Pharmaceutical Research

, Volume 32, Issue 3, pp 1028–1044 | Cite as

Peripherally Cross-Linking the Shell of Core-Shell Polymer Micelles Decreases Premature Release of Physically Loaded Combretastatin A4 in Whole Blood and Increases its Mean Residence Time and Subsequent Potency Against Primary Murine Breast Tumors After IV Administration

  • Rajesh R. Wakaskar
  • Sai Praneeth R. Bathena
  • Shailendra B. Tallapaka
  • Vishakha V. Ambardekar
  • Nagsen Gautam
  • Rhishikesh Thakare
  • Samantha M. Simet
  • Stephen M. Curran
  • Rakesh K. Singh
  • Yuxiang Dong
  • Joseph A. Vetro
Research Paper



Determine the feasibility and potential benefit of peripherally cross-linking the shell of core-shell polymer micelles on the premature release of physically loaded hydrophobic drug in whole blood and subsequent potency against solid tumors.


Individual Pluronic F127 polymer micelles (F127 PM) peripherally cross-linked with ethylenediamine at 76% of total PEO blocks (X-F127 PM) were physically loaded with combretastatin A4 (CA4) by the solid dispersion method and compared to CA4 physically loaded in uncross-linked F127 PM, CA4 in DMSO in vitro, or water-soluble CA4 phosphate (CA4P) in vivo.


X-F127 PM had similar CA4 loading and aqueous solubility as F127 PM up to 10 mg CA4 / mL at 22.9 wt% and did not aggregate in PBS or 90% (v/v) human serum at 37°C for at least 24 h. In contrast, X-F127 PM decreased the unbound fraction of CA4 in whole blood (fu) and increased the mean plasma residence time and subsequent potency of CA4 against the vascular function and growth of primary murine 4T1 breast tumors over CA4 in F127 PM and water-soluble CA4P after IV administration.


Given that decreasing the fu is an indication of decreased drug release, peripherally cross-linking the shell of core-shell polymer micelles may be a simple approach to decrease premature release of physically loaded hydrophobic drug in the blood and increase subsequent potency in solid tumors.


drug delivery peripheral shell cross-linking pluronic F127 poloxamer 407 polymer nanocarriers premature drug release vascular disrupting agents 


4 T1

Breast tumor epithelial cells from BALB/c mice

4 T1-Luc

4 T1 cells that stably express luciferase


Atomic force microscopy


Combretastatin A4


Combretastatin A4 Phosphate / Fosbretabulin disodium


Dynamic light scattering


Dimethyl sulfoxide


N,N’-Disuccinimidyl carbonate


Electric cell-substrate impedance sensing




Pluronic F127 (Poloxamer 407)

F127 PM

Pluronic F127 polymer micelles


Unbound fraction of drug in whole blood


Human umbilical vein endothelial cells


In vivo imaging system


DSC-activated Pluronic F127


DSC-activated Pluronic F127 polymer micelles

X-F127 PM

Pluronic F127 polymer micelle shell cross-linked with ED at 76%



This work was supported by NIH COBRE grant 2P20GM103480-06 (Nebraska Center for Nanomedicine) (RRW, RKS, JAV), NIH 1U54CA163120-01 grant (RKS), and UNMC Predoctoral Fellowships (SPRB, ST, VVA). The Nanoimaging Core Facility was supported by the NIH (SIG program), the UNMC Program of Excellence (POE), and the Nebraska Research Initiative (NRI). The Authors would also like to acknowledge Todd A. Wyatt, PhD and the VA Nebraska-Western Iowa Health Care System Research Service for providing access to and assistance with the Electric Cell Impedance Sensing apparatus.

