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Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System
将化学稳定的疏水siRNA加载到细胞外囊泡中用于治疗中枢神经系统疾病   

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Molecular Therapy
Oct 2016

Abstract

Efficient delivery of oligonucleotide therapeutics, i.e., siRNAs, to the central nervous system represents a significant barrier to their clinical advancement for the treatment of neurological disorders. Small, endogenous extracellular vesicles were shown to be able to transport lipids, proteins and RNA between cells, including neurons. This natural trafficking ability gives extracellular vesicles the potential to be used as delivery vehicles for oligonucleotides, i.e., siRNAs. However, robust and scalable methods for loading of extracellular vesicles with oligonucleotide cargo are lacking. We describe a detailed protocol for the loading of hydrophobically modified siRNAs into extracellular vesicles upon simple co-incubation. We detail methods of the workflow from purification of extracellular vesicles to data analysis. This method may advance extracellular vesicles-based therapies for the treatment of a broad range of neurological disorders.

Keywords: RNA interference (RNA干扰), Hydrophobically modified siRNA (疏水改性siRNA), Extracellular vesicles (细胞外囊泡), Therapy (疗法), Neurological disorder (神经系统障碍)

Background

siRNAs are one type of oligonucleotide therapeutics, a new class of drugs directly targeting messenger RNAs (mRNAs) to prevent the expression of proteins leading to disease phenotypes. The therapeutic application of siRNAs is extremely promising as siRNAs can be designed to target any gene, including genes not ‘druggable’ with small molecules or protein-based therapies. The progress made in the chemistry of oligonucleotide therapeutics enables the design of fully stabilized hydrophobically modified siRNAs (hsiRNAs, modified with 2’-O-Methyl or 2’-Fluoro as well as phosphorothioates and sense strand covalently conjugated to cholesterol), which promote cellular self-internalization of hsiRNAs and maintain an ability to be efficiently loaded into the RNA-induced silencing complex (RISC) (Byrne et al., 2013; Khvorova and Watts, 2017). A cholesterol conjugate, linked to the 3’ end of the passenger strand, is essential for rapid cellular membrane association (Byrne et al., 2013; Alterman et al., 2015). The single-stranded phosphorothioate tail promotes cellular internalization (Geary et al., 2015). We recently demonstrated that hsiRNAs bind cellular membranes within seconds after treatment, enter cells and promote potent gene silencing in vitro (Byrne et al., 2013; Alterman et al., 2015; Ly et al., 2017). However, upon local bolus injection in vivo in mouse brain, hsiRNA spread and efficacy are limited to the region surrounding the site of administration (Alterman et al., 2015). Whereas hsiRNAs remain therapeutically promising due to potent and specific gene silencing, their delivery to the brain hampers their advancement for the treatment of diseases in the central nervous system.

Endogenously produced extracellular vesicles mediate intercellular transfer of lipids, proteins, and RNAs between cells over short and long distances, thus playing a crucial role in health and disease (Distler et al., 2005; Muralidharan-Chari et al., 2010). The ability of extracellular vesicles to carry functional RNAs has attracted considerable interest to their use as novel vehicles to transport and deliver RNA-based therapeutics. The cargo includes siRNAs or other oligonucleotide therapeutics (Tetta et al., 2013). Strategies used to load RNA-based therapeutics into extracellular vesicles include electroporation (Alvarez-Erviti et al., 2011; Ohno et al., 2013) or overexpression of miRNAs in extracellular vesicle-producing cells (Kosaka et al., 2012; Ohno et al., 2013; Mizrak et al., 2013). Though both strategies have been able to promote the transfer of siRNA-loaded extracellular vesicles into target cells and the silencing of the target gene, they cannot be controlled or scaled up for clinical-stage manufacturing (Kooijmans et al., 2013). Moreover, electroporation compromises the integrity of extracellular vesicles (Kooijmans et al., 2013). We demonstrated the efficient loading of extracellular vesicles with hsiRNAs without modifying the vesicle size distribution, concentration and integrity. hsiRNA-loaded extracellular vesicles were shown to induce gene silencing of the target gene, huntingtin mRNA, in vitro in mouse primary neurons, and in vivo in mouse brain (Didiot et al., 2016).

Here, we describe a method exploring the ability of hsiRNAs to bind membranes to promote their loading into extracellular vesicles. The co-incubation of hsiRNAs with extracellular vesicles purified from cell culture conditioned medium, promotes loading into extracellular vesicles. We provide details on our methods for extracellular vesicles purification, loading of extracellular vesicles with hsiRNAs, size, charge and integrity characterization of hsiRNA-loaded extracellular vesicles, as well as in vivo testing of hsiRNAs-loaded extracellular vesicles in mouse brain, and data analysis. This technology may promote the loading of several other classes of oligonucleotide therapeutics (i.e., antisense, splice-switching oligonucleotides, sterically blocking oligonucleotides, aptamers and others) to extracellular vesicles, thus providing a significant leap forward to advance multiple classes of oligonucleotide therapeutics for the treatment of diseases in the brain. Subsequently, exploiting the natural properties of extracellular vesicles to functionally transport small RNAs (Valadi et al., 2007; Pegtel et al., 2010; Wang et al., 2010) offers a strategy for improving the in vivo distribution of and cellular uptake of oligonucleotide therapeutics (Zomer et al., 2010; El Andaloussi et al., 2013; Kooijmans et al., 2012; Lasser, 2012; Lee et al., 2012; Pan et al., 2012; Marcus and Leonard, 2013; Nazarenko et al., 2013; Didiot et al., 2016).

Materials and Reagents

  1. Tips (from 0.2 µl to 1,000 µl) (VWR)
  2. Paper towel
  3. Serological pipettes individually wrapped (from 5 ml to 50 ml) (Olympus)
  4. 0.22-μm filter-sterilization system (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 567-0020 )
  5. Tissue culture treated multilayer flask–T500 cm2 triple flask (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 132867 )
  6. 50 ml conical centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339652 )
  7. Vacuum-connected Pasteur pipette
  8. 1.7 ml microcentrifuge tubes (Genesee Scientific, catalog number: 22-282 )
  9. Aluminum foil
  10. UV-transparent, flat-bottom 96-well plate (Corning, catalog number: 3635 )
  11. 1 ml syringe (BD, catalog number: 309659 )
  12. Parafilm (Bemis, Parafilm M®)
  13. Whatman No. 1 filter paper (GE Healthcare, Whatman, catalog number: 10010155 )
  14. 30 G ½ needle
  15. Electron microscopy grids (Electron Microscopy Sciences, catalog number: FCF2010-Ni ) and clean forceps to manipulate the grid
  16. Grid storage box (Electron Microscopy Sciences, catalog number: 71156 )
  17. Mouse (FVB/NJ) (THE JACKSON LABORATORY, catalog number: 001800 )
  18. 200-proof ethanol (Decon Labs, catalog number: 2805M )
  19. Fetal bovine serum (FBS) (Mediatech, catalog number: 35-010-CV )
  20. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190250 )
  21. Protease inhibitor cocktail stock solution (Sigma-Aldrich, catalog number: P8340 )
  22. Cy3-labeled or biotinylated cholesterol-conjugated hsiRNAs (produced in-house)
  23. Cy3-OO-PNA strands, fully complementary to the hsiRNA guide strand (20 nucleotides long) (PNA Bio)
  24. Peptide-nucleic acid (PNA) hybridization assay (developed by Axolabs, Kulmbach, Germany)
  25. RIPA buffer (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 89900 )
  26. Proteinase K (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25530049 )
  27. Glycine (Sigma-Aldrich, catalog number: G7126 )
  28. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153 )
  29. Saponin (Sigma-Aldrich, catalog number: S1252 )
    Note: This product has been discontinued.
  30. Glutaraldehyde (Polysciences, catalog number: 01909-10 )
  31. Avertin (Sigma-Aldrich, catalog number: T48402 )
  32. QuantiGene 2.0 Assay Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: QS0011 )
  33. Quantigene 2.0 Probesets (Varies by gene)
  34. Dulbecco’s modified Eagle medium (DMEM)
  35. MSC culture media (ATCC, catalog number: PCS-500-041 )
  36. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  37. Tris base (TRIZMA) (Sigma-Aldrich, catalog number: T6066 )
  38. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758 )
  39. Ethylenediaminetetraacetate acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  40. Sodium hydroxide (NaOH) (~50 ml of NaOH) (Sigma-Aldrich, catalog number: 72068 )
  41. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  42. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  43. Sodium phosphate monobasic monohydrate (NaH2PO4·H2O)
  44. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S3139 )
  45. Acetonitrile (50% acetonitrile solution, diluted in distilled H2O) (Fisher Scientific, catalog number: A998-4 )
  46. Sodium perchlorate monohydrate (NaClO4·xH2O) (Fisher Scientific, catalog number: S490-500 )
  47. Methyl cellulose (Sigma-Aldrich, catalog number: M6385 )
  48. PFA powder (Sigma-Aldrich, catalog number: P6148 )
  49. Uranyl acetate (Electron Microscopy Sciences, catalog number: 22400 )
  50. Oxalic acid
  51. Tris-HCl (pH 8.5) (Fisher Scientific, catalog number: BP153 )
  52. Ammonium hydroxide (NH4OH) (Fisher Scientific, catalog number: A669-212 )
  53. Cell culture medium (see Recipes)
  54. 1 M sucrose (see Recipes)
  55. 1 M Tris-HCl (see Recipes)
  56. 0.5 M EDTA (see Recipes)
  57. 3 M KCl (see Recipes)
  58. 10% SDS (see Recipes)
  59. 0.1 M sodium phosphate buffer (see Recipes)
  60. HPLC buffer A (see Recipes)
  61. HPLC buffer B (see Recipes)
  62. Glutaraldehyde, 1% (v/v) (see Recipes)
  63. Methyl cellulose, 2% (w/v) (see Recipes)
  64. Paraformaldehyde (PFA), 2% and 4% (w/v) (see Recipes)
  65. Uranyl acetate (4% w/v), pH 4 (see Recipes)
  66. Uranyl-oxalate, pH 7 (see Recipes)
  67. Methyl cellulose-UA, pH 4 (see Recipes)

Equipment

  1. Tissue culture hood
  2. Soft brush
  3. Ultracentrifuge 70 ml polycarbonate bottles (Beckman Coulter, catalog number: 355655 )
  4. Fixed-angle Ti45 ultracentrifuge rotor (Beckman Coulter, model: Type 45 Ti , catalog number: 339160)
  5. Refrigerated ultracentrifuge (Beckman Coulter, model: Optima XE )
  6. Refrigerated benchtop centrifuge (Beckman Coulter, model: Allegra® X-15R )
  7. Micropipettes from 0.5 µl to 1 ml (Labnet International, model: BioPetteTM Plus )
  8. Fixed-angle TLA-110 rotor (Beckman Coulter, model: TLA-110 , catalog number: 366735)
  9. 1.5 ml microcentrifuge adapters (Beckman Coulter, catalog number: 360951 )
  10. Refrigerated benchtop ultracentrifuge (Beckman Coulter, model: OptimaTM MAX-TL )
  11. Orbital thermo-shaker for 1.5 ml microtube (Grant Instruments, model: PHMT series )
  12. Plate reader spectrophotometer (Tecan Trading, model: Infinite® M1000 Pro )
  13. Stereotactic frame (KOPF INSTRUMENTS, model: Model 963 )
  14. HPLC system with fluorescent detector with autosampler (Agilent Technologies, model: HPLC 1100 series )
  15. Hamilton syringe (Hamilton, model: Gastight #1002 )
  16. Extracellular vesicles size distribution and concentration reader (Malvern Instruments, model: NanoSight NS300 )
  17. Extracellular vesicles charge reader (Malvern Instruments, model: Zetasizer Nano NS )
  18. Glass micro-electrophoresis cuvette Dip Cell kit (Malvern Instruments, catalog number: ZEN1002 )
  19. Transmission electron microscope (JEOL, model: JEM-1011 )
    Note: This product has been discontinued.
  20. ALZET® osmotic pumps (DURECT, ALZET, catalog numbers: 1003D and 1007D )
  21. Water bath at 37 °C
  22. Microscissors (Fine Science Tools, catalog numbers: 14060-10 ; 14002-12 )
  23. Set of two forceps (Fine Science Tools, catalog number: 11251-30 )
  24. Vibratome (Leica Biosystems, model: Leica VT1000 S)
  25. Autoplate washer (BioTek Instruments, model: ELx405 )
  26. Tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HeracellTM 150i )
  27. Pipet-aid (Drummond Scientific, model: Portable Pipet-Aid® XP )
  28. Tissue culture phase-contrast inverted microscope (Motic, model: AE2000 )
  29. Anion exchange column (Thermo Fisher Scientific, Thermo ScientificTM, model: DNAPacTM PA100 )
  30. Heat block (Fisher Scientific, model: IsotempTM 2050FS )
  31. µPlate carrier (Beckman Coulter, model: SX4750 )
  32. -86 °C freezer (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM 900 Series )
  33. Biological safety cabinet connected to vacuum (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series Class II , Type A2)

Software

  1. Nanoparticles Tracking Analysis (NTA) software
  2. Microsoft Office Excel (Microsoft Pack Office)
  3. GraphPad Prism 6 software (GraphPad Software, Inc.)

