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Apr 2020
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Peptide-mediated Targeting of Nanoparticles with Chemical Cargoes to Chloroplasts in Arabidopsis Plants
多肽介导的化学物质纳米粒对拟南芥叶绿体的靶向作用   

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Abstract

Plant nanobiotechnology is a flourishing field that uses nanomaterials to study and engineer plant function. Applications of nanotechnology in plants have great potential as tools for improving crop yield, tolerance to disease and environmental stress, agrochemical delivery of pesticides and fertilizers, and genetic modification and transformation of crop plants. Previous studies have used nanomaterials functionalized with chemicals, including biocompatible polymers with charged, neutral, or hydrophobic functional groups, to improve nanomaterial uptake and localization in plant cells. Recently, the use of biorecognition motifs such as peptides has been demonstrated to enable the targeted delivery of nanoparticles in plants (Santana et al., 2020). Herein, we describe a bio-protocol to target nanoparticles with chemical cargoes to chloroplasts in plant leaves and assess targeting efficiency using advanced analytical tools, including confocal microscopy and elemental analysis. We also describe the use of isothermal titration calorimetry to determine the affinity of nanomaterials for their chemical cargoes. Nanotechnology-based methods for targeted delivery guided by conserved plant molecular recognition mechanisms will provide more robust plant bioengineering tools across diverse plant species.


Graphic abstract:



Targeted delivery of nanomaterials with chemical cargoes to chloroplasts enabled by plant biorecognition


Keywords: Drug delivery (药物输送), Nanoparticle imaging (纳米粒子成像), Peptide (肽), Agrochemicals (农药)

Background

Nanomaterials have enabled improved diagnostic tools, drug delivery, bioengineering, and tissue regeneration platforms for mammalian systems (Das et al., 2014; Li et al., 2016; Patra et al., 2018). Applications of nanotechnology in plant bioengineering and nano-enabled agriculture have recently emerged (Kah et al., 2019; Lowry et al., 2019). The use of plant nanobiotechnology has great potential in developing valuable diagnostic and therapeutic tools for improving crop management, resistance to diseases and environmental stresses, targeting the delivery of agrochemicals, and genetic bioengineering tools (Wang et al., 2016; Yin et al., 2018; Giraldo et al., 2019; Wang et al., 2019; Santana et al., 2020).


Currently, the delivery of chemicals in plants leads to unintended alterations in plant function and environmental pollution from chemical leaching (Nagajyoti et al., 2010; Smith and Gilbertson, 2018; Lowry et al., 2019). Nanotechnology approaches have relied on size, surface charge, and hydrophobicity modifications to tune their distribution in plant cells (Asati et al., 2010; Wong et al., 2016; Wu et al., 2017; Demirer et al., 2019; Hu et al., 2020); however, these approaches based on chemical coatings cannot target specific plant subcellular compartments with high precision. Nanoparticle functionalization with targeting peptide recognition motifs enables plant molecular machinery to guide the nanomaterials to plant organelles in vivo with high specificity (Santana et al., 2020).


Herein, we present a protocol for synthesizing, characterizing, and detecting a nanomaterial platform that targets the delivery of chemicals to chloroplasts in wild-type Arabidopsis thaliana (Col-0) using a peptide recognition motif that is relatively conserved in dicotyledonous plants. We describe techniques for the synthesis and characterization of targeted quantum dots using UV-vis spectroscopy, Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and transmission electron microscopy (TEM). We outline methods to image and quantitate the nanoparticles in plant chloroplasts using advanced analytical tools with high resolution, including confocal microscopy and inductively coupled plasma mass spectrometry (ICP-MS). These nanomaterials target the delivery of chemicals to the chloroplast, allowing tuning of their function, e.g., redox state, with higher specificity and efficiency than chemicals alone.


The use of quantum dots coated with biorecognition moieties to target the delivery of chemicals to chloroplasts can be extended to sustainable nanomaterials for the targeted delivery of genetic elements, nanosensors, nutrients, or pesticides across multiple plant species.

Materials and Reagents

  1. Centrifugal filter (Merck Millipore, Amicon Ultra 15, molecular weight cut-off, 10,000 Da)

  2. Arabidopsis thaliana seeds (ecotype Columbia seed stock source CS60000)

  3. NaBH4, purum p.a., ≥96% (gas-volumetric) (Sigma-Aldrich, catalog number: 71320-25G, CAS Number 16940-66-2)

  4. Succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-MAL linker, Thermo Fisher Scientific, U.S.A.)

  5. Perfluorodecalin (Acros Organics, 25 g, 90% mixture of cis and trans, CAS 306-94-5)

  6. Tellurium powder, Te; 99.8%, (Sigma-Aldrich, catalog number: 266418-25G)

  7. Ethanol 200 proof (Fisher Scientific, Acros organics 61509-0040 4L)

  8. Molecular grade H2O (Corning, catalog number: 46-000-CM)

  9. Cadmium chloride hydrate (Sigma-Aldrich catalog number: 529575)

  10. Mercaptopropionic acid (Sigma-Aldrich, catalog number: M5801-100G)

  11. Sodium hydroxide solution (50% w/w certified; Fisher Scientific, catalog number: SS254-1, 1L, CAS number 1310-73-2)

  12. 1 ml NORM-JECT® (4010-2000V0)

  13. 1-Ethyl-3-[3-dimethylaminopropyl] carboamide hydrochloride, EDC, 5 g (G-Biosciences, catalog number: BC 25-5)

  14. N-Hydroxysuccinimide, NHS, 25 g (Thermo Scientific, catalog number: 24500)

  15. TES buffer (Sigma Life Science, catalog number: T1375-25G)

  16. 3-Aminophenylboronic acid hydrochloride (Sigma-Aldrich, catalog number: 410705-1G)

  17. L-Ascorbic acid (Fisher Chemical, catalog number: A61-100)

  18. Methyl viologen (Acros organics, catalog number: A227320010)

  19. Centrifugal filter (Merck Millipore, Amicon Ultra 15, molecular weight cut-off, 10,000 Da, catalog number: UFC901024)

  20. NaOH 1% solution

  21. HCl 1% solution

  22. Mono-(6-ethanediamine-6-deoxy)-beta-cyclodextrin (PONOCO enterprise, CAVCON, catalog number: 60984-63-6) (store at -4°C)

  23. NHS-PEG4-Maleimide, SM-PEG pegylated crosslinker (NHS-PEG4-MAL) (Thermo Scientific, catalog number: 22107) (store at -20°C)

  24. RbcS targeting peptide was designed from Rubisco small subunit transit peptide sequence (GenBank: OAP15425): MASSMLSSATMVGGC (1432.72 g/mol) (Genscript) (store at -20°C)

  25. Carolina microscope observation gel (Corning, 2984-75x25)

  26. Cheesecloth grade 50 (VWR International, catalog number: 470150-438 (PK))

  27. 1× chilled sucrose buffer (pH 7.3) (see Recipes)

Equipment

  1. Erlenmeyer flasks (200 ml)

  2. Fume hood

  3. -20°C freezer

  4. Plastic plant growth inserts (T.O. plastics st-10804)

  5. Hot plate/stir plate (IKA RCT basic safety control magnetic stirrer, RCT BS001)

  6. Bath sonicator (Elmasonic p, P-30H, #101-3737)

  7. UV-vis spectrophotometer (Shimadzu 2600, UV-2600 EN)

  8. Malvern 1600 zetasizer (Nano S, ZEN1600)

  9. Folded capillary zeta cell cuvette (Malvern Panalytics, DTS1070)

  10. Disposable cuvettes (Spectrum Laboratory Products, 330-10304P5)

  11. Quartz open top cuvette 10 mm (Starna Cells Inc, 18-Q-10)

  12. FTIR spectrometer (Bruker Alpha I)

  13. TEM (Philips FEI Technai 12 microscope)

  14. Leica SP5 Confocal Microscope

  15. Malvern isothermal titration calorimeter (G.E. Healthcare, MicroCal ITC200 instrument)

  16. ICP-MS (Agilent 7700x)

  17. Grinder or macerator (Intertek KWG-100A)

Software

  1. ImageJ

Procedure

  1. Plant growth

    1. Germinate Arabidopsis thaliana seeds (ecotype Columbia seed stock source CS60000) in (2.5” × 2.5” × 3”) pots filled with soil containing 1% marathon (OHP, Inc., Marathon 1% Granular, 5 lbs., 985490.0) and 1% osmocote (Classic 3-4 Month 14-14-4 Fertilizer 50 lb., E90550).

    2. Grow plants in Adaptis A1000 growth chambers (Conviron MODEL No. A1000, SERIAL No. 150031) set to 200 μmol m−2 s−1 photosynthetic active radiation (PAR), 24 ± 1°C, 60% humidity, and a 14 h/10 h day/night regime.

