Construction of protein-crosslinked nanogels with vitamin B₆ bearing polysaccharide

Abstract

Protein-crosslinked nanogels were prepared by introducing vitamin B₆ (pyridoxal) to hydrophilic polysaccharide. The pH dependence of Schiff base formation was utilized to generate a pH-sensitive nanogel system. This approach represents a new method to prepare hybrid nanogels crosslinked with biomolecules.

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Nanogels are nanometre-sized hydrogel nanoparticles (<100 nm) with three-dimensional networks of crosslinked polymer chains.^1–4 They have attracted growing interest over the recent years because of their potential biomedical applications, including in drug delivery systems and bioimaging. Bioactive compounds such as drugs, proteins and DNA/RNA can be trapped within their nanospace via the polymer networks of nanogels. Moreover, nanogels rapidly respond to microenvironmental factors such as temperature and pH because of their nano-scaled dimension. These properties are useful for the controlled release of bioactive compounds. Our research group has been developing self-assembled nanogels. For example, hydrophobized polysaccharides such as cholesterol-bearing pullulan (CHP) form stable amphiphilic nanogels in water by self-association of the hydrophobic groups, which form physical crosslinks.⁴CHP nanogels trap proteins and show molecular chaperone-like activity.⁵ Furthermore, CHP nanogels are useful for protein delivery, such as in cancer vaccine and cytokine therapy.^6,7

To develop nanogels that are intended for biomedical applications, we must develop novel crosslinking methods with acceptable biodegradability and biocompatibility. Accordingly, numerous methods to form crosslinks, other than exploiting hydrophobic interactions, have been investigated to prepare nanogels appropriate for specific applications. For example, researchers have prepared nanogels using electrostatic interactions⁸ or using host–guest interactions with cyclodextrin.⁹ Photoresponsive nanogels based on a spiropyrane-modified pullulan¹⁰ and pH-responsive nanogels comprised of ethylenediaminetetraacetic acid-bearing chitosan have also been reported.¹¹ These nanogels developed by exploiting a wide range of intermolecular forces show great potential for the preparation of novel stimulus-responsive nanomaterials.

Biomolecules, including oligopeptides,^12,13proteins,¹⁴antibodies¹⁵ and oligonucleotides,¹⁶ have been widely used as crosslinkers for the preparation of macroscale hydrogels other than nanogels. One of the main advantages of biomolecule-crosslinked hydrogels is that their response to stimuli is controlled and modulated by the biomolecules.¹⁷ Despite the promising utility of crosslinked biomolecules, there are few examples of biomolecule-crosslinked nanogels, perhaps because of the difficulty in conjugating the biomolecules with polymer chains in an aqueous environment.

In this study, vitamin B₆ (pyridoxal), which easily reacts with amine derivatives under ambient conditions, was introduced to a polysaccharide to prepare a novel nanogel crosslinked with a protein containing several amino groups (Fig. 1). Vitamin B₆ is a coenzyme for vitamin B₆-dependent enzymes.¹⁸ One of the important chemical characteristics of the pyridoxal moiety is that it acts as an active aldehyde to form a Schiff base between the amine and the pyridoxal formyl group. The Schiff base is also considered to be a dynamic covalent bond that is regulated by external environmental factors, such as pH. This property enables us to control the association and dissociation of the pyridoxal with amine derivatives by changing the pH of the aqueous solution. By using the Schiff base, polysaccharides modified with pyridoxal can be held together to form nanogels that crosslink with biomolecules containing amino groups, such as proteins. Here we report the preparation of pyridoxal 5′-phosphate-bearing pullulan (PLPP, Fig. 2) that was generated by click chemistry. We also characterise the PLPP nanogels that formed crosslinks with lysozyme containing six lysine residues as a model protein.

Fig. 1 Schematic representation of the protein-crosslinked nanogel formed by self-assembly of PLPP and protein.

Fig. 2 Chemical structure of pyridoxal phosphate-bearing pullulan (PLPP).

