Pd miditrap column

The above method for PDLLA-b-PAA modification
was also employed for polyglutamic acid
(PGA, Sigma-Aldrich, P4761, 20.5 kDa), heparin (Sigma-Aldrich, H3393,
18 kDa), and chondroitin sulfate A (CSA, Sigma-Aldrich, C9819) modification.
10 mg/mL PGA, 20 mg/mL heparin, or 20 mg/mL CSA was prepared in MES
buffer. 21.4 mg of AMBS or 19.4 mg of AMSA was added and dissolved
per mL of sample. Eight aliquots of 6.6 mg of EDC-HCl were added over
a period of 6–8 h, while sonicating the solution for 1 min
before addition of a new aliquot. The samples were purified by sequential
SEC using first PD MidiTrap column (GE Healthcare) equilibrated in
phosphate buffer (0.1 M phosphate (Sigma-Aldrich), 0.05 M NaCl (VWR),
pH 7.4) and second PD 10 (GE Healthcare) equilibrated in PBS. The
samples were passed through 0.22 μm syringe filters for sterilization.
The degree of modification was determined using UV–vis spectroscopy
(SpectraMax M5, Molecular Devices) using the characteristic AMBS peak
and assuming no loss of PGA or heparin during purification. The degree
of modification was confirmed by ¹H NMR in D₂O using a JEOL 400 MHz spectrometer, assuming 1 available COOH per
heparin disaccharide unit.

Films of 4:1 mol % DPPC:CH were
prepared by adding 265 μL of a 10 mg/mL DPPC (Avanti, 850355P-200MG)
stock in chloroform and 34 μL of a 10 mg/mL CH stock in chloroform
(Sigma) to a glass vial. For Et and NH₂ loaded particles, 1 μmol of Et or NH₂ in either chloroform
or THF, respectively, at 1 mg/mL was added to glass vial. chloroform
was removed by a stream of nitrogen above the solution surface for
10 min, followed by 1 h of vacuum desiccation. SO₃ and chloroquine loaded liposomes were rehydrated
with either 1 mL of 1 mM SO₃ in
DPBS (no Ca, no Mg, Sigma) or 50 mM chloroquine in ultrapure water.
After 5–6 freeze–thaw cycles, the suspension was extruded
through a 100 nm pore-size membrane using an Avanti mini-extruder.
Liposomes were purified by running 2× SEC columns through a PD
MidiTrap column (GE Healthcare) equilibrated DPBS.

Empty polymersomes and polymersomes
loaded with 2 mM Et and COOMe were prepared,
as described previously. Samples then underwent buffer exchange to
deuterated PBS for SANS measurements (Gibco PBS tablets (ThermoFisher
Scientific) dissolved in D₂O). Samples in PBS were passed
through a PD MidiTrap column (GE Healthcare) equilibrated in deuterated
PBS. All of the measurements were performed at the ZOOM beamline of
the ISIS Pulsed Neutron Source at the Rutherford Appleton Laboratory,
Didcot, UK. A sample changer and 2 mm path length quartz cuvette cells
were used. The beamline was configured with L1 = L2 = 4 m, where L1
is the source to sample distance and L2 is the sample to detector
distance, yielding a scattering variable (Q) range
of 0.004 to 1 Å^–1. Samples were measured at
15 μAmps (SANS) and 5 μAmps (TRANS) at 25 °C.
SANS data were reduced with MantidPlot.^{53 (link)} SasView v5.0.4. (http://www.sasview.org/) was employed to fit the experimental data over a q range of 0.01 < q < 0.1 Å^–1 with a polydispersity of 0.1 on the radius and shell thickness.
Scattering length density (sld) of core, shell, and solvent were set
to to 6.3, 4, and 6.3 × 10^–6/Ų, respectively.