AAPS PharmSciTech

, 20:317 | Cite as

Selectivity Enhancement of Paclitaxel Liposome Towards Folate Receptor-Positive Tumor Cells by Ligand Number Optimization Approach

  • Mahendra Kumar Prajapati
  • Aniketh Bishnu
  • Pritha Ray
  • Pradeep R. VaviaEmail author
Research Article


The present work aims to develop folate-targeted paclitaxel liposome (F-PTX-LIP), which will selectively target tumor cells overexpressing folate receptor (FR) and leave normal cells. Liposomes were prepared by thin-film hydration method followed by post-insertion of synthesized ligand 1,2-distearoyl-sn-glycero-phosphoethanolamine-polyethyleneglycol 2000-folic acid (DSPE-PEG2000-FA) on the outer surface of the liposome. The synthesized ligand was evaluated for in vivo acute toxicity in Balb/c mice. Developed liposomal formulations were characterized using transmission electron microscopy (TEM) and small-angle neutron scattering (SANS). We have investigated the effect of ligand number on cell uptake and cytotoxicity by confocal laser scanning microscopy (CLSM), competitive inhibition and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. Compared to lung adenocarcinoma cells (A549), uptake in human ovarian carcinoma cells (SKOV3) was 2.2- and 1.2-fold higher for liposome with 480 and 240 ligand number respectively. Competitive inhibition experiment shows that prior incubation of SKOV3 cells with free folic acid significantly reduced the cell uptake of F-PTX-LIP with 480 ligand number (480 F-PTX-LIP) by 2.6-fold. 480 F-PTX-LIP displays higher cytotoxicity than free drug and PTX liposome. Moreover, it specifically targets the cells with higher folate receptor expression. Optimized 480 F-PTX-LIP formulation can be potentially useful for the treatment of folate receptor-positive tumors.


nanotechnology liposome paclitaxel DSPE-PEG2000-Folate ligand number folate receptor targeting 



folate receptor




paclitaxel liposome


folate-targeted paclitaxel liposome


1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol 2000


1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol 2000-folic acid


particle size


polydispersity index


zeta potential

% EE

percent drug entrapment efficiency

% DL

percent drug loading


transmission electron microscopy


small-angle neutron scattering


differential scanning calorimetry


confocal laser scanning microscopy


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


FR-targeted liposomes


folate receptor-targeted liposomes with no ligand


blank liposome

240 F-LIP

folate receptor-targeted liposomes with 240 ligand per liposome

480 F-LIP

folate receptor-targeted liposomes with 480 ligand per liposome


folate receptor-targeted paclitaxel liposomes with 240 ligand per liposome


folate receptor-targeted paclitaxel liposomes with 480 ligand per liposome


polyethylene glycols


soya phosphatidylcholine LIPOID S100










dynamic light scattering


Fourier transform infrared


position-sensitive detector


Roswell Park Memorial Institute


proton nuclear magnetic resonance


mean fluorescence intensity



The authors would like to acknowledge the Council of Scientific and Industrial Research (CSIR), New Delhi, and Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Mumbai, for providing fellowship. They are also thankful to AICTE-NAFETIC for providing research facilities.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12249_2019_1531_MOESM1_ESM.docx (767 kb)
ESM 1 (DOCX 767 kb)


