Pharmaceutical Research

, 35:13 | Cite as

Optimization of Weight Ratio for DSPE-PEG/TPGS Hybrid Micelles to Improve Drug Retention and Tumor Penetration

  • Ya Jin
  • Zimei Wu
  • Caibin Li
  • Weisai Zhou
  • John P. Shaw
  • Bruce C. Baguley
  • Jianping LiuEmail author
  • Wenli ZhangEmail author
Research Paper



To enhance therapeutic efficacy and prevent phlebitis caused by Asulacrine (ASL) precipitation post intravenous injection, ASL-loaded hybrid micelles with size below 40 nm were developed to improve drug retention and tumor penetration.


ASL-micelles were prepared using different weight ratios of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol-2000 (DSPE-PEG2000) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) polymers. Stability of micelles was optimized in terms of critical micelle concentration (CMC) and drug release properties. The encapsulation efficiency (EE) and drug loading were determined using an established dialysis-mathematic fitting method. Multicellular spheroids (MCTS) penetration and cytotoxicity were investigated on MCF-7 cell line. Pharmacokinetics of ASL-micelles was evaluated in rats with ASL-solution as control.


The ASL-micelles prepared with DSPE-PEG2000 and TPGS (1:1, w/w) exhibited small size (~18.5 nm), higher EE (~94.12%), better sustained in vitro drug release with lower CMC which may be ascribed to the interaction between drug and carriers. Compared to free ASL, ASL-micelles showed better MCTS penetration capacity and more potent cytotoxicity. Pharmacokinetic studies demonstrated that the half-life and AUC values of ASL-micelles were approximately 1.37-fold and 3.49-fold greater than that of free ASL.


The optimized DSPE-PEG2000/TPGS micelles could serve as a promising vehicle to improve drug retention and penetration in tumor.


asulacrine CMC drug retention hybrid micelles multicellular spheroids penetration 





Asulacrine micelles


Confocal laser scanning microscope


Critical micelle concentration


Drug loading


Dulbecco’s modified Eagle’s media


Extracellular matrix


Encapsulation efficiency


Fetal bovine serum


Fluorescein isothiocyanate isomer I


Multicellular tumor spheroids


3-(4, 5-Dimethylthia-zol-2-yl) -2, 5-diphenyltetrazolium bromide


Polydispersity index


Transmission electron microscopy



This study was financially supported by the National Science Foundation Grant of China (No. 81503005), the Natural Science Fundation of Jiangsu Province (No. BK20140669), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and National Science and Technology Major Project (No. 2017YFA0205400). The authors declare no competing financial interest.


