Article

Cytosolic Delivery of Functional Proteins In Vitro through Tunable Gigahertz Acoustics

Article

Cytosolic Delivery of Functional Proteins In Vitro through Tunable Gigahertz Acoustics

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Abstract

Intracellular delivery is essential to therapeutic applications such as genome engineering and disease diagnosis. Current methods lack simple, non-invasive strategies, and are often hindered by long incubation time or high toxicity. Hydrodynamic approaches offer rapid and controllable delivery of small molecules, but thus far have not been demonstrated for delivering functional proteins. In this work, we developed a robust hydrodynamic approach based on gigahertz (GHz) acoustics to achieve rapid and non-invasive cytosolic delivery of biologically active proteins. With this method, GHz-based acoustic devices trigger oscillations through a liquid medium (acoustic streaming) generating shear stress on the cell membrane and inducing transient nanoporation. This mechanical effect enhances membrane permeability and enables cytosolic access to cationic proteins without disturbing their bioactivity. We evaluated the versatility of this approach through delivery of cationic fluorescent proteins to a range of cell lines, all of which displayed equally efficient delivery speed (≤ 20 minutes). Delivery of multiple enzymatically active proteins with functionality related to apoptosis or genetic recombination further demonstrated the relevance of this method.

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... 3−5 Despite the success of current protein products that mostly address extracellular targets, efficient cytosolic delivery of bioactive proteins still remains a major challenge. 6−8 Several physical methods have been developed, mainly based on the in vitro induction of transient and rapid cell membrane permeabilization such as electroporation, 9,10 acoustic fields, 11 membrane perforating nanowires, 12 or microfluidic constrictions. 13,14 The emergence of nanotechnology shed light on carrier-dependent protein delivery, 8 where attention has been dedicated to numerous platforms such as inorganic or organic nanoparticles, 15−18 nanogels, 19,20 polymers, 21−30 lipid nanoformulations, 31−33 cell-derived vesicles, 34 and cell-penetrating peptides (CPPs). ...
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It has been postulated that the segregation of nucleus and cytoplasm supported the development of increased organismal complexity. For example, separating transcription and translation allows for mRNA splicing, while the sequestration of genomic DNA supports the innate immune system's ability to equate cytoplasmic DNA with pathogens. Consistent with the importance of nucleocytoplasmic compartmentalization in a broad array of cellular processes, defects in maintaining discrete nuclear and cytoplasmic compartments, either due to loss of nuclear pore complex integrity, disrupted nuclear transport or ruptures of the nuclear envelope, lead to cellular dysfunction, cell death and disease. Here, we discuss recent insights into how loss of compartmentalization can arise as well as the consequences for cellular and organismal homeostasis.
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Hierarchical organization of macromolecules through self-assembly is a prominent feature in biological systems. Synthetic fabrication of such structures provides materials with emergent functions. Here we report the fabrication of self-assembled superstructures through co-engineering of recombinant proteins and nanoparticles. These structures feature a highly-sophisticated level of multi-layered hierarchical organization of the components: individual proteins and nanoparticles co-assemble to form discrete assemblies that collapse to form granules, which then further self-organize to generate superstructures of hundreds of nanometer size. The components within these superstructures are dynamic and spatially reorganize in response to environmental influences. The precise control over the molecular organization of building blocks imparted by this protein-nanoparticle co-engineering strategy provides strategy for creating hierarchical hybrid materials.
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Efficient delivery of genes and therapeutic agents to the interior of the cell is critical for modern biotechnology. Herein, a new type of chemical-free cell poration method— hypersonic poration—is developed to improve the cellular uptake, especially the nucleus uptake. The hypersound (≈GHz) is generated by a designed piezoelectric nano-electromechanical resonator, which directly induces normal/shear stress and “molecular bombardment” effects on the bilayer membranes, and creates reversible temporal nanopores improving the membrane permeability. Both theory analysis and cellular uptake experiments of exogenous compounds prove the high delivery efficiency of hypersonic poration. Since target molecules in cells are accumulated with the treatment, the delivered amount can be controlled by tuning the treatment time. Furthermore, owing to the intrinsic miniature of the resonator, localized drug delivery at a confined spatial location and tunable arrays of the resonators that are compatible with multiwell plate can be achieved. The hypersonic poration method shows great delivery efficacy combined with advantage of scalability, tunable throughput, and simplification in operation and provides a potentially powerful strategy in the field of molecule delivery, cell transfection, and gene therapy.
