Polyelectrolyte complex (PEC) capsules/beads are very important for biotechnological applications such as drug delivery and bacterial whole-cell biocatalyst development. The very beam-sensitive bio-polymer capsules are laboratory produced as a uniform with a controlled shape, size, membrane thickness, permeability and mechanical resistance [1]. PEC capsules are very sensitive to any treatment and samples could be inspected in their fully native and functional state to prevent any misinterpretation. Characterization and study of PEC capsules properties is possible using thermodynamically stabile and fully wet state, precisely reached after very slow changing of conditions in the specimen chamber of ESEM. The morphological study using low current ESEM was already presented [2]. The internal structure can be in solvent, semisolid or solid state, depend on capsule type and manufacturing process [3], nevertheless it was not described in its native state yet. Study of inner part as well as surface morphology of PEC capsules using classical SEM or cryo-SEM can be misleading due to requirement of dry resp. freeze sample. The aim of this work is in-situ study of internal structure of PEC capsules in fully wet state and demonstration of state of matter of PEC capsules core.

PEC capsules has been produced by air-stripping nozzle via polyelectrolyte complexation (20 min) of sodium alginate and cellulose sulphate (CS) as polyanions, poly(methylene-co-guanidine) as a polycation, CaCl₂ as a gelling agent and NaCl as an antigelling agent [1] without the use of a multiloop reactor. Due to the high beam sensitivity of samples and its relatively big size (800 μm in diameter), a combination of our published method [4] and special improvement of our ionization detector of SE were used. The gentle and slow sample chamber pumping procedure [4] and our ionization detector of SEs [2] (beam current up to 40 pA) enhanced for larger field of view (850 μm) were combined Samples were observed in conditions of vapor pressure 684 Pa, stage temperature 2°C, humidity 97%, acc. voltage 20 kV and probe current 35 pA.

Fully wet and well preserved PEC capsule with visible surface microstructure is presented in Fig. 1A. PEC capsules are very sensitive to beam impact which was used to in-situ disruption of outer shell. Afterwards the liquid core slowly rose by capillary action on the PEC capsules wall simultaneously with capsule collapsing due to its emptying, see Fig. 1B. Due to different temperatures between the sample and the Peltier cooling stage, the liquid core was dried and crystalized on the PEC capsule surface, see Fig. 1C. First results provide promising information leading to statement that the inner structure of this type of PEC capsules is viscous liquid.

[1] A Schenkmayerová et al., Applied Biochemistry and Biotechnology 174 (5) (2014), p. 1834.

[2] V Neděla, et al., Nuclear Instrumentation and Methodology A 645 (2011), p. 79.

[3] Q-X Wu et al., Mar. Drugs 12 (2014), p. 6236.

[4] E Tihlaříková, V Neděla and M Shiojiri, Microscopy and Microanalysis 19 (2013), p. 914.

This work was supported by the Grant Agency of the Czech Republic: grant No. GA 14-22777S and LO1212 together with the European Commission (ALISI No. CZ.1.05/2.1.00/01.0017).

Figures:

Fig 1 PEC capsules with immobilized recombinant cells E. coli (10% wt). A) The PEC capsule before initiation and the same bed; B) after observation; bar is 200 μm. C) Detail of inner substance crystalized on the PEC capsule surface, bar is 20 μm.

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