Supplementary material

11095_2014_1515_Fig12_ESM.gif (52 kb)
Fig. S1

Key peak assignments for 1H-NMR spectra of NHS-activated F127 alone or F127 polymer micelles with exclusively monovalent or divalent (cross-linked) conjugation of ethylenediamine (ED). (A) NHS-activated F127 (NHS-F127) or polymer micelles of NHS-F127 reacted with (B) a 5/1 molar ratio of ED/NHS-F127 that produced only monovalent ED conjugation to PEO blocks of F127 PM (Monovalent ED) or (C) a 1/1 molar ratio of ED/NHS-F127 that produced only divalent ED conjugation (i.e., cross-linking) between PEO blocks of F127 PM (Divalent ED) were compared by 1H-NMR (500 MHz, CDCl3, 1 mg/mL). NHS-F127: δ 1.01–1.26 (m, 195H, peak 5), 2.85 (s, 8H, peak 2), 3.25–3.82 (m, ca. 991H, peak 3), 4.46 (t, J = 5.2 Hz, 4H, peak 1); F127 PM with monovalent and/or divalent ED conjugation: δ 1.01–1.26 (m, 195H, peak 5), 3.12 (brs, peak 4), 3.25–3.82 (m, ca. 991H, peak 3), 4.22 (brs, peak 1A). The proton peak at 4.22 ppm (peak 1A) represents both monovalent and divalent ED conjugation and the proton peak at 3.12 ppm (peak 4) represents monovalent ED conjugation alone. (GIF 52.3 kb)

11095_2014_1515_MOESM1_ESM.tif (987 kb)
High Resolution Image (TIFF 987 kb)
11095_2014_1515_Fig13_ESM.gif (39 kb)
Fig. S2

Effect of peripherally cross-linking the shell of F127 PM on the pharmacokinetics of physically loaded CA4 after IV administration in 4 T1 tumor-bearing mice. Uncross-linked F127 polymer micelles (F127 PM) or F127 PM individually cross-linked with ED at 76% of total PEO blocks (X-F127 PM) were loaded with CA4 at 22.9 wt% as described in Fig. 5. 4 T1 cells stably expressing luciferase (4 T1-Luc) were injected SQ into the mammary fat pad of female BALB/c mice and grown until ~100 mm3 before treatment. Water-soluble CA4P (closed circles) or CA4 loaded in F127 PM (closed squares) or X-F127 PM (cross-hatched squares) was then injected IV (1 mg / kg) and the average concentration of CA4 in plasma ± SEM (n = 6 mice / time point) was determined by LC-MS/MS. (GIF 39.4 kb)

11095_2014_1515_MOESM2_ESM.tif (99 kb)
High Resolution Image (TIFF 98.9 kb)
11095_2014_1515_MOESM3_ESM.docx (13 kb)
ESM 3 (DOCX 13 kb)
11095_2014_1515_MOESM4_ESM.docx (14 kb)
ESM 4 (DOCX 13.9 kb)
11095_2014_1515_MOESM5_ESM.docx (15 kb)
ESM 5 (DOCX 15 kb)
11095_2014_1515_MOESM6_ESM.docx (14 kb)
ESM 6 (DOCX 14 kb)