    3. Water the plants once every three days.

    4. For all experiments, use Arabidopsis thaliana Col-0 plants that are 3 weeks old (Figure 1).



      Figure 1. Leaves of 3-week-old Arabidopsis thaliana plants (Col-0) were used for nanoparticle infiltration


  2. Synthesis of quantum dots

    1. To synthesize quantum dots (QDs), prepare a colloidal solution of 0.01 g CdCl2 and 40 µl mercaptopropionic acid in 50 ml molecular grade water.

    2. Label this solution A, add a stir bar, and stir the solution at 500 rpm and room temperature.

    3. Next, adjust the pH of solution A to 11.4 with 1 M NaOH added dropwise. Place solution A on a hot plate set to 100°C and allow to reflux. Stir solution A at 700 rpm.

    4. Meanwhile, label a 20-ml glass vial with a cap as solution B.

    5. Add 0.05 g NaBH4 and 0.02 g tellurium powder into the 20-ml vial labeled solution B.

    6. Add 600 µl 50% ethanol into the 20-ml vial and add a stir bar. Make sure to dispense the ethanol gently into the glass vial labeled solution B.

    7. Keep the solution lightly capped to avoid air entering the reaction (Figure 2A).

    8. Place solution B onto a hot plate set to 70°C and stir at 300 rpm.

    9. Allow solution B to react for 5-10 min. The solution will turn from a dark black color to purple-blue, producing NaHTe for later use (Figure 2A-2B).

    10. Immediately after the color change (Figure 2B), use a pipet tip to collect 150 µl freshly prepared solution B and quickly dispense directly into solution A.

    11. Allow the mixture to react for 5 min under reflux conditions with vigorous stirring (700 rpm) (Figure 2C).

    12. An increase in fluorescence of the mixture can be monitored when excited under U.V. light (375 nm) (Figure 2D).



      Figure 2. Synthesis steps for mercaptopropionic-coated quantum dots. A. Image of a 20-ml vial labeled solution B filled with NaBH4 and tellurium powder in 50% ethanol. B. Final product NaHTe after the reaction. C. Formation of MPA-QD crystals in an Erlenmeyer flask containing solutions A and B under reflux. D. Image of fluorescent MPA-QDs under U.V. light excitation (375 nm).


    13. Stop the reaction by removing it from the hot plate after 5 minutes and cooling it to room temperature. The emission of QD could be tuned to a specific wavelength by adjusting the reaction time (Table 1).


      Table 1. QD synthesis reaction time versus emission peak wavelength

      Reaction time (min) Emission peak wavelength (nm)
      1 524
      3 537
      5 552


    14. The resulting solution contains fluorescent Cd/Te-Cd/S core quantum dots functionalized with mercaptopropionic acid with terminal carboxyl groups on the outer shell. Label this solution as MPA-QD.


  3. Formation of 3-aminophenylboronic acid (APBA)-capped QDs (APBA-QD)

    1. Prepare APBA-QDs by reacting the MPA-QD terminal carboxyl group with 3-aminophenylboronic acid using the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) activated reaction method.

    2. First, determine the concentration of QDs (see section F-M).

    3. Bath sonicate MPA-QDs for 30 min at 80% power and 37 Hz.

    4. Dilute the MPA-QD solution to 1 µM in 10 mM TES buffer at pH 7.0.

    5. Add NHS (2000 nmol) dissolved in molecular grade H2O to 1 nmol MPA-QD in 10 mM TES buffer at pH 7.0.

    6. Next, add EDC/HCl (2000 nmol) dissolved in molecular grade H2O into 1 nmol MPA-QD in 10 mM TES buffer at pH 7.0.

    7. Gently stir the mixture (500 rpm) for 15 min at room temperature.

    8. Add 80 μl 25 mM APBA dissolved in molecular grade H2O to the activated MPA-QD solution to generate aminophenylboronic acid-functionalized quantum dots (APBA-QD). Allow the reaction to stir (500 rpm) for 3 h at room temperature.

    9. Wash the APBA-QD solution twice with molecular grade H2O to remove the excess APBA using a centrifugal filter (Merck millipore, Amicon Ultra 15, molecular weight cut-off, 10,000 Da). Set the centrifuge to 2,360 × g for 10 min.

    10. Bath sonicate the APBA-QD solution for 30 min at 80% power at 37 Hz to break down any agglomerated particles.


  4. Synthesis of β-cyclodextrin-capped QD

    1. Suspend the resulting APBA-QD in 10 ml TES buffer 10 mM pH 10.4.

    2. Add 1 μmol β-cyclodextrin (β-CD, Cavcon) dissolved in molecular grade H2O (0.5 ml) to the APBA-QD solution.

    3. Allow the mixture to react overnight at room temperature with stirring set at 500 rpm.

    4. Remove the excess of β-cyclodextrin by washing through a centrifugal filter (Merck millipore, Amicon Ultra 15, molecular weight cut-off, 10,000 Da). Centrifuge at 2,360 × g for 10 min.

    5. Bath sonicate the resulting solution for 30 min at 80% power and 37 Hz.

    6. Suspend the β-cyclodextrin-coated quantum dots (CD-QD) in 10 ml 10 mM TES pH 7.5.

    7. Confirm CD-QD formation by collecting the FTIR spectrum (see section K).


  5. Preparation of peptide-conjugated β-CD-capped QD

    1. To conjugate the RbcS targeting peptide to CD-QD nanoparticles, first dissolve succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol] ester (NHS-PEG4-MAL linker, Thermo Fisher Scientific, U.S.A.) in DMSO to make a 200 mM stock solution.

    2. Next, add 5 μl (1 μmol) NHS-PEG4-MAL stock solution to 1 μM CD-QDs in 10 ml final volume.

    3. Incubate the mixture at room temperature for 1 h and stir at 500 rpm to yield MAL-PEG4-QD.

    4. Remove excess NHS-PEG4-MAL by washing the mixture through a centrifugal filter (Merck Millipore, Amicon Ultra 15, molecular weight cut-off, 10,000 Da) with molecular grade H2O.

    5. Resuspend the MAL-PEG4-QD in 10 ml 10 mM TES pH 8.0.

    6. A peptide sequence from the Rubisco small subunit (RbcS) was used for targeting nanomaterials to the chloroplast. The peptide was synthesized by Genscript containing the amino acid sequence MASSMLSSATMVGGC.

    7. RbcS chloroplast-targeting peptide was dissolved in 5% DMSO diluted with TES buffer pH 8.0 to 10 mg·ml-1 (equivalent to 7 mM).

    8. Finally, add 0.143 ml (1 μmol) RbcS chloroplast-targeting peptide to the resulting MAL-PEG4-QD.

    9. React for 1 h at room temperature and stir at 500 rpm to form chloroplast-targeting peptide-functionalized QD (Chl-QD).

    10. Remove excess peptide and reactants by washing at least twice through a centrifugal filter (Merck Millipore, Amicon Ultra 15, molecular weight cut-off, 10,000 Da) with molecular grade H2O loaded onto a centrifuge set to 2,360 × g for 10 min.

      Notes:

      1. Make sure not to completely wash away the buffer from the centrifugal filter. Keep Chl-QD in solution during washing.

      2. The resulting Chl-QD can be stored for up to one week without significant aggregation.

    11. Bath sonicate the Chl-QD solution for 30 min at 80% power and 37 Hz to break down any agglomerated particles.

    12. Characterize the Chl-QD using the methods described below (see section F).

    13. Measure the hydrodynamic size, zeta potential, and fluorescence.

    14. Confirm the formation of Chl-QD by collecting the FTIR spectrum.


  6. Characterization of quantum dots (MPA-QD)

    The resulting nanomaterial absorbance, size, zeta potential, and fluorescence emission (under 405 nm excitation) can be characterized accordingly.


  7. UV-vis spectroscopy

    1. Measure the UV-vis absorption spectrum of QDs on a Shimadzu UV-2600 spectrophotometer (Figure 3A).

    2. The spectrophotometer settings are scanning range from 200 to 700 nm, interval of 0.5 nm, and integration time of 0.1 s.

    3. Collect a background spectrum using molecular grade H2O in a 1-ml quartz spectrophotometer cuvette (10 mm × 4 mm).

    4. Take 10 µl prepared MPA-QD and dilute it into 990 µl molecular grade H2O.

    5. Place the sample in a quartz cuvette to record the UV-vis absorbance from 200 to 700 nm.

    6. Record the peak absorbance value. The typical absorption peak of green fluorescent QD is 500-510 nm (Figure 3A).


  8. Fluorescence emission

    1. Measure the fluorescence of MPA-QD with the PTI QuantaMaster 600 fluorometer (Figure 3B).

    2. Prepare the MPA-QD solution (200 nM) in molecular grade H2O.

    3. Fill 3 ml solution into a quartz fluorescence cuvette and insert the cuvette into the holder of the PTI QuantaMaster 600 fluorometer.