Procedure

  1. Extracellular vesicles production and purification
    Notes:
    1. The purification of extracellular vesicles is shown in Figure 1.


      Figure 1. Flowchart of extracellular vesicle production: Extracellular vesicles (microvesicles and exosomes) are purified from conditioned media via differential ultracentrifugation

    2. All steps that involve opening the ultracentrifuge bottles must be performed in a tissue culture hood. To sterilize the bottles, wash the bottles and their lids with soap using a soft brush and rinse carefully with sterile water to remove any trace of soap. In a tissue culture hood, cleaned bottles and their lids are sterilized in a 70% ethanol bath and dried on a paper towel under UV light for a final sterilization. Polycarbonate bottles are sensitive to ethanol and UV light, so avoid leaving the bottles in 70% ethanol for longer than 2 min and UV for longer than 30 min.
    3. The extracellular vesicle pellets are transparent brown and very small, which make them difficult to detect. Using a permanent marker, label the side of each 70 ml ultracentrifugation bottle to orient the bottles in the rotor and to indicate where to look for the extracellular vesicle pellet. All 70 ml bottles should be full to the bottle shoulder (~65 ml) for high-speed centrifugation. Otherwise the bottle will collapse and damage the rotor. If not filled to the shoulder, complete the volume with PBS.

    1. Preparing extracellular vesicle-depleted medium and cell culture
      Note: Extracellular vesicles are naturally present in body fluid, including the fetal bovine serum (FBS) usually used to supplement cell culture medium. Thus, prior any use, the cell culture medium, from which the extracellular vesicles are purified, is depleted of the serum-extracellular vesicles by overnight ultracentrifugation to minimize the contamination.
      1. For each cell line, prepare the recommended medium supplemented with 40% (v/v) FBS. Depleted medium supplemented with 40% FBS allows preparation of a large volume of extracellular vesicle-free culture medium in a single round of centrifugation.
      2. Transfer 65 ml of medium in 70 ml bottles using a 50 ml serological pipette.
      3. Place the bottles, with the label facing up, in a pre-cooled Type 45 Ti rotor (stored at 4 °C) and pre-cool the ultracentrifuge to 4 °C.
      4. Ultracentrifuge for at least 18 h at 100,000 x g, 4 °C.
      5. To filter sterilize the supernatant, transfer the contents of each bottle into a vacuum-connected 0.22 μm filter-sterile rapid-flow disposable unit using a 50 ml serological pipette. At the bottom of the bottle, leave 2-3 ml of medium containing higher concentration of brown sediments. The pellet is brown and very sticky and will not detach from the edge of the bottle (see Note 1).
      6. Store the sterilized extracellular vesicle-depleted medium, diluted or not, up to 4 weeks at 4 °C.
      7. Prior to cell culture, dilute the extracellular vesicle-depleted medium with additional medium to obtain medium with the recommended concentration of FBS (usually 10% FBS) required for the cell line. If necessary, supplement the medium with any additional nutrients and/or antibiotics.
    2. Production and purification of extracellular vesicles
      Notes:
      1. Extracellular vesicles are purified from conditioned medium by a method adapted from Thery et al. (2006) combining sequential centrifugation and filtration steps. All extracellular vesicles quality controls were performed following the experimental requirements recommended by the International Society of Extracellular Vesicles (Lotvall et al., 2014; Thery et al., 2006). 
      2. In vivo experiments need a significant concentration of extracellular vesicles for efficient silencing. To collect large quantities of extracellular vesicles, we purify extracellular vesicles from conditioned medium from adherent cells in culture. The yield of purified extracellular vesicles correlates with the increase of medium volume. Thus, to produce large volume of conditioned medium and to scale up the concentration of extracellular vesicles, we use T500 cm2 triple flasks.

      1. In multilayer flasks, grow adherent cells of interest in the regular recommended medium until they reach 70% to 80% confluency. We observed that 60 ml is the minimal volume of medium that can be used in T500 cm2 triple flask.
      2. Pour the culture medium in a liquid trash, as serological pipette cannot be used to remove liquid from multilayer flasks. Using a 50 ml serological pipette, replace the medium with a similar volume of recommended EV-depleted medium.
      3. Incubate the cells for 48 h at 37 °C, 5% CO2 and 100% humidity.
      4. Collect the medium and proceed to extracellular vesicle purification as followed. If the cells are not 100% confluent, replace the collected medium with fresh extracellular vesicle-depleted medium, incubate for 48 h and repeat the extracellular vesicle purification as followed.
      5. Harvest the culture medium containing extracellular vesicles in 50 ml centrifuge conical tubes.
      6. Centrifuge at 300 x g for 10 min at room temperature to pellet and eliminate the floating cells and large debris.
      7. Transfer the supernatant into new 50 ml centrifuge conical tubes using 50 ml serological pipette. Leave 2-3 ml of medium in the bottom of the tube to avoid disturbing the pellet. Discard the pellet.
      8. Centrifuge the supernatant at 10,000 x g, 4 °C for 30 min to isolate large vesicles or microvesicles.
      9. Pour and filter sterilize the supernatant through a vacuum-connected sterile 0.22 μm filter unit to further eliminate larger vesicles.
        Note: The 10,000 g pellet, containing microvesicles (300 nm to 1 µm), is white and sticky and can be used for additional experiments. If microvesicles are needed, completely remove the remaining medium forming a drop at the bottom of the bottle using a vacuum-connected Pasteur pipette. Using a micropipette, suspend the pellet in 1 ml of PBS. The pellet should detach easily with the flow of PBS. Centrifuge the collected microvesicles at 10,000 x g, 4 °C for 30 min, as an additional wash step, and suspend the pellet in 100 µl of PBS.
      10. Using a 50 ml serological pipette, transfer the filtered medium in 70 ml polycarbonate bottles.
        Note: Mark the bottles with a permanent marker and place the bottles in the centrifuge with the mark facing away from the center. This will help the localization of the pellet later.
      11. Ultracentrifuge for at least 70 min at 100,000 x g, 4 °C in a pre-cooled Type 45 Ti rotor.
      12. Aspirate completely the supernatant from all bottles using a vacuum-connected Pasteur pipette.
        Note: Remaining medium will fall down the wall of the bottle and form a final drop of supernatant that has to be aspirated by vacuum-connected Pasteur pipette.
      13. Detect the extracellular vesicles pellet on the bottom of the bottle. The pellet is brownish transparent in color and approximately 5 mm in diameter. 
        Note: The pellet will be localized to the marked side of the bottle.
      14. To concentrate the extracellular vesicles, suspend all the pellets in the same 1 ml PBS using a micropipette. With the tip, scrape the bottle in and around the pellet to recover a maximum of extracellular vesicles.
      15. Transfer the suspended pellet into 1.5 ml microcentrifuge tubes. Carefully recover the remaining drops of extracellular vesicle-containing PBS forming at the bottom of each bottle.
      16. Place the 1.5 ml tubes in the tube holders in a pre-cooled TLA-110 rotor with the tube-cap orientated to find the pellet.
      17. Centrifuge for at least 70 min at 100,000 x g, 4 °C in a tabletop ultracentrifuge.
      18. Resuspend the pellet in 100 µl PBS, 1x protease inhibitor cocktail and 10 mM sucrose for further experiments. Store at 4 °C for a short-term use and -80 °C for long term storage. Avoid repeated freezing and thawing (see Notes 2 and 3).
        Note: For proteomic or lipidomic analysis, store the pellet as is at -80 °C.

  2. Preparation and characterization of hsiRNA-loaded extracellular vesicles
    1. Loading of extracellular vesicles with hsiRNAs.
      The loading of extracellular vesicles with hsiRNA is performed as shown in Figure 2.


      Figure 2. Flowchart of exosome loading procedure: co-incubation of Cy3-hsiRNA and extracellular vesicles results in Cy3-hsiRNA-loaded extracellular vesicles that pellet by ultracentrifugation

