Lipase stability and lipid microparticle preparation

As described in the first part of this chapter, lipid microparticles and other lipid based drug depot systems were incubated in lipase solution, in order to determine lipid degradation rates of the applied systems. When working with enzymes such as lipase, it is crucial to thoroughly characterize these bio-catalysers in order to achieve a constant and reproducible activity.

Since enzymes are generally proteins - with an amino acid range from only 62 [1] to several hundred or thousand amino acids [2] – these macromolecules comprise the risk of inactivation and activity loss. Enzyme activity and catalysis is a result of a complex process, involving structural changes thereby exposing the active site of this macromolecule comprising the amino acids serine, aspartate and histidine which form the catalytic triad [3-5]. Enzyme folding and interfacial activation can be impaired by several factors such as ion strength, heat or cold and mechanical forces [6]. Therefore, for long term experiments as described in 4.2 enzyme stability has to be evaluated. In the first part of this section the experiments of the conducted lipase stability test are outlined. The second part deals with the preparation of lipid microparticles featuring a defined particle size.

Materials and methods Materials

All lipids (Dynasan 118 and Dynasan 116) used in these experiments were purchased from Sasol GmbH, Witten, Germany. All lipases and all other chemicals were purchased from Sigma–Aldrich, Deisenhofen, Germany.

Methods

Lipase activity assay

Lipase activity was tested with a trioleate emulsion. The emulsion was prepared freshly for each test by mixing 4.5 ml aqueous polyvinyl alcohol solution (2%) with 1.5 ml triolein. This mixture was then emulsified at 0°C with an Ultra Turrax® for 2 min at 15,000 rpm. 500µl of this emulsion and 400µl PBS buffer 0.01 M pH 7.4 then were incubated in a 2 ml Eppendorf cap for 10 min at 37°C in a water bath. 100µl of lipase solution was added and the mixture was incubated again at 37°C. After 10 min the reaction was stopped by adding 1 ml of a MeOH:acetone:methylene chloride mixture (1:1:1 v/v). Before derivatization and HPLC analysis free fatty acids produced in the triglyceride cleavage were extracted using the method already described in the first part of this chapter. In order to achieve a standardized quality of the used trioleate emulsion, oil droplet size was determined with static laser light scattering.

CHAPTER TWO In vitro degradation studies of lipid based drug depot systems ___________________________________________________________________________

Stability study

In order to investigate on the stability of the lipases (Lipase amano M, lipoprotein lipase from pseudomonas sp, lipase from candida rugosa and lipase from rhizoma oryzae) used for the degradation studies lipase solutions of known concentrations were incubated in PBS pH 7.4 at 37° for 8 days in TopPac® vials. Every day a specified amount of the incubated solution was withdrawn and tested with the trioleate emulsion method described above. Preliminary to the start of this study the activity of all lipase was determined and the resulting level of activity was set to 100% as a starting point reference.

Lipid microparticle preparation

Lipid based microparticles were prepared as described in chapter 2. As 2 different particle sizes were required for the experiments (10µm and 100µm mean particle diameter) process conditions, i.e. the rotation speed of the Ultra Turrax® (TP 18/10, Stauffen, Germany) had to be adjusted in order to obtain the particle size needed for the experiments. Therefore in preliminary experiments the suitability of the applied method has been tested. Rotation speed of the Ultra Turrax® was controlled externally with a continuously variable 9-step TRIAC-controller (Messner, Dettenhausen, Germany). In order to validate the preparation of distinct particles sizes lipid microparticles were prepared using rotation speed settings from 1 to 9 on the TRIAC-controller. Preliminary to the mixing step the rotation tools were adjusted to the same temperature as the lipid melt in a temperature controlled water bath in order to avoid lipid solidification. Rotation time was set to 30 sec. After stabilizing the obtained particles in the ice cold aqueous phase the particles were collected and particle size was determined with static light scattering.

Particle size distribution

Size of lipid microparticles and oil droplets of prepared emulsions was determined using a Horiba LA 950 particle size distribution analyzer (Retsch Technology GmbH, Haan, Germany).

Results and discussion Lipase stability

As expected lipase activity was decreasing with increasing time of storage at 37° in aqueous media. The tested enzymes exhibited different stabilities. Lipoprotein lipase from pseudomonas sp. (LPL) exhibited the highest stability in aqueous media (Fig. 1). After 3 days of incubation almost 80% of the initial activity was measured. However after 8 days activity level was decreased to 20%. Comparable to LPL was the obtained stability data for lipase from rhizoma oryzae (RO). After 3 days activity was decreased to 80% and after 8 days 20%

of residual activity was measured.

