The effect of Bovine serum albumin concentration on the size of MAA particle size was observed. BSA macro aggregates were prepared by heating at 70 °C for 60-120 seconds having pH of the solution at 4.6. From higher concentration to lower concentration of albumin was taken respectively and size of the MAA particle was measured. 30mg, 20mg, 10mg and 5mg of BSA was taken and the size of MAA was observed.
Different concentrations of BSA were taken and macro aggregates were prepared by using the microwave heating. It was observed that increasing the concentration of Albumin increases the size of the aggregates. With increasing the concentration of BSA the increase in the size of MAA was observed. Ilyas et al (16) also observed that increasing the concentration of HSA the larger macro aggregates are obtained and vice versa. Petriev (17) also observed increase in the size of albumin microspheres by increasing the concentration of albumin.
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The distribution of the macro aggregate size was not affected by the presence of 68Ga. The binding of 68Ga to albumin MAA is not known. Green et al. (18) hypothesized that 68Ga adsorbs on the surface of the MAA particles. The exact mechanism behind the binding of 68Ga with MAA is not yet known. In our experiment we observed that there is not so much difference in the particle size of MAA formed with and without 68Ga. It can be proposed that 68Ga comes in interspaces between the albumin particles which are denatured and aggregated and binds there.
Increasing the temperature of Microwave heating increases the Size of MAA formed. It was observed by measuring the size distribution of MAA formed that at 70°C the particles were in the size range of 1-100um, while at 80C the size range of the MAA formed was between 1- >150um.
The temperature effects on the structure of albumin. At temperature below 70 °C there is a reversible denaturation due to formation of intermolecular β structure. Above 75 °C irreversible denaturation occurs which increases the aggregation of particles. (19)
It was observed in the previous experiments that sodium acetate effects the labeling of 68Ga with MAA, but what are its effects on the size of MAA is not known. But in our experiment at higher concentration of sodium acetate the particle size was smaller while at higher concentration and at higher time of heating of sodium acetate with MAA, the larger sized particles were obtained.
Albumin has a helical structure. At higher heating time the albumin starts to denature and lose its α-helix structure. When it is heated at 80 °C for longer time the denaturation is irreversible and unfolding of α-helix occurs and it is irreversible.
Wetzel et al. (19) also found the unfolding phenomenon with albumin. He found that up to 68 °C the denaturation is reversible but at 80°C there is 40% irreversible denaturation. This denaturation effects on the size of MAA formed. Longer heating time increases the possibility of higher denaturation and higher particle size and vice versa. In our experiment we also observed that increasing the heating time while keeping albumin concentration and other parameters fixed, the increase in the size of particles were observed. This may be due to the unfolding of the chains of albumin molecules and due to higher degree of denaturation. The above experiments show that 600 seconds is the required heating time for the required size of particles to be obtained.
One pot and two pot method of preparation of MAA was also done to evaluate which method is better to make the required size MAA. In one pot method the preparation and labeling of MAA was done all in the same pot and with only one time heating in the microwave, while in two pot methods first the MAA were formed and then they were labeled with radionuclide. Two pot methods require two times heating in the microwave heating chamber. We observed with that experiment that one pot method is good to keep the particle size to a required size because in two pot method we also obtained larger particles which are not required in our experiments. Also the higher radioactivity incorporation was observed in the case of one pot experiment than two pot method of preparation.
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Experiment was done to see the effects of heating time on the 68Ga incorporation in the HSA MAA and BSA MAA. In Human serum albumin MAA preparation there was a steady increase in the 68Ga incorporation was observed and with increasing heating time higher incorporation was observed. This may be due to the higher degree of denaturation and more timing for the 68Ga to get adsorbed on the surface of MAA. The binding of 68Ga to MAA is still not clear but Green et al. (18) proposed that 68Ga get adsorbed on the surface of MAA particle after hydrolysis to insoluble form of gallium hydroxide. It is known that albumin has the carrying capacity to bind with Zinc and other metals but what is the mechanism of binding of 68Ga with denatured albumin MAA is still unknown. But we can hypothesized that 68Ga is get incorporated on the surface of MAA formed and also between the spaces between aggregated albumin particles. In our experiment the higher the heating time the higher the incorporation of 68Ga is observed. It may be due to higher degree of denaturation in MAA at higher heating time that 68Ga is incorporated more.