      1. Into a 1.5 ml microcentrifuge tube, mix required concentration of hsiRNAs (3,000-5,000 molecules of hsiRNA per vesicle) and extracellular vesicles resuspended in 100 µl PBS by gently pipetting up and down with micropipette (see Notes 4 and 5).
        Note: It is advised to determine the concentration of the extracellular vesicles first (see step B4). If the concentration of hsiRNA is above 100 µM, the volume of hsiRNA solution needed to reach the 3,000-5,000 hsiRNA per vesicle ratio will be negligible (less than 10 µl).
      2. Incubate the mix in a thermo-shaker for at least 60 min at 37 °C and shake at 500 rpm. If the oligonucleotide is fluorescently labeled, protect the tube from light using aluminum foil.
      3. Place the 1.5 ml tubes in the tube holder in a pre-cooled TLA-110 rotor with the tube-cap oriented to find the pellet.
      4. Centrifuge for at least 70 min at 100,000 x g, 4 °C in a tabletop ultracentrifuge. Unloaded hsiRNAs remain in the supernatant and hsiRNA-loaded extracellular vesicles form a pellet.
      5. Remove and save the supernatant into a 1.5 ml centrifugation for further measurement. The quantification of hsiRNA remaining in the supernatant can be used to estimate the efficiency of loading (see steps B2 and B3).
      6. Resuspend the pellet in required volume of medium or artificial CSF for cell based assays, or for in vivo brain injection, respectively. Resuspended hsiRNA-loaded extracellular vesicles are immediately used for further experiment without being stored. Alternatively extracellular vesicles can be stored at -80 °C in 0.1 M sucrose. We observed that storage did not affect the number or activity of vesicles for up to 6 months.
      7. Save an aliquot of hsiRNA-loaded extracellular vesicles for quantification of hsiRNAs and extracellular vesicles concentration.
    2. Determination of hsiRNA concentration by direct quantification of fluorescently labeled hsiRNAs.
      Two methods are used to estimate the loading efficiency of hsiRNAs in extracellular vesicles (Figure 1) (see Notes 6 and 7). First, directly measuring the Cy3 fluorescence in the hsiRNA-extracellular vesicles pellet. We observed that extracellular vesicles pellets may display autofluorescent signal, thus may confound measurement of Cy3 fluorescence coming from loaded hsiRNA. Therefore we recommend a second strategy: calculating the difference between the total amount of Cy3-hsiRNAs originally added to the sample and the amount of Cy3-hsiRNAs remaining in the supernatant (unloaded) after incubation with extracellular vesicles and ultracentrifugation. To determine an accurate concentration of hsiRNAs, prepare negative controls (PBS only with no hsiRNA) and a hsiRNAs calibration curve into the well of a UV-transparent, flat-bottom 96-well plate. The minimal volume of sample usually used in 96-well plate is 50 µl. For the calibration curve make a 1:2 dilution series of hsiRNA, spanning amounts comparable to what was loaded into the extracellular vesicles.
      1. For the negative control, dispense 50 µl of PBS (blank) in the first well of the plate (well #A1).
      2. In the following well (well #A2), prepare a Cy3 fluorescent positive control stock solution. Dilute a known concentration of Cy3 labeled hsiRNAs in 100 µl PBS. Use twice the amount of hsiRNA than what was used in the hsiRNA-extracellular vesicles sample. Dilute 1:2 in PBS to get an 8-points calibration curve.
      3. Dispense 50 µl of PBS into three following wells (wells #A3, A4 and A5). To make a series dilution, transfer 50 µl of the stock solution (well #A2) into the next well (well #A3) containing 50 µl of PBS and mix gently with a micropipette.
      4. Using a fresh micropipette tip, transfer 50 µl (from well #A3) into the next well (well #A4) and proceed until the last well of the calibration curve.
      5. Transfer 50 µl of supernatant (containing unloaded hsiRNAs) and pellet (containing hsiRNA-extracellular vesicles) of each sample into the plate.
      6. Measure the Cy3 fluorescence of each sample using a spectrophotometer plate-reader at 547 nm excitation and 570 nm emission wavelengths.
      7. Using the calibration curve, calculate the Cy3 fluorescence in both the pellet and supernatant samples.
      8. The Cy3 fluorescence of the pellet sample gives a direct estimate of hsiRNAs concentration in the hsiRNA-extracellular vesicles sample.
      9. As a second method to estimate the concentration of hsiRNAs in the pellet, calculate the difference between the total amount Cy3-hsiRNAs originally added to the sample and the amount of Cy3 fluorescence remaining in the supernatant.
    3. Determination of hsiRNA concentration by peptide-nucleic acid hybridization assay
      To confirm the concentration of hsiRNAs obtained by measuring the Cy3 fluorescence using a spectrophotometer, we determine the hsiRNAs guide strand concentration (not Cy3-labeled) in samples using a peptide-nucleic acid (PNA) hybridization assay (developed by Axolabs, Kulmbach, Germany) (Roehl et al., 2011). PNAs are single strand analogues of DNA with the sugar-phosphate backbone replaced by a polyamide backbone. Therefore, free of any charge, PNAs bind to their complementary strand with a very high affinity, forming double-stranded DNA-RNA structures. PNA assay is a more accurate but also more laborious measurement of hsiRNA concentration in the pellet than spectrophotometric fluorescence measurement. Furthermore, the samples used for PNA assay is lysed, and cannot be used in downstream experiments.
      1. Dilute hsiRNA-extracellular vesicles sample in 100 µl of RIPA buffer containing 2 mg/ml Proteinase K in a 1.5 ml microcentrifuge tube.
      2. Sonicate for 15 min to lyse the vesicles at room temperature.
      3. To prepare a calibration curve, add a known amount of hsiRNA duplex into lysed extracellular vesicles sample.
      4. Add 20 µl of 3 M KCl and mix gently with a micropipette to precipitate SDS (component of RIPA buffer).
      5. Centrifuge for 15 min at 5,000 x g at room temperature to pellet insoluable membrane matieral and precipitated SDS.
      6. Transfer the cleared supernatant containing the hsiRNAs guide strands in a new 1.5 ml microcentrifuge tube.
      7. Dilute the supernatant to obtain a concentration below 5 pmol hsiRNA/100 µl sample.
      8. Add 5 pmol (or an amount exceeding the amount of hsiRNA loaded into extracellular vesicles) of Cy3-labeled PNA strands fully complementary to guide strand of hsiRNA and mix gently with the micropipette.
      9. To anneal the PNA strands to the hsiRNA guide strands, incubate for 15 min at 95 °C (to melt hsiRNA duplexes) followed by an incubation at 50 °C (to anneal PNA to hsiRNA guide strands, replacing sense strands) for 15 min and cool to room temperature.
      10. Transfer the samples in a 96-well plate or in vials compatible with your HPLC system.
      11. Inject the samples containing PNA-hsiRNA guide strand hybrids into HPLC anion exchange column, automatically using an autosampler or manually using a Hamilton syringe. The buffer A and buffer B used for the mobile phase are described in the Recipes section. Optimize the speed of gradient between 10% buffer B to 100% buffer B according to your HPLC system and anion exchange column. An example of protocol is described in Table 1. The elution time will be determined by the negative charge of the hsiRNA guide strands and not influenced by the neutral PNA.
      12. Read the Cy3 fluorescence at 550 nm excitation and 570 nm emission wavelengths. The signal is coming from the Cy3 label on the PNA. Integrate the peak corresponding to Cy3-PNA-hsiRNA guide strand hybrid.
      13. Using the calibration curve, calculate the hsiRNA guide strand concentration.

        Table 1. Table describing the HPLC standard operating procedure for Cy3-PNA-hsiRNA hybrid concentration estimation


    4. Characterization of extracellular vesicles size distribution and concentration
      For each sample, the concentration and size of vesicles are determined by recording and analyzing the Brownian motion of particles in PBS and using a NanoSight NS300 system and a Nanoparticles Tracking Analysis (NTA) software (see Note 8).
      1. Dilute 1 to 5 µl of sample in 1 ml PBS (dilution ratio 1:200 to 1:1,000) at room temperature in a 1.5 ml microtube and mix gently using 1 ml micropipette.
      2. Inject the diluted sample in the NanoSight NS300 system using a 1 ml syringe. Use a new, clean syringe for each sample. Before injecting a new sample, flush out the previous sample to empty the tubing from vesicles.
      3. Adjust camera shutter and gain manually. To accurately compare the concentration of different samples, use similar camera shutter and gain values.
      4. Monitor the sample in duplicate for 30 to 60 sec.
      5. Process the recorded videos with NTA software to determine the particle size and concentration.
      6. Calculate the number of hsiRNAs loaded per extracellular vesicle by dividing the total amount of hsiRNAs in the sample by the number of extracellular vesicles detected by NTA, assuming normal distribution.
    5. Characterization of extracellular vesicles surface charge
      The hsiRNA-EV surface charges are determined by Laser Doppler Micro-electrophoresis (DLM) using a Zetasizer Nano. The particles charge is obtained by applying an electric field on the sample and measuring the velocity of the particles present in the sample. The surface charge of the particles is directly correlated to their velocity.
      1. Dilute 1 to 5 µl of sample in 1 ml distilled H2O (dilution ratio 1:200 to 1:1,000) at room temperature in a 1.5 ml microtube and mix gently using 1 ml micropipette. We noticed that diluting the sample in PBS provoked the burn of the solution that induces false measurements.
      2. Transfer the diluted sample in a universal glass micro-electrophoresis cuvette. A minimum volume of 700 µl of sample is required to completely immerse the electrodes. However do not overfill the cuvette as this will produce a thermal gradient within the sample and reduce the accuracy of the measurement.
      3. Slowly insert the Dip cell avoiding the formation of any bubble between the sample and the electrodes. For more detail, follow the procedure recommended by the manufacturer (Zetasizer Nano accessories guide). Clean the Dip cell electrodes thoroughly with distilled water between each measurement.
      4. Create a standard operating procedure following the manufacturer’s instructions (Zetasizer Nano User Manual).
      5. Monitor the sample charges in duplicate.
      6. The instrument gives an absolute value of the particles charge. We observed that hsiRNA-loaded extracellular vesicles have more negative charges than non-loaded extracellular vesicles.
    6. Analysis of extracellular vesicles and hsiRNA-loaded extracellular vesicles integrity by electron microscopy.
      To detect hsiRNAs bound to extracellular vesicles by electron microscopy, we use biotinylated hsiRNA and streptavidin immune-gold particles. Samples and grids used for electron microscopy are prepared at room temperature unless otherwise specified.
      1. To fix the sample, add an equal volume of 4% paraformaldehyde to the hsiRNA-loaded extracellular vesicle sample and incubate for 2 h. For example, add 50 µl of 4% paraformaldehyde to 50 µl of hsiRNA-extracellular vesicle sample.
      2. Drop 3 µl of fixed hsiRNA-EV aliquots onto EM grids
      3. Incubate for 20 min in a dry environment to let the membrane adsorb.
      4. To wash the grid, drop 100 µl of PBS on a sheet of Parafilm and transfer the grids upside down to the drop of PBS.
        Note: Subsequent steps are performed similarly, transferring the grid upside down from drop to drop of reagents on a sheet of Parafilm.
      5. Transfer the grid to a 100 µl drop of 50 mM glycine/PBS and incubate for 5 min.
      6. Transfer the grid to a 100 µl drop of 5% BSA/PBS for 10 min to block the sample.
        Note: The addition of 0.1% saponin to the reagent will permeabilize the extracellular vesicles to enable the detection of any intraluminal hsiRNA molecule.
      7. Wash the grid twice in 100 µl PBS drop.
      8. Incubate the grid with 50 µl drop of 6-nm or 10-nm streptavidin immune-gold particles diluted 1:10 to 1:20 in 0.5% BSA/PBS for 1 h in presence or absence of 0.1% saponin.
      9. Wash the grid three times in 100 µl PBS drop.
      10. Incubate the grid in 1% glutaraldehyde for 5 min.
      11. Wash the grid eight times in water for 2 min each wash.
      12. Transfer the grid in uranyl oxalate pH 7 for 5 min to contrast the sample.
      13. Contrast and embed the samples in a 9:1 solution of respectively 1% methyl cellulose and 4% uranyl acetate for 10 min on ice.
      14. Remove the excess liquid with a Whatman No. 1 filter paper and let the grids air-dry for 5 to 10 min.
      15. Observe the grid in a transmission electron microscope at 60-80 kV.
      16. Store the grid in grid storage boxes at room temperature. Grids can be stored indefinitely.