Figure 1:Stability data for lipase from pseudomonas sp. (a) and lipase from rhizoma oryzae (b)

Figure 2:Stability data for lipase amano M (a) and lipase from candida rugosa (b)

Lipase amano M was shown to be very unstable under the tested conditions (Fig 2). After 3 days the activity levels was beneath 20% and after 8 days of incubation no activity was measurable anymore. Lipase from candida rugosa (CRG) was slightly better. After 3 days of incubation approximately 23% of the primordial activity was detected. After 8 days the activity level dropped to values of about 13%.According to the results generated in these stability tests, lipoprotein lipase from pseudomonas sp. and lipase from rhizoma oryzae were

CHAPTER TWO In vitro degradation studies of lipid based drug depot systems ___________________________________________________________________________

selected for the long term degradation studies. Further on, as a consequence of these results, it was decided to change the lipase-containing buffer media every 3 days in order to maintain high lipase activity in the setup. According to these results the lipase activity level are almost kept constant for a 3 day cycle.

Trioleate emulsion

The oil droplet size of the obtained trioleate emulsions was in a narrow size range (0.5 µm to 25 µm) with an average droplet size of approximately 4 µm.

0 2 4 6 8 10 12 14

0,51 0,67 0,88 1,15 1,51 1,98 2,6 3,41 4,47 5,87 7,7 10,1 13,2 17,4 22,8 29,9 39,2 51,5 67,5 88,6 116 diameter [µm]

q(%)

oil droplet size

Figure 3:Typical size distribution of oil droplets generated during the preparation of a trioleate emulsion

Microparticle preparation

It was shown that the attached TRIAC-controller enabled the controlled preparation of microparticles. As seen in Fig. 4 the particles size and the rotation speed of the emulsification tool correlate closely in a certain range. As expected, with lower rotation velocity larger particles are obtained. It can be stated that this method it is not suitable to produce particles with a mean particle size smaller than 1-2 µm as the particle size didn’t decrease significantly when using the settings 7 to 9. Unfortunately, it was not possible to obtain exact data concerning the number of revolutions with the TRIAC-controller attached. However it can be stated that level 9 corresponds to 20,000 rotations per minute.

0 20 40 60 80 100 120 140

9 8 7 6 5 4 3 2 1

TRIAC setting

mean particle size [µm]

Figure 4:Size of microparticles correlated to settings of the TRIAC controller attached to the emulsification tool

Summary

It was shown that lipases designated for lipid depot system degradation studies exhibit different properties concerning their stability in aqueous environment. As a result of these experiments lipase from pseudomonas sp and lipase from rhizoma oryzae were employed in the degradation experiments described in chapter 1. Accordingly samples were drawn every three days in order to quantify the FFA amount and to refresh the lipase in the incubation set up. In addition to that a microparticle preparation method was presented which allows obtaining microparticles of a certain size range by controlling the rotation speed of the emulsification tool.

CHAPTER TWO In vitro degradation studies of lipid based drug depot systems ___________________________________________________________________________

References

1. Chen, L.H., et al., 4-Oxalocrotonate tautomerase, an enzyme composed of 62 amino acid residues per monomer. J. Biol. Chem., 1992. 267(25): p. 17716-17721.

2. Smith, S., The animal fatty acid synthase: one gene, one polypeptide, seven enzymes.

FASEB J., 1994. 8(15): p. 1248-1259.

3. Brady, L., et al., A serine protease triad forms the catalytic centre of a triacylglycerol lipase. Nature, 1990. 343(6260): p. 767-770.

4. Brumlik, M.J. and J.T. Buckley, Identification of the catalytic triad of the

lipase/acyltransferase from Aeromonas hydrophila. J. Bacteriol., 1996. 178(7): p.

2060-2064.

5. Emmerich, J., et al., Human lipoprotein lipase. Analysis of the catalytic triad by site-directed mutagenesis of Ser-132, Asp-156, and His-241. J. Biol. Chem., 1992. 267(6):

p. 4161-4165.

6. Iyer, P.V. and L. Ananthanarayan, Enzyme stability and stabilization--Aqueous and non-aqueous environment. Process Biochemistry, 2008. 43(10): p. 1019-1032.