Protocols


1. Conjugation of EGF ligand onto liposomes Protocol

STEP 1: Thiolation of EGF Protocol

1A : Preparation of 1x PBS with 5mM EDTA (50mL)

Reagents and apparatus:

  1. From 500mL 1x PBS stock solution, aliquot 30 mL into a 50 mL Falcon tube using a graduated cylinder or serological pipette
  2. On an analytical balance, weigh out 73.06 mg of EDTA (MW: 292.24 g/mol) into a weigh boat
  3. Transfer EDTA salt to the 50 mL Falcon tube and rinse weigh boat with ~10 mL of 1x PBS
  4. Vortex solution for 1 min or until EDTA is dissolved
  5. Adjust final volume of Falcon tube to 50 mL with PBS

1B: Thiolation

Reagents and Apparatus:

  1. Prepare 4x1 mL of 23.87 uM stock solution of EGF
    • Add 1 mL of PBS/EDTA solution to a 15 mL Falcon tube
    • In the EGF bottle, add 1 mL of the PBS/EDTA solution using a P1000 pipettor
    • Gently, pipette the solution up and down 10 times to solubilize the protein
    • Transfer the 1 mL of solution to Falcon tube containing 1 mL of PBS/EDTA and rinse pipette tip by pipetting up and down
    • With a fresh 1 mL of PBS/EDTA solution, rinse the protein bottle by pipetting up and down and transfer to Falcon tube
    • Add an additional 378 uL of PBS/EDTA solution to the Falcon tube for a final volume of 3.378 mL and a final concentration of 23.87 uM
    • Place on orbital shaker to mix and dissolve
    • Aliquot into 4x 1 mL tubes and freeze in -20°C
  2. Prepare 19.72mL of 368.41 uM stock solution of Traut’s Reagent
    • Weigh out 1 mg of Traut’s Reagent in a weigh boat on an analytical balance
    • In a 50 mL Falcon tube, transfer 10 mL of 5 mM PBS/EDTA solution
    • Transfer 1 mg of Traut’s Reagent to tube and wash weight boat with remaining 9.72 mL
    • Place on orbital shaker to mix and dissolve
  3. In a 1.5 mL microcentrifuge tube, pipette 1000 uL of 23.87 uM EGF stock solution
  4. Add 260 uL of 368.41uM Traut’s Reagent to the microcentrifuge tube containing EGF
  5. Gently mix and centrifuge at 6000 rpm for 5 seconds to ensure liquid is located at the bottom of the tube
  6. Incubate for 1 hour at room temperature
  7. Take sample for thiol and protein assay (volume: remainder in 378 uL)

STEP 2: Buffer Exchange Via Dialysis

Reagents and Apparatus:

Dialysis Volume: 600 uL Dialysis Length = 1.1mL/cm for 11.5 mm tubing

  1. Cut ~6 mm + extra handling length of 11.5 mm dialysis tubing
  2. Soak tubing in MiliQ water for 10-15min (make sure inside is washed too)
  3. Use dialysis clamps by folding one end of tubing and pinching tubing in the clamp
  4. Open other end of tubing by gently rubbing w/ fingers. Pipet small amount of water or buffer into tubing, check for tears or leaks
  5. Rinse tubing with MilliQ water and perform a final rinse with the buffer
  6. Fill tubing with thiolated EGF sample by pipetting gently into the tubing
  7. Remove all small air bubbles, clamp or tie off open end and leave a little slack in tube if sample has high salt concentration
  8. Place tubing in 10mL large beaker or Erlenmeyer flask
  9. Fill beaker w/ 1x PBS. Add stir bar, place on stir plate in a cold room.
    • Buffer volume should be 10-20x volume of sample
  10. Change buffers at least once for changes between samples being dialyzed and the new buffer
    • Check dialysis situation a few times during the first half hour of stirring to make sure that the bag is not moving too quickly
    • To be more precise, can perform 2 to 4 buffer changes
    • First buffer change can take place 4 hours after starting
    • Second buffer change dialyze overnight.
  11. Remove the dialysis bag from the flask, and carefully remove the closure(s). Cut the bag right beneath the upper knot, and carefully remove your sample from the bag by pipetting. Work over a funnel and beaker just in case bag slips from your hand.
  12. Take sample for thiol and protein assay (volume: 126 uL)

STEP 3: Thioether Bonding of Thiolated EGF with Liposome

Reagents and Apparatus:

  1. In a microcentrifuge tube, combine 1100 uL of thiolated EGF with 1000 uL of 30 mg/mL liposomes (split into two tubes; 550 uL EGF + 500 uL liposome in one tube)
  2. Incubate overnight at room temperature on an orbital shaker
  3. Take sample for maleimide assay (volume: 100 uL)

