Pharmaceutical Research

, Volume 26, Issue 4, pp 926–935 | Cite as

A Novel Camptothecin Derivative Incorporated in Nano-Carrier Induced Distinguished Improvement in Solubility, Stability and Anti-tumor Activity Both In Vitro and In Vivo

  • Min Han
  • Cai-Xia He
  • Qiu-Li Fang
  • Xiao-Chun Yang
  • Yuan-Yuan Diao
  • Dong-Hang Xu
  • Qiao-Jun He
  • Yong-Zhou Hu
  • Wen-Quan Liang
  • Bo Yang
  • Jian-Qing Gao
Research Paper



An oil/water nanoemulsion was developed in the present study to enhance the solubility, stability and anti-tumor activity of a novel 10-methoxy-9-nitrocamptothecin (MONCPT).

Materials and Methods

MONCPT nanoemulsion was prepared using Lipoid E80 and cremophor EL as main emulsifiers by microfluidization. The droplet size of the nanoemulsion was measured by dynamic light scattering. In vitro drug release was monitored by membrane dialysis. Kinetics of MONCPT transformed into carboxylic salt was performed in phosphate buffer at different pH. Hemolysis of MONCPT nanoemulsion was conducted in rabbit erythrocytes. Solubilization character of MONCPT in nanoemulsion was experimented using Nile red as a solvatochromic probe. In vitro cytotoxicity of the nanoemulsion was measured in A549 and S180 cells using Sulforhodamine B protein stain method, and suppression rate of tumor growth was investigated in S180-bearing mice. The cell cycle effects of MONCPT nanoemulsion on S180 cells were analyzed by flow cytometry. Distribution of the nanoemulsion in A549 cells and S180-bearing mice were also investigated by fluorescence image.


MONCPT is incorporated in the nanoemulsion in form of lactone with concentration of 489 µg/ml, more than 200 folds higher than that in water. Experiments using Nile red as a solvatochromic probe indicated that more MONCPT might be located in the interfacial surfactant layer of the nanoemulsion than that in discrete oil droplet or continuous aqueous phase. Nanoemulsion could release MONCPT in a sustained way, and it was further shown to notably postpone the hydrolysis of MONCPT with longer hydrolysis half-life time (11.38 h) in nanoemulsion at pH 7.4 than that of MONCPT solution (4.03 h). No obvious hemolysis was caused by MOCPT nanoemulsion in rabbit erythrocytes. MONCPT nanoemulsion showed a marked increase in cytotoxic activity, 23.6 folds and 28.6 folds in S180 cells and A549 cells respectively via arresting the cell at G2 phase, compared to that induced by MONCPT injection. It correlated well to the in vivo anti-tumor activity of MONCPT nanoemulsion with suppression rate of 93.6%, while that of MONCPT injection was only 24.2% at the same dosage. Moreover, nanoemulsion exhibited enhanced capability of delivering drug into malignant cell’s nucleus in vitro and induced drug accumulation in tumor in S180-bearing mice using in vivo imaging.


The nanoemulsion prepared exhibited an improved MONCPT solubility, stability and anti-tumor activity, providing a promising carrier for cancer chemotherapy using MONCPT.


10-Methoxy-9-nitrocamptothecin (MONCPT) anti-tumor fluorescence nanoemulsion nile red 



The study was supported in part by Scientific Research Fund of Ministry of Health-Medical Science Critical Technological Program of Zhejiang Province, by the Health Bureau of Zhejiang Province Foundation (No. WKJ2008-2-029), Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents, and Zhejiang Provincial Natural Science Foundation of China (Y207259).