    4. The fluorometer settings are 5 nm slit size, 1 nm step size, and 0.1 s integration time.

    5. Collect the emission spectra of MPA-QD following excitation at 405 nm (Figure 3B).


  9. Determination of nanomaterial concentration

    1. Determine the concentration of the solution using the following equations according to the peak absorbance and size.

      c=Abs/(L×ϵ)

      ε=10043×d2.12 (Yu et al., 2003)

      where,

      c is the QD concentration in M,

      Abs is the peak absorbance at 500-510 nm,

      L is the path length (1 cm),

      𝜖 is the extinction coefficient in L mol-1 cm-1,

      d is the diameter of QD 10-9 meters.


  10. Hydrodynamic size

    1. Measure the hydrodynamic size using a Malvern Zetasizer Nano S (Figure 2C) (model 1600).

    2. The zetasizer settings we set for water as the solvent, a temperature of 20°C, material refractive index of 1.350, and a material absorbance of 1.000. Repeat the measurement 3 times.

    3. Place 1 ml diluted MPA-QD in a 4-ml disposable cuvette. Insert the cuvette into the zetasizer for size measurements.

    4. Measure the particle size distribution and take the average volume-based particle size distribution. A summary table will be displayed alongside the graph, which can help to determine the peak distribution of particles and the average from each measurement (Figure 3C).


  11. FTIR spectroscopy

    1. To collect the FTIR spectrum of nanomaterials, take a 2-ml centrifuge tube, add 500 µl 3 µM MPA-QD, and suspend in 1.5 ml ethanol at a 3:1 (v/v) ratio mixture.

    2. Centrifuge at a max speed of 847 × g to precipitate MPA-QD into a pellet.

    3. Remove the supernatant and allow it to air dry in a fume hood overnight.

    4. A small dry pellet should form at the bottom of the centrifuge tube, which is used for subsequent FTIR spectroscopy using a Bruker Alpha II FTIR spectrophotometer or equivalent.


  12. Zeta potential

    1. Measure the zeta potential of MPA-QD with a Malvern Zetasizer (Nano Z.S.) (Figure 2D).

    2. Prepare the MPA-QD solution (200 nM) in molecular grade H2O.

    3. Fill about 0.7 ml solution into a folded capillary zeta cell cuvette (Malvern Panalytics, DTS1070) with a 1-ml syringe and insert the cuvette into the zetasizer (Nano Z.S.).

    4. The zetasizer (Nano Z.S.) settings are water as the solvent, a temperature of 20°C, material refractive index of 1.350, material absorbance of 1.000, Hückel approximation, and a measurement repeat of 5.

    5. Record the zeta potential and calculate the average and standard deviation (Figure 3D).



      Figure 3. Characterization of MPA-QD and Chl-QD. A. UV-vis absorption spectra. B. Fluorescence excitation and emission spectra. C. Hydrodynamic size distribution. D. Zeta potential.


  13. Transmission electron microscopy (TEM) of MPA-QDs

    1. Load one drop (about 2 μl) MPA-QD onto the TEM grid (ultrathin carbon film on lacey carbon support film, 400 mesh, Cu, Ted Pella).

    2. Allow the droplet to air dry.

    3. Image the MPA-QD on the prepared grid with a Philips FEI Tecnai 12 microscope operated at an accelerating voltage of 120 kV.

    4. Analyze the images obtained by TEM using ImageJ by measuring the particle diameter using the line segment tool (Figure 4A). Outline over 100 particles in each image and calculate the average particle diameter. To obtain lattice spacing of nanoparticles using the line segment tool (Figures 4A-4B), select one single particle clearly showing the lattice fringe, measure the distance between two planes that are separated by (at least) 10 lattice spacings, and calculate the lattice spacing by dividing the measured distance by 10.



      Figure 4. The ImageJ line segment tool was used to measure particle diameter and lattice spacing in TEM images


    5. Calculate the average size of MPA-QD with statistical analysis on more than 100 particles and measure the lattice spacing (Figure 4B).


  14. Loading chemicals into Chl-QD

    1. To load chemicals into Chl-QD, make a 1 ml stock solution of 0.1 mM methyl viologen or ascorbic acid in molecular grade H2O.

    2. Add 100 µl 0.1 mM methyl viologen or ascorbic acid to 1 ml 200 nM Chl-QD in TES buffer pH 7.0.

    3. Allow the mixture to incubate for 30 min and wash once through a centrifugal filter (Merck millipore, Amicon Ultra 15, molecular weight cut-off, 10,000 Da) with molecular grade H2O to remove excess molecules.

    4. Determine the Chl-QD nanoparticle concentration loaded with chemicals, methyl viologen or ascorbic acid (MV-Chl-QD or Asc-Chl-QD, respectively), using the Beer-Lambert law, which allows determination of the unknown concentration of sample mixture to be calculated by measuring its absorbance.

    5. Make a set of standards in triplicate using a fixed concentration of Chl-QD (200 nM) and a gradient concentration of methyl viologen or ascorbic acid ranging from 0 to 100 µM.

    6. Record the max absorbance value of each standard mixture between 260 and 265.5 nm.

    7. Use the known concentration of the standards and their respective absorbance values to generate a standard curve and determine the dilution factor (slope of the line).

    8. Using the Beer–Lambert law, determine the unknown concentration of chemicals loaded into the 200 nM Chl-QDs.

    9. Next, measure and record the initial absorbance of the Chl-QD sample without chemicals and the mixed chemical cargo-Chl-QD complex (from Step 3) after washing.

    10. Use the Beer-Lambert law to determine the concentration of the chemicals mixed with the Chl-QD samples.

    11. The final dosage of chemicals infiltrated into plants with 200 nM Chl-QD should be approximately 60 µM methyl viologen or 60 µM ascorbic acid in 1 ml TES buffer pH 7.0.


  15. Measurement of the affinity of QDs (MPA-QD and CD-QD) for the chemicals (methyl viologen and ascorbic acid) using isothermal titration calorimetry (ITC)

    Isothermal titration calorimetry (ITC) was performed using a MicroCal ITC200 instrument (G.E. Healthcare) and MPA-QD and cyclodextrin-coated QDs (CD-QD).

    1. Prepare a 10 mM TES buffer pH 7.3.

    2. Prepare CD-QD (0.5 μM) and methyl viologen (25 mM) stock solutions with TES buffer 10 mM pH 7.3.

    3. Thoroughly clean the reference cell, sample cell, and the injection syringe with molecular grade H2O.

    4. Load 0.3 ml CD-QD in 10 mM TES buffer pH 7.3 solution into the reference and sample cells.

    5. Load methyl viologen solution into the calorimeter injection syringe and ensure that no bubbles are present.

    6. Set up the instrument as follows: Temperature of 25°C, injection volume of 2 μl, 21 injections, time intervals between two consecutive injections of 180 s, reference compensation power of 5 μcal/s.

    7. Run the measurement to obtain the thermogram and plot the thermal power against time.

    8. Display the thermal power peaks corresponding to each chemical injection.

    9. Use Origin (MicroCal) to integrate the thermal power peaks and normalize according to the amount of injected methyl viologen in moles to obtain the enthalpy changes, and plot the enthalpy changes against the molar ratios of injected methyl viologen to CD-QD in the sample cell to obtain the binding isotherm.

    10. Fit the binding isotherm raw data using a one-set-of-sites binding model to generate the best-fit curve and change in enthalpy of CD-QD interacting with methyl viologen (Figure 5A-5B).

    11. Record the thermodynamic parameters of methyl viologen binding to CD-QD, including the number of binding sites on CD-QD (n), association constant (Ka, M-1), dissociation constant (Kd, M), enthalpy change (ΔH, cal mol-1), and entropy change (ΔS, cal mol-1 K-1) (Figure 5A-5B).

    12. Use the same method to determine the thermodynamic parameters for the binding between methyl viologen and MPA-QD, ascorbic acid and CD-QD, and ascorbic acid and MPA-QD, respectively.



      Figure 5. Isothermal titration calorimetry (ITC) of cyclodextrin-coated quantum dots (CD-QD) with chemical cargoes. A. The change in enthalpy of CD-QD interacting with methyl viologen and ascorbic acid. B. Thermal parameters that can be extrapolated from ITC, including enthalpy change (ΔH, cal mol-1), stoichiometry (n), and binding affinity (Kd).


  16. Determination of nanoparticle bound and unbound chemicals in solution


    1. Use the thermodynamic parameters, such as the number of binding sites (n), dissociation constant (Kd), and concentration of chemical ligand acquired from the ITC data analysis, to determine the bound and unbound fractions of chemicals in cyclodextrin-coated nanoparticles.

    2. Use the following equation to determine the bound and unbound fractions:



      Where [Abound] and [A] is the concentration of bound and unbound chemicals in solution, respectively; n is the number of binding sites on QD; [QD]0 is the initial QD concentration; and Kd is the dissociation constant between QD and the chemicals.


