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. 2017 Jul 13;7:5303. doi: 10.1038/s41598-017-05525-4

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Identification of doxorubicin immobilised on His-tagged VLNPs. (a and b) Doxorubicin (DOX) was detected by measuring absorbance at 495 nm of the fractions obtained from sucrose gradients. (a) DOX, NTA-DOX, HisHBcAg nanoparticles incubated with NTA-DOX in the absence of Zn²⁺ [HisHBcAg + (NTA-DOX)] stayed on top of the sucrose gradients after centrifugation. HisHBcAg nanoparticles (HisHBcAg) were not detected at A₄₉₅. HisHBcAg nanoparticles incubated with Zn²⁺ (HisHBcAg + Zn²⁺) showed a negligible absorbance at 495 nm. (b) HisHBcAg nanoparticles conjugated non-covalently with NTA-DOX (HisHBcAg-NTA-DOX) and HisHBcAg nanoparticles conjugated covalently with folic acid (FA) and non-covalently with NTA-DOX (FA-HisHBcAg-NTA-DOX) migrated into the sucrose gradient, and the immobilised DOX was detected with A₄₉₅. (c and d) Native agarose gel electrophoresis of the HisHBcAg nanoparticles conjugated non-covalently with DOX. The same gel was visualised under (c) ultraviolet (UV) illumination, and (d) stained with Coomassie Brilliant Blue (CBB).