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


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.

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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.

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.


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

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

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

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  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).

    CAS  Article  Google Scholar 

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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.

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Authors and Affiliations



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

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Correspondence to Leaf Huang.

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Competing interests

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

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

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Key references using this protocol

Guo, J. et al. ACS Nano 14, 5075–5089 (2020):

Guo, J. et al. Mol. Cancer 20, 10 (2021):

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Guo, J., Huang, L. Formulation of two lipid-based membrane–core nanoparticles for FOLFOX combination therapy. Nat Protoc (2022).

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