Nonionic Block Copolymers Assemble on the Surface of Protein

Aug 9, 2012 - *E-mail: [email protected]. Cite this:Langmuir 28, ... Niu , Yong Huang. Advanced Healthcare Materials 2015 4 (10.1002/adhm.v4.3), 413-...
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Nonionic Block Copolymers Assemble on the Surface of Protein Bionanoparticle Zhi Liu,† Jingxia Gu,† Man Wu,† Shidong Jiang,† Dayong Wu,† Qian Wang,‡ Zhongwei Niu,*,† and Yong Huang† †

National Research Centre of Engineering Plastics, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100084, China ‡ Department of Chemistry and Biochemistry and Nanocenter, University of South Carolina, 631 Sumter Street, Columbia, South Carolina 29208, United States S Supporting Information *

ABSTRACT: Efficient delivery of therapeutic proteins to a target site remains a challenge due to rapid clearance from the body. Here, we selected tobacco mosaic virus (TMV) as a model protein system to investigate the interactions between the protein and a nonionic block copolymer as a possible protecting agent for the protein. By varying the temperature, we were able to obtain core−shell structures based on hydrophobic interactions among PO blocks and noncovalent interactions between TMV and EO blocks. The protein−polymer interactions were characterized by dynamic light scattering and isothermal titration calorimetry. This study establishes principles for the possible design of clinically useful protein delivery systems.

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INTRODUCTION Therapeutic proteins represent currently a significant part of new pharmaceuticals.1 The high biological activity and specificity of proteins compared to more conventional, low molecular weight drugs often allows for a better treatment of diseases. Despite their potential medical applications, effective delivery of the proteins to a target site remains a challenge due to rapid clearance from the body due to the combination of proteolysis in the bloodstream, liver clearance, and starvation by the immune system. Direct covalent conjugation of poly(ethylene glycol) (PEG) on the surface of proteins (known as PEGylation) has been widely used to increase the stability and plasma half-life of proteins.2−4 However, a drawback with covalent protein PEGylation is that biological activity may be compromised by the chemical modification.5 To overcome this obstacle, an alternative strategy based on noncovalent charge−charge interactions to coat proteins with polyion complex (PIC) micelles has been recently developed.6 However, deficiencies of this method, such as cytotoxicity of PIC micelles, is a limiting factor for clinical applications.7−9 Here, we demonstrate a simple approach to coat protein bionanoparticles with nonionic block copolymer Pluronics F127 via noncovalent interaction. Tobacco mosaic virus (TMV), a classic example of a self-assembled rod-like protein cylinder, was selected here as a model system to investigate the interactions between the protein and nonionic block copolymer. Native TMV particles consist of 2130 identical protein subunits arranged helically around genomic single-stranded RNA. TMV is 300 nm long and 18 nm in diameter, with a 4 nm cylindrical cavity along the central core.10 Triblock copolymers of poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene © 2012 American Chemical Society

oxide) (PEO−PPO−PEO), referred to as Pluronics or Polaxamers, are FDA approved biocompatible polymers which are widely used in the biomedical applications.11,12 As demonstrated in Scheme 1, when mixing TMV with block copolymer F127 in solution above the critical micelle temperature (CMT) of F127, micelles of F127 can adsorb onto the surface of TMV. The transition from micelle to capsule-like structure can generate a stable polymer coating on the surface of TMV. Scheme 1. Cartoon Illustrating the Formation of the Possible Assembly Structure

Received: February 28, 2012 Revised: August 7, 2012 Published: August 9, 2012 11957

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Figure 1. TEM images of (a) TMV, (b) coated structures resulting from mixing 0.1 mg/mL TMV with 2.5 mg/mL F127 in pH 7.8 potassium phosphate buffer, and (c) TMV/F127 solution with Hep-β-CD; the inset shows a cartoon of a possible assembly structure. (d) Schematic representation of the structure of the α-CD/F127 and Hep-β-CD/F127 pseudopolyrotaxanes.

