1 Introduction
2.1 Materials used during this study
Hydroxypropyl methacrylate (HPMA) 97 %, ethylene glycol dimethacrylate 98 % (EGDMA), ethyl α-bromoisobutyrate (EBiB) 98 %, α-bromoisobutyryl bromide 98 %, methoxypolyethylene glycol average mol wt 750, 2000, 5000 g mol-1, Cu(I)Cl
>99 %, 2,2,’-bipyridyl >99 %, aluminium oxide (activated, basic), Dowex Marathon exchange resin, Celite, Oil Red O, ibuprofen >98 %, pyrene > 99 %, 4- (dimethylamino) pyridine >98 %, triethylamine (TEA) >99 %, Amberlyst resin, anisole (anhydrous) 99.7 %, methanol (HPLC grade), tetrahydrofuran (HPLC grade),
n-hexane, acetone (HPLC grade), toluene (anhydrous), dialysis tubing (benzoylated) molecular weight cut off = 2000 g mol-1, Whatman 200 nm PTFE syringe filters were all purchased from Sigma-Aldrich and used without any further purification. CDCl3 and d6-DMSO solvents were purchased from GOSS Scientific. Lopinavir
was purchased from LGM Pharmaecuticals (Chicago).
2.2 Equipment utilised during the characterisation of linear and
branched polymers and nanoparticles formed from the polymers
2.2.1
1H NMR Spectroscopy
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H NMR spectra were recorded in either DMSO-d6, CDCl3 or D2O using a 400 MHz
Brüker Avance spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) with respect to an internal reference of tetramethylsilane.
NMR spectroscopy can be used to calculate Mn values using end-group analysis
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comparing the strength of end-group signals to signals originating from the polymer backbone, the ratio of end group: repeat unit can be calculated, (DPn). Samples were
prepared by dissolving approximately 0.05 g of product in 1-2 mL of solvent.
This method is subject to inaccuracy, especially as chain length increases and the end-group signal becomes weaker. For copolymers, the solvent also has a great influence, where solubility of copolymer components may be different. However, with appropriate solvent choice, end-group analysis can be a useful technique allowing determination of composition and Mn of complex structures such.
2.2.2 Gel Permeation Chromatography
Triple detection gel permeation chromatography (GPC) (either HPLC grade tetrahydrofuran (THF) stabilized with 2,6-di-tert-butyl-4-methylphenol, or HPLC grade acetone eluent) with a flow rate of 1 mL min-1 was performed using a Malvern Viscotek 270 Max instrument using T6000M x's 2 + guard column set. All samples were dissolved at 5 mg mL-1 and passed through a 200 nm syringe filter prior to injection (100µL) with a run time of 60 minutes.
GPC (also known as size exclusion chromatography) analysis relies on different hydrodynamic sizes of polymer molecules in solution which are separated. Columns are packed with porous beads, and dilute samples of polymer are injected into these columns. Depending on polymer sizes in solution, chains are able to penetrate the beads and are eventually eluted. Larger polymer chains are less able to diffuse through pores than small chains, therefore they pass through columns faster. The smallest polymer molecules will penetrate the most pores, and are therefore eluted at longer retention times. Detector signals are plotted against elution volume or time. Elution volume for a given molecule is given by: Ve = V0 + KeVi,where Ve is elution
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volume, V0 is void volume; i.e. the volume between the beads. Vi is the internal
volume (i.e. the volume inside porous beads) and Ke is a coefficient defined by the
partition of the polymer molecules between voids and the beads. If all chains are large chains are excluded from all bead pores, Ke = 0 and Ve = V0. If all chains are
very small and penetrate all bead pores, Ke = 1 and Ve = V0 + Vi. For a polymer
sample to be separated, elution volume must lie between the limits i.e. Vi<Ve< V0 +
Vi.
Triple detection GPC uses a concentration detector (refractive index detector (RI)) which is based on deflection of light passing through a dual component flow cell (one containing a reference solvent and the other containing solvent and sample). The difference in refractive index can be used to determine sample concentration with respect to time during polymer separation; plotted as a detector response vs.
elution volume (or time). For light scattering detectors to determine molecular weight, they use the Rayleigh Equation:
Where the intensity of scattered light is equal to an optical constant (K) multiplied by concentration (C) and molecular weight (M). To record the intensity of scattered light the measuring angle must be 0°; since this is not possible, angles close to 0° are used. Low angle light scattering is performed at angles typically >10 °. Right angle light scattering data (at 90°) is used in conjunction to viscosity measurements to correct the angle back to 0°. The viscometer measures intrinsic viscosity of samples, which may allow determination of molecular size.
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2.2.3 Dynamic light scattering (DLS)
DLS studies of nanoparticle dispersions were performed using a Malvern Zetasizer Nano ZS equipped with a 4 mW He–Ne, 633 nm at a temperature of 25 °C and using plastic disposable cuvettes for aqueous dispersions. Glass cuvettes were used for solutions containing organic solvents. Malvern Zetasizer software version 6.20 was used for data analysis using the instruments automatic optimisation settings. It should be noted that measurements were taken directly from the nanoparticle dispersions without any additional filtration or centrifugation.
2.2.3.1 Zeta potential measurements
Carried out at 1 mg mL-1, at 25 °C, pH of 6.5, using disposable capillary zeta cells; measurements were obtained using the instruments automatic optimisation settings.
2.2.4 Scanning Electron Microscopy (SEM)
SEM images were recorded using a Hitachi S-4800 FE-SEM at 3 kV. Aqueous nanoparticle solutions (50 mL, containing approximately 0.1 mg mL-1 polymer) were pipetted onto aluminium stubs. Clinical tissue was used to lightly dab the solution and the stubs were left to dry for approximately 3 hours. The dry samples were gold coated for 2 minutes at 15 mA using a sputter-coater (EMITECH K550X) prior to imaging.