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Development of ionic liquid-coated PLGA nanoparticles for applications in intravenous drug delivery

Nature Protocols volume 18, pages 2509–2557 (2023)Cite this article

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Abstract

Polymeric nanoparticles (NPs) are a promising platform for medical applications in drug delivery. However, their use as drug carriers is limited by biological (e.g., immunological) barriers after intravenous administration. Ionic liquids (ILs), formed from bulky asymmetric cations and anions, have a wide variety of physical internal and external interfacing properties. When assembled on polymeric NPs as biomaterial coatings, these external-interfacing properties can be tuned to extend their circulation half-life when intravenously injected, as well as drive biodistribution to sites of interest for selective organ accumulation. In our work, we are particularly interested in optimizing IL coatings to enable red blood cell hitchhiking in whole blood. In this protocol, we describe the preparation and physicochemical and biological characterization of choline carboxylate IL-coated polymeric NPs. The procedure is divided into five stages: (1) synthesis and characterization of choline-based ILs (1 week); (2) bare poly(lactic-co-glycolic acid) (50:50, acid terminated) Resomer 504H (PLGA) NP assembly, modified from previously established protocols, with dye encapsulation (7 h); (3) modification of the bare particles with IL coating (3 h); (4) physicochemical characterization of both PLGA and IL-PLGA NPs by dynamic light scattering, 1H nuclear magnetic resonance spectroscopy, and transmission electron microscopy (1 week); (5) ex vivo evaluation of intravenous biocompatibility (including serum-protein resistance and hemolysis) and red blood cell hitchhiking in whole BALB/c mouse blood via fluorescence-activated cell sorting (1 week). With practice and technique refinement, this protocol is accessible to late-stage graduate students and early-stage postdoctoral scientists.

Key points

  • Ionic liquids (ILs) are organic salts that stay in liquid form below 100 °C. Examples have been found that promote drug solubility, permeability, and stability. Choline-based ILs are prepared and used to coat polymeric nanoparticles to improve their circulation half-life and biodistribution

  • The biological mechanism is red blood cell hitchiking. The composition of the IL coating can be screened for optimal red blood cell binding of the particles.

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Fig. 1: Experimental guidance for the preparation and synthesis of IL-PLGA DiD NPs and chemical characterization.
Fig. 2: Example range of choline carboxylate IL physical appearance, and representative 1H NMR spectrum of choline 2-hexenoate.
Fig. 3: Appearance, EE (%) and stability of bare PLGA versus IL-coated PLGA DiD-encapsulated NPs (NPs).
Fig. 4: Representative DLS results of IL-coated versus bare PLGA NPs.
Fig. 5: DLS characterization of bare and IL-coated DiD NPs made via the alternative sonication method.
Fig. 6: 1H NMR (top) spectrum and TEM (bottom) characterization of PLGA NPs coated with choline 2-hexenoate29,30,45.
Fig. 7: Representative enhancement of PLGA NP shelf-life stability by choline carboxylate IL29.
Fig. 8: Anticipated results of IL-PLGA NP protein resistance in neat mouse serum over 1 h (ref. 45).
Fig. 9: Sample SDS–PAGE gel for IL-PLGA NP serum protein resistance29.
Fig. 10: Sample RBC hemolysis induced by IL-PLGA NPs30.
Fig. 11: Representative IL-PLGA versus bare PLGA NP RBC hitchhiking in whole BALB/c mouse blood by FACS.
Fig. 12: Representative SEM visualization of IL-PLGA NP RBC hitchhiking in vitro.

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Data availability

The datasets generated during the study have been uploaded to FigShare (https://doi.org/10.6084/m9.figshare.c.6279060.v2).

References

  1. Lü, J. M. et al. Current advances in research and clinical applications of PLGA-based nanotechnology. Expert Rev. Mol. Diagn. 9, 325–341 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Park, J. et al. Enhancement of surface ligand display on PLGA nanoparticles with amphiphilic ligand conjugates. J. Control. Release 156, 109–115 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Manoochehri, S. et al. Surface modification of PLGA nanoparticles via human serum albumin conjugation for controlled delivery of docetaxel. Daru. https://doi.org/10.1186/2008-2231-21-58 (2013).

  4. Amo, L. et al. Surface functionalization of PLGA nanoparticles to increase transport across the BBB for Alzheimer’s disease. Appl. Sci. 11, 4305 (2021).

