Received 24th March 2010, Accepted 14th July 2010 www.rsc.org/loc | Lab on a Chip 2010, 10, 2741–2748 Biomolecular-motor-based autonomous delivery of lipid vesicles as nano- or microscale reactors on a chip Satoshi Hiyama,*Yuki Moritani, Riho Gojo, Shoji Takeuchic and Kazuo Sutoh* Received 24th March 2010, Accepted 14th July 2010 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo 2010/11/25

Kinesin on immobilized microtubule (bead assay) Active transport… Biomolecular motor based on chip system, two type geometrical arrangements… Kinesin on immobilized microtubule (bead assay) Microtubule on immobilized kinesin (gliding assay) Microfabricated silicon, glasses, quantum dots, proteins, DNAs, RNAs… To this study, targeted delivery of liposomes was not done Liposomes are artificially prepared vesicles made of lipid bilayer. Liposomes can be filled with drugs, and used to deliver drugs for cancer and other diseases. Liposomes can be prepared by disrupting biological membranes, for example by sonication and its inner diameter can be controlled from nanometer to micrometer. 2010/11/25

2008, 4, No. 4, 410–415 N-[γ-maleimidobutyryloxy] succinimide ester-sulfo GMBS Microbeads of 1 μm diameter was used. Autonomous Loading, Transport, and Unloading of Specified Cargoes by Using DNA Hybridization and Biological Motor-Based Motility Biomolecular-Motor-Based Nano- or Microscale Particle Translocations on DNA Microarrays 2010/11/25

For the study five type of DNA sequences are used… The spacer was introduced to prevent the collapse of ssDNAs onto the attached surfaces and to keep the single-stranded sticky end free. The sequences used in the sticky end regions were selected from a set of orthogonal base sequences, designed according to theoretical and experimental considerations to minimize mismatched hybridization and to enable rapid hybridization at 25 0C. 2010/11/25

For the study three type of liposomes are used… Materials… For the study three type of liposomes are used… Giant liposomes (diameters ranged from 1 mm to 5 mm) were used to clearly visualize the loading, transport, and unloading processes. Three types of large liposomes (590, 300, and 180 nm in diameter), were used to perform statistical analyses of loading and unloading efficiencies. Small liposomes ( diameter 100 nm), were used to confirm the versatility of our proposed system. 2010/11/25

Using fluorescent microscopy. Giant liposomes. Loading … Using fluorescent microscopy. Giant liposomes. Average gliding speed of 0.31 µm s-1 Some MTs loaded multiple liposomes onto a single MT. Hydrophilic fluorescent dye, calcein, was encapsulated (see 65 s and 115 s). 2010/11/25

Large liposome having 300 nm diameter… To know the effect of the number of ssDNAs attached to a single liposome….. Large liposome having 300 nm diameter… It may be because of MT–liposome aggregations… In five distinct microscopic fields… Result: Loading of reactor liposomes are due to the DNA hybridization. There should be an optimum number of liposome -attached ssDNAs for efficient loading and transport of reactor -liposomes. 2010/11/25

Effect of the size of liposomes… The number of generated liposomes decreased as the size of the liposomes increased Larger reactor-liposomes should be superior to smaller reactor-liposomes in the transport efficiency of encapsulated molecules since it has larger inner volume. labeled with 100 ssDNAs complementary to those attached to the MTs 2010/11/25

Using fluorescent microscopy. Giant liposomes. Unloading… Using fluorescent microscopy. Giant liposomes. The liposome loaded MTs (in a separate test tube) injected into a flow cell where ssDNAs complementary to those in the liposomes were micro-arrayed as unloading sites. Partial DNA hybridizations are not as strong as complete DNA hybridizations. 2010/11/25

Unloading by selective DNA hybridization…??? Ten distinct microscopic fields showing nine unloading site. Monodispersed large liposomes (300 nm diameter) used. Most of the cargo-liposomes were unloaded within the first 30 min which is faster than microspheres cargo case and may be due to non-specific interactions b/w MTs and liposomes were efficiently suppressed . Without ATP the number of liposomes on the unloading sites did not increase over time, which shows that unloading was by active transport using gliding MTs not by passive transport by simple Brownian motions. Partial DNA hybridization is stable. Result:- The number of liposomes on the non-complementary unloading sites did not increase over time shows that unloading of liposomes is by selective DNA hybridization. 2010/11/25

Unloading process may be due to … …strand exchange reaction, a spontaneous transition from a relatively stable state to a more stable state …and also be possible in the context of a mechanical analysis. Assuming that a cargo-liposome has free 23-base ssDNAs that have not yet hybridized with 15-base ssDNAs of an MT and the transported liposome passes through a complementary unloading site, the free 23-base ssDNAs initially hybridize with those of the unloading site and the transported liposome is trapped at the site. Cholesterol-modified 23-base ssDNAs that are hybridized with 15-base ssDNAs of the MT are then extracted from the liposomal membrane due to the exerted force by the kinesin molecules (up to ca. 7 pN per single kinesin molecule). This event occurs prior to strand exchange reactions and liposomal deformation, thereby resulting in the completion of the liposome unloading. Unloading efficiency… Liposomes ( 300 nm in diameter) labeled with 50 ssDNAs complementary to the MTs used. 70 cargo-liposomes transported across the unloading sites of 5 mm diameter . 66 cargo-liposomes were trapped and unloaded from gliding MTs, But 4 cargo-liposomes shaved the edges of unloading sites and continued to be transported. The rate of unloading was 94% and may vary dependent on the experimental conditions such as the strand length used in the DNA hybridization, ssDNA concentrations of the unloading sites, and gliding speed of MTs etc. 2010/11/25

Conclusion… The feasibility of selective, autonomous and parallel loading, transport, and unloading of cargo-liposomes on a chip was demonstrated. In this study, ssDNA-labeled MTs gliding on kinesin-coated surfaces acted as cargo transporters and that ssDNA-labeled nano- or microscale cargo-liposomes were loaded/ unloaded onto/from gliding MTs without bursting at loading reservoirs/micropatterned unloading sites where ssDNAs were micro arrayed. The result shows that MT motility and DNA hybridization are versatile processes towards autonomous delivery of reactor-liposomes, as has been demonstrated for nano- and microspheres. thank you 2010/11/25 A.R