Self-assembly hollow nanosphere for enzyme encapsulation

Abstract

This paper studies a kind of self-assembled hollow nanosphere for L-asparaginase encapsulation, and it was found that the hollow spheres not only enable a high loading of enzyme, but also show semi-permeability which could prevent the enzyme from leaving while allowing substrates and products to pass through to maintain the enzyme activity, and the encapsulated L-asparaginase showed significantly higher stability.

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Construction of supramolecular architectures by self-assembly or self-organization is an attractive subject for scientists. In recent years, great progress has been made in fabricating supramolecular objects with novel phase morphologies, architectures and functions.^1–4 One kind of targeting material is hollow spherical aggregates, which have great potential for encapsulation of large quantities of guest molecules or large sized guests within the “empty” core domain.^5,6 Since Jenekhe and Chen reported that a rigid-coil copolymer could self-assemble into hollow spheres directly in their selective solvent,^7,8 over the last ten years, this novel approach has aroused many researchers' interest in using rigid-coil systems to construct hollow spheres. For example, Jiang and coworkers reported that hydrogen-bonding complexes of combined rod-like and coil-like polymers could also self-assemble into hollow spheres directly.^9–13 However, these self-assembly hollow spheres lack biocompatibility and non-biodegradability because the rod-like blocks are π-conjugated long chains formed by synthesis method.

Our group recently reported a novel approach to construct hollow spheres by inclusion alginate-graft-poly(ethylene glycol) (Alg-g-PEG) and α-cyclodextrin (α-CD), in which the rod-like blocks are formed by self-assembly of α-CD with poly(ethylene glycol) (PEG).¹⁴ Because of having biocompatibility and degradability, Alg-g-PEG/α-CD hollow spheres may serve as an alternative to small-molecule surfactant vesicles or polymer micelles as drug delivery vehicles. In this paper, the work is extended to investigate the encapsulation behavior of enzymes within Alg-g-PEG/α-CD hollow spheres. To our surprise, the Alg-g-PEG/α-CD hollow spheres not only enabled a greater loading of enzymes due to having cavities, but also showed semi-permeability which could prevent the enzyme from leaving to stabilize the enzyme while allowing substrates and products to pass through to maintain the enzyme activity.

L-Asparaginase was selected as a model enzyme, because it is an effective antitumor agent against leukaemia but it has a short half-life in vivo and side effects limit its clinical use.¹⁵ In order to improve the enzyme efficiency and reduce the immune response and toxicity, the native L-asparaginase is often chemically modified or administered by a drug delivery system. But chemical methods suffer from severe reactive conditions and always result in a large loss of enzymatic activity.^16,17 Although loading L-asparaginase into a drug delivery system could reduce the side effects and increase the enzyme efficiency,¹⁸ immunological response and toxicity still exist when L-asparaginase is released into the blood. Our experiments show that entrapping the L-asparaginase in Alg-g-PEG/α-CD hollow spheres could effectively prevent the enzyme from leaking into the blood and maintain enzyme activity.

The Alg-g-PEG solution with L-asparaginase was added dropwise to the α-CD solution in water and the mixed solution gradually became slightly turbid. Our previous paper has demonstrated that α-CD molecules should thread along the PEG chains to form rod-like inclusion blocks and form hollow spheres due to the propensity of parallel packing of the rod-like inclusion block grafted on the alginate chains. It should be noted that the enzyme cannot form aggregates with Alg-g-PEG (Fig. S4, ESI†) or be included in α-CD molecules when the enzyme was mixed with Alg-g-PEG solution or α-CD solution separately, and both α-CD and Alg-g-PEG have no effect on the activity of the enzyme (Table S1, S2 and S3, ESI†). So the absorbance change in solution resulted from the formation of Alg-g-PEG/α-CD aggregation. The Alg-g-PEG/α-CD aggregation was separated by centrifugation and the amount of enzyme in the supernatant was significantly decreased, which indicated that the enzyme was entrapped in the Alg-g-PEG/α-CD spheres when it was added into solution containing both Alg-g-PEG and α-CD. The encapsulation procedure was shown in Fig. 1.

