Skip to main content

Advertisement

SpringerLink
Log in

A Simple and Improved Active Loading Method to Efficiently Encapsulate Staurosporine into Lipid-Based Nanoparticles for Enhanced Therapy of Multidrug Resistant Cancer

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

This study was aimed at developing a new active loading method to stably encapsulate staurosporine (STS), a water insoluble drug, into lipid-based nanoparticles (LNPs) for drug targeting to tumors.

Methods

A limited amount of DMSO was included during the active loading process to prevent precipitation and facilitate the loading of insoluble STS into the aqueous core of a LNP. The drug loading kinetics under various conditions was studied and the STS-LNPs were characterized by size, drug-to-lipid ratio, drug release kinetics and in vitro potency. The antitumor efficacy of the STS-LNPs was compared with free STS in a mouse model.

Results

The drug loading efficiency reached 100% within 15 min of incubation at a drug-to-lipid ratio of 0.31 (mol) via an ammonium gradient. STS formed nano-aggregates inside the aqueous core of the LNPs and was stably retained upon storage and in the presence of serum. A 3-fold higher dose of the STS-LNPs could be tolerated by BALB/c mice compared with free STS, leading to nearly complete growth inhibition of a multidrug resistant breast tumor, while free STS only exhibited moderate activity.

Conclusion

This simple and efficient drug loading method produced a stable LNP formulation for STS that was effective for cancer treatment.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

AUC:

Area under the concentration curve

CHOL:

Cholesterol

Cl:

Clearance

DMEM:

Dulbecco’s Modified Eagle’s medium

DSPC:

1,2-distearoyl-sn-glycero-3-phosphatidylcholine

DSPE-PEG:

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly ethyleneglycol)-2000

EPR-effect:

Enhanced permeability and retention effect

FBS:

Fetal bovine serum

LNPs:

Lipid-based nanoparticles

MDR:

Multi-drug resistance

MWCO:

Molecular weight cut-off

PDI:

Polydispersity index

STS:

Staurosporine

t1/2 :

Half life

UPLC:

Ultra-performance liquid chromatography

UPLC-MS/MS:

Ultra-performance liquid chromatography-tandem mass spectrometer

References

  1. Schwartz GK, Redwood SM, Ohnuma T, Holland JF, Droller MJ, Liu BC. Inhibition of invasion of invasive human bladder carcinoma cells by protein kinase C inhibitor staurosporine. J Natl Cancer Inst. 1990;82:1753–6.

    Article  CAS  PubMed  Google Scholar 

  2. Akinaga S, Gomi K, Morimoto M, Tamaoki T, Okabe M. Antitumor activity of UCN-01, a selective inhibitor of protein kinase C, in murine and human tumor models. Cancer Res. 1991;51:4888–92.

    CAS  PubMed  Google Scholar 

  3. Li T, Christensen SD, Frankel PH, Margolin KA, Agarwala SS, Luu T, et al. A phase II study of cell cycle inhibitor UCN-01 in patients with metastatic melanoma: a California Cancer Consortium trial. Investig New Drugs. 2012;30:741–8.

    Article  Google Scholar 

  4. Allen TM, Cullis PR. Liposomal drug delivery systems: from concept to clinical applications. Adv Drug Deliv Rev. 2013;65:36–48.

    Article  CAS  PubMed  Google Scholar 

  5. Hwang SH, Maitani Y, Qi XR, Takayama K, Nagai T. Remote loading of diclofenac, insulin and fluorescein isothiocyanate labeled insulin into liposomes by pH and acetate gradient methods. Int J Pharm. 1999;179:85–95.

    Article  CAS  PubMed  Google Scholar 

  6. Avnir Y, Ulmansky R, Wasserman V, Even-Chen S, Broyer M, Barenholz Y, et al. Amphipathic weak acid glucocorticoid prodrugs remote-loaded into sterically stabilized nanoliposomes evaluated in arthritic rats and in a Beagle dog: a novel approach to treating autoimmune arthritis. Arthritis Rheum. 2008;58:119–29.

    Article  CAS  PubMed  Google Scholar 

  7. Mayer LD, Bally MB, Cullis PR. Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient. Biochim Biophys Acta. 1986;857:123–6.

    Article  CAS  PubMed  Google Scholar 

  8. Gubernator J. Active methods of drug loading into liposomes: recent strategies for stable drug entrapment and increased in vivo activity. Expert Opin Drug Deliv. 2011;8:565–80.

    Article  CAS  PubMed  Google Scholar 

  9. Drummond DC, Noble CO, Guo Z, Hong K, Park JW, Kirpotin DB. Development of a highly active nanoliposomal irinotecan using a novel intraliposomal stabilization strategy. Cancer Res. 2006;66:3271–7.

