S. Snima is grateful to Council of Scientific and Industrial Research (CSIR), India, for providing Senior Research Fellowship (09/963 (0030)/2 K 13-EMK-I) for carrying out her research work. We thank Dr. A.K.K. Unni, Dr. P. Reshmi, and Mr. Sunil Kumar O. R for the support and help they extended to carry out in vivo experiments in the Central animal house facility, Amrita Institute of Medical Science and Research centre, Kerala, India. We are also grateful to Mrs. Sreerekha P. R and Dr. G. Siddaramana Gowd for there helps in FACS analysis and in vivo NIR imaging. We thank Amrita Centre for Nanosciences and Molecular Medicine for the infrastructure support.


  1. 1.
    Siegel R, Ward E, Brawley O, Jemal A. Cancer statistics. CA Cancer J Clin. 2011;61(4):212–36.PubMedCrossRefGoogle Scholar
  2. 2.
    Pollak M. Insulin and insulin-like growth factor signalling in neoplasia. Nat Rev Cancer. 2008;8(12):915–28.PubMedCrossRefGoogle Scholar
  3. 3.
    Pollak M. The insulin and insulin-like growth factor receptor family in neoplasia: an update. Nat Rev Cancer. 2012;12(3):159–69.PubMedGoogle Scholar
  4. 4.
    Zakikhani M, Dowling R, Fantus IG, Sonenberg N, Pollak M. Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells. Cancer Res. 2006;66(21):10269–73.PubMedCrossRefGoogle Scholar
  5. 5.
    Hardie DG. AMP-activated protein kinase as a drug target. Annu Rev Pharmacol Toxicol. 2007;47:185–210.PubMedCrossRefGoogle Scholar
  6. 6.
    Hawley SA, Ross FA, Chevtzoff C, Green KA, Evans A, Fogarty S, et al. Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation. Cell Metab. 2010;11(6):554–65.PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Gotlieb WH, Saumet J, Beauchamp MC, Gu J, Lau S, Pollak MN, et al. In vitro metformin anti-neoplastic activity in epithelial ovarian cancer. Gynecol Oncol. 2008;110(2):246–50.PubMedCrossRefGoogle Scholar
  8. 8.
    Zhuang Y, Miskimins WK. Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1. J Mol Signal. 2008;3:18.PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Luo Q, Hu D, Hu S, Yan M, Sun Z, Chen F. In vitro and in vivo anti-tumor effect of metformin as a novel therapeutic agent in human oral squamous cell carcinoma. BMC Cancer. 2012;12:517.PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Ben Sahra I, Laurent K, Loubat A, Giorgetti-Peraldi S, Colosetti P, Auberger P, et al. The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level. Oncogene. 2008;27(25):3576–86.PubMedCrossRefGoogle Scholar
  11. 11.
    Liu B, Fan Z, Edgerton SM, Deng XS, Alimova IN, Lind SE, et al. Metformin induces unique biological and molecular responses in triple negative breast cancer cells. Cell Cycle. 2009;8(13):2031–40.PubMedCrossRefGoogle Scholar
  12. 12.
    Alimova IN, Liu B, Fan Z, Edgerton SM, Dillon T, Lind SE, et al. Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro. Cell Cycle. 2009;8(6):909–15.PubMedCrossRefGoogle Scholar
  13. 13.
    Vazquez-Martin A, Oliveras-Ferraros C, Menendez JA. The antidiabetic drug metformin suppresses HER2 (erbB-2) oncoprotein overexpression via inhibition of the mTOR effector p70S6K1 in human breast carcinoma cells. Cell Cycle. 2009;8(1):88–96.PubMedCrossRefGoogle Scholar
  14. 14.
    Jalving M, Gietema JA, Lefrandt JD, de Jong S, Reyners AK, Gans RO, et al. Metformin: taking away the candy for cancer? Eur J Cancer. 2010;46(13):2369–80.PubMedCrossRefGoogle Scholar
  15. 15.
    Kim HG, Hien TT, Han EH, Hwang YP, Choi JH, Kang KW, et al. Metformin inhibits P-glycoprotein expression via the NF-κB pathway and CRE transcriptional activity through AMPK activation. Br J Pharmacol. 2011;162(5):1096–108.PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Dowling RJ, Zakikhani M, Fantus IG, Pollak M, Sonenberg N. Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Res. 2007;67(22):10804–12.PubMedCrossRefGoogle Scholar
  17. 17.
    Morgensztern D, McLeod HL. PI3K/Akt/mTOR pathway as a target for cancer therapy. Anticancer Drug. 2005;16(8):797–803.CrossRefGoogle Scholar
  18. 18.
    Kisfalvi K, Eibl G, Sinnett-Smith J, Rozengurt E. Metformin disrupts crosstalk between G protein-coupled receptor and insulin receptor signaling systems and inhibits pancreatic cancer growth. Cancer Res. 2009;69(16):6539–45.PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Rozengurt E, Sinnett-Smith J, Kisfalvi K. Crosstalk between Insulin/Insulin-like growth factor-1 receptors and G protein-coupled receptor signaling systems: a novel target for the antidiabetic drug metformin in pancreatic cancer. Clin Cancer Res. 2010;16(9):2505–11.PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Bao B, Wang Z, Ali S, Ahmad A, Azmi AS, Sarkar SH, et al. Metformin inhibits cell proliferation, migration and invasion by attenuating CSC function mediated by deregulating miRNAs in pancreatic cancer cells. Cancer Prev Res. 2012;5(3):355–64. Philadelphia, Pa.CrossRefGoogle Scholar
  21. 21.