    Article  Google Scholar 

  5. Rezvantalab, S. et al. PLGA-based nanoparticles in cancer treatment. Front. Pharmacol. 9, 1260 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hashemi, M., Shamshiri, A., Saeedi, M., Tayebi, L. & Yazdian-Robati, R. Aptamer-conjugated PLGA nanoparticles for delivery and imaging of cancer therapeutic drugs. Arch. Biochem. Biophys. 691, 108485 (2020).

    Article  CAS  PubMed  Google Scholar 

  7. Dang, Y. & Guan, J. Nanoparticle-based drug delivery systems for cancer therapy. Smart Mater. Med. 1, 10–19 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Chung, T. W., Tsai, Y. L., Hsieh, J. H. & Tsai, W. J. Different ratios of lactide and glycolide in PLGA affect the surface property and protein delivery characteristics of the PLGA microspheres with hydrophobic additives. J. Microencapsul. 23, 15–27 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Keles, H., Naylor, A., Clegg, F. & Sammon, C. Investigation of factors influencing the hydrolytic degradation of single PLGA microparticles. Polym. Degrad. Stab. 119, 228–241 (2015).

    Article  CAS  Google Scholar 

  10. Amjadi, I., Rabiee, M. & Hosseini, M. S. Anticancer activity of nanoparticles based on PLGA and its co-polymer: in-vitro evaluation. Iran. J. Pharm. Res. 12, 623–634 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Gentile, P., Chiono, V., Carmagnola, I. & Hatton, P. V. An overview of poly(lactic-co-glycolic) acid (PLGA)-based biomaterials for bone tissue engineering. Int. J. Mol. Sci. 15, 3640–3659 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Suk, J. S., Xu, Q., Kim, N., Hanes, J. & Ensign, L. M. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv. Drug Deliv. Rev. https://doi.org/10.1016/j.addr.2015.09.012 (2016).

    Article  PubMed  Google Scholar 

  13. Nie, S. Editorial: understanding and overcoming major barriers in cancer nanomedicine. Nanomedicine 5, 523–528 (2010).

    Article  PubMed  Google Scholar 

  14. Wilhelm, S. et al. Analysis of nanoparticle delivery to tumours. Nat. Rev. Mater. 1, 16014 (2016).

    Article  CAS  Google Scholar 

  15. Rosenblum, D., Joshi, N., Tao, W., Karp, J. M. & Peer, D. Progress and challenges towards targeted delivery of cancer therapeutics. Nat. Commun. 9, 1410 (2018).

  16. Ferrari, R., Sponchioni, M., Morbidelli, M. & Moscatelli, D. Polymer nanoparticles for the intravenous delivery of anticancer drugs: the checkpoints on the road from the synthesis to clinical translation. Nanoscale 10, 22701–22719 (2018).

    Article  CAS  PubMed  Google Scholar 

  17. Barui, A. K., Oh, J. Y., Jana, B., Kim, C. & Ryu, J. Cancer‐targeted nanomedicine: overcoming the barrier of the protein corona. Adv. Ther. 3, 1900124 (2020).

  18. Foroozandeh, P. & Aziz, A. A. Insight into cellular uptake and intracellular trafficking of nanoparticles. Nanoscale Res. Lett. https://doi.org/10.1186/S11671-018-2728-6 (2018).

  19. Hoang Thi, T. T. et al. The importance of poly(ethylene glycol) alternatives for overcoming PEG immunogenicity in drug delivery and bioconjugation. Polymers 12, 298 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Zhang, P., Sun, F., Liu, S. & Jiang, S. Anti-PEG antibodies in the clinic: current issues and beyond PEGylation. J. Control. Release 244, 184–193 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Garay, R. P., El-Gewely, R., Armstrong, J. K., Garratty, G. & Richette, P. Antibodies against polyethylene glycol in healthy subjects and in patients treated with PEG-conjugated agents, Expert Opin. Drug Deliv. https://doi.org/10.1517/17425247.2012.720969 (2012).

  22. López-Royo, T. et al. Encapsulation of large-size plasmids in PLGA nanoparticles for gene editing: comparison of three different synthesis methods. Nanomater 11, 2723 (2021).

  23. Caparica, R. et al. Anticancer activity of rutin and its combination with ionic liquids on renal cells. Biomolecules 10, 233 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Tahara, Y., Morita, K., Wakabayashi, R., Kamiya, N. & Goto, M. Biocompatible ionic liquid enhances transdermal antigen peptide delivery and preventive vaccination effect. Mol. Pharm. 17, 3845–3856 (2020).

    Article  CAS  PubMed  Google Scholar 

  25. Tanner, E. E. L. et al. Design principles of ionic liquids for transdermal drug delivery. Adv. Mater. 31, 1901103 (2019).