    Article  CAS  PubMed  Google Scholar 

  10. Fujisawa T, Miyai H, Hironaka K, Tsukamoto T, Tahara K, Tozuka Y, et al. Liposomal diclofenac eye drop formulations targeting the retina: formulation stability improvement using surface modification of liposomes. Int J Pharm. 2012;436:564–7.

    Article  CAS  PubMed  Google Scholar 

  11. Belliveau NM, Huft J, Lin PJ, Chen S, Leung AK, Leaver TJ, et al. Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA. Mol Ther–Nucleic Acids. 2012;1, e37.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Avnir Y, Turjeman K, Tulchinsky D, Sigal A, Kizelsztein P, Tzemach D, et al. Fabrication principles and their contribution to the superior in vivo therapeutic efficacy of nano-liposomes remote loaded with glucocorticoids. PLoS One. 2011;6, e25721.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ramsay E, Alnajim J, Anantha M, Taggar A, Thomas A, Edwards K, et al. Transition metal-mediated liposomal encapsulation of irinotecan (CPT-11) stabilizes the drug in the therapeutically active lactone conformation. Pharm Res. 2006;23:2799–808.

    Article  CAS  PubMed  Google Scholar 

  14. 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;473:528–35.

    Article  CAS  PubMed  Google Scholar 

  15. Pisal PB, Joshi MA, Padamwar MN, Patil SS, Pokharkar VB. Probing influence of methodological variation on active loading of acetazolamide into nanoliposomes: biophysical, in vitro, ex vivo, in vivo and rheological investigation. Int J Pharm. 2014;461:82–8.

    Article  CAS  PubMed  Google Scholar 

  16. Lipka D, Gubernator J, Filipczak N, Barnert S, Suss R, Legut M, et al. Vitamin C-driven epirubicin loading into liposomes. Int J Nanomedicine. 2013;8:3573–85.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Gubernator J, Chwastek G, Korycinska M, Stasiuk M, Grynkiewicz G, Lewrick F, et al. The encapsulation of idarubicin within liposomes using the novel EDTA ion gradient method ensures improved drug retention in vitro and in vivo. J Control Release. 2010;146:68–75.

    Article  CAS  PubMed  Google Scholar 

  18. Pinto AC, Angelo S, Moreira JN, Simoes S. Development, characterization and in vitro evaluation of single or co-loaded imatinib mesylate liposomal formulations. J Nanosci Nanotechnol. 2012;12:2891–900.

    Article  CAS  PubMed  Google Scholar 

  19. Yang Y, Ma Y, Wang S. A novel method to load topotecan into liposomes driven by a transmembrane NH(4)EDTA gradient. Eur J Pharm Biopharm. 2012;80:332–9.

    Article  CAS  PubMed  Google Scholar 

  20. Gubernator J, Lipka D, Korycinska M, Kempinska K, Milczarek M, Wietrzyk J, et al. Efficient human breast cancer xenograft regression after a single treatment with a novel liposomal formulation of epirubicin prepared using the EDTA ion gradient method. PLoS One. 2014;9, e91487.

    Article  PubMed  PubMed Central  Google Scholar 

  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;54:987–92.

    CAS  PubMed  Google Scholar 

  22. Barenholz Y. Doxil(R)—the first FDA-approved nano-drug: lessons learned. J Control Release. 2012;160:117–34.

    Article  CAS  PubMed  Google Scholar 

  23. Drummond DC, Noble CO, Guo Z, Hayes ME, Park JW, Ou CJ, et al. Improved pharmacokinetics and efficacy of a highly stable nanoliposomal vinorelbine. J Pharmacol Exp Ther. 2009;328:321–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mukthavaram R, Jiang P, Saklecha R, Simberg D, Bharati IS, Nomura N, et al. High-efficiency liposomal encapsulation of a tyrosine kinase inhibitor leads to improved in vivo toxicity and tumor response profile. Int J Nanomedicine. 2013;8:3991–4006.

    PubMed  PubMed Central  Google Scholar 

  25. Roy A, Murakami M, Ernsting MJ, Hoang B, Undzys E, Li SD. Carboxymethylcellulose-based and docetaxel-loaded nanoparticles circumvent P-glycoprotein-mediated multidrug resistance. Mol Pharm. 2014;11:2592–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Patel KJ, Tannock IF. The influence of P-glycoprotein expression and its inhibitors on the distribution of doxorubicin in breast tumors. BMC Cancer. 2009;9:356.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Zhigaltsev IV, Winters G, Srinivasulu M, Crawford J, Wong M, Amankwa L, et al. Development of a weak-base docetaxel derivative that can be loaded into lipid nanoparticles. J Control Release. 2010;144:332–40.

