Bioassay of Lipophilized Insulin

Insulin is a protein frequently used in cell culture due to its well known ability to enhance cell proliferation and differentiation. To prove the biological activity of modified insulin we investigated the influence of insulin and lipo-insulin 1.5 on the proliferation of bovine chondrocytes.

Hence, four different concentrations of each protein were added to the cell culture, while molar amounts of insulin and lipophilized insulin were kept equal in corresponding groups.

Proteins were supplied as solutions in IGF-buffer. While native insulin dissolved in a short period of time, lipo-insulin 1.5 gave a turbid solution. As control, only IGF-buffer was supplied to one cell group. After cultivation for eight days and subsequent harvest, cells were counted and the obtained numbers served as proliferation markers. Resulting cell numbers are

Chapter 7 Synthesis and Characterization of Lipophilized Insulin

0 0,4 0,8 1,2 1,6 2

0.1 1.0 10 50 0.1 1.0 10 50 Ctr.

rel. cell number

Insulin Lipo-Insulin 1.5

Figure 8: Effects of insulin and lipo-insulin 1.5 on cell numbers after eight days of cultivation. Four different protein concentrations of each protein were evaluated. In the insulin group figures represent ng/ml of the protein. Molar amounts of supplemented insulin and lipophilized insulin were equal in corresponding groups. n = 5 with error bars representing the standard deviations from the mean.

As expected, insulin shows a concentration dependent positive effect on the proliferation of bovine chondrocytes. Especially in groups with higher amounts of insulin (10 ng/ml and 50 ng/ml) a distinct increase in cell number compared to the control group can be observed. In contrast, lipophilized insulin shows a somewhat incoherent tendency. Lower lipo-insulin 1.5 concentrations (Lipo-Insulin 1.0) hint at enhancement of proliferation. But higher lipophilized insulin concentrations show a definite decrease in cell number. This effect might be due to the presence of undissolved substance in cell culture wells. Directly after addition of the protein we could observe with 100-fold magnification small fragments of lipophilized insulin, which were still present after two days of cultivation. Endocytosis of such particles might lead to over-stimulation and death of the cells, as it has already been shown, that at high concentrations insulin shows not an antiapoptotic, but a toxic effect on cells [255]. Higher amounts of lipophilized insulin were also associated with a higher decrease in cell numbers.

Chapter 7 Synthesis and Characterization of Lipophilized Insulin Thus, the necessity was concluded to completely dissolve the lipophilized insulin before application to cells.

In a first approach solubility enhancers were tested for their ability to promote protein solubility: Pluronic F68, Tween 80, PEG 400. But even in concentrations as high as 10% (m/V), which are far from typically employed values, these surface-active agents fell short of the goal. Hence a decision was made in favor of biocompatible organic solvents. We settled for N-methyl-2-pyrrolidone a solvent applicated in other cell culture models in concentrations as high as 10% [256].

0

Figure 9: Effects of insulin and lipo-insulin 1.5 on cell numbers after fourteen days of cultivation in the presence of the proteins. Three different protein concentrations were evaluated. In the insulin group figures represent ng/ml of the protein and molar amounts of insulin and lipophilized insulin were equal in corresponding groups. As control served groups supplemented with both solvents [Ctr.] or with no additional supplements [No solvent]. n = 6 with error bars representing the standard deviations from the mean.

In the following experiment insulin was dissolved in IGF-buffer, whereas lipophilized insulin was dissolved in the organic solvent. Nonetheless, each group was supplemented with both

Chapter 7 Synthesis and Characterization of Lipophilized Insulin served groups supplemented with both solvents or without solvents. Cells were cultured for eight or fourteen days and counted after harvest. Exemplarily, relative cell numbers counted after fourteen days of cultivation and serving as marker for cell growth are shown in Figure 9.

This graph shows very clearly that N-methyl-2-pyrrolidon interferes with cell proliferation.

Even though, absolute cell numbers slightly increased in each well compared to initially seeded cell numbers, neither insulin nor lipophilized insulin showed under these conditions a positive effect on cell proliferation.

Having identified the organic solvent as the reason for diminished cell growth, measures were undertaken to reduce the total amount of solvent used, hoping that lower concentrations would be tolerated better. 2 mg of lipophilized insulin could be dissolved in as little as 10 µ l of N-methyl-2-pyrrolidon. Unfortunately, the substance precipitated upon insertion into the cell culture medium leading to the problems mentioned above.

The problem of reduced solubility, encountered here on the way to bioactivity testing, often occurs with lipophilized proteins. Not only solubility in water might be a problem of protein-fatty acid complexes, but also their bioactivity can be diminished compared to the original protein. The question of reduced bioavailability must be assessed separately for each processed protein, as it is often caused by deteriorated receptor binding ability, either through sterical hindrance or removal of essentially needed functional groups, which depend on the modification site. Moreover, fundamental features like hydrophilicity or conformation might be changed.

Insulin binds to the insulin receptor [IR] and to a much lesser extent to the insulin-like growth factor [IGF] receptor. The insulin receptor is a member of the Type II tyrosine kinase receptor family consisting of two extracellular α-subunits and two transmembrane β-subunits with intrinsic tyrosine kinase activity. In contrast to most growth factor receptors, IR is dimeric even if not activated. Upon binding of insulin, the tyrosine kinase activity is increased and a high affinity pocket for e.g. SH2 domains containing proteins is formed. Among other adaptor proteins, IR substrate 1 or 2 are needed for activation of downstream cascades. The regulatory effects thereof affected fall into two categories: metabolic effects of short or long duration on

Chapter 7 Synthesis and Characterization of Lipophilized Insulin the uptake, transport, intermediary metabolism and storage of small food molecules, and mitogenic effects realized at gene level (Scheme 2).

Scheme 2: The insulin-signaling system affects numerous intracellular processes. The small