  1. 1.
    Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature. 2000;407(6801):249–57.CrossRefPubMedGoogle Scholar
  2. 2.
    Milosevic M, Fyles A, Hedley D, Hill R. The human tumor microenvironment: invasive (needle) measurement of oxygen and interstitial fluid pressure. Semin Radiat Oncol. 2004;14(3):249–58.CrossRefPubMedGoogle Scholar
  3. 3.
    Pluen A, Boucher Y, Ramanujan S, Mckee TD, Gohongi T, Di TE, et al. Role of tumor-host interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors. Proc Natl Acad Sci U S A. 2001;98(8):4628–33.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Kim TH, Mount CW, Gombotz WR, Pun SH. The delivery of doxorubicin to 3-D multicellular spheroids and tumors in a murine xenograft model using tumor-penetrating triblock polymeric micelles. Biomaterials. 2010;31(28):7386–97.CrossRefPubMedGoogle Scholar
  5. 5.
    Jang SH, Wientjes MG, Lu D, Au JL. Drug delivery and transport to solid tumors. Pharm Res. 2003;20(9):1337–50.CrossRefPubMedGoogle Scholar
  6. 6.
    Lim HJ, Masin D, Mcintosh NL, Madden TD, Bally MB. Role of drug release and liposome-mediated drug delivery in governing the therapeutic activity of liposomal mitoxantrone used to treat human A431 and LS180 solid tumors. J Pharmacol Exp Ther. 2000;292(1):337–45.PubMedGoogle Scholar
  7. 7.
    Lim HJ, Masin D, Madden TD, Bally MB. Influence of drug release characteristics on the therapeutic activity of liposomal mitoxantrone. J Pharmacol Exp Ther. 1997;281(1):566–73.PubMedGoogle Scholar
  8. 8.
    Paxton JW, Kim SN, Whitfield LR. Pharmacokinetic and toxicity scaling of the antitumor agents amsacrine and CI-921, a new analogue, in mice, rats, rabbits, dogs, and humans. Cancer Res. 1990;50(9):2692–7.PubMedGoogle Scholar
  9. 9.
    Schneider E, Darkin SJ, Lawson PA, Ching L-M, Ralph RK, Baguley BC. Cell line selectivity and DNA breakage properties of the antitumour agent N-[2-(dimethylamino) ethyl] acridine-4-carboxamide: role of DNA topoisomerase II. Eur J Cancer Clin Oncol. 1988;24(11):17831789–71790.CrossRefGoogle Scholar
  10. 10.
    Baguley BC. The possible role of electron-transfer complexes in the antitumour action of amsacrine analogues. Biophys Chem. 1990;35(2–3):203–12.CrossRefPubMedGoogle Scholar
  11. 11.
    Covey JM, Kohn KW, Kerrigan D, Tilchen EJ, Pommier Y. Topoisomerase II-mediated DNA damage produced by 4′-(9-acridinylamino)-methanesulfon-m-anisidide and related acridines in L1210 cells and isolated nuclei: relation to cytotoxicity. Cancer Res. 1988;48(4):860–5.PubMedGoogle Scholar
  12. 12.
    Fyfe D, Price C, Langley R, Pagonis C, Houghton J, Osborne L, et al. A phase I trial of amsalog (CI-921) administered by intravenous infusion using a 5-day schedule. Cancer Chemother Pharmacol. 2001;47(4):333–7.CrossRefPubMedGoogle Scholar
  13. 13.
    Sklarin NT, Wiernik PH, Grove WR, Benson L, Mittelman A, Maroun JA, et al. A phase II trial of CI-921 in advanced malignancies. Investig New Drugs. 1992;10(4):309–12.CrossRefGoogle Scholar
  14. 14.
    Afzal A, Sarfraz M, Wu Z, Wang G, Sun J. Integrated scientific data bases review on asulacrine and associated toxicity. Crit Rev Oncol Hematol. 2016;104:78–86.CrossRefPubMedGoogle Scholar
  15. 15.
    Zhang W, Wang G, See E, Shaw JP, Baguley BC, Liu J, et al. Post-insertion of poloxamer 188 strengthened liposomal membrane and reduced drug irritancy and in vivo precipitation, superior to PEGylation. J Control Release. 2015;203:161–9.CrossRefPubMedGoogle Scholar
  16. 16.
    Zhang W, Falconer JR, Baguley BC, Shaw JP, Kanamala M, Xu H, et al. Improving drug retention in liposomes by aging with the aid of glucose. Int J Pharm. 2016;505(1–2):194–203.CrossRefPubMedGoogle Scholar
  17. 17.
    Greish K, Sawa T, Fang J, Akaike T, Maeda H. SMA-doxorubicin, a new polymeric micellar drug for effective targeting to solid tumours. J Control Release. 2004;97(2):219–30.CrossRefPubMedGoogle Scholar
  18. 18.
    Lukyanov AN, Hartner WC, Torchilin VP. Increased accumulation of PEG-PE micelles in the area of experimental myocardial infarction in rabbits. J Control Release. 2004;94(1):187–93.CrossRefPubMedGoogle Scholar
  19. 19.
    Attia ABE, Ong ZY, Hedrick JL, Lee PP, Ee PLR, Hammond PT, et al. Mixed micelles self-assembled from block copolymers for drug delivery. Curr Opin Colloid In. 2011;16(3):182–94.CrossRefGoogle Scholar