  3. Treatment of mouse central nervous system with hsiRNA-loaded extracellular vesicles
    Note: All animal procedures are performed according to your facility Institutional Animal Care and Use Committee regulations (IACUC). For the validation of hsiRNATarget-extracellular vesicles dependent silencing of target mRNA in vivo, control groups include mice treated with artificial CSF, extracellular vesicles alone, hsiRNANTC-extracellular vesicles and hsiRNATarget alone. (NTC: non-targeting control) (see Note 9).
    1. Prior to any treatment in the brain, animals are deeply anesthetized by intraperitoneal injection of 1.2% Avertin.
    2. Place the anesthetized mouse in the stereotactic frame (see Note 10).
      1. For direct injection, mice receive microinjections by stereotactic placement into the right striata. The coordinates relative to bregma are 1.0 mm anterior, 2.0 mm lateral, and 3.0 mm ventral.
      2. For brain infusion, we use ALZET® osmotic pumps that infuse the sample for 3 days (flow rate of 1 µl/h) or for 1 week (flow rate of 0.5 µl/h). Using a 30 G ½ needle attached to a 1 ml syringe, prefill the ALZET® pumps with the required volume of sample: 100 µl for both 3 days and 1 week pumps, following the manufacturer’s instructions. Incubate the pump overnight at 37 °C in a water bath. The next day, perform the mouse surgery to implant the ALZET® pumps in mouse brain.
    3. Upon 3 days or 7 days of treatment, mice are euthanized by intraperitoneal injection of 1 ml Avertin.
    4. Lay the mouse on its back and clean the abdominal surface with 70% ethanol to sterilize the area.
    5. Using microscissors, make a vertical incision in the abdominal muscles to open the abdominal cavity of the mouse.
    6. In the mouse heart, still beating, place a needle connected to the peristaltic pump to infuse room temperature PBS for 5 min to remove the blood from the brain.
    7. Using microscissors, carefully cut the skull of the mouse in half from the neck to the forehead. Carefully remove the two sides of the skull.
    8. Using forceps beneath the brain, carefully lift up and remove the brain from the skull.
    9. Process the brains accordingly to further experiments.
    10. mRNAs are unstable and degraded rapidly after the mouse anesthesia. Thus, for an accurate mRNA quantification and evaluation of in vivo silencing efficiency, the brains are processed immediately after harvest (Alterman et al., 2015; Coles et al., 2016; Didiot et al., 2016). Brains are cut in 300 µM coronal sections in PBS at -1 to 0.5 °C using a vibratome according to the manufacturer’s instructions. The temperature of the PBS should be stable around 0 ± 0.2 °C). If the temperature of the PBS increases, the brains soften and become more difficult to cut.
    11. Depending on the size of the brain region treated, collect a minimum of 3 biopsies, representing technical replicates, per animal. The 4th section from the front of the brain (olfactory bulbs) corresponds to the beginning of the striatum, which is also visually confirmed. The next 3 consecutive coronal sections (5th, 6th and 7th) are selected to collect the 2 mm biopsies from the striatal region easily identified in the mouse brain. The position of the biopsy are strictly fixed and the samples are always collected from the same region of the brain for all animals.
    12. Brain samples are processed for mRNA quantification following the method from Alterman et al., 2015; Coles et al., 2016; Didiot et al., 2016.
    13. The target and control mRNAs are quantified using the QuantiGene® 2.0 assay as recommended by the manufacturer protocol (Affymetrix). The amount of target mRNA is always normalized to the amount of the control housekeeping mRNA (Coles et al., 2016) (see Note 11).

Data analysis

Data are processed using Microsoft Office Excel and analyzed using GraphPad Prism 6 software.
The levels of targeted and housekeeping genes in the biopsies are independently measured using the QuantiGene® bDNA assay. The average of the 3 technical replicates represents the mRNA expression value per animal (see Notes 12 and 13).

  1. For each plate and each individual probe set, average the values obtained in the DLM (blank) wells and subtract this average from all sample wells.
  2. Divide the target gene luminescence value by the housekeeping gene luminescence value to normalize the target gene expression.
  3. Average the normalized untreated cell values.
  4. Divide each individual normalized well by the average of the untreated wells and multiply by 100 to calculate the percent of target gene expression relative to untreated cells.
  5. If compounds are tested in replicate, average the replicates and calculate the standard deviation.
  6. For dose response analysis, graph concentration-dependent IC50 curves using a non-lineal regression curve fit, log(inhibitor) vs. response–variable slope (four parameters). If necessary, set the lower limit of the curve at zero, and the upper limit of the curve at 100.
  7. The statistical analysis for the in vivo silencing experiments is performed using standard package in GraphPad Prism software. Compare efficacy of target sequences to the non-targeting control (NTC) to determine statistical significance. Differences in all comparisons are considered significant at P-values less than 0.05 compared with the NTC group.
  8. When the injections or infusions are performed unilaterally, the analysis is performed comparing ipsilateral or contralateral values groups (artificial CSF, extracellular vesicles only, hsiRNAtarget only and hsiRNAtarget-extracellular vesicles) to the hsiRNANTC-extracellular vesicles group.

Notes

  1. After ultracentrifugation performed to make extracellular vesicle-depleted medium or purify extracellular vesicles or hsiRNA-loaded extracellular vesicles, it is important to remove the supernatant quickly to avoid the pellet to dissolve back in the supernatant.
  2. We find that it is best to prepare fresh extracellular vesicles for each functional in vitro and in vivo assay.
  3. Purified extracellular vesicles are kept at 4 °C for a maximum of 48 h prior any functional assay.
  4. Particular attention should be given to the hsiRNAs modification pattern. Functionally efficient hsiRNA, loaded or not in extracellular vesicles, requires extensive chemical stabilization. Chemical modification of the ribose with 2’-O-Methyl and 2’-Fluoro result in a significant increase of the hsiRNA-resistance to nuclease degradation, providing higher stability in vitro and in vivo (Byrne et al., 2013). Unmodified or partially modified hsiRNAs will undergo nucleases degradation affecting hsiRNA-extracellular vesicles functional efficacy. Nucleases can potentially be present in the extracellular vesicles sample purified from cell culture medium.
  5. We noticed that extracellular vesicles purified from different cell sources (e.g., glioblastoma-, mesenchymal stem cell- or umbilical-derived extracellular vesicles) show similar ability to be loaded with hsiRNAs.
  6. The loading of extracellular vesicles with hsiRNA is saturatable with maximum of ~3,000-5,000 hsiRNA loaded per individual vesicle. However, incubation of extracellular vesicles with higher concentration of hsiRNAs slightly increase the number of hsiRNAs per extracellular vesicles. With increase in the hsiRNA/vesicle loading ratio, there is a slight increase in surface negative charge indicative that of the hsiRNAs association with the vesicle membrane. Thus, it is possible that too much loading might interfere with native extracellular vesicles cellular-uptake mechanism.
  7. The co-incubation of cholesterol-conjugated hsiRNAs with extracellular vesicles provides a robust, efficient and highly reproducible method for loading extracellular vesicles with chemically synthesized oligonucleotides. We observed that the cholesterol is essential for loading as non-cholesterol conjugated hsiRNAs did not associate with extracellular vesicles (Coles et al., 2016). We used the same procedure to load extracellular vesicles with siRNAs conjugated to various hydrophobic moiety (e.g., DHA). We observed that the more hydrophobic is the conjugate, the more efficient is the loading (data not published).
  8. The measurement of protein concentration of purified extracellular vesicles by Bradford assay represents an additional reproducible approach to quantify extracellular vesicles.
  9. The hsiRNAHTT, targeting the Huntingtin gene, and hsiRNANTC (NTC: non-targeting control) used to develop this method were designed and synthesized in our laboratory (Alterman et al., 2015). hsiRNAHTT silencing efficiency was confirmed without or with loading into extracellular vesicles in mouse brain (Coles et al., 2016).
  10. Bollus injection and pumps loaded with hsiRNA-extracellular vesicles samples are performed by stereotaxic surgery. Stereotaxic surgery allows an optimized delivery of oligonucleotide therapeutics in the mouse brain with excellent spatiotemporal control; essentially any brain region of choice can be targeted to silence any gene at any postnatal developmental stage up to adulthood. This clean and highly reproducible procedure, makes it an attractive approach for delivery of a therapeutic agent in the brain. The entire procedure can be completed in 2 h for 2 mice.
  11. hsiRNA-loaded extracellular vesicles are stable and loaded hsiRNAs are resistant to nucleases. However, only a fraction of the loaded hsiRNA is internalized into the vesicle lumen, and another fraction associates to the outer membrane. At this point, we cannot define which of the extracellular vesicles-associated oligonucleotides (surface bound or internalized) are functionally active. Further studies are essential to determine the optimal loading, long-term stability, and in vivo behavior of the hsiRNA-loaded extracellular vesicles.
  12. Based on preliminary data, the level of target mRNA expression variation from animal to animal has ~15% error, which theoretically requires n = 8 animals to be able to detect 20-30% gene modulation with 80% confidence. For in vivo validation of hsiRNA-extracellular vesicles sample efficacy using pump-based infusion, the mice groups are powered with n = 8 to n = 10 mice.
  13. The pump implantation requires extensive surgery that can result in uncontrolled animal death during the procedure and account for the difference in number of animals for different groups.

Recipes

Note: For all recipes and at any step of the procedure use Milli-Q-purified water.

  1. Cell culture medium
    Note: Recommended culture medium supplemented with FBS and antibiotics.


  2. 1 M sucrose
    Dissolve 34.23 g of sucrose in 100 ml of distilled H2O
    Filter sterilize with 0.22 µm syringe filter and store at 4 °C
  3. 1 M Tris-HCl
    1. Dissolve 121 g of Tris base (TRIZMA) in 700 ml of distilled H2O
    2. Adjust the pH to 8.5 with HCl
    3. Fill up to volume 1 L with distilled H2O
    4. Sterilize by autoclaving and store at room temperature
  4. 0.5 M EDTA
    1. Add 186.1 g of EDTA in 800 ml of distilled H2O
    2. Adjust the pH to 8 with NaOH (~50 ml of NaOH)
    3. Bring the volume to 1 L with distilled H2O
    4. Mix on a magnetic stirrer
    5. Sterilize by autoclaving and store at room temperature
  5. 3 M KCl
    Dissolve 22.368 g of KCl in 100 ml of distilled H2O and mix thoroughly
  6. 10% SDS
    Dissolve 10 g of SDS in 80 ml of distilled H2O. Adjust the volume to 100 ml with distilled water
  7. 0.1 M sodium phosphate buffer
    Dissolve 3.1 g of NaH2PO4·H2O and 10.9 g of Na2HPO4 (anhydrous) to distilled H2O to make a volume of 1 L. This buffer can be stored for up to 1 month at 4 °C
  8. HPLC buffer A
    50 v/v % acetonitrile, 50 v/v % water, containing 25 mM Tris-HCl (pH 8.5) and 1 mM EDTA in water
  9. HPLC buffer B
    Buffer A containing 800 mM NaClO4
  10. Glutaraldehyde, 1% (v/v)
    Dilute EM-grade glutaraldehyde fixatives (commercially available as 8%, 25% or 70% aqueous solutions; Sigma-Aldrich) in 0.1 M sodium phosphate buffer, pH 7.4, to the appropriate dilution. Store up to 6 months at -20 °C or up to 1 week at 4 °C after thawing
    Note: The buffers used to prepare fixatives need to have a good buffering capacity to maintain a pH of about 7.4 during fixation.
  11. Methyl cellulose, 2% (w/v)
    1. Heat 196 ml of distilled water to 90 °C and add 4 g methyl cellulose with stirring
    2. Rapidly cool on ice with stirring, until the solution has reached 10 °C. Continue slow stirring overnight at 4 °C
    3. Stop stirring and allow the solution ‘ripen’ for 3 days at 4 °C. Bring to a final volume of 200 ml with water
    4. Centrifuge using polycarbonate centrifuge bottles with cap assemblies 95 min at 100,000 x g, 4 °C
    5. Collect the supernatant and store up to 3 months at 4 °C
  12. Paraformaldehyde (PFA), 2% and 4% (w/v)
    1. Dissolve 4 g of PFA powder in 90 ml of 0.1 M sodium phosphate buffer and heat to 65 °C while stirring. If needed, add drops of 1 N NaOH until the solution becomes clear
    2. Bring to 100 ml with 0.1 M sodium phosphate buffer. Cool and filter. Aliquot and store at -20 °C. Use thawed aliquots immediately and do not refreeze
    3. To make 2% paraformaldehyde, dilute 4% paraformaldehyde with 0.1 M sodium phosphate buffer
  13. Uranyl acetate (4% w/v), pH 4
    1. Weigh 2 g of uranyl acetate and dissolve in 50 ml distilled water. Store up to 4 months at 4 °C protected from light
    2. Just before use, filter the amount needed of uranyl solution with a 0.22-μm syringe filter

      CAUTION: Consult with the institutional Radiation Safety Office for guidelines concerning proper handling and disposal of radioactive material. The uranyl acetate crystals are difficult to dissolve and it may be necessary to use a rotating wheel for mixing.

