参见作者原研究论文

本实验方案简略版
Jul 2020
Advertisement

本文章节


 

Atomic Force Microscopy to Characterize Ginger Lipid-Derived Nanoparticles (GLDNP)
原子力显微镜研究生姜脂质纳米颗粒(GLDNP)   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

We have demonstrated that a specific population of ginger-derived nanoparticles (GDNP-2) could effectively target the colon, reduce colitis, and alleviate colitis-associated colon cancer. Naturally occurring GDNP-2 contains complex bioactive components, including lipids, proteins, miRNAs, and ginger secondary metabolites (gingerols and shogaols). To construct a nanocarrier that is more clearly defined than GDNP-2, we isolated lipids from GDNP-2 and demonstrated that they could self-assemble into ginger lipid-derived nanoparticles (GLDNP) in an aqueous solution. GLDNP can be used as a nanocarrier to deliver drug candidates such as 6-shogaol or its metabolites (M2 and M13) to the colon. To characterize the nanostructure of GLDNP, our lab extensively used atomic force microscopy (AFM) technique as a tool for visualizing the morphology of the drug-loaded GLDNP. Herein, we provide a detailed protocol for demonstrating such a process.

Keywords: Atomic force microscopy (原子力显微镜), Ginger lipid-derived nanoparticles (生姜衍生脂质纳米粒), Colon-targeted drug delivery (结肠靶向给药), 6-shogaol (6-姜烯酚), Metabolites of 6-shogaol (6-姜烯酚的代谢物)

Background

Developing new drug-based therapeutic approaches against Intestinal Bowel Disease (IBD) must overcome numerous challenges, including potential off-target effects, large-scale production costs, and the need to ensure tissue-specific delivery, systemic safety, and low toxicity. Our group and others have recently demonstrated that artificially synthesized nanoparticles could target low doses of drugs (e.g., siRNAs, proteins, or peptides) to colonic tissues or colonic immune cells, such as macrophages (Ulbrich and Lamprecht, 2010; Chen et al., 2017). However, these synthetic NPs to date have two major limitations: i) each constituent of the synthesized nanoparticle must be examined for potential in vivo toxicity before clinical application; and ii) the production scale is limited. The use of nanoparticles derived from natural sources may overcome these limitations. In this context, we reported that a special population of ginger-derived nanoparticles (GDNP-2) could reduce colitis and colitis-associated colon cancer (Zhang et al., 2016). Naturally occurring GDNP-2 is also safer and cheaper than synthetic NPs. We further identified 6-shogaol as a major candidate that may account for the anti-inflammatory and anti-cancer activities of GDNP-2 (Yang et al., 2020). To construct a nanocarrier that is more clearly defined than GDNP-2, we characterized ginger lipid-derived nanoparticles (GLDNP) and demonstrated that they could be used as a natural carrier to deliver natural anti-inflammatory drug candidates such as 6-shogaol, M2, or M13 to the colon and reduced colitis in mice (Yang et al., 2020).


Atomic force microscopy (AFM) is a versatile and powerful technique to characterize the morphology of nanoscale and submicron structured materials. It has been widely used to visualize different types of NPs, including metal-, inorganic- (non-metallic), and organic-NPs. AFM has the advantages of simplicity in sample preparation and no need for electric conducting treatment (Morris et al., 2010). In previous studies, our group and others have extensively used AFM to analyze the morphology and structure of GLDNP (Zhang et al., 2017; Wang et al., 2019; Sung et al., 2020; Yang et al., 2020). However, no attempt has been taken to document the procedure of sample preparation and AFM parameter setting. In the following protocol, we will use the GLDNP as the specimen to demonstrate the process of obtaining AFM pictures for nanostructure characterization.


Materials and Reagents

  1. Pipette tips 0.1-10 μl, 1-200 μl and 100-1,000 μl (Sorenson Bioscience, catalog numbers: 70600 , 70520 , 70540 )

  2. Powder-free gloves (Denville Scientific, catalog number: G4162 )

  3. 50 ml conical tubes (Denville Scientific, catalog number: C1062-P )

  4. Phosphate-buffered Saline (PBS) (Corning, catalog number: 21-040-CV )

  5. Methanol (Sigma-Aldrich, catalog number: 34860-1L-R )

  6. Potassium chloride (KCl, Millipore, catalog number: 7447-40-7 )

  7. Dichloromethane (Sigma-Aldrich, catalog number: 650463-1L )

  8. Deionized-distilled water (ddH2O)

  9. Mica sheet (Electron Microscopy Sciences, catalog number: 71855-15 )

  10. 1 M KCl solution (see Recipes)

Equipment

  1. Pipettes 0.5-10 μl, 10-100 μl and 100-1,000 μl (Eppendorf, model: Research® Plus , Variable Adjustable Volume Pipettes)

  2. Milli-Q advantage A10 water purification system (Millipore-Sigma, catalog number: C10117 )

  3. Glass separatory funnel (Southern Labware, model: 3964-3)

  4. Centrifuge (Thermo Fisher Scientific, model: Sorvalis ST16R )

  5. Vortexer (Scientific Industries, model: 200-SI0236 )

  6. Rotary evaporator (Buchi, model: R-210 )

  7. Vacuum pump (Buchi, model: V-700 )

  8. Vacuum controller (Buchi, model: V-800 )

  9. Heating bath (Buchi, model: B-491 )

  10. Evaporating flask (Buchi, catalog number: Z402982 )

  11. Notebook computer (ThinkPad, model: T570 )

  12. CoreAFM controller (Nanosurf, model: CoreAFM controller)

  13. CoreAFM system (Nanosurf, model: CoreAFM system)

  14. Isostage 300 controller (Nanosurf, model: Isostage 300 controller)

  15. CoreAFM tool set (Nanosurf, model: CoreAFM tool set)

Software

  1. CoreAFM control software (Version 3.10.0, https://www.nanosurf.net/en/software/coreafm)

Procedure

  1. Preparation of GLDNP

    Please refer to our published bio-protocols Sung et al. (2019 and 2020).