Article
Genome editing through the delivery of CRISPR/Cas9-ribonulceoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge. Here we report a remarkably high efficient (~90%) direct cytoplasmic/nuclear delivery of Cas9 protein complexed with a guide RNA (sgRNA) through the co-engineering of Cas9 protein and carrier nanoparticles. This construct provides effective (~30%) gene editing efficiency and opens up opportunities in studying genome dynamics.
Article
In this research, a thermo-responsive drug release system was synthesized, which encapsulated the magnetic nanoparticles Fe3O4 and the drug model 5-fluorouracil with thermo-sensitive polymer PNIPAM. Mesoporous SiO2 was used as the channel of drug release, which could enhance the rate of drug loading and reduce the loss of drug. Chitosan (CHI) is a natural cationic linear polymer. The results showed the successful coating of chitosan and rhodamine 6G on the surface of SiO2 sphere. The intermolecular interactions of the nanocomposites were confirmed by Fourier transform infrared spectroscopy. Rhodamine 6G (R6G) is a typical fluorochrome, which could be applied for the cell imaging. Fluorescent imaging studies by confocal laser scanning microscopy (CLSM) indicated that the prepared nanocomposites Fe3O4/PNIPAM/5-Fu@mSiO2-CHI/R6G could specifically target to tumor cells. Therefore, our work showed great potential in drug delivery and cancer therapy.
Article
Protein-based therapeutics have made a significant impact in the treatment of a variety of important human diseases. However, given their intrinsically vulnerable structure and susceptibility to enzymatic degradation, many therapeutic proteins such as enzymes, growth factors, hormones, and cytokines suffer from poor physicochemical/biological stability and immunogenicity that may limit their potential benefits, and in some cases limit their utility. Furthermore, when protein therapeutics are developed for intracellular targets, their internalization and biological activity may be limited by inefficient membrane permeability and/or endosomal escape. Development of effective protein delivery strategies is therefore essential to further enhance therapeutic outcomes to enable widespread medical applications. This review discusses the advantages and limitations of marketed and developmental-stage protein delivery strategies, and provides a focused overview of recent advances in nanotechnology platforms for the systemic delivery of therapeutic proteins. In addition, we also highlight nanoparticle-mediated non-invasive administration approaches (e.g., oral, nasal, pulmonary, and transdermal routes) for protein delivery.
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Combination cancer therapy has attracted considerable attentions due to its advantages of enhancing therapeutic efficacy, decreasing possibility of drug resistance and reducing side effects over monotherapy. Coencapsulation of multiple anticancer agents in one single nanocarrier can optimize their pharmacokinetic profiles and biodistribution behaviors in a spatiotemporal co-delivery fashion, resulting in a more efficient synergistic antitumor activity, compared with the conventional cocktail-based drug mixtures. The images of HE-stained tumor tissue presented that a massive remission of tumor cells occurred after the mice were treated with TRAIL/DOX-fGO. The fluorescence images obtained from the in situ TUNEL staining showed that the highest level of the Alexa Fluor 488-stained apoptotic DNA fragmentation (green) in the tumor tissue of the mice receiving TRAIL/DOX-fGO, suggesting that the pronounced inhibition on tumor growth was attributed mainly to the elevated apoptosis induced by TRAIL/DOX-fGO. Additionally, the histologic images of other organs, such as heart, liver, spleen, lung, kidney, collected from the mice treated with TRAIL/DOXfGO exhibited no obvious pathological abnormalities in all the studied normal organs compared with those treated with PBS.