  14. Uranyl-oxalate, pH 7
    1. Mix 4% uranyl acetate, pH 4 with 0.15 M solution of oxalic acid (0.945 g in 50 ml distilled water), in a 1:1 ratio
    2. Adjust the pH to 7 by adding 25% (w/v) NH4OH in drops to prevent formation of insoluble precipitates
    3. Store in the dark up to 1 month at 4 °C
  15. Methyl cellulose-UA, pH 4
    9 parts 2% methyl cellulose and 1 part 4% uranyl acetate mixed just before use

Acknowledgments

We thank the members of the Khvorova and Aronin Laboratories, NIH Extracellular RNA Consortium and CHDI Foundation Inc. for helpful discussions. This work is supported in part by grants from the NIH UH2-TR000888 and UH3-4UH3TR000888-03, NIH NS38194 and CHDI Foundation (Research Agreement A-6119). The authors declare no conflict of interest.

References

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简介

将寡核苷酸治疗剂即siRNAs有效递送到中枢神经系统代表了治疗神经系统疾病的临床进展的显着障碍。 小的内源性细胞外囊泡显示能够在细胞(包括神经元)之间传输脂质,蛋白质和RNA。 这种天然的贩运能力使细胞外囊泡成为寡核苷酸即siRNA的递送载体的潜力。 然而,缺乏用寡核苷酸载体装载细胞外囊泡的稳健和可扩展的方法。 我们描述了在简单共孵育后将疏水修饰的siRNA加载到细胞外囊泡中的详细方案。 我们详细介绍了从细胞外囊泡纯化到数据分析的工作流程。 该方法可以促进基于细胞外基于囊泡的疗法用于治疗广泛的神经障碍。
【背景】siRNA是一种类型的寡核苷酸治疗剂,一类新的直接靶向信使RNA(mRNA)的药物,以防止导致疾病表型的蛋白质的表达。 siRNA的治疗应用是非常有希望的,因为siRNA可以被设计为靶向任何基因,包括不能用小分子或基于蛋白质的疗法“可药用”的基因。在寡核苷酸治疗剂的化学中取得的进展使得能够设计完全稳定的疏水改性的siRNA(hsiRNA,用2'-O-甲基或2'-氟以及硫代磷酸酯和共价键合到胆固醇的有义链修饰),其促进细胞hsiRNA的自我内化,并保持有效负载在RNA诱导的沉默复合体(RISC)中的能力(Byrne等,2013; Khvorova和Watts,2017)。与乘客链的3'末端连接的胆固醇缀合物对于快速细胞膜缔合是必需的(Byrne等,2013; Alterman等,2015)。单链硫代磷酸酯尾促进细胞内化(Geary等,2015)。我们最近证实了hsiRNA在治疗后数秒内结合细胞膜,进入细胞并促进体外有力的基因沉默(Byrne等,2013; Alterman等,2015; Ly等,2017)。然而,在小鼠脑内局部推注体内,hsiRNA扩散和功效限于给药部位周围的区域(Alterman等,2015)。而由于有力和特异性的基因沉默,hsiRNA保持治疗上有希望,它们向大脑的传递阻碍了其在中枢神经系统中治疗疾病的进步。
内源性产生的细胞外囊泡介导短距离和长距离细胞之间的脂质,蛋白质和RNAs的细胞间转移,从而在健康和疾病中发挥关键作用(Distler等,2005; Muralidharan-Chari等,2010)。细胞外囊泡携带功能性RNA的能力已经引起了人们对其作为运输和递送基于RNA的治疗剂的新型载体的兴趣。货物包括siRNA或其他寡核苷酸治疗剂(Tetta等,2013)。用于将基于RNA的治疗剂加载到细胞外囊泡中的策略包括电穿孔(Alvarez-Erviti等,2011; Ohno等,2013)或miRNA在细胞外小泡生成细胞中的过表达(Kosaka等,2012; Ohno et al。等等,2013; Mizrak等,2013)。虽然这两种策略都能够促进siRNA加载的细胞外囊泡转移到靶细胞和靶基因的沉默,但是它们不能被控制或扩大用于临床阶段制造(Kooijmans等,2013)。此外,电穿孔损害细胞外囊泡的完整性(Kooijmans等,2013)。我们证明了细胞外囊泡与hsiRNA的有效负载,而不改变囊泡大小分布,浓度和完整性。显示hsiRNA负载的细胞外囊泡诱导目标基因,小鼠原代神经元体外的huntingtin mRNA,和小鼠脑内体内的基因沉默(Didiot等,2016)。
在这里,我们描述了一种探索hsiRNA结合膜以促进其加载到细胞外囊泡中的能力的方法。将hsiRNA与从细胞培养条件培养基中纯化的胞外囊泡共孵育,促进负载到细胞外囊泡中。我们提供了细胞外囊泡纯化方法,hsiRNA细胞外囊泡的加载,hsiRNA负载的细胞外囊泡的大小,电荷和完整性表征以及小鼠脑中载有hsiRNA的胞外囊泡的体内测试的细节,以及数据分析。该技术可以促进几种其他类型的寡核苷酸治疗剂(即,反义,剪接切换寡核苷酸,空间封闭寡核苷酸,适体和其他)加载到细胞外囊泡,从而提供了显着的跨越式,以推进多种类型的寡核苷酸治疗剂治疗脑部疾病。随后,利用细胞外囊泡的自然特性来功能性转运小RNA(Valadi等,2007; Pegtel等,2010; Wang等,2010)提供了一种改善体内分布和细胞摄取的策略寡核苷酸治疗(Zomer等,2010; El Andaloussi等,2013; Kooijmans等,2012; Lasser,2012; Lee等,2012; Pan等,2012; Marcus and Leonard,2013; Nazarenko等人,2013; Didiot等,2016)。

关键字:RNA干扰, 疏水改性siRNA, 细胞外囊泡, 疗法, 神经系统障碍

材料和试剂

  1. 提示(0.2μl至1,000μl)(VWR)
  2. 纸巾
  3. 血清学移液器单独包裹(从5毫升到50毫升)(奥林巴斯)
  4. 0.22微米过滤灭菌系统(Thermo Fisher Scientific,Thermo Scientific TM,目录号:567-0020)
  5. 组织培养处理的多层烧瓶-T500cm 3双烧瓶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:132867)
  6. 50ml锥形离心管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:339652)
  7. 真空连接巴斯德吸管
  8. 1.7ml微量离心管(Genesee Scientific,目录号:22-282)
  9. 铝箔
  10. 紫外线透明的平底96孔板(Corning,目录号:3635)
  11. 1 ml注射器(BD,目录号:309659)
  12. 石蜡膜(Bemis,Parafilm M ®
  13. Whatman 1号滤纸(GE Healthcare,Whatman,目录号:10010155)
  14. 30 G½针
  15. 电子显微镜网格(电子显微镜科学,目录号:FCF2010-Ni)和清洁镊子操纵网格
  16. 网格存储盒(Electron Microscopy Sciences,目录号:71156)
  17. 鼠标(FVB/NJ)(杰克逊实验室,目录号:001800)
  18. 200瓶乙醇(Decon Labs,目录号:2805M)
  19. 胎牛血清(FBS)(Mediatech,目录号:35-010-CV)
  20. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190250)
  21. 蛋白酶抑制剂鸡尾草储备液(Sigma-Aldrich,目录号:P8340)
  22. Cy3标记或生物素化胆固醇结合的hsiRNA(内部生产)
  23. 与HsiRNA指导链(20个核苷酸长)完全互补的Cy3-OO-PNA链(PNA Bio)
  24. 肽 - 核酸(PNA)杂交测定(由Axolabs,Kulmbach,Germany开发)
  25. RIPA缓冲液(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:89900)
  26. 蛋白酶K(Thermo Fisher Scientific,Invitrogen TM,目录号:25530049)
  27. 甘氨酸(Sigma-Aldrich,目录号:G7126)
  28. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
  29. 皂苷(Sigma-Aldrich,目录号:S1252)
    注意:本产品已停产。
  30. 戊二醛(Polysciences,目录号:01909-10)
  31. Avertin(Sigma-Aldrich,目录号:T48402)
  32. QuantiGene 2.0测定试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:QS0011)
  33. Quantigene 2.0 Probesets(由基因变化)
  34. Dulbecco改良Eagle培养基(DMEM)
  35. MSC培养基(ATCC,目录号:PCS-500-041)
  36. 蔗糖(Sigma-Aldrich,目录号:S0389)
  37. Tris碱(TRIZMA)(Sigma-Aldrich,目录号:T6066)
  38. 盐酸(HCl)(Sigma-Aldrich,目录号:H1758)
  39. 乙二胺四乙酸二钠盐(EDTA)(Sigma-Aldrich,目录号:E6758)
  40. 氢氧化钠(NaOH)(〜50ml NaOH)(Sigma-Aldrich,目录号:72068)
  41. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  42. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  43. 磷酸二氢钠一水合物(NaH 2 PO 4·2H 2 O)
  44. 磷酸二氢钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S3139)
  45. 乙腈(50%乙腈溶液,在蒸馏H 2 O中稀释)(Fisher Scientific,目录号:A998-4)
  46. 高氯酸钠一水合物(NaClO 4·xH 2 O)(Fisher Scientific,目录号:S490-500)
  47. 甲基纤维素(Sigma-Aldrich,目录号:M6385)
  48. PFA粉末(Sigma-Aldrich,目录号:P6148)
  49. 乙酸铀酯(Electron Microscopy Sciences,目录号:22400)
  50. 草酸
  51. Tris-HCl(pH8.5)(Fisher Scientific,目录号:BP153)
  52. 氢氧化铵(NH 4 OH)(Fisher Scientific,目录号:A669-212)
  53. 细胞培养基(参见食谱)
  54. 1 M蔗糖(见食谱)
  55. 1M Tris-HCl(参见食谱)
  56. 0.5 M EDTA(参见食谱)
  57. 3 M KCl(见配方)
  58. 10%SDS(参见食谱)
  59. 0.1 M磷酸钠缓冲液(见配方)
  60. HPLC缓冲液A(参见食谱)
  61. HPLC缓冲液B(参见食谱)
  62. 戊二醛,1%(v/v)(参见食谱)
  63. 甲基纤维素,2%(w/v)(见配方)
  64. 2%和4%(w/v)的多聚甲醛(PFA)(见配方)
  65. 乙酸异丙酯(4%w/v),pH 4(参见食谱)
  66. 草酸铀酸盐,pH 7(参见食谱)
  67. 甲基纤维素-UU,pH4(参见食谱)