    Note: Stored GLDNP (in 1× PBS) can be enriched after ultracentrifugation (30,000 × g, 4 °C, and 45 min) and remove the supernatant.


  2. Setting up the AFM (see Note 1)

    1. Assemble the Nanosurf CoreAFM system according to the steps in the operating instructions of CoreAFM. The AFM equipment after assembly is shown in Figure 1.



      Figure 1. Assembly of the Nanosurf CoreAFM system. A. The assembled Nanosurf CoreAFM system, which contains CoreAFM system, Isostage 300 controller, CoreAFM controller, and a laptop, according to the instructions. B. The CoreAFM toolset (see Note 2). C. Enlarged photo of CoreAFM in its opened configuration.


    2. Switch on the Nanosurf CoreAFM system (see Note 3).

    3. Insert a Nanosurf Dyn190AI dynamic mode cantilever in the cantilever holder, ensuring that the tip is not damaged and attaching the cantilever holder to the magnets at the bottom of the CoreAFM scan head (see Note 3).


  3. Loading GLDNP samples for AFM imaging

    1. Dilute GLDNP solution in ddH2O to 1 mg/ml.

    2. Deposit 2 μl of nanoparticle sample to mica sheet (Figure 2A).

      Note: As a drug delivery vehicle, the precise dose-response is one of the factors that must be considered. For different samples, the solution is usually diluted from high concentration to low concentration, while the volume of the solution is continuously reduced. According to the imaging effect of AFM, the most suitable solution concentration and volume are finally selected. The concentration of GLDNP samples used for imaging in this procedure should be > 0.01 μg/ml.

    3. Dry the sample at room temperature (RT) for 2 h.

    4. Gently rinse the mica sheet with 5 μl of distilled water three times (Figure 2B).

      Note: First, drop distilled water gently into the middle of the mica sheet using the pipette, and then use filter paper to absorb water from the edge of the mica sheet. Since GLDNP samples are stored in PBS, the salt particles from PBS will affect the imaging effect of AFM, so the purpose of gently rinsing the mica sheet is to remove the salt in GLDNP samples.

    5. Dry the sample at RT for 2 h again.

    6. Leave the mica sheet for 30 min at RT until the sample becomes flat.

      Note: To judge whether a sample is dried and becomes flat, we can place the mica sheet in a vertical position, and if we observe no sign of flow from the sample spot, it generally means that the sample is flat and dried. The drying and flatness of the sample on the mica sheet will affect the imaging effect of AFM.

    7. Fix the mica sheet on the sample magnet in the center of the standard sample holder (Figure 2C).

    8. Close the CoreAFM scan head lid, and the three scan head feet will align with the corresponding sample stage pillars.



      Figure 2. Loading the GNDLN sample to the mica sheet and fixing it on the sample magnet. A. The process of depositing 2 μl of nanoparticle sample to a mica sheet. B. The process of gently rinsing the mica sheet with distilled water and adsorbing water from the edge of the mica sheet with filter paper. C. The process of fixing the mica sheet on the sample magnet of the standard sample holder (see Note 3).


  4. AFM imaging of the GLDNP samples

    1. Checking the laser position and quality (see Note 3).

      1. Attach the CoreAFM system to the CoreAFM controller, and start the equipment and software.

      2. In the CoreAFM control software, open the Laser Alignment dialog (Figure 3A) and switch the CoreAFM to the top view camera (Figure 3B) using the Camera selector (Figure 3C).

        Note: The Laser Alignment dialog displays the current position of the AFM laser spot on the detector and the used laser power. The dialog is opened by clicking the “Laser Align” button in the Preparation group of the Acquisition tab.

      3. Use the laser alignment tool that came with the CoreAFM system to turn the screws in a clockwise or counterclockwise direction (Figure 3C). An optimally aligned laser would result in Figure 3A.

        Note: CoreAFM system comes with 4 holes in the scan head’s top cover that provide access to the laser and detector alignment screws that change different aspects of the laser beam’s optical pathway (Figure 3C). Open the scan head lid to an angle of approximately 30°, and you should now see a red laser spot somewhere on the sample holder or sample holder platform (Figure 3C).



      Figure 3. Checking the laser position and quality. A. The menu of CoreAFM control software application and the proofread completed Laser Alignment dialog. This graphical area shows where the deflected laser beam hits the photodiode detector. A green spot anywhere within the area enclosed by the dotted square means that the cantilever deflection detection system (consisting of laser, cantilever, and detector) is properly aligned. If the laser spot falls outside this area, it will become red, meaning that the alignment does not allow proper measurements to take place. B. Top view image of a cantilever and its chip structure. C. Physical photos of the CoreAFM scan head and the sample holder.


