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WP2 - Heterophase Microcontainers

Two different chemtainer addressing systems have been developed.  One is based on electrostatic addresses and results in both address-specific assembly of vesicle chemtainers and subsequent fusion, tunable through address titration (Sunami et al, 2010 and Caschera et al, 2011). The second system is a standardized and exportable DNA address system protocol has been developed at SDU.  This has been used successfully on site at SDU, WISb and now at RUBa and is enjoyed by the consortium.  This system has been demonstrated to be compatible with DNA address-mediated assembly of vesicle chemtainers, DNA computing, solid supports, and addressed microfluidic platforms. The protocol and assembly of addressed vesicles has been published in the first year in PLoS One (Hadorn and Eggenberger Hotz, 2010).  A follow-up publication on the use of this DNA address system with oil droplet chemtainers has been published in PNAS (Hadorn et al, 2012). In addition, during the third year of the project, WP2 has been able to make significant progress on executing chemtainer fusion fission cycles, (Caschera et al 2012).

Substantial progress had been achieved through collaboration of WP2 with WP3 on DNA address anchoring systems, with WP4 on DNA computing, with WP5 on implementation in microfluidics and WP6 on theory, modeling and analysis.

Address systems for chemtainers

For the charged address system, SDUa has shown a proof of principle programmed chemtainer fusion system using vesicles during the first year of MATCHIT in close collaboration with the group of Tetsuya Yomo at Osaka University.  In this system vesicles of POPC are prepared with different contents.  The vesicles are then decorated with either a net negative or positive charge.  The vesicles are then mixed together and were shown to aggregate and fuse depending on the charge-charge interaction.  The positive fusion signal was demonstrated using a fluorescent reporter and quencher system.  These results were published in two peer reviewed publications (Sunami et al, 2010 and Caschera et al, 2011).

For the DNA-based address system, SDUa has developed a protocol for effectively labeling chemtainers (precisely vesicles, oil droplets and emulsions) without disturbing the integrity or utility of the chemtainer.  This is done by first forming the chemtainer using a low molar percentage of a biotinylated lipid (bPEG2000-DSPE) in the presence of POPC.  A biotinylated-ssDNA address of choice is then allowed to complex with streptavidin in a separate preparation.  The ssDNA address bound to streptavidin is the introduced to and incubated with the chemtainer containing the biotinylated lipid.  After careful washing, we have shown that we can produce chemtainers with any DNA address of choice.  These addresses are stable (i.e. no significant exchange between chemtainers seen) and are able to coordinate address specific hybridizaton and assembly of chemtainer structures, see Figure 2.1. Therefore we are satisfied with this system for effectively addressing our chemtainers and this was published Hadorn and Eggenberger Hotz, 2010). We recently showed that this DNA anchoring and addressing system is suitable not only for vesicle chemtainers but also for oil dropelts as we reported in PNAS in 2012.

Figure 2.1: DNA-addressing system for oil droplet chemtainers. Specific detail of emulsion droplet aggregation. (A) Scheme of the surface functionalization of a binary mixture of emulsion droplet (ED) populations functionalized with complementary biotinylated single stranded DNA (btn-ssDNA) oligonucleotides. The surface of the EDs was functionalized with btn-ssDNA oligonucleotides using streptavidin (Strept.) as a connecting element. Both Strept. populations were fluorescently modified by Alexa Fluor (AF), resulting in ED (Left) labeled green (AF488) and ED (Right) labeled red (AF532). (B) Representative fluorescence micrograph of a binary mixture of EDs  functionalized with complementary btn-ssDNA oligonucleotides. θ1 and θ2, angles of contact; R1 and R2, radii of EDs. (Scale bar: 5 μm.).  Image from Hadorn et al, PNAS 2012.

Chemtainer replication cycles

Figure 2.2: A two-generation oil droplet in water replication cycle. Generation 0: a 20 μl nitrobenzene droplet with sudan black B (blue) and CTAB is added to 100 μl 5 mM oleate pH 12.  After droplet fission 1, 10ul of the divided droplets are transferred to a chamber containing 100 μl 20 mM CTAB pH 12 and a 10 μl nitrobenzene droplet with oil red O (red).  Fusion between the droplets is induced by adding 5 M NaCl. Generation 1: after fusion 1, the process is reiterated, see text and Movie S3. The diameter of each frame is 2.0 cm. Figure derived from Cashera et al, 2012.