  1. 1.
    Singla AK, Garg A, Aggarwal D. Paclitaxel and its formulations. Int J Pharm. 2002;235:179–92.CrossRefGoogle Scholar
  2. 2.
    Zhang Z, Mei L, Feng S-S. Paclitaxel drug delivery systems. Expert Opin Drug Deliv. 2013;10:325–40.CrossRefGoogle Scholar
  3. 3.
    Kampan NC, Madondo MT, McNally OM, Quinn M, Plebanski M. Paclitaxel and its evolving role in the management of ovarian cancer. Biomed Res Int. 2015;2015:1–21.CrossRefGoogle Scholar
  4. 4.
    Sgadari C, Toschi E, Palladino C, Barillari G, Carlei D, Cereseto A, et al. Mechanism of paclitaxel activity in Kaposi’s sarcoma. J Immunol. 2000;165:509–17.CrossRefGoogle Scholar
  5. 5.
    Perez EA. Paclitaxel in breast cancer. Women’s Health. 2006;2:11–21.Google Scholar
  6. 6.
    Yoshizawa Y, Kono Y, Ogawara K, Kimura T, Higaki K. PEG liposomalization of paclitaxel improved its in vivo disposition and anti-tumor efficacy. Int J Pharm. 2011;412:132–41.CrossRefGoogle Scholar
  7. 7.
    Yang T, Cui F, Choi M, Cho J. Enhanced solubility and stability of PEGylated liposomal paclitaxel: in vitro and in vivo evaluation. Int J Pharm. 2007;338:317–26.CrossRefGoogle Scholar
  8. 8.
    Gelderblom H, Verweij J, Nooter K, Sparreboom A, Cremophor EL. The drawbacks and advantages of vehicle selection for drug formulation. Eur J Cancer. 2001;37:1590–8.CrossRefGoogle Scholar
  9. 9.
    Szebeni J, Muggia FM, Alving CR. Complement activation by Cremophor EL as a possible contributor to hypersensitivity to paclitaxel: an in vitro study. J Natl Cancer Inst. 1998;90:300–6.CrossRefGoogle Scholar
  10. 10.
    Gogaté US, Schwartz PA, Agharkar SN. Effect of unpurified Cremophor EL on the solution stability of paclitaxel. Pharm Dev Technol. 2009;14:1–8.CrossRefGoogle Scholar
  11. 11.
    Maas B, Huber C, Krgmer I. Plasticizer extraction of Taxol®—infusion solution from various infusion devices. Pharm World Sci. 1996;18:78–82.CrossRefGoogle Scholar
  12. 12.
    Turánek J. Liposomal paclitaxel formulations. J Control Release. 2012;163:322–34.CrossRefGoogle Scholar
  13. 13.
    Perche F, Torchilin VP. Recent trends in multifunctional liposomal nanocarriers for enhanced tumor targeting. J Drug Deliv. 2013;2013:1–32.CrossRefGoogle Scholar
  14. 14.
    Henderson IC, Bhatia V. Nab-paclitaxel for breast cancer: a new formulation with an improved safety profile and greater efficacy. Expert Rev Anticancer Ther. 2007;7:919–43.CrossRefGoogle Scholar
  15. 15.
    Green MR, Manikhas GM, Orlov S, Afanasyev B, Makhson AM, Bhar P, et al. Abraxane®, a novel Cremophor®-free, albumin-bound particle form of paclitaxel for the treatment of advanced non-small-cell lung cancer. Ann Oncol. 2006;17:1263–8.CrossRefGoogle Scholar
  16. 16.
    Ma P, Mumper RJ. Paclitaxel nano-delivery systems: a comprehensive review. J Nanomed Nanotecghnol. 2013;4:1–35.Google Scholar
  17. 17.
    Zhang JA, Anyarambhatla G, Ma L, Ugwu S, Xuan T, Sardone T, et al. Development and characterization of a novel Cremophor EL free liposome-based paclitaxel (LEP-ETU) formulation. Eur J Pharm Biopharm. 2005;59:177–87.CrossRefGoogle Scholar
  18. 18.
    Maurer N, Fenske DB, Cullis PR. Developments in liposomal drug delivery systems. Expert Opin Biol Ther. 2001;1:923–48.CrossRefGoogle Scholar
  19. 19.
    Immordino ML, Dosio F, Cattel L. Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. Int J Nanomedicine. 2006;1:297–315.CrossRefGoogle Scholar
  20. 20.
    Cheng Z, Al Zaki A, Hui JZ, Muzykantov VR, Tsourkas A. Multifunctional nanoparticles: cost versus benefit of adding targeting and imaging capabilities. Science (80-). 2012;338:903–10.CrossRefGoogle Scholar
  21. 21.
    Wilhelm S, Tavares AJ, Dai Q, Ohta S, Audet J, Dvorak HF, et al. Analysis of nanoparticle delivery to tumours. Perspectives (Montclair). 2016;1:1–12.Google Scholar
  22. 22.
    Li J, Wang F, Sun D, Wang R. A review of the ligands and related targeting strategies for active targeting of paclitaxel to tumours. J Drug Target. 2016;24:590–602.CrossRefGoogle Scholar
  23. 23.
    Pattni BS, Torchilin VP. Targeted drug delivery: concepts and design. Target Drug Deliv Concepts Des. 2015.Google Scholar