    2. Configuring measurement parameters of the CoreAFM (see Note 3).

      1. In the menu of CoreAFM control software, click the “Air” to select the measurement environment from the Measurement environment drop-down menu (Figure 4A).

      2. Click the “Static Force” to select the desired operating mode from the Operating mode drop-down menu (Figure 4B).

      3. Click the “ACL-A” to select the desired cantilever type from the Cantilever selector drop-down menu (Figure 4C).

      4. Click the “Frequency Sweep” button that opens the Vibration frequency search dialog. Click the “Find Vibration Frequency” button in this dialog (Figure 4D). Leave the dialog by clicking “OK”.

        Note: After the automatic frequency search is finished, you should see a clean resonance curve and a marker showing the vibration frequency.



      Figure 4. Configuring measurement parameters of the CoreAFM. A-C. Set the Measurement environment to “Air” (A), set Operating mode to “Static Force” (B) and select “ACL-A” as cantilever in the Cantilever selector (C). D. The menu of CoreAFM control software application and the proofread completed Vibration frequency search dialog.


    3. Approaching the GLDNP samples (see Note 3).

      1. Observe the distance between tip and sample in the side view (Figure 5A).

      2. While observing the tip-sample distance, click and hold the “Advance” button in the Approach group of the Acquisition tab until the tip is close enough to the sample (Figure 5B).

      3. Click the “Approach” button in the Approach group of the Acquisition tab (Figure 5B). Click the “OK” button (Figure 5C).

        Note: In this last step, this sample automatically approaches the tip until a given setpoint is reached.



      Figure 5. Approaching the GNDLN samples. A. A side view image of a cantilever. B. The “Advance” and “Approach” buttons in the Approach group of the Acquisition tab. C. When the Setpoint is reached, the “Approach done” message is displayed.


    4. Starting a measurement (see Note 3).

      1. Select the initial scan parameters in the Imaging dialog as follows (Figure 6A):

        Image Scan Size = 10.00 μm

        Time/Line = 0.78 s

        Points/Line = 256

        Note: The other values on the master panel are the values automatically filled in by the instrument upon the finishing of probe tuning; thus, no changes of these values are needed.

      2. Click the “Start” button in the Imaging group of the Acquisition tab and start scanning for 1 min to stabilize the cantilever and adjust the instrument to surrounding environmental vibrations (Figure 6B).

        Note: Manually lower the “Setpoint” value until surface features start to appear on the height image. Imaging optimization can be achieved by adjusting “Setpoint”, “P-Gain”, and “I-Gain” values (see Note 4). Finally, the “Image Scan Size” can be decreased to 5.00 μm or 4.00 μm for adjusting the scan size of the image, and the “Points/Line” can be increased to 512 or even 1,024 (see Note 5) for more pixels in each image thus enhance the image quality.

      3. Once satisfied with the image quality, click the “Start” button and restart the scan with optimized parameters. After scanning, click the “Stop” button in the Imaging group of the Acquisition tab (Figure 6B).

        Note: Adjust the positioning screws of the CoreAFM system (see Figure 1C) so that the cantilever can be positioned in different locations of the mica sheet surface for each sample. Then click the “Prescan” button in the Imaging group of the Acquisition tab (Figure 6B). When the desired position is located by pre-scanning, click the “Start” button for scanning.



      Figure 6. Starting and storing the measurement of the GLDNP samples. A. Select the initial scan parameters and adjust the “Setpoint”, “P-Gain”, and “I-Gain” values to enhance the image quality in the Imaging dialog. B. The “Prescan”, “Start” and “Stop” buttons in the Imaging group of the Acquisition tab. C. The “Capture” button in the Imaging toolbar.


    5. Storing the measurement and working with documents.

      1. By default, each completed measurement is automatically stored (temporarily) on your computer so that it can be used later. You can also take snapshots of measurements still in progress by clicking the “Capture” button (Figure 6C).

      2. The captured document will remain open in the document space of the CoreAFM control software (Figure 7A). Adding or removing a chart, or setting chart parameters is all performed in the Chart Properties dialog (Figure 7B).

      3. Click the “Add Chart” button (“+” in Figure 7B) to create a copy of the currently selected or active chart and add it to the active window in the last position. Click the “Remove Chart” button (“-” in Figure 7B) to remove the currently active chart.

      4. Selects the chart type (Line graph, Color map, 3D view, XY Line Graph, or Force Curve graph) to be used for the display of the measurement data from the “Type” of Chart Parameters (Figure 7B). Figure 7A shows three different chart types of the GLDNP samples.

      5. Use the Chart Properties dialog to set the various parameters of the corresponding chart type of the GLDNP samples.

      6. After setting, click "Close" button (Figure 7B) to close the Chart Properties dialog and click the “Save icon” (Figure 7A) to save the documents.



    Figure 7. Different chart types of the GLDNP samples and the Chart Properties dialog. A. (i) Color map, (ii) 3D view and (iii) Line graph of the GLDNP samples, and (iv) the “Chart Properties” button. B. The Charts Properties dialog is used to set all chart properties that influence data display by the respective chart.

Data analysis

  1. We used the above process to perform AFM imaging of the GLDNP samples (Figures 8A-8C).

    Select the final scan parameters in the Imaging dialog for the GLDNP as follows:

    Image Scan Size = 5.00 μm

    Time/Line = 0.78 s

    Points/Line = 256

    In Figures 8A-8C, AFM images showed that GLDNPs are nano-sized particles and have a spherical shape and presented a size-homogenized appearance.