  17. Nanoparticle and chemical delivery into plant leaves

    1. Dilute Chl-QD to 200 nM (0.17 mg ml-1) in 10 mM TES buffer pH 7.0.

    2. Load Chl-QDs with 60 µM methyl viologen or ascorbic acid (see section N).

    3. Take a 1-ml NORM-JECT needleless syringe and fill with 100 μl Chl-QD solution loaded with methyl viologen or ascorbic acid.

    4. Gently press the tip of the syringe up against the abaxial side of the leaf and hold the index finger up against the adaxial side for support (Figure 6A).

    5. Slowly perfuse each plant leaf by gently depressing the plunger. Gently remove any excess solution with Kimwipes (Figure 6B).



      Figure 6. Nanoparticle and chemical cargo delivery into plant leaves. Nanomaterials are delivered through the leaf lamina using a needleless syringe. A. Gently press the bottom of the syringe up against the abaxial side of the leaf and hold the index finger up against the adaxial side. B. After infiltration with nanoparticles, the leaf appears darker as the particle solution fills the mesophyll space (dashed circle).


    6. Place the plant on a lab bench and incubate for 15 min before placing the plant back into the growth chamber.


  18. Confocal fluorescence microscopy sample preparation

    1. Take a pea-sized amount of Carolina observation gel, make a thin film (~1 mm in thickness) on a microscope slide (Corning 2984-75x25), and create a chamber using a cork border (diameter 8 mm) to remove a circular section from the film center (Figure 7A).

    2. Take a leaf punch from the treated leaf with a cork border (diameter 6 mm) and incubate the leaf disk in 0.5 ml 10 µM DHE (Thermo Fisher Scientific, U.S.A.) in 10 mM TES buffer pH 7.0 for 30 minutes.

    3. Immerse the DHE-incubated leaf disk in perfluorodecalin (Acros Organics, 25 g, 90% mixture of cis and trans, CAS 306-94-5) (Figure 7A-7B) filled in the gel chamber and seal it with a coverslip. The sample is ready for confocal fluorescence microscopy imaging.



      Figure 7. Preparation of a microscopy slide for confocal imaging of nanoparticles in leaf samples. A. Leaf disk samples were placed inside a gel chamber and mounted on glass slides for confocal imaging. B. The gel chamber containing a leaf disk was filled with perfluorodecalin.


  19. Confocal fluorescence microscopy

    1. Load the leaf sample on the Leica laser scanning confocal microscope TCS SP5 (Leica Microsystems, Germany).

    2. Set up the microscope as follows: 40× wet objective (HCX PL APO CS 40.0 × 1.10 WATER UV, Leica Microsystems, Germany); 405 nm laser excitation for QDs; 514 nm for DHE; z-stack section thickness of 2 µm; line average of 4; PMT detection range of 500-550 nm for QDs, 580-615 nm for DHE, and 720-780 nm for chloroplast autofluorescence.

    3. Scan to locate a flat-leaf surface region of interest and image the sample.

    4. Collect the QD and DHE signals separately to avoid overlap between the excitation of the DHE dye and the emission detection range of QDs.

    5. Image three or more leaf discs for each plant and take images of two different regions per leaf disc for confocal analysis (Figure 8).



      Figure 8. Confocal images of nanoparticles in plant leaves. Confocal images of mesophyll cells from leaves infiltrated with 10 mM TES buffer pH 7.0, MPA-QD, and Chl-QD. Images show the degree of overlap of the QD fluorescence signal with chloroplast autofluorescence. Scale bars: 50 µm.


  20. Chloroplast isolation for nanoparticle detection by confocal microscopy and ICP-MS

    1. Infiltrate the plant leaf tissue with approximately 100 µl 500 nM Chl-QD in TES buffer 10 mM pH 7.3.

    2. Allow the plant leaves to incubate for 6 h at room temperature.

    3. Collect approximately 8 g leaf tissue from 3-week-old Arabidopsis thaliana plants treated with buffer control or Chl-QD from 5-6 plants per treatment. Macerate the leaf tissue in a grinder or macerator with 1× chilled sucrose buffer (Recipe 1).

    4. Grind the tissue by pulsing (3-4 pulses) for 5-s intervals until the leaf tissue is homogenized into a slurry (Figure 9A).

    5. Strain the slurry through 4 layers of cheesecloth into a glass beaker on ice. Place the filtered flowthrough into a 50-ml centrifuge tube and centrifuge twice with 1× sucrose buffer at 3,082 × g for 10 min (Figure 9B).

    6. Remove the supernatant each time and refill with a new sucrose buffer solution.

    7. A pellet should form at the bottom of the centrifuge tube (Figure 9B).



      Figure 9. Chloroplast isolation schematic. Chloroplasts were isolated from 3-week-old Arabidopsis thaliana plants. A. Centrifuge tube filled with macerated leaf homogenate in a sucrose buffer. B. Pellet of intact chloroplasts. C. Confirmation of the colocalized Chl-QDs in isolated chloroplasts by confocal microscopy. The QD signal is visualized in green and the chloroplast autofluorescence in magenta. Scale bar: 20 µm. D. Detection and quantitation of QD elements (Cd and Te) detected in chloroplasts by ICP-MS analysis.


    8. Following chloroplast isolation, a small stab sample of chloroplasts was placed on a glass slide to detect quantum dot fluorescence within extracted chloroplasts using confocal microscopy (see section S for settings) (Figure 9C-9D).

    9. Remove the supernatant (sucrose buffer and damaged chloroplasts) from the tube. Allow pelleted chloroplasts to air dry in a fume hood for more than 24 h. A small green sticky pellet will form.

    10. Once the pellet is dry, place the tube in a -20°C freezer for 1 h until the pellet solidifies to allow easy removal from the centrifuge tube and preparation for ICP-MS analysis.


  21. Inductively coupled plasma mass spectrometry (ICP-MS)

    1. Following chloroplast isolation, dry the sample pellets (~0.1 g) in air for 48 h.

    2. Place the air-dried samples in 50-ml polypropylene digestion tubes and digest with a solution of 5% HNO3/1% HCl/1% H2O2 v/v. Digest the samples in 1 ml HNO3/0.4 ml HCl while heating at 115°C for 5 min using a heat block (DigiPREP System; S.C.P. Science, Champlain, NY). Add 0.4 ml H2O2 and incubate for an additional 10 min.

    3. Dilute the solution and analyze the samples by ICP-MS (Agilent 7700x ICP-MS) to quantitate the Cd and Te content. Report individual element concentrations in μg g-1 as in Figure 8D (element mass in μg per g dry chloroplast).

Data analysis

Confocal fluorescence microscopy image analysis with Fiji (ImageJ)

  1. To analyze the images, open the image in the Fiji (ImageJ) software (Figure 10A-10C).

  2. Using the line tool, draw six transect lines on an image set to be analyzed evenly. Save the lines to the region of interest (ROI) manager (Figure 10A).

  3. Measure the corresponding fluorescence intensity profiles for the QD and DHE fluorescence and chloroplast autofluorescence channels across each of the six ROI line sections. Using the Fiji software, select Analyze > Plot profile (Figure 10B).

  4. Count the number of overlapping peaks between the QD and DHE channels over chloroplast fluorescence, and calculate the colocalization percentage (Figure 10B-10C).



    Figure 10. Stepwise schematic for colocalization analysis between targeted nanomaterials (Chl-QD) and chloroplasts in leaf mesophyll cells. A. Confocal fluorescence microscopy image analysis using the Fiji (ImageJ) software. B. Plots of normalized fluorescence intensity profiles collected by confocal microscopy and processed in Fiji (ImageJ). C. Calculated colocalization percentage of overlapping fluorescence intensity peaks.

Notes

  1. If air enters, the reaction solution will turn from blue to black, thereby oxidizing the catalyst and causing the inefficient formation of quantum dot crystals.

  2. Solution B should exhibit a color change as the reaction progresses, turning black-blue to pink-purple (Figure 2B).

  3. EDC is hygroscopic and will degrade in molecular grade H2O; make sure EDC is prepared fresh.

  4. QD solution can be filtered through a 20-nm filter to remove agglomeration.

  5. QDs tend to aggregate at a pH lower than 6.5.

  6. Make sure not to completely wash away the buffer from the 10 K filter column. Keep APBA-QD in solution during washing.

Recipes

  1. 1× chilled sucrose buffer (pH 7.3)

    28 mM Na2HPO4

    22 mM KH2PO4

    2.5 mM MgCl2

    400 mM sucrose

    10 mM KCl

    pH 7.3

Acknowledgments

This material is based upon work supported by the National Science Foundation under Grant No. 1817363 to J.P.G. Students C.C. and H.T., and Postdoc P.H. were supported by the National Science Foundation under Grant No. CHE-2001611, the NSF Center for Sustainable Nanotechnology. The CSN is part of the Centers for Chemical Innovation Program. This protocol was based on our previous publication in Nature Communications (Santana et al., 2020).

