2.4 Experimental
2.4.3 Synthetic Procedures
2.4.3.1 Synthesis of (propanoic acid)yl butyl trithiocarbonate (PABTC)20
A 50% w/w sodium hydroxide solution (9.68 g NaOH, 0.242 mol, 1.1 eq) in water was added to a mixture of butanethiol (20 g, 0.22 mol, 1 eq) dissolved in acetone (11 mL). Water (40 mL) was added and the solution was stirred for 30 min at room temperature. Carbon disulphide (17.32 g, 0.228 mol, 1.025 eq) was added and the orange solution was stirred for 30 minutes at room temperature, then cooled in ice below 10°C. 2- Bromopropionic acid (34.9 g, 0.228 mol, 1.025 eq) was added slowly, monitoring the temperature, and subsequently a further 19.36 g of 50% w/w sodium hydroxide solution was added. The reaction mixture was left to stir for 18 h at ambient temperature. 200 mL of water was added to the reaction mixture, cooled in ice, and a 10 M solution of HCl was added dropwise until the pH reached between 2-3. The resulting precipitate was filtered, washed with water, and recrystallised in hot hexane to afford 36.53 g of 2- (((butylthio)carbonothioyl)thio)propanoic acid. Yield = 70%. 1H NMR (400 MHz,
CDCl3) δH 6.06 (br, 1H, CO2H), 4.86 (q, J = 7.4 Hz, 1H, SCH), 3.37 (t, J = 7.4 Hz, 2H, CH2S), 1.69 (quint, J = 7.5 Hz, 2H, CH2CH2S), 1.63 (d, J = 7.4 Hz, 3H, SCHCH3), 1.43 (sext, J = 7.5 Hz, 2H, CH3CH2CH2), 0.94 (t, J = 7.3 Hz, 3H, CH3CH2). 13C NMR (100 MHz, 298 K CDCl3) δc 175.4 (COOH), 47.2 (SCH), 37.1(CH2S), 29.88 (CH2CH2S), 22.1(CH3CH2CH2), 16.4 (CH3CH), 13.6 (CH3CH2CH2). FTIR (cm-1): 3093, 2958, 2929, 2871, 2362, 2340, 1454, 1412, 1285, 1230, 1200, 1089, 1059, 912, 861. MS (ESI) m/z 237.0 [M-H], 238 [M-]
2.4.3.2 P(PEGA)8 (MRA-PEGA) synthesis
PABTC (0.31 g, 1.30 x 10-3 mol), PEGA (5 g, 10.4 x 10-3 mol) and ACVA (from a pre-
made stock solution in 1,4-dioxane) (18 mg, 6.51 x 10-5 mol) were dissolved in 4.9 mL
1,4-dioxane in a 25 mL round bottom flask equipped with a magnetic stirrer bar. The solution was fitted with an appropriately sized rubber septum, and purged with nitrogen for 20 minutes. The round bottom flask was subsequently immersed in an oil bath preheated to 70°C and stirred for 3 h. The reaction vessel was cooled to ambient temperature and opened to oxygen to quench further polymerisation. The polymer was purified by precipitation into a mixture of 20% hexane and 80% diethyl ether (v/v), collected, and the precipitation repeated once more. Finally, the precipitated polymer was
82 dissolved in DCM, transferred to a 20 mL vial, the DCM evaporated and dried in a vacuum oven overnight at 40°C to yield P(PEGA)8 as a yellow viscous liquid (4.5 g).
2.4.3.3 Macro-RAFT agent synthesis
Both MRA-nBA and MRA-tBA macro-RAFT agents were synthesized with the following general procedure, as an example P(PEGA)8-P(n-BA)8 (MRA-nBA) is
described. n-Butyl acrylate (0.25 g, 1.95 x 10-3 mol) and 0.68 mL of a 5 mg ml-1 ACVA
stock solution in 1,4-dioxane (3.4 mg, 1.21 x 10-5 mol) were added to MRA-PEGA(0.94
g, 2.43 x 10-4). The polymerisation mixture was purged with nitrogen for 20 minutes and
heated to 70°C for 3 h. The resulting polymer solution was cooled to room temperature and subsequently purified by precipitation in hexane. The yellow viscous liquid was re- dissolved in dichloromethane and the precipitation was repeated once more. Finally, the solvent was evaporated under reduced pressure to yield the di-block macro-RAFT agent as a yellow viscous liquid (0.95 g). 1H NMR spectra and SEC chromatograms can be
found above (Figure 2.2 and Figure 2.1) respectively.
