Cationic liposomes loaded with carbonic anhydrase were used to analyze the influence of 130 GHz irradiation pulse modulated at low frequencies, on the diffusion rate of the substrate p-nitrophenyl acetate across the lipid bilayer.
Additionally, changes on kinetic parameters, such as the Michaelis-Menten constant and maximum reaction rate of both free carbonic anhydrase and loaded in liposome were examined to address the possible effects of 130 GHz irradiation on the enzyme activity of carbonic anhydrase itself. The effect of 130 GHz exposure on carbonic anhydrase reaction rate was also studied before and after proteolytic digestion of carbonic anhydrase sticking onto external surface of liposomes.
Thermal effects are also investigated.
| Exposure | Parameters |
|---|---|
|
Exposure 1:
130 GHz
Modulation type:
pulsed
Exposure duration:
continuous for 30 s plus 2 min
|
|
|
Exposure 2:
150 GHz
Modulation type:
CW
Exposure duration:
continuous for 30 s plus 2 min
|
|
| Frequency | 130 GHz |
|---|---|
| Type | |
| Exposure duration | continuous for 30 s plus 2 min |
| Exposure source | |
|---|---|
| Chamber | The exposure system was located in a temperature-controlled room (~22 °C). The temperature at the bottom of the cuvette containing the liposome suspension was continuously monitored and recorded during the irradiation time. |
| Setup | The sample cell of a CARY 50 spectrophotometer was modified to directly deliver the radiation to one side of the cuvette (1 cm wide x 3 cm high x 0.5 cm thick) containing the liposomes in a final volume of 1.5 ml. The radiation generated by the FEL was sent through a copper light-pipe with 2.5 cm diameter. An electroformed copper horn was used to match the circular cross-section of the light-pipe to the rectangular cross-section of the cuvette focussing the beam to a spot size of 0.5 cm². The radiation was strongly attenuated while passing through the liposome suspension, and the reflection from the opposite wall of the cuvette was negligible. |
| Sham exposure | A sham exposure was conducted. |
| Additional info | The radiation was turned on and, after 30 s, the p-NPA was added and stirred before carrying out the kinetic measurement over 2 min. The initial stirring of the liposomes maintained them in suspension and avoided any sedimentation during irradiation. |
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| power density | 5 mW/cm² | average over time | calculated | - | min |
| power density | 17 mW/cm² | average over time | calculated | - | max |
| Frequency | 150 GHz |
|---|---|
| Type | |
| Exposure duration | continuous for 30 s plus 2 min |
| Modulation type | CW |
|---|
| Exposure source |
|
|---|---|
| Setup | The IMPATT diode was connected to the exposure system by means of an additional focusing cone. |
| Sham exposure | A sham exposure was conducted. |
| Additional info | For comparison purposes, CW irradiation at 150 GHz was performed under identical experimental conditions. |
| Measurand | Value | Type | Method | Mass | Remarks |
|---|---|---|---|---|---|
| power density | 6.2 mW/cm² | average over time | calculated | - | - |
The data indicate that interactions can take place between lipid bilayer and 130 GHz pulsed irradiation affecting its permeability, and that this is not simply caused by heating. 130 GHz expsoure pulse modulated at low frequencies (5, 7, or 10 Hz) can enhance the permeability of cationic enzyme-containing liposomes.
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