Poly(N-isopropylacrylamide)-block-poly(acrylic acid), PNIPAAm-b-PAA, with low polydispersity was prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization in methanol. The block copolymers respond to both temperature and pH stimuli. The behavior of the doubleresponsive block copolymers in solution was investigated by dynamic light scattering, temperature-sweep NMR, cryogenic transmission electron microscopy, and IR spectroscopy. The block copolymers form micelles in aqueous solutions in dependence of pH and temperature. Cloud point measurements indicated the formation of larger aggregates at pH 4.5 and temperatures above the lower critical solution temperature (LCST) of PNIPAAm. The solution behavior is strongly influenced by hydrogen bonding interactions between the NIPAAm and acrylic acid blocks.
The principles of RAFT polymerization were applied to the polymerization of N-isopropylacrylamide (NIPAAm), which was carried out in the presence of the dithiocarbamates benzyl 1-pyrrolecarbodithioate and cumyl 1-pyrrolecarbodithioate, respectively, as chain transfer agents in 1,4-dioxane at 60 °C. A kinetic investigation using in situ FT-NIR spectroscopy shows very long induction periods which depend on the nature and concentration of the chain transfer agent. The resulting polymers have polydispersity indices M w/Mn < 1.3 and have been investigated by MALDI-TOF mass spectrometry, GPC, NMR, and UV spectroscopy. The expected end group signals for chain transfer agent (CTA) and initiator could be identified together with fragmentation of the dithioester end group under MALDI conditions. The number-average molecular weights obtained by MALDI-TOF MS are significantly lower than those obtained by GPC with polystyrene calibration. With the use of the abovementioned dithiocarbamates, new thiocarbonylthio compounds have been applied in the RAFT polymerization of N-isopropylacrylamide.
Block copolymers containing stimuli-responsive segments provide important new opportunities for controlling the activity and aggregation properties of protein-polymer conjugates. We have prepared a RAFT block copolymer of a biotin-terminated poly(N-isopropylacrylamide) (PNIPAAm)-b-poly(acrylic acid) (PAA). The number-average molecular weight (M(n)) of the (PNIPAAm)-b-(PAA) copolymer was determined to be 17.4 kDa (M(w)/M(n) = 1.09). The PNIPAAm block had an M(n) of 9.5 kDa and the poly(acrylic acid) (PAA) block had an M(n) of 7.9 kDa. We conjugated this block copolymer to streptavidin (SA) via the terminal biotin on the PNIPAAm block. We found that the usual aggregation and phase separation of PNIPAAm-SA conjugates that follow the thermally induced collapse and dehydration of PNIPAAm (the lower critical solution temperature (LCST) of PNIPAAm is 32 degrees C in water) is prevented through the shielding action of the PAA block. In addition, we show that the cloud point and aggregation properties (as measured by loss in light transmission) of the [(PNIPAAm)-b-(PAA)]-SA conjugate also depended on pH. At pH 7.0 and at temperatures above the LCST, the block copolymer alone was found to form particles of ca. 60 nm in diameter, while the bioconjugate exhibited very little aggregation. At pH 5.5 and 20 degrees C, the copolymer alone was found to form large aggregates (ca. 218 nm), presumably driven by hydrogen bonding between the -COOH groups of PAA with other -COOH groups and also with the -CONH- groups of PNIPAAm. In comparison, the conjugate formed much smaller particles (ca. 27 nm) at these conditions. At pH 4.0, however, large particles were formed from the conjugate both above and below the LCST (ca. 700 and 540 nm, respectively). These results demonstrate that the aggregation properties of the block copolymer-SA conjugate are very different from those of the free block copolymer, and that the outer-oriented hydrophilic block of PAA shields the intermolecular aggregation of the block copolymer-SA bioconjugate at pH values where the -COOH groups of PAA are significantly ionized.
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