在脉冲激光激发金纳米粒子集成光电响应脂质体及其微泡空化的测量的合成
Summary
这个协议描述了金纳米颗粒集成光响应的脂质体与市售材料的简单制备方法。这也说明了如何在脉冲激光治疗测量合成脂质体的微泡空化进程。
Abstract
Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.
Introduction
可能使用外部的刺激是一个有吸引力的方式为客户提供药物以最大的特异性和最小的不利影响spatial-，temporal-和剂量控制的时装来触发药物释放。之间广泛的外源刺激响应系统（光，磁场，超声波，微波辐射），光触发的平台是有吸引力的，因为它们的非侵入性，简便性和适应性的诊所。在过去的十年1广泛的研究已经提供了各种平台技术，例如近红外光负责金的涂有智能聚合物（Au）的纳米笼，用药物，3和自组装porphysome纳米囊泡共轭2对光不稳定的，聚合物纳米颗粒（纳米颗粒）。4但是这些技术仍处于发展的临床前阶段，要求参与发起和续的工艺参数清醒的认识和优化滚动药物的释放。
一种用于制备这种系统的最简单和方便的方法是对金纳米粒子与热敏感脂质体5,6，这两者都是在市场上广泛使用，并已在临床前和甚至临床试验被广泛地研究集成。尽管金纳米粒子的深层组织活化的在其电浆波长的限制，相对于近红外活化的Au纳米结构（例如，纳米笼），在小动物或用于人类局部递送时使用该系统仍然保持很大的希望。 7中有金纳米粒子与脂质体相结合的用于光触发释放一些早期的努力。8-11虽然大多集中在材料的新颖性，需要解决可访问性和可扩展性的问题。此外，在使用这些纳米载体释放机构的报告仍然有限。
这里，在制造光响应脂质体，同时装载有药物和亲水性金纳米粒子已经描述。钙黄绿素被用作模型化合物以评价包封效率和系统的释放曲线。另外，在这个系统中，由金纳米粒子吸收的光消散至以热的形式的周围的微环境，从而增加在局部温度。空气微泡是在激光加热过程中产生的，并引起的脂质体（图1）的机械破坏。微泡空化的机制是由水听器测量证实。
Protocol
Representative Results
Discussion
薄膜水化是用于制备脂质体的常规方法。有机溶剂（在这种情况下，氯仿）首先用于溶解脂质，然后取出在旋转蒸发器于37℃，以产生在烧瓶的脂质薄膜。此脂质膜用含有60mM的钙黄绿素和5纳米金纳米粒子水溶液水合。在水合过程中，将温度维持在大约50℃，将该烧瓶不断受到烧瓶旋转搅拌。该步骤中的关键是在其中的蒸发和水合分别进行温度的选择。 DPPC，脂质体的主要成分，所述的相变温度（T …
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是部分由一线学术研究经费由新加坡教育部（RG十二分之六十四到CX）和纳米医学台大研究所西北支持。
Materials
| 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) | Avanti Polar Lipids (Alabama, US) | 850355P | Powder, Store at -20 °C |
| 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC) | Avanti Polar Lipids (Alabama, US) | 855675P | Powder, Store at -20 °C |
| 1, 2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG2000) | Avanti Polar Lipids (Alabama, US) | 880120P | Powder, Store at -20 °C |
| Gold Nanoparticles | Sigma Aldrich | 752568-100mL | 5nm particles, stabilized at 0.1mM PBS |
| Calcein | Sigma Aldrich | C0875-10g | 60mM, pH 7.4 – adjusted using NaOH |
| phosphate buffered saline (PBS) | Sigma Aldrich | P5493 | 0.1 mM, pH 7.4 |
| Double distilled water | Millipore Milli-DI water purification system | ||
| Triton X100 | Sigma, Life Sciences | X-100 | To disrupt the liposomes to calculate total encapsulation |
| Rotavapor | Buchi (Switzerland) | R 210 | Used for Lipososme preparation |
| Heating bath | Buchi (Switzerland) | B 491 | Used for Lipososme preparation |
| Vacuum Controller | Buchi (Switzerland) | V-850 | Used for Lipososme preparation |
| Vacuum Pump | Buchi (Switzerland) | V-700 | Used for Lipososme preparation |
| Recirculation bath with temperature controller | Polyscience | Used for Lipososme preparation | |
| Mini-extruder assembly with heating block | Avanti Polar Lipids (Alabama, US) | 610000 | Used for extrusion of liposomes |
| Syringes, 1000 uL | Avanti Polar Lipids (Alabama, US) | 610017 | Used for extrusion of liposomes |
| Polycarbonate filter membrane, 200nm | Whatmann | 800281 | Used for extrusion of liposomes |
| Filter Support | Avanti Polar Lipids (Alabama, US) | 610014 | Used for extrusion of liposomes |
| PD 10 Desalting coulumns, Sephadex G-25 medium | GE Healthcare, Life sciences | 17-0851-01 | Used to purify the liposomes |
| Centrifuge | Sigma Laboratory Centrifuges | 3K30 | Used to concentrate the liposomal solution |
| Rotor | Sigma | 19777-H | Used to concentrate the liposomal solution |
| Zetasizer | Nano ZS Malvern | Used for the determination of liposome size and zetapotential | |
| UV- Visible Spectrophotometer | Shimadzu | UV-2450 | Used to measure the absorbance of the samples |
