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Dual mode bioreactions on polymer nanoparticles covered with phosphorylcholine group
Colloids and Surfaces B: Biointerfaces 50 (2006) 55–60
Dual mode bioreactions on polymer nanoparticles covered with phosphorylcholine group Tomomi Ito a , Junji Watanabe a , Madoka Takai a , Tomohiro Konno a , Yasuhiko Iwasaki b , Kazuhiko Ishihara a,∗ b
a Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
Received 2 December 2005; received in revised form 23 March 2006; accepted 5 April 2006 Available online 27 April 2006
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. © 2006 Published by Elsevier B.V. Keywords: Polymer nanoparticles; Phospholipid polymer; Bioconjugation; Bioreaction
1. Introduction Polymer nanoparticles are widely used in the life science fields for separation technologies, histological studies, clinical diagnostic systems and drug delivery [1–3]. 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
∗
Corresponding author. Tel.: +81 3 5841 7124; Fax: +81 3 5841 8647. E-mail address: [email protected] (K. Ishihara).
0927-7765/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.colsurfb.2006.04.006
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
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T. Ito et al. / Colloids and Surfaces B: Biointerfaces 50 (2006) 55–60
Fig. 1. Chemical structures of PMBN.
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.
2.2. Preparation of polymer nanoparticles The polymer nanoparticles having both PC groups and pnitrophenyl ester groups on the surface were prepared by solvent evaporation and interfacial precipitation techniques in aqueous medium, the same method as previously reported [6]. A brief explanation is as follows. The PLA (Mw = 2 × 104 , Wako Pure Chemical Industries, Osaka, Japan) was used as a core polymer material. In a glass bottle, 40 mL of an aqueous solution containing the PMBN (10 mg/mL) was placed, and the resultant mixture stirred at 400 rpm with cooling in an ice bath. The PLA (20 mg) was dissolved in 2.0 mL of methylene chloride. The PLA solution was then dropped into a PMBN aqueous solution. The mixture was sonicated using a probe-type generator (Sonifier 250, Branson, USA) for 30 min and kept under reduced pressure for 2 h to evaporate the methylene chloride. The formed polymer nanoparticles (PMBN/PLA-NP) were fractionated by centrifugation at 10,300 × g at 4 ◦ C for 30 min (AllegraTM 21R Centrifuge, Beckman Coulter, Palo Alto, USA). The PMBN/PLA-NP as a precipitate was resuspended with distilled water and centrifuged again under the same conditions. This procedure was repeated three times to completely remove any free PMBN. The particle size and size distribution of the PMBN/PLA-NP were determined by a dynamic light scattering measurement (DLS, Otsuka Electronics, Osaka, Japan) and observed using an atomic force microscope (AFM, SPI-3800, Seiko instrument, Chiba, Japan). The surface elemental analysis and surface potential measurement of the PMBN/PLA-NP were carried out by X-ray photoelectron spectroscopy (XPS, ESCA-200, Scienta, Uppsala, Sweden) and laser-doppler electrophoresis (ELS 8000, Otsuka Electronics), respectively. After 0.5 N NaOH was added to the PMBN/PLA-NP suspension, the PMBN/PLA-NP was precipitated by centrifugation. The UV adsorption of the supernatant was measured using a UV spectrophotometer (V-650, Jasco, Tokyo, Japan) at 400 nm to determine the amount of the active ester group on the PMBN/PLA-NP.
2. Materials and methods 2.1. 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 poured into diethyl ether to precipitate the polymer, and then the polymer was collected and dried in vacuo. Using 1 H NMR, each mole fraction unit of PMBN was determined. Fig. 1 and Table 1 shows the chemical structures and synthetic results of the PMBN, respectively. All reagents and solvents were purified by conventional methods. Table 1 Characterization of PMBN Monomer unit composition (mol%)
Yield (%)
Mw b
Solubility in waterc
5
78
6.2 × 104
++
In polymera
In feed
PMBN
Time (h)
MPC
BMA
MEONP
MPC
BMA
MEONP
40
55
10
32
62
6.0
[Monomer] = 1.0 mol/L, [AIBN] = 10 mmol/L. Reaction temperature at 60 ◦ C. Precipitated by diethyl ether/chloroform = 8/2. a Determined by 1 H NMR. b Weight-averaged molecular weight(Mw) was determined by GPC in water/methanol = 3/7, PEO standard. c Solubility was determined at 1 mg/mL polymer concentration.
