含脂脂的微流体生产-温度敏感脂质体
Summary
该协议提出了使用交错的刺骨微流体器装置制备热敏脂质体的优化参数。这还允许将多索鲁比辛和丁氨酸绿色共同封装到脂质体中，并产生光热触发的多索鲁比素释放，用于控制/触发药物释放。
Abstract
所提出的协议支持高通量连续制备低温敏感脂质体（LTSLs），能够加载化疗药物，如多索鲁比辛（DOX）。为此，将乙醇脂质混合物和硫酸铵溶液注射到交错的刺骨微混剂（SHM）微流体装置中。SHM 可快速混合该解决方案，为脂质体自组装提供均匀的溶剂环境。收集的脂体首先退火，然后透析去除残留的乙醇。通过使用尺寸排除色谱，通过外部溶液的缓冲液交换，建立了硫酸铵 pH 梯度。然后，DOX 以高封装效率（> 80%）远程加载到脂体中。获得的脂质体大小均匀，Z平均直径为100nm。它们能够在轻度高热（42°C）的情况下，通过温度触发的封装DOX的突发释放。Indocyanine 绿色 （ICG） 也可以共同加载到脂质体中，用于近红外激光触发 DOX 释放。微流体方法可确保 LTSL 的高通量、可重复性和可扩展的制备。
Introduction
LTSL制剂是一种临床相关的脂质体产品，已开发用于提供化疗药物多索鲁比辛（DOX），并允许在临床上达到的轻度高热（T = 41°C）1高效突发药物释放。LTSL配方包括1，2-二甲酰-sn-甘油-3-磷胆碱（DPPC），脂质脂1-stearoyl-2-羟基-sn-甘油-3-磷脂酰胆碱（MSPC;M 代表”单体”） 和 PEGylates 脂质 1，2-二聚氨酯-sn-甘油-3-磷乙醇胺-N-甲氧（聚乙烯乙二醇）-2000+ （DSPE-PEG2000）。达到相变温度（Tm = 41 °C）时，脂质脂和DSPE-PEG2000一起促进膜孔的形成，导致药物2的爆裂释放。LTSL的制备主要采用批量自上而下的方法，即脂质膜水化和挤出。重新制备具有相同特性和足够数量的大批量用于临床应用仍然具有挑战性3。
微流体学是一种新兴的制备脂体技术，提供可调纳米颗粒大小、可重现性和可扩展性3。一旦优化制造参数，吞吐量可以通过并行化进行扩展，其性能与台秤3、4、5中准备的属性相同。微流体与传统的散装技术相比，一个主要优势是能够处理小液体体积，在空间和时间上具有高可控性，通过小型化，允许更快的优化，同时以连续和自动化的方式运行6。使用微流体装置生产脂质体是通过自下而上的纳米沉淀方法实现的，这种方法更具有时间和能效，因为同质化过程（如挤出和声波）是不必要的。通常，脂质的有机溶液（如乙醇）与混合无溶剂（如水和亲水有效载荷）混合。当有机溶剂与非溶剂混合时，脂质的溶解度降低。脂质浓度最终达到临界浓度，在沉淀过程触发7。脂质的纳米沉淀物最终成种，并接近于脂质体。决定脂质体大小和均匀性的主要因素是非溶剂体与溶剂之间的比率（即水与有机流动率;FRR）和溶剂环境的均匀性，在脂质自组装成脂质体8。
因此，微流体中的高效流体混合对于制备同质脂质体至关重要，混合器的各种设计已应用于不同的应用9。交错的刺骨微混频器 （SHM） 代表新一代无源混合器之一，可实现高通量（在 mL/min 范围内），具有低稀释系数。这优于传统的微流体流体动力混合装置8，10。SHM已经图案的刺骨槽，它迅速混合流体通过混沌对流9，11。SHM 的短混合时间刻度（< 5 ms，小于 10–100 ms 的典型聚合时间刻度）允许脂质自组装发生在均匀的溶剂环境中，产生尺寸均匀分布为3、12的纳米颗粒。
然而，与传统的脂质体制剂相比，由于胆固醇8缺乏，LTSL的制备并不简单，没有胆固醇8，脂质二层就容易受到乙醇引起的三位一体13、14、15的相互作用的影响。到目前为止，残留乙醇在脂质体微流体生产过程中的影响还没有得到很好的理解。大多数报告的配方本质上对间念（含胆固醇或不饱和脂质）16具有抵抗力，这与LTSL不同，它们既饱和又不含胆固醇。
此处介绍的协议使用 SHM 为温度触发释放药物输送准备 LTSL。在所提出的方法中，我们通过动态光散射（DLS）确保微流体制备的LTLS是纳米尺寸（100nm）和均匀（分散<0.2）。此外，我们使用跨膜硫酸铵梯度法（也称为远程装载）17封装了 DOX，以验证 LTSL 脂质双层体的完整性。DOX 的远程加载需要脂质体保持 pH 梯度，以实现高封装效率 （EE），如果没有完整的脂质双层，这种情况不太可能发生。在这种方法中，与典型的微流体脂质体制备方案不同，在去除乙醇之前，需要退火步骤，以实现远程加载能力;即恢复脂质双层的完整性。
如前所述，在形成LTSL期间同时封装有效载荷的初始解决方案中，也可以引入亲水和疏水性有效载荷。作为概念验证，一种FDA批准的近红外荧光染料（也是一种有前途的光热剂）被引入到初始脂质混合物中，并成功共同加载到LTSLs中。808 nm 激光器用于照射 DOX/ICG 加载 LTSL，并在 5 分钟内成功诱导多氟辛克光热触发的 DOX 爆发释放。
所有仪器和材料均可上市，即使用即用，无需定制。由于配制LTSL的所有参数都进行了优化，按照此协议，对微流体没有事先了解的研究人员也可以准备LTSL，作为热敏药物输送系统的基础。
Protocol
Representative Results
Discussion
提出的协议描述了使用交错的刺骨微混频器（SHM）制备低温敏感脂质体（LTSLs）。LTSL10配方可在临床上达到的42°C高热温度下，在5分钟内释放多索鲁比辛。Indocyanine 绿色 （ICG） 也可以共同加载用于光热加热触发 DOX 的释放。该方法依赖于：（i） 在SHM11中乙醇和硫酸铵溶液的快速混沌混合所提供的均质溶剂环境下，将磷脂自行组装成脂质体;（二） 脂质体退火，以保持多药装载所?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢英国前列腺癌（CDF-12-002奖学金）和工程和物理科学研究委员会（EPSRC）（EP/M008657/1）的资助。
Materials
| 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) | Lipoid | PC 16:0/16:0 (DPPC) | |