    Li W, Yuan Y, Huang L, Qiao M, Zhang Y. Metformin alters the expression profiles of microRNAs in human pancreatic cancer cells. Diabetes Res Clin Pract. 2012;96(2):187–95.PubMedCrossRefGoogle Scholar
  22. 22.
    Soares HP, Ni Y, Kisfalvi K, Sinnett-Smith J, Rozengurt E. Different patterns of Akt and ERK feedback activation in response to rapamycin, active-site mTOR inhibitors and metformin in pancreatic cancer cells. PLoS One. 2013;8(2):e57289.PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Nair V, Pathi S, Jutooru I, Sreevalsan S, Basha R, Abdelrahim M, et al. Metformin inhibits pancreatic cancer cell and tumor growth and downregulates Sp transcription factors. Carcinogenesis. 2013. doi: 10.1093/carcin/bgt231.PubMedGoogle Scholar
  24. 24.
    Graham GG, Punt J, Arora M, Day RO, Doogue MP, Duong JK, et al. Clinical pharmacokinetics of metformin. Clin Pharmacokinet. 2011;50(2):81–98.PubMedCrossRefGoogle Scholar
  25. 25.
    Snima KS, Jayakumar R, Unnikrishnan AG, Nair SV, Lakshmanan VK. O-Carboxymethyl chitosan nanoparticles for metformin delivery to pancreatic cancer cells. Carbohydr Polym. 2012;89(3):1003–7.PubMedCrossRefGoogle Scholar
  26. 26.
    Bolen S, Feldman L, Vassy J, Wilson L, Yeh HC, Marinopoulos S, et al. Systematic review: comparative effectiveness and safety of oral medications for type 2 diabetes mellitus. Ann Intern Med. 2007;147(6):386–99.PubMedCrossRefGoogle Scholar
  27. 27.
    Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Investig. 2001;108(8):1167–74.PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF, Lachin JM, Walker EA, et al. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med. 2002;346(6):393–403.PubMedCrossRefGoogle Scholar
  29. 29.
    Li D, Yeung SC, Hassan MM, Konopleva M, Abbruzzese JL. Antidiabetic therapies affect risk of pancreatic cancer. Gastroenterology. 2009;137(2):482–8.PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Lakshmanan VK, Snima KS, Bumgardner JD, Nair Shantikumar V, Jayakumar R. Chitosan-based nanoparticles in cancer therapy. Chitosan Biomaterials I Adv Polym Sci. 2011;243:55–91. doi: 10.1007/12_2011_132.CrossRefGoogle Scholar
  31. 31.
    Khanna A, Mahalingam K, Chakrabarti D, Periyasamy G. Ets-1 expression and gemcitabine chemoresistance in pancreatic cancer cells. Cell Mol Biol Lett. 2011;16(1):101–13.PubMedCrossRefGoogle Scholar
  32. 32.
    Hanna RK, Zhou C, Malloy KM, Sun L, Zhong Y, Gehrig PA, et al. Metformin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and modulation of the mTOR pathway. Gynecol Oncol. 2012;125(2):458–69.PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    López-Ruiz P, Rodriguez-Ubreva J, Cariaga AE, Cortes MA, Colás B. SHP-1 in cell-cycle regulation. Anticancer Agents Med Chem. 2011;11(1):89–98.PubMedCrossRefGoogle Scholar
  34. 34.
    Huang H, Dong X, Kang MX, Xu B, Chen Y, Zhang B, et al. Novel blood biomarkers of pancreatic cancer-associated diabetes mellitus identified by peripheral blood-based gene expression profiles. Am J Gastroenterol. 2010;105(7):1661–9.PubMedCrossRefGoogle Scholar
  35. 35.
    Ashokan A, Gowd GS, Somasundaram VH, Bhupathi A, Peethambaran R, Unni AK, et al. Multifunctional calcium phosphate nano-contrast agent for combined nuclear, magnetic and near-infrared in vivo imaging. Biomaterials. 2013;34(29):7143–57.PubMedCrossRefGoogle Scholar
  36. 36.
    Gopal V, Mandal V, Mandal SC. Biochemical investigation of standardized wattakaka volubilis leaf petroleum ether cold macerated extract against experimentally induced diabetes in the Rat. Pharmacologia. 2013;4(5):391–9.CrossRefGoogle Scholar
  37. 37.
    Severino C, Brizzi P, Solinas A, Secchi G, Maioli M, Tonolo G. Low-dose dexamethasone in the rat: a model to study insulin resistance. Am J Physiol Endocrinol Metab. 2002;283(2):E367–73.PubMedGoogle Scholar
  38. 38.
    Yin M, van der Horst IC, van Melle JP, Qian C, van Gilst WH, Silljé HH, et al. Metformin improves cardiac function in a nondiabetic rat model of post-MI heart failure. Am J PhysiolHeart Circ physiol. 2011;301(2):H459–68.CrossRefGoogle Scholar
  39. 39.
    Jian MY, Alexeyev MF, Wolkowicz PE, Zmijewski JW, Creighton JR. Metformin-stimulated AMPK-α1 promotes microvascular repair in acute lung injury. Am J Physiol Lung Cell Mol Physiol. 2013;305(11):L844–55.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • K. S. Snima
    • 1
  • R. Jayakumar
    • 1
  • Vinoth-Kumar Lakshmanan
    • 1
  1. 1.Amrita Centre for Nanosciences and Molecular MedicineAmrita Institute of Medical Sciences and Research centreKochiIndia

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