2.1. Influence of Organic Solvent Species and Emulsifying Operation on Catalase Activity
Experimental constraints such as sonication and organic solvent might disturb the activity of catalase. Different organic solvents decreased catalase activity to varying extents with acetone/DCM (1:1) causing the lowest loss in activity among the three solvents tested, regardless of whether sonication or vortex was used (
Table 1). Therefore, acetone/DCM (1:1) was used as dissolvent of catalase. This was also supported by a study of Gander
et al. who found that acetone did not disturb the structure of protein [
3] and it was often used for the fractionation of plasmatic proteins. The choice of methylene chloride was rational as it has always been used for nanoparticle preparation [
8], and served as the solvent for acetone. It was found that susceptibility to the denaturing action of DCM is dependent on the protein type during the primary emulsification step [
9]. SDS-PAGE and circular dichroism spectroscopy analysis showed that loading into SLN neither induced catalase fragmentation nor significantly changed in secondary structure (data not shown).
Emulsification was an important step for preparation of SLN. Emulsion from vortexing was found to be less stable than from sonication. However, occurrence of cavitation [
10] and increased interaction between enzyme and organic solvent might disturb the enzyme conformation during sonication. Sonication operation was optimized before preparation of SLN. During the two steps of sonication, it was found that activity loss was mainly induced by the first step
(Table 2). Thus, the sonication time during the second step is preferable to be extended for ample emulsifying effects. So we used the first step of 20 s and a second step of 30 s for emulsifying.
2.3. Production and Characterization of Lipid Nanoparticles
Table 4 shows that volume of outer aqueous phase and lecithin:triglyceride ratio affected the size distribution of the nanoparticles. The size decreased with increase of the lecithin:triglyceride ratio. Increasing lecithin concentrations might favor the creation of additional w/o interfaces, then the smaller particles form. But this tendency no longer continues when the radius of curvature of the interface reaches a particularly low value, this might be due to shaping of other structures like lecithin multilayer.
Table 4 shows that when 5% of lecithin/triglyceride was used, polydispersity decreased depending on decreasing of outer aqueous phase volume; in agreement with that reported by Garcıa-Fuentes
et al. [
12]. Two milliliters of outer aqueous phase resulted in lowest polydispersity (0.322–0.354). Smaller outer aqueous phase volumes might receive a higher energy input per gram of coarse emulsion as found in preparation of SLN with high-speed homogenizers [
13]. In
Table 4, increasing amounts of lecithin resulted in higher polydispersity, due to the possible formation of multiple lecithin layers [
14] or other structures such as liposomes [
13].
To modify the surface characteristics, Poloxamer 188 or PVA were incorporated in outer aqueous phase during the second emulsification process (
Table 5). These surfactants could attach to and coat SLN surface via its hydrophobic segments and increase SLN stability.
Table 5 shows that coating with surfactants significantly increased zeta potential of SLN. This might be due to extension of particles near plane [
15].
2.4. Catalase Loading and Release from the Nanoparticles
Drug loading and releasing properties are key factors determining application efficiency of SLNs.
Table 6 shows that Poloxmer 188 coating was better than PVA in increasing encapsulation efficiency, which might reduce the w/o interfacial tension and enhance enzyme-lipid affinity. In general, the lipid matrixes used in SLN resulted in sustained enzyme release profiles, probably due to the nature of the lipid matrix itself and to the affinity of the enzyme to some formulation components [
16].
For Poloxmer 188 coated SLN, lecithin:triglyceride ratio greatly influenced catalase releasing (
Figure 1). Lowest cumulative release during 20 h was reached when 5% of lecithin:triglyceride ratio was used. Lecithin:triglyceride ratio might change the polymorphic state of the lipid constitutes, inducing a difference in catalase solubility in the lipid matrix, and change drug incorporation [
17]. Nevertheless, while the mechanisms controlling drug release of hydrophobic molecules were being elucidated [
18], very little work has been carried out on the release of hydrophilic macromomolecules. Almeida
et al. [
19] had achieved a 0.03% (w/w) loading of lysozyme in lipid nanoparticles and Garcıa-Fuentes
et al. [
3,
16] achieved >90% loading of calcitonin and cumulative release of about 4% within 6 h. Encapsulation efficiency of catalase in our study reached its maximum of about 77.9% at 20 h, which needs further improvement. Recently, Liu
et al. have prepared insulin loaded-SLN [
20]. They employed sodium cholate and soybean phosphatidylcholine to improve the liposolubility of insulin and reached high entrapment efficiency. Encapsulation efficiency of catalase in SLN might be enhanced by using sodium cholate as co-emulsifier and needs to be studied further.
2.5. The TEM Image of Particles Prepared by Different Lipid Matrix
As shown in
Figure 2, triglyceride based SLN was more round than that of monoglyceride (control). The probable reason was that triglyceride was a nonpolar molecule, but monoglyceride was a polar molecule. When the polar group of the enzyme had contact with the monoglyceride, the molecules of monoglyceride moved and some particles contacted each other, which caused the morphology changing of the particle. In contrast, the morphology of the particle prepared with the triglyceride was more stable, which is why triglyceride is the lipid matrix most commonly used.
2.7. Protection of Catalase in Nanocarriers from Proteolysis
The time course of the proteolytic loss of catalase activity was determined (
Figure 5). Free catalase lost about 70% of its activity after 1 h incubation, and fell below measurable levels at 5 h; in contrast, SLN loaded catalase retained 48% of its initial activity at 5 h, and reached a plateau corresponding to 30% of its initial activity and remained at a stable level for at least ~24 h. The loss of activity in the SLN is believed to be associated with the catalase that is either surface bound or is released into the aqueous medium. These results indicate that catalase loaded in SLN was protected against proteolysis, yet was still capable of degrading H
2O
2 diffusing through the polymer shell. Dziubla
et al. have loaded catalase into polymer nanocarriers containing poly (ethylene glycol) -
b-poly (lactic-glycolic acid) (PEG- PLGA) [
21]. Catalase in nanocarriers stably retained 25–30% of H
2O
2-degrading activity for at least 18 h in a proteolytic environment. Poloxmer 188 coating and lipid matrix in catalase-loaded SLN might be more effective to prevent protease diffusion in nanoparticles than PEG-PLGA.