  1. 1.
    Allen LV, Popovich NG, Ansel HC. Parenterals. In. Ansel’s pharmaceutical dosage forms and drug delivery systems. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins; 2011. p. 432–3.Google Scholar
  2. 2.
    Williams HD, Trevaskis NL, Charman SA, Shanker RM, Charman WN, Pouton CW, et al. Strategies to address low drug solubility in discovery and development. Pharmacol Rev. 2013;65(1):315–499.CrossRefPubMedGoogle Scholar
  3. 3.
    Teicher BA. Anticancer Drug Development Guide: Preclinical Screening, Clinical Trials, and Approval: Humana Press; 1997.Google Scholar
  4. 4.
    Yalkowsky SH. Techniques of Solubilization of Drugs: Books on Demand; 1993.Google Scholar
  5. 5.
    Batrakova EV, Bronich TK, Vetro JA, Kabanov AV. Polymeric Micelles as Drug Carriers. In: Torchilin VP, (Ed). Nanoparticulates as Drug Carriers London: Imperial College Press; 2006. p. 57–93.Google Scholar
  6. 6.
    Torchilin VP. Structure and design of polymeric surfactant-based drug delivery systems. J Control Release: Off J Control Release Soc. 2001;73(2–3):137–72.CrossRefGoogle Scholar
  7. 7.
    Kataoka K, Harada A, Nagasaki Y. Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv Drug Deliv Rev. 2001;47(1):113–31.CrossRefPubMedGoogle Scholar
  8. 8.
    Kabanov AV, Alakhov VY. Pluronic block copolymers in drug delivery: from micellar nanocontainers to biological response modifiers. Crit Rev Ther Drug Carrier Syst. 2002;19(1):1–72.CrossRefPubMedGoogle Scholar
  9. 9.
    Adams ML, Lavasanifar A, Kwon GS. Amphiphilic block copolymers for drug delivery. J Pharm Sci. 2003;92(7):1343–55.CrossRefPubMedGoogle Scholar
  10. 10.
    Duncan R. The dawning era of polymer therapeutics. Nat Rev Drug Discov. 2003;2(5):347–60.CrossRefPubMedGoogle Scholar
  11. 11.
    Bulmus V, Woodward M, Lin L, Murthy N, Stayton P, Hoffman A. A new pH-responsive and glutathione-reactive, endosomal membrane-disruptive polymeric carrier for intracellular delivery of biomolecular drugs. J Control Release. 2003;93(2):105–20.CrossRefPubMedGoogle Scholar
  12. 12.
    Veronese FM, Morpurgo M. Bioconjugation in pharmaceutical chemistry. Farmaco. 1999;54(8):497–516.CrossRefPubMedGoogle Scholar
  13. 13.
    D’Souza AJ, Topp EM. Release from polymeric prodrugs: linkages and their degradation. J Pharm Sci. 2004;93(8):1962–79.CrossRefPubMedGoogle Scholar
  14. 14.
    Bae Y, Fukushima S, Harada A, Kataoka K. Design of environment-sensitive supramolecular assemblies for intracellular drug delivery: Polymeric micelles that are responsive to intracellular pH change. Angew Chem Int Ed. 2003;42(38):4640–3. S4640/4641-S4640/4611.CrossRefGoogle Scholar
  15. 15.
    Torchilin VP. Micellar nanocarriers: pharmaceutical perspectives. Pharm Res. 2007;24(1):1–16.CrossRefPubMedGoogle Scholar
  16. 16.
    Hurter PN, Hatton TA. Solubilization of polycyclic aromatic hydrocarbons by poly (ethylene oxide-propylene oxide) block copolymer micelles: effects of polymer structure. Langmuir. 1992;8(5):1291–9.CrossRefGoogle Scholar
  17. 17.
    Nagarajan R, Ganesh K. Comparison of solubilization of hydrocarbons in (PEO-PPO) diblock versus (PEO-PPO-PEO) triblock copolymer micelles. J Colloid Interface Sci. 1996;184(2):489–99.CrossRefPubMedGoogle Scholar
  18. 18.
    Xing L, Mattice WL. Strong solubilization of small molecules by triblock-copolymer micelles in selective solvents. Macromolecules. 1997;30(6):1711–7.CrossRefGoogle Scholar
  19. 19.
    Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res. 1986;46(12 Pt 1):6387–92.PubMedGoogle Scholar
  20. 20.