Competing interests

There is a pending U.S. patent entitled "Compositions and methods for chloroplast genetic and biochemical engineering in plants" (16/218.429) that is based on this work. All authors of this Bio-protocol manuscript, J.P.G., I.S., and P.H., are inventors in this patent, including Gregory M. Newkirk (University of California, Riverside) and Hong Hong Wu (Huazhong Agricultural University). Specific aspects outlined in this protocol are covered in the patent application, including methods for the targeted delivery of nanomaterials to chloroplasts using rationally designed guiding peptides.

References

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简介

[摘要]植物纳米生物技术是一个蓬勃发展的领域,它利用纳米材料来研究和设计植物功能。纳米技术在植物中的应用作为提高作物产量、对疾病和环境压力的耐受性、农药和化肥的农用化学品输送以及作物植物的基因改造和转化的工具具有巨大的潜力。先前的研究已经使用化学物质功能化的纳米材料,包括具有带电、中性或疏水性官能团的生物相容性聚合物,以改善纳米材料在植物细胞中的吸收和定位。近来,采用生物识别的图案,如肽已经证明,以使该 在植物中靶向递送纳米颗粒(Santana等,2020)。在此,我们描述了一种生物协议,将含有化学货物的纳米颗粒靶向植物叶片中的叶绿体,并使用先进的分析工具(包括共聚焦显微镜和元素分析)评估靶向效率。我们还描述了使用等温滴定量热法来确定的亲和力的纳米材料为它们的化学货物。由保守的植物分子识别机制引导的基于纳米技术的靶向递送方法将为不同植物物种提供更强大的植物生物工程工具。

图文摘要:


化学货物纳米材料的靶向递送到叶绿体使植物生物识别


[背景]纳米材料为哺乳动物系统提供了改进的诊断工具、药物输送、生物工程和组织再生平台(Das等人,2014 年;Li等人,2016 年;Patra等人,2018 年)。纳米技术在植物生物工程和纳米功能农业中的应用已经最近EMERG ED (陈嘉庚等人,2019;洛瑞等人,2019)。植物纳米生物技术的使用在开发有价值的诊断和治疗工具方面具有巨大的潜力,以改善作物管理、对疾病和环境胁迫的抵抗力、针对农用化学品的递送以及基因生物工程工具(Wang等人,2016 年;Yin等人, 2018;Giraldo等人,2019 年;Wang等人,2019 年;Santana等人,2020 年)。

目前,植物中化学物质的输送导致植物功能的意外改变和化学浸出造成的环境污染(Nagajyoti等人,2010 年;Smith 和 Gilbertson,2018 年;Lowry等人,2019 年)。纳米技术方法依赖于尺寸、表面电荷和疏水性修饰来调整它们在植物细胞中的分布(Asati等,2010;Wong等,2016;Wu等,2017;Demirer等,2019;Hu等人,2020 年);^ h H但是,这些方法基于化学涂料无法针对高精度特定的植物亚细胞器。具有靶向肽识别基序的纳米颗粒功能化使植物分子机制能够以高特异性将纳米材料引导至体内植物细胞器(Santana等,2020)。

在此,我们提出了一个用于合成、表征和检测纳米材料平台的协议,该平台使用在双子叶植物中相对保守的肽识别基序,将化学物质输送到野生型拟南芥(Col-0)中的叶绿体。我们日为划线技术的焦油的合成和表征使用UV- geted量子点v是小号pectroscopy,傅里叶变换红外光谱(FTIR),d ynamic升飞行小号cattering(DLS),和吨ransmission Ë lectron米icroscopy(TEM )。我们概述的方法进行成像和孔定量泰特使用先进的分析工具以高分辨率在植物叶绿体中的纳米颗粒,包括CONF OCAL显微镜和电感耦合等离子体质谱(ICP-MS)。这些纳米材料的化学品输送定位到叶绿体,使它们的功能,调谐例如,氧化还原状态,具有较高的特异性和效率比单独的化学物质。

使用涂覆有生物识别部分定位到叶绿体输送化学品的量子点的小号可以扩展到可持续为纳米材料的遗传元件,靶向递送纳米传感器,营养物,或农药在多个植物物种。

关键字:药物输送, 纳米粒子成像, 肽, 农药



材料和试剂


离心过滤器(默克密理博,Amicon Ultra 15,截留分子量,10,000 Da)
拟南芥拟南芥种子(哥伦比亚生态型种子储备源CS60000)
NaBH 4 ,purum pa,≥96%(气体体积)(Sigma-Aldrich,目录号:71320-25G,CAS 编号 16940-66-2)
琥珀酰亚胺基-[(N- maleimidopropionamido )-四甘醇] 酯(NHS-PEG4-MAL 接头,Thermo Fisher Scientific,美国)
全氟萘烷(Acros Organics,25 g,90%顺式和反式混合物,CAS 306-94-5)
碲粉,Te ;99.8%,(Sigma-Aldrich,目录号:266418-25G)
乙醇 200 证明(Fisher Scientific, Acros Organics 61509-0040 4L)
分子级 H 2 O(康宁,目录号:46-000-CM)
氯化镉水合物(Sigma-Aldrich 目录号:529575)
巯基丙酸(Sigma-Aldrich,目录号:M5801-100G)
氢氧化钠溶液(50% w/w 认证;Fisher Scientific,目录号:SS254-1,1L,CAS 号 1310-73-2)
1毫升NORM-JECT ® (4010-2000V0)
1-乙基-3- [3-二甲基氨基丙基]羧酰胺盐酸盐,EDC,将5g (GB iosciences ,目录号:BC 25-5 )
N-羟基琥珀酰亚胺,NHS,25 g(Thermo Scientific,目录号:24500)
TES 缓冲液(Sigma Life Science,目录号:T1375-25G)
3-氨基苯基硼酸盐酸盐(Sigma-Aldrich,目录号:410705-1G)
L-抗坏血酸(Fisher Chemical,目录号:A61-100)
甲基紫精(Acros Organics,目录号:A227320010)
离心过滤器(Merck M illipore,Amicon Ultra 15,截留分子量,10 , 000 Da,目录号:UFC901024)
NaOH 1% 溶液
盐酸 1% 溶液
中号ono-(6乙二胺-6-脱氧)-β-环糊精(PONOCO企业,CAVCON,目录号:60984-63-6)(存储在-4℃ )
NHS-PEG 4 -马来酰亚胺,SM-PEG p egylated交联剂(NHS-PEG4-MAL)(Thermo Scientific的,目录号:22107)(在-20商店℃下)
RBCS靶向肽从Rubisco小亚基转运肽序列(设计GenBank登录:OAP15425):MASSMLSSATMVGGC(1432.72克/摩尔)(金斯瑞(在-20商店)℃下)
卡罗莱纳显微镜观察凝胶 (Corning , 2984-75x25)
粗棉布 50 级(VWR International,目录号:470150-438(PK))
1 ×冷冻蔗糖缓冲液(pH 7.3)(见食谱)


设备


锥形瓶(200 毫升)
通风柜
-20°C 冰箱
塑料植物生长插入物(TO 塑料 st-10804)
热板/搅拌板(IKA RCT 基本安全控制磁力搅拌器,RCT BS001)
巴斯超声波仪( Elmasonic p, P-30H, #101-3737)
UV- v是分光光度计(Shimadzu 2600、UV-2600 EN)
Malvern 1600 z etasizer (Nano S, ZEN1600)
折叠Ç apillary Ž ETA Ç ELL比色池(马尔文Panalytics ,DTS1070)
一次性比色皿(频谱大号aboratory P RODUCTS,330-10304P5)
石英开口顶部试管10毫米(Starna细胞我NC ,18-Q-10)
FTIR 光谱仪 (Bruker Alpha I)
TEM(飞利浦 FEI Technai 12 显微镜)
Leica SP5 共聚焦显微镜
马尔文我sothermal吨itration Ç alorimeter(GE医疗集团,MicroCal ITC200仪)
ICP-MS(安捷伦 7700x)
研磨机或浸渍机 (Intertek KWG-100A)


小号oftware


图像J


程序


植物生长
发芽拟南芥thalian一个种子(哥伦比亚生态型种子储备源CS60000)在(2.5” × 2.5” × 3” )盆中填充有含有1%马拉松土壤(OHP公司,马拉松1%颗粒,5磅,985490.0)和1% osmocote (经典 3-4 个月 14-14-4 肥料 50 磅,E90550)。
植物生长在Adaptis A1000生长室(Conviron型号A1000,序列号为150031)被设置为200微摩尔米-2小号-1 p hotosynthetic有效辐射(PAR),24± 1℃,湿度60%,和一个14 h / 10 h 白天/黑夜制度。
水在每三天一次的植物。
对于所有实验,使用3 周龄的拟南芥Col-0 植物(图 1)。