T. Ito et al. / Colloids and Surfaces B: Biointerfaces 50 (2006) 55–60
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2.3. Conjugation of biomolecules on PMBN/PLA-NP The fluorecein isothyocyanate (FITC) labeled anti-mouse IgG (antibody: 100 g/mL, Sigma–Aldrich, F0257) or alkaline phosphatase (AP) (enzyme: 2 U/mL, Mw = 1.4 × 105 , alkaline phosphatase calf intestine, CALBIOCHEM, Darmstadt, Germany) was suspended with PMBN/PLA-NP in Tris–HCl buffer solution (pH 8.0) for 24 h at 4 ◦ C. The suspension was centrifuged at 10,300 × g for 30 min at 4 ◦ C. After centrifuging, the conjugation amounts of the antibody on the PMBN/PLA-NP were measured by a fluorescence spectrophotometer (FP-6500, Jasco, Tokyo, Japan). Double conjugation of two biomolecules to the PMBN/PLANP was also conducted using the same previously described procedure. The PMBN/PLA-NP were added to the antigen and enzyme mixture solution (pH 8.0) and reacted for 24 h at 4 ◦ C. After centrifuging, the PMBN/PLA-NP was thoroughly reacted with 10 mM glycine for 24 h at 4 ◦ C for complete reaction with the remaining active ester groups. 2.4. Bioreaction of biomolecules on PMBN/PLA-NP To the suspension of the PMBN/PLA-NP after bioconjugation with the antibody, a solution containing various concentrations of an antigen (Mouse-IgG, Rockland Immunochemicals, Gilbertsville, USA) was added and reacted for 24 h at 4 ◦ C. The reaction was observed by the change in the transmittance of the suspension at 550 nm. For the enzymatic reaction, the substrate for the AP, 4-methylumbelliferyl phosphate (MUP; Mw = 256.2, excitation 355 nm, Molecular Probes, Leiden, The Netherlands), was added and reacted for 24 h at 37 ◦ C. After the reaction, the fluorescence intensity was measured at 460 nm. 3. Results and discussion 3.1. 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 result, we determined a suitable composition of the PMBN for the surface modification of the polymer nanoparticles during their preparation. 3.2. Characterization of the PMBN/PLA-NP The PMBN/PLA-NP could be prepared by solvent evaporation and interfacial precipitation methods. The core polymer, PLA, was dissolved in methylene chloride, but was insoluble in water. The droplets of the PLA solution were suspended in the
Fig. 2. AFM image of the PMBN/PLA-NP.
aqueous solution along with the PMBN aggregate. The methylene chloride was then evaporated and the PLA was precipitated at the interface faced on aqueous phase. At this interface, the PMBN chains and PLA chains should make entanglements. Thus, a stable coating layer of PMBN was formed on the surface of the PLA nanoparticles. Fig. 2 shows an AFM image of the PMBN/PLA-NP. Based on the AFM observation and DLS measurement, we determined the size of the PMBN/PLA-NP, which was ca. 260 nm. The diameter and size distribution of the PMBN/PLANP averaged d = 259.7 nm and the polydispersity index was α = 1.4 × 10−1 . The size distribution of the PMBN/PLA-NP suggested a monodispersion as calculated from the cumulant method. The XPS analysis indicated that the PMBN/PLA-NP had specific peaks attributed the component atoms, such as a phosphorus peak at 135 eV, a nitrogen peak at 403 eV, an oxygen peak at 530 eV and a strong carbon peak attributed to the methyl or methylene groups at 285 eV (data not shown). For the PLA nanoparticles without the PMBN coating, there were no phosphorus and nitrogen peaks [6]. The zeta-potential of the PMBN/PLA-NP was −5 mV, whereas that of the PLA nanoparticles was −60 mV. The strong negative value became nearly zero. This is due to the electrically neutral property of the PC group by formation of an inner salt between the phosphate anion and trimethyl ammonium cation. Thus, it was revealed that the surface of the PMBN/PLA-NP was covered with PC groups. The dispersion stability depended on the surface hydrophilicity of the nanoparticles. The PC group is extremely hydrophilic, therefore, the PMBN/PLA-NP had a good dispassion stability in an aqueous medium. The formation of p-nitrophenol by hydrolysis of the active ester group of PMBN/PLA-NP was measured. We observed the UV absorbance at 400 nm, which is attributed to the pnitrophenoxy anion when the PMBN/PLA-NP was added to an alkaline solution. The p-nitrophenyl ester groups might be located on the surface of the PMBN/PLA-NP. Based on these results, the PMBN/PLA-NP possessed both PC groups and active ester groups near the surface.
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Fig. 3. Fluorescence intensity of conjugated antibody on the PMBN/PLA-NP. The fluorescence intensity on (—) PMBN/PLA-NP conjugated with antibody; (– – –) PMBN/PLA-NP when the active ester groups were hydrolyzed.
