1 Assembly of liposomes

1.1 GUVs (Giant Unilamellar Vesicles):

Materials:

Procedure:

  1. Cleaning the Electroformation Vials
    • Sonicate vials and Platinum caps in 50% ethanol for 20 minutes
    • Rinse the chambers with methanol and followed by rinse with chloroform
    • Dry completely

  2. Lipid Mixture and Electroformation
         
    Component Volume (μL)
    DOPC (25 mg/mL) 3.8
    Biotin (25 mg/mL) 2 (1:10 dilution)
    2:1 Chloro-methanol 90
    DiI (25 mg/mL) 4

    • Prepare 1 mg/ml lipids mixture in a polypropylene vial. Prepare 5% biotin solution by 1:10 dilution in 2:1 chloro-methanol solvent.
    • Spread 5 μL of lipid mixture on different platinum wires. Slowly pipette the lipid on the wires to make an even layer.
    • Evaporate chloroform completely: place the platinum wires in vacuum chamber for ~10 minutes
    • Fill the chambers with 350 μL sucrose solution
    • Insert the platinum wires into the chambers and seal the chambers with electrical tape
    • Place the chambers in heat block and set the temperature to 68°C
    • Start the alternating current: 10 Hertz, 2 volts for 2 hours
    • Decrease the current to 2 Hertz for 30 minutes

1.2 LUVs (Large Unilamellar Vesicles):

Materials:

Procedure:

  1. Formation of multi-lamellar vesicles
    • Using a glass pipette tip, pipette 1 mg/mL of 10 mg/mL of DOPC into a cryo-vial
    • With a gentle flow of argon or nitrogen gas, evaporate the chloroform carefully
    • Place the vial in a vacuum oven at room temperature. Dry for 10-15 minutes
    • Hydrate the lipid film with 1x PBS to form multi-lamellar vesicles
  2. Formation of LUVs
    • Freeze and thaw: (Repeat 3 times) Sonicate the solution for ~5 minutes and freeze in liquid nitrogen.
    • Extrusion:Put together the extrusion kit (Avanti lipids) using a 200 nm polycarbonate membrane
    • Push the PBS through the extruder into a waste beaker using a Hamilton syringe
    • Add the second syringe on the other side of the extruder and perform extrusion.(21 times)
    • Carefully push the liposomes into a fresh Eppendorf tube and Store at 4°C

1.3 Peptide Synthesis:

Materials:

  • Reactive amino group with 9-fluorenylmethoxycarbonyl (Fmoc)
  • 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronoium hexafluorphosphate (HBTU)
  • Acetic Anhydride
  • O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorphosphat (HATU)
  • 1-Hydroxy-7-azabenzotriazol (HOAT)
  • Diisopropylethylamin (DIPEA)
  • TFA/TIS/water/DTT (90v/v):5(v/v):2.5(v/v):2.5(m/v))
  • Milli-Q water/acetonitrile (1:1)
  • Automated solid-phase peptide synthesizer (ResPep SL, Intavis)

Procedure:

  1. Synthesis
         
    Peptides Sequences
    Poly-Arginine (PolyR) RRRRRRRRR
    Transportan (Trans) GWTCNSAGYLLGKINLKALAALAKKIL

    • Prepare peptide sequences using standard Fmoc chemistry on a solid-phase with HBTU activation on an automated solid-phase peptide synthesizer
    • Couple each amino acid twice with 5-fold excess for good quality
    • Cap all non-reacted amino acids with acetic anhydride
  2. Biotinylation
    • After the final cycle, biotintylation of the resin bound peptides can be conducted via HATU, HOAT, and DIPEA mediated coupling
    • Cleave the biotinylated peptides from the resin with TFA/TIS/water/DTT (90v/v):5(v/v):2.5(v/v):2.5(m/v)) for 2 hours
    • After final peptide precipitation, wash with ice-cold diethyl ether
  3. Purification of the peptides
    • Solve precipitated peptide in MilliQ water/acetonitrile (1:1)
    • Purification: reverse-phase high pressure liquid chromatography (HPLC) on a Semi-preparative HPLC with C18 column
    • During purification, the peptide will attach to the solid phase of the column and is eluted by the mobile phase over 25 min at 4mL/min according to its hydrophobic profile
  4. Sample Analysis
    • The mobile phase is represented by a gradient of 0.1% TFA in water (100% to 5%) and 0.1% TFA in acetonitrile (5% to 100%)
    • The fraction containing the product can be identified using UV detector at 220 nm
    • Further analysis: analytical reverse phase Ultra-high Pressure Liquid Chromatography (UPLC) with UV detector and analytical C18 column
    • Sequence specificity can be confirmed on the base of the molecular weight
    • The fraction containing the product was lyophilized and stored at -20°C

2 Purification Techniques

Purification of DNA-Encapsulated liposomes using dialysis

Materials:

  • 10 kDa membrane filtration tube

Procedure:

  • Dialysis: High encapsulation frequency of the DNA inside the vesicles was obtained by performing rotatory filtration dialysis
    • Membrane filtration tube of pore size 10 kDa was used to filter ssDNA (MW: 9,864 Da)
    • Suspend 100 μL of sample in the suspended media (PBS) with the surface of the filtration tube touching the meniscus
    • Mix the sample with magnetic stirrer at 400 rpm
    • Change the buffer every 4 hours
    • Repeat the cycle 6 times

3 Modifications

3.1 Biotinylation of liposomes

Materials:

Procedure:

  • Calculate the volume of both biotinylated lipids and DOPC needed to synthesize 2 mg/mL lipids in 1 mL
  • Follow the same method previously described for GUV and LUV synthesis

3.2 DiI staining of liposomes

Materials:

Procedure:

  1. Incorporate 25 μM of DiI stain in with both the biotinylated lipids and DOPC for a final concentration of 2 mg/ml in 1mL
  2. Follow the same method previously described of GUV and LUV synthesis

3.3 DNA encapsulation in Liposomes

Materials:

  • 100 μM of ssDNA

Procedure:

  1. Add 1 μL of 100 μM single-stranded DNA to 40 μL of 25 mg/mL DOPC
  2. Add 458 μL of PBS to create a final volume of 500 μL
  3. Follow the same method previously described for LUV synthesis

4 Measurements and Imaging

4.1 DLS

  1. Set the Malvern Zetasizer Nano Software to include sample parameters such as PBS buffer and refractive index of the different lipids
  2. Create a 2:100 dilution of liposomes and PBS by adding 2 μL of the liposomes to 98 μL of PBS
  3. Add 60 μL of the diluted sample to a Malvern Instruments low-volume quartz batch cuvette
  4. Place the cuvette in the sample holder and close the lid
  5. Label the sample file and click measure
  6. Make sure the sample equilibrates and temperature is constant at 25°C

4.2 UV vis

  1. Set the wavelength measurements to 260 nm for nucleic acid absorption and 280 nm for protein absorption (due to Tryptophan)
  2. Place 1.2 μL of clean milliQ water and measure the blank
  3. Place 1.2 μL of an undiluted sample of DNA encapsulated dialyzed liposomes
  4. Measure and record the absorbance values at 260 nm and 280 nm
  5. Wipe both the upper and lower optical surfaces with a Kimwipe

4.3 Wide-field Microscopy of GUVs

  1. Fill the wells of microscopy chambers with 300 μL BSA (1 mg/mL) and wait for 1 hour for complete coating
  2. Wash 5-10 times with distilled/sterilized water
  3. Add 150 μL of GUV suspension to the wells
  4. Add 200 μL of PBS onto GUV suspension in the wells and mix the solution gently.
  5. Using Wide-Field Microscopy, set the magnification to 40x, excitation to 530 nm (green light), emission to 570 nm (yellow light)

4.4 STORM imaging of LUVs stained with DiI

  1. Prepare 5 wells of PEGylated chambers on a glass slide and store in the fridge until the samples are ready
  2. Wash the chambers with neutravidin for surface immobilization of the sample
  3. Dilute the samples to 100 pM and flush the chamber with 20 μL
  4. Pat the other end of the chamber with a Kimwipe™ to avoid spilling every time flushing occurs
  5. Flush the chamber with 20 μL PBS once
  6. Repeat for each sample
  7. Place the slide on the stage of the microscope and If the images demonstrate that the sample is too concentrated, flush with PBS once more

4.5 Cryo-TEM

  1. 2µl of sample in 1x FB was deposited on a holey carbon support film attached to a copper grid.
  2. After rapid freezing, the sample was loaded to a Gatan cryo transfer holder at -130°C to prevent ice crystallization.

5 Bursting of liposomes and uptake

5.1 Bursting

  1. Prepare a 5 μL sample containing 2.5 μL of 4000 nM peptides and 2.5 μL of 200 nM quantum dots
  2. Let the sample incubate for at least 15 minutes to ensure full conjugation of the peptides to the QDs
  3. After incubation, add 5 μL of the prepared liposomes
  4. Create three different ratios of peptides/QDs to liposomes: 1:1, 1:2, and 1:10
  5. From those three samples, take 2 μL from each and add to 3 separate Eppendorf tubes containing 98 μL of PBS, creating a 2:100 dilution
  6. Add 60 μL of the diluted samples to the DLS cuvette
  7. Place the cuvette in the DLS sample holder and measure

5.2 Uptake

  1. Prepare a solution of peptides and quantum dots containing 200 nM quantum dots and 20 nM peptides, creating a 10-fold quantum dot excess
  2. Incubate for at least 15 minutes to allow for full conjugation of the peptides to the QDs
  3. Follow the same steps for bursting for the preparation of the ratios and the DLS measurements