图1 3的叶片-周-旧拟南芥拟南芥植物(Col-0中)被用来对纳米颗粒infiltrat离子


量子点的合成
要合成量子点 (QD),请在 50 毫升分子级水中制备0.01 克 CdCl 2和 40 微升巯基丙酸的胶体溶液。
标记此溶液A ,添加搅拌棒,并小号TIR的在500rpm溶液和室温。
接下来,滴加1 M NaOH将溶液 A 的 pH 值调节至 11.4 。将溶液 A 放在设置为 100 °C的热板上并允许回流。以 700 rpm 的速度搅拌溶液 A。
另一方面,标号20 -毫升玻璃小瓶用盖子作为溶液B.
将 0.05 g NaB H 4和 0.02 g 碲粉加入 20 - ml 小瓶标记的溶液 B 中。
将600 µl 50% 乙醇加入20 - ml 小瓶中并加入搅拌棒。确保分配的乙醇轻轻地进入了玻璃小瓶标记解决方案B.
保持溶液轻轻盖上,以避免空气进入反应(图 2A)。
将溶液 B 放在设置为 70 °C的热板上,并以 300 rpm 的速度搅拌。
允许溶液B至REA CT 5 - 10分钟。该解决方案会由一个暗黑色到蓝紫色,产生NaHTe供以后使用(图2A - 2 B)。
立即颜色变化(F后igure 2B),使用吸管尖收集150μl的新鲜LY分配直接制得的溶液B和迅速进入溶液A.
允许该混合物在剧烈搅拌下(700转)(图2C)在回流条件下反应5分钟。
在荧光的增加的混合物可当在UV光下(375纳米)(图2D)激发来监测。




FIGUR ê2.合成步骤为巯基-涂覆的量子点。A.图像一个20 -填充用NaBH毫升小瓶标记溶液B 4和吨在50%乙醇ellurium粉末。B.反应后的最终产物NaHTe 。C.在包含溶液 A 和 B 的锥形烧瓶中在回流下形成 MPA-QD 晶体。D.在紫外光激发 (375 nm) 下荧光 MPA-QD 的图像。


5 分钟后将其从热板上取出并冷却至室温以停止反应。通过调整反应时间,可以将 QD 的发射调谐到特定波长(表 1)。


表 1. QD 合成反应时间与发射峰波长


所得溶液包含用巯基丙酸官能化的荧光 Cd/ Te - Cd/ S 核量子点,在外壳上具有末端羧基。将此解决方案标记为 MPA-QD。




3-氨基苯基硼酸 (APBA) 的形成-封端量子点 (APBA-QD)
使用 1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC) 和 N-羟基琥珀酰亚胺(NHS) 激活反应方法,通过 MPA-QD 末端羧基与 3-氨基苯基硼酸反应制备 APBA-QD 。
首先,确定量子点的浓度(见F - M部分)。
巴斯在 80% 功率和 37 Hz 下对 MPA-QD 进行超声处理 30 分钟。
在 pH 7.0 的10 mM TES 缓冲液中将 MPA-QD 溶液稀释至 1 μM 。
在 pH 7.0 的 10 mM TES 缓冲液中,将溶解在分子级 H 2 O 中的NHS(2000 nmol)添加到 1 nmol MPA-QD。
接下来,在 pH 7.0 的 10 mM TES 缓冲液中,将溶解在分子级 H 2 O 中的EDC/HCl(2000 nmol)添加到 1 nmol MPA-QD 中。
在室温下轻轻搅拌混合物 (500 rpm) 15 分钟。
添加80 μ升25毫APBA溶解在分子等级H 2 O到活化的MPA-QD溶液,以生成氨基苯酸官能化的量子点(APBA-QD)。让反应在室温下搅拌 (500 rpm) 3 小时。
用分子级 H 2 O清洗 APBA-QD 溶液两次,使用离心过滤器(默克微孔,Amicon Ultra 15,分子量截止,10 , 000 Da)去除多余的 APBA 。将离心机设置为 2,360 × g 10 分钟。
浴在 37 Hz 下以 80% 的功率对 APBA-QD 溶液进行超声处理 30 分钟,以分解任何结块的颗粒。


β-的合成Ç yclodextrin皑皑的QD
将产生的 APBA-QD 悬浮在 10 ml TES 缓冲液 10 mM pH 10.4 中。
将 1 μmol β-环糊精(β-CD,Cavcon )溶解在分子级 H 2 O(0.5 ml)中添加到 APBA-QD 溶液中。
让混合物在室温下反应过夜,搅拌速度设置为 500 rpm。
通过离心过滤器(默克微孔,Amicon Ultra 15,截留分子量,10 , 000 Da)洗涤去除过量的 β-环糊精。以 2,360 × g离心10 分钟。
浴在 80% 的功率和37 Hz 下对所得溶液进行 30 分钟的超声处理。
暂停β环糊精-在10ml的10mM TES pH为7.5涂覆的量子点(CD-QD)。
确认CD-QD的形成通过收集FTIR光谱(参见小号挠度ķ )。


肽偶联 β-CD 封顶 QD 的制备
缀合的rbcS基因靶向肽到CD-QD纳米颗粒制品,首先溶解琥珀酰亚胺- [(N-马来酰亚胺基) -四甘醇]酯(NHS-PEG4-MAL连接体,赛默飞世尔科技,USA)在DMSO中,制成200毫米原液.
              接下来,添加5 μ升(1微摩尔)NHS-PEG4-MAL原液1 μ中号在10毫升的最终体积CD-量子点。
在室温下将混合物孵育 1 小时,并以 500 rpm 的速度搅拌以产生 MAL-PEG 4 - QD。
通过离心过滤器 (Merck M illipore, Amicon Ultra 15, 截留分子量, 10 , 000 Da) 用分子级 H 2 O洗涤混合物,去除多余的 NHS-PEG 4 -MAL 。
在 10 ml 10 mM TES pH 8.0 中重悬 MAL-PEG 4 -QD。
来自 Rubisco 小亚基 ( RbcS )的肽序列用于将纳米材料靶向叶绿体。该肽通过合成金斯瑞含有氨基酸序列MASSMLSSATMVGGC。
RBCS叶绿体-靶向肽溶于5%DMSO与TES缓冲液pH 8.0至10mg稀释·毫升-1 (相当于7毫米)。
最后,添加0.143毫升(1微摩尔)的rbcS基因叶绿体-靶向肽到所得的MAL-PEG 4 -QD。
在室温下反应 1 小时并在 500 rpm 下搅拌以形成叶绿体-靶向肽功能化 QD ( Chl -QD)。
至少两次通过离心过滤通过洗涤除去过量的肽和反应物(默克中号illipore,的Amicon超15,分子量截止,10 ,000道尔顿)用分子级ħ 2 ö加载到离心分离机设置为2360 ×克为10 分钟。
ñ OTES:


确保不要完全洗掉离心过滤器上的缓冲液。在洗涤过程中将Chl- QD保持在溶液中。
得到的Chl -QD 可以储存长达 1 周而不会出现明显聚集。
以 80% 的功率和37 Hz 的频率对Chl- QD 溶液进行超声处理 30 分钟,以分解任何聚集的颗粒。
表征叶绿素使用方法-QD描述如下(参见小号挠度˚F )。
测量流体动力学大小、zeta 电位和荧光。
通过收集 FTIR 光谱确认Chl- QD的形成。


量子点的表征 (MPA-QD)
结果荷兰国际集团的纳米材料的吸光度,尺寸,zeta电位,和(下405nm激发)的荧光发射可以相应地表征。


UV- v是光谱
在 Shimadzu UV-2600 分光光度计上测量 QD 的 UV-vis 吸收光谱(图3A)。
分光光度计设置为扫描范围从 200 到 700 nm,间隔为0.5 nm,积分时间为0. 1 s。
使用分子级 H 2 O 在 1 - ml 石英分光光度计比色皿 (10 毫米× 4 毫米) 中收集背景圆形光谱。
取 10 µl 制备好的MPA-QD 并将其稀释成 990 µl分子级 H 2 O。
将样品放入石英比色皿中,记录 200至700 nm的 UV-vis 吸光度。
记录峰值吸光度值。绿色荧光QD的典型吸收峰为500 - 510nm的(图3A) 。


荧光发射
测量MPA-QD的与PTI荧光QuantaMaster 600 FL uorometer (图3B)。
在分子级 H 2 O 中制备 MPA-QD 溶液 (200 nM ) 。
将 3 ml 溶液填充到石英荧光比色皿中,并将比色皿插入 PTI QuantaMaster 600荧光计的支架中。
荧光计设置为 5 nm 狭缝大小、1 nm 步长和 0.1 s 积分时间。
收集MPA-QD的发射光谱以下激发在405nm处(图3B) 。