Article
Regulation of nuclear import is fundamental to eukaryotic biology. The majority of nuclear import pathways are mediated by importin-cargo interactions. Yet not all nuclear proteins interact with importins, necessitating the identification of a general importin-independent nuclear import pathway. Here, we identify a code that determines importin-independent nuclear import of ankyrin repeats (ARs), a structural motif found in over 250 human proteins with diverse functions. AR-containing proteins (ARPs) with a hydrophobic residue at the 13th position of two consecutive ARs bind RanGDP efficiently, and consequently enter the nucleus. This code, experimentally tested in 17 ARPs, predicts the nuclear-cytoplasmic localization of over 150 annotated human ARPs with high accuracy and is acquired by the most common familial melanoma-associated CDKN2A mutation, leading to nuclear accumulation of mutant p16ink4a. The RaDAR (RanGDP/AR) pathway represents a general importin-independent nuclear import pathway and is frequently used by AR-containing transcriptional regulators, especially those regulating NF-κB/p53.
Article
Functional studies in neurons often require controllable simultaneous delivery of different molecules to individual cells within networks. Microinjection represents a suitable and alternative method to deliver cDNAs, oligonucleotides, siRNAs, peptides or antibodies for expression, expression knockdown or loss-of-function studies, respectively. Moreover, molecules can be systematically applied to individual neurons in a controlled manner without affecting neighbouring cells. Establishment of microinjection is often complicated and time consuming. Here we describe a simple and reliable protocol for molecular cell biologists to establish injection of various molecules (ng to μg range) to living neurons in a reasonable period of time.
Article
The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, intracellular delivery of molecules and transfection with plasmid DNA by electroporation is presented using a novel microneedle electrode array designed for the targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50% of cells and transfection of 12% of cells with a gene for green fluorescent protein is demonstrated at high cell viability. It is concluded that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA, and other biopharmaceuticals.
Article
New nanocarrier platforms based on natural biological building blocks offer great promises in revolutionalizing medicine. The usage of specific protein cage structures: virus-like particles (VLPs) for drug packaging and targetted delivery is summarized here. Versatile chemical and genetic modifications on the outer surfaces and inner cavities of VLPs facilitate the preparation of new materials that could meet the biocompatibility, solubility and high uptake efficiency requirements for drug delivery. A full evaluation on the toxicity, bio-distribution and immunology of these materials are envisaged to boost their application potentials.
Article
Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. The potency of functional delivery in some cases can exceed that of other current methods for macromolecule delivery, including the use of cell-penetrating peptides such as Tat and adenoviral delivery vectors. This chapter summarizes methods for engineering supercharged proteins, optimizing cell penetration, identifying naturally occurring supercharged proteins, and using these proteins for macromolecule delivery into mammalian cells.
Article
We have developed a strategy for chromosome engineering in embryonic stem (ES) cells that relies on sequential gene targeting and Cre-loxP site-specific recombination. Gene targeting was first used to integrate loxP sites at the desired positions in the genome. Transient expression of Cre recombinase was then used to mediate the chromosomal rearrangement. A genetic selection relying on reconstruction of a selectable marker from sequences co-integrated with the loxP sites allowed detection of cells containing the Cre-mediated rearrangement. A programmed translocation between the c-myc and immunoglobulin heavy chain genes on chromosomes 15 and 12 was created by this method. This strategy will allow the design of a variety of chromosome rearrangements that can be selected and verified in ES cells or activated in ES cell-derived mice.
Article
Intrahepatic bile ducts (BD) are a critical target of injury in the postischemic liver. Decreased vascular perfusion causes characteristic changes in the morphology of the ductular epithelia including a loss of secondary membrane structures and a decrease in plasma membrane surface area. Using adenosine triphosphate (ATP) depletion of cultured normal rat cholangiocytes (NRC) to model ischemic ducts, the present studies examined the fate of apical membrane proteins to determine whether membrane recycling might contribute to rapid functional recovery. Apical proteins, including gamma-glutamyl transpeptidase (GGT), Na(+)-glucose cotransporter (SGLT1), and apically biotinylated proteins, were not shed into the luminal space during ATP depletion. Instead, labeling of surface proteins after ATP depletion showed a significant decrease in GGT and SGLT1, consistent with membrane internalization. Similarly, z-axis confocal microscopy of biotinylated apical proteins also showed protein internalization. During ATP recovery, SGLT1 transport activity remained profoundly depressed even after 24 hours of recovery, indicating that the function of the internalized apical proteins is not rapidly recovered. These studies suggest that the membrane internalization in ATP-depleted cholangiocytes is a unidirectional process that contributes to prolonged functional deficits after restoration of normal cellular ATP levels. This sustained decrease in transport capacity may contribute to the development of ductular injury in postischemic livers.