  2. We prepared the synthetic material Poly(lactic-co-glycolic Acid) (PLGA)-based nanoparticles, then used the above process and set the same parameters to obtain the AFM image of PLGA samples (Figures 8D-8F), and compared it with GLDNP. Through the comparison of the images, we can observe that PLGA nanoparticles are also spherical with a size-homogenized appearance, and their particle size is slightly smaller than GLDNP. PLGA nanoparticles show a certain degree of aggregation on mica sheet compared to GLDNP.



    Figure 8. AFM characterization of the GLDNP samples and the poly lactic-co-glycolic acid (PLGA) samples (as control). A-C. The Color map (A), 3D View (B), and the data information of image scan (C) for AFM characterization of GLDNP. D-F. The Color map (D), 3D View (E), and the data information of image scan (F) for AFM characterization of PLGA.

Notes

  1. All procedures here are described for a Nanosurf CoreAFM system. For different models and brands of AFM, the protocol will need to be adapted according to the manufacturer’s instructions.

  2. The content of the toolset depends on the options included in the user's order. It minimally contains the following items:

    Ground cable

    Cantilever tweezers: (103A C.A.)

    Cantilever exchange tool

    Laser alignment tool: Allen key 1.5-mm (ballpoint hex) with a screwdriver handle

    Allen key 5-mm: used for the CoreAFM transportation locks

    Standard sample holder

    Cantilever holder liquid-air flat

  3. Prepare and operate the CoreAFM system and use this system to measure the brief program. Video is available at the product official website (https://www.nanosurf.net/en/products/coreafm-the-essence-of-atomic-force-microscopy). It will take about 4 h 30 min to prepare nanoparticles-loaded mica sheet for AFM imaging, and 15-20 min for AFM debugging and photography. Therefore, AFM imaging excluding data analysis can be completed within 5 h. In particular, it should be noted that the main limitation of the above procedure is that AFM imaging can only be done on dry samples in an indoor air environment, and AFM imaging of prepared samples (loaded on mica sheet) usually needs to be completed within a limited time (no more than 6 h). In addition, the minimum resolvable particle size of the sample is about 10 nm by the CoreAFM system.

  4. Use a low “P-Gain” and “I-Gain” in the beginning to protect the cantilever. These values can be elevated in a later scan for better image quality.

  5. “Points/Line” determines the number of pixel points on each line and the number of lines scanned in the image. In most cases, this value should be kept the same.

Recipes

  1. 1 M KCl solution

    Dissolve 0.745 g potassium chloride (KCl) in 10 ml of ddH2O and mix well

Acknowledgments

This work is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (RO1-DK-116306 and RO1-DK-107739 to D.M.), the Department of Veterans Affairs (Merit Award BX002526 to D.M.), and the Crohn’s and Colitis Foundation of America. D.L. is a recipient of the Research Fellowship Award from the Crohn’s and Colitis Foundation of America (Award Number #689659). D.M. is a recipient of the Senior Research Career Scientist Award from the Department of Veterans Affairs (BX004476). This protocol is based on our previously published study (Yang et al., 2020).

Competing interests

The authors declare no conflicts of interest within the work.

References

  1. Chen, Q., Xiao, B. and Merlin, D. (2017). Nanotherapeutics for the treatment of inflammatory bowel disease. Expert Rev Gastroenterol Hepatol 11(6): 495-497.
  2. Morris, V. J., Kirby, A. R. and Gunning, A. P. (2010). Atomic Force Microscopy for Biologists. 2nd edition. Imperial College Press, ISBN-10: 184816467X.
  3. Sung, J., Yang, C., Viennois, E., Zhang, M. and Merlin, D. (2019). Isolation, Purification, and Characterization of Ginger-derived Nanoparticles (GDNPs) from Ginger, Rhizome of Zingiber officinale. Bio-protocol 9(19): e3390.
  4. Sung, J., Yang, C., Collins, J. F. and Merlin, D. (2020). Preparation and Characterization of Ginger Lipid-derived Nanoparticles for Colon-targeted siRNA Delivery. Bio-protocol 10(14): e3685.
  5. Ulbrich, W. and Lamprecht, A. (2010). Targeted drug-delivery approaches by nanoparticulate carriers in the therapy of inflammatory diseases. J R Soc Interface 7 Suppl 1: S55-66.
  6. Wang, X., Zhang, M., Flores, S. R., Woloshun, R. R., Yang, C., Yin, L., Xiang, P., Xu, X., Garrick, M. D., Vidyasagar, S., Merlin, D. and Collins, J. F. (2019). Oral gavage of ginger nanoparticle-derived lipid vectors carrying Dmt1 siRNA blunts iron loading in murine hereditary hemochromatosis. Mol Ther 27(3): 493-506.
  7. Yang, C., Zhang, M., Lama, S., Wang, L. and Merlin, D. (2020). Natural-lipid nanoparticle-based therapeutic approach to deliver 6-shogaol and its metabolites M2 and M13 to the colon to treat ulcerative colitis. J Control Release 323: 293-310.
  8. Zhang, M., Viennois, E., Prasad, M., Zhang, Y., Wang, L., Zhang, Z., Han, M. K., Xiao, B., Xu, C., Srinivasan, S. and Merlin, D. (2016). Edible ginger-derived nanoparticles: A novel therapeutic approach for the prevention and treatment of inflammatory bowel disease and colitis-associated cancer. Biomaterials 101: 321-340.
  9. Zhang, M., Wang, X., Han, M. K., Collins, J. F. and Merlin, D. (2017). Oral administration of ginger-derived nanolipids loaded with siRNA as a novel approach for efficient siRNA drug delivery to treat ulcerative colitis. Nanomedicine (Lond) 12(16): 1927-1943.

