Thermoresponsive Poly(N,N′-dimethylacrylamide)-Based Diblock Copolymer Worm Gels via RAFT Solution Polymerization: Synthesis, Characterization, and Cell Biology Applications

RAFT solution polymerization is used to polymerize 2-hydroxypropyl methacrylate (HPMA). The resulting PHPMA precursor is then chain-extended using N,N′-dimethylacrylamide (DMAC) to produce a series of thermoresponsive PHPMA-PDMAC diblock copolymers. Such amphiphilic copolymers can be directly dispersed in ice-cold water and self-assembled at 20 °C to form spheres, worms, or vesicles depending on their copolymer composition. Construction of a pseudo-phase diagram is required to identify the pure worm phase, which corresponds to a rather narrow range of PDMAC DPs. Such worms form soft, free-standing gels in aqueous solution at around ambient temperature. Rheology studies confirm the thermoresponsive nature of such worms, which undergo a reversible worm-to-sphere on cooling below ambient temperature. This morphological transition leads to in situ degelation, and variable temperature 1H NMR studies indicate a higher degree of (partial) hydration for the weakly hydrophobic PHPMA chains at lower temperatures. The trithiocarbonate end-group located at the end of each PDMAC chain can be removed by treatment with excess hydrazine. The resulting terminal secondary thiol group can form disulfide bonds via coupling, which produces PHPMA-PDMAC-PHPMA triblock copolymer chains. Alternatively, this reactive thiol group can be used for conjugation reactions. A PHPMA141-PDMAC36 worm gel was used to store human mesenchymal stem cells (MSCs) for up to three weeks at 37 °C. MSCs retrieved from this gel subsequently underwent proliferation and maintained their ability to differentiate into osteoblastic cells.


Figure
Figure S1. 1 H NMR spectra recorded for the MePETTC RAFT agent and the PHPMA141 precursor.

Figure S2 .
Figure S2.Oscillatory rheology data obtained for PHPMA148-PDMAC39 worms of varying copolymer concentration at (a) 20 °C and (b) 37 °C.Oscillatory rheology data obtained for a 10% w/w aqueous dispersion of PHPMA148-PDMAC39 worms as a function of temperature in (c) the absence and (d) the presence of PBS.

Figure S4 .
Figure S4.Digital images of freeze-dried PHPMA141-PDMAC36 diblock copolymer powder recorded before and after end-group removal.

Figure S5 .
Figure S5. 1 H NMR spectra to confirm successful RAFT end-group removal for the PHPMA141-PDMAC36 diblock copolymer.

Figure S6 .
Figure S6.Linear calibration curve obtained for the Picogreen assay used to determine the mass of DNA after MSC encapsulation within worm gels.

Figure S7 .
Figure S7.Mass of DNA recorded for MSCs retrieved from two worm gels after encapsulation for 21 days at 37 °C.

Figure S8 .
Figure S8.Fluorescence microscopy images obtained for a live/dead assay.

Figure S9 .
Figure S9.Fluorescence microscopy control images obtained for the immunocytochemical detection of Ki-67.

Figure S2 .
Figure S2.Oscillatory rheology data obtained (a) at 20 ℃ and (b) at 37 ℃ for a series of PHPMA148-PDMAC39 worms of varying copolymer concentration.[N.B.G' data are denoted by solid circles (green for 20 ℃ and light blue for 37 ℃) and G'' data are denoted by solid squares (red for 20 ℃ and blue for 37 ℃)].(c) Variable temperature oscillatory data obtained during heating (G' data denoted by red solid circles, and G'' data denoted by light red solid squares) and cooling (G' data denoted by blue solid circles, and G'' data denoted by light blue solid squares) for a 10% w/w aqueous dispersion of PHPMA148-PDMAC39 worms.(d) Variable temperature oscillatory data obtained during heating (G' data denoted by red solid circles, and G'' data denoted by light red solid squares) and cooling (G' data denoted by blue solid circles, and G'' data denoted by light blue solid squares) for a 10% w/w aqueous dispersion of PHPMA148-PDMAC39 worms in the presence of PBS.All oscillatory rheology experiments were conducted at a strain of 1.0% and an angular frequency of 1.0 rad s -1 .

Figure S4 .
Figure S4.Digital photographs recorded for freeze-dried PHPMA141-PDMAC36 diblock copolymer powder before and after end-group removal.Note the change in color from pale yellow (left vial) to white (right vial).

Figure
Figure S5. 1 H NMR spectra (CD3OD) recorded (a) an as-synthesized PHPMA141-PDMAC36 diblock copolymer and (b) the same copolymer after removal of its RAFT end-groups by using a twenty-fold excess of propylamine at 20 ℃.

Figure S6 .
Figure S6.Linear calibration curve obtained for the Picogreen assay used to determine the amount of DNA in MSCs retrieved from 6% PHPMA135-PGMA55 gels or 4% PHPMA141-PDMAC39 gels.Fluorescence was recorded at 85% gain using an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

Figure S8 .
Figure S8.Fluorescence microscopy images recorded for live/dead staining of MSCs retrieved after 14 days encapsulation within a 4% w/w PHPMA141-PDMAC36 worm gel (image A) or a 6% w/w PHPMA135-PGMA55 worm gel (image B). Green fluorescence indicates uptake of the CMFDA (live stain) within the cytoplasm of viable MSCs.Red fluorescence indicates the uptake of propidium iodide (dead stain) within the nuclei of dead/dying MSCs.Scale bar = 100 µm in each case.

Figure S9 .
Figure S9.Fluorescence microscopy images recorded for the immunocytochemical detection of Ki-67.In this control experiment, the staining protocol was performed for actively proliferating monolayer cultures of MSCs in the absence of the primary antibody to Ki-67.Image A shows the blue fluorescence from the nuclear stain, DAPI.Image B confirms that no green fluorescence is observed with the secondary antibody when the primary (nonfluorescent) antibody to Ki67 is omitted.This lack of fluorescence demonstrates that the binding between the Ki67 antibody and the secondary fluorescent antibody is specific -there is no non-specific binding with the secondary fluorescent antibody.Image C is a merged image combining image A and image B. Scale bar = 200 µm in each case.