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In our experiment, α-CD or hep-β-CD was added into the TMV/F127 solution (CTMV = 0.1 mg/mL, CF127 = 2.5 mg/mL), with a molar ratio of α-CD:EO = 1:2 or hep-β-CD:PO = 1:2. In the case of α-CD, no polymer coating can be visualized (data not shown). Since α-CD has strong interaction with EO units of F127 in solution, it will compete with TMV to interact with F127 (the EO block) and prohibit the adsorption of F127 on the surface of TMV. As for hep-β-CD, a denser layer with a thickness around 16− 18 nm on the surface of TMV can be clearly visualized, as shown in Figure 1c. The AFM image also clearly shows the formation of a thick layer of F127 capsule on the surface of TMV (Figure S2b, Supporting Information). It has been observed that the addition of hep-β-CD can trigger the breakup of micelles below 50 °C.17,18 Therefore, upon addition of hep-β-CD into the TMV/F127 solution, it is likely that pseudopolyrotaxanes formed spontaneously in our situation (illustrated in the inset of Figure 1c), resulting in threading of PO units through hep-β-CD rings. This can further contribute to the larger thickness increase (16−18 nm) compared with the TEM observation in Figure 1b (around 11 nm) where the cyclodextrin was not present. This result also indicated the formation of an EO−PO−EO sandwich-like structure on the surface of TMV (Figure 1c, inset): one section of the hydrophilic EO chains adsorbs on the surface of TMV, and the other section dissolves in water with PO parts in the middle phase. To probe the initial self-assembly behavior of F127 with TMV under different conditions, dynamic light scattering (DLS) experiments were performed, which demonstrated temperature-dependent dynamic adsorption behavior. As shown in Figure 2a, when dissolving 2.5 mg/mL F127 in potassium phosphate buffer (pH 7.8) at 35 °C, the PO block progressively loses its hydrated forms, resulting in increasing PO−PO interactions; thus, the aggregation of F127 leads to the formation of micelles. A distinct peak at a radius of about 10 ±

RESULTS AND DISCUSSION As shown in Figure 1a, the diameter of native TMV is around 18 nm after negative staining with 2% uranium acetate. A clear polymer layer with a thickness around 11 nm on the surface of TMV can be clearly visualized in Figure 1b, after mixing 0.1 mg/mL TMV and 2.5 mg/mL F127 in 0.01 M potassium phosphate buffer solution at about 30 °C, which is higher than the CMT of F127 at this concentration (the CMT of F127 at 1 mg/mL is 30 °C). The coating can be attributed to the adsorption of micelles of F127 onto the surface of TMV. No obvious coating was found when TMV and F127 were mixed at 25 °C (data not shown). Similarly, using the same protocol, another nonionic block copolymer P123 (EO19−PO69− EO19) could also assemble with TMV and form a thin layer on the surface of TMV (Figure S1, Supporting Information). The polarity of the nanoparticle surface can determine which of the hydrophilic or hydrophobic blocks adsorb to the surface of the particle, just as the polarity of the solvent determines the formation of the outer shell and the inner core of micelles. As the surface of TMV in phosphate buffer at pH 7.8 is hydrophilic and anionic (the isoelectric point of TMV is around 3.4), our hypothesis is that the hydrophilic block of the Pluronics adsorbs on the surface of TMV through noncovalent interaction (a weaker hydrophobic interaction or hydrogen bonding) between EO blocks and coat proteins of TMV. In order to verify this hypothesis and explore the potential selfassembly mechanism, different kinds of cyclodextrins (CDs), including α-CD and heptakis (2,6-di-O-methyl)-β-CD (hep-βCD), were used as selective threading reagents to bind selectively to either PEO or PPO blocks. CDs have attracted particular interest for their ability to form inclusion complexes through noncovalent interactions with a variety of molecular guests that can fit into the cavity.13,14 It has been reported that α-CD can form complexes with EO units,15 whereas β-CD would have a higher affinity with the bulkier PO units in aqueous solution (illustrated in Figure 1d).16 11958