In our previous research, a surface covered with a PC group has an excellent bio/blood compatibility, that is, the suppression of non-specific protein adsorption and following thrombus formation, and immunological reactions [13–19]. That is, if the PMBN/PLA-NP was in contact with protein molecules in the aqueous medium, the protein molecules can selectively react with active ester groups by amide bond formation. This is a very important feature for controlling the conjugation density of the proteins. 3.3. Single bioconjugation with antibody on PMBN/PLA-NP Fig. 3 shows the fluorescence spectra of the suspension of the FITC-labeled antibody conjugated with PMBN/PLA-NP and that with nanoparticles after hydrolysis of the active ester groups. The hydrolysis process could inactivate the PMBN/PLA-NP for bioconjugation. Non-specific physical adsorption of the antibody could then be evaluated. The fluorescence intensity based on the FITC-antibody on the PMBN/PLA-NP was significantly higher when compared with that after hydrolysis. PMBN/PLANP sufficiently reacted in the Tris–HCl buffer solution. Thus, it clearly showed that the antibody could react with the active ester group on the surface of the nanoparticles. Since the MPC polymer suppressed protein adsorption, these data corresponded to the previous results [5]. Based on the fluorescence intensity observed on the FITC-labeled antibody conjugated with PMBN/PLA-NP, the amount of the antibody conjugated was determined. It was 2.34 g/mg-nanoparticles that corresponded to a 27% consumption of the active ester group. This consumption value of the active ester group was small due to the high molecular weight of the antibody. Using the antibody conjugated-PMBN/PLA-NP, the aggregation behavior by the addition of the antigen was investigated. Fig. 4 shows the change in the transmittance of the suspension containing nanoparticles with the reaction time after the addition of the antigen. For the antibody conjugated nanoparticles, the transmittance at 550 nm increased with time, but the change depended on the amount of antigen added to the suspension. The change in the transmittance was due to the aggregation of the nanoparticles. This means that the antibody conjugated on the nanoparticles could react with the antigen through an antigen–antibody reaction.
Fig. 4. Transmittance of the PMBN/PLA-NP conjugated antibody and enzyme after addition of the antigen for 24 h. The fluorescence intensity on () ((PMBN/PLA-NP)/antibody)/antigen (0.167 × 10−9 mol); () ((PMBN/PLA-NP)/antibody)/antigen (1.67 × 10−9 mol); () PMBN/PLANP/antigen (1.67 × 10−9 mol); (䊉) PMBN/PLA-NP.
The aggregation was very quickly initiated after addition of the antigen, and it reached an equilibrium state within 4 h under this condition. When a higher antigen concentration was added, the rate of aggregation of the antibody conjugated nanoparticles increased. Thus, the antibody conjugated on the PMBN/PLANP functioned well. One unexpected phenomenon was observed on non-conjugated nanoparticles. We could not clearly explain it at the present time. However, the following consideration was raised. Since the active ester groups on the nanoparticles were deactivated by hydrolysis, carboxylic groups were generated on the nanoparticles. These carboxylic groups might interact with the antigen and a small aggregation of nanoparticles was formed. We also carried out the enzyme conjugation on the PMBN/PLA-NP. The enzyme used in this study was AP. The reacted condition was the same condition as the antibody conjugation. As shown in Fig. 5, in the absence of the enzyme, the fluorescence intensity based on the enzymatic reaction product was quite low. However, for the AP conjugated on the PMBN/PLANP case, a strong fluorescence intensity was observed. Thus, the AP could react with the substrate and form a product due to the enzymatic reaction even though these conjugated on the
Fig. 5. The fluorescence intensity of the enzymatic reaction product on PMBN/PLA-NP/AP. The substrate for AP was MUP.
T. Ito et al. / Colloids and Surfaces B: Biointerfaces 50 (2006) 55–60
nanoparticles. In our previous articles, we applied the PMBNbased polymers to conjugate enzymes, such as acetylcholine esterase (its function is hydrolysis of the carboxylic ester bond), choline oxydase (its function is oxidation), horseradish peroxydase (its function is reduction) and luciferase [6–8]. These various functional enzymes could also work well on the PMBNbased polymers. In addition to these enzymes, phosphatase (its function is hydrolysis of the phospholic ester bond) also functioned well on the surface. Thus, it is now concluded that the PMBN-based polymers have the possibility to conjugate every functional enzyme. Vertegel et al. reported the effect of the nanoparticle size on the adsorbed proteins [20]. They performed the adsorption of the enzyme lysozyme on SiO2 particles with 4–100 nm diameters. The structure and function of the adsorbed lysozyme on the nanoparticles are strongly dependent on the size of the nanoparticles. A less significant change in the structure and activity of the lysozyme adsorbed on smaller nanoparticles was observed. However, in our case, proteins conjugated on 200 nm particles had good functions. This is due to the effects of the PC groups on the nanoparticles. We already reported that the conformational change in protein adsorbed on the MPC polymers and poly(2-hydroxyethyl methacrylate (HEMA)) were determined using circular dichroism (CD) spectroscopic measurement [21]. The CD spectrum of BSA adsorbed on PMB with 0.30 MPC mole fraction was almost the same as that in PBS. The negative ellipticity at 222 nm of BSA adsorbed on the MPC polymers increased with a decrease in MPC composition, and then became almost zero in the case of BSA adsorbed on poly(HEMA). The secondary structure of adsorbed protein was determined to calculate the ␣-helix content. The ␣-helix content of BSA, which is assumed to be the “native” secondary structure, was 54%. On the PMB with 0.30 MPC mole fraction surface of the ␣-helix content of adsorbed BSA was almost the same as that of native proteins. However, it decreased by contact with poly(HEMA). We suggested that the secondary structure of proteins adsorbed on the MPC polymer surface was maintained at the same level as that of the original state. Thus, the PMBN/PLA-NP provided a good platform to conjugate proteins.