| 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) | Lipoid | PE 18:0/18:0-PEG 2000 (MPEG 2000-DSPE) |
|
| 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (MSPC) | Avanti Polar Lipid | 855775P-500MG | Distributed by Sigma-Adrich; also known as Lyso 16:0 PC (Not to be confused with 14:0/18:0 PC, which is also termed MSPC) |
| 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) | Sigma-Aldrich | H3375-100G | |
| Adapters, Female Luer Lock to 1/4"-28UNF | IDEX Health & Science | P-624 | Requires 2 units. For the inlets |
| Adapters, Union Assembly, 1/4"-28UNF | IDEX Health & Science | P-630 | Requires 2 units. (One unit included 2 nuts and 2 ferrules) |
| Ammonium Sulfate ((NH4)2SO4) | Sigma-Aldrich | 31119-1KG-M | |
| Bijou vial | VWR | 216-0980 | 7 mL, clear, polystyrene vial |
| Centrifugal Filter Unit | Sigma-Aldrich | UFC801008 | 10 kDa MWCO, Amicon Ultra-4 Centrifugal Filter Unit |
| Centrifuge | ThermoFisher Scientific | Heraeus Megafuge 8R | With HIGHConic III Fixed Angle Rotor |
| Cuvette | Fisher Scientific | 11602609 | Disposable polystyrene cuvette, low volume, for DLS measurement |
| Dialysis Kit – Pur-A-Lyzer Maxi | Sigma-Aldrich | PURX12015-1KT | 12-14 kDa MWCO |
| Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich | 34943-1L-M | |
| DLS Instrument | Malvern Panalytical | Zetasizer Nano ZS90 | |
| Doxorubicin Hydrochloride (DOX) | Apollo Scientific | BID0120 | |
| DSC Instrument | TA Instruments | TA Q200 DSC | |
| DSC Tzero Hermetic Lids | TA Instruments | 901684.901 | For DSC measurement |
| DSC Tzero Pans | TA Instruments | 901683.901 | For DSC measurement |
| DSC Tzero Sample Press Kit | TA Instruments | 901600.901 | For DSC measurement |
| Ethanol | VWR | 20821.330 | Absolute, ≥99.8% |
| FC-808 Fibre Coupled Laser System | CNI Optoelectronics Tech | FC-808-8W-181315 | FOC-01-B Fiber Collimator included. |
| Ferrule, 1/4"-28UNF to 1/16" OD | IDEX Health & Science | P-200 | For the outlet |
| Fibre Optic Temperature Probe | Osensa | PRB-G40 | |
| Glass Staggered Herringbone Micromixer (SHM) | Darwin Microfluidics | Herringbone Mixer – Glass Chip | |
| Heating Tape | Omega | DHT052020LD | Can be replaced by other syringe heater such as "HTC" or "SRT series" for slower heating. Manual wiring to a 3-pin plug required for 240V models |
| Indocyanine Green | Adooq | A10473-100 | Distributed by Bioquote Limited (U.K.) |
| Luer-lock Syringe, 5 mL | VWR | 613-2043 | Hanke Sass Wolf SOFT-JECT 3-piece syringes, O.D. 12.45 mm |
| Microplate Reader | BMG Labtech | FLUOstar Omega | Installed with 485 nm (exictation) and 590 nm (emission) filters |
