Membrane Formation in Polymersomes
Polymersomes are unilamellar vesicals produced from amphiphilic synthetic block copolymers. The diameter of polymersomes ranges from 50 nm up to 50 µm. In comparison to liposomes, natural vesicals formed by lipid membranes, the polymer membranes of polymersomes provide a much stroger physical barrier and can be used for almost complete encapsulation and protection of sensitive molecules.
In order to better unserstand and eventually tune the membrane properties of polymersones, the formation and porperties of the polymersome membrane have been probed on the molecular level by NMR methods. The challenging point of this study is the very low amount of relatively rigid polymer material in an aqueous environment.
Both overall rotation of the polymersomes as well as local polymer modes in the polymersome membrane will determine T1 and T2 relaxation times of the polymer membrane material and thus determine the spectral resolution of the NMR spectra. The main challenge for NMR studies of poylsmersomes, however, is the very low polymer concentration in highly concentrated polymersome solutions. Assuming a dense cubic packing of polymersomes with a diameter 40 µm and a membrane thickness of 40 nm leads to polymer concentration of 1.56 g/L, which leads to realistic sample with less 0.1 g polymer/L.
Due to this very low polymer concentration, even in sample prepared with highly deuterated D2O the residual water signal is orders of magnitudes stronger than the polymer signal. The most efficient water suppression sequences like e.g. excitation sculping rely on relatively long shaped gradient pulses are well suited for samples with long T2 times. The signals of the dynamically restricted polymer memebrane material, however, have short T2 times and therefore would be signifiantly reduces and filtered by these pulse sequences. The best NMR results have been obtained applying water signal pre-saturation techniques, which may lead to NOE based intensity distortions but do not act as T2 filter for the broad polymer signals.
The spectral resolution of the NMR spectra can slightly be improved using high-resolution magic angle spinning (HR-MAS) NMR methods. Dispite significant centrifugal forces in the MAS rotor, the large polymersomes were not distroyed during the HR-MAS measurements is confirmed by inspection of several sample under the optical microscope after the HR-MAS NMR measurements in 4 mm MAS rotors and a spinning frequency of 5 kHz for serveral hours.
The 1H HR-MAS NMR spectra of polymersomes obtained from oleyl alcohol and from toluene indicate that a significant amount of oley alcohol stays in the membrane material and diffuses with the polymersomes, while toluene molecules are completely removed from the polymersome membrane and diffuse much faster than the polymersomes as seen the duffision odered spectrocopy (DOSY) NMR, where the NMR signals of the different components are correlated with their translational diffusion coefficient. When comparing the NMR signals of the different polymersomes, the signals of the polymersomes from oleyl alcohol are significantly more narrow. This indicates that oleyl alcohol remaining in the polymersome membrane material acts as softener for the membrane. The increased molecular mobility of the membrane polymer facilitates the membrane transfection of smaller molecules.
Literature and References