Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Formulation of two lipid-based membrane–core nanoparticles for FOLFOX combination therapy

Nature Protocols volume 17, pages 1818–1831 (2022)Cite this article

Subjects

Abstract

FOLFOX is a combination of folinic acid (FnA), 5-fluorouracil (5-Fu) and oxaliplatin (OxP). It has been used as the standard treatment for colorectal cancer (CRC) and hepatocellular carcinoma (HCC). This treatment is effective, but its high toxicity is dose limiting, and the drugs need to be taken for a long time. To lower the toxicity so that higher doses can be administered with minimal side effects, two lipid-based membrane–core (MC) nanoformulations, Nano-Folox and Nano-FdUMP, have recently been developed by using the nanoprecipitation technique. The combination of Nano-Folox (containing platinum drug and FnA) and Nano-FdUMP (containing fluorine drug) significantly improves the antitumor effect against CRC and HCC relative to FOLFOX (the combination of free drugs), resulting in long-term survival of animals without significant toxic signs. Here, we describe two formulation protocols. First, for Nano-Folox, a Pt(DACH)•FnA nanoprecipitate is formed by [Pt(DACH)(H2O)2]2+ (the active form of OxP) and FnA2−, and the resultant nanoprecipitate is encapsulated inside the lipid nanoparticles (NPs) modified with the PEGylated aminoethyl anisamide (AEAA, a targeting ligand for sigma-1 receptor overexpressing on CRC and HCC). Second, for Nano-FdUMP, FdUMP (the active metabolite of 5-Fu) is entrapped inside the amorphous Ca3(PO4)2 nanoprecipitate, and the resultant Ca3(PO4)2•FdUMP nanoprecipitate is encapsulated into the AEAA-targeted PEGylated lipid NPs. The procedures for Nano-Folox and Nano-FdUMP take ~17 h and ~4 h, respectively (~17 h if they are prepared simultaneously). Procedures for the physicochemical (~30 h) and cytotoxic (~54 h) characterization are also described.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Schematic representation of Nano-Folox formulated in microemulsions by using the nanoprecipitation technique.
Fig. 2: Schematic representation of Nano-FdUMP developed in microemulsions by using the nanoprecipitation technique.
Fig. 3: The characteristic outline of Nano-Folox and Nano-FdUMP.
Fig. 4: Experimental outline for formulation of Nano-Folox.
Fig. 5: Experimental outline for formulation of Nano-FdUMP.
Fig. 6: Physicochemical and cytotoxic characterization for Nano-Folox and Nano-FdUMP.
Fig. 7: The concept of Nano-FOLFOX.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the references listed in Related links. Further information is available from the corresponding author upon request.

References

  1. Ghosh Chaudhuri, R. & Paria, S. Core/shell nanoparticles: classes, properties, synthesis mechanisms, characterization, and applications. Chem. Rev. 112, 2373–2433 (2012).

    Article  CAS  Google Scholar 

  2. Guo, J. & Huang, L. Membrane-core nanoparticles for cancer nanomedicine. Adv. Drug Deliv. Rev. 156, 23–39 (2020).

    Article  CAS  Google Scholar 

  3. Li, J., Chen, Y. C., Tseng, Y. C., Mozumdar, S. & Huang, L. Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery. J. Control. Release 142, 416–421 (2010).

    Article  CAS  Google Scholar 

  4. Li, J., Yang, Y. & Huang, L. Calcium phosphate nanoparticles with an asymmetric lipid bilayer coating for siRNA delivery to the tumor. J. Control. Release 158, 108–114 (2012).

    Article  CAS  Google Scholar 

  5. Goodwin, T. J. et al. Liver specific gene immunotherapies resolve immune suppressive ectopic lymphoid structures of liver metastases and prolong survival. Biomaterials 141, 260–271 (2017).

    Article  CAS  Google Scholar 

  6. Hu, K. et al. Quercetin remodels the tumor microenvironment to improve the permeation, retention, and antitumor effects of nanoparticles. ACS Nano 11, 4916–4925 (2017).

    Article  CAS  Google Scholar 

  7. Hu, M. et al. Relaxin gene delivery mitigates liver metastasis and synergizes with check point therapy. Nat. Commun. 10, 2993 (2019).

    Article  Google Scholar 

  8. Allegra, C. J. et al. Bevacizumab in stage II-III colon cancer: 5-year update of the National Surgical Adjuvant Breast and Bowel Project C-08 trial. J. Clin. Oncol. 31, 359–364 (2013).

    Article  CAS  Google Scholar 

  9. Qin, S. et al. Randomized, multicenter, open-label study of oxaliplatin plus fluorouracil/leucovorin versus doxorubicin as palliative chemotherapy in patients with advanced hepatocellular carcinoma from Asia. J. Clin. Oncol. 31, 3501–3508 (2013).

    Article  CAS  Google Scholar 

  10. Kuebler, J. P. et al. Oxaliplatin combined with weekly bolus fluorouracil and leucovorin as surgical adjuvant chemotherapy for stage II and III colon cancer: results from NSABP C-07. J. Clin. Oncol. 25, 2198–2204 (2007).