Article
The aim of this study was to increase the skin penetration of two drugs, granisetron hydrochloride and diclofenac sodium, using a microelectronic device based on an ablation of outer layers of skin using radiofrequency high-voltage currents. These radiofrequency currents created an array of microchannels across the stratum corneum deep into the epidermis. The percutaneous penetration studies were first performed in vitro using excised full thickness porcine ear skin. An array of 100 microelectrodes/cm(2) was used in these studies. The skin permeability of both molecules was significantly enhanced after pretreatment with the radiofrequency microelectrodes, as compared to the delivery through the untreated control skin. Steady state fluxes of 41.6 micro g/cm(2)/h (r=0.997) and 23.0 micro g/cm(2)/h (r=0.989) were obtained for granisetron and diclofenac, respectively. The enhanced transdermal delivery was also demonstrated in vivo in rats. It was shown that diclofenac plasma levels in the pretreated rats reached plateau levels of 1.22+/-0.32 micro g/ml after 3 h to 1.47+/-0.33 micro g/ml after 6 h, as compared to 0.16+/-0.04 micro g/ml levels obtained after 6 h in untreated rats. Similarly, application of granisetron patches (3% in crosslinked hydrogel) onto rats' abdominal skin pretreated with radiofrequency electrodes resulted in an averaged peak plasma level of 239.3+/-43.7 ng/ml after 12 h, which was about 30 times higher than the plasma levels obtained by 24-h passive diffusion of the applied drug. The results emphasize, therefore, that the new transdermal technology is suitable for therapeutic delivery of poorly penetrating molecules.
Article
Microfluidics-based cell assays offer high levels of automation and integration, and allow multiple assays to be run in parallel, based on reduced sample volumes. These characteristics make them attractive for studies associated with drug discovery. Controlled delivery of drug molecules or other exogenous materials into cells is a critical issue that needs to be addressed before microfluidics can serve as a viable platform for drug screening and studies. In this study, we report the application of hydrodynamic focusing for controlled delivery of small molecules into cells immobilized on the substrate of a microfluidic device. We delivered calcein AM which was permeant to the cell membrane into cells, and monitored its enzymatic conversion into fluorescent calcein during and after the delivery. Different ratios of the sample flow to the side flow were tested to determine how the conditions of hydrodynamic focusing affected the delivery. A 3D numerical model was developed to help understand the fluid flow, molecular diffusion due to hydrodynamic focusing in the microfluidic channel. The results from the simulation indicated that the calcein AM concentration on the outer surface of a cell was determined by the conditions of hydrodynamic focusing. By comparing the results from the simulation with those from the experiment, we found that the calcein AM concentration on the cell outer surface correlated very well with the amount of the molecules delivered into the cell. This suggests that hydrodynamic focusing provides an effective way for potentially quantitative delivery of exogenous molecules into cells at the single cell or subcellular level. We expect that our technique will pave the way to high-throughput drug screening and delivery on a microfluidic platform.
Article
The killer lymphocyte protease granzyme A (GzmA) triggers caspase-independent target cell death with morphological features of apoptosis. We previously showed that GzmA acts directly on mitochondria to generate reactive oxygen species (ROS) and disrupt the transmembrane potential (DeltaPsi(m)) but does not permeabilize the mitochondrial outer membrane. Mitochondrial damage is critical to GzmA-induced cell death since cells treated with superoxide scavengers are resistant to GzmA. Here we find that GzmA accesses the mitochondrial matrix to cleave the complex I protein NDUFS3, an iron-sulfur subunit of the NADH:ubiquinone oxidoreductase complex I, after Lys56 to interfere with NADH oxidation and generate superoxide anions. Target cells expressing a cleavage site mutant of NDUFS3 are resistant to GzmA-mediated cell death but remain sensitive to GzmB.