In-situ 초음파가 인지질 다층막의 은 나노 입자 형성에 미치는 영향

Abstract

본 연구에서는 초음파 에너지가 인지질 다층막에 어떤 영향을 미치는지와 인지질 다층막의 형성된 나노 입자의 자가 정렬(Self-assembly)과 입자 크기의 정제(Particle refinement)로의 응용 가능성에 대해 알아보고자 한다. 초음파 에너지는 지질 막의 순간적인 물리적 변화를 가져온다. 이러한 초음파 에너지의 바이오 생체막으로의 응용은 Sonoporation 혹은 Cellular sonication이라고 이미 바이오 의료 분야에서 많이 적용되어 왔다. 초음파를 이용한 기계적 에너지는 세포막의 투과성을 변화시켜 DNA 같은 큰 분자들을 세포 안으로 쉽게 이동시킬 수 있어 유전자 치료, 약물 전달 등에 많이 응용되고 있다. 온도와 습도에 따라 Lα상(Liquid-Crystalline phase) 혹은 HII상(Inverted Hexagonal phase)으로 존재하는 DOPE 다층막내의 은 나노 입자를 합성시키기 위해 Thermal Evaporator를 통하여 은 증착을 하였다. 이 때, 은 증착 과정에서 초음파 Tranducer를 이용하여 가해주는 in-situ 초음파 에너지가 DOPE 다층막안의 은 나노 입자 형성에 미치는 영향에 대해 알아보았다. in-situ 초음파로 인해 DOPE 분자들이 물리적 변화를 일으키면서 은 나노 입자의 공간적인 배열과 입자 크기 분포를 변화시켰고, DOPE 다층막을 Lα상에서 HII상으로 상전이를 일으켰다. 실온에서 단단한 gel 상을 갖고 있어 은 증착을 통한 나노 입자의 핵생성이 제한적이었던 DPPC의 경우 DOPE와 혼합하여 in-situ 초음파를 가해줄 경우 은 나노 입자 형성이 잘 되는 DOPE 부분과 잘 섞이면서 은 나노 입자 형성이 되는 영역이 증가하였다. 서로 다른 상을 갖고 있어 다층막 내에서 명확하게 분리되어 존재하던 DPPC와 DOPE가 in-situ 초음파의 세기가 세고, 주파수가 높을수록 더 잘 혼합되는 모습을 관찰할 수 있었다. 본 연구를 통해 초음파 에너지를 이용하여 지질 막 내의 은 나노 입자의 크기와 형태를 제어할 수 있는 새로운 방법을 제시하고자 한다. 나노의학 분야에서 응용되는 금, 은과 같은 귀금속 나노 입자들은 순환 혈관계 혹은 세포 안에서 유체 혹은 반 유체 환경에서 사용된다. 이번 연구에서 초음파를 이용하여 반 유체 환경인 지질 막 내에서의 나노 입자를 제어할 수 있다는 가능성을 보여줬다. 그리고 세포막이 DPPC와 같은 포화 지질과 DOPE와 같은 불포화 지질로 구성되어 있다는 점에서 초음파 에너지가 세포막과 같은 생체막에서 물리적으로 어떤 영향을 미칠 수 있는지에 대해서도 알 수 있었다.| In this research, that the possibility of using ultrasound energy was explored to drive the particle-refinement and the self-assembly of the nanoparticles embedded in a phospholipid membrane as ultrasonic energy can temporarily induce the physical properties of a lipid membrane. For example, ultrasound energy has been applied to vector-free drug / gene delivery (sonoporation) in which the focused ultrasound is used to locally increase the membrane permeability. The effect of in-situ application of ultrasonic waves on the Ag nanoparticles spontaneously produced inside 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) membrane, which can exist in two different phases: a lamellar Lα phase and an inverted hexagonal HII phase depending on temperature and humidity, was studied by placing the substrate membrane on a ultrasonic transducer during the metal deposition. Application of the ultrasonic vibration promoted spatial ordering of the deposited nanoparticles due to the induced phase transition from Lα to HII for DOPE. Arising from the agitation effect, particle size refinement, which depended on the amplitude of the ultrasonic vibration, was observed. It was also shown that 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) membrane, which suppresses the Ag nucleation due to its stiff gel phase at room temperature, can be made locally permeable to incident Ag atoms by introducing DOPE molecules into the DPPC membrane as the Ag nanoparticles preferentially nucleated in the DOPE-rich region. Application of ultrasonic vibration with increasingly higher amplitude or frequency made the Ag nanoparticles uniformly distributed in the DPPC membrane, suggesting that the permeability of the DPPC membrane can be temporarily increased without permanently damaging the membrane by addition of liquid crystalline lipids and subsequent application of ultrasonic waves. As noble metal nanoparticles used in nanomedical applications are often subjected to fluidic or semi-fluidic environment such as in the circulatory system or in the cells, this research suggests that ultrasonic waves may be used to remotely modify the morphology of such particles. In addition, since a cell membrane is typically composed of mixture of saturated (e.g. DPPC) and unsaturated (e.g. DOPE) lipids, this research provides a direct evidence that the physical state of a cell membrane can be largely modified during application of moderate ultrasonic waves.; In this research, that the possibility of using ultrasound energy was explored to drive the particle-refinement and the self-assembly of the nanoparticles embedded in a phospholipid membrane as ultrasonic energy can temporarily induce the physical properties of a lipid membrane. For example, ultrasound energy has been applied to vector-free drug / gene delivery (sonoporation) in which the focused ultrasound is used to locally increase the membrane permeability. The effect of in-situ application of ultrasonic waves on the Ag nanoparticles spontaneously produced inside 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) membrane, which can exist in two different phases: a lamellar Lα phase and an inverted hexagonal HII phase depending on temperature and humidity, was studied by placing the substrate membrane on a ultrasonic transducer during the metal deposition. Application of the ultrasonic vibration promoted spatial ordering of the deposited nanoparticles due to the induced phase transition from Lα to HII for DOPE. Arising from the agitation effect, particle size refinement, which depended on the amplitude of the ultrasonic vibration, was observed. It was also shown that 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) membrane, which suppresses the Ag nucleation due to its stiff gel phase at room temperature, can be made locally permeable to incident Ag atoms by introducing DOPE molecules into the DPPC membrane as the Ag nanoparticles preferentially nucleated in the DOPE-rich region. Application of ultrasonic vibration with increasingly higher amplitude or frequency made the Ag nanoparticles uniformly distributed in the DPPC membrane, suggesting that the permeability of the DPPC membrane can be temporarily increased without permanently damaging the membrane by addition of liquid crystalline lipids and subsequent application of ultrasonic waves. As noble metal nanoparticles used in nanomedical applications are often subjected to fluidic or semi-fluidic environment such as in the circulatory system or in the cells, this research suggests that ultrasonic waves may be used to remotely modify the morphology of such particles. In addition, since a cell membrane is typically composed of mixture of saturated (e.g. DPPC) and unsaturated (e.g. DOPE) lipids, this research provides a direct evidence that the physical state of a cell membrane can be largely modified during application of moderate ultrasonic waves.