  1. 1.
    C. H. Takimoto, J. Wright, and S. G. Arbuck. Clinical applications of the camptothecins. Biochim. Biophys. Acta. 1400:107–119 (1998).PubMedGoogle Scholar
  2. 2.
    O. Lavergne, and D. C. Bigg. The other camptothecins: recent advances with camptothecin analogues other than irinotecan and topotecan. Bull. Cancer. 12:51–58 (1998).Google Scholar
  3. 3.
    U. Vanhoefer, W. Achterrath, S. Cao, S. Seeber, and Y. M. Rustum. Irinotecan in the treatment of colorectal cancer: clinical overview. J. Clin. Oncol. 19:1501–1518 (2001).PubMedGoogle Scholar
  4. 4.
    Z. S. Cao, K. Armstrong, M. Shaw, E. Petry, and N. Harris. Nitration of camptothecin with various inorganic nitrate salts in concentrated sulfuric acid: a new preparation of anticancer drug 9-nitrocamptothecin. Synthesis. 12:1724–1730 (1998). doi: 10.1055/s-1998-2207.CrossRefGoogle Scholar
  5. 5.
    M. C. Wani, A. W. Nicholas, and M. E. Wall. Plant antitumor agents .23. Synthesis and antileukemic activity of camptothecin analogues. J. Med. Chem. 29:2858–2863 (1986). doi: 10.1021/jm00161a035.CrossRefGoogle Scholar
  6. 6.
    M. Gao, K. D. Miller, G. W. Sledgeb, and Q. H. Zheng. Radiosynthesis of carbon-11-labeled camptothecin derivatives as potential positron emission tomography tracers for imaging of topoisomerase I in cancers. Bioorg. Med. Chem. Lett. 15:3865–3869 (2005). doi: 10.1016/j.bmcl.2005.05.108.PubMedCrossRefGoogle Scholar
  7. 7.
    X. C. Yang, P. H. Luo, B. Yang, and Q. J. He. Antiangiogenesis response of endothelial cells to the antitumour drug 10-methoxy-9-nitrocamptothecin. Pharmacol. Res. 54:334–340 (2006). doi: 10.1016/j.phrs.2006.06.001.PubMedCrossRefGoogle Scholar
  8. 8.
    P. H. Luo, Q. J. He, X. G. He, Y. Z. Hu, W. Lu, Y. Y. Cheng, and B. Yang. Potent antitumor activity of 10-methoxy-9-nitrocamptothecin. Mol. Cancer. Ther. 54:962–968 (2006). doi: 10.1158/1535-7163.MCT-05-0385.CrossRefGoogle Scholar
  9. 9.
    C. J. Thomas, N. J. Rahier, and S. M. Hecht. Camptothecin: current perspectives. Bioorg. Med. Chem. 12:1585–1604 (2004). doi: 10.1016/j.bmc.2003.11.036.PubMedCrossRefGoogle Scholar
  10. 10.
    J. J. Zhou, J. Liu, and B. Xu. Relationship between lactone ring forms of HCPT and their antitumor activities. Acta. Pharmacol. Sin. 22:827–830 (2001).PubMedGoogle Scholar
  11. 11.
    R. P. Hertzberg, M. J. Caranfa, K. G. Holden, D. R. Jakas, G. Gallagher, M. R. Mattern, S. M. Mong, J. O. Bartus, R. K. Johnson, and W. D. Kingsbury. Modification of the hydroxy lactone ring of camptothecin: inhibition of mammalian topoisomerase I and biological activity. J. Med. Chem. 32:715–720 (1989). doi: 10.1021/jm00123a038.PubMedCrossRefGoogle Scholar
  12. 12.
    S. Kawakami, F. Yamashita, and M. Hasida. Disposition characteristics of emulsions and incorporated drugs after systemic or local injection. Adv. Drug Deliv. Rev. 45:77–88 (2000). doi: 10.1016/S0169-409X(00)00102-2.PubMedCrossRefGoogle Scholar
  13. 13.