纳米材料浓度的测定
根据峰值吸光度和大小,使用以下方程确定溶液的浓度。






(余等,2003)


w ^这里,


c是 M 中的 QD 浓度,


Abs是 500-510 nm 处的峰值吸光度,


L是路径长度(1 厘米),


𝜖是以 L mol -1 cm -1为单位的消光系数,


d是直径10 -9米的QD 。


水动力尺寸
使用 Malvern Zetasizer Nano S (图 2C)(型号 1600)测量流体动力学尺寸。
所述ž etasizer我们对水设定为溶剂,设置一个温度的20 ℃下,材料的折射率的1.350,和材料吸光度的1.000。重复的测量3次。
地方1毫升在4稀释MPA-QD -毫升一次性试管。将比色皿插入z etasizer进行尺寸测量。
测量粒度分布并取基于平均体积e 的粒度分布。汇总表将曲线图中,它可以帮助一起显示,以确定从每个测量颗粒的峰值分布和平均(图3C) 。


红外光谱
为了收集纳米材料的FTIR光谱,取2 -毫升离心管中,添加500μl的3μMMPA-QD,并在1.5毫升悬浮乙醇T A 3:1(V / V)比的混合物。
离心机以最大转速的847 ×克到沉淀MPA-QD成粒料。
取出上清液,使其在通风橱中风干过夜。
离心管底部应形成干燥的小颗粒,用于随后使用 Bruker Alpha II FTIR 分光光度计或等效设备进行 FTIR 光谱分析。


Zeta电位
使用 Malvern Zetasizer (Nano ZS)测量 MPA-QD 的 zeta 电位(图 2D)。
在分子级 H 2 O 中制备 MPA-QD 溶液 (200 nM ) 。
约0.7毫升的溶液加入到填充折叠ç apillary Ž ETA Ç ELL试管(马尔文Panalytics用1个,DTS1070)-毫升注射器和插入反应杯插入Ž etasizer (钠无ZS) 。
所述ž etasizer (纳米ZS)设置水作为溶剂,一个温度的20 ℃下,材料的折射率的1.350,材料吸收的1.000,休克尔近似,并且一个测量重复的5。
记录 zeta 电位并计算平均值和标准偏差(图 3D)。




Figu重新3. MPA-QD与表征叶绿素-QD 。A. 紫外可见吸收光谱。B. 荧光激发和发射光谱。C. 流体动力学尺寸分布。D. Zeta 电位。


MPA-QD 的透射电子显微镜 (TEM)
将一滴(约 2 μl )MPA-QD 加载到 TEM 网格(花边碳支撑膜上的超薄碳膜,400 目,Cu,Ted Pella)。
让液滴风干。
使用 Philips FEI Tecnai 12 显微镜在 120 kV 的加速电压下对准备好的网格上的 MPA-QD 进行成像。
通过使用线段工具测量粒径,使用 ImageJ分析TEM获得的图像(图 4A)。在每个图像中勾勒出 100 多个粒子并计算平均粒径。为了获得纳米颗粒的晶格间距小号使用的线段工具(图小号4A - 4 B),选择一个单个颗粒清楚地示出的晶格条纹,测量距离2个PL之间由(至少)10个点阵间距分离ANES,并通过将测量的距离除以 10 来计算晶格间距。




图4.该ImageJ的线段工具被用于测量粒径和晶格间距在TEM图像


通过对 100 多个粒子的统计分析计算 M PA-QD的平均尺寸并测量晶格间距(图 4B)。


将化学品装入Chl -QD
到负载的化学品我n要叶绿素-QD,使在分子等级H a的0.1mM的甲基紫精或抗坏血酸1毫升原液2 O.
将 100 µl 0.1 mM 甲基紫精或抗坏血酸添加到 1 ml 200 nM Chl -QD 的 TES 缓冲液 pH 7.0 中。
让混合物孵育 30 分钟,并通过离心过滤器(默克微孔,Amicon Ultra 15,截留分子量,10 , 000 Da)用分子级 H 2 O洗涤一次以去除多余的分子。
确定叶绿素装有化学品-QD纳米颗粒浓度,甲基紫精或抗坏血酸(MV-叶绿素-QD或升序-叶绿素-QD,分别地),使用啤酒-朗伯定律,这允许样品混合物的未知浓度的测定对通过测量其吸光度来计算。
使用固定浓度的Chl- QD (200 nM ) 和0到100 µM的甲基紫精或抗坏血酸的梯度浓度,制作一组一式三份的标准。
记录每个标准混合物在 260和265.5 nm之间的最大吸光度值。
使用已知浓度的标准和它们各自的绝对orbance值以产生标准曲线,并确定稀释因子(直线的斜率)。
使用比尔-朗伯定律,确定化学物质的未知浓度加载我n要在200个纳米叶绿素-QDs。
接着,测量并记录初始吸光度的的叶绿素-QD样品不使用化学品和混合化学cargo-叶绿素-QD络合物(从小号TEP 3)洗涤后。
使用的啤酒-兰伯特法律来确定与混合化学物质的浓度的叶绿素-QD样本。
用 200 nM Chl -QD渗入植物中的化学品的最终剂量应该是大约 60 µM 甲基紫精或 60 µM 抗坏血酸在 1 ml TES 缓冲液 pH 7.0 中。


的测量的affinit Y的量子点(MPA-QD和CD-QD)用于化学品(甲基紫精和抗坏血酸)使用等温滴定量热法(ITC)
等温滴定量热法(ITC)被使用进行MicroCal ITC200仪器(GE Healthcare)和MPA-QD和Ç yclodextrin -涂覆的量子点(CD-QD) 。


准备 10 mM TES 缓冲液 pH 7.3。
用 TES 缓冲液 10 mM pH 7.3制备 CD-QD (0.5 μM ) 和甲基紫精(25 mM) 库存溶液。
使用分子级 H 2 O彻底清洁参比池、样品池和进样针。
加载0.3毫升CD-QD在10mM TES缓冲液,pH 7.3溶液到参考和样品细胞小号。
加载甲基紫精溶液进入量热计注射器,并确保其没有气泡存在。
温度:设置仪器如下的25 ℃下,进样体积的2 μ升,21次注射,两个连续的喷射之间的时间间隔的180秒,参考补偿功率的5 μcal /秒。
运行测量以获得热谱图并绘制热功率与时间的关系图。
显示每个化学品注入对应的热功率峰值。
使用 Origin ( MicroCal ) 对热功率峰值进行积分并根据注入的甲基紫精的摩尔量进行归一化以获得焓变,并将焓变与样品池中注入的甲基紫精与 CD-QD 的摩尔比作图以获得结合等温线。
使用一组站点结合模型拟合结合等温线原始数据,以生成最佳拟合曲线和 CD-QD 与甲基紫精相互作用的焓变化(图5A-5 B)。
记录热力学参数的甲基紫精结合CD-QD,包括CD-QD(n)的缔合常数的结合位点的(K数一个中,M -1 ),解离常数(ķ d ,M),焓变( ΔH, cal mol -1 ) 和熵变 (ΔS, cal mol -1 K -1 ) (图5A-5 B ) 。
使用相同的方法分别确定甲基紫精和 MPA-QD、抗坏血酸和 CD-QD以及抗坏血酸和 MPA-QD之间结合的热力学参数。




图5的等温滴定量热法环糊精(ITC)-涂覆的量子点小号化学货物(CD-QD)。A.在交互CD-QD的焓的变化荷兰国际集团用甲基紫精和抗坏血酸。B. Ť可以从ITC外推有源冰箱参数,包括焓变(ΔH,CAL摩尔-1 ),化学计量(n),并结合亲和力(的Kd )。


测定溶液中纳米颗粒结合和未结合的化学物质
使用热力学参数,例如结合位点(N),解离常数(数量ķ d ),以及从ITC数据分析获取的化学配位体的浓度,以确定结合的和未结合的部分小号在环糊精的化学品-涂覆纳米粒子.
使用以下等式来确定有界和无界分数s:






其中和 分别是溶液中结合和未结合化学物质的浓度;是 Q 上结合位点的数量是初始 QD 浓度;和是QD之间的解离常数的化学品。 



纳米粒子和化学物质输送到植物叶子中
在 10 mM TES 缓冲液 pH 7.0 中将Chl -QD稀释至 200 nM (0.17 mg ml -1 )。
加载叶绿素-QDs用60μM甲基紫精或抗坏血酸(参见小号挠度N)。
取1 -毫升NORM-JECT无针注射器和填充用100 μ升叶绿素装载有甲基紫精或抗坏血酸-QD溶液。
轻轻地将注射器的尖端压在叶子的背面,并将食指靠在正面以支撑(图 6A)。
轻轻按下柱塞,慢慢灌注每片植物叶子。用Kimwipes轻轻去除任何多余的溶液(图 6B)。