    Article  Google Scholar 

  26. Shi, Y. et al. Oral delivery of sorafenib through spontaneous formation of ionic liquid nanocomplexes. J. Control. Release 322, 602–609 (2020).

    Article  CAS  PubMed  Google Scholar 

  27. Banerjee, A. et al. Ionic liquids for oral insulin delivery. Proc. Natl Acad. Sci. USA 115, 7296–7301 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Halayqa, M., Pobudkowska, A., Domańska, U. & Zawadzki, M. Studying of drug solubility in water and alcohols using drug-ammonium ionic liquid-compounds. Eur. J. Pharm. Sci. 111, 270–277 (2018).

    Article  CAS  PubMed  Google Scholar 

  29. Hamadani, C. M., Goetz, M. J., Mitragotri, S. & Tanner, E. E. L. Protein-avoidant ionic liquid (PAIL)–coated nanoparticles to increase bloodstream circulation and drive biodistribution. Sci. Adv. 6, eabd7563 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Hamadani, C. M. et al. Improved nanoformulation and biofunctionalization of linear-dendritic block copolymers with biocompatible ionic liquids. Nanoscale https://doi.org/10.1039/D2NR00538G (2022).

  31. Hu, C. M. J., et al. Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.1106634108 (2011).

  32. Makadia, H. K. & Siegel, S. J. Poly lactic-co-glycolic acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers 3, 1377–1397 (2011).

    Article  CAS  PubMed  Google Scholar 

  33. Govender, T., Stolnik, S., Garnett, M. C., Illum, L. & Davis, S. S. PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug. J. Control. Release 57, 171–185 (1999).

    Article  CAS  PubMed  Google Scholar 

  34. Haque, S. et al. Suggested procedures for the reproducible synthesis of poly(d,l-lactideco-glycolide) nanoparticles using the emulsification solvent diffusion platform. Curr. Nanosci. 14, 448–453 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Astete, C. E. & Sabliov, C. M. Synthesis and characterization of PLGA nanoparticles. J. Biomater. Sci. Polym. Ed. 17, 247–289 (2006).

  36. Hernández-Giottonini, K. Y. et al. PLGA nanoparticle preparations by emulsification and nanoprecipitation techniques: effects of formulation parameters. RSC Adv. 10, 4218–4231 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Kizilbey, K. Optimization of rutin-loaded PLGA nanoparticles synthesized by single-emulsion solvent evaporation method. ACS Omega 4, 555–562 (2019).

    Article  CAS  Google Scholar 

  38. McCall, R. L. & Sirianni, R. W. PLGA nanoparticles formed by single- or double-emulsion with vitamin E-TPGS. J. Vis. Exp. https://doi.org/10.3791/51015 (2013).

  39. Alshamsan, A. Nanoprecipitation is more efficient than emulsion solvent evaporation method to encapsulate cucurbitacin I in PLGA nanoparticles. Saudi Pharm. J. 22, 219–222 (2014).

    Article  PubMed  Google Scholar 

  40. Bilati, U., Allémann, E. & Doelker, E. Nanoprecipitation versus emulsion-based techniques or the encapsulation of proteins into biodegradable nanoparticles and process-related stability issues. AAPS PharmSciTech https://doi.org/10.1208/pt060474 (2005)

  41. Shkodra-Pula, B. et al. Encapsulation of the dual FLAP/mPEGS-1 inhibitor BRP-187 into acetalated dextran and PLGA nanoparticles improves its cellular bioactivity. J. Nanobiotechnol. 18, 73 (2020).

    Article  CAS  Google Scholar 

  42. Huang, W. & Zhang, C. Tuning the size of poly(lactic-co-glycolic acid) (PLGA) nanoparticles fabricated by nanoprecipitation. Biotechnol. J. 13, 1700203 (2018).

    Article  Google Scholar 

  43. Whittington, N. C. & Wray, S. Suppression of red blood cell autofluorescence for immunocytochemistry on fixed embryonic mouse tissue. Curr. Protoc. Neurosci. 81, 2.28.1–2.28.12 (2017).

    Article  CAS  PubMed  Google Scholar 

  44. Zhang, Y. et al. Near-infrared fluorescent thienothiadiazole dyes with large Stokes shifts and high photostability. J. Org. Chem. 82, 5597–5606 (2017).

    Article  CAS  PubMed  Google Scholar 

  45. Hamadani, C. M. A Novel Chemotherapeutic Nanoparticle Drug Delivery System: Surface Functionalization of Polymeric Nanoparticles with Protein-Phobic Ionic Liquid to Enhance Drug Bioavailability in Systemic Circulation. MSc thesis, Harvard Univ. (2020).

