129
15. Hexane (>99%) (see Note 4).
16. Dimethylformamide (DMF) (99.9+%, special grade for peptide synthesis with less than 50 ppm of active impurities) (see
Note 5) is the polymerization solvent.
17. 1,6-Diaminohexane (98%) is the initiator of the polymerization (see Note 6).
3 Methods
1. Use size-exclusion chromatography (SEC) to determine the
M n and M w /M n values. Perform the analysis using a SEC
equipment composed of 600 high-pressure liquid chromatographic pump, Ultrastyragel columns, a differential refractometer detector, and a precision PD light scattering detector at
60 °C featuring two detectors at 15° and 90° (TALLS). Use a
DMF solution containing 0.1 N LiBr as an eluent at a rate of
1 mL/min, operating at 60 °C.
2. Perform nuclear magnetic resonance spectroscopy (
1
H NMR),
300 MHz. Take the spectra of the N-carboxyanhydrides
(NCAs) in CDCl 3 at room temperature.
3. Perform Fourier transform infrared (FTIR) spectroscopy measurements in KBr pellets at room temperature, in the range of
450–4000 cm
−1
.
4. Perform circular dichroism with an instrument containing
thermo-stabilizing system. Use cell of 1 mm Quartz Suprasil.
The aqueous solution concentration of the PBLG/PHIS
polymer with a ratio of the monomeric units 50/50 is
3.3 × 10
−4
g/mL, while the aqueous solution concentration of
the PBLG/PHIS polymer with a ratio of the monomeric units
70/30 at every pH is 2.5 × 10
−4
g/mL. Perform the adjustment of pH through addition of diluted HCl or NaOH.
5. Perform scanning electron microscopy (SEM)-EDS of the
hydrogels using carbon grids. Prepare the samples first by
quick deep freezing of a small amount of the hydrogels with
liquid nitrogen, followed by freeze-drying to remove water.
Make sputter coating of the samples with gold prior to the
measurements.
6. Perform enzymatic degradation-fluorescence measurements
using 10 M multimode microplate reader and 96-well black
microplates. To measure the enzymatic degradation of the
hydrogel by either trypsin or leucine aminopeptidase (LAP),
mix 2 mg of hydrogel with 0.1 nM trypsin or 100 nM LAP in
PBS and incubate at 37 °C for 1 or 24 h, respectively. At the
end of the incubation, mix a sample of 10 μL from the reaction with 90 μL of a solution of fluorescamine containing
3.1 Instrumentation
Nanostructured Hydrogels for Controlled Drug Delivery
15. Hexane (>99%) (see Note 4).
16. Dimethylformamide (DMF) (99.9+%, special grade for peptide synthesis with less than 50 ppm of active impurities) (see
Note 5) is the polymerization solvent.
17. 1,6-Diaminohexane (98%) is the initiator of the polymerization (see Note 6).
3 Methods
1. Use size-exclusion chromatography (SEC) to determine the
M n and M w /M n values. Perform the analysis using a SEC
equipment composed of 600 high-pressure liquid chromatographic pump, Ultrastyragel columns, a differential refractometer detector, and a precision PD light scattering detector at
60 °C featuring two detectors at 15° and 90° (TALLS). Use a
DMF solution containing 0.1 N LiBr as an eluent at a rate of
1 mL/min, operating at 60 °C.
2. Perform nuclear magnetic resonance spectroscopy (
1
H NMR),
300 MHz. Take the spectra of the N-carboxyanhydrides
(NCAs) in CDCl 3 at room temperature.
3. Perform Fourier transform infrared (FTIR) spectroscopy measurements in KBr pellets at room temperature, in the range of
450–4000 cm
−1
.
4. Perform circular dichroism with an instrument containing
thermo-stabilizing system. Use cell of 1 mm Quartz Suprasil.
The aqueous solution concentration of the PBLG/PHIS
polymer with a ratio of the monomeric units 50/50 is
3.3 × 10
−4
g/mL, while the aqueous solution concentration of
the PBLG/PHIS polymer with a ratio of the monomeric units
70/30 at every pH is 2.5 × 10
−4
g/mL. Perform the adjustment of pH through addition of diluted HCl or NaOH.
5. Perform scanning electron microscopy (SEM)-EDS of the
hydrogels using carbon grids. Prepare the samples first by
quick deep freezing of a small amount of the hydrogels with
liquid nitrogen, followed by freeze-drying to remove water.
Make sputter coating of the samples with gold prior to the
measurements.
6. Perform enzymatic degradation-fluorescence measurements
using 10 M multimode microplate reader and 96-well black
microplates. To measure the enzymatic degradation of the
hydrogel by either trypsin or leucine aminopeptidase (LAP),
mix 2 mg of hydrogel with 0.1 nM trypsin or 100 nM LAP in
PBS and incubate at 37 °C for 1 or 24 h, respectively. At the
end of the incubation, mix a sample of 10 μL from the reaction with 90 μL of a solution of fluorescamine containing
3.1 Instrumentation
Nanostructured Hydrogels for Controlled Drug Delivery