DNA-Mediated Self-Assembly of Artificial Vesicles

Although multicompartment systems made of single unilamellar vesicles offer the potential to outperform single compartment systems widely used in analytic, synthetic, and medical applications, their use has remained marginal to date. On the one hand, this can be attributed to the binary character of the majority of the current tethering protocols that impedes the implementation of real multicomponent or multifunctional systems. On the other hand, the few tethering protocols theoretically providing multicompartment systems composed of several distinct vesicle populations suffer from the readjustment of the vesicle formation procedure as well as from the loss of specificity of the linking mechanism over time.In previous studies, we presented implementations of multicompartment systems and resolved the readjustment of the vesicle formation procedure as well as the loss of specificity by using linkers consisting of biotinylated DNA single strands that were anchored to phospholipid-grafted biotinylated PEG tethers via streptavidin as a connector. The systematic analysis presented herein provides evidences for the incorporation of phospholipid-grafted biotinylated PEG tethers to the vesicle membrane during vesicle formation, providing specific anchoring sites for the streptavidin loading of the vesicle membrane. Furthermore, DNA-mediated vesicle-vesicle self-assembly was found to be sequence-dependent and to depend on the presence of monovalent salts.This study provides a solid basis for the implementation of multi-vesicle assemblies that may affect at least three distinct domains. (i) Analysis. Starting with a minimal system, the complexity of a bottom-up system is increased gradually facilitating the understanding of the components and their interaction. (ii) Synthesis. Consecutive reactions may be implemented in networks of vesicles that outperform current single compartment bioreactors in versatility and productivity. (iii) Personalized medicine. Transport and targeting of long-lived, pharmacologically inert prodrugs and their conversion to short-lived, active drug molecules directly at the site of action may be accomplished if multi-vesicle assemblies of predefined architecture are used

Spatial and spatiotemporal variation in metapopulation structure affects population dynamics in a passively dispersing arthropod

The spatial and temporal variation in the availability of suitable habitat within metapopulations determines colonization-extinction events, regulates local population sizes and eventually affects local population and metapopulation stability. Insights into the impact of such a spatiotemporal variation on the local population and metapopulation dynamics are principally derived from classical metapopulation theory and have not been experimentally validated. By manipulating spatial structure in artificial metapopulations of the spider mite Tetranychus urticae, we test to which degree spatial (mainland-island metapopulations) and spatiotemporal variation (classical metapopulations) in habitat availability affects the dynamics of the metapopulations relative to systems where habitat is constantly available in time and space (patchy metapopulations). Our experiment demonstrates that (i) spatial variation in habitat availability decreases variance in metapopulation size and decreases density-dependent dispersal at the metapopulation level, while (ii) spatiotemporal variation in habitat availability increases patch extinction rates, decreases local population and metapopulation sizes and decreases density dependence in population growth rates. We found dispersal to be negatively density dependent and overall low in the spatial variable mainland-island metapopulation. This demographic variation subsequently impacts local and regional population dynamics and determines patterns of metapopulation stability. Both local and metapopulation-level variabilities are minimized in mainland-island metapopulations relative to classical and patchy ones

Light-Switchable Membrane Permeability in Giant Unilamellar Vesicles

: In this work, giant unilamellar vesicles (GUVs) were synthesized by blending the natural phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with a photoswitchable amphiphile (1) that undergoes photoisomerization upon irradiation with UV-A (E to Z) and blue (Z to E) light. The mixed vesicles showed marked changes in behavior in response to UV light, including changes in morphology and the opening of pores. The fine control of membrane permeability with consequent cargo release could be attained by modulating either the UV irradiation intensity or the membrane composition. As a proof of concept, the photocontrolled release of sucrose from mixed GUVs is demonstrated using microscopy (phase contrast) and confocal studies. The permeability of the GUVs to sucrose could be increased to ~4 × 10-2 μm/s when the system was illuminated by UV light. With respect to previously reported systems (entirely composed of synthetic amphiphiles), our findings demonstrate the potential of photosensitive GUVs that are mainly composed of natural lipids to be used in medical and biomedical applications, such as targeted drug delivery and localized topical treatments

The future of layer-by-layer assembly: A tribute to ACS Nano associate editor Helmuth Möhwald

Layer-by-layer (LbL) assembly is a widely used tool for engineering materials and coatings. In this Perspective, dedicated to the memory of ACS Nano associate editor Prof. Dr. Helmuth Möhwald, we discuss the developments and applications that are to come in LbL assembly, focusing on coatings, bulk materials, membranes, nanocomposites, and delivery vehicles

Biomimetic engineering of colloidal nanoarchitectures with "in vitro" and "in vivo" functionality