设备

  1. 组织文化罩
  2. 软刷
  3. 超离心机70毫升聚碳酸酯瓶(Beckman Coulter,目录号:355655)
  4. 固定角度Ti45超速离心机转子(Beckman Coulter,型号:45 Ti,目录号:339160)
  5. 冷冻超速离心机(Beckman Coulter,型号:Optima XE)
  6. 冷藏式台式离心机(Beckman Coulter,型号:Allegra X-15R)
  7. 0.5微升至1毫升的微量移液器(Labnet International,型号:BioPette Plus)
  8. 固定角度TLA-110转子(Beckman Coulter,型号:TLA-110,目录号:366735)
  9. 1.5ml微量离心机适配器(Beckman Coulter,目录号:360951)
  10. 冷藏台式超速离心机(Beckman Coulter,型号:Optima TM MAX-TL)
  11. 用于1.5 ml微管的轨道式热振动筛(Grant Instruments,型号:PHMT系列)
  12. 读卡器分光光度计(Tecan Trading,型号:Infinite ® M1000 Pro)
  13. 立体镜框架(KOPF INSTRUMENTS,型号:963型)
  14. 带有自动进样器荧光检测器的HPLC系统(Agilent Technologies,型号:HPLC 1100系列)
  15. 汉密尔顿注射器(Hamilton,型号:Gastight#1002)
  16. 细胞外囊泡大小分布和浓度读取器(Malvern Instruments,型号:NanoSight NS300)
  17. 细胞外囊泡电荷读数器(Malvern Instruments,型号:Zetasizer Nano NS)
  18. 玻璃微电泳比色杯Dip Cell kit(Malvern Instruments,目录号:ZEN1002)
  19. 透射电子显微镜(JEOL,型号:JEM-1011)
    注意:本产品已停产。
  20. 渗透泵(DURECT,ALZET,目录号:1003D和1007D)
  21. 水浴37°C
  22. Microscissors(Fine Science Tools,目录号:14060-10; 14002-12)
  23. 一套两个镊子(精细科学工具,目录号:11251-30)
  24. Vibratome(Leica Biosystems,型号:Leica VT1000 S)
  25. 自动洗板机(BioTek Instruments,型号:ELx405)
  26. 组织培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heracell TM 150i)
  27. 吸管(Drummond Scientific,型号:Portable Pipet-Aid ® XP)
  28. 组织培养相差倒置显微镜(Motic,型号:AE2000)
  29. 阴离子交换柱(Thermo Fisher Scientific,Thermo Scientific TM,型号:DNAPac TM,PA100)
  30. 热块(Fisher Scientific,型号:Isotemp TM 2050FS)
  31. μPlate载体(Beckman Coulter,型号:SX4750)
  32. -86℃冷冻机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma< sup>> 900系列)
  33. 连接真空的生物安全柜(Thermo Fisher Scientific,Thermo Scientific TM,型号:1300系列II类,A2型)

软件

  1. 纳米粒子跟踪分析(NTA)软件
  2. Microsoft Office Excel(Microsoft Pack Office)
  3. GraphPad Prism 6软件(GraphPad Software,Inc.)

程序

  1. 细胞外囊泡的生产和净化
    注意:
    1. 细胞外囊泡的纯化如图1所示。


      图1.细胞外囊泡生产流程图:通过差速超速离心从条件培养基中纯化细胞外囊泡(微泡和外来体)

    2. 涉及打开超速离心瓶的所有步骤必须在组织培养罩中进行。要对瓶子进行消毒,使用软刷子用肥皂清洗瓶子和盖子,并用无菌水冲洗干净,以清除任何痕量的肥皂。在组织培养罩中,将清洁的瓶子及其盖子在70%乙醇浴中灭菌,并在纸巾上在UV光下干燥以进行最终灭菌。聚碳酸酯瓶对乙醇和紫外线敏感,所以避免将瓶子放在70%乙醇中超过2分钟,紫外线超过30分钟。
    3. 细胞外囊泡是透明的棕色,非常小,这使得它们难以检测。使用永久性标记,标记每个70毫升超速离心瓶的一侧以将瓶子定向在转子中,并指示在哪里寻找细胞外囊泡。所有70毫升瓶子都应满满瓶肩(约65毫升),以进行高速离心。否则瓶子会塌陷并损坏转子。如果没有填充到肩膀,用PBS完成体积。

    1. 准备细胞外消化细胞的培养基和细胞培养物 注意:细胞外囊泡天然存在于体液中,包括通常用于补充细胞培养基的胎牛血清(FBS)。因此,在任何使用之前,细胞外囊泡从其中纯化的细胞培养基通过过夜超速离心而耗尽血清 - 细胞外囊泡,以最小化污染。
      1. 对于每个细胞系,准备补充有40%(v/v)FBS的推荐培养基。补充有40%FBS的贫血培养基允许在单次离心中制备大量的细胞外无囊泡培养基。
      2. 使用50ml血清移液管将65ml培养基转移到70ml瓶中
      3. 将标签面朝上的瓶子放入预冷45型转子转子(储存在4°C),并将超速离心机预冷却至4°C。
      4. 超速离心机在10万x g,4℃下至少18小时。
      5. 为了过滤消毒上清液,使用50ml血清移液管将每个瓶子的内容物转移到真空连接的0.22μm过滤器 - 无菌快速流动的一次性装置中。在瓶的底部,留下2-3ml含较高浓度棕色沉积物的培养基。颗粒是棕色的,很粘,不会从瓶子的边缘脱落(见注1)。
      6. 将经灭菌的细胞外小泡细胞培养基储存至4周不饱和。
      7. 在细胞培养之前,用另外的培养基稀释细胞外小泡细胞培养基,以获得培养基,推荐浓度为细胞系所需的FBS(通常为10%FBS)。如有必要,补充培养基与任何额外的营养和/或抗生素。
    2. 细胞外囊泡的生产和纯化
      注意:
      1. 通过改良自Thery等人的方法从条件培养基中纯化细胞外小泡。 (2006)组合顺序离心和过滤步骤。所有细胞外囊泡质量控制按照国际细胞外囊泡学会推荐的实验要求进行(Lotvall等,2014; Thery et al。,2006)。 
      2. 体内实验需要显着浓度的细胞外囊泡以进行有效的沉默。为了收集大量的细胞外囊泡,我们从培养物中粘附细胞的条件培养基中纯化细胞外囊泡。纯化细胞外囊泡的产量与培养基体积的增加有关。因此,为了产生大量的条件培养基并且扩大细胞外囊泡的浓度,我们使用T500cm 2的三烧瓶。

      1. 在多层烧瓶中,在常规推荐的培养基中生长感兴趣的贴壁细胞,直到它们达到70%至80%的汇合度。我们观察到,60毫升是可以用于T500厘米三重烧瓶的介质的最小体积。
      2. 将培养基倒入液体垃圾中,因为血清移液管不能用于从多层烧瓶中去除液体。使用50ml血清移液管,用相似体积的推荐的EV-贫血培养基更换培养基。
      3. 在37℃,5%CO 2和100%湿度下孵育细胞48小时。
      4. 收集培养基并进行细胞外囊泡纯化,如下。如果细胞不是100%融合,则用新鲜的细胞外消化细胞的培养基替换收集的培养基,孵育48小时,然后重复细胞外囊泡纯化。
      5. 将含有细胞外囊泡的培养基收集在50ml离心锥形管中
      6. 在室温下以300 x g离心10分钟,沉淀并除去浮游细胞和大碎片。
      7. 使用50ml血清移液管将上清液转移到新的50ml离心锥形管中。在管的底部留下2-3ml培养基,以避免干扰沉淀。丢弃颗粒。
      8. 将上清液以10,000 x g,4℃离心30分钟,以分离大泡囊或微泡。
      9. 通过真空连接的无菌0.22μm过滤器单元倒出并过滤消毒上清液,以进一步消除较大的囊泡。
        注意:含有微泡(300 nm至1μm)的10,000 g沉淀物是白色和粘稠的,可用于其他实验。如果需要微泡,则使用真空连接的巴斯德吸管将瓶子底部的残留介质完全去除。使用微量移液管将沉淀悬浮在1ml PBS中。沉淀物应与PBS的流动容易分离。将收集的微泡以10,000xg,4℃离心30分钟,作为另外的洗涤步骤,并将沉淀悬浮于100μlPBS中。
      10. 使用50ml血清移液管,将过滤的培养基转移到70ml聚碳酸酯瓶中。
        注意:用永久性标记标记瓶子,并将瓶子放置在离心机中,标记面向中心。这将有助于稍后本地化颗粒。
      11. 在预冷45型Ti转子中,超离心机在100,000 x g,4℃下至少70分钟。
      12. 使用真空连接的巴斯德吸管将所有瓶子的上清液完全吸出。
        注意:剩余的介质将落在瓶子的壁上,形成必须通过真空连接的巴斯德吸管吸出的最后一滴上清液。
      13. 检测瓶子底部的细胞外囊泡。颗粒的颜色为棕色透明,直径约5毫米。
        注意:颗粒将被定位到瓶子的标记侧。
      14. 为了浓缩细胞外囊泡,使用微量移液管将所有颗粒悬浮在相同的1ml PBS中。用尖端,刮擦球囊内和周围的瓶子,以恢复最大的细胞外囊泡。
      15. 将悬浮的颗粒转移到1.5ml微量离心管中。仔细回收在每个瓶底部形成的含细胞外小泡的PBS的剩余液滴
      16. 将1.5毫升的管子放入管子的预冷TLA-110转子中,并将管帽取向,找到沉淀物。
      17. 在桌面超速离心机中离心至少70分钟,以100,000 x g,4℃离心。
      18. 将沉淀重悬于100μlPBS,1x蛋白酶抑制剂混合物和10mM蔗糖中用于进一步的实验。储存于4°C进行短期使用,-80°C长期储存。避免反复冻融(见注2和3)。
        注意:对于蛋白质组学或脂质分析,将沉淀物保存在-80℃。