    Danson S, Ferry D, Alakhov V, Margison J, Kerr D, Jowle D, et al. Phase I dose escalation and pharmacokinetic study of pluronic polymer-bound doxorubicin (SP1049C) in patients with advanced cancer. Br J Cancer. 2004;90(11):2085–91.PubMedCentralPubMedGoogle Scholar
  21. 21.
    Gabizon A, Catane R, Uziely B, Kaufman B, Safra T, Cohen R, et al. Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes. Cancer Res. 1994;54(4):987–92.PubMedGoogle Scholar
  22. 22.
    Savic R, Eisenberg A, Maysinger D. Block copolymer micelles as delivery vehicles of hydrophobic drugs: micelle-cell interactions. J Drug Target. 2006;14(6):343–55.CrossRefPubMedGoogle Scholar
  23. 23.
    Matsumura Y, Hamaguchi T, Ura T, Muro K, Yamada Y, Shimada Y, et al. Phase I clinical trial and pharmacokinetic evaluation of NK911, a micelle-encapsulated doxorubicin. Br J Cancer. 2004;91(10):1775–81.CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    van Cornelus FN. Covalently cross-linked amphiphilic block copolymer micelles. Soft Matter. 2011;7(7):3246–59.CrossRefGoogle Scholar
  25. 25.
    Thurmond II KB, Kowalewski T, Wooley KL. Water-soluble knedel-like structures: the preparation of shell-cross-linked small particles. J Am Chem Soc. 1996;118(30):7239–40.CrossRefGoogle Scholar
  26. 26.
    O’Reilly RK, Hawker CJ, Wooley KL. Cross-linked block copolymer micelles: functional nanostructures of great potential and versatility. Chem Soc Rev. 2006;35(11):1068–83.CrossRefPubMedGoogle Scholar
  27. 27.
    Read ES, Armes SP. Recent advances in shell cross-linked micelles. Chem Commun (Cambridge, U K). 2007(29):3021–3035.Google Scholar
  28. 28.
    Li Y, Xiao K, Luo J, Xiao W, Lee JS, Gonik AM, et al. Well-defined, reversible disulfide cross-linked micelles for on-demand paclitaxel delivery. Biomater. 2011;32(27):6633–45.CrossRefGoogle Scholar
  29. 29.
    Lee S-Y, Kim S, Tyler JY, Park K, Cheng J-X. Blood-stable, tumor-adaptable disulfide bonded mPEG-(Cys)4-PDLLA micelles for chemotherapy. Biomater. 2013;34(2):552–61.CrossRefGoogle Scholar
  30. 30.
    Koo AN, Min KH, Lee HJ, Lee SU, Kim K, Kwon IC, et al. Tumor accumulation and antitumor efficacy of docetaxel-loaded core-shell-corona micelles with shell-specific redox-responsive cross-links. Biomater. 2012;33(5):1489–99.CrossRefGoogle Scholar
  31. 31.
    Kwon SH, Kim SY, Ha KW, Kang MJ, Huh JS, Im TJ, et al. Pharmaceutical evaluation of genistein-loaded pluronic micelles for oral delivery. Arch Pharm Res. 2007;30(9):1138–43.CrossRefPubMedGoogle Scholar
  32. 32.
    Aliabadi HM, Brocks DR, Mahdipoor P, Lavasanifar A. A novel use of an in vitro method to predict the in vivo stability of block copolymer based nano-containers. J Control Release: Off J Control Release Soc. 2007;122(1):63–70.CrossRefGoogle Scholar
  33. 33.
    Zhao D, Richer E, Antich PP, Mason RP. Antivascular effects of combretastatin A4 phosphate in breast cancer xenograft assessed using dynamic bioluminescence imaging and confirmed by MRI. FASEB J. 2008;22(7):2445–51.CrossRefPubMedGoogle Scholar
  34. 34.
    Ambardekar VV, Wakaskar RR, Sharma B, Bowman J, Vayaboury W, Singh RK, et al. The efficacy of nuclease-resistant Chol-siRNA in primary breast tumors following complexation with PLL-PEG(5K). Biomater. 2013;34(20):4839–48.CrossRefGoogle Scholar
  35. 35.
    Cooney MM, van Heeckeren W, Bhakta S, Ortiz J, Remick SC. Drug insight: vascular disrupting agents and angiogenesis–novel approaches for drug delivery. Nat Clin Pract Oncol. 2006;3(12):682–92.CrossRefPubMedGoogle Scholar
  36. 36.
    Horsman MR, Siemann DW. Pathophysiologic effects of vascular-targeting agents and the implications for combination with conventional therapies. Cancer Res. 2006;66(24):11520–39.CrossRefPubMedGoogle Scholar