    Article  CAS  PubMed  Google Scholar 

  28. Tagami T, Ernsting MJ, Li SD. Efficient tumor regression by a single and low dose treatment with a novel and enhanced formulation of thermosensitive liposomal doxorubicin. J Control Release. 2011;152:303–9.

    Article  CAS  PubMed  Google Scholar 

  29. de Smet M, Langereis S, van den Bosch S, Grull H. Temperature-sensitive liposomes for doxorubicin delivery under MRI guidance. J Control Release. 2010;143:120–7.

    Article  PubMed  Google Scholar 

  30. Meyer T, Regenass U, Fabbro D, Alteri E, Röusel J, Möller M, et al. A derivative of staurosporine (CGP 41 251) shows selectivity for protein kinase C inhibition and In vitro anti-proliferative as well as In vivo anti-tumor activity. Int J Cancer. 1989;43:851–6.

    Article  CAS  PubMed  Google Scholar 

  31. Burkholder T, Foltz C, Karlsson E, Linton CG, Smith JM. Health evaluation of experimental laboratory mice. Curr Protoc Mouse Biol. 2012;2:145–65.

    PubMed  PubMed Central  Google Scholar 

  32. Roy A, Ernsting MJ, Undzys E, Li S-D. A highly tumor-targeted nanoparticle of podophyllotoxin penetrated tumor core and regressed multidrug resistant tumors. Biomaterials. 2015;52:335–46.

    Article  CAS  PubMed  Google Scholar 

  33. Dos Santos N, Cox KA, McKenzie CA, van Baarda F, Gallagher RC, Karlsson G, et al. pH gradient loading of anthracyclines into cholesterol-free liposomes: enhancing drug loading rates through use of ethanol. Biochim Biophys Acta. 1661;2004:47–60.

    Google Scholar 

  34. Tschaikowsky K, Brain JD. Staurosporine encapsulated into pH-sensitive liposomes reduces tnf production and increases survival in rat endotoxin shock. Shock. 1994;1:401–7.

    Article  CAS  PubMed  Google Scholar 

  35. Yamauchi M, Kusano H, Nakakura M, Kato Y. Reducing the impact of binding of UCN-01 to human alpha1-acid glycoprotein by encapsulation in liposomes. Biol Pharm Bull. 2005;28:1259–64.

    Article  CAS  PubMed  Google Scholar 

  36. Sur S, Fries AC, Kinzler KW, Zhou S, Vogelstein B. Remote loading of preencapsulated drugs into stealth liposomes. Proc Natl Acad Sci U S A. 2014;111:2283–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Shigehiro T, Kasai T, Murakami M, Sekhar SC, Tominaga Y, Okada M, et al. Efficient drug delivery of Paclitaxel glycoside: a novel solubility gradient encapsulation into liposomes coupled with immunoliposomes preparation. PLoS One. 2014;9, e107976.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Modi S, Xiang TX, Anderson BD. Enhanced active liposomal loading of a poorly soluble ionizable drug using supersaturated drug solutions. J Control Release. 2012;162:330–9.

    Article  CAS  PubMed  Google Scholar 

  39. Joguparthi V, Xiang TX, Anderson BD. Liposome transport of hydrophobic drugs: gel phase lipid bilayer permeability and partitioning of the lactone form of a hydrophobic camptothecin, DB-67. J Pharm Sci. 2008;97:400–20.

    Article  CAS  PubMed  Google Scholar 

  40. Park K. Active liposomal loading of a poorly soluble ionizable drug. J Control Release. 2012;162:475.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments and Disclosures

This work was funded by grants from the Canadian Institutes of Health Research (MOP-119471 and PPP-130153). S.D. Li is a recipient of a Coalition to Cure Prostate Cancer Young Investigator Award from the Prostate Cancer Foundation and a New Investigator Award from Canadian Institutes of Health Research (MSH-130195).

Author information

Authors and Affiliations

  1. Faculty of Pharmaceutical Science, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada

    Wei-Lun Tang, Weihsu Claire Chen, Aniruddha Roy & Shyh-Dar Li

  2. Drug Discovery Program, Ontario Institute for Cancer Research, 101 College Street, Suite 800, Toronto, Ontario, M5G 0A3, Canada

    Wei-Lun Tang, Aniruddha Roy, Elijus Undzys & Shyh-Dar Li

Authors
  1. Wei-Lun Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Weihsu Claire Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aniruddha Roy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elijus Undzys
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shyh-Dar Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shyh-Dar Li.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. 1

(DOCX 61 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tang, WL., Chen, W.C., Roy, A. et al. A Simple and Improved Active Loading Method to Efficiently Encapsulate Staurosporine into Lipid-Based Nanoparticles for Enhanced Therapy of Multidrug Resistant Cancer. Pharm Res 33, 1104–1114 (2016). https://doi.org/10.1007/s11095-015-1854-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-015-1854-4

KEY WORDS

Search

Navigation