  46. Brenner, J. S. et al. Red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magnitude. Nat. Commun. 9, 2684 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Evans, B. C. et al. Ex vivo red blood cell hemolysis assay for the evaluation of pH-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs. J. Vis. Exp. https://doi.org/10.3791/50166 (2013).

  48. Gindri, I. M. et al. Preparation of TiO2 nanoparticles coated with ionic liquids: a supramolecular approach. ACS Appl. Mater. Interfaces 6, 11536–11543 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Pourjavadi, A., Hosseini, S. H., Doulabi, M., Fakoorpoor, S. M. & Seidi, F. Multi-layer functionalized poly(ionic liquid) coated magnetic nanoparticles: highly recoverable and magnetically separable brønsted acid catalyst. ACS Catal. 2, 1259–1266 (2012).

    Article  CAS  Google Scholar 

  50. Adumitrăchioaie, A., Tertis, M., Cernat, A., Sandulescu, R. & Cristea, C. Electrochemical methods based on molecularly imprinted polymers for drug detection: a review. Int. J. Electrochem. Sci. 13, 2556–2576 (2018).

    Article  Google Scholar 

  51. Zhang, R., Gao, R., Gou, Q., Lai, J. & Li, X. Precipitation polymerization: a powerful tool for preparation of uniform polymer particles. Polymers 14, 1851 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Takahashi, C. et al. Optimization of ionic liquid-incorporated PLGA nanoparticles for treatment of biofilm infections. Mater. Sci. Eng. C. 97, 78–83 (2019).

    Article  CAS  Google Scholar 

  53. Abid, Z. et al. Investigation of mucoadhesion and degradation of PCL and PLGA microcontainers for oral drug delivery. Polymers 11, 1828 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Procedures developed and optimized in this protocol are supported by a PhRMA Foundation grant and Sigma Xi Student grant G0315202198510166.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Mississippi, Oxford, MS, USA

    Christine M. Hamadani, Gaya S. Dasanayake, Meghan E. Gorniak, Mercedes C. Pride, Wake Monroe, Claylee M. Chism, Rebekah Heintz, Ethan Jarrett, Gagandeep Singh, Sara X. Edgecomb & Eden E. L. Tanner

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Contributions

Under the supervision of E.E.L.T., C.M.H developed the original experimental procedure and data, with updated troubleshooting, data and optimization/experimental advice for all sections, and wrote the paper with contributions from all coauthors. G.S.D., M.E.G., C.M.C. and W.M. helped develop and optimize the separation procedure of all blood components when isolating RBCs in whole blood (Box 3). G.S.D. contributed experimental development, procedure, text, data and troubleshooting advice for the alternative sonication synthesis. M.E.G. contributed optimization of experimental development, text and troubleshooting data, and advice for all aspects of NP synthesis with encapsulation of DiD dye, NMR quantitative characterization, optimization of RBC biocompatibility and optimization of RBC hitchhiking procedure/FACS gating analysis. M.C.P contributed optimization of experimental development and experimental setup for the ‘whip-in’ (displayed in Fig. 1c) method of stirring IL-PLGA DiD NP synthesis. W.M. and R.H. provided critical feedback on experimental method procedures and supplied data used for EE, DiD NP characterization and experimental setup figures (Figs. 1 and 3). W.M. provided troubleshooting supplementary data and created the supplementary video with assistance from G.S. G.S. provided critical feedback on NMR characterization of ILs and quantitative NMR characterization and provided further optimization of the sonication NP synthesis method. C.M.C. and E.J. synthesized and provided some of the ILs displayed in Fig. 2 from Table 1. C.M.C. provided the ‘Timing’ section in the manuscript. S.X.E. provided critical text formatting, procedure organization and troubleshooting section numbering, troubleshooting and critical callouts. E.E.L.T. provided resources, supervision, funding, draft editing and project administration.

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Correspondence to Eden E. L. Tanner.

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Hamadani, C. M. et al. Sci Adv. 6, eabd7563 (2020): https://doi.org/10.1126/sciadv.abd7563

Hamadani, C. M. et al. Nanoscale 14, 6021–6036 (2022): https://doi.org/10.1039/d2nr00538g

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Hamadani, C.M., Dasanayake, G.S., Gorniak, M.E. et al. Development of ionic liquid-coated PLGA nanoparticles for applications in intravenous drug delivery. Nat Protoc 18, 2509–2557 (2023). https://doi.org/10.1038/s41596-023-00843-6

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