图6. 纳米颗粒和化学物质输送到植物叶子中。纳米材料使用无针注射器通过叶层输送。A.轻轻地将注射器底部压在叶的背面,并用食指抵住正面。B.用纳米颗粒浸润后,随着颗粒溶液填充叶肉空间(虚线圆圈),叶子看起来更暗。


将植物放在实验室工作台上,孵育 15 分钟,然后再将植物放回生长室。


共聚焦荧光显微镜样品制备
取豌豆大小d量的 Carolina 观察凝胶,在显微镜载玻片(Corning 2984-75x25)上制作薄膜(约 1 毫米厚),并使用软木边框(直径 8 毫米)创建一个腔室以去除薄膜中心的圆形截面(图 7A)。
从带有软木边框(直径 6 毫米)的处理过的叶子上取下叶片,并将叶盘在 0.5 毫升 10 µM DHE(Thermo Fisher Scientific,美国)中在 10 mM TES 缓冲液 pH 7.0 中孵育30 分钟。
浸入DHE -在孵育的叶盘全氟萘烷(ACROS有机物25克,90%的混合物的顺式和反式,CAS 306-94-5) (图7A- 7 B)填充在凝胶室ED和盖玻片密封它. 该样品已准备好用于共聚焦荧光显微镜成像。




图7.制备一个用于叶样品中的纳米颗粒的共焦成像显微载玻片上。A. 叶盘样品放置在凝胶室中并安装在载玻片上进行共聚焦成像。B.含有叶盘的凝胶室充满全氟萘烷。


共聚焦荧光显微镜
将叶子样品加载到 Leica 激光扫描共聚焦显微镜 TCS SP5(Leica Microsystems,德国)上。
设置显微镜如下:40 ×湿物镜(HCX PL APO CS 40.0 × 1.10 WATER UV,Leica Microsystems,德国);用于 QD 的 405 nm 激光激发;DHE 为 514 nm;z-stack 切片厚度为2 µm;线平均值为4;PMT d etection范围的500 -对于量子点550纳米,580 - 615nm处为DHE和720 -叶绿体自发荧光为780nm。
扫描以定位感兴趣的平叶表面区域并对样本进行成像。
分别收集QD和DHE信号,以避免重叠之间的的激励的DHE染料和QD的发射检测范围。
为每个植物成像三个或更多叶盘,并为每个叶盘拍摄两个不同区域的图像以进行共聚焦分析(图 8)。




图 8. 植物叶片中纳米粒子的共聚焦图像。来自用 10 mM TES 缓冲液 pH 7.0、MPA-QD 和Chl -QD 浸润的叶子的叶肉细胞的共聚焦图像。图像显示的重叠程度的的与叶绿体自发荧光QD荧光信号。比例尺:50 µ m。


通过共聚焦显微镜和 ICP-MS 进行纳米颗粒检测的叶绿体分离
渗透的植物的叶组织用约100μl的500 nM的叶绿素-QD在TES缓冲液10mM pH为7.3。
允许在植物叶片孵育在室温下6小时。
收集大约8克叶组织从3 -周-旧拟南芥用缓冲液对照或处理的植物的叶绿素-QD从5 -每个处理6株植物。在研磨机或浸渍机中用 1 ×冷冻蔗糖缓冲液浸渍叶组织(配方 1 )。
研磨通过脉冲的组织(3 - 5周4个脉冲)-秒的间隔,直至叶组织均化成浆料(图9A)。
应变的通过4层粗棉布浆料注入冰上的玻璃烧杯中。放置的过滤器编通流成50 -与毫升离心管中并离心两次1 ×蔗糖缓冲液中在3082 ×克10分钟(图9B)。
除去所述上清液每个时间和再充填以新的蔗糖缓冲液中。
离心管底部应形成颗粒(图 9B)。




图 9. 叶绿体分离示意图。叶绿体小号从3分离-周-老拟南芥植物。一个。在蔗糖缓冲液中装有浸渍叶匀浆的离心管。B. 完整叶绿体颗粒。所述的C.确认共定位叶绿素在分离的叶绿体-QDs š通过共聚焦显微镜。该QD信号在绿色和可视化的洋红色叶绿体自发荧光。比例尺:20 µ m。D.检测和孔定量吨QD元件(Cd和的通货膨胀碲)中通过ICP-MS分析叶绿体检测。


以下叶绿体隔离,叶绿体小刺样品放置在载玻片上,以检测提取chloroplas内的量子点荧光使用共聚焦显微镜TS(见节小号用于设定)(图9C - 9 d)。
除去的上清液(蔗糖缓冲液和损伤的叶绿体小号从管)。允许沉淀叶绿体小号空气干燥在一个通风橱中超过24小时。将形成一个小的绿色粘性颗粒。
一旦粒料干燥,放置管在-20 ℃下冷冻1个小时直到沉淀凝固以允许容易祛瘀人从离心管和prepar通货膨胀用于ICP-MS分析。


电感耦合等离子体质谱 (ICP-MS)
叶绿体分离后,将样品颗粒(~0.1 克)在空气中干燥 48 小时。
放置风干样品中50 -毫升聚丙烯消化管并用5%HNO的溶液消化3 /1%的HCl / 1%H 2 ö 2 V / V。消化的在1ml HNO样品3 /0.4毫升的HCl,同时使用啊在115℃下加热5分钟,EA吨块(的DigiPREP系统; SCP科学,普兰,NY)。添加 0.4 ml H 2 O 2并再孵育 10 分钟。
稀释溶液,并分析通过ICP-MS(安捷伦7700x ICP-MS)对孔定量样品泰特Cd和碲的内容。以μg g -1为单位报告单个元素浓度,如图 8D(元素质量为μg /g 干叶绿体)。


数据分析


斐济共聚焦荧光显微镜图像分析 ( ImageJ)


要分析图像,请在斐济 (ImageJ) 软件中打开图像(图 10A- 10 C)。
使用线条工具,在要均匀分析的图像集上绘制六条样线。保存该行利息(ROI)管理的区域(图10A)。
测量六个 ROI 线部分中每一个的 QD 和 DHE 荧光和叶绿体自发荧光通道的相应荧光强度分布。使用斐济软件,选择分析 > 绘图配置文件(图 10B)。
计数之间的重叠峰的数目的QD和DHE通道超过叶绿体荧光,并计算所述共定位百分数(图10B - 10 C)。




图10. 目标纳米材料 ( Ch l -QD) 和叶肉细胞中叶绿体之间共定位分析的逐步示意图。A.利用共聚焦荧光显微镜图像分析的斐济(ImageJ的)软件。B的通过共聚焦显微镜收集标准化荧光强度分布图解ÿ和斐济(ImageJ的)进行处理。C. 计算重叠荧光强度峰的共定位百分比。


笔记


如果空气进入,反应溶液会由蓝色变为黑色,从而氧化催化剂,导致量子点晶体的形成效率低下。
随着反应的进行,溶液 B 应呈现颜色变化,从黑蓝色变为粉紫色(图 2B)。
EDC 具有吸湿性,会在分子级 H 2 O 中降解;确保 EDC 是新鲜制备的。
QD溶液可通过20被过滤-纳米过滤器,以除去结块。
量子点倾向于在 pH 值低于 6.5 时聚集。
确保不要从 10 K 过滤器列中完全冲洗掉缓冲液。在洗涤过程中将 APBA-QD 保持在溶液中。


[R ecipes


1 ×冷冻蔗糖缓冲液(pH 7.3)
28 mM Na 2 HPO 4


22 mM KH 2 PO 4


2.5 毫米氯化镁2


400 mM 蔗糖


10毫米氯化钾


酸碱度 7.3


致谢


这种材料是基于由下格兰特1817363号美国国家科学基金会,以支持JPG工作的学生CC和HT和P ostdoc PH被下批准号CHE-2001611,美国国家科学基金会中心可持续纳米技术国家科学基金会的支持。CSN 是化学创新计划中心的一部分。该协议基于我们之前在Nature Communications上发表的文章(Santana等人,2020 年)。


利益争夺


有标题为“组合物和叶绿体方法遗传和生化工程植物未决的美国专利小号”(16 / 218.429)基于这方面的工作。这种生物的协议,所有原稿的作者,JPG,是和PH ,在这个专利发明,包括格雷戈里米纽柯克(大学加州,滨江)和中国香港·吴(华中农业大学)。在这个协议中所概述的特定方面在所述专利申请中,包括用于方法的纳米材料的使用合理设计的引导肽的叶绿体靶向递送。


参考


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引用:Santana, I., Hu, P., Jeon, S., Castillo, C., Tu, H. and Giraldo, J. P. (2021). Peptide-mediated Targeting of Nanoparticles with Chemical Cargoes to Chloroplasts in Arabidopsis Plants. Bio-protocol 11(12): e4060. DOI: 10.21769/BioProtoc.4060.
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