简介

[摘要]我们已经证明,特定人群的生姜纳米颗粒(GDNP-2)可以有效地靶向结肠,减少结肠炎,并减轻结肠炎相关的结肠癌。天然存在的GDNP-2包含复杂的生物活性成分,包括脂质,蛋白质,miRNA和生姜的次生代谢产物(姜油和松香油)。为了构建比GDNP-2更明确定义的纳米载体,我们从GDNP-2中分离出脂质,并证明它们可以自组装成在水溶液中生姜脂质衍生的纳米颗粒(GLD NP )。GLD NP可用作纳米载体,以将诸如6-shogaol或其代谢产物(M2和M13)之类的药物念珠菌递送至结肠。Ť Ó表征纳米结构GLDNP ,邻乌尔实验室中广泛使用原子力显微镜(AFM)技术作为一种工具 可视化载药的GLDN P的形态。在此,我们提供了一个详细的协议来演示这种过程。


[背景]开发新的基于药物的肠道肠道疾病(IBD)治疗方法必须克服众多挑战,包括潜在的脱靶效应,大规模生产成本以及确保组织特异性递送,全身安全性和低毒性的需求。我们的组和其他最近已经证明,人工合成的纳米颗粒可以定位低剂量的药物(例如,siRNA的,蛋白质,或肽),以结肠组织或结肠免疫细胞,例如巨噬细胞(奥博锐和兰普雷克特,2010 ;陈等人, 2017 )。然而,迄今为止,这些合成NP具有两个主要局限性:i)在临床应用之前必须检查合成纳米颗粒的每种成分的潜在体内毒性;ii)生产规模有限。使用天然来源的纳米颗粒可以克服这些限制。在这种情况下,我们报道了特殊的生姜纳米颗粒(GDNP -2)可以减少结肠炎和结肠炎相关的结肠癌(Zhang et al。,201 6 )。天然存在的GDNP-2比合成NP更安全,更便宜。我们进一步确定6-shogaol是可能解释GDNP-2抗炎和抗癌活性的主要候选药物(Yang等,2020)。为了构建比GDNP-2更明确定义的纳米载体,我们表征了生姜脂质衍生的纳米颗粒(G LDNP ),并证明了它们可以用作天然载体,以提供天然抗炎药物候选物,例如6-shogaol, M2,M13或在小鼠中结肠和降低的结肠炎(杨等人,2020)。

原子力显微镜(AFM)是一种通用且功能强大的技术,可表征纳米级和亚微米级结构材料的形态。它已被广泛用于可视化不同类型的NP ,包括金属,无机(非金属)和有机NP。AFM具有简单的样品制备的优点和不需要电传导处理(莫里斯等人,2010 )。在先前的研究中,我们的研究小组和其他研究者广泛地使用AFM分析G LDNP的形态和结构(Zhang等人,2017; Wang等人,2019; Sung等人,2020 ; Yang等人,2020) 。但是,尚未尝试记录样品制备和AFM参数设置的过程。在以下协议中,我们将使用G LDNP作为标本来演示获得用于纳米结构表征的AFM图片的过程。

关键字:原子力显微镜, 生姜衍生脂质纳米粒, 结肠靶向给药, 6-姜烯酚, 6-姜烯酚的代谢物



材料和试剂


1.移液器吸头0.1-10μl,1-200μl和100-1,000μl(Sorenson Bioscience,目录号:70600、70520、70540)     
2.无粉手套(Denville Scientific,目录号:G4162)     
3. 50 ml锥形管(Denville Scientific,目录号:C1062-P)     
4.磷酸盐缓冲盐水(PBS)(Corning,目录号:21-040-CV)     
5,       甲醇(Sigma-Aldrich,目录号:34860-1L-R)

6.氯化钾(氯化钾,密理博,目录号:7447-40-7)     
7.二氯甲烷(西格玛奥德里奇,目录号:650463-1L)     
8.去离子蒸馏水(ddH 2 O)     
9.云母片(电子显微镜科学,目录号:71855-15)     
10. 1 M KCl溶液(请参阅配方)   

设备


吸管小号0.5-10微升,10-100微升和100-1,000微升仪(Eppendorf,型号:研究® P的LU,可变可调体积移液管)
Milli-Q Advantage A10净水系统(Millipore- S Sigma,目录号:C10117)
玻璃分液漏斗(Southern Labware,型号:3964-3)
离心机(Thermo Fisher Scientific,型号:Sorvalis ST16R)
Vortexer(科学工业,型号:200-SI0236)
旋转蒸发仪(Buchi,型号:R-210)
真空泵(Buchi,型号:V-700)
真空控制器(Buchi,型号:V-800)
加热浴(Buchi,型号:B-491)
蒸发瓶(Buchi,目录号:Z402982)
笔记本电脑(ThinkPad,型号:T570)
11。    CoreAFM控制器(Nanosurf,型号:CoreAFM控制器)