    P. P. Constantinides, K. J. Lambert, A. K. Tustian, B. Schneider, S. Lalji, W. Ma, B. Wentzel, D. Kessler, D. Worah, and S. C. Quay. Formulation development and antitumor activity of filter-sterilizable emulsion of paclitaxel. Pharm. Res. 17:175–182 (2000). doi: 10.1023/A:1007565230130.PubMedCrossRefGoogle Scholar
  14. 14.
    J. Rossi, S. Giasson, M. N. Khalid, P. Delmas, C. Allen, and J. C. Leroux. Long-circulation poly (ethylene glycol)-coated emulsions to target solid tumors. Eur. J. Pharm. Biopharm. 67:329–338 (2007). doi: 10.1016/j.ejpb.2007.03.016.PubMedCrossRefGoogle Scholar
  15. 15.
    K. Zurowska-Pryczkowska, M. Sznitowska, and S. Janicki. Studies on the effect of pilocarplne incorporation into a submicron emulsion on the stability of the drug and the vehicle. Eur. J. Pharm. Biopharm. 47:255–260 (1999). doi: 10.1016/S0939-6411(98)00098-8.PubMedCrossRefGoogle Scholar
  16. 16.
    M. J. Lawrence, and G. D. Rees. Microemulsion-based media as novel drug delivery systems. Adv. Drug Deliv. Rev. 45:89–121 (2000). doi: 10.1016/S0169-409X(00)00103-4.PubMedCrossRefGoogle Scholar
  17. 17.
    O. Sonneville-Aubrun, J. T. Simonnet, and F. l’Alloret. Nanoemulsions: a new vehicle for skincare products. Adv. Colloid Interface Sci. 108–109:145–149 (2004). doi: 10.1016/j.cis.2003.10.026.PubMedCrossRefGoogle Scholar
  18. 18.
    L. A. Pires, R. Hegg, C. J. Valduga, S. R. Graziani, D. G. Rodrigues, and R. C. Maranhao. Use of cholesterol-rich nanoparticles that bind to lipoprotein receptors as a vehicle to paclitaxel in the treatment of breast cancer: pharmacokinetics, tumor uptake and a pilot clinical study. Cancer Chemother Pharmacol., in press (2008)
  19. 19.
    D. Attivi. Formulation of insulin-loaded polymeric nanoparticles using response surface methodology. Drug DeV. Ind. Pharm. 31:179–189 (2005). doi: 10.1081/DDC-200047802.PubMedCrossRefGoogle Scholar
  20. 20.
    L. Copolovici, and U. Niinemets. Salting-in and salting-out effects of ionic and neutral osmotica on limonene and linalool Henry’s law constants and octanol/water partition coefficients. Chemosphere. 69:621–629 (2007). doi: 10.1016/j.chemosphere.2007.02.066.PubMedCrossRefGoogle Scholar
  21. 21.
    M. Y. Levy, and S. Benita. Drug release from submicronized o/w emulsion: new in vitro kinetic evaluation model. Int. J. Pharm. 66:29–37 (1990). doi: 10.1016/0378-5173(90)90381-D.CrossRefGoogle Scholar
  22. 22.
    M. B. Moshe, S. Magdassi, Y. Cohen, and L. Avram. Structure of microemulsions with Gemini surfactant studied by solvatochromic probe and diffusion NMR. J. Colloid Interface Sci. 276:221–226 (2004). doi: 10.1016/j.jcis.2004.03.015.PubMedCrossRefGoogle Scholar
  23. 23.
    P. Skehan, D. Scudiero, R. Storeng, A. Monks, J. McMahon, D. Vistica, J. T. Warren, H. Bokesch, S. Kenney, and M. R. Boyd. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Nat. Cancer Inst. 82:1107–1112 (1990). doi: 10.1093/jnci/82.13.1107.PubMedCrossRefGoogle Scholar
  24. 24.
    S. S. Rane, and B. D. Anderson. What determines drug solubility in lipid vehicles: is it predictable. Adv. Drug Del. Rev. 60:638–656 (2008). doi: 10.1016/j.addr.2007.10.015.CrossRefGoogle Scholar