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3.4. Double conjugation of proteins on PMBN/PLA-NP Next, both the antibody and enzyme were conjugated on the same PMBN/PLA-NP. In this step, it was clarified that the proteins conjugated on the nanoparticles could independently react with their own specific target molecule. The conjugation procedure was very simple, that is, the PMBN/PLA-NP was added to the protein solution mixture. The confirmation of double conjugation of the antibody and enzyme was carried out. The PMBN/PLA-NP conjugated with both the antibody and enzyme (PMBN–PLA-NP/AP/antibody) were suspended, and the antigen was added to the suspension. Fig. 6 shows the results of the antigen–antibody reaction. The PMBN/PLANP/AP/antibody could aggregate by the addition of the antigen similar to the single conjugation with the antibody. In Fig. 6 (on the left side figures), the fluorescence spectra of the PMBN/PLA-NP/AP/antibody suspension are demonstrated. As shown in Fig. 6 (on the right side figures), we observed precipitation of the PMBN/PLA-NP/AP/antibody when the antigen was added. The fluorescence spectrum of the precipitate indicated that the antibody was included in the precipitate (Part A in Fig. 6(a)). At the same time, we observed a weak fluorescence intensity in the supernatant of the suspension (Part B in Fig. 6(a)). This is due to the remaining PMBN/PLANP/AP/antibody. However, the degree of precipitation versus the total nanoparticles was ca. 86%, which was calculated from the fluorescence intensity. A highly efficient reaction was confirmed. We added the enzyme substrate (MUP) to the suspension of PMBN/PLA-NP/AP/antibody after addition of the antigen. As shown in Fig. 7, the fluorescence intensity of the enzymatic reaction product was observed after the antigen addition. Although a low fluorescence was observed in the case of the PMBN/PLA-NP and PMBN/PLA-NP/antibody, a strong fluorescence was observed in the other PMBN/PLA-NP/AP and PMBN/PLA-NP/AP/antibody. It was almost the same level as that of the single conjugation of the enzyme on the PMBN/PLA-NP. Based on these results, it is clearly realized that both the antigen and enzyme were conjugated on the PMBN/PLA-NP and these proteins independently functioned very well.
Fig. 6. Fluorescence intensity of antibody conjugating the PMBN/PLA-NP with enzyme. The fluorescence intensity on A (—): ((PMBN/PLANP)/antibody/AP)/antigen; B (– – –): the top clear layer of ((PMBN/PLA-NP/antibody/AP)/antigen); C (· · · · · ·): ((PMBN/PLA-NP)/AP)/antigen.
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References
Fig. 7. The fluorescence intensity of the product of enzymatic reaction on PMBN/PLA-NP/AP/antibody with antigen.
In the near future, we will conjugate an antigen against a specific protein on the cell membrane and enzyme that can react with a specific polysaccharide chain to digest it on the PMBN/PLANP. 4. 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).