The same above experiment was also repeated with bovine serum albumin MAA but at 600 seconds heating time there was less incorporation was observed.
This is a preliminary data about theses albumin MAA and more experiments are needed to further validate these results in case of bovine serum albumin MAA.
3.1.8 Effect of Pre-washed MAA on labeling with 68Ga
In order to check the incorporation of 68Ga in pre-washed MAA and without washing MAA we conducted this experiment. First the albumin MAA was formed and then they were purified by washing two times with 1ml of deionized water each time. Then these pre-washed MAA were labeled with 68Ga and that data was recorded. The same experiment was repeated but this time there was no washing of the MAA was done. The formed MAA were subjected to labeling with 68Ga and data was recorded. The experimental results were compared and it was observed that there is slight higher incorporation of 68Ga in pre-washed MAA but the difference is not so high to be considered. It was also observed that % purity of the pre-washed MAA was higher than its counterpart. It is a preliminary data and further work is needed to further evaluate these results. Green et al. (18) observed higher labeling yield with Pulmolite MAA with pre-washed MAA, but it was not tested with other MAA formulations.
Further experiment was done to evaluate which method is better to separate the small sized particles from required size MAA. Centrifugation and filtration method was adopted. It was observed that centrifugation method is a better way to separate the small sized particles and it also has higher 68Ga incorporation, while in case of filtration method it was difficult to get higher incorporation in MAA, the particle size was also not uniform with this method.
3.1.10 Pre-stirring and non-pre-stirring method
To check the effect of stirring of albumin with radioactivity and then preparation of MAA was checked by comparing the results of labeling yield and size distribution in the experiments. We compared the data obtained from pre-stirring and non-pre-stirring method. It was observed that pre-stirring has relatively higher labeling yield with radioactivity but the particle size of the MAA formed was smaller than the non-pre-stirring method.
To see the effect of different pH of the solution and formation of MAA was observed by varying the pH of the solution from 3.6 to 5. It was observed that more than 90% of the radioactivity bound to MAA at pH 3.6 and at pH 4.6 but it was nothing at pH 5. The particle size of the MAA formed was much reduced at pH 3.6 while it was of required size at pH 4.6.
Peters described in his experiments that the when there is change in pH of the protein solution of albumin then net charge on the HSA is disturbed and attractive and repulsive forces exit which effects on the size of particles formed (20).
To separate the smaller particle from larger particle size this experiment was done. It was observed that 15 seconds centrifugation is enough to remove the smaller sized particles from the larger ones. It is less time consuming and convenient to use than 5 minutes centrifugation.
3.1.14 Stability of BSA and HSA MAA
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The stability studies of the bovine serum albumin MAA and human serum albumin MAA was done. It was observed that BSA and HSA MAA were stable in acetate buffer for 1 month at 4 °C. The stability was checked after 1 week period and after 1 month period. The size distribution of MAA was measured and it was observed that there was a no such degradation of the MAA.
A quick method of bovine and human serum albumin macro aggregate preparation and labeling with 68Ga for imaging of lung perfusion was developed by using microwave heating. The particle size distribution was optimized to meet the required range of 1-100 µm with > 90 % of 10-100 µm. The macro aggregates were stable in the buffer for at least one month at 4 °C.
All the steps of preparation of MAA and labeling of MAA with 68Ga were performed in a single pot within less than an hour time.
Parameters such as heating time, temperature, pH, stirring rate as well as pre-washing, one pot and two pot methods were investigated in order to optimize the particle size distribution and labeling efficiency.
The labeling efficiency was higher and particle size distribution was narrower for human serum albumin macro aggregates as compared to bovine serum albumin macro aggregates.