  25. 25.
    C. Malcolmson, C. Satra, S. Kantaria, A. Sidhu, and M. J. Lawrence. Effect of oil on the level of solubilization of testosterone propionate into nonionic oil-in-water microemulsions. J. Pharm. Sci. 87:109–116 (1998). doi: 10.1021/js9700863.PubMedCrossRefGoogle Scholar
  26. 26.
    H. Kunieda, N. Masuda, and K. Tsubone. Comparison between phase behavior of anionic dimeric (Gemini-Type) and monomeric surfactants in water and water-oil. Langmuir. 16:6438–6444 (2000). doi: 10.1021/la0001068.CrossRefGoogle Scholar
  27. 27.
    F. Liu, and D. Liu. Long-circulating emulsions (oil-in-water) as carriers for lipophilic drugs. Pharm. Res. 12:1060–1064 (1995). doi: 10.1023/A:1016274801930.PubMedCrossRefGoogle Scholar
  28. 28.
    D. F. Zhong, K. Li, J. H. Xu, Y. Du, and Y. F. Zhang. Pharmacokinetics of 9-nitro-20(S)-camptothecin in rats. Acta Pharmacol. Sin. 24:256–262 (2003).PubMedGoogle Scholar
  29. 29.
    P. K. Ghosh, and R. S. R. Murthy. Microemulsions: a potential drug delivery system. Curr. Drug Deliv. 3:167–180 (2006). doi: 10.2174/156720106776359168.PubMedCrossRefGoogle Scholar
  30. 30.
    J. B. Tagne, S. Kakumanu, D. Ortiz, T. Shea, and R. J. Nicolosi. A nanoemulsion formulation of tamoxifen increases its efficacy in a breast cancer cell line. Mol. Pharm. 5:280–286 (2008). doi: 10.1021/mp700091j.PubMedCrossRefGoogle Scholar
  31. 31.
    T. Masuda, H. Akita, T. Nishio, K. Niikura, K. Kogure, K. Ijiro, and H. Harashima. Development of lipid particles targeted via sugar-lipid conjugates as novel nuclear gene delivery system. Biomaterials. 29:709–723 (2008). doi: 10.1016/j.biomaterials.2007.09.039.PubMedCrossRefGoogle Scholar
  32. 32.
    W. Junping, K. Takayama, T. Nagai, and Y. Maitani. Pharmacokinetics and antitumor effects of vincristine carried by microemulsions composed of PEG-lipid, oleic acid, vitamin E and cholesterol. Int. J. Pharm. 251:13–21 (2003). doi: 10.1016/S0378-5173(02)00580-X.PubMedCrossRefGoogle Scholar
  33. 33.
    B. A. Teicher. Molecular targets and cancer therapeutics: discovery, development and clinical validation. Drug Resist Updat. 3:67–73 (2000). doi: 10.1054/drup.2000.0123.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Min Han
    • 1
  • Cai-Xia He
    • 1
  • Qiu-Li Fang
    • 1
  • Xiao-Chun Yang
    • 2
  • Yuan-Yuan Diao
    • 1
  • Dong-Hang Xu
    • 3
  • Qiao-Jun He
    • 2
  • Yong-Zhou Hu
    • 4
  • Wen-Quan Liang
    • 1
  • Bo Yang
    • 2
  • Jian-Qing Gao
    • 1
  1. 1.Institute of Pharmaceutics, College of Pharmaceutical SciencesZhejiang UniversityHangzhouChina
  2. 2.Institute of Pharmacology & Toxicology and Biochemical Pharmacy, College of Pharmaceutical SciencesZhejiang UniversityHangzhouChina
  3. 3.2nd Affiliated Hospital of Zhejiang University School of MedicineHangzhouChina
  4. 4.Institute of Material Medica, College of Pharmaceutical SciencesZhejiang UniversityHangzhouChina

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