[1] M. Sugano, N.K. Egilmez, S.J. Yokota, F.A. Chen, J. Harding, S.K. Huang, R.B. Bankert, Cancer Res. 60 (2000) 6942. [2] C. Hebert, K. Norris, J.J. Sauk, J. Drug Target. 11 (2003) 101. [3] A.H. Blair, T.I. Ghose, J. Immunol. Methods 59 (1983) 29. [4] T. Konno, K. Ishihara, Trans. Mater. Res. Soc. Jpn. 26 (2001) 897. [5] T. Konno, K. Kurita, Y. Iwasaki, N. Nakabayashi, K. Ishihara, Biomaterials 22 (2001) 1883. [6] T. Konno, J. Watanabe, K. Ishihara, Biomacromolecules 5 (2004) 342. [7] J.W. Park, S. Kurosawa, J. Watanabe, K. Ishihara, Anal. Chem. 76 (2004) 2649. [8] J. Watanabe, K. Ishihara, Biomacromolecules 7 (2006) 171. [9] A. Kobata, J. Amano, Immunol. Cell Biol. 83 (2005) 429. [10] Y. Iwasaki, E. Tabata, K. Kurita, K. Akiyoshi, Bioconjugate Chem. 16 (2005) 567. [11] K. Ishihara, T. Ueda, N. Nakabayashi, Polym. J. 22 (1990) 355. [12] M.V. Solovskij, E.F. Panarin, O.P. Gorbunova, E.V. Korneeva, N.A. Petuhkova, N.A. Michajlova, G.M. Pavlov, Eur. Polym. J. 36 (2000) 1127. [13] K. Ishihara, H. Oshida, Y. Endo, T. Ueda, A. Watanabe, N. Nakabayashi, J. Biomed. Mater. Res. 26 (1992) 1543. [14] K. Ishihara, Y. Iwasaki, J. Biomater. Appl. 13 (1998) 111. [15] S. Sakaki, N. Nakabayashi, K. Ishihara, J. Biomed. Mater. Res. 47 (1999) 523. [16] K. Ishihara, E. Ishikawa, Y. Iwasaki, N. Nakabayashi, J. Biomater. Sci. Polym. Ed. 10 (1999) 1047. [17] T. Moro, Y. Takatori, K. Ishihara, T. Konno, Y. Takigawa, T. Matsushita, U.I. Chung, K. Nakamura, H. Kawaguchi, Nature Mater. 3 (11) (2004) 829. [18] S. Sawada, S. Sakaki, Y. Iwasaki, N. Nakabayashi, K. Ishihara, J. Biomed. Mater. Res. 64A (2003) 411. [19] J. Patel, Y. Iwasaki, K. Ishihara, J. Anderson, J. Biomed. Mater. Res. 73A (2005) 359. [20] A.A. Vertegel, R.W. Siegel, J.S. Dordick, Langmuir 20 (2004) 6800. [21] K. Ishihara, H. Nomura, T. Mihara, K. Kurita, Y. Iwasaki, N. Nakabayashi, J. Biomed. Mater. Res. 39 (1998) 323.
Dual mode bioreactions on polymer nanoparticles covered with phosphorylcholine group Tomomi Ito a , Junji Watanabe a , Madoka Takai a , Tomohiro Konno a , Yasuhiko Iwasaki b , Kazuhiko Ishihara a,∗ b
a Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
Received 2 December 2005; received in revised form 23 March 2006; accepted 5 April 2006 Available online 27 April 2006
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. © 2006 Published by Elsevier B.V. Keywords: Polymer nanoparticles; Phospholipid polymer; Bioconjugation; Bioreaction
1. Introduction Polymer nanoparticles are widely used in the life science fields for separation technologies, histological studies, clinical diagnostic systems and drug delivery [1–3]. 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
∗
Corresponding author. Tel.: +81 3 5841 7124; Fax: +81 3 5841 8647. E-mail address: [email protected] (K. Ishihara).
0927-7765/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.colsurfb.2006.04.006
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
56
T. Ito et al. / Colloids and Surfaces B: Biointerfaces 50 (2006) 55–60
Fig. 1. Chemical structures of PMBN.
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.
2.2. Preparation of polymer nanoparticles The polymer nanoparticles having both PC groups and pnitrophenyl ester groups on the surface were prepared by solvent evaporation and interfacial precipitation techniques in aqueous medium, the same method as previously reported [6]. A brief explanation is as follows. The PLA (Mw = 2 × 104 , Wako Pure Chemical Industries, Osaka, Japan) was used as a core polymer material. In a glass bottle, 40 mL of an aqueous solution containing the PMBN (10 mg/mL) was placed, and the resultant mixture stirred at 400 rpm with cooling in an ice bath. The PLA (20 mg) was dissolved in 2.0 mL of methylene chloride. The PLA solution was then dropped into a PMBN aqueous solution. The mixture was sonicated using a probe-type generator (Sonifier 250, Branson, USA) for 30 min and kept under reduced pressure for 2 h to evaporate the methylene chloride. The formed polymer nanoparticles (PMBN/PLA-NP) were fractionated by centrifugation at 10,300 × g at 4 ◦ C for 30 min (AllegraTM 21R Centrifuge, Beckman Coulter, Palo Alto, USA). The PMBN/PLA-NP as a precipitate was resuspended with distilled water and centrifuged again under the same conditions. This procedure was repeated three times to completely remove any free PMBN. The particle size and size distribution of the PMBN/PLA-NP were determined by a dynamic light scattering measurement (DLS, Otsuka Electronics, Osaka, Japan) and observed using an atomic force microscope (AFM, SPI-3800, Seiko instrument, Chiba, Japan). The surface elemental analysis and surface potential measurement of the PMBN/PLA-NP were carried out by X-ray photoelectron spectroscopy (XPS, ESCA-200, Scienta, Uppsala, Sweden) and laser-doppler electrophoresis (ELS 8000, Otsuka Electronics), respectively. After 0.5 N NaOH was added to the PMBN/PLA-NP suspension, the PMBN/PLA-NP was precipitated by centrifugation. The UV adsorption of the supernatant was measured using a UV spectrophotometer (V-650, Jasco, Tokyo, Japan) at 400 nm to determine the amount of the active ester group on the PMBN/PLA-NP.