Fig. 1 Construction of hollow spheres and the process of enzyme encapsulation.

The morphology of the particles with entrapped L-asparaginase and corresponding particles without L-asparaginase were explored by transmission electron microscopy (TEM). As shown in Fig. 2a, an obvious contrast between the central and outer part of particle without entrapping L-asparaginase was observed, which is typical of hollow spheres as reported for different kinds of hollow particles.^7–13 After the introduction of L-asparaginase into the system, the presence of these enzyme molecules has no effect of the self-assembly procedure, suggesting that a typical host–guest recognition also plays an important role in the formation of hollow spheres and encapsulation of enzyme into the hollow spheres. In Fig. 2b, no bright domains can be found within the spheres with entrapped L-asparaginase. In contrast, the cores of spheres were a little darker than the outlines of spheres. This result was due to the fact that the hollow spheres are filled with enzyme molecules. Fig. 2c shows the hydrodynamic diameter (Dh) distribution of the particles with entrapped L-asparaginase in an aqueous solution obtained by light scattering. It can be seen that the particles show a narrow size distribution and the average Dh is about 467 nm. The diameter of the particles in Fig. 2b (around 200 nm) is little smaller than that from Dh, which may result from the shrinkage of particles during the process of solvent evaporation in the sample preparation.

Fig. 2 (a) TEM image of the Alg-g-PEG/α-CD particle without L-asparaginase; (b) TEM image of the Alg-g-PEG/α-CD particles with entrapped L-asparaginase, and (c) hydrodynamic diameter distribution of Alg-g-PEG/α-CD particles with entrapped L-asparaginase.

The enzyme entrapment potential of the Alg-g-PEG/α-CD hollow spheres was investigated with a L-asparaginase loading of 1–2 mg. As shown in Table 1, the encapsulation efficiency was increased with the concentrations of Alg-g-PEG increasing. This maybe due to the fact that the amount of hollow spheres increased as the concentration of polymer increased. But when the concentration of Alg-g-PEG was above 0.375%, the encapsulation efficiency almost keep stable. The leakage of L-asparaginase from the Alg-g-PEG/α-CD hollow spheres was tested after the hollow spheres were kept in a Tris buffer at 4 °C for 48 h. It is shown that the leakage efficiency is low for all the samples (below 10%).

Table 1 The encapsulation efficiency, leakage efficiency of nanospheres

Entry	Concentrations of Alg-g-PEG	Encapsulation efficiency (%)	Leakage efficiency (%)
1	0.065	37.2	6.1
2	0.125	45.4	6.3
3	0.187	49.7	7.3
4	0.250	72.4	1.3
5	0.375	81.9	5.4
6	0.500	80.1	1.3

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The activities of the entrapped L-asparaginase in sample 4 were calculated according to the literature.^19,20 The results showed that the activity of entrapped enzyme was 85.5 ± 5.5% (average value of three times determination) of the free enzyme activity. This result indicates that: through the self-assembly of Alg-g-PEG and α-CD, the L-asparaginase could be entrapped in semi-permeable hollow spheres effectively, which prevent the enzyme from diffusion into the surrounding solution while allowing substrates and products to pass through to undertake a enzymatic catalytic reaction (Scheme 1) to preserve the activity. Since the enzyme exerts its action in the hollow spheres and the antigen determinants of the enzyme may be masked by a shell of spheres, this encapsulated L-asparaginase has good potential for medical applications. Because of avoiding ultrasonication, organic solvents, or different additives for preparation of L-asparaginase in Alg-g-PEG/CD, the enzyme maintained most of the activity. The slight activity decrease of the entrapped enzyme compared with free one maybe attributed to the transport barrier resulting from the shells of the hollow spheres.

Scheme 1 A schematic of the enzymatic catalytic reaction.