  24. 24.
    Fernández M, Javaid F, Chudasama V. Advances in targeting the folate receptor in the treatment/imaging of cancers. Chem Sci. 2018;9:790–810.CrossRefGoogle Scholar
  25. 25.
    Saul JM, Annapragada AV, Bellamkonda RV. A dual-ligand approach for enhancing targeting selectivity of therapeutic nanocarriers. J Control Release. 2006;114:277–87.CrossRefGoogle Scholar
  26. 26.
    Bandyopadhyay A, Fine RL, Demento S, Bockenstedt LK, Fahmy TM. The impact of nanoparticle ligand density on dendritic-cell targeted vaccines. Biomaterials. 2011;32:3094–105.CrossRefGoogle Scholar
  27. 27.
    Alkilany AM, Zhu L, Weller H, Mews A, Parak W, Barz M, et al. Ligand density on nanoparticles: a parameter with critical impact on nanomedicine. Adv Drug Deliv Rev. 2019.Google Scholar
  28. 28.
    Liu H, Doane TL, Cheng Y, Lu F, Srinivasan S, Zhu J-J, et al. Control of surface ligand density on PEGylated gold nanoparticles for optimized cancer cell uptake. Part Part Syst Charact. 2015;32:197–204.CrossRefGoogle Scholar
  29. 29.
    Elias DR, Poloukhtine A, Popik V, Tsourkas A. Effect of ligand density, receptor density, and nanoparticle size on cell targeting. Nanomed Nanotechnol Biol Med. 2013;9:194–201.CrossRefGoogle Scholar
  30. 30.
    Cho HY, Lee CK, Lee YB. Preparation and evaluation of PEGylated and folate-PEGylated liposomes containing paclitaxel for lymphatic delivery. J Nanomater. 2015:1–10.Google Scholar
  31. 31.
    Committee for Medicinal Products for Human Use (CHMP)—guideline on the evaluation of anticancer medicinal products in man. Eur Med Agency. 2017;44:1–43.Google Scholar
  32. 32.
    Berlot G. Monitoring of hemostasis. Hemocoagulative Probl Crit Ill Patient. 2011;1–238.Google Scholar
  33. 33.
    Fromell K, Andersson M, Elihn K, Caldwell KD. Nanoparticle decorated surfaces with potential use in glycosylation analysis. Colloids Surf B Biointerfaces. 2005;46:84–91.CrossRefGoogle Scholar
  34. 34.
    Montanari JAM, Bucci PL, Alonso SV, De Biomembranas L, De Quilmes UN, Peña RS, et al. A model based in the radius of vesicles to predict the number of unilamellar liposomes. Int J Res Pharm Chem. 2014;4:484–9.Google Scholar
  35. 35.
    Nilsson T, Lundin CR, Nordlund G, Ädelroth P, Von Ballmoos C, Brzezinski P. Lipid-mediated protein-protein interactions modulate respiration-driven ATP synthesis. Sci Rep Nature Publishing Group. 2016;6:1–11.CrossRefGoogle Scholar
  36. 36.
    Wu J, Liu Q, Lee RJ. A folate receptor-targeted liposomal formulation for paclitaxel. Int J Pharm. 2006;316:148–53.CrossRefGoogle Scholar
  37. 37.
    Xiang G, Wu J, Lu Y, Liu Z, Lee RJ. Synthesis and evaluation of a novel ligand for folate-mediated targeting liposomes. Int J Pharm. 2008;356:29–36.CrossRefGoogle Scholar
  38. 38.
    Yang X, Li Y, Li M, Zhang L, Feng L, Zhang N. Hyaluronic acid-coated nanostructured lipid carriers for targeting paclitaxel to cancer. Cancer Lett. 2013;334:338–45.CrossRefGoogle Scholar
  39. 39.
    Aswal VK, Goyal PS. Small-angle neutron scattering diffractometer at Dhruva reactor. Curr Sci. 2000;79:947–53.Google Scholar
  40. 40.
    Jan BY, Pedersen S. Resolution function and flux at the sample for small-angle X-ray scattering calculated in position-angle-wavelength space. J Appl Crystallogr. 1991;24:893–909.CrossRefGoogle Scholar
  41. 41.
    Foroozandeh P, Aziz AA. Insight into cellular uptake and intracellular trafficking of nanoparticles. Nanoscale Res Lett. 2018;13.Google Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2019

Authors and Affiliations

  • Mahendra Kumar Prajapati
    • 1
  • Aniketh Bishnu
    • 2
  • Pritha Ray
    • 2
  • Pradeep R. Vavia
    • 1
    Email author
  1. 1.Center for Novel Drug Delivery Systems, Department of Pharmaceutical Sciences and Technology, Institute of Chemical TechnologyUniversity Under Section 3 of UGC Act – 1956, Elite Status and Center of Excellence – Govt. of Maharashtra, TEQIP Phase III FundedMumbaiIndia
  2. 2.Advance Centre for Treatment, Research and Education in Cancer, Tata Memorial CentreKhargharIndia

Personalised recommendations