Giant unilamellar vesicles formed by hybrid films of agarose and lipids display altered mechanical properties

AbstractGiant unilamellar vesicles (GUVs) are presumably the current most popular biomimetic membrane model. Preparation of GUVs in physiological conditions using the classical electroformation method is challenging. To circumvent these difficulties, a new method was recently reported, by which GUVs spontaneously swell from hybrid films of agarose and lipids. However, agarose is left encapsulated in the vesicles in different amounts. In this work, we thoroughly characterize the mechanical properties of these agarose-GUVs in response to electric pulses, which induce vesicle deformation and can lead to membrane poration. We show that the relaxation dynamics of deformed vesicles, both in the presence and absence of poration, is significantly slowed down for agarose-GUVs when compared to agarose-free GUVs. In the presence of poration, agarose polymers prevent complete pore closure and lead to high membrane permeability. A fraction of the vesicles were found to encapsulate agarose in the form of a gel-like meshwork. These vesicles rupture and open up after electroporation and the meshwork is expelled through a macropore. When the agarose-GUVs are heated above the melting temperature of agarose for 2 h before use, vesicle response is (partially) recovered due to substantial release of encapsulated agarose during temperature treatment. Our findings reveal potential artifactual behavior of agarose-GUVs in processes involving morphological changes in the membrane as well as poration

To close or to collapse : the role of charges on membrane stability upon pore formation

Resealing of membrane pores is crucial for cell survival. We study membrane surface charge and medium composition as defining regulators of membrane stability. Pores are generated by electric field or detergents. Giant vesicles composed of zwitterionic and negatively charged lipids mixed at varying ratios are subjected to a single strong electric pulse. Charged vesicles are prone to catastrophic collapse transforming them into tubular structures. The spectrum of destabilization responses includes the generation of long-living submicroscopic pores and partial vesicle bursting. The origin of these phenomena is related to the membrane edge tension, which governs pore closure. This edge tension significantly decreases as a function of the fraction of charged lipids. Destabilization of charged vesicles upon pore formation is universal – it is also observed with other poration stimuli. Disruption propensity is enhanced for membranes made of lipids with higher degree of unsaturation. It can be reversed by screening membrane charge in the presence of calcium ions. We interpret the observed findings in light of theories of stability and curvature generation and discuss mechanisms acting in cells to prevent total membrane collapse upon poration. Enhanced membrane stability is crucial for the success of electroporation-based technologies for cancer treatment and gene transfer.Competing Interest StatementThe authors have declared no competing interest

Pengembangan Agrowisata Di Desa Wisata Tulungrejo Kota Batu, Jawa Timur

Meningkatnya konsumsi jasa dalam bentuk komoditas wisata bagi sebagian masyarakat di Indonesia telah menjadi salah satu kebutuhan selaras dengan meningkatnya pendapatan masyarakat. Hal ini melatar-belakangi suatu penelitian yang telah dila-kukan untuk mengidentifikasi potensi perta-nian dalam agrowisata Desa Wisata Tulung-rejo, menentukan jenis atraksi yang dapat dijadikan sebagai atraksi utama dan atraksi penunjang, mengidentifikasi harapan pe-ngunjung terhadap agrowisata, dan menen-tukan upaya pengembangan agrowisata berdasarkan prioritas potensi agrowisata di Desa Tulungrejo. Penelitian ini dilaksana-kan di Desa Tulungrejo, Kecamatan Bumiaji, Kota Batu pada bulan Januari-Mei 2013. Penelitian bersifat deskriptif. Metode yang digunakan antara lain observasi se-cara langsung, wawancara, dan kuesioner. Dilakukan juga analisis SWOT untuk me-nentukan upaya pengembangan agro-wisata. Potensi pertanian agrowisata Desa Tulungrejo terdiri dari budidaya apel, jamur tiram, sayur dan bunga krisan. Atraksi yang dapat dijadikan sebagai atraksi utama adalah atraksi jamur tiram dan atraksi lainnya sebagai atraksi penunjang. Sebesar 74,51% pengunjung mengharapkan adanya perbaikan akses menuju lokasi agrowisata. Upaya pengembangan yang dapat dila-kukan adalah pembuatan jadwal kunjungan, pembuatan brosur, perbaikan akses/jalan menuju lokasi agrowisata, penambahan ke-ragaman atraksi, perbaikan kualitas fasi-litas, dan penambahan jumlah fasilitas

Assessing membrane material properties from the response of giant unilamellar vesicles to electric fields

Knowledge of the material properties of membranes is crucial to understanding cell viability and physiology. A number of methods have been developed to probe membranes in vitro, utilizing the response of minimal biomimetic membrane models to an external perturbation. In this review, we focus on techniques employing giant unilamellar vesicles (GUVs), model membrane systems, often referred to as minimal artificial cells because of the potential they offer to mimick certain cellular features. When exposed to electric fields, GUV deformation, dynamic response and poration can be used to deduce properties such as bending rigidity, pore edge tension, membrane capacitance, surface shear viscosity, excess area and membrane stability. We present a succinct overview of these techniques, which require only simple instrumentation, available in many labs, as well as reasonably facile experimental implementation and analysis

Electrically addressable vesicles: Tools for dielectrophoresis metrology

Dielectrophoresis (DEP) has emerged as an important tool for the manipulation of bioparticles ranging from the submicron to the tens of microns in size. Here we show the use of phospholipid vesicle electroformation techniques to develop a new class of test particles with specifically engineered electrical propserties to enable identifiable dielectrophoretic responses in microfabricated systems. These electrically addressable vesicles (EAVs) enable the creation of electrically distinct populations of test particles for DEP. EAVs offer control of both their inner aqueous core and outer membrane properties; by encapsulating solutions of different electrolyte strength inside the vesicle and by incorporating functionalized phospholipids containing poly(ethylene glycol) (PEG) brushes attached to their hydrophilic headgroup in the vesicle membrane, we demonstrate control of the vesicles’ electrical polarizabilities. This combined with the ability to encode information about the properties of the vesicle in its fluorescence signature forms the first steps toward the development of EAV populations as metrology tools for any DEP-based microsystem.National Institutes of Health (U.S.) (Grant RR199652)National Institutes of Health (U.S.) (Grant EB005753)Merck/CSBi (Fellowship)Solomon Buchsbaum AT&T Research Fun