Nanogel preparation and characterization

1.       Prepare an organic phase: dissolve 356 mg dioctyl sulfosuccinate sodium salt and 688 mg polyoxyethylene lauryl ether (Brij L4) in 5 ml of hexane in a 20 ml vial

2.       Prepare an aqueous phase: dissolve 45 mg acrylamide, 23 mg thrombin-cleavable peptide (TCP) cross-linker and 8 mg clot-targeted peptide in 370 μl of PBS solution in Eppendorf tube and add 100 μl of the HV solution (2000 U/ml) and 30 μl of 20% ammonium persulfate solution

3.       Place the 20 ml vial with the organic phase in an ice-water bath on a magnetic stirrer with high-speed stirring

4.       Add the aqueous phase into the organic phase by a pipette drop by drop

5.       Add 20 μl of N,N,N’,N’-tetramethylethylenediamine to the mixed solution

6.       Stir the mixed solution with high speed under ice-water bath for 2 hours

7.       Transfer the solution to a 100 ml round-bottom flask and remove the organic solvent using a vacuum rotary evaporator

8.       Add 5 ml ethanol to the flask to suspend the residual product

9.       Transfer the suspension to a 15 ml centrifugation tube and centrifuge at 10000 rpm for 5 minutes

10.    Remove the supernatant, add 5 ml ethanol, resuspend the pellet by pipetting and centrifuge at 10000 rpm for 5 minutes

11.    Repeat the ethanol washing procedure three times

12.    Dry the pellet under vacuum overnight to obtain solid nanogel powder

13.    Suspend 15 mg powder in 3 ml PBS and disperse the nanogel solution using a probe-type sonicator

14.    Filter the nanogel solution through the strainers with the pore size of 800, 450 and 220 nm successively

15.    Place the filtrate in a dialysis bag (MWCO 100 KDa) and dialyze against PBS for 24 hours

Use Zetasizer to measure the particle size and zeta potential of the nanogel

1.       Dilute the nanogel solution ten times.

2.       Place the nanogel solution in the corresponding container and measure the particle size and zeta potential using Zetasizer (Malvern ZS90), respectively

Use transmission electron microscope (TEM) to observe the morphology of nanogel.

1.       Drop the nanogel solution onto a TEM copper grid (300 mesh) and stand for 3 minutes

2.       Remove the nanogel solution by a filter paper

3.       Drop 2% phosphotungstic acid solution onto the copper grid and stand for 3 minutes

4.       Remove the phosphotungstic acid solution by a filter paper

5.       Dry the copper grid

6.       Observe the morphology of nanogel using TEM (Hitachi HT7700)