Table 2.5. Summary of polymerisation conditions for Macro-RAFT agents. All reactions were performed at [M] = 1M, T = 3 h at 70°C using ACVA as thermal initiator and 1,4-dioxane as the solvent.
2.4.3.4 RAFT emulsion polymerisation procedure
Nanoparticles of different sizes and core compositions were prepared by altering various conditions, full details can be found in Table 2.6. As an example, P[(PEGA)8-b-(t-BA)8-
b-(t-BA)200] was prepared as follows. 1.43 mL of a 10 mg mL-1 sodium hydroxide stock
solution (14.3 mg, 3.6 x 10-4 mol) was added to a suspension of ACVA (50 mg, 1.8 x 10- 4 mol) in water (8.57 mL) and stirred for 30 min to ensure full solubility. P[(PEGA)
8-b-
(t-BA)8] (MRA-tBA) (0.145 g, 2.85 x 10-5 mol) was dissolved in 7.71 mL of water, in a
25 mL round bottomed flask and equipped with a magnetic stirrer. 1.45 mL of the above ACVA stock solution was added, the vial fitted with a rubber septum, and the solution was deoxygenated with dinitrogen gas for 20 minutes. Deoxygenated t-BA (0.83 mL, 2.14 x10-3 mol) was added via syringe and the polymerisation mixture was immersed in
Monomer RAFT agent [M]0/[RAFT]0 [CTA]0/[I]0 Conversion (%)
MRA-PEGA PEGA PABTC 9 20 91
MRA-nBA n-BA MRA-PEGA 8 20 96
83 a 70°C oil bath and stirred for 3 h at 400 RPM. Monomer conversion was determined via
gravimetric techniques.
Table 2.6. Synthetic conditions used to generate P[(PEGA)8-b-(n-BA)8-b-(n-BA)n]and P[(PEGA)8-b-(t-
BA)8-b-(t-BA)n] nanoparticles (where n = 200, 150, 100, 75 and 50) usingRAFT emulsion polymerisation.
Product Macro-RAFT agent Monomer [CTA][M]0/
0
[CTA]0/
[I]0 (mol L[M] -1)
P[(PEGA)8-b-(n-BA)8-(n-BA)200] MRA-nBA n-BA 200 1.1 0.57
P[(PEGA)8-b-(n-BA)8-(n-BA)150] MRA-nBA n-BA 150 1.1 0.428
P[(PEGA)8-b-(n-BA)8-(n-BA)100] MRA-nBA n-BA 100 1.1 0.285
P[(PEGA)8-b-(n-BA)8-(n-BA)75] MRA-nBA n-BA 75 1.1 0.214
P[(PEGA)8-b-(n-BA)8-(n-BA)50] MRA-nBA n-BA 50 1.1 0.143
P[(PEGA)8-b-(t-BA)8-(n-BA)200] MRA-tBA t-BA 200 1.1 0.57
P[(PEGA)8-b-(t-BA)8-(n-BA)150] MRA-tBA t-BA 150 1.1 0.428
P[(PEGA)8-b-(t-BA)8-(n-BA)100] MRA-tBA t-BA 100 1.1 0.285
P[(PEGA)8-b-(t-BA)8-(n-BA)75] MRA-tBA t-BA 75 1.1 0.214
P[(PEGA)8-b-(t-BA)8-(n-BA)50] MRA-tBA t-BA 950 1.1 0.143
All polymerisations were performed in aqueous conditions, with [macro-RAFT]0 = 2.85 mM and reached
full (>99%) monomer conversion.
2.4.3.5 PEG binding assay
Tannic acid was diluted to a concentration of 10 µg mL-1 in deionised water. Nanoparticle
suspensions were diluted with deionised water to a concentration of 10 µg mL-1. 1 mL of
the nanoparticle suspensions were transferred to a polystyrene cuvette and placed in the UV-VIS spectrometer. Absorbance measurements were recorded once every 1 s. After 1 min, 250 µL of the tannic acid solution was added via micropipette and mixed briefly without allowing the pipette tip into the detection window. The absorption was monitored for a further 4 minutes.
2.4.3.6 Encapsulation of Cyanine 7.5
Cy7.5 (1 mg) was added to 1 mL of 10% DMF in THF and sonicated until the powder had fully dissolved. 100 µL of the Cy7.5 solution was added to 900 µL of nanoparticle suspension and shaken for 1 h. The suspension was then dialysed (3500-5000 Da MWCO) against pure water for 48 h to remove residual DMF and THF. Loaded particles were used immediately after encapsulation for in vivo fluorescence studies.
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