    Article  CAS  Google Scholar 

  11. Tournigand, C. et al. OPTIMOX1: a randomized study of FOLFOX4 or FOLFOX7 with oxaliplatin in a stop-and-Go fashion in advanced colorectal cancer—a GERCOR study. J. Clin. Oncol. 24, 394–400 (2006).

    Article  CAS  Google Scholar 

  12. Guo, J., Yu, Z., Das, M. & Huang, L. Nano codelivery of oxaliplatin and folinic acid achieves synergistic chemo-immunotherapy with 5-fluorouracil for colorectal cancer and liver metastasis. ACS Nano 14, 5075–5089 (2020).

    Article  CAS  Google Scholar 

  13. Guo, J. et al. Two nanoformulations induce reactive oxygen species and immunogenetic cell death for synergistic chemo-immunotherapy eradicating colorectal cancer and hepatocellular carcinoma. Mol. Cancer 20, 10 (2021).

    Article  CAS  Google Scholar 

  14. Gustavsson, B. et al. A review of the evolution of systemic chemotherapy in the management of colorectal cancer. Clin. Colorectal Cancer 14, 1–10 (2015).

    Article  CAS  Google Scholar 

  15. Banerjee, R., Tyagi, P., Li, S. & Huang, L. Anisamide-targeted stealth liposomes: a potent carrier for targeting doxorubicin to human prostate cancer cells. Int. J. Cancer 112, 693–700 (2004).

    Article  CAS  Google Scholar 

  16. Liu, Y. et al. Nanocarrier-mediated immunogenic chemotherapy for triple negative breast cancer. J. Control. Release 323, 431–441 (2020).

    Article  CAS  Google Scholar 

  17. Chen, Y. et al. Vasodilator hydralazine promotes nanoparticle penetration in advanced desmoplastic tumors. ACS Nano 13, 1751–1763 (2019).

    CAS  PubMed  Google Scholar 

  18. Das, M., Shen, L., Liu, Q., Goodwin, T. J. & Huang, L. Nanoparticle delivery of RIG-I agonist enables effective and safe adjuvant therapy in pancreatic cancer. Mol. Ther. 27, 507–517 (2019).

    Article  CAS  Google Scholar 

  19. Johnstone, T. C., Suntharalingam, K. & Lippard, S. J. The next generation of platinum drugs: targeted Pt(II) agents, nanoparticle delivery, and Pt(IV) prodrugs. Chem. Rev. 116, 3436–3486 (2016).

    Article  CAS  Google Scholar 

  20. Guo, S. et al. Lipid-coated cisplatin nanoparticles induce neighboring effect and exhibit enhanced anticancer efficacy. ACS Nano 7, 9896–9904 (2013).

    Article  CAS  Google Scholar 

  21. Guo, S. et al. Turning a water and oil insoluble cisplatin derivative into a nanoparticle formulation for cancer therapy. Biomaterials 35, 7647–7653 (2014).

    Article  CAS  Google Scholar 

  22. Guo, S. et al. Co-delivery of cisplatin and rapamycin for enhanced anticancer therapy through synergistic effects and microenvironment modulation. ACS Nano 8, 4996–5009 (2014).

    Article  CAS  Google Scholar 

  23. Miao, L., Guo, S., Zhang, J., Kim, W. Y. & Huang, L. Nanoparticles with precise ratiometric co-loading and co-delivery of gemcitabine monophosphate and cisplatin for treatment of bladder cancer. Adv. Funct. Mater. 24, 6601–6611 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by an NIH grant (CA198999, to L.H.). We thank O. Gololobova at the University of North Carolina at Chapel Hill for assistance with ICP-MS and thank Z. Cong at Jilin University for assistance with HPLC.

Author information

Authors and Affiliations

  1. School of Pharmaceutical Sciences, Jilin University, Changchun, China

    Jianfeng Guo

  2. Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA

    Leaf Huang

Authors
  1. Jianfeng Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Leaf Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization and writing and editing, L.H. and J.G.; funding acquisition, L.H. Both authors read and approved the final manuscript.

Corresponding author

Correspondence to Leaf Huang.

Ethics declarations

Competing interests

L.H. is a consultant for PDS Biotechnology, Samyang Biopharmaceutical Co. and Stemirna. J.G. declares no competing interests.

Peer review

Peer review information

Nature Protocols thanks James Moon, Guangyu Zhu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Guo, J. et al. ACS Nano 14, 5075–5089 (2020): https://doi.org/10.1021/acsnano.0c01676

Guo, J. et al. Mol. Cancer 20, 10 (2021): https://doi.org/10.1186/s12943-020-01297-0

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Guo, J., Huang, L. Formulation of two lipid-based membrane–core nanoparticles for FOLFOX combination therapy. Nat Protoc 17, 1818–1831 (2022). https://doi.org/10.1038/s41596-022-00698-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-022-00698-3

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research