STEP 4: Cap Free Maleimide Ligands

Reagents:

Perform in fume hood (b-mercaptoethanol gives off a foul odor)

  1. In the liposome solution, add 86.35 uL (150x molar excess) of b-mercaptoethanol
  2. Vortex for 5 second to mix and centrifuge at 6000 rpm for 5 seconds to ensure liquid is located at the bottom of the tube
  3. Incubate for 1 hour at room temperature
  4. Take sample for thiol, maleimide, protein assay and DLS sizing

Step 5: Remove b-mercaptoethanol from Liposome


2. Assay Protocols

2A: Protein Assay

Preparation of the Micro BCA Working Reagent (WR)

  1. Use the following formula to determine the total volume of WR required:

Example: for the standard Test Tube Procedure with 3 unknowns and 2 replicates of each sample:

Note: 150µL of WR is required for each sample in the Microplate Procedure.

  1. Prepare WR by mixing 25 parts of Micro BCA Reagent MA and 24 parts Reagent MB with 1 part of Reagent MC (25:24:1, Reagent MA:MB:MC). For the above example, combine 12.5mL of Reagent MA and 12.0mL Reagent MB with 0.5mL of Reagent MC.

Note: When Reagent MC is initially added to Reagents MA and MB, turbidity occurs that quickly disappears upon mixing to yield a clear-green solution. Prepare sufficient volume of WR based on the number of samples to be assayed. The WR is stable for one day when stored in a closed container at room temperature (RT). It is not necessary to protect the solution from light.

Microplate Procedure

  1. Pipette 150 µl of each standard or unknown sample replicate into a microplate well.
  2. Add 150 µl of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds. 3. Cover plate and incubate at 37°C for 2 hours.
  3. Cool plate to RT.
  4. Measure the absorbance at or near 562 nm on a plate reader.
    • Wavelengths from 540-590 nm have been used successfully with this method.
    • Because plate readers use a shorter light path length than cuvette spectrophotometers, 562 nm readings are lower with the Microplate Procedure than with the Test Tube Procedure. Consequently, the lower limit of detection is greater (2.0 µg/ml) in the Microplate Procedure.
  5. Subtract the average 562 nm absorbance reading of the Blank standard replicates from the 562 nm reading of all other individual standard and unknown sample replicates.
  6. Prepare a standard curve by plotting the average Blank-corrected 562 nm reading for each BSA standard vs. its concentration in µg/ml. Use the standard curve to determine the protein concentration of each unknown sample.

Note: If using curve-fitting algorithms associated with a microplate reader, a four-parameter (quadratic) or best-fit curve will provide more accurate results than a purely linear fit. If plotting results by hand, a point-to-point curve is preferable to a linear fit to the standard points

Preparation of Diluted Albumin (BSA Standards)

Vial Standard Solution (uL) Buffer (uL) Final BSA Concentration (ug/mL)
A 100 900 200
B 200 of A 800 40
C 500 of B 500 20
D 500 of C 500 10
E 500 of D 500 5
F 500 of E 500 2.5
G 400 of E 600 1
H 500 of G 500 0.5
I 0 500 0

2B: Thiol and Maleimide Assay

Desired Samples to Be Tested

Preparation of Cysteine Standard Curve

Table 1: Standard Curve Dilutions

Tube Standard (uL) PBS (uL) Final Concentration (mM)
1 0 500 0
2 100 of 3 400 0.05
3 750 of 4 750 0.125
4 750 of 5 750 0.250
5 750 of 6 750 0.500
6 1000 of 7 500 1.000
7 1047 of 2.15 mM Std. 453 1.500
8 500 of 2.15 mM Std. 0 2.150

Assay Protocol

Preparation of Assay Reagents

Maleimide Assay Preparation

Table 2: Maleimide Assay Sample Prep

Sample Sample Volume (uL) Assay Buffer (uL) MEA (uL) Sample Concentration
Mal 1 88 264 88 1:5 dil
Mal 2 44 308 88 1:10 dil

Thiol Assay Preparation

Table 3: Thiol Assay Sample Prep

Sample Sample Volume (uL) Assay Buffer (uL) Sample Concentration
Thiol 1 88 352 1:10 dil
Thiol 2 44 396 1:10 dil

Loading Plate with Samples

Maleimide Sample Absorbance:


3. Conjugation of APOE ligand onto liposomes

STEP 1: Creating DSPE-PEG-CONHNH2 linker

Materials:

Equipment/Techniques:

1A: Synthesis of DSPE-PEG-2000-SH

  1. In a small glass vial, reconstitute 25 mg of DSPE-PEG2000-Amine in 500 µL of chloroform.
  2. Vortex mixture and store at -20°C. This will be the stock chloroform solution of DSPE-PEG2000-Amine (17.918 mM)
  3. In a small glass vial, reconstitute 25 mg of Traut’s reagent in 3 mL methanol to make up a stock Traut’s solution of 60.56 mM
  4. Vortex mixture and aliquot into five 1.5 mL microcentrifuge tubes. Store at -20°C.
    For a 5 mg batch of DSPE-PEG-Amine
  5. Thaw an aliquot each of the stock solutions by placing them on ice and occasionally vortexing if necessary. Alternatively, you can flick the tube to help facilitate the thawing. Centrifuge the vial before opening to bring all the liquid down.
  6. Weigh a small, empty glass vial and record this weight
  7. In a small glass vial, pipette 100 µL of stock chloroform solution
  8. In the same glass vial, add 15 µL of stock Traut’s solution.
  9. In the same glass vial, add 95 µL of methanol to bring the final volume up to 210 µL. The final concentration of DSPE-PEG-Amine should be around 8.5238 mM, while the final concentration of Traut’s should be around 4.3258 mM
  10. Mix the solution by slowly pipetting up and down, and then vortexing for around 30 seconds.
  11. Stir the mixture at room temperature for 2 hours in the hood by occasionally turning over the tube. Keep checking the the tube until the chloroform has evaporated completely. Expect to see dried powder.
  12. Dissolve remaining mixture in around 250 µL to 300 µL of 1x PBS. Pipette up and down to mix and then vortex for around 30 seconds.
  13. Dialyze mixture using a cellulose ester membrane overnight (see step 1.2)
  14. Collect the dialysate the next morning and concentrate using the Savant SpeedVac Concentrator.
  15. Record final weight of solid product after concentration.
    Note: theoretical yield using 5 mg of dspe-peg-amine = 4.98 mg (using MW: 2781.1429 g/mol . Assume 100% yield for the next step and use around 4.5 mg to be conservative/ account for possible losses. We can change this calculation depending on actual yield.

STORAGE OPTIONS:

1B: Dialysis of DSPE-PEG2000-SH

Reagents:

Apparatus:

  1. Cut 6 mm + extra handling length of dialysis tubing and soak tubing in MiliQ water for 10-15min
  2. Use dialysis clamps by folding one end of tubing and pinching tubing in the clamp
  3. Open other end of tubing by gently rubbing w/ fingers. Pipette a small amount of water into tubing to check for leaks
  4. Rinse tubing with MilliQ water, final rinse w/ the buffer
  5. Fill tubing w/ 210 µL DSPE-PEG2000-SH sample by pipetting gently into the tubing
  6. Remove all but small air bubble. Clamp or tie off open end. Leave a little slack in tube if sample has high salt concentration
  7. Place tubing in 10mL large beaker or Erlenmeyer
  8. Fill beaker w/ 1x PBS. Add stir bar, place on stir plate in a cold room. Buffer volume should be 10-20x volume of sample
  9. Change buffers
  10. Remove the dialysis bag from the flask, and carefully remove the closure(s). Cut the bag right beneath the upper knot, and carefully remove your sample from the bag by pipetting.
  11. Take sample for thiol assay and H-NMR

1C: Synthesis of DSPE-PEG-2000-CONHNH2

  1. In the same glass vial where DSPE-PEG-SH is stored (there should be around 4.5-5 mg (1.61 µmol) of DSPE-PEG2k-SH in the vial), add 1 mL of 1x PBS (pH 7.4) to redissolve the DSPE-PEG2k-SH to give a final concentration of 1.6 mM
  2. Vortex the vial to mix contents.
  3. In a separate tube, weigh out 3.5 mg of MPBH (around 9.89 µmol; 353.8 g/mol MW of MPBH) and dissolve in 1 mL 1x PBS (~10 mM).
  4. Add this to the 1.5 mL microcentrifuge tube containing the DSPE-PEG2k-SH solution made in step 2. Vortex the tubes to mix contents.
  5. Stir mixture at room temperature for 4 hours using a microcentrifuge tube rotator or rocker.
  6. Insert into cellulose ester bag (1 kDa MWCO) against water overnight for dialysis
  7. Concentrate dialysate
  8. Dissolve freeze-dried solid in chloroform at ~5 mg/mL and store at -80 degrees celsius

1D: TNBSA Assay

Reagents:

  1. Calculate the amount needed from DSPE-PEG2k-CONHNH2 to be added to a total volume of 0.5 mL 0.1M sodium bicarbonate with the following concentrations: 40 µg/ml, 160 µg/mL and 400 µg/mL.
  2. Calculate amount of glycine to be added to a total of 0.5 mL of sodium bicarbonate such that the final concentration would be: 0 µg/mL, 5 µg/mL, 10 µg/ml, 15 ug/mL, 20 µg/mL, 25 ug/mL, 30 ug/mL
  3. Add calculated amounts of buffer to labelled microcentrifuge tubes
  4. Add the specified amounts of glycine to standard tubes, vortex each for 5 seconds
  5. Add the specified amounts of DSPE-PEG2k-CONHNH2 to the sample tubes, vortex each one for 20 seconds
  6. Add 250 µL of TNBSA solution to each sample tube, vortex for 5 seconds
  7. Incubate at 37°C for two hours
  8. Add 250 µL of SDS solution and and 125 µL of 1 N HCL to samples, vortex for 10 seconds
  9. Using a plate reader read the absorbance at 335 nm
  10. Create a standard curve and calculate the hydrazone bond content in each sample

STEP 2: Oxidizing APOE with Na-periodate (Aldehyde activation)

Note: This should be performed simultaneously with step 1.3 when ready to form DSPE-PEG-HZ-APOE, in order to be efficient with time.

2A: APOE buffer exchange

SIGMA APOE: The APOE from Sigma is in solution (50 mM Ammonium bicarbonate)

  1. Using a 100K Amicon centrifuge filter, perform a buffer exchange with 1X PBS + 1% BSA to purify the protein and get rid of anything toxic.
  2. Using a 100K Amicon centrifuge filter, perform a second buffer exchange with oxidation buffer to get a final concentration of around 0.5 - 1 mg/mL

2B: Sodium metaperiodate oxidation of APOE (time-sensitive)

  1. In a dark room, weigh out 2.15 mg of Na-periodate in an amber vial/microcentrifuge tube covered in foil.
    Note: Na-periodate is light sensitive
  2. Add 500 µL of oxidation buffer to the amber vial (~20mM final concentration). Stir mixture for around 10 mins
  3. Quench the reaction by removing free aldehydes: Add 5 µL of 40mM sodium borohydride into the PCR tube and mix contents using vortex
  4. Purify by gel filtration using a desalting spin column in 100 uL PBS and use product immediately

STEP 3: APOE Conjugation to DSPE-PEG-CONHNH2

3A: Protein DSPE-PEG-CONHNH2 titration

  1. Test the following ratios by adding the following ratios into a microcentrifuge tube:
    • add 50 µg ApoE in PBS to 2 µg DSPE-PEG-CONHNH2
    • add 25 ug ApoE in PBS to 2 ug DSPE-PEG-CONHNH2
    • add uL ApoE in PBS to 2 ug DSPE-PEG-CONHNH2

3B: APOE conjugation to DSPE-PEG-CONHNH2

  1. In proportions appropriate for the intended conjugation as determined by the above titration, combine solutions of DSPE-PEG-CONHNH2 and oxidized ApoE
  2. Incubate reaction mixture for 2 hours at room temperature
  3. Buffer exchange the PBS with water to remove any excess unconjugated DSPE-PEG-CONHNH2
  4. Evaluate conjugation product via MALDI

STEP 4: Acid-release Tests

Incubation & Preliminary Steps

  1. Prepare lactic acid with pH 4.0, 4.5 and 5.0
  2. Incubate the ApoE-Conjugated DSPE-PEG-CONHNH2 with lactic acid at different pHs for 1 hour at room temperature in a microcentrifuge tube
  3. Add contents of microcentrifuge tube to a 10 kDa filtration tube and spin at 12,000 RCF for 5 minutes
  4. Collect the bottom and top fractions, label each fraction and store for analysis using MALDI-MS.

STEP 5: Incorporation of Micelle into Liposome

  1. At 60°C, mix the micelle formed from lipid films into liposomes (3% PEG) at a ratio of 5% micellar lipids to liposomal lipids for 1 hour.
  2. Purification of incorporated micelles via dialysis


Supplementary Protocols

0.1M Sodium Bicarbonate Buffer (pH 8.2)

Equipment:

Reagents:

  1. Dissolve 8.401g Sodium bicarbonate in 1000 mL distilled water, mix well
  2. Adjust to pH 8.2 using small amounts of either strong acid or base as needed

Coupling Buffer: 0.1M sodium phosphate, 0.15M NaCl (pH 7.2)

Equipment:

Reagents:

  1. Add 3.1g of NaH2PO4•H2O, 10.9g of Na2HPO4 (anhydrous), and 8.766g NaCl to distilled H2O to make a volume of 1L
  2. Adjust pH as needed

Oxidation Buffer: 0.1M sodium acetate (pH 5.5)

  1. Weigh out 13.6 grams of trihydrate (anhydrous sodium acetate) and dissolve in 1L dH2O to make 0.1M sodium acetate (stock solution)
  2. Add 19 mL of 0.1M acetic acid to 181 mL of 0.1M sodium acetate stock to bring to pH 5.5
  3. Verify pH with pH meter

Methods

Calculations