12. CoreAFM系统(Nanosurf,型号:CoreAFM系统) 
13. Isostage 300控制器(Nanosurf,型号:Isostage 300控制器)               
14. CoreAFM工具集(Nanosurf,型号:CoreAFM工具集) 

软件


CoreAFM控制软件(版本3.10.0,https: //www.nanosurf.net/en/software/coreafm)

程序


的准备 摹LDNP
请参考我们的出版b IO-协议宋等人。(2019年和2020年)。

注意:超速离心(30,000 × g,4°C和45分钟)后,可以富集储存的GLDNP(在1 × PBS中)并除去上清液。           

设置AFM(请参阅注释1)
组装Nanosurf CoreAFM系统 根据CoreAFM操作说明中的步骤进行操作。组装后的AFM设备如图1所示。

图1.组装 Nanosurf CoreAFM系统。答:根据说明,组装好的Nanosurf CoreAFM系统包含CoreAFM系统,Isostage 300控制器,CoreAFM控制器和便携式计算机。B. CoreAFM工具集(请参阅注释2)。C.处于打开状态的CoreAFM的放大照片。


打开Nanosurf CoreAFM系统(请参阅注释3)。
将Nanosurf Dyn190AI动态模式悬臂插入悬臂支架中,确保尖端没有损坏,并将悬臂支架连接到CoreAFM扫描头底部的磁铁上(请参阅注3)。

载入中 用于AFM成像的GLDNP样品
         将ddH 2 O中的GLDNP溶液稀释至1 mg / ml。
         将2μl纳米颗粒样品沉积到云母片上(图2A)。
注意:作为药物输送工具,精确的剂量反应是必须考虑的因素之一。对于不同的样品,通常将溶液从高浓度稀释到低浓度,同时不断减少溶液的体积。根据成像AFM的效果,最适合溶液的浓度和体积是FINA LLY选择。的浓度GLDNP样品在此过程中用于成像应> 0.01 μ克/米升。

         在室温(RT)下干燥样品2小时。
         用5μl蒸馏水轻轻冲洗云母片3次(图2B)。
注意:首先,滴蒸馏水轻轻放入云母的中间片使用的p ipette,然后用滤纸从云母的边缘吸收水分片。由于GLDNP样品小号被储存在PBS中,盐颗粒从PBS将影响AFM的成像效果,所以目的克ently RINS我纳克的云母片是去除盐GLDNP样品小号。

         再次在室温下干燥样品2小时。
         将云母片在室温放置30分钟,直到样品变平。
注意:要判断样品是否干燥并且变平,我们可以将云母片放在垂直位置,如果我们没有观察到样品点有水流的迹象,则通常意味着样品已变平并干燥。在样品上的干燥度和平整度的MI CA片会影响AFM的成像效果。

将云母片固定在标准样品架中央的样品磁体上(图2C)。
关闭CoreAFM扫描头盖,三个扫描头支脚将与相应的样品台支柱对齐。

图2.加载GNDLN样品到所述云母片和它在样品上磁体固定。A.将2μl纳米颗粒样品沉积到云母片上的过程。B.轻轻漂洗过程的云母片用蒸馏水和从边缘吸附水的所述云母片用滤纸。C.将云母片固定在标准样品架的样品磁体上的过程(请参见注释3)。                         

GLDNP样品的AFM成像
检查激光的位置和质量(请参阅注释3)。
将CoreAFM系统连接到CoreAFM控制器,然后启动设备和软件。
在CoreAFM控制软件中,打开“激光对准”对话框(图3A),然后使用“摄像机”选择器(图3C)将CoreAFM切换到顶视图摄像机(图3B )。
注意:所述的激光对准对话框显示的当前位置的的AFM激光点上的检测器和所述用过的激光功率。通过单击“采集”选项卡的“准备”组中的“激光对准”按钮,可以打开该对话框。

使用CoreAFM系统随附的激光对准工具以顺时针或逆时针方向旋转螺钉(图3C)。最佳对准的激光将产生图3A。
注意:CoreAFM系统在扫描头的顶盖上带有4个孔,可用来接近激光和检测器对准螺钉,从而改变了激光束光路的不同方面(图3C)。将扫描头盖打开到大约30°的角度,现在您应该在样品架或样品架平台上的某个位置看到红色激光点(图3C)。



图3.检查激光器的位置和质量。答:              菜单的CoreAFM控制软件应用程序和校对完成激光校准对话框。该图形区域显示偏转的激光束撞击光电二极管检测器的位置。虚线框包围的区域内的任何地方都有绿点,表示悬臂挠度检测系统(由激光,悬臂和检测器组成)已正确对齐。如果激光光斑落在该区域之外,它将变成红色,这意味着对准不允许进行正确的测量。B.悬臂及其芯片结构的俯视图。C. CoreAFM扫描头和样品架的物理照片。           

配置CoreAFM的测量参数(请参见注释3)。
在CoreAFM控制软件的菜单中,单击“空气”以从“测量环境”下拉菜单中选择测量环境(图4A)。
单击“静态力”以从“操作模式”下拉菜单中选择所需的操作模式(图4B)。
单击“ ACL-A”,从“悬臂选择器”下拉菜单中选择所需的悬臂类型(图4C)。
单击“频率扫描”按钮,将打开“振动频率搜索”对话框。单击此对话框中的“查找振动频率”按钮(图4D)。单击“确定”退出对话框。
注意:自动频率搜索完成后,您应该看到一条清晰的共振曲线和一个显示振动频率的标记。