We first used click chemistry to prepare PLPP from pyridoxal 5′-phosphate modified with an alkyne (PLP-alkyne) and pullulan (M_w = 100 000) grafted with azides (pullulan-N₃). To prepare PLP-alkyne, p-ethynylaniline was diazotised by sodium nitrate and then reacted with pyridoxal by an azo coupling reaction.¹⁹ Next, pullulan was activated by 1,1′-carbonyldiimidazole and reacted with 3-azidepropylamine to yield pullan-N₃.²⁰ The product was identified by ¹H NMR and we determined that 6.8 azide groups were introduced per 100 glucose units. Finally, PLPP was prepared by a 1,3-cycloaddition reaction of PLP-alkyne with pullan-N₃ in the presence of sodium L-ascorbate and copper(II) sulfate as a catalyst. The conjugation of pyridoxal to pullulan was confirmed by the FT-IR spectrum for the products, which confirmed the disappearance of the azide peak (2100 cm⁻¹). The degree of substitution of PLP coupled to the polysaccharides per 100 glucose units was determined to be 3.8 based on the integrated areas of glucopyranosyl rings and the decrease in the number of ethynylaniline groups.

To examine the reactivity of aldehyde on pullulan-conjugated PLP, we determined the formation of a Schiff base using dopamine as a simple model compound with a primary amine group. The binding behaviour of the pullulan-embedded PLP with dopamine was examined by electronic absorption spectroscopy at 25 °C. The PLPP dissolved in water at pH 8.0 had an absorption maximum of 481 nm. Adding dopamine to the solution changed the UV spectra of PLPP, reflecting the formation of a Schiff base between the amino group of the dopamine and the formyl group of the pyridoxal in PLPP. The titration isotherms were obtained by monitoring the absorbance at 481 nm and were applied to determine the apparent binding constant (3.7 × 10³ M⁻¹) (Fig. S4, ESI†). This result indicates that pullulan was modified by pyridoxal bearing an active aldehyde.

Then, the binding of PLPP with protein as a biomolecular crosslinker to yield protein-crosslinked nanogels was investigated. Lysozyme, one of the most extensively studied proteins, was used as a model protein. It has a molecular weight of 14 300, an isoelectric point of 10.9, and is composed of 129 amino acid residues, 4 sulfide bridges, and 6 lysine residues. Adding lysozyme to the PLPP solution at pH 8.0 significantly changed the UV spectra, as shown in Fig. 3. The UV titration of PLPP with lysozyme (Fig. S5, ESI†) clearly shows the formation of a Schiff base with an apparent binding constant of 7.7 × 10⁴ M⁻¹, which is higher than that of dopamine. This is likely because the electrostatic interaction between cationic lysozyme and anionic PLPP at pH 8.0 increases the apparent binding constant, although multivalent interactions might also be involved in the enhanced binding. In contrast, the binding constant at pH 5.0 was <10² M⁻¹, much weaker than that at pH 8.0. In general, the formation of a Schiff base is dependent on the pH of the solution. These results indicate that the formation of a Schiff base can be controlled by changing the environmental pH.

Fig. 3 Change in the UV spectra of PLPP (0.2 mM, as a molar concentration of PLP embedded in pullulan) following the addition of lysozyme at pH 8.0 (a) and pH 5.0 (b). Concentrations in mmol dm⁻³: [HEPES], 100; [NaCl], 100.

The interaction between lysozyme and PLPP was also evaluated by size exclusion chromatography. After mixing PLPP with lysozyme for 3 min, the original peak corresponding to free lysozyme disappeared, while the magnitude of the peak corresponding to PLPP increased (Fig. S6, ESI†). These results suggest that a significant amount of lysozyme interacts with PLPP, and we estimated that >80% of the lysozyme added to the solution formed complexes with PLPP. However, the elution profile of lysozyme did not change when we mixed lysozyme with PLPP at pH 5.0/25 °C for 5 min. Fig. 4 shows the amount of lysozyme that bounds to PLPP as a function of the lysozyme concentration and shows that the amount of bound lysozyme increased with increasing the lysozyme concentration. In contrast, lysozyme hardly complexed with PLPP at pH 5.0, even at the highest concentration of lysozyme tested. By changing the pH from 8 to 5, lysozyme that interacted with PLPP at pH 8 was released from the complex at pH 5 (for example, 12%, after 2 h). PLPP contains 24 vitamin B₆ moieties in a polysaccharide chain, whereas lysozyme contains 6 lysine residues per protein. Therefore, each PLPP molecule can bind to 4 lysozyme molecules if each vitamin B₆ moiety reacts with a lysine group. At pH 8.0, each PLPP molecule could bind to 5 lysozymes, as shown in Fig. 4. This indicates that a considerable number of lysine and vitamin B₆ molecules formed Schiff bases to generate the inter- or intramolecular crosslinks.