  2. hsiRNA负载细胞外囊泡的制备和表征
    1. 用hsiRNA载体细胞外囊泡 用hsiRNA加载细胞外囊泡如图2所示。


      图2.外来体加载程序的流程图Cy3-hsiRNA和细胞外囊泡的共孵育导致通过超速离心沉淀的Cy3-hsiRNA胞外囊泡

      1. 将1.5ml微量离心管中混合所需浓度的hsiRNA(3,000-5,000分子的每个囊泡)和胞外囊泡重悬于100μlPBS中,用微量吸管轻轻移液(见注释4和5)。
        注意:建议首先确定细胞外囊泡的浓度(参见步骤B4)。如果hsiRNA的浓度高于100μM,则每个囊泡比达到3,000-5,000个hsiRNA所需的hsiRNA溶液的体积可以忽略不计(少于10μl)。
      2. 在37℃下将混合物在热振荡器中孵育至少60分钟,并以500rpm摇动。如果寡核苷酸被荧光标记,则使用铝箔保护管免受光线影响
      3. 将1.5毫升管放在管夹中,放入预先冷却的TLA-110转子中,并将管帽取向以找到颗粒。
      4. 在桌面超速离心机中离心至少70分钟,以100,000 x g,4℃离心。未加载的hsiRNA保留在上清液中,加载hsiRNA的细胞外囊泡形成颗粒
      5. 取出并将上清液保存在1.5ml离心中进一步测定。上清液中保留的hsiRNA的定量可用于估计加载效率(见步骤B2和B3)。
      6. 将沉淀重悬在所需体积的培养基或人造脑脊液中,用于基于细胞的测定,或分别用于体内注射。重新悬浮的hsiRNA负载的细胞外小泡立即用于进一步实验而不被储存。或者细胞外囊泡可以在-80℃下储存在0.1M蔗糖中。我们观察到存储不影响囊泡数量或活动长达6个月。
      7. 保存hsiRNA负载的胞外囊泡的等分试样,用于定量hsiRNA和细胞外囊泡浓度。
    2. 通过直接定量荧光标记的hsiRNA测定hsiRNA浓度 使用两种方法来估计细胞外囊泡中hsiRNA的负载效率(图1)(见注释6和7)。首先,直接测量hsiRNA - 细胞外囊泡中的Cy3荧光。我们观察到细胞外囊泡可能会显示自身荧光信号,从而可能会混淆来自加载的hsiRNA的Cy3荧光的测量。因此,我们推荐第二个策略:计算最初添加到样品中的Cy3-hsiRNA的总量与上清液中剩余的Cy3-hsiRNA的量(未加载)与细胞外囊泡和超离心孵育之间的差异。为了确定hsiRNA的准确浓度,可以将准备阴性的对照(PBS只有没有hsiRNA)和hsiRNA的校准曲线制成紫外透明的平底96孔板的孔。通常在96孔板中使用的最小体积的样品是50μl。对于校准曲线,制备1:2稀释系列的hsiRNA,跨越量与加载到细胞外囊泡中的量相当。
      1. 对于阴性对照,在板的第一口(孔#A1)中分配50μlPBS(空白)。
      2. 在以下孔(井#A2)中,制备Cy3荧光阳性对照储备溶液。在100μlPBS中稀释已知浓度的Cy3标记的hsiRNA。使用hsiRNA的数量是hsiRNA-细胞外囊泡样品中使用的两倍。在PBS中稀释1:2,得到8点校准曲线。
      3. 将50μlPBS分配到三个以下孔(孔#A3,A4和A5)中。为了进行系列稀释,将50μl储备溶液(孔#A2)转移到含有50μlPBS的下一个孔(well#A3)中,并用微量移液管轻轻混合。
      4. 使用新鲜的微量吸头,将50μl(来自井#A3)转移到下一个井(井#A4)中,并进行到校准曲线的最后一个井。
      5. 将每个样品的50μl上清液(含有未加载的hsiRNA)和沉淀(含有hsiRNA-细胞外囊泡)转移到板中。
      6. 使用分光光度计读板器在547nm激发和570nm发射波长测量每个样品的Cy3荧光。
      7. 使用校准曲线计算沉淀和上清液样品中的Cy3荧光。
      8. 颗粒样品的Cy3荧光显示了hsiRNA-细胞外囊泡样品中hsiRNAs浓度的直接估计。
      9. 作为评估沉淀中hsiRNA浓度的第二种方法,计算最初添加到样品中的总Cy3-hsiRNA与上清液中残留的Cy3荧光的量之间的差异。
    3. 通过肽 - 核酸杂交分析测定hsiRNA浓度
      为了确认通过使用分光光度计测量Cy3荧光获得的hsiRNA的浓度,我们使用肽 - 核酸(PNA)杂交测定(由Axolabs,Kulmbach,Germany开发)确定样品中的hsiRNA引导链浓度(不是Cy3标记的) )(Roehl等人,2011)。 PNA是DNA的单链类似物,糖 - 磷酸骨架被聚酰胺主链取代。因此,没有任何电荷,PNA以非常高的亲和力结合到它们的互补链上,形成双链DNA-RNA结构。 PNA测定是比分光光度法荧光测量更准确,但也更费力地测量颗粒中的hsiRNA浓度。此外,用于PNA测定的样品被裂解,不能在下游实验中使用。
      1. 在含有2mg/ml蛋白酶K的100μlRIPA缓冲液的1.5ml微量离心管中稀释hsiRNA-胞外囊泡样品。
      2. 超声处理15分钟,在室温下溶解囊泡
      3. 为了制备校准曲线,将已知量的hsiRNA双链体添加到裂解的胞外囊泡样品中
      4. 加入20μl3 M KCl,并用微量吸管轻轻混合,沉淀SDS(RIPA缓冲液组分)。
      5. 在室温下以5,000×g离心15分钟,以沉淀不溶性膜并沉淀SDS。
      6. 将含有hsiRNA引导链的清除的上清液转移到新的1.5ml微量离心管中。
      7. 稀释上清液以获得低于5pmol的hsiRNA /100μl样品的浓度。
      8. 加入完全互补于hsiRNA的引导链的Cy3标记的PNA链的5pmol(或超过加载到细胞外囊泡中的hsiRNA的量)的量,并与微量移液管轻轻混合。
      9. 为了将PNA链退火至hsiRNA指导链,在95℃孵育15分钟(以融合hsiRNA双链体),然后在50℃孵育(将PNA退火至hsiRNA引导链,取代有义链)15分钟,很酷到室温。
      10. 将样品转移到96孔板或与HPLC系统相容的小瓶中。
      11. 将含有PNA-hsiRNA指导链杂交体的样品注入HPLC阴离子交换柱,自动使用自动进样器或手动使用汉密尔顿注射器。用于流动相的缓冲液A和缓冲液B在食谱部分中有描述。根据您的HPLC系统和阴离子交换柱,优化10%缓冲液B至100%缓冲液B之间的梯度速度。方案的一个例子如表1所示。洗脱时间将由hsiRNA引导链的负电荷确定,不受中性PNA的影响。
      12. 读取550nm激发和570nm发射波长的Cy3荧光。信号来自PNA上的Cy3标签。整合对应于Cy3-PNA-hsiRNA引导链杂交体的峰。
      13. 使用校准曲线计算hsiRNA导向链浓度
        表1.描述Cy3-PNA-hsiRNA杂交浓度估计的HPLC标准操作程序的表


    4. 细胞外囊泡大小分布和浓度的表征
      对于每个样品,通过记录和分析PBS中的颗粒的布朗运动并使用NanoSight NS300系统和纳米粒子跟踪分析(NTA)软件(参见注8)来确定囊泡的浓度和大小。
      1. 在1.5ml微量管中,在室温下,在1ml PBS(稀释比1:200至1:1000)中稀释1至5μl样品,并使用1ml微量移液管轻轻混合。
      2. 使用1 ml注射器将稀释样品注入NanoSight NS300系统。对每个样品使用新的干净的注射器。在注入新样品之前,先冲洗以前的样品,以清除囊泡中的管道。
      3. 调整相机快门并手动增益。要准确比较不同样品的浓度,请使用类似的相机快门和增益值
      4. 将样品一式两份监测30至60秒。
      5. 使用NTA软件处理录制的视频,以确定粒度和浓度。
      6. 通过将样品中hsiRNA的总量除以NTA检测到的细胞外囊泡数,假定正态分布,计算每个细胞外囊泡加载的hsiRNA数量。
    5. 细胞外囊泡表面电荷的表征
      通过使用Zetasizer Nano的激光多普勒微电泳(DLM)测定hsiRNA-EV表面电荷。通过在样品上施加电场并测量样品中存在的颗粒的速度来获得颗粒电荷。颗粒的表面电荷与其速度直接相关。
      1. 在1.5ml微量管中,在1ml蒸馏的H 2 O(稀释比1:200至1:1000)中稀释1至5μl样品,并使用1ml微量移液管轻轻混合。我们注意到,在PBS中稀释样品会引起溶液的燃烧,导致误诊。
      2. 将稀释的样品转移到万能玻璃微电泳试管中。要求最小体积的700μl样品完全浸没电极。但是,不要过度填充反应杯,因为样品中会产生热梯度并降低测量精度。
      3. 缓慢地插入浸泡池,避免在样品和电极之间形成任何气泡。有关详细信息,请按照制造商推荐的步骤( Zetasizer Nano配件指南)。每次测量之前,用蒸馏水彻底清洗Dip细胞电极。
      4. 按照制造商的说明创建标准操作程序( Zetasizer Nano用户手册)。
      5. 一式两份地监控样本费用。
      6. 仪器给出了颗粒电荷的绝对值。我们观察到,hsiRNA负载的细胞外囊泡比非负载的细胞外囊泡具有更多的负电荷。
    6. 通过电子显微镜分析细胞外囊泡和hsiRNA负载的细胞外囊泡完整性 通过电子显微镜检测与细胞外囊泡结合的hsiRNA,我们使用生物素化的hsiRNA和链霉亲和素免疫金颗粒。用于电子显微镜的样品和网格在室温下制备,除非另有说明。
      1. 为了固定样品,将等体积的4%多聚甲醛加入到载有hsiRNA的胞外囊泡样品中并孵育2小时。例如,向50μl的hsiRNA-细胞外囊泡样品中加入50μl的4%多聚甲醛。
      2. 将3μl固定的hsiRNA-EV等分试样放入EM网格上
      3. 在干燥的环境中孵育20分钟使膜吸附。
      4. 要清洗网格,将100μlPBS放在一片石蜡膜上,并将网格上下颠倒转移到PBS一滴。
        注意:后续步骤类似地执行,将网格从一滴Parafilm的一滴试剂上下颠倒转移。
      5. 将网格转移到100微升的50mM甘氨酸/PBS中并孵育5分钟
      6. 将网格转移到100微升5%BSA/PBS中10分钟以阻止样品。
        注意:向试剂中加入0.1%的皂苷将使细胞外囊泡渗透,以检测任何腔内hsiRNA分子。
      7. 在100μlPBS滴下洗两次网格。
      8. 在存在或不存在0.1%皂角苷的情况下,将0.5μlBSA/PBS中以1:10至1:20稀释的5微升6-nm或10-nm链霉亲和素免疫金颗粒孵育1小时。
      9. 在100μlPBS滴中洗涤网格三次。
      10. 在1%戊二醛中孵育网格5分钟
      11. 在水中洗涤网格8次,每次洗涤2分钟。
      12. 将网格转移到草酸铀酰盐酸盐pH7中5分钟,以对比样品
      13. 将样品与1%甲基纤维素和4%乙酸双氧铀的9:1溶液对比并将其包埋在冰上10分钟。
      14. 用Whatman 1号滤纸去除多余的液体,让网格空气干燥5〜10分钟
      15. 观察60-80kV透射电子显微镜中的栅格
      16. 将电网在室温下存放在电网储存箱中。网格可以无限期存储。

  3. 用hsiRNA负载的细胞外囊泡治疗小鼠中枢神经系统
    注意:所有的动物手术都是根据您的设施进行的。机构动物护理和使用委员会条例(IACUC)。为了验证目标mRNA在体内的hsiRNA - 细胞内小泡依赖性沉默,对照组包括用人造CSF,单独的细胞外小泡,hsiRNA和NTC - 细胞内小泡处理的小鼠和hsiRNA Target 。 (NTC:非定位控件)(见注9)。
    1. 在大脑中进行任何治疗之前,通过腹膜内注射1.2%的Avertin将动物深度麻醉。
    2. 将麻醉的老鼠置于立体定向框架中(见注10)。
      1. 对于直接注射,小鼠通过立体定向放置在正确的条纹中接受微注射。相对于母体的坐标为前方1.0mm,侧面2.0mm,腹侧3.0mm
      2. 对于脑输注,我们使用注射样品3天(流速1μl/h)或1周(流速为0.5μl/h)的ALZET超声波泵。使用连接到1 ml注射器的30 G½针,按照制造商的说明,为所需体积的样品预填充ALZET ®泵:100μl,用于3天和1周的泵。在37℃下在水浴中孵育过夜。第二天,进行小鼠手术,将小鼠脑中的ALZET ®泵植入。
    3. 治疗3天或7天后,通过腹膜内注射1ml Avertin将小鼠安乐死。
    4. 将鼠标放在其背面,用70%乙醇清洁腹部表面以对该区域进行消毒。
    5. 使用微型剪刀,在腹部肌肉中进行垂直切口以打开小鼠腹腔。
    6. 在小鼠的心脏中,仍然跳动,将针头连接到蠕动泵,将室温PBS注入5分钟,以从脑中除去血液。
    7. 使用微型剪刀,将颈部的颅骨从颈部小心地切割到前额。仔细取出颅骨的两侧。
    8. 使用大脑下方的镊子,小心地抬起头脑并从头骨中取出大脑
    9. 相应地处理大脑以进一步实验。
    10. mRNA在小鼠麻醉后不稳定并迅速降解。因此,对于准确的mRNA定量和评价体内沉默效率,收获后立即处理脑(Alterman等人,2015; Coles等人,2016; Didiot等人,2016)。根据制造商的说明书,使用vibratome,在-1至0.5℃的PBS中,在300μM冠状切片中切割大脑。 PBS的温度应稳定在0±0.2°C附近)。如果PBS的温度升高,则脑软化并变得更难切割。
    11. 根据所治疗的大脑区域的大小,每只动物至少收集3个活组织检查,代表技术重复。大脑前部(嗅球)的第4个部分对应于纹状体的开始,这也是视觉上确认的。选择接下来的3个连续的冠状切片(5 th ,6 th 和7 th )来容易地从纹状体区域收集2mm活组织检查在小鼠脑中识别。活检的位置是严格固定的,并且样品总是从所有动物的脑的相同区域收集。
    12. 根据Alterman等人,2015年的方法处理脑样品用于mRNA定量; Coles 等,2016; Didiot等,2016年。
    13. 使用制造商协议(Affimetrix)推荐的使用QuantiGene 2.0测定法定量靶和对照mRNA。目标mRNA的总量总是归一化到控制管家的mRNA的数量(Coles等人,2016)(参见附注11)。