图4.配置Core AFM的测量参数。交流电 将测量环境设置为“空气”(A),将操作模式设置为“静态力”(B),然后在“悬臂选择器” (C)中选择“ ACL-A”作为悬臂。D. CoreAFM控制软件应用程序的菜单和校对完成的“振动频率搜索”对话框。           

接近荷兰国际集团的GLDNP样本(见注3)。
在侧视图中观察吸头和样品之间的距离(图5A)。
在观察吸头样品距离的同时,单击并按住“采集”选项卡的“进路”组中的“前进”按钮,直到吸头足够靠近样品为止(图5B)。
单击“获取”选项卡的“进场”组中的“进场”按钮(图5B)。单击“确定”按钮(图5C)。
注意:在最后一步中,此样本将自动接近吸头,直到达到给定的设定值。



图5.接近GNDLN样本。A.悬臂的侧视图图像。B. “前进”和“进场”,但在“获取”选项卡的“进场”组中占很多。C.达到设定点时,显示“已完成处理”消息。


开始测量(请参阅注释3)。
在“成像”对话框中选择初始扫描参数,如下所示(图6A):
图像扫描尺寸= 10.00μm

时间/线= 0.78 s

点/线= 256

注意:在主面板上的其他值是在完成探针调优后仪器自动填写的值。因此,不需要更改这些值。

点击“开始”按钮,在成像组的的取样选项卡并开始扫描,持续1分钟,以稳定悬臂和仪器调整到周围环境vibrati组件(图6B)。
注意:手动降低“设定值”值,直到表面特征开始出现在高度图像上。可以通过调整“设定值”,“ P增益”和“ I增益”值来实现成像优化(请参见注4)。最后,可以将“图像扫描尺寸”减小到5.00μm或4.00μm以调整图像的扫描尺寸,并且“点/线”可以增加到512或什至1,024(见注5)f个或更多像素。因此,每个图像中的图像质量都得到了提高。

对图像质量满意后,单击“开始”按钮,然后使用优化的参数重新开始扫描。扫描后,单击“采集”选项卡的“成像”组中的“停止”按钮(图6B)。
注意:调整CoreAFM系统的定位螺钉(请参见图1C),以使每个样品的悬臂可以放置在云母片表面的不同位置。然后单击“采集”选项卡的“成像”组中的“ Prescan ”按钮(图6B)。通过预扫描找到所需位置后,单击“开始”按钮进行扫描。



图6.挑动克和存储在所述的测量GLDNP样品。答:选择初始扫描参数并调整“设定值”,“ P增益”和“ I增益”值以提高“成像”对话框中的图像质量。B. “采集”选项卡的“成像”组中的“预扫描” ,“开始”和“停止”按钮。C.在“捕获”按钮中的成像工具栏。


存储测量值并使用文档。
默认情况下,每个完成的测量都会自动(临时)存储在您的计算机上,以便以后使用。您还可以通过单击“捕获”按钮来拍摄仍在进行的测量的快照(图6C)。
Ť他获取的文档将保持打开在CoreAFM控制软件(图7A)的文件空间。添加或删除图表或设置图表参数全部在“图表属性”对话框中执行(图7B)。
点击(“+“添加图表”按钮中的”图7B) ,以创建当前所选或活性图表的副本,并将其在添加到活动窗口的最后位置。点击“删除图表”按钮(图7B),以删除当前激活的图表。
选择的图表类型(线图,颜色映射,3维视图,XY线图,或力曲线图),以用于将测量数据从“类型”显示的图表参数(图7B)。图7A示出了3个不同图表TY PES 的所述GLDNP样品。
使用图表属性对话框来设置相应的图表的类型的各种参数的GLDNP样品
设置完成后,单击“关闭”按钮(图7B),以ç失去图表属性对话框,然后单击“ Ş AVE图标”(图7A)保存文档。

图7. d ifferent图表类型的GLDNP样品和所述图表属性对话框。一种。 (ⅰ)颜色图,(ⅱ)3D视图和(iii)次的线图ë GLDNP样品,和(IV )的“图表属性”按钮。B.图表属性对话框用于设置所有图表属性,这些属性会影响相应图表的数据显示。


数据分析


我们使用上述方法来执行的原子力显微镜成像GLDNP样品(图小号8A - 8 C) 。
在“成像”对话框中为GLDNP选择最终扫描参数,如下所示:

图像扫描尺寸= 5 .00μm

时间/线= 0.78 s

点/线= 256

我Ñ Figu重新小号8A- 8 C,AFM图像显示,GLDNP小号是纳米尺寸的颗粒和具有一球形形状,并且提出了一个大小均质的外观。

我们制备的合成材料P聚(乳酸-共-乙醇酸)(PLGA)系纳米颗粒,然后使用上述方法和设置相同的参数,以获得样品PLGA(图的AFM图像小号8D- 8 F)和与GLDNP进行了比较。通过图像比较,我们可以观察到PLGA纳米颗粒也是球形的,具有尺寸均一的外观,并且其粒径略小于GLDNP。与GLDNP相比,PLGA纳米颗粒在云母片上显示出一定程度的聚集。