  37. 37.
    Giaever I, Keese CR. Monitoring fibroblast behavior in tissue culture with an applied electric field. Proc Natl Acad Sci U S A. 1984;81(12):3761–4.CrossRefPubMedCentralPubMedGoogle Scholar
  38. 38.
    Lundien MC, Mohammed KA, Nasreen N, Tepper RS, Hardwick JA, Sanders KL, et al. Induction of MCP-1 expression in airway epithelial cells: role of CCR2 receptor in airway epithelial injury. J Clin Immunol. 2002;22(3):144–52.CrossRefPubMedGoogle Scholar
  39. 39.
    Zudaire E, Cuesta N, Murty V, Woodson K, Adams L, Gonzalez N, et al. The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers. J Clin Invest. 2008;118(2):640–50.PubMedCentralPubMedGoogle Scholar
  40. 40.
    Tiruppathi C, Malik AB, Del Vecchio PJ, Keese CR, Giaever I. Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function. Proc Natl Acad Sci U S A. 1992;89(17):7919–23.CrossRefPubMedCentralPubMedGoogle Scholar
  41. 41.
    Grosios K, Holwell SE, McGown AT, Pettit GR, Bibby MC. In vivo and in vitro evaluation of combretastatin A-4 and its sodium phosphate prodrug. Br J Cancer. 1999;81(8):1318–27.CrossRefPubMedCentralPubMedGoogle Scholar
  42. 42.
    Galbraith SM, Chaplin DJ, Lee F, Stratford MR, Locke RJ, Vojnovic B, et al. Effects of combretastatin A4 phosphate on endothelial cell morphology in vitro and relationship to tumour vascular targeting activity in vivo. Anticancer Res. 2001;21(1A):93–102.PubMedGoogle Scholar
  43. 43.
    Hug TS. Biophysical methods for monitoring cell-substrate interactions in drug discovery. Assay Drug Dev Technol. 2003;1(3):479–88.CrossRefPubMedGoogle Scholar
  44. 44.
    Ryu J, Jeong YI, Kim IS, Lee JH, Nah JW, Kim SH. Clonazepam release from core-shell type nanoparticles of poly(epsilon-caprolactone)/poly (ethylene glycol)/poly (epsilon-caprolactone) triblock copolymers. Int J Pharm. 2000;200(2):231–42.CrossRefPubMedGoogle Scholar
  45. 45.
    Alexandridis P, Holzwarth JF, Hatton TA. Micellization of Poly (ethylene oxide)-Poly (propylene oxide)-Poly (ethylene oxide) Triblock Copolymers in Aqueous Solutions: Thermodynamics of Copolymer Association. Macromolecules. 1994;27(9):2414–25.CrossRefGoogle Scholar
  46. 46.
    Chen H, Kim S, Li L, Wang S, Park K, Cheng JX. Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Forster resonance energy transfer imaging. Proc Natl Acad Sci U S A. 2008;105(18):6596–601.CrossRefPubMedCentralPubMedGoogle Scholar
  47. 47.
    Hinderling PH. Red blood cells: a neglected compartment in pharmacokinetics and pharmacodynamics. Pharmacol Rev. 1997;49(3):279–95.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Rajesh R. Wakaskar
    • 2
  • Sai Praneeth R. Bathena
    • 2
  • Shailendra B. Tallapaka
    • 2
  • Vishakha V. Ambardekar
    • 3
  • Nagsen Gautam
    • 2
  • Rhishikesh Thakare
    • 2
  • Samantha M. Simet
    • 4
  • Stephen M. Curran
    • 2
  • Rakesh K. Singh
    • 1
    • 5
  • Yuxiang Dong
    • 2
  • Joseph A. Vetro
    • 1
    • 2
  1. 1.Center for Drug Delivery and NanomedicineUniversity of Nebraska Medical CenterOmahaUSA
  2. 2.Department of Pharmaceutical Sciences, College of PharmacyUniversity of Nebraska Medical CenterOmahaUSA
  3. 3.Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc.Frederick National Laboratory for Cancer ResearchFrederickUSA
  4. 4.Department of Pulmonary, Critical Care, Sleep, & Allergy, College of MedicineUniversity of Nebraska Medical CenterOmahaUSA
  5. 5.Department of Pathology and MicrobiologyUniversity of Nebraska Medical CenterOmahaUSA

Personalised recommendations