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SCIENCE CHINA Materials, Volume 60, Issue 6: 563-570(2017) https://doi.org/10.1007/s40843-017-9053-y

Tuning the molecular size of site-specific interferon-polymer conjugate for optimized antitumor efficacy

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  • ReceivedMay 10, 2017
  • AcceptedMay 22, 2017
  • PublishedJun 2, 2017

Abstract

The covalent attachment of protein-resistant polymers to therapeutic proteins is a widely used method for extending their in vivo half-lives; however, the effect of molecular weight of polymer on the in vitro and in vivo functions of protein-polymer conjugates has not been well elucidated. Herein we report the effect of molecular weight of poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) on the in vitro and in vivo properties of C-terminal interferon-alpha (IFN)-POEGMA conjugates. Increasing the molecular weight of POEGMA decreased the in vitro activity of IFN-α but increased its thermal stability and in vivo pharmacokinetics. Intriguingly, the in vivo antitumor efficacy of IFN-α was increased by increasing the POEGMA molecular weight from ca. 20 to 60 kDa, but was not further increased by increasing the molecular weight of POEGMA from ca. 60 to 100 kDa due to the neutralization of the improved pharmacokinetics and the reduced in vitro activity. This finding offers a new viewpoint on the molecular size rationale for designing next-generation protein-polymer conjugates, which may benefit patients by reducing administration frequency and adverse reactions, and improving therapeutic efficacy.


Funded by

grants from the National Natural Science Foundation of China(21274043,21534006)


Acknowledgment

This study was financially supported by Grants from the National Natural Science Foundation of China (21274043 and 21534006).


Interest statement

The authors declare that they have no conflict of interest.


Contributions statement

Gao W and Wang G designed the project; Wang G and Hu J synsthesized and purified the samples and carried out all the analyses; Hu J completed the H&E staining, biochemistry and hematological investigations; Wang G and Hu J wrote the paper with support from Gao W. All authors contributed to the general discussion.


Author information

Jin Hu received her bachelor’s degree in biomedical engineering from Nanchang University, China, in 2012. She is currently a PhD candidate at the Department of Biomedical Engineering, School of Medicine, Tsinghua University. Her research is focused on the synthesis of site-specific polymer-protein conjugates and their pharmacological properties.


Weiping Gao is a professor at the Department of Biomedical Engineering, School of Medicine, Tsinghua University. He received his PhD degree from Peking University in 2004. From 2005 to 2007, he worked as a researcher at Kyoto University, Japan, and from 2007 to 2011, he was a postdoctoral associate at the Department of Biomedical Engineering and Center for Biologically Inspired Materials and Material Systems, Duke University, USA. His research interests include the synthesis of polymer-biomolecule conjugates and the application of nanotechnology in biomedicine.


Supplement

Supplementary information

Materials, full experimental details including synthesis, physicochemical characterization, in vitro cytotoxicity and thermal stability, pharmacokinetics, in vivo antitumor efficacy and systemic toxicity. Supporting data including SDS-PAGE analysis, pharmacokinetic parameters, the change of mouse body weight post administration, H&E staining, clinical biochemistry and hematological parameters for mice, are available in the online version of the paper.


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  • Figure 1

    Synthesis and physicochemical characterization of IFN-POEGMA. (a) Synthetic route of C-terminal IFN-POEGMA conjugates. ATRP initiator was site-specifically attached to the C-terminus of IFN-α by sortase A mediated ligation, followed by in situ ATRP of OEGMA from IFN-Br to yield IFN-POEGMA. (b) GPC traces of the ATRP reaction mixtures. (c) GPC traces of the purified IFN-POEGMA conjugates. (d) DLS analyses of the IFN-POEGMA conjugates, where Rh represents hydrodynamic radius. (e) CD analyses of the IFN-POEGMA conjugates.

  • Figure 2

    The in vitro anti-proliferative activity and thermal stability of IFN-POEGMA. (a) The in vitro cytotoxicity of IFN-POEGMA. (b) The relative activity of IFN-POEGMA as a function of molecular weight. (c) The thermal stability (in terms of relative activity retention) of IFN-POEGMA after incubation at 50°C, where the number represents the molecular weight of POEGMA. (d) CD spectra of IFN-α and IFN-POEGMA after incubation for 24 h at 50°C.

  • Figure 3

    The in vivo pharmacokinetic profiles of IFN-POEGMA. (a) The plasma IFN concentration as a function of time post injection. (P< 0.001 for IFN-POEGMA vs. IFN-α). (b) The distribution and terminal half-life as a function of molecular weight, where the number represents the molecular weight of POEGMA. (c) The area under curve and central clearance as a function of molecular weight, where the number represents the molecular weight of POEGMA. Data are shown as mean ± SD (n = 3).

  • Figure 4

    The in vivo antitumor efficacy of IFN-POEGMA. (a) Inhibition of tumor growth after weekly treatment, as indicated by the arrows. (***P < 0.001 for IFN-POEGMA 20 kDa vs. IFN-α, ****P < 0.0001 for IFN-POEGMA 60 and 100 kDa vs. IFN-α). (b) Survival curves of mice. The vertical dashed line indicates the ending date of treatment. (***P < 0.001 for IFN-POEGMA vs. IFN-α). (c) Histological evaluation of tumors. Data are shown as mean ± SD (n = 5–8).

  •    The molecular weights, polydispersities and yields of the IFN-POEGMA conjugates

    Sample

    Number-average molecular weight (Mn) (kDa)

    Molar mass dispersity

    Yield (%)

    IFN-POEGMA 20 kDa

    23.6

    1.29

    38.4

    IFN-POEGMA 60 kDa

    64.3

    1.35

    63.3

    IFN-POEGMA 100 kDa

    104.7

    1.32

    79.5

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