| Microplate, 96-well, Black, Flat-bottom | ThermoFisher Scientific | 611F96BK | For fluorescence measurement in microplate reader |
| Microplate, 96-well, Clear, Flat-bottom | Grenier | 655101 | For absorbance measurement microplate reader |
| Nut, 1/4"-28UNF to 1/16" OD | IDEX Health & Science | P-245 | For the outlet |
| PC to Pump Network Cable for Aladdin, 7ft | World Precision Instruments | NE-PC7 | Optional: Syringe pumps can be operated manually |
| Pump control software – SyringePumpPro Software License for 2 | World Precision Instruments | SYRINGE-PUMP-PRO-02 | Optional: Syringe pumps can be operated manually |
| Pump to Pump Network Cable for Aladdin, 7 ft | World Precision Instruments | NE-NET7 | Optional: Syringe pumps can be operated manually |
| Size exclusion chromatography (SEC) column | GE Life Science | 17085101 | Sephadex G-25 resin in PD-10 Desalting Columns |
| Sodium chloride (NaCl) | Sigma-Aldrich | 31434-1KG-M | |
| Sodium hydroxide (NaOH) | Sigma-Aldrich | S5881-500G | |
| Syringe Pumps & Cable (DUAL-PUMP-NE-1000) | World Precision Instruments | ALADDIN2-220/AL1000-220 | |
| Thermostat Temperature Controller | Inkbird | ITC-308 | Can be replaced by other syringe heater kit/thermostat |
| Triton X-100 | Sigma-Aldrich | X100-100ML | |
| Tubing, ETFE (1/16" OD) | IDEX Health & Science | 1516 | |
| USB To RS-232 Converter | World Precision Instruments | CBL-USB-232 | Optional: For computer without RS-232 port |
| Water Bath | Grant Instruments Ltd. | JB Nova 12 |
References
- Needham, D., Park, J., Wright, A. M., Tong, J. Materials characterization of the low temperature sensitive liposome (LTSL): effects of the lipid composition (lysolipid and DSPE-PEG2000) on the thermal transition and release of doxorubicin. Faraday Discussions. 161, 515-534 (2013).
- Ickenstein, L. M., Arfvidsson, M. C., Needham, D., Mayer, L. D., Edwards, K. Disc formation in cholesterol-free liposomes during phase transition. Biochimica et Biophysica Acta – Biomembranes. 1614 (2), 135-138 (2003).
- Valencia, P. M., Farokhzad, O. C., Karnik, R., Langer, R. Microfluidic technologies for accelerating the clinical translation of nanoparticles. Nature Nanotechnology. 7 (10), 623-629 (2012).
- Chen, D., et al. Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. Journal of the American Chemical Society. 134 (16), 6948-6951 (2012).
- Forbes, N., et al. Rapid and scale-independent microfluidic manufacture of liposomes entrapping protein incorporating in-line purification and at-line size monitoring. International Journal of Pharmaceutics. 556, 68-81 (2019).