Biomimetic engineering opens unprecedented possibilities of combining biomolecules (i.e. proteins, DNA, polysaccharides) with synthetic materials (i.e. synthetic polymers). This combination results in unique hybrid systems with functionalities that mimic processes in living organisms. While the translational value of functional biomimetically engineered structures is of exceptional importance in fields such as technology, engineering, chemistry, biology and medicine, due to the properties the structures inherit from both the synthetic and bio-materials, the understanding of how biomimetically engineered systems self-assemble and function is equally important, as it gives insight in how non-living systems progressed to living organisms. Some of the most prominent examples of functional biomimics include polymersome based catalytic nanocompartments, multicompartment systems that mimic cellular organization and artificial organelles. In this thesis, the focus lies on understanding and applying the fundamental principles of biomimetic engineering by equipping colloidal nanoarchitectures (soft polymer nanoparticles and hollow sphere polymersomes) with functional biomolecules (transmembrane proteins and enzymes). First, the most important questions are addressed – why do polymer nanoarchitectures present ideal building blocks for creating novel biomimics, how do biomimics self-assemble in solution, which methods are most frequently used for their characterization, and where the applications of biomimics are in technology and medicine. Both colloidal and 2D supported/free standing polymeric nanoarchitectures structures are discussed in order to familiarize the reader with the wide range of nanoarchitectures that can be formed by polymers, however the focus primarily rests on biomimetic design of colloidal nanoarchitectures, as their colloidal nature favours them as therapeutic agents that can act on the cellular level. To develop a pH responsive protein delivery agent, a biomimetic approach is applied in equipping self-assembled poly(ethylene glycol)-b-poly(methylcaprolactone)-b-poly(2- (N,Ndiethylamino)ethyl methacrylate) (PEG-b-PMCL-b-PDMAEMA) polymer nanoparticles with a therapeutic enzyme, acid sphingomyelinase. Due to the electrostatic interactions between the negatively charged enzyme and the positively charged PDMAEMA groups present in the nanoparticle corona, the biomimetically engineered nanoparticles display a distinct protein localization on their corona and a pH dependent release behavior of the attached protein. The application of the self-assembled system as a very efficient delivery agent for catalytically active biomolecules is demonstrated in human epithelial HeLa cells. Next, a more complex nanoscale biomimic - a pH triggered catalytic nanocompartment - is built by biomimetically engineering the nanoarchitecture of poly(2-methyl-2-oxazoline)- block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b- PMOXA) polymersomes. Aqueous cavities of polymersomes are loaded with horseradish peroxidase while a chemically modified Outer membrane protein F (OmpF) is reconstituted in polymersome membranes. The chemical modification of OmpF transforms the otherwise unspecific pore into a selective and pH responsive pore, through which molecules can only diffuse once the attached molecular cap blocking the pore is cleaved. Hence, once the modified OmpF is functionally reconstituted in polymersome membranes it allows the developed biomimic to present an on demand catalytic activity. As a first proof of concept of a pH responsive catalytic nanocompartment the system demonstrates that a spatial control of a reaction inside a nanocompartment can be achieved and supports the further development of complex reaction spaces that can act in an analogous manner to cellular compartments, where in situ reactions are modulated by a plethora of responsive proteins. Finally, biomimetically engineered polymersomes are designed for an in vitro and in vivo application as artificial organelles. In order to mimic processes taking place in lipid membranes of cellular organelles, polymersome membranes are equipped with a genetically and chemically modified OmpF. The structural modifications done at the rim of the OmpF pore, limit the OmpF permeability to small molecular weight molecules, but make it capable of responding to the presence of small signaling molecules. When the modified OmpF is reconstituted in membranes of enzyme loaded polymersomes it prevents the enzyme to access enzymatic substrates. However, the presence of of glutathione, which for example is found in abundant concentrations in the cytoplasm, readily cleaves the chemical modification of OmpF and opens the pore, thereby allowing the encapsulated enzyme to catalyze a reaction. The responsiveness of the self-assembled system to glutathione, abundantly present in the cytoplasm, makes the developed biomimic a suitable candidate for intracellular functionality as an artificial organelle. To demonstrate this, we not only show that the system is functional in the cellular microenvironment of human epithelial HeLa cells but also that it is robust enough to function in vivo in Zebrafish embryos

NanoJanus and Nanosatellite Assembly for Biomolecular Delivery and Cancer Therapeutics