  20. 20.
    Owen SC, Chan DPY, Shoichet MS. Polymeric micelle stability. Nano Today. 2012;7(1):53–65.CrossRefGoogle Scholar
  21. 21.
    Sawant RR, Sawant RM, Torchilin VP. Mixed PEG–PE/vitamin E tumor-targeted immunomicelles as carriers for poorly soluble anti-cancer drugs: improved drug solubilization and enhanced in vitro cytotoxicity. Eur J Pharm Biopharm. 2008;70(1):51–7.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Hui T, Chen D, Jiang M. A one-step approach to the highly efficient preparation of core-stabilized polymeric micelles with a mixed shell formed by two incompatible polymers. Macromolecules. 2005;38(13):5834–7.CrossRefGoogle Scholar
  23. 23.
    Kim SH, Tan JP, Nederberg F, Fukushima K, Yang YY, Waymouth RM, et al. Mixed micelle formation through stereocomplexation between enantiomeric poly (lactide) block copolymers. Macromolecules. 2008;42(1):25–9.CrossRefGoogle Scholar
  24. 24.
    Zhou W, Li C, Wang Z, Zhang W, Liu J. Factors affecting the stability of drug-loaded polymeric micelles and strategies for improvement. J Nanopart Res. 2016;18(9):275.CrossRefGoogle Scholar
  25. 25.
    Dabholkar RD, Sawant RM, Mongayt DA, Devarajan PV, Torchilin VP. Polyethylene glycol-phosphatidylethanolamine conjugate (PEG-PE)-based mixed micelles: some properties, loading with paclitaxel, and modulation of P-glycoprotein-mediated efflux. Int J Pharm. 2006;315(2):148–57.CrossRefPubMedGoogle Scholar
  26. 26.
    Wei Z, Hao J, Yuan S, Li Y, Juan W, Sha X, et al. Paclitaxel-loaded Pluronic P123/F127 mixed polymeric micelles: formulation, optimization and in vitro characterization. Int J Pharm. 2009;376(1–2):176–85.CrossRefPubMedGoogle Scholar
  27. 27.
    Wang AT, Liang DS, Liu YJ, Qi XR. Roles of ligand and TPGS of micelles in regulating internalization, penetration and accumulation against sensitive or resistant tumor and therapy for multidrug resistant tumors. Biomaterials. 2015;53:160–72.CrossRefPubMedGoogle Scholar
  28. 28.
    Gill KK, Kaddoumi A, Nazzal S. Mixed micelles of PEG(2000)-DSPE and vitamin-E TPGS for concurrent delivery of paclitaxel and parthenolide: enhanced chemosenstization and antitumor efficacy against non-small cell lung cancer (NSCLC) cell lines. Eur J Pharm Sci. 2012;46(1–2):64–71.CrossRefPubMedGoogle Scholar
  29. 29.
    Yan H, Wei P, Song J, Jia X, Zhang Z. Enhanced anticancer activity in vitro and in vivo of luteolin incorporated into long-circulating micelles based on DSPE-PEG2000 and TPGS. J Pharm Pharmacol. 2016;68(10):1290–8.CrossRefPubMedGoogle Scholar
  30. 30.
    Jin Y, Zhang Z, Zhao T, Liu X, Jian L. Mixed micelles of doxorubicin overcome multidrug resistance by inhibiting the expression of P-glycoprotein. J Biomed Nanotechnol. 2015;11(8):1330.CrossRefPubMedGoogle Scholar
  31. 31.
    Wang Y, Fan W, Dai X, Katragadda U, Mckinley D, Teng Q, et al. Enhanced tumor delivery of gemcitabine via PEG-DSPE/TPGS mixed micelles. Mol Pharm. 2014;11(4):1140–50.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Liang J, Wu WL, Xu XD, Zhuo RX, Zhang XZ. pH responsive micelle self-assembled from a new amphiphilic peptide as anti-tumor drug carrier. Colloid Surface B. 2014;114(8):398–403.CrossRefGoogle Scholar
  33. 33.
    Chen KH, Sabatino MD, Albertini B, Passerini N, Kett VL. The effect of polymer coatings on physicochemical properties of spray-dried liposomes for nasal delivery of BSA. Eur J Pharm Sci. 2013;50(3–4):312–22.CrossRefPubMedGoogle Scholar
  34. 34.
    Xu WH, Han M, Dong Q, Fu ZX, Diao YY, Liu H, et al. Doxorubicin-mediated radiosensitivity in multicellular spheroids from a lung cancer cell line is enhanced by composite micelle encapsulation. Int J Nanomedicine. 2012;2012(default):2661–71.Google Scholar
  35. 35.
    Zhang W, Wang G, Falconer JR, Baguley BC, Shaw JP, Liu J, et al. Strategies to maximize liposomal drug loading for a poorly water-soluble anticancer drug. Pharm Res. 2015;32(4):1451–61.CrossRefPubMedGoogle Scholar
  36. 36.
    See E, Zhang W, Liu J, Svirskis D, Baguley BC, Shaw JP, et al. Physicochemical characterization of asulacrine towards the development of an anticancer liposomal formulation via active drug loading: stability, solubility, lipophilicity and ionization. Int J Pharm. 2014;473(1–2):528–35.CrossRefPubMedGoogle Scholar
  37. 37.