2. Materials and methods 2.1. 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 poured into diethyl ether to precipitate the polymer, and then the polymer was collected and dried in vacuo. Using 1 H NMR, each mole fraction unit of PMBN was determined. Fig. 1 and Table 1 shows the chemical structures and synthetic results of the PMBN, respectively. All reagents and solvents were purified by conventional methods. Table 1 Characterization of PMBN Monomer unit composition (mol%)
Yield (%)
Mw b
Solubility in waterc
5
78
6.2 × 104
++
In polymera
In feed
PMBN
Time (h)
MPC
BMA
MEONP
MPC
BMA
MEONP
40
55
10
32
62
6.0
[Monomer] = 1.0 mol/L, [AIBN] = 10 mmol/L. Reaction temperature at 60 ◦ C. Precipitated by diethyl ether/chloroform = 8/2. a Determined by 1 H NMR. b Weight-averaged molecular weight(Mw) was determined by GPC in water/methanol = 3/7, PEO standard. c Solubility was determined at 1 mg/mL polymer concentration.
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2.3. Conjugation of biomolecules on PMBN/PLA-NP The fluorecein isothyocyanate (FITC) labeled anti-mouse IgG (antibody: 100 g/mL, Sigma–Aldrich, F0257) or alkaline phosphatase (AP) (enzyme: 2 U/mL, Mw = 1.4 × 105 , alkaline phosphatase calf intestine, CALBIOCHEM, Darmstadt, Germany) was suspended with PMBN/PLA-NP in Tris–HCl buffer solution (pH 8.0) for 24 h at 4 ◦ C. The suspension was centrifuged at 10,300 × g for 30 min at 4 ◦ C. After centrifuging, the conjugation amounts of the antibody on the PMBN/PLA-NP were measured by a fluorescence spectrophotometer (FP-6500, Jasco, Tokyo, Japan). Double conjugation of two biomolecules to the PMBN/PLANP was also conducted using the same previously described procedure. The PMBN/PLA-NP were added to the antigen and enzyme mixture solution (pH 8.0) and reacted for 24 h at 4 ◦ C. After centrifuging, the PMBN/PLA-NP was thoroughly reacted with 10 mM glycine for 24 h at 4 ◦ C for complete reaction with the remaining active ester groups. 2.4. Bioreaction of biomolecules on PMBN/PLA-NP To the suspension of the PMBN/PLA-NP after bioconjugation with the antibody, a solution containing various concentrations of an antigen (Mouse-IgG, Rockland Immunochemicals, Gilbertsville, USA) was added and reacted for 24 h at 4 ◦ C. The reaction was observed by the change in the transmittance of the suspension at 550 nm. For the enzymatic reaction, the substrate for the AP, 4-methylumbelliferyl phosphate (MUP; Mw = 256.2, excitation 355 nm, Molecular Probes, Leiden, The Netherlands), was added and reacted for 24 h at 37 ◦ C. After the reaction, the fluorescence intensity was measured at 460 nm. 3. Results and discussion 3.1. 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 result, we determined a suitable composition of the PMBN for the surface modification of the polymer nanoparticles during their preparation. 3.2. Characterization of the PMBN/PLA-NP The PMBN/PLA-NP could be prepared by solvent evaporation and interfacial precipitation methods. The core polymer, PLA, was dissolved in methylene chloride, but was insoluble in water. The droplets of the PLA solution were suspended in the
Fig. 2. AFM image of the PMBN/PLA-NP.
aqueous solution along with the PMBN aggregate. The methylene chloride was then evaporated and the PLA was precipitated at the interface faced on aqueous phase. At this interface, the PMBN chains and PLA chains should make entanglements. Thus, a stable coating layer of PMBN was formed on the surface of the PLA nanoparticles. Fig. 2 shows an AFM image of the PMBN/PLA-NP. Based on the AFM observation and DLS measurement, we determined the size of the PMBN/PLA-NP, which was ca. 260 nm. The diameter and size distribution of the PMBN/PLANP averaged d = 259.7 nm and the polydispersity index was α = 1.4 × 10−1 . The size distribution of the PMBN/PLA-NP suggested a monodispersion as calculated from the cumulant method. The XPS analysis indicated that the PMBN/PLA-NP had specific peaks attributed the component atoms, such as a phosphorus peak at 135 eV, a nitrogen peak at 403 eV, an oxygen peak at 530 eV and a strong carbon peak attributed to the methyl or methylene groups at 285 eV (data not shown). For the PLA nanoparticles without the PMBN coating, there were no phosphorus and nitrogen peaks [6]. The zeta-potential of the PMBN/PLA-NP was −5 mV, whereas that of the PLA nanoparticles was −60 mV. The strong negative value became nearly zero. This is due to the electrically neutral property of the PC group by formation of an inner salt between the phosphate anion and trimethyl ammonium cation. Thus, it was revealed that the surface of the PMBN/PLA-NP was covered with PC groups. The dispersion stability depended on the surface hydrophilicity of the nanoparticles. The PC group is extremely hydrophilic, therefore, the PMBN/PLA-NP had a good dispassion stability in an aqueous medium. The formation of p-nitrophenol by hydrolysis of the active ester group of PMBN/PLA-NP was measured. We observed the UV absorbance at 400 nm, which is attributed to the pnitrophenoxy anion when the PMBN/PLA-NP was added to an alkaline solution. The p-nitrophenyl ester groups might be located on the surface of the PMBN/PLA-NP. Based on these results, the PMBN/PLA-NP possessed both PC groups and active ester groups near the surface.