Nanotechnology has been utilized widely in medical fields to improve the treatment and diagnosis of several diseases. One of the key players to drive medical nanotechnology forward is nanoparticles, which have been intensively studied and used as a tool for imaging, drug delivery, and disease treatments. Gold and iron oxide, undoubtedly, are on the short list of the nanoparticles used in medical nanotechnology due to their biocompatibility, tunable surface, and unique physico-chemical properties. In this dissertation, we developed novel nanostructures using gold, iron oxide nanoparticles and polymers for various applications including Janus motors, vaccine delivery, and controlled drug release. We generated an asymmetrical Janus nanostructure using thermo-cleavable polymer, gold, and iron oxide nanoparticles for photothermal enhancement and nano motors through an active rotational motion. Gold/iron oxide Janus nanoparticles (JNS) are developed by a seed-mediated self-assembly using a thermo-cleavable polymer facilitating the process. The formed JNS strongly displays an asymmetrical photothermal effect to activate a rotational motion and enhances photothermia resulting in significant cell killing effects under weak near-infrared (NIR) light exposure. In addition, the JNS displays distinct active rotational motion under NIR laser light due to the temperature gradient at its surface, which can be used potentially as Janus motors for drug delivery in the future. We next harnessed the same thermo-cleavable polymer used in JNS formation for controlled drug release under NIR laser light irradiation. The iron oxide nanoparticles (IONP) were first encapsulated in the thermo-cleavable polymeric micelles with doxorubicin (Dox), a chemotherapeutic drug. After NIR trigger, the polymer is cleaved due to heat transfer from the IONP resulting in the release of doxorubicin from the micelles. This study demonstrated that the thermo-cleavable polymer could be used as a smart material for controlled drug release. We also generated another type of secondary structure, a “gold/iron oxide nanosatellite”, using poly (- methacryloxypropyl trimethoxysilane) -b- poly (ethylene oxide) polymer (MPS-b-PEO). This nanosatellite structure, in which IONP is a central core and surrounded by multiple gold nanoparticles as satellites, is used for delivering antigens and an adjuvant for HPV+ head and neck cancer treatment. These nanosatellites deliver high surface density of E7/E6 oncogenic peptides and cyclic- guanosine-adenosine monophosphate (cGAMP) adjuvant to antigen presenting cells (APC) and further activate type I interferon (IFN-I) response. The nanosatellite vaccine also promotes antigen specific CD8+ T cells to infiltrate the tumors and inhibits tumor growth in an HPV+ head and neck tumor model when used as a single therapy or in combination therapy with an anti PD-L1 antibody. Nanosatellites offer many advantages for antigenic peptide and adjuvant delivery such as having a larger surface area, higher antigenic peptide density, higher cell uptake, and lower systemic elimination. This thesis presents the versatile developments and applications of gold/iron oxide nanostructures (Janus and Nanosatellite) which have advantages for drug and vaccine delivery in the future.PHDPharmaceutical SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140893/1/kanokwas_1.pd

Biomimetic membranes as a technology platform: Challenges and opportunities

Biomimetic membranes are attracting increased attention due to the huge potential of using biological functional components and processes as an inspirational basis for technology development. Indeed, this has led to several new membrane designs and applications. However, there are still a number of issues which need attention. Here, I will discuss three examples of biomimetic membrane developments within the areas of water treatment, energy conversion, and biomedicine with a focus on challenges and applicability. While the water treatment area has witnessed some progress in developing biomimetic membranes of which some are now commercially available, other areas are still far from being translated into technology. For energy conversion, there has been much focus on using bacteriorhodopsin proteins, but energy densities have so far not reached sufficient levels to be competitive with state-of-the-art photovoltaic cells. For biomedical (e.g., drug delivery) applications the research focus has been on the mechanism of action, and much less on the delivery ‘per se’. Thus, in order for these areas to move forward, we need to address some hard questions: is bacteriorhodopsin really the optimal light harvester to be used in energy conversion? And how do we ensure that biomedical nano-carriers covered with biomimetic membrane material ever reach their target cells/tissue in sufficient quantities? In addition to these area-specific questions the general issue of production cost and scalability must also be treated in order to ensure efficient translation of biomimetic membrane concepts into reality

Advanced 3D cell culture techniques in micro-bioreactors, Part II: Systems and applications

In this second part of our systematic review on the research area of 3D cell culture in micro-bioreactors we give a detailed description of the published work with regard to the existing micro-bioreactor types and their applications, and highlight important results gathered with the respective systems. As an interesting detail, we found that micro-bioreactors have already been used in SARS-CoV research prior to the SARS-CoV2 pandemic. As our literature research revealed a variety of 3D cell culture configurations in the examined bioreactor systems, we defined in review part one “complexity levels” by means of the corresponding 3D cell culture techniques applied in the systems. The definition of the complexity is thereby based on the knowledge that the spatial distribution of cell-extracellular matrix interactions and the spatial distribution of homologous and heterologous cell–cell contacts play an important role in modulating cell functions. Because at least one of these parameters can be assigned to the 3D cell culture techniques discussed in the present review, we structured the studies according to the complexity levels applied in the MBR systems