Intracellular delivery by membrane disruption: Mechanisms, strategies, and concepts

© 2018 American Chemical Society. Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo typesñYsmall molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications and challenges to implementation. Throughout, we highlight specific mechanisms of membrane disruption and suggest areas in need of further experimentation. We hope the concepts discussed in our review inspire scientists and engineers with further ideas to improve intracellular delivery

DESIGN, MICROFABRICATION, AND TESTING OF ALL-PMMA, NANOPORE-BASED ELECTROPHORETIC FLOW DETECTORS FOR BIOMEDICAL APPLICATIONS

ABSTRACT Detection of and discrimination between different nanoparticles and biomolecules are vital steps in analytical, biochemical, and diagnostic biomedical procedures used in life sciences. Synthetic micro/nanopores in solid-state membranes form an emerging class of single-molecule detectors capable of detecting and probing the properties of particles and biomolecules with high throughput and resolution: The particles or biomolecules to be analyzed are added to an electrolyte solution in one of the two reservoirs of the detector system separated by a thin membrane containing a single micro/nanopore. An outer electric field induces an open-pore ionic current (Iopen) through the pore, dragging the particles with itself. Transient changes occur when a particle slightly smaller than the pore translocates through the pore. This electrical signal can be analyzed to derive information regarding to the particle or biomolecule size and even its morphology, concentration in the solution, and the affinity for the pore. Many detectors are based on self-assembled, naturally occurring protein pores in lipid bilayer membranes. Most solid-state pore-based detectors reported in literature use artificial pores in silicon nitride or silicon oxide membranes. Applying polymers as a membrane potentially offers advantages over the aforementioned types, including good electrical insulation, improved wettability thanks to higher hydrophilicity, and long-term stable yet low-cost and disposable devices. The present study aims at exploiting such advantages by developing the proof-of-concept for a single-material, all-polymer, nanopore detector allowing the continuous variation of target pore size in the range from micrometers to a few nanometers for best pore size adaption to the biomolecules to be investigated. The research comprises materials selection, system design, development of a fabrication and assembly sequence, device fabrication, and functional device testing. Poly (methyl methacrylate) (PMMA) was selected as it combines advantageous microfluidic properties know from competing materials, such as polyimide, polystyrene, polycarbonate, or polyethylene terephthalate, with outstanding micropatterning capabilities. The membrane thickness is set to be 1 µm, based on a compromise between robustness during fabrication and operation on one side, and electrochemical performance on the other. After spincoating the membrane onto a sacrificial wafer, pores with diameters of typically several hundred nanometers are patterned by electron beam lithography. In combination with thermal post processing leading to polymer reflow, diameters one order of magnitude smaller can be achieved. The present study focuses on 450 nm and 22 nm pores, respectively. Besides these pores fabricated in a top-down approach, self-assembled -hemolysin protein pores of 1.5 nm diameter are integrated in a combined top-down and bottom-up approach so that single digit, double digit, and triple digit nanometer pores are available. Systems integration is achieved by capillary-forced based release from the sacrificial substrate and the application of UV-initiated glue. Test sequences proved and qualified the device functionality: Electrical characterization was performed in aqueous KCl electrolyte solution. The devices exhibit a stable, time-independent ionic current. The current-voltage curves are linear and scale with the electrolyte concentration. System verification was performed using silica nanospheres of 100 nm and 150 nm diameter as known test particles. Translocation through a 450 nm pore induced current blockades for about 1 ms with an amplitude of 30 pA to 55 pA for 100 nm particles and in excess of 70 pA for 150 nm particles. This is in close agreement with results obtained by a mathematical model used in this study. Biomolecules relevant to many life science applications, double-stranded DNA (dsDNA) and bovine serum albumin (BSA) were subsequently analyzed to prove the device concept. Post-processed pores of 22 nm diameter were used at 600 mV driving voltage and 0.1 molar electrolyte in a slightly acidic regime of pH = 6. Typical current blockade amplitudes for complete translocations of dsDNA are Iblock = 22 pA for a translocation time of tD = 0.2 ms, and an almost threefold current blockade (Iblock = 60 pA) for the larger BSA molecules, respectively. The results demonstrate that the PMMA-based nanopores are sensitive enough to not only detect translocating biomolecules, but to also sense them by distinguishing between different biomolecules. The molecule-specific and distinct translocation signals through the pores using both, standardized silica nanoparticles and biomolecules of different dimensions, prove the concept of an all-PMMA electrophoretic flow detector with adjustable pore diameters. Devices with pore diameters covering three orders of magnitude in the nanometer range were successfully built, tested, and characterized. The results suggest such detectors are promising candidates for biomolecule detecting applications

Aerospace medicine and biology: A cumulative index to the continuing bibliography of the 1973 issues

A cumulative index to the abstracts contained in Supplements 112 through 123 of Aerospace Medicine and Biology A Continuing Bibliography is presented. It includes three indexes: subject, personal author, and corporate source

Construction of recombinant adenoviruses encoding skeletal troponin C protein and expression analyses in transduced cardiac myocytes

Troponin C is a regulatory protein of the myofilament which binds to calcium to trigger the process of contraction. This protein exists in two isoforms, skeletal and cardiac, which are spatially and temporally regulated. Work in this project builds the primary stage of a long-term project, for using the gene transfer method to overexpress the skeletal isoform of Troponin C in cardiomyocytes. The long-term aim is to achieve complete or partial substitution of the native cardiac isoform and study the effects on contractile force produced, in normal and ischemic cardiomyocytes, both in vitro and in vivo. This project has involved designing, constructing and analyzing expression of adenoviral gene transfer vectors overexpressing the sTnC isoform. Several adenoviral vectors were generated with the wild type sTnC gene under the control of muscle-specific promoters. To facilitate analysis of protein expression and its subcellular localization, the sTnC protein was tagged with epitope tags and adenovirus generated, with this gene under the control of constitutive (CMV) and cardiac-specific (HCA) promoters. Epitope-tagged adenoviruses were expressed in vitro using mouse fibroblast (NIH3T3) cells and analyzed by western blot analysis, showing successful constitutive expression. Recombinant adenoviruses containing epitope-tagged-sTnC under the control of the human cardiac actin promoter showed cardiac-specific expression in cultured cardiomyocytes, in situ, using immunocytochemistry. The constitutively-expressing sTnC adenoviral vector showed successful expression in cardiomyocytes in culture, using northern blot analysis. A range of adenoviral vectors have been successfully generated, and constitutive and tissue-specific expression has been established for some of these vectors. Successes attained in this project have established the initial requirements to achieve the long-term goal to alter calcium sensitivity of myofilaments, by overexpression of sTnC isoform in cardiomyocytes, both in vitro and in vivo

2020, UMaine News Press Releases

This is a catalog of press releases put out by the University of Maine Division of Marketing and Communications between January 2, 2020 and December 15, 2020

Maine State Government Administrative Report 1984-1985

https://digitalmaine.com/me_annual_reports/1011/thumbnail.jp