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Fig. 3. Fluorescence intensity of conjugated antibody on the PMBN/PLA-NP. The fluorescence intensity on (—) PMBN/PLA-NP conjugated with antibody; (– – –) PMBN/PLA-NP when the active ester groups were hydrolyzed.
In our previous research, a surface covered with a PC group has an excellent bio/blood compatibility, that is, the suppression of non-specific protein adsorption and following thrombus formation, and immunological reactions [13–19]. That is, if the PMBN/PLA-NP was in contact with protein molecules in the aqueous medium, the protein molecules can selectively react with active ester groups by amide bond formation. This is a very important feature for controlling the conjugation density of the proteins. 3.3. Single bioconjugation with antibody on PMBN/PLA-NP Fig. 3 shows the fluorescence spectra of the suspension of the FITC-labeled antibody conjugated with PMBN/PLA-NP and that with nanoparticles after hydrolysis of the active ester groups. The hydrolysis process could inactivate the PMBN/PLA-NP for bioconjugation. Non-specific physical adsorption of the antibody could then be evaluated. The fluorescence intensity based on the FITC-antibody on the PMBN/PLA-NP was significantly higher when compared with that after hydrolysis. PMBN/PLANP sufficiently reacted in the Tris–HCl buffer solution. Thus, it clearly showed that the antibody could react with the active ester group on the surface of the nanoparticles. Since the MPC polymer suppressed protein adsorption, these data corresponded to the previous results [5]. Based on the fluorescence intensity observed on the FITC-labeled antibody conjugated with PMBN/PLA-NP, the amount of the antibody conjugated was determined. It was 2.34 g/mg-nanoparticles that corresponded to a 27% consumption of the active ester group. This consumption value of the active ester group was small due to the high molecular weight of the antibody. Using the antibody conjugated-PMBN/PLA-NP, the aggregation behavior by the addition of the antigen was investigated. Fig. 4 shows the change in the transmittance of the suspension containing nanoparticles with the reaction time after the addition of the antigen. For the antibody conjugated nanoparticles, the transmittance at 550 nm increased with time, but the change depended on the amount of antigen added to the suspension. The change in the transmittance was due to the aggregation of the nanoparticles. This means that the antibody conjugated on the nanoparticles could react with the antigen through an antigen–antibody reaction.
Fig. 4. Transmittance of the PMBN/PLA-NP conjugated antibody and enzyme after addition of the antigen for 24 h. The fluorescence intensity on () ((PMBN/PLA-NP)/antibody)/antigen (0.167 × 10−9 mol); () ((PMBN/PLA-NP)/antibody)/antigen (1.67 × 10−9 mol); () PMBN/PLANP/antigen (1.67 × 10−9 mol); (䊉) PMBN/PLA-NP.
The aggregation was very quickly initiated after addition of the antigen, and it reached an equilibrium state within 4 h under this condition. When a higher antigen concentration was added, the rate of aggregation of the antibody conjugated nanoparticles increased. Thus, the antibody conjugated on the PMBN/PLANP functioned well. One unexpected phenomenon was observed on non-conjugated nanoparticles. We could not clearly explain it at the present time. However, the following consideration was raised. Since the active ester groups on the nanoparticles were deactivated by hydrolysis, carboxylic groups were generated on the nanoparticles. These carboxylic groups might interact with the antigen and a small aggregation of nanoparticles was formed. We also carried out the enzyme conjugation on the PMBN/PLA-NP. The enzyme used in this study was AP. The reacted condition was the same condition as the antibody conjugation. As shown in Fig. 5, in the absence of the enzyme, the fluorescence intensity based on the enzymatic reaction product was quite low. However, for the AP conjugated on the PMBN/PLANP case, a strong fluorescence intensity was observed. Thus, the AP could react with the substrate and form a product due to the enzymatic reaction even though these conjugated on the
Fig. 5. The fluorescence intensity of the enzymatic reaction product on PMBN/PLA-NP/AP. The substrate for AP was MUP.