Fig. 4 Number of bound lysozyme molecules per PLPP molecule as a function of the concentration of lysine residues in lysozyme at pH 8.0 (closed circle), 7.0 (closed diamond), 6.0 (closed square) and 5.0 (closed triangle), as determined by HPLC. Concentrations in mmol dm⁻³: [HEPES], 100; [NaCl], 100.

The above results suggest that lysozymes bearing multiple plural binding sites held together the polysaccharidevia the formation of Schiff base at pH 8.0 to generate nanogels. To confirm the formation of nanogels through the complexation of PLPP and lysozyme, we performed dynamic light-scattering (DLS) measurements (Table 1). The hydrodynamic diameter (D_hy) of the PLPP aqueous solution (1 mg ml⁻¹) could not be determined using DLS because of the low light-scattering intensity of the solution. This suggests that PLPP was molecularly dispersed in water and did not associate with macromolecules in these conditions. In the presence of lysozyme at pH 8.0, the D_hy was 20–30 nm, indicating the formation of nanogels. In contrast, the mixture containing unmodified pullulan at pH 8.0 did not provide measurable light scattering. The nanogel formation was also analysed by transmission electron microscopy (TEM). Negatively stained TEM images of PLPP in the presence of lysozyme confirmed the formation of monodisperse nanogels, as shown in Fig. 5. The size of the nanogel corresponded to the D_hy determined by DLS. The reason for the formation of the nanoparticle is not clear yet. Effective coating of the protein by the polysaccharide chain with PLP may stop further aggregation.

Table 1 Hydrodynamic diameters (D_hy) of PLPP and pullulan in the presence and absence of lysozyme at 25 °C^a

Sample			D hy/nm^e
[PLP]^b	[Pullulan]^c	[Lys in lysozyme]^d
a Hydrodynamic diameters were determined by dynamic light scattering measurements. b The concentration of PLP in PLPP is given in mmol dm^−3. The concentration of the parent pullulan is given in parentheses in mmol dm^−3. c The concentration of pullulan is given in mmol dm^−3. d The concentration of lysine residues in lysozyme is given in mmol dm^−3. e The standard deviation of three independent measurements is given in parenthesis. f The Dhy could not be determined because of the low light-scattering intensity of the solution.
0.2 (0.01)	0	0	n.d.^f
0.2 (0.01)	0	0.15	21.8 (0.2)
0.2 (0.01)	0	0.30	22.4 (0.3)
0.2 (0.01)	0	0.60	27.9 (1.6)
0	0.01	0	n.d.^f
0	0.01	0.60	n.d.^f

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Fig. 5 Negatively stained TEM images of the PLPP nanogel at pH 8.0. Concentrations in mmol dm⁻³: [PLPP], 0.1; [lysozyme], 0.2; [HEPES], 100; [NaCl], 100.

Conclusions

In summary, pyridoxal-modified pullulan was prepared to yield protein-crosslinked nanogels via the formation of Schiff base between the pyridoxal moiety and the protein's amino groups. The pH dependence of the formation of Schiff base was utilized to construct a pH-sensitive nanogel system. To the best of our knowledge, this is the first report describing the preparation of protein-crosslinked nanogels. These pH-sensitive hybrid nanogels upon Schiff base formation with various biomolecules are useful for a nanocarrier in drug delivery system. For example, acid sensitive nanogels with proteins would be applied for cytosolic protein delivery. Biomedical applications are under investigation in our laboratory.

Acknowledgements

The present work was supported by a Grant-in-Aid for Scientific Research from the Japanese Government (No. 20240047) and by the Global Center of Excellence Program at the International Research Center for Molecular Science in Tooth and Bone Diseases at Tokyo Medical and Dental University.

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Footnote

† Electronic supplementary information (ESI) available: Materials, instruments, synthesis of compounds, UV titration, and HPLC profiles for complexation. See DOI: 10.1039/c1py00100k

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