数据分析

数据使用Microsoft Office Excel进行处理,并使用GraphPad Prism 6软件进行分析。
使用QuantiGene bDNA测定法独立测量活组织检查中靶向和管家基因的水平。 3个技术重复的平均值代表每只动物的mRNA表达值(见注12和13)。

  1. 对于每个板和每个单独的探针组,平均在DLM(空白)孔中获得的值,并从所有样品孔中减去该平均值。
  2. 将目标基因发光值用管家基因发光值除以标准化靶基因表达
  3. 平均归一化未处理的细胞值。
  4. 将每个单独的标准化除以未处理孔的平均值,并乘以100,以计算相对于未处理细胞的靶基因表达的百分比。
  5. 如果化合物在复制中进行测试,则平均复制并计算标准偏差
  6. 对于剂量反应分析,使用非线性回归曲线拟合,log(抑制剂)对响应 - 可变斜率(四个参数)的图表浓度依赖性IC 50 曲线。如果需要,将曲线的下限设置为零,曲线上限为100.
  7. 使用GraphPad Prism软件中的标准包进行静态实验的体内统计分析。比较目标序列与非靶向对照(NTC)的功效以确定统计学显着性。与NTC组相比,所有比较的差异被认为在P - 值小于0.05的显着性。
  8. 当单次进行注射或输注时,仅进行分析,比较同侧或对侧值组(仅人工CSF,胞外囊泡,hsiRNA 靶标 和hsiRNA - 细胞内囊泡)到hsiRNA - 细胞内囊泡组。

笔记

  1. 在进行超速离心以使细胞外小泡耗尽的培养基或纯化细胞外小泡或hsiRNA负载的细胞外囊泡之后,重要的是快速去除上清液以避免沉淀物溶解回上清液。
  2. 我们发现,最好在体外实验中为每个功能性体外制备新鲜的细胞外囊泡。
  3. 在任何功能测定前,将纯化的细胞外囊泡保持在4℃最多48小时
  4. 应特别注意hsiRNA修饰模式。功能有效的hsiRNA,负载或不在胞外囊泡,需要广泛的化学稳定。用2'-O-甲基和2'-氟进行核糖的化学修饰导致hsiRNA对核酸酶降解的抗性的显着增加,在体内提供更高的稳定性和//B>(Byrne等人,2013))。未修饰或部分修饰的hsiRNA将经历影响hsiRNA-细胞外囊泡功能功效的核酸酶降解。核细胞可能存在于从细胞培养基中纯化的胞外囊泡样品中
  5. 我们注意到,从不同细胞来源(例如,胶质母细胞瘤,间充质干细胞或脐带来源的细胞外囊泡)纯化的细胞外囊泡显示出与hsiRNA相似的载体能力。
  6. 用hsiRNA负载细胞外囊泡可饱和,每个囊泡最多装载约3,000-5,000个hsiRNA。然而,使用较高浓度的hsiRNA的细胞外囊泡的孵育稍微增加每个细胞外囊泡的hsiRNA的数量。随着hsiRNA /囊泡负载率的增加,表面负电荷略有增加,表明hsiRNA与囊泡膜结合。因此,太多的负载可能会干扰天然胞外囊泡细胞摄取机制
  7. 胆固醇结合的hsiRNA与细胞外囊泡的共孵育为化学合成的寡核苷酸加载细胞外囊泡提供了鲁棒,有效和高度可重复的方法。我们观察到胆固醇对于加载而言非常重要,因为非胆固醇结合的hsiRNA与细胞外囊泡无关(Coles等人,2016)。我们使用相同的程序来加载细胞外囊泡,其中siRNA与各种疏水部分缀合(例如,DHA)。我们观察到缀合物越疏水,加载效率越高(数据未公布)。
  8. 通过Bradford测定法测定纯化的细胞外囊泡的蛋白质浓度代表了量子化细胞外囊泡的另外可重复的方法。
  9. hsiRNA ,靶向Huntingtin 基因和hsiRNA (NTC:用于开发该方法的非靶向对照在我们的实验室中设计和合成(Alterman等人,2015)。 hsiRNA 沉默效率在小鼠脑中没有或加载到细胞外囊泡中被证实(Coles等人,2016)。
  10. 通过立体定位手术进行Bollus注射和装载有hsiRNA-细胞外囊泡样品的泵。立体定向手术允许优化的时空控制小鼠脑中寡核苷酸治疗的优化递送;基本上任何选择的脑区域都可以针对任何产后发育阶段直到成年期的任何基因沉默。这种清洁和高度可重现的方法使其成为在脑中递送治疗剂的有吸引力的方法。 2只小鼠可以在2小时内完成整个手术。
  11. hsiRNA负载的细胞外囊泡是稳定的,并且加载的hsiRNA对核酸酶具有抗性。然而,只有一部分加载的hsiRNA被内化到囊泡腔中,另一部分与外膜相关联。在这一点上,我们不能定义细胞外囊泡相关的寡核苷酸(表面结合或内化)在功能上是活跃的。进一步的研究对于确定负载hsiRNA的胞外囊泡的最佳载量,长期稳定性和体内的行为至关重要。
  12. 基于初步数据,从动物到动物的靶mRNA表达变化水平具有〜15%的误差,理论上要求n = 8动物能够以80%置信度检测20-30%的基因调控。对于使用基于泵的输注的hsiRNA-细胞外囊泡样品效能的体内验证,小鼠组用n = 8至n = 10只小鼠供电。
  13. 泵植入需要大量的手术,这可能导致手术过程中动物死亡不受控制,并说明不同组别的动物数量的差异。

食谱

注意:对于所有食谱,并且在本步骤的任何步骤中使用Milli-Q纯化水。

  1. 细胞培养基
    注意:补充有FBS和抗生素的推荐培养基。


  2. 1 M蔗糖
    将34.23g蔗糖溶解在100ml蒸馏水中的二氧化碳 用0.22μm注射器过滤器过滤消毒,并在4°C下储存
  3. 1M Tris-HCl
    1. 将121g Tris碱(TRIZMA)溶解在700ml蒸馏水中的二氧化O /
    2. 用HCl将pH调节至8.5
    3. 用蒸馏水H 2填充至体积1升
    4. 通过高压灭菌灭菌并在室温下储存
  4. 0.5 M EDTA
    1. 在800ml蒸馏的H 2 O 2中加入186.1g EDTA
    2. 用NaOH(〜50 ml NaOH)调节pH至8
    3. 使用蒸馏H 2将O体积调节至1升
    4. 混合磁力搅拌器
    5. 通过高压灭菌灭菌并在室温下储存
  5. 3 M KCl
    将22.368g KCl溶于100ml蒸馏水中,并彻底混合,
  6. 10%SDS
    将10g SDS溶解在80ml蒸馏的H 2 O中。用蒸馏水将体积调整至100ml
  7. 0.1M磷酸钠缓冲液
    溶解3.1g NaH 2 PO 4·H 2 O和10.9g Na 2 HPO 3, 4(无水)至蒸馏的H 2 O,使体积为1L。该缓冲液可以在4℃下储存长达1个月
  8. HPLC缓冲液A
    50v/v%乙腈,50v/v%水,含有25mM Tris-HCl(pH8.5)和1mM EDTA水溶液
  9. HPLC缓冲液B
    缓冲液A含有800mM NaClO 4
  10. 戊二醛,1%(v/v)
    在0.1M磷酸钠缓冲液(pH 7.4)中稀释EM级戊二醛固定剂(以8%,25%或70%水溶液Sigma-Aldrich商购)至合适的稀释液。在-20°C下储存6个月或在解冻后在4°C下储存最多1周 注意:用于制备固定剂的缓冲液需要具有良好的缓冲能力,以在固定期间保持约7.4的pH。
  11. 甲基纤维素,2%(w/v)
    1. 将196ml蒸馏水加热至90℃,并搅拌加入4g甲基纤维素
    2. 在搅拌下在冰上快速冷却,直到溶液达到10℃。继续在4℃下缓慢搅拌过夜
    3. 停止搅拌并使溶液在4℃下"成熟"3天。带水至200毫升的最终体积
    4. 离心机使用聚碳酸酯离心机瓶盖组件95分钟,100,000 x g,4°C
    5. 收集上清液,并在4°C下储存3个月
  12. 2%和4%(w/v)的多聚甲醛(PFA)
    1. 将4g PFA粉末溶于90ml 0.1M磷酸钠缓冲液中,加热至65℃同时搅拌。如果需要,加入1N NaOH水溶液,直到溶液变澄清
    2. 用0.1M磷酸钠缓冲液加至100ml。冷却和过滤。等分并储存于-20°C。立即使用解冻等分试样,不要重新冻结
    3. 制备2%多聚甲醛,用0.1M磷酸钠缓冲液稀释4%多聚甲醛
  13. 乙酸铀酯(4%w/v),pH 4
    1. 称量2g乙酸双氧铀并溶于50ml蒸馏水中。在4°C下保存4个月免受光照
    2. 就在使用之前,用0.22μm注射器过滤器
      过滤铀酰溶液所需的量

      注意:请咨询机构的辐射安全办公室,了解有关放射性物质的妥善处理和处理的准则。乙酸铀酰酯晶体难以溶解,可能需要使用旋转轮进行混合

  14. 草酸铀酯,pH 7
    1. 混合4%乙酸铀酰,pH4,用0.15M草酸溶液(0.945g,在50ml蒸馏水中),以1:1的比例
    2. 通过加入25%(w/v)NH 4 OH水溶液将pH调节至7以防止形成不溶性沉淀物
    3. 在黑暗中存放1个月,4°C
  15. 甲基纤维素-UU,pH 4
    9份2%甲基纤维素和1份4%乙酸双氧铀酸盐混合使用前

致谢

感谢Khvorova和Aronin实验室,NIH胞外RNA联盟和CHDI基金会的成员进行有益的讨论。这项工作部分由NIH UH2-TR000888和UH3-4UH3TR000888-03,NIH NS38194和CHDI基金会(研究协议A-6119)的资助部分支持。作者宣称没有利益冲突。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Haraszti, R. A., Coles, A., Aronin, N., Khvorova, A. and Didiot, M. (2017). Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System. Bio-protocol 7(12): e2338. DOI: 10.21769/BioProtoc.2338.
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