Dual mode bioreactions on polymer nanoparticles covered with phosphorylcholine group

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Abstract

We investigated the preparation of polymer nanoparticles covered with phosphorylcholine (PC) groups and the immobilization of proteins in order to observe dual mode bioreactions on the nanoparticles. For the surface modification on the nanoparticles, a water-soluble amphiphilic phospholipid polymer with PC groups as a hydrophilic moiety was synthesized. In this polymer, an active ester group, which can immobilize proteins, was introduced. Using the phospholipid polymer as a solubilizer, poly(l-lactic acid) nanoparticles were prepared from its methylene chloride solution in an aqueous medium by the solvent evaporation method. The diameter of the nanoparticles was ca. 200 nm and the surface was covered with the PC groups and active ester groups. Proteins could immobilize on the nanoparticles under mild conditions by the reaction between the active ester group and amino group in the proteins. Both an antibody and enzyme were immobilized on the nanoparticles and bioreactions such as the antigen/antibody reaction and enzymatic reaction were observed. When an antigen was added to the suspension of the nanoparticles, aggregation of the nanoparticles occurred and then they precipitated. Also, the enzymatic reaction proceeded well when the enzyme substrate was added to the suspension. Based on these results, we provided polymer nanoparticles functionalized with both the antibody and enzyme, and the dual mode bioreactions could occur. We concluded that the novel polymer nanoparticles could be used for nano-/micro-scaled diagnostic and medical treatment systems.

Introduction

Polymer nanoparticles are widely used in the life science fields for separation technologies, histological studies, clinical diagnostic systems and drug delivery [1], [2], [3]. We are continuously investigating the preparation of polymer nanoparticles covered with phosphorylcholine (PC) groups to obtain excellent bio/blood compatibility and stability in an aqueous medium including plasma [4]. To cover the surface of the polymer nanoparticles, we prepared a water-soluble amphiphilic phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)) (PMB) [5]. Since the PMB formed a polymer aggregate in the aqueous medium, it functioned as a good solubilizer for hydrophobic compounds. Thus, we could prepare polymer nanoparticles by solvent evaporation and interfacial precipitation techniques from an organic solvent containing a core polymer in aqueous medium containing the PMB. Moreover, the introduction of active ester units to the PMB was made possible for reactions with biomolecules [6]. We conjugated biomolecules such as a protein enzyme or antibody on the polymer nanoparticles and revealed the good performance of these biomolecules even if they were located on the solid surface [7], [8].

Another viewpoint of biorecognition between the MPC polymer and living cells has been reported. On the cell membrane, carbohydrate and polysaccharide chains play an important role in molecular recognition from the outer medium and signal transport into the cells [9]. The incorporation of unnatural carbohydrates provides an opportunity to study the specific contributions of sialic acid and its N-acyl side chains to the sialic acid-dependent ligand–receptor interactions at a submolecular level. The MPC polymer surfaces with hydrazide groups, which can selectively react with unnatural ketone-containing carbohydrate as a cell surface tag, controlled the cell attachment [10].

From these fundamental research results, we propose novel diagnostic and medical treatment systems using polymer nanoparticles, that is, the polymer nanoparticles can selectively bind target cells, and enzymes conjugated on the polymer nanoparticles react with specific polysaccharide chains on the cell membrane. If the specific polysaccharide chains are digested by the enzymatic reaction, the cell cannot survive. In this study, the preparation of polymer nanoparticles covered with the PC groups and double bioconjugation with the antibody and enzyme on one polymer nanoparticle was carried out. Dual mode bioreactions, that is, aggregation of the polymer nanoparticles by the addition of an antigen and enzymatic reaction by the addition of an enzyme substrate were investigated.

Section snippets

Synthesis of the MPC polymer

MPC was synthesized by a previously reported method [11]. BMA was reagent grade and used after vacuum distillation (bp 68.5 °C/32 mmHg). p-Nitrophenyloxycarbonyl polyethyleneglycol methacrylate (MEONP) was synthesized by a previously reported method [6]. Poly(MPC-co-BMA-co-MEONP) (PMBN) was synthesized by a conventional radical polymerization technique using 2,2′-azobisisobutyronitrile (AIBN) as an initiator [12]. The polymerization was carried out at 60 °C for 5 h. The reaction mixture was then

Characterization of PMBN

The PMBN was synthesized using a conventional radical polymerization technique. The characterizations of PMBN are summarized in Table 1. The compositions of each monomer unit in the polymer were in good agreement with their compositions in the feed. The obtained PMBN was water-soluble, but had an amphiphilic nature because the hydrophobic BMA units were introduced and its composition was above 60 mol%. In our previous article, we reported other PMBNs with different compositions. Based on this

Conclusion

We prepared novel polymer nanoparticles which can conjugate protein including an antibody and enzyme. After conjugation, the proteins worked well and we observed dual mode bioreactions against the target molecules. Thus, we concluded that the polymer nanoparticles could be used to make a new diagnostic system and a medical treatment system.

Acknowledgements

The present research was supported in part by a Grant for the 21st Century COE Program “Human-Friendly Materials based on Chemistry” from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (16650098).

References (21)

  • T. Konno et al.

    Biomaterials

    (2001)
  • M.V. Solovskij et al.

    Eur. Polym. J.

    (2000)
  • M. Sugano et al.

    Cancer Res.

    (2000)
  • C. Hebert et al.

    J. Drug Target.

    (2003)
  • A.H. Blair et al.

    J. Immunol. Methods

    (1983)
  • T. Konno et al.

    Trans. Mater. Res. Soc. Jpn.

    (2001)
  • T. Konno et al.

    Biomacromolecules

    (2004)
  • J.W. Park et al.

    Anal. Chem.

    (2004)
  • J. Watanabe et al.

    Biomacromolecules

    (2006)
  • A. Kobata et al.

    Immunol. Cell Biol.

    (2005)
There are more references available in the full text version of this article.

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