パルス130GHz放射が脂質膜透過性に及ぼす影響を、ジパルミトイルホスフェチジルコリン(DPPC)、コレステロール、ステアリルアミンを含有したカチオン性リポソームを用いて調査した。カルボニックアンヒドラーゼ(CA)をリポソーム内に封入し、基質p-ニトロフィニルアセテート(p-NPR)を大量の水相に添加した。脂質二重層を浸透してきたp-NPA分子は内部のCAの触媒作用で生成物質に変換される。完全なリポゾームを透過したp-NPAの自己拡散率は非常に低いので、ΔA/minで表されるCA反応率は膜透過性の変化の追跡に用いられる。5、7、10Hzの低周波数でパルス変調された130GHzを、17mW/cm2までの時間平均入射強度(IAV)でばく露した影響を、DPPCリポソームの相転移温度よりも低い室温(22℃)で調べた。IAVのすべての実験値において、CA封入リポソームにおける酵素反応率の目立った上昇が、パルス繰り返し率7Hzの場合に生じた。代表的には、ΔA/min＝0.0026±0.0010(n＝11)からΔA/min＝0.0045±0.0013(n＝12)(P＜0.0005)への上昇が、IAV＝7.7mW/c㎡において生じた。また、7Hzのパルス変調130GHzの影響は、パルミトイルオレオイル・ホスファチジル・コリン(POPC)で形成されたカチオン性リポソームにおいても観察されたが、その時の室温(22℃)はPOPCリポソームの相転移温度を上回るものである。
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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.
| 周波数 | 130 GHz |
|---|---|
| タイプ |
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| ばく露時間 | continuous for 30 s plus 2 min |
| ばく露の発生源／構造 |
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|---|---|
| チャンバの詳細 | 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. |
| ばく露装置の詳細 | 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 information | 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. |
| 周波数 | 150 GHz |
|---|---|
| タイプ |
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| ばく露時間 | continuous for 30 s plus 2 min |
| Modulation type | CW |
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| ばく露の発生源／構造 |
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|---|---|
| ばく露装置の詳細 | The IMPATT diode was connected to the exposure system by means of an additional focusing cone. |
| Sham exposure | A sham exposure was conducted. |
| Additional information | For comparison purposes, CW irradiation at 150 GHz was performed under identical experimental conditions. |
| 測定量 | 値 | 種別 | Method | Mass | 備考 |
|---|---|---|---|---|---|
| 電力密度 | 6.2 mW/cm² | average over time | 計算値 | - | - |
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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