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nanoparticles. In our previous articles, we applied the PMBNbased polymers to conjugate enzymes, such as acetylcholine esterase (its function is hydrolysis of the carboxylic ester bond), choline oxydase (its function is oxidation), horseradish peroxydase (its function is reduction) and luciferase [6–8]. These various functional enzymes could also work well on the PMBNbased polymers. In addition to these enzymes, phosphatase (its function is hydrolysis of the phospholic ester bond) also functioned well on the surface. Thus, it is now concluded that the PMBN-based polymers have the possibility to conjugate every functional enzyme. Vertegel et al. reported the effect of the nanoparticle size on the adsorbed proteins [20]. They performed the adsorption of the enzyme lysozyme on SiO2 particles with 4–100 nm diameters. The structure and function of the adsorbed lysozyme on the nanoparticles are strongly dependent on the size of the nanoparticles. A less significant change in the structure and activity of the lysozyme adsorbed on smaller nanoparticles was observed. However, in our case, proteins conjugated on 200 nm particles had good functions. This is due to the effects of the PC groups on the nanoparticles. We already reported that the conformational change in protein adsorbed on the MPC polymers and poly(2-hydroxyethyl methacrylate (HEMA)) were determined using circular dichroism (CD) spectroscopic measurement [21]. The CD spectrum of BSA adsorbed on PMB with 0.30 MPC mole fraction was almost the same as that in PBS. The negative ellipticity at 222 nm of BSA adsorbed on the MPC polymers increased with a decrease in MPC composition, and then became almost zero in the case of BSA adsorbed on poly(HEMA). The secondary structure of adsorbed protein was determined to calculate the ␣-helix content. The ␣-helix content of BSA, which is assumed to be the “native” secondary structure, was 54%. On the PMB with 0.30 MPC mole fraction surface of the ␣-helix content of adsorbed BSA was almost the same as that of native proteins. However, it decreased by contact with poly(HEMA). We suggested that the secondary structure of proteins adsorbed on the MPC polymer surface was maintained at the same level as that of the original state. Thus, the PMBN/PLA-NP provided a good platform to conjugate proteins.
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3.4. Double conjugation of proteins on PMBN/PLA-NP Next, both the antibody and enzyme were conjugated on the same PMBN/PLA-NP. In this step, it was clarified that the proteins conjugated on the nanoparticles could independently react with their own specific target molecule. The conjugation procedure was very simple, that is, the PMBN/PLA-NP was added to the protein solution mixture. The confirmation of double conjugation of the antibody and enzyme was carried out. The PMBN/PLA-NP conjugated with both the antibody and enzyme (PMBN–PLA-NP/AP/antibody) were suspended, and the antigen was added to the suspension. Fig. 6 shows the results of the antigen–antibody reaction. The PMBN/PLANP/AP/antibody could aggregate by the addition of the antigen similar to the single conjugation with the antibody. In Fig. 6 (on the left side figures), the fluorescence spectra of the PMBN/PLA-NP/AP/antibody suspension are demonstrated. As shown in Fig. 6 (on the right side figures), we observed precipitation of the PMBN/PLA-NP/AP/antibody when the antigen was added. The fluorescence spectrum of the precipitate indicated that the antibody was included in the precipitate (Part A in Fig. 6(a)). At the same time, we observed a weak fluorescence intensity in the supernatant of the suspension (Part B in Fig. 6(a)). This is due to the remaining PMBN/PLANP/AP/antibody. However, the degree of precipitation versus the total nanoparticles was ca. 86%, which was calculated from the fluorescence intensity. A highly efficient reaction was confirmed. We added the enzyme substrate (MUP) to the suspension of PMBN/PLA-NP/AP/antibody after addition of the antigen. As shown in Fig. 7, the fluorescence intensity of the enzymatic reaction product was observed after the antigen addition. Although a low fluorescence was observed in the case of the PMBN/PLA-NP and PMBN/PLA-NP/antibody, a strong fluorescence was observed in the other PMBN/PLA-NP/AP and PMBN/PLA-NP/AP/antibody. It was almost the same level as that of the single conjugation of the enzyme on the PMBN/PLA-NP. Based on these results, it is clearly realized that both the antigen and enzyme were conjugated on the PMBN/PLA-NP and these proteins independently functioned very well.
Fig. 6. Fluorescence intensity of antibody conjugating the PMBN/PLA-NP with enzyme. The fluorescence intensity on A (—): ((PMBN/PLANP)/antibody/AP)/antigen; B (– – –): the top clear layer of ((PMBN/PLA-NP/antibody/AP)/antigen); C (· · · · · ·): ((PMBN/PLA-NP)/AP)/antigen.
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References
Fig. 7. The fluorescence intensity of the product of enzymatic reaction on PMBN/PLA-NP/AP/antibody with antigen.
In the near future, we will conjugate an antigen against a specific protein on the cell membrane and enzyme that can react with a specific polysaccharide chain to digest it on the PMBN/PLANP. 4. 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).
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