- Dittrich, P. S., Manz, A. Lab-on-a-chip: microfluidics in drug discovery. Nature Reviews Drug Discovery. 5 (3), 210-218 (2006).
- Capretto, L., Carugo, D., Mazzitelli, S., Nastruzzi, C., Zhang, X. Microfluidic and lab-on-a-chip preparation routes for organic nanoparticles and vesicular systems for nanomedicine applications. Advanced Drug Delivery Reviews. 65 (11-12), 1496-1532 (2013).
- Cheung, C. C. L., Al-Jamal, W. T. Sterically stabilized liposomes production using staggered herringbone micromixer: Effect of lipid composition and PEG-lipid content. International Journal of Pharmaceutics. 566, 687-696 (2019).
- Suh, Y. K., Kang, S. A. Review on Mixing in Microfluidics. Micromachines. 1 (3), 82-111 (2010).
- Jahn, A., Vreeland, W. N., Gaitan, M., Locascio, L. E. Controlled Vesicle Self-Assembly in Microfluidic Channels with Hydrodynamic Focusing. Journal of the American Chemical Society. 126 (9), 2674-2675 (2004).
- Stroock, A. D. Chaotic Mixer for Microchannels. Science. 295 (5555), 647-651 (2002).
- Belliveau, N. M., et al. Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA. Molecular Therapy – Nucleic Acids. 1 (8), 37 (2012).
- Patra, M., et al. Under the influence of alcohol: The effect of ethanol and methanol on lipid bilayers. Biophysical Journal. 90 (4), 1121-1135 (2006).
- Komatsu, H., Rowe, E. S., Rowe, E. S. Effect of Cholesterol on the Ethanol-Induced Interdigitated Gel Phase in Phosphatidylcholine: Use of Fluorophore Pyrene-Labeled Phosphatidylcholine. Biochemistry. 30 (9), 2463-2470 (1991).
- Lu, J., Hao, Y., Chen, J. Effect of Cholesterol on the in Lysophosphatidylcholine Formation of an Interdigitated Gel Phase and Phosphatidylcholine Binary. Journal of Biochemistry. 129 (6), 891-898 (2001).
- Vanegas, J. M., Contreras, M. F., Faller, R., Longo, M. L. Role of unsaturated lipid and ergosterol in ethanol tolerance of model yeast biomembranes. Biophysical Journal. 102 (3), 507-516 (2012).
- Haran, G., Cohen, R., Bar, L. K., Barenholz, Y. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochimica et Biophysica Acta (BBA) – Biomembranes. 1151 (2), 201-215 (1993).
- Sadeghi, N., et al. Influence of cholesterol inclusion on the doxorubicin release characteristics of lysolipid-based thermosensitive liposomes. International Journal of Pharmaceutics. 548 (2), 778-782 (2018).
- Lawaczeck, R., Kainosho, M., Chan, S. I. The formation and annealing of structural defects in lipid bilayer vesicles. Biochimica et Biophysica Acta (BBA) – Biomembranes. 443 (3), 313-330 (1976).
- Komatsu, H., Okada, S. Ethanol-induced aggregation and fusion of small phosphatidylcholine liposome: participation of interdigitated membrane formation in their processes. BBA – Biomembranes. 1235 (2), 270-280 (1995).
- Marsh, D., Bartucci, R., Sportelli, L. Lipid membranes with grafted polymers: physicochemical aspects. Biochimica et Biophysica Acta (BBA) – Biomembranes. 1615 (1-2), 33-59 (2003).
- Hood, R. R., Vreeland, W. N., DeVoe, D. L. Microfluidic remote loading for rapid single-step liposomal drug preparation. Lab on a Chip. 14 (17), 3359-3367 (2014).
- Dalwadi, G., Benson, H. A. E., Chen, Y. Comparison of diafiltration and tangential flow filtration for purification of nanoparticle suspensions. Pharmaceutical Research. , (2005).
- Roces, C., Kastner, E., Stone, P., Lowry, D., Perrie, Y. Rapid Quantification and Validation of Lipid Concentrations within Liposomes. Pharmaceutics. 8 (3), 29 (2016).
- Kim, S. -. H., Kim, J. W., Kim, D. -. H., Han, S. -. H., Weitz, D. A. Enhanced-throughput production of polymersomes using a parallelized capillary microfluidic device. Microfluidics and Nanofluidics. 14 (3-4), 509-514 (2013).