    Rapoport N. Physical stimuli-responsive polymeric micelles for anti-cancer drug delivery. Prog Polym Sci. 2007;32(8–9):962–90.CrossRefGoogle Scholar
  38. 38.
    Permprasert J, Devahastin S. Evaluation of the effects of some additives and pH on surface tension of aqueous solutions using a drop-weight method. J Food Eng. 2005;70(2):219–26.CrossRefGoogle Scholar
  39. 39.
    Ravindra P. A critical review: surface and interfacial tension measurement by the drop weight method. Chem Eng Commun. 2007;195(8):889–924.Google Scholar
  40. 40.
    Yip D, Cho CH. A multicellular 3D heterospheroid model of liver tumor and stromal cells in collagen gel for anti-cancer drug testing. Biochem Biophys Res Commun. 2013;433(3):327–32.CrossRefPubMedGoogle Scholar
  41. 41.
    Zhang W, Li C, Baguley BC, Zhou F, Zhou W, Shaw JP, et al. Optimization of the formation of embedded multicellular spheroids of MCF-7 cells: how to reliably produce a biomimetic 3D model. Anal Biochem. 2016;515:47–54.CrossRefPubMedGoogle Scholar
  42. 42.
    O'brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem. 2000;267(17):5421–6.CrossRefPubMedGoogle Scholar
  43. 43.
    Rampersad SN. Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors. 2012;12(9):12347–60.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Van HH, Baumans V, Brandt CJ, Hesp AP, Sturkenboom JH, van Lith HA. Orbital sinus blood sampling in rats as performed by different animal technicians: the influence of technique and expertise. Lab Anim. 1998;32(4):377–86.CrossRefGoogle Scholar
  45. 45.
    Parasuraman S, Raveendran R, Kesavan R. Blood sample collection in small laboratory animals. J Pharmacol Pharmacother. 2010;1(2):87–93.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Ganta S, Paxton JW, Baguley BC, Garg S. Formulation and pharmacokinetic evaluation of an asulacrine nanocrystalline suspension for intravenous delivery. Int J Pharm. 2009;367(1–2):179–86.CrossRefPubMedGoogle Scholar
  47. 47.
    Ernsting MJ, Murakami M, Roy A, Li SD. Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles. J Control Release. 2013;172(3):782–94.CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Zhu H, Chen H, Zeng X, Wang Z, Zhang X, Wu Y, et al. Co-delivery of chemotherapeutic drugs with vitamin E TPGS by porous PLGA nanoparticles for enhanced chemotherapy against multi-drug resistance. Biomaterials. 2014;35(7):2391–400.CrossRefPubMedGoogle Scholar
  49. 49.
    Qian J, Gao X. Triblock copolymer-encapsulated nanoparticles with outstanding colloidal stability for siRNA delivery. ACS Appl Mater Interfaces. 2013;5(8):2845–52.CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Sezgin Z, Yüksel N, Baykara T. Preparation and characterization of polymeric micelles for solubilization of poorly soluble anticancer drugs. Eur J Pharm Biopharm. 2006;64(3):261–8.CrossRefPubMedGoogle Scholar
  51. 51.
    Mi Y, Liu Y, Feng SS. Formulation of Docetaxel by folic acid-conjugated d-α-tocopheryl polyethylene glycol succinate 2000 (Vitamin E TPGS(2k)) micelles for targeted and synergistic chemotherapy. Biomaterials. 2011;32(16):4058–66.CrossRefPubMedGoogle Scholar
  52. 52.
    Chen L, Xiao Z, Meng Y, Zhao Y, Han J, Su G, et al. The enhancement of cancer stem cell properties of MCF-7 cells in 3D collagen scaffolds for modeling of cancer and anti-cancer drugs. Biomaterials. 2012;33(5):1437–44.CrossRefPubMedGoogle Scholar
  53. 53.
    Lee J, Lilly GD, Doty RC, Podsiadlo P, Kotov NA. In vitro toxicity testing of nanoparticles in 3D cell culture. Small. 2009;5(10):1213–21.PubMedGoogle Scholar
  54. 54.
    Wan F, You J, Sun Y, Zhang XG, Cui FD, YZ D, et al. Studies on PEG-modified SLNs loading vinorelbine bitartrate (I): preparation and evaluation in vitro. Int J Pharm. 2008;359(1–2):104–10.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ya Jin
    • 1
  • Zimei Wu
    • 2
  • Caibin Li
    • 1
  • Weisai Zhou
    • 1
  • John P. Shaw
    • 2
  • Bruce C. Baguley
    • 3
  • Jianping Liu
    • 1
    Email author
  • Wenli Zhang
    • 1
    Email author
  1. 1.Department of PharmaceuticsChina Pharmaceutical UniversityNanjingPeople’s Republic of China
  2. 2.School of PharmacyUniversity of AucklandAucklandNew Zealand
  3. 3.Auckland Cancer Society Research CentreUniversity of AucklandAucklandNew Zealand

Personalised recommendations