To further investigate the differences between the free enzymes and entrapped enzymes, their Michaelis constants were determined. Fig. 3 shows the Lineweaver–Burk plots of these two enzymes. Two linear equations of the entrapped L-asparaginase and free L-asparaginase were obtained from the plots, y = 10.779x + 1.5038 and y = 8.0313x + 1.7283, respectively. The Michaelis constant (Km) of the L-asparaginase encapsulated in hollow spheres (4.65 × 10⁻³ M) was a 1.54-fold decrease of the native enzyme (7.17 × 10⁻³ M). This indicates that the affinity between L-asparaginase and its substrate L-asparagine was increased when L-asparaginase was encapsulated in the hollow spheres.

Fig. 3 Lineweaver–Burk plots for free L-asparaginase and L-asparaginase entrapped in the hollow spheres (sample 4). The data in the figure were average values of three-repeated measurements.

For storage and application of L-asparaginase in a physiological environment, it is important to examine the stability of the L-asparaginase in the hollow spheres (sample 4).

The effect of pH on the activity of the free and encapsulated L-asparaginase was determined by adjusting the reaction medium at a fixed pH value ranging from 4 to 12. As shown in Fig. 4, the free L-asparaginase showed maximum activity at pH 8.6, while the optimal pH of the encapsulated enzyme was much wider, in a range of pH 7.0–11.0. These results indicate the encapsulated L-asparaginase in hollow spheres stabilized the enzymatic activity over a broader pH range.

Fig. 4 Effect of the reaction pH on the activities of the entrapped and free L-asparaginase.

In a weakly acidic environment (pH = 5), the relative activity of entrapped and free L-asparaginase decreased with increasing incubation time. But the entrapped enzyme was clearly stabilized over the free enzyme (Fig. 5). After 270 min in the pH 5 solution, the remaining activity of the entrapped enzyme was 83.05%, while that of free enzyme was 55.36%. Large enzyme molecules are physically confined in a small space of the core, which maybe result in that the three-dimensional structure of the entrapped enzyme is difficult to change, and then pH stability of L-asparaginase is improved.

Fig. 5 pH stability of the free and entrapped L-asparaginase (pH 5).

The activities of the free and entrapped L-asparaginase were measured by maintaining the reaction medium in a thermal bath at a constant temperature from 17 to 85 °C. Fig. 6 shows the relative activity as the temperature of the entrapped L-asparaginase reaches its highest value at 45 °C, and the entrapped L-asparaginase showed almost maximum activity in the range 35–65 °C. The free enzyme shows maximum activity at 65 °C. Above the optimum temperature, both free and encapsulated enzymes are gradually deactivated with increasing temperature. These results indicate that the encapsulated L-asparaginase in hollow spheres stabilizes the enzymatic activity over a wider temperature range.

Fig. 6 Effect of the reaction temperature on the activities of the entrapped and free L-asparaginase.

Conclusions

The anti-leukemic enzyme L-asparaginase was encapsulated in Alg-g-PEG/α-CD hollow spheres directly in aqueous solution through the self-assembly of Alg-g-PEG and α-CDs. Although protected in hollow spheres, the encapsulated enzyme can still exert its activity and the affinity between the substrate and encapsulated enzyme was lightly increased. Furthermore, the encapsulation of L-asparaginase widened the optimum reactive temperature and pH range of the enzyme, and the encapsulated L-asparaginase showed significantly higher stability in an acidic environment as compared to the native enzyme. We suggest that the encapsulation method presented in this paper represents an approach to the preservation of biological activity of protein drugs for medical purposes.

Acknowledgements

This work was financed by National Natural Science Foundation of China (NSFC Grant Nos. 30600148 and 50703025).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental section for synthesis of the PEG-branched alginate and spectra, preparation of the hollow spheres, encapsulation procedure, determination the activity of the encapsulated and free L-asparaginase, L-asparaginase encapsulation efficiency, activity recovery of the encapsulated L-asparaginase and the measurement of Michaelis constant. See DOI: 10.1039/b925747k

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