We developed one chemtainer specifically, the oil droplet, towards a complete fission and fusion cycle.  Fission has been explored using physical instabilities induced in the chemtainer oil droplet.  This is a particularly good system for fission as interfacial dynamics coupled with fluid dynamics can lead to spontaneous fission under non-equilibrium conditions.  The principle is based on non-equilibrium dynamics of catanoinic systems.  As the system approaches equilibrium, surface instabilities lead to flow and spontaneous fission. SDUa coupled this with droplet fusion to show a rudimentary chemtainer droplet cycle including chemical iodination reaction induced by chemtainer fusion, Figure 2.2. This work has recently been published (Caschera et al, 2012).

Chemtainer stability and hierarchy

We explored the conditions that would allow chemtainers to release or share contents.  We completed a basic study of how a series a salts effect the production and stability of POPC-based vesicles.  These are the same vesicles we use for DNA addressing.  The determination of salt effects will aid us in developing protocols for chemtainer content release and sharing.  The data from this investigation was published in Nature Scientific Reports (Hadorn et al, 2011); and a peer-reviewed proceedings paper showing how this concept in the context of the MATCHIT goals can be used in the next generation of drug therapy was published in Biomedical Engineering Systems and Technologies (Hadorn and Hotz, 2011).

Finally we have developed a hierarchical chemtainer within chemtainer system for membrane computing.  Assembly of vesicles of one type within a parent vesicle of another type is now possible and programmable, see Figure 2.3.  The internal daughter vesicles and external parent vesicle contain anchors for DNA addressing (Hadorn et al, 2012b).

Figure 2.3: Vesicles within vesicles for membrane computing.  Large parent vesicles (green, B) were prepared selectively with smaller daughter vesicles inside (red, C). The efficiency and programmability of this process is evident in microscopy (A). Scale bar: 25 μm. From Hadorn et al, 2012b.


Hadorn M, Boenzli E, Sørensen KT, Fellermann H, Eggenberger Hotz P, Hanczyc MM. 2012a. Specific and Reversible DNA-directed Self-Assembly of Oil-in-Water Emulsion Droplets. PNAS, doi: 10.1073/pnas.1214386109.
Caschera F, Rasmussen S, Hanczyc MM.  2012. An Oil Droplet Division-Fusion Cycle, ChemPlusChem, DOI: 10.1002/cplu.201200275.
Hadorn M, Boenzli E, Eggenberger Hotz P, Hanczyc MM. 2012b. Hierarchical Unilamellar Vesicles of Controlled Compositional Heterogeneity, PLoS ONE, 7 (11) e50156.
Caschera F, Bernardino de la Serna J, Loffler PMG, Rasmussen TE, Hanczyc MM, Bagatolli LA, Monnard P-A. Stable Vesicles Composed of Monocarboxylic or Dicarboxylic Fatty Acids and Trimethylammonium Amphiphiles. Langmuir, 27 (23), 14078–14090. (2011a)
A Quantitative Analytical Method to Test for Salt Effects on Giant Unilamellar Vesicles / Hadorn, M.; Boenzli, E. & Eggenberger, P. H. Scientific Reports 1 (168) (2011)
Mode Switching and Collective Behavior in Chemical Oil Droplets. / Horibe, N.; Kobayashi, K.; Hanczyc, M. and Ikegami, T.Entropy 2011, 13(3), 709-719 (2011).
Caschera F, Sunami T, Matsuura T, Suzuki H,. Hanczyc MM, Yomo T. Programmed Vesicle Fusion Triggers Gene Expression. Langmuir 2011, 27, 13082–13090. (2011b)
Hadorn, M. & Hotz, P.E., Encapsulated Multi-vesicle Assemblies of Programmable Architecture: Towards Personalized Healthcare. in Biomedical Engineering Systems and Technologies, edited by A. Fred, J. Filipe, & H. Gamboa (Springer-Verlag Berlin, Berlin, 2011), Vol. 127, pp. 141-151. (2011)
Hadorn M and Eggenberger Hotz P. DNA-Mediated Self-Assembly of Artificial Vesicles. Plos One 5(3):e9886. (2010).
Sunami T, Caschera F, Morita Y, Toyota T, Nishimura K, Matsuura T, Suzuki H, Hanczyc MM, Yomo T. Detection of Association and Fusion of Giant Vesicles Using a Fluorescence-Activated Cell Sorter, Langmuir 26(19), 15098–15103. (2010).