| Fluorescent Spectrofluorometer | Molecular Devices | SpectraMax M5 | Used to measure the fluorescence emission of the samples |
| Nd:YAG Laser | NewWave Research | 532 nm; Maximum power: 17mJ; Width: 406 ns; Used for sample irradiation | |
| HNR Hydrophone | ONDA | HNR-1000 | 1000 mm diameter and 450 nV/Pa sensitivity, Proper working frequency range: 0.25-10 MHz; Calibration: 50 mV/Bar; Used to measure the acoustic signals |
| Digital Osciloscope | LECORY – Wave Runner 64Xi-A | Frequency: 600 MHz; Max sample rate : 10 Gs/s (at two channel); Used to record the measured acoustic signals |
References
- McCoy, C. P., et al. Triggered drug delivery from biomaterials. Expert Opin. Drug Deliv. 7 (5), 605-616 (2010).
- Yavuz, M. S., et al. Gold nanocages covered by smart polymers for controlled release with near-infrared light. Nat. Mater. 8 (12), 935-939 (2009).
- Gohy, J. F., Zhao, Y. Photo-responsive block copolymer micelles: design and behavior. Chem. Soc. Rev. 42 (17), 7117-7129 (2013).
- Lovell, J. F., et al. Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents. Nat. Mater. 10 (4), 324-332 (2011).
- Needham, D., Dewhirst, M. W. The development and testing of a new temperature-sensitive drug delivery system for the treatment of solid tumors. Adv. Drug Deliv. Rev. 53 (3), 285-305 (2001).
- Landon, C. D., Park, J. Y., Needham, D., Dewhirst, M. W. Nanoscale drug delivery and hyperthermia: the materials design and preclinical and clinical testing of low temperature-sensitive liposomes used in combination with mild hyperthermia in the treatment of local cancer. Open Nanomed. J. 3, 38-64 (2011).
- Sykes, E. A., Dai, Q., Tsoi, K. M., Hwang, D. M., Chan, W. C. Nanoparticle exposure in animals can be visualized in the skin and analysed via skin biopsy. Nat. Commun. 5, 3796 (2014).
- Paasonen, L., et al. Gold nanoparticles enable selective light-induced contents release from liposomes. J. Control. Release. 122 (1), 86-93 (2007).
- Wu, G., et al. Remotely Triggered Liposome Release by Near-Infrared Light Absorption via Hollow Gold Nanoshells. J. Am. Chem. Soc. 130 (26), 8175-8177 (2008).
- Leung, S. J., Kachur, X. M., Bobnick, M. C., Romanowski, M. Wavelength-Selective Light-Induced Release from Plasmon Resonant Liposomes. Adv. Funct. Mater. 21 (6), 1113-1121 (2011).
- Volodkin, D. V., Skirtach, A. G., Möhwald, H. Near-IR Remote Release from Assemblies of Liposomes and Nanoparticles. Angew. Chem. Int. Ed. 48 (10), 1807-1809 (2009).
- Mills, J. K., Needham, D. Lysolipid incorporation in dipalmitoylphosphatidylcholine bilayer membranes enhances the ion permeability and drug release rates at the membrane phase transition. Biochim. Biophys. Acta. 1716 (2), 77-96 (2005).
- Jiang, K., Smith, D. A., Pinchuk, A. O. Size-dependent Photothermal Conversion Efficiencies of Plasmonically Heated Gold Nanoparticles. J. Phys. Chem. C. 117 (51), 27073-27080 (2013).
- Chongsiriwatana, N., Barron, A., Giuliani, A., Rinaldi, A. C. Comparing bacterial membrane interaction of antimicrobial peptides and their mimics. Antimicrobial Peptides. 618, 171-182 (2010).
- Egerev, S., et al. Acoustic signals generated by laser-irradiated metal nanoparticles. Appl. Opt. 48 (7), C38-C45 (2009).
- González, M. G., Liu, X., Niessner, R., Haisch, C. Strong size-dependent photoacoustic effect on gold nanoparticles by laser-induced nanobubbles. Appl. Phys. Lett. 96, 174104 (2010).