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The primarily spherical micelles will then reassemble into cylindrical capsule-like forms. As the temperature decreases, desorption of F127 occurs because the EO chains experience a rehydration process, while PO chains change from a hydrophobic microenvironment to a hydrophilic one at lower temperature.22 Therefore, when lowering the temperature to 25 °C, TMV restores to its original appearance and the average hydrodynamic radius transfers back to around 70 nm. F127 copolymers exist as individual unimers in aqueous solution again. If the temperature was increased to 35 °C again, the hydrodynamic radius increased to 90 nm, and this behavior could be repeated many times (Figure S5, Supporting Information). Isothermal titration calorimetry (ITC) was used to characterize the binding affinity between F127 with TMV at different temperature. ITC is a widely used technique to determine the binding energetics of biological processes, including protein− ligand binding, protein−carbohydrate binding, protein−lipid binding, antigen−antibody binding, and protein−protein binding.23−25 As shown in Figure 3, ITC results clearly show the

Figure 2. Hydrodynamic radius measured by DLS in pH 7.8 phosphate buffer solution of (a) F127 alone at 35 °C, (b) TMV alone at 35 °C, and (c) TMV/F127 at 35 °C, respectively. CF127 = 2.5 mg/mL, and CTMV = 0.1 mg/mL.

1 nm, which can be attributed to the formation of F127 micelles, and a small peak corresponding to the hydrodynamic radius ⟨Rh⟩ of F127 unimer at a radius of about 2 ± 0.5 nm are also visualized.19 While at 25 °C, which is below the CMT of F127 (the CMT of F127 at 2.5 mg/mL is 28 °C20), both EO and PO chains have been proved to be in a hydrated form at low temperature, and only one peak at a radius of about 2 ± 0.5 nm can be visualized (Figure S3a, Supporting Information). The ⟨Rh⟩ of TMV (0.1 mg/mL in pH 7.8 potassium phosphate buffer) is approximately 70 ± 5 nm, which is independent of temperature (Figure 2b and Figure S3b, Supporting Information). When mixing TMV and F127 (CTMV = 0.1 mg/mL, CF127 = 2.5 mg/mL) at 35 °C, two distinct peaks at a radius of about 10 ± 1 and 90 ± 5 nm can be clearly seen (Figure 2c). The peak at radius 10 ± 1 nm can be attributed to the excess micelles of F127, which are not adsorbed onto the surface of TMV. The peak at a radius of about 90 ± 5 nm compared with 70 nm for TMV alone (Figure 2b) and the broader distribution provide evidence of the formation of the F127/TMV complex. As the temperature reaches 45 °C, the size of the objects also increases correspondingly, with an average hydrodynamic radius of more than 100 ± 5 nm (Figure S4, Supporting Information). The formation of F127/TMV complex is a temperature-dependent process. As the temperature is increased beyond the CMT, the hydrophilic EO chains will dehydrate because of the breakage of the hydrogen bonds with water molecules,21 which should promote interactions between EO chains and TMV coat proteins. Therefore, copolymer micelles can adsorb on the surface of TMV. As more and more micelles adsorb, the local concentration of F127 on the surface of TMV also increases.