图8 。的AFM表征吨他GLDNP样品和聚乳酸-共-乙醇酸(PLGA)的样品(作为对照)。交流电 彩色图(A),3D视图(B)和图像扫描的数据信息(C),用于GLDNP的AFM表征。DF。彩色图(D),3D视图(E)和用于PLGA的AFM表征的图像扫描(F)的数据信息。


笔记


这里描述了Nanosurf CoreAFM系统的所有过程。对于AFM的不同型号和品牌,将需要根据协议对协议进行调整。制造商的说明。
工具集的内容取决于用户订单中包含的选项。它至少包含以下各项:
接地线

悬臂镊子:(103A CA )

悬臂交换工具

激光对准工具:内六角扳手1.5毫米(球形六角头),带螺丝刀手柄

5毫米内六角扳手:用于CoreAFM运输锁

标准样品架

钙ntilever支架液体-空气平

准备并操作 使用CoreAFM系统并测量该系统的简要程序。V IDEO是可以在产品官方网站( https://www.nanosurf.net/en/products/coreafm-the-essence-of-atomic-force-microscopy)。准备用于AFM成像的负载纳米粒子的云母片大约需要4小时30分钟,而用于AFM调试和照相则需要15-20分钟。因此,可以在5小时内完成不包括数据分析的AFM成像。 我Ñ特别,它应当指出的是 的主要限制在一个波夫过程是,AFM成像只能在干燥样品来进行在室内空气的环境中,并制备的样品的原子力显微镜成像(装载在云母片),通常需要是在有限的时间内(不超过6小时)完成。另外,通过CoreAFM系统,样品的最小可分辨粒度为约10nm 。
使用低“P增益”的d在开始“I增益”,以保护悬臂。这些值可以在以后的扫描中提高,以获得更好的图像质量。
“积分/线”确定小号的像素点数量小号上的每个线和所述图像中的扫描线的数量。在大多数情况下,该值应保持不变。

菜谱


1 M KCl溶液
将0.745 g氯化钾(KCl)溶解在10 ml的ddH 2 O中,并充分混合


致谢


这项工作得到了美国糖尿病,消化与肾脏疾病研究所的支持(DM的RO1-DK-116306和RO1-DK-107739),退伍军人事务部(DM的优异奖BX002526)以及克罗恩氏和结肠炎基金会的支持美国。D. L.是美国克罗恩氏和结肠炎基金会(Research Fellowship Award)的获得者(奖号#689659)。DM是退伍军人事务部(BX004476)颁发的“高级研究职业科学家奖”的获得者。该协议基于我们先前发表的研究(Yang等,2020)。


利益争夺


作者声明该作品内无利益冲突。


参考


Chen,Q.,Xiao,B. and Merlin,D.(2017年)。用于治疗炎症性肠病的纳米疗法。Gastroenterol Hepatol专家评论11(6):495-497。
莫里斯(VJ),柯比(Kirby)和加宁(AP)(2010)。生物学家用的原子力显微镜。第二版银行足球比赛。帝国大学出版社,ISBN-10:184816467X。
Sung,J.,Yang,C.,Viennois,E.,Zhang,M.和Merlin,D.(2019)。姜的根茎生姜中的生姜纳米颗粒(GDNP)的分离,纯化和表征。生物- p rotoc醇9(19):e3390。
Sung,J.,Yang,C.,Collins,JF和Merlin,D.(2020年)。结肠靶向siRNA递送的生姜脂质纳米颗粒的制备和表征。生物- p rotoc醇10(14):e3685。
Ulbrich,W.和Lamprecht,A.(2010年)。纳米颗粒载体在炎性疾病治疗中的靶向药物递送方法。JR Soc接口7增补1:S55-66。
Wang,X.,Zhang,M.,Flores,SR,Woloshun,RR,Yang,C.,Yin,L.,Xiang,P.,Xu,X.,Garrick,MD,Vidyasagar,S.,Merlin,D 。和Collins,JF(2019)。携带Dmt1 siRNA的生姜纳米颗粒脂质载体的口服管饲法可钝化鼠遗传性血色病中的铁负荷。Mol Ther 27(3):493-506。
Yang,C.,Zhang,M.,Lama,S.,Wang,L.和Merlin,D.(2020年)。基于天然脂质纳米颗粒的治疗方法,可将6-松果酚及其代谢产物M2和M13输送至结肠,以治疗溃疡性结肠炎。J Control版本323:293-310。
Zhang,M.,Viennois,E.,Prasad,M.,Zhang,Y.,Wang,L.,Zhang,Z.,Han,MK,Xiao,B.,Xu,C.,Srinivasan,S.和Merlin ,D.(2016年)。可食用的生姜纳米颗粒:预防和治疗炎症性肠病和结肠炎相关癌症的新型治疗方法。生物材料101:321-340。
Zhang,M.,Wang,X.,Han,MK,Collins,JF and Merlin,D.(2017年)。口服含siRNA的生姜纳米脂质作为有效siRNA药物递送治疗溃疡性结肠炎的新方法。纳米医学(Lond)12(16):1927-1943。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Long, D., Yang, C., Sung, J. and Merlin, D. (2021). Atomic Force Microscopy to Characterize Ginger Lipid-Derived Nanoparticles (GLDNP). Bio-protocol 11(7): e3969. DOI: 10.21769/BioProtoc.3969.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。