Figure 3. ITC thermograph recorded for titration of F127 aqueous solution (CF127 = 0.4 mM) into TMV (CTMV = 0.01 mM) aqueous solution in potassium phosphate buffer (0.01 M, pH 7.8) at 35 °C after background subtraction of the formation of micelle. (For experimental details and raw ITC data, see the Supporting Information and Figures S6−S9.)

binding of F127 with TMV at 35 °C after background subtraction. However, no obvious uptake or release of heat can be detected upon titration of F127 into TMV at 25 °C after background subtraction, which indicates that the interaction between F127 and the surface of TMV is very weak at lower temperature. Previous studies have shown that the salt concentration can affect the aggregation behavior of the F127 copolymer.26,27 Here, we chose 50 and 500 mM NaCl aqueous solution to study the effect of different salt concentrations on the adsorption behavior of F127 on the TMV surface. At high salt concentration, i.e., 500 mM NaCl, the salt screened the surface charge of TMV and caused the aggregation of TMV with F127 due to the depletion force, which is independent of temperature. For the 50 mM NaCl, two distinct peaks at a radius of about 2 ± 0.5 and 70 ± 5 nm can be clearly seen in DLS at 25 °C (Figure 4a). The peak at radius 2 ± 0.5 nm can be attributed to the unimer of F127, and the hydrodynamic radius of TMV is approximately 70 ± 5 nm. At 35 °C, the peak at a radius of 100 ± 5 nm is assigned to the F127/TMV complex, which is a little larger than the size of the F127/TMV complex without NaCl (Figure 4b). However, as shown in 11959

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are larger than either ferritin or F127 alone at this temperature, indicating the formation of ferritin/F127 complexes. Therefore, this assembly method has the potential to be a general strategy to coat protein-based bionanoparticles with a biocompatible polymer layer.

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CONCLUSIONS To conclude, we have demonstrated a simple, effective method to decorate TMV with Pluronics F127, to our knowledge, the first case of such nonionic block copolymer based nanocapsule formation on the surface of protein-based nanoparticles. Also, this work may help us to understand the interaction between polymer and biological molecules and open a new direction for protein delivery systems. Furthermore, the shell of polymer nanocapsules may be further modified to alter the functionality of the nanocapsules for many other applications in nanomedicine.

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Figure 4. Hydrodynamic diameters measured by DLS in pH 7.8 phosphate buffer solution (with salt concentration: 50 mM NaCl) of (a) TMV/F127 at 25 °C and (b) TMV/F127 at 35 °C, respectively. Concentrations of polymer and TMV: CF127 = 2.5 mg/mL and CTMV = 0.1 mg/mL.

ASSOCIATED CONTENT

S Supporting Information *

Experimental details, images of TEM and AFM, and DLS and ITC data. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure S12 (Supporting Information) and Figure 5, the ITC result shows that obvious changes of thermodynamic character-

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Zhenzhong Yang and Prof. Yongming Chen for helpful discussions. This work was supported by the National Natural Science Foundation of China (Grant Nos. 21074143 and 91027030) and Hundred Talents Program of the Chinese Academy of Sciences.

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Figure 5. ITC thermographs recorded for titration of F127 aqueous solution (CF127 = 0.4 mM) into TMV (CTMV = 0.01 mM) aqueous solution in potassium phosphate buffer (pH 7.8, with salt concentration: 50 mM NaCl) at 35 °C after background subtraction.

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ization between F127 and TMV can only be detected at higher temperature, and there were no meaningful differences in the ITC data obtained at different salt concentrations. Therefore, the result indicates that temperature is the main factor influencing the adsorption of micelles on the surface of TMV, while high salt concentration may favor the adsorption of F127 on the surface of TMV. To extend this method to a different protein-based nanoparticle, F127 was found to self-assemble with spherical ferritin nanoparticles (Figures S15 and S16, Supporting Information). The ⟨Rh⟩ of ferritin (0.1 mg/mL in pH 7.8 potassium phosphate buffer) according to DLS is approximately 7 ± 1 nm, which is independent of temperature (Figure S17a and b, Supporting Information). When ferritin and F127 are mixed at 25 °C, objects having a radius of approximately 10 ± 1 nm are observed (Figure S17c, Supporting Information). However, the size of the objects increases to about 15 nm at 35 °